Epigenetic Regulation of Apoptosis during Development and Tumorigenesis in Drosophila

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Epigenetic Regulation of Apoptosis during Development and Tumorigenesis in Drosophila
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Zhang, Can
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University of Florida
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Medical Sciences, Genetics (IDP)
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Zhou, Lei
Committee Members:
Resnick, James L
Harfe, Brian D
Bungert, Jorg
Terada, Naohiro

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apoptosis -- drosophila -- epigenetics -- irer -- myc
Genetics (IDP) -- Dissertations, Academic -- UF
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Abstract:
The balance between proliferation and apoptosis is essential for tissue homeostasis during animal development. Induction of apoptosis, especially in the context of elevated oncogene expression and overproliferation, has been considered as a major cell-autonomous tumor suppression mechanism. At the same time, emerging evidence suggests that cellular competition–induced apoptosis of cells with lower fitness reflects a surveillance mechanism to optimize the organismal fitness, and is likely also implicated in the process of tumor development. Our previous study has characterized an intergenic region, the IRER (Irradiation Responsive Enhancer Region), required for mediating DNA damage–induced expression of multiple pro-apoptotic genes and cell death. Interestingly, the DNA accessibility of this region, and consequently the responsiveness of the pro-apoptotic genes to DNA damage, is controlled by histone modification. In this study, we provided evidence indicating that the IRER functions as a locus control region for determining cell numbers during development and for the induction of pro-apoptotic genes in response to oncogenic stress. Animals lacking the IRER are viable but have superfluous cells in multiple tissues. The regulatory function of the IRER is also required for the induction of reaper and the compensatory apoptosis following dMyc–induced overproliferation to prevent hyperplasia. Therefore, similar to mammalian systems, overproliferation-induced apoptosis overlaps with the DNA damage response in Drosophila. Cell competition is a “social” behavior which eliminates viable but suboptimal cells via apoptosis. Some growth factors, such as dMyc, can transform cells into super competitors and induce apoptosis in neighboring cells with lower levels of dMyc. We show that the IRER is required for dMyc-induced non-cell autonomous apoptosis by controlling the expression of pro-apoptotic gene hid. In addition,cells mutant for neoplastic tumor suppressor scribble are out competed by surrounding wild type cells through apoptosis.We found that the IRER is required for pro-apoptotic gene expression and cell death in scrib RNAi cells. In the absence of the IRER, the scrib RNAicells generated in larval epithelium are able to escape the competitive stress and grow into neoplastic tumors in adult flies. Moreover, the chromatin conformation of IRER is responsible to the cellular levels of scrib, and is de-repressed in scrib RNAi cells. The increased accessibility of the IRER may render cells more sensitive to competitive stress and favor apoptosis in loser cells. Taken together, our studies provide the mechanistic link between epigenetic regulation of apoptosis and tumorigenesis.
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In the series University of Florida Digital Collections.
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Includes vita.
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by Can Zhang.
Thesis:
Thesis (Ph.D.)--University of Florida, 2012.
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Adviser: Zhou, Lei.
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1 EPIGENETIC REGULATION OF APOPTOSIS DURING DEVELOPMENT AND TUMORIGENESIS IN DROSOPHILA By CAN ZHANG A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2012

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2 2012 Can Zhang

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3 In loving memory of my father

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4 ACKNOWLEDGMENTS Completing my Ph.D. degree has probably been the most challenging task I have undertaken in the first 29 years of my life. I have experienced my best and wor st memories on this remarkable journey. It would not have been possible for me to complete this doctoral dissertati on without the help of the following individuals. First ly I give my utmost gratitude to my father Chuanxin Zhang and my mother Yongqin Shen for bringing me in to the world and educating me with love and courage. I could never have reach ed this far without their parenthood. No such word exists that can express my deepest love and enormous appreciation to them. I would like to thank my advisor Dr. Lei Zhou for his continuous support of my Ph.D. study His patience, inspiration and motivation made my achievements possible He has show n me the curiosity, enthusiasm and persistence a real scientist should carry, and he always encourage s me t o overcome all obstacles an d gui d e s me to become independent in every aspect of my life. I also want to express a special thanks to my dissertation committee: Dr. Jorg Bungert, Dr. Brian Harfe, Dr. James Resnick, and D r. Naohiro Terada, for their valuable suggestions inspiration and continuous support My sincere thanks also go to the formal and current members of Zhou lab oratory especially Dr. Yanp ing Zhang, Dr. Nianwei Lin, Bo Liu, Guangyao Li Hannah Wang, John Pan g, Michelle Chung and Denis Titov f or their kind and patient help. I also would like to thank my IDP classmates and best friends, including Weiyi Ni, Shuibin Lin, Chen Ling, Huiming Xia, Yue Liu and Robert Ng, as well as their families. I am incredibly for tunate to have met them in Gainesville and become lifetime friends. They have filled my life with so much happiness and made my research career much less painful.

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5 Last and most importantly, I want to thank m y husband, Bing Yao, whose love and encourageme nt allow ed me to finish this journey. He always has an unwavering faith in me when I experience tough moments. His company makes my life more d elightful and meaningful. Bing, I hope this work makes you proud.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 9 LIST OF FIGURES ................................ ................................ ................................ ........ 10 LIST OF ABBREVIATIONS ................................ ................................ ........................... 12 ABSTRACT ................................ ................................ ................................ ................... 13 CHAPTER 1 BACKGROUND AND INTRODUCTION ................................ ................................ 15 Regulation of Apoptosis in Drosophila ................................ ................................ .... 15 The Apoptosis Regulatory Pathway in Drosophila ................................ ............ 15 Developmental Cell Deat h in Drosophila ................................ .......................... 16 DNA Damage induced Cell Death in Drosophila ................................ .............. 17 Development al Control of Tissue Growth in Drosophila ................................ ......... 19 Organ Size Control ................................ ................................ ........................... 20 Hippo pathway ................................ ................................ ........................... 20 EGFR pathway ................................ ................................ ........................... 21 Cell Competition ................................ ................................ ............................... 22 Extra Sex Combs, Chromatin, and Cellular Identity ................................ ................ 24 Identi fication of PcG and TrxG Proteins as Chromatin Regulators ................... 24 Chromatin Structures and Epigenetic Regulations ................................ ........... 26 PcG/TrxG Mediate d Chromatin Modifications ................................ .................. 27 Epigenetic Regulation of Apoptosis ................................ ................................ ........ 29 Dysregulation of PcG Targeting ................................ ................................ ....... 29 Epigenetic Silencing of Pro apoptotic Genes ................................ ................... 30 2 MONITORING DNA ACCESSIBILITY AND EPIGENETIC STATUS IN INDIVIDUAL CELLS AND LIVE ANIMALS WITH A FLUORESCENT REPORTER ................................ ................................ ................................ ............ 37 Introduction ................................ ................................ ................................ ............. 37 Materials and Methods ................................ ................................ ............................ 39 Generation of IRER{ubi DsRed} ................................ ................................ ....... 39 Fly Handling ................................ ................................ ................................ ..... 40 DNase I Sensitivity Assay and Chromatin Immunoprecipitation (ChIP) ............ 40 Immunofluorescence ................................ ................................ ........................ 41 Flourometric Measurement of DsRed Expression ................................ ............ 41 Results ................................ ................................ ................................ .................... 42

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7 Inserting the ubi DsRed Reporter into IRER via Homologous Recombination ................................ ................................ .............................. 42 Verifying the Responsiveness of the Reporter to Changes in DNA Accessibility ................................ ................................ ................................ ... 44 Monitoring Cell and Tissue Specific Epigenetic Changes Using IRER{ubi DsRed} ................................ ................................ ................................ .......... 46 Monitoring Epigenetic S tatus of IRER at Organismal Level ............................. 49 Discussions ................................ ................................ ................................ ............. 51 Epigenetic Regulation with Cellular Resolution ................................ ................ 52 Limitations of the Strategy ................................ ................................ ................ 53 3 AN EPIGENETICALLY REGULATED INTERGENIC REGION CONTROLS ORGAN SIZE AND MEDIATES DMYC INDUCED APOPTOSIS IN DROSOPHILA ................................ ................................ ................................ ........ 64 Introduction ................................ ................................ ................................ ............. 64 Materials and Methods ................................ ................................ ............................ 66 Drosophila Strains and C ulture ................................ ................................ ......... 66 Clone Induction ................................ ................................ ................................ 66 Immunohistochemistry and Microscopy ................................ ............................ 66 Wing Size Measurement ................................ ................................ .................. 67 Gene Expression Analysis ................................ ................................ ................ 67 Statistics ................................ ................................ ................................ ........... 68 Bioinformatics ................................ ................................ ................................ ... 68 Results ................................ ................................ ................................ .................... 69 IRER Mediates DNA damage induced Pro apoptotic Gene Expression in Post embryonic Tissues ................................ ................................ ................ 69 The cis Regulatory Function of IRER is Required for Tissue Homeostasis and Organ Size Control ................................ ................................ ................. 70 IRER is Required for the Induct ion of Apoptosis following dMyc i nduced Overproliferation ................................ ................................ ............................ 75 Cells with Relatively Open IRER are More Sensitive to dMyc induced Cell Death ................................ ................................ ................................ ............ 77 Cells Lacking the cis Regulatory Function of IRER have the Propensity to Overgrow and are Resistant to Stress induced Cell Death. .......................... 79 The RHG Genomic Regulatory Block Contains Con sensus Myc Binding Sites ................................ ................................ ................................ .............. 80 Discussions ................................ ................................ ................................ ............. 81 IRER Serves as the Gatekeeper for Overproliferation Induced Apoptosis ....... 82 Functional Significance of Epigenetic Regulation of IRER in Development and in Tumorigenesis ................................ ................................ .................... 85 4 A STRESS RESPONSIVE REGULATORY REGION IS REQUIRED FOR COMPETITION INDUCED APOPTOSIS ................................ ............................. 108 Introduction ................................ ................................ ................................ ........... 108 Materials and Methods ................................ ................................ .......................... 110

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8 Drosophila Strains and Culture ................................ ................................ ....... 110 Clone Induction ................................ ................................ .............................. 110 Microscopy and Immunohistochemistry ................................ .......................... 110 Gene Expression Analysis ................................ ................................ .............. 110 Results ................................ ................................ ................................ .................. 111 IRER is Required for Super Co mpetition induced Elimination of Loser Cells 111 IRER is Required for Super Competition induced hid Expression in Loser Cells ................................ ................................ ................................ ............ 111 IRER is Required for the Elimination of scrib Knockdown Cells ..................... 112 Disruption of Cell Polarity led to de Repression of IRER ................................ 114 Discussions ................................ ................................ ................................ ........... 115 Myc induced Cell Autonomous Apoptosis vs. Cell Non autonomous Apoptosis ................................ ................................ ................................ .... 115 Epigenetic Regulation and Cell Comp etition ................................ .................. 116 5 DISCUSSION AND PERSPECTIVE ................................ ................................ ..... 127 Coordinated Transcriptional Regulation of the RHG Genes by Locus Control Regions ................................ ................................ ................................ ............. 127 Epigenetic Regulation and Cancer ................................ ................................ ....... 128 Is Cell Competition Relevant to Cancer? ................................ .............................. 130 Unraveling Epigenetic Regulation and Tumorigenesis in Drosophila .................... 132 Perspectives ................................ ................................ ................................ ......... 134 What Mechanism(S) Contr ols the Epigenetic Regulation of IRER? ............... 135 Which Signaling Pathways Regulate the Function IRER? .............................. 135 What is the Relevance of our Study to Human Research? ............................ 136 LIST OF REFEREN CES ................................ ................................ ............................. 138 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 154

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9 LIST OF TABLES T able page 1 1 Many histone modifiers are evolutionary conserved and implicated in tumorigenesis. ................................ ................................ ................................ .... 35 2 1 List of RNAi li nes used for the screen of epigenetic modifiers of IRER. ............. 63 3 1 Drosophila strains used in this study ................................ ................................ .. 89

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10 LIST OF FIGURES Figure page 1 1 Apoptotic pathways in Drosophila mammals and C.elegans ............................ 32 1 2 PcG and TrxG proteins regulate gene expression by modulating chromatin structure. ................................ ................................ ................................ ............ 33 1 3 Schematic diagram summarizing previous findings regarding the IRER. .......... 34 2 1 Generation of IRER{ubi DsRed}. ................................ ................................ ........ 55 2 2 The expression of IRER{ubi DsRed} from embryos and larvae. ......................... 56 2 3 Verification of the IRER{ubi DsRed} reporter for DNA accessibility and epigenetic status of IRER. ................................ ................................ ................. 57 2 4 Monitoring the change of epigenetic status of IRER in wing discs. .................... 58 2 5 The expression pattern of the ubi DsRed reporter is lar gely controlled by the local chromosome environment. ................................ ................................ ......... 59 2 6 Su(var)3 9 knockdown induced DsRed expression is not due to abnormal activation of the ubiquitin promoter. ................................ ................................ .... 60 2 7 The expression of IRER{ubi DsRed} is responsible to HDAC. .......................... 61 2 8 Monitoring the epigenetic status of IRER in different genetic backgrounds and in response to dietary restriction (DR). ................................ ....................... 62 3 1 IRER mediates DNA damage induced reaper and hid expression in the developing wing discs. ................................ ................................ ........................ 90 3 2 IRER is required for the control of imaginal disc size. ................................ ....... 91 3 3 The cis regulatory function of IRER is required for organ size control ............... 92 3 4 Differentially increased compartment size in Df(IRER) wings. ............................ 94 3 5 Homozygous Df(IRE R) embryos have enlarged central nervous systems. ........ 95 3 6 Decreased expression levels of reaper in homozygous Df(IRER) embryos. ..... 96 3 7 IRER is required for dMyc induced cell death. ................................ .................. 97 3 8 Cell autonomous role of IRER regulates dMyc induced overgrowth. ................ 99 3 9 Cells with an open IRER are more sensitive to dMyc induced cell death. ....... 100

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11 3 10 Su(var)3 9 is required for the epigenetic silencing of IRER in wing discs. ........ 101 3 11 Epigenetic silencing of IRER is responsive to stress. ................................ ....... 102 3 12 Cells lacking IRER have the propensity to overgrown and are resistant to stress induced cell death. ................................ ................................ ............... 103 3 13 Df(IRER) clones overgrow their twin spots in the eye discs. ............................ 105 3 14 High order chromatin organization brings IRER into the proximity of multiple RHG gene promoters. ................................ ................................ ...................... 106 3 15 Diagram su mmarizing the mechanism of IRER ................................ ............... 107 4 1 IRER is r equired for super competition induced elimination of loser cells. ..... 120 4 2 IRER is required for super competition induced hid expression in loser cells. 121 4 3 IRER is required for the elimination of scrib knockdown cells. ......................... 122 4 4 Scrib knockdown leads to differentiation defect s of photoreceptor cells. ......... 123 4 5 Scrib knockdown phenotype in eyes. ................................ .............................. 124 4 6 IRER is required for scrib RNAi i nduced reaper and hid expression from eye discs. ................................ ................................ ................................ ................ 125 4 7 Disruption of cell polarity leads to de repression of IRER. ............................... 126

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12 LIST OF ABBREVIATION S AEL After egg lay ing ChIP Chromatin Immuno precipitation CNS Central nervous system Cyc C yclin DIAP Drosophila inhibitor of apoptosis protein H3K9me3 Trimethylated histone 3 lysine 9 H3K27me3 Trimethylated histone 3 lysine 27 Hdac1 Histone deacetylase 1 HMTase H istone me thyltransferase IBM IAP binding motif IRER Irradiation responsive enhancer region JNK c Jun amino terminal kinase NDR Nuclear Dbf2 Related NTS Neoplastic tumor suppressor PcG Polycomb group PRE Polycomb response elements PUMA p53 upregulated modulator of apoptosis QPCR Quantitative polymerase chain reaction Su(Hw) Suppressor of h airy w ing Su(var)3 9 Suppressor of variegation 3 9 TNF T umor necrosis factor trxG T rithorax group TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling UAS Upstream activation sequence

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13 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Part ial Fulfillment of the Requirements for the Degree of Doctor of Philosophy EPIGENETIC REGULATION OF APOPTOSIS DURING DEVELOPMENT AND TUMORIGENESIS IN DROSOPHILA By Can Zhang August 2012 Chair: Lei Zhou Major: Medical Sciences -Genetics The balance be tween proliferation and apoptosis is essential for tissue homeostasis during animal development. Induction of apoptosis, especially in the context of elevated oncogene expression and overproliferation, has been considered as a major cell autonomous tumor s uppression mechanism. At the same time, emerging evidence suggests that cellular competi tion induced apoptosis of cells with lower fitness reflects a surveillance mechanism to optimize the organismal fitness, and is likely also implicated in the process of tumor development Our previous study has characterized an intergenic region, the IRER ( I rradiation R esponsive E nhancer R egion), re quired for mediating DNA damage induced expression of multiple pro apoptot ic genes and cell death Interestingly, the DNA a ccessibility of this region, and consequently the responsiveness of the pro apopt otic genes to DNA damage, i s contr olled by histone modification. In this study, we provided evidence indicating that the IRER functions as a locus control region for determini ng cell numbers during development and for the induction of pro apoptotic genes in response to oncogenic stress Animals lacking the IRER are viable but have superfluous c ells in multiple tissues. The regulatory function of the IRER is also required for th e induction of

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14 reaper and the compensatory apoptosis following d Myc induced overproliferation to prevent hyperplasia. Therefore, similar to mammalian systems, overproliferation induced apoptosis overlaps with the DNA damage response in Drosophila Cell c eliminate s viable but suboptimal cells via apoptosis. Some growth factors, such as dMyc, can transform cells into super compe titors and induce apoptosis in neighboring cells with lower levels of dMyc. We show that th e IRER is re quired for dMyc induced non cell autonomous apoptosis by controlling the expression of pro apoptotic gene hid In addition, cells mutant for neoplastic tumor suppressor scribble are out competed by surrounding wild type cells through apoptosis We found that the IRER is required for pro apoptotic gene expression and cell death in scrib RNAi cells. In the absence of the IRER, t he scrib RNAi cells ge nerated in larval epitheliu m are able to escape the competitive stress and grow into neoplastic tumor s in adult flies. Moreover, the chromatin conformation of IRER is responsible to the cellular levels of scrib and is de repressed in scrib RNAi cells. The increased accessibility of the IRER may ren der cells more sensitive to competitive stress and favor ap optosis in loser cells. Taken together, o ur studies provide the mechanistic link between epigenetic regulation of apoptosis and tumorigenesis

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15 CHAPTER 1 BACKGROUND AND INTRO DUCTION Regulation of A poptosis in Drosophila Apoptosis is a tightly regulated an d conserved process of c ell suicide in mult icellular organisms. The apoptotic cells are c haracterized by a series of morphological changes including cell shrinkage, chromatin condensation and nuclear fragmentation ( Kerr et al ., 1972 ) During normal development, a poptosis helps to remove cells that are no longer needed to sculpt organs. In adult tissues, cell death balances perfectly with cell division to adjust cell number and maintain organ size. In addition, the damaged an d potentially dangerous cells need to be removed via apoptosis. Genetic or epigenetic deregulation of this physiological process leads to severe developmental defects an d diseases such as tumors and neurodegenerat ive disease ( Baehrecke, 2002 ) Drosophila h as made a great contribu tion to our understandi ng of the regulation of cell death practicability in large scaled genetic screen s low gene redundancy, and the availability of powerful genetic tools. The Apoptosis Regulatory Pathway in Drosophila The compon ents and mechanisms of apoptosis are highly conserved from flies to human s (Figure 1 1 ). In general, the final execution of ap optosis requires activation of c aspases such as the initiator caspase D ronc and Dredd ( Chen et al., 1998 ; Quinn et al., 2000 ; Xu et al., 2005 ) and the effector caspases Drice and D cp 1 ( Xu et al., 2006 ) However, the fact that caspases are ubiquitously expressed in all living cells suggests caspases are simultaneously subject to negative regulation ( Kornbluth and Whi te, 2005 ) Indeed, t he inhibitor of apoptosis proteins (IAPs) are identified as brakes on

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16 death inhibit caspase activation and therefore suppress apoptosis ( Salvesen and Duckett, 2002 ; Vaux and Silke, 2005 ) In fruit flies Drosop hila IAP 1 (Diap 1) plays an essential role in protect ing cells from inappropriate caspase activation and constitutive apoptosis A ll cells die synchronously in embryos lacking functional Diap1 ( Chandraratna et al., 2007 ; Goyal et al., 2000 ; Wang et al., 1999 ) On the other hand, in response to apoptotic signals or developmental cues a group of pro apoptotic proteins are transcriptionally upregulated in doomed cells to inhibit IAP and induce apoptosis ( Song and S teller, 1999 ) Four pro apoptotic genes including reaper ( White et al., 1994 ) head involution defective ( hid) ( Grether et al., 1995 ) grim ( Chen et al., 1996 ) and sickle ( Christich et al., 2002 ; Srinivasula et al., 2002 ; Wing et al., 2002 ) have been identified in the Drosophila genome through genetic screens and microarray studies Coincidently, all four pro apoptotic genes reside in a highly conserved ~350 kb genetic regulatory block share the same transcription orientation and are often coordinate ly regulated during apoptosis ( Lin et al., 2009 ) The proper transcriptional and translational re gulation of pro apoptotic genes is essential for triggering apopto sis in cells destined to die. Developmental Cell D eath in Drosophila Apoptosis plays crucial roles throughout the entire Drosophila development The earliest apoptotic event s start at embry onic stage 11 (about 7 hours after egg laying (AEL)) in the precephalic region and quickly spreads to other segments by stage 12 14 (8 10 hours AEL) ( Abrams et al., 1993 ) It has been well appreciated that the segmental repeated pattern of apoptosis helps to establish the precise border of segmentation in the late stage embryo ( Pazdera et al., 1998 ) Indeed, t he temporal and spatial pattern of embryonic cell death associates with and contributes to certain developmental events

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17 such as head involution and germ band retraction In post emb ryonic tissues such as the eye imaginal discs, cell death occuri ng between 24 and 30 hours after pupal formation (APF) is required to remov e surplus interommatidial cells; d isruption of this process leads to a rough eye phenotype ( Wolff and Ready, 1991 ) During metamorphosis, larval tissu es are reorganized and develop into adult structure s. Many larval structures such as salivary glands, are no longer needed and are removed by ecdysone mediated programmed cell death (PCD) ( Jiang et al., 1997 ) In newly emerged adult flies, PCD is initiated to remove wing epithelial cells at the time of wing spreading ( Kimura et al., 2004 ) In addition to these normal developmental events apoptosi s also plays a ro le in removing cell s incorrectly specified during development ( Werz et al., 2005 ) DNA Damage induced Cell Death in Drosophila Although c ells h ave evolved with a series of mechanisms to preserve genome stability, the integrity of DNA is still frequently challenged by a wide variety of insults Cells are faced with the decis ion to either undergo DNA repair to correct the damage, or initiate apopto sis to eliminate themselves from the organism ( Friedberg, 1995 ) Defects in the appropriate a poptotic response may lead to an accumulation of cells carrying mutations and genomic instability which can potentially have devastating consequences for the organism such as tumorigenesis Ionizing radiation is able to induce single and double strand breaks in DNA and such DNA lesions need to be repaired by appropriate repair mechanisms or be eliminated through apoptosis. A high dosage of irradiation (e.g 40Gy) reliably i nduces apoptosis within 20 minutes in early stage Drosophila embryos which involves the induction of pro apoptotic genes r eaper hid and sickl e ( Brodsky et al., 2004 ; White et

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18 al., 1994 ; Zhang et al., 2008 ) Although the imaginal discs from third in star larvae show very little developmental apoptosis ( Milan et al., 1997 ) they respond w ith elevated levels of apoptosis as soon as 4 hours after irradiation, as recognized by TUNEL and cleaved Caspase 3 immunostaining ( Perez Garijo et al., 2004 ) Because of its ability to elicit a rapid apoptotic response and its broad applicatio n to cancer therapy, ionizing radiation has been widely used as a tool to study the molecular mechanisms and signal transductions of DNA damage induced cell death. Abundant evidence suggests that transcriptional mechanisms play crucial roles in regulating DNA damage induced apoptosis. The genetic requirement of transcription factors such as P53 in irradiation induced cell death underscores the importance of the transcriptional machinery Like its mammalian ortholog, the Drosophila P53 (DmP53) is also requir ed for the DNA damage response Following irradiation and many other types of stress, cellular levels of DmP53 increase dramatically In addition, irradiation induced immediate apoptosis is totally abolished in flies DmP53 null mutant s ( Brodsky et al., 2004 ; Sogame et al., 2003 ) Genetic and microarray analysis performed on Drosophila embryos indicate that al l irradiation responsive genes are direct or indirect transcriptional targets of DmP53, including pro apoptotic genes reaper hid and sickle ( Brodsky et al., 2004 ) While P53 is required for a rapid induction of cell death following DNA damage, P53 in dependent mechanisms do exist in both mammals and fruit flies albeit with a reduced and delayed response ( Urist et al., 2004 ; Wichmann et al., 2006 ) In flies, DmP53 inde pend ent apoptosis proves to be hid dependent It works in parallel with the DmP53 dependent pathwa y to limit DNA damage induced aneuploidy during development ( McNamee and Brodsky, 2009 )

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19 In addition to the P53 pathway, the evolutionarily conserved c Jun N terminal kinase (JNK) signal cascade also plays a crucial role in stress response JNK activity, when monitored in vivo with an enhancer trap line of puc lacZ, is strongly induced in the wing discs by i rradiation (puc, puckered is a downstream target of the JNK pathway ) Moreover, radiation induced a poptosis i s greatly attenuated when the JNK pathway is blocked suggesting the vital role of JNK in irradiation induced apoptosis ( McEwen and Peifer, 2005 ) In addition to irradiation stress JNK also regulates a poptosis in response to many other stresses, such as UV exposure ( Jassim et al., 2003 ) ectopic morphogens expression ( Adachi Yamada et al., 1999 ) and growth factor withdrawal ( Luo et al., 2007 ) Development al Control of Tissue Growth in Drosophila How to achieve proper organ size is a fundamental question in developmental biology. Cell growth, cell division, and cell death have to be coordinately regulated by physiological cues to maintain homeostasis in multicellular organisms ( Conlon and Raff, 1999 ) Genetic studies from invertebrates and vertebrates strongly sug gest the existence of an intrinsic signal that stops growth at the right time during development. One piece of evid ence comes from a famous liver regeneration test performed in 1930s: when two thirds of a rat liver was removed the remaining part of the liver regenerate d and eventually mad e up the lost mass ( Michalopoulos and D eFrances, 1997 ) Since then, t he molecular mechanisms of organ size control have been intensively studied during the past decade and conspicuous progress has been made. This has expand ed our knowledge of how cell growth and cell death are balanced in nor mal development, and how a disturbed balance contributes to d isease development

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20 Organ Size Control Hippo pathway Genetic screens in Drosophila led to the identification of an evolutionarily conserved Hippo signaling pathway which is essential for organ size control ( Pan, 2010 ) The core components of this pathway include the Ste20 like kinases Hippo (Hpo) and its co activator Salvador (Sav) the NDR family protein kinase Warts (Wts) and its adaptor protein Mob as a tumor suppressor ( M ats ) and the transcriptional co activator Yorkie (Yki). These proteins function in a kinase cas c ade to control organ size Hpo and Sav physically interact with each other to phosphorylate and activate Wts, which in turn phosphorylates Yki with the help of Mats. The non phosphorylated Yki is able to enter the nucleus to mediate transcription of its target genes, such as the growth regulator dMyc, the cell cycle p romoting factor cycE, the apoptosis inhibitor Diap1, and the anti apoptotic microRNA bantam and others Upon phosphorylation, Yki stalls in the cytoplasm and thus fails to activate target gene transcription. Therefore, Yki is a growth promoter whereas Sav/ Hpo/Wts function as tumor suppressors to simultaneously restrict proliferation and promote apoptosis. Indeed, most components of the Hippo pathway were originally identified as hyperplastic tumor suppressors. Clones of Hpo, Sav or Wts mutant cells display increased proliferation and are relatively resistant to apoptosis. Consequently, animals carrying these mutant clones develop enlarged tissues without differentiation defects ( Halder and Johnson, 2011 ; Hariharan and Bilder, 2006 ; Pan, 2010 ; Zhao et al., 20 11 ) Studies over the past few years have uncovered several upstream regulators that positively regulate the Hippo pathway, including the cytoskeleton protein s Merlin (Mer) and Expanded (Ex) ( Hamaratoglu et al., 2006 ) their binding partner Kibra ( Baumgartner

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21 et al., 2010 ; Genevet et al., 2010 ; Yu et al., 2010 ) the trans membrane protein s Crumbs (Crb) ( Chen et al. 2010 ; Robinson et al., 2010 ) Fat (Ft) and Dachsous (Ds) ( Bennett and Harvey, 2006 ; Cho et al., 2006 ; Silva et al., 2006 ; Willecke et al., 2006 ) It is interesting to notice that unlike the co re components in the Hippo pathway (i.e. Sav/Hpo/Wts), mutations in each of these upstream regulator s fail to induce a strong overgrown phenotype, suggesting that they may co operate with each other to regulate Hippo signal The list of upstream regulators was further expanded by including a group of cell polarity proteins, such as the atypical protein kinase C (aPKC) and the Scribble (Scrib) Lethal giant larvae (Lgl) Disc large (Dlg) protein complex ( Chen et al., 2010 ; Grzeschik et al., 2010 ; Ling et al., 2010 ; Robinson et al., 2010 ) The se pla y ers could regu late the Hippo pathway by affecting the cellular localization of Hpo and the transcriptional activity of Yki. These studies implicate the cooperation of cell polarity, cell contact and cell growth in organ size control Further studies are required to uncover the molecular mechanisms and biological significance of how polarity factors regulate cell growth via the Hippo signal ing pathway EGFR pathway M any organs develop to appropriate and reproducibl e size in spite of a considerable plasticity in cell growth rates. This suggests the organ size regulation migh t employ certain secreted extra cellular signals such as morphogens from neighboring tissues or compartments. Such a mechanism has been nicely cha racterized and shown to control compartment size in Dr osophila embryos Spi/EGF R is secreted from neighboring anterior compartment s in limited amount s and its up taken by cells in posterior compartment s stimulates their growth and proliferation. However, t he increased cell proliferation eventually causes a drop of cellular levels of Spi which in

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22 turn leads to reduced cell size and compensatory apoptosis, leaving the entire compartment size unchanged ( Parker, 2006 ) The EGFR signal promotes cell surv ival by activating a downstream MAP kinase signal, which can phosphorylate and inactivate Hid protein to block Hid ind uced apoptosis ( Bergmann et al., 1998 ) Therefore, insufficiency of Spi/EGFR signal may trigger apoptosis by releasing Hid activity. Thus, in organs where cell survival is growth factor dependent, apoptosis regulation presumably plays a key role in controlling organ size. Cell Competition Recent studies of flies have shed light on how proliferation signal s and death signal s are coupled to determine cell fates in a growing tissue In one study, de la Cova and colleagues reported that local expression of proto oncoge ne dMyc is sufficient to induce non cell autonomous apoptosis in neighboring wild type cell s, through a process called cell competition Cell competition happens when cells with different fitness meet each other in the same compartment, and results i n the expanding of stronger cells at t he expense of the suboptimal pop ulation ( de Beco et al., 2012 ) The removal of viable but less fitted cells through cell competition may reflect a process of positive selection which ensure s accumulation of cells with higher fitness in the organism The first evidence of cell competition was obtained from a series of Minute (M) mutations targeting ribosome p rotein co ding genes. C ells heterozygous for Minute (M/+) have defects in ribosomal f unction and are vigorous ly out competed by wild type cells ( Morata and Ripoll, 1975 ) When clones of M/+ cells are induced in otherwise wild type wing discs, the M/+ cells fail to be recovered from the adult wing due to disproportional elimination by apoptosis ( Moreno et al., 2002a ) Cell competition also happens under many other circumstances. For example, mutations of Hpo/Sav/Wats or over

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23 expression of Yki can transform cells into super compet itors who out compete surrounding wild type cells. In addition, mutations of several Hippo pathway components, including hpo sav mats warts ex and fat allow further survival of M/ cells, suggesting them as important regulators for cell competition ( Tyler et al., 2007 ) It has been postulated that Hippo induced competition is attributable to the differential expression of its target gene dMyc, a hypothesis which needs to be further verifie d. Nonetheless, the discovery that imbalances in the Hippo pathway affects cell competition shed lights on the involvement of cell competition in growth regulation and tumor progression. One consequence of cell competition is the eliminat ion of loser cells via apoptosis, which requires the activation of JNK signal. However, the contribution of JNK to loser cell death varies depending on situation I n Minute or dMyc induced cell competitio n, JNK seems to play a minor role in triggering cell death, as evidenced by continued loser cell death even when the JNK signal is totally blocked ( de la Cova et al., 2004 ; Moreno and Basler, 2004 ; Moreno et al., 2002a ) Conversely JNK is absolutely required and responsible for eliminating cells mutan t for neoplastic tumor suppressor s (NTS s) The Drosophila TNF homologue Eiger (Egr) is up regulated from neighboring wild type cells and /or hemocytes, which is further taken up by the NTS mutant cells to activate JNK signal ( Igaki et al., 2009 ) In the absence of Eiger or JNK, these NTS mutant cells are able to escap e from competitive stress and grow into neoplastic tumor. Recent studies have revealed additional r oles of JNK in inducing cel l death following competition First, JNK is able to enhance Hippo activity in NTS mutant clones, which leads to decreased expression of Hippo target genes including dMyc The reduced dMyc signal could in

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24 turn boost their elimina tion ( Chen et al., 2012 ) In addition, JNK may also play a role in promoting the compensatory proliferation from winner cells ( Fan and Bergmann, 2008 ; Ryoo et al., 2004 ) Although many advances have been made in our understanding of the molecular mechanisms of cell compet ition, there are still a lot of unanswered questions. For example, how the cells are identified as winner and loser statu s when they confront each other, what is the physiological significan ce of adapting cell competition, whether there is a common cause o f competition in different situations, how the activated JNK signal is transfor med to death consequence, etc. U nderstanding these question s will help us to draw a more comprehensive picture of the competition phenomenon, and more importantly, to transform our knowledge to clinical research and disease treatment Extra Sex Combs, Chromatin, and Cellular I dentity Identification of PcG and TrxG Proteins as Chromatin Regulators The accurate placement of segmental structures along the anterior posterior axis o f the animal body is defined b y the highly conserved homeotic (Hox) gene family Dysregulation of Hox gene s lead to homeotic tr ansformation, that is transformation of one bo dy segment into another ( Pearson et al., 2005 ) Therefore, in order to maintain cellular identity, the Hox gene expression pattern has to be properly established and tightly mainta ined In Drosophila the Hox gene expression is initiated by transient transcriptional factors encoded by gap genes and pair rule genes in early embryogenesis. However, the transient transcription factors disappear soon after turning on the Hox genes. Deve lopment of an organism requires a cellular system to remember the appropriate Hox gene expression status ascribed to individual cell.

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25 Genetic analysis unraveled two groups of gene s affecting cellular identity : P olycomb group (PcG) and T rithorax group (T rxG). The first two PcG genes Extra sex combs ( Esc ) and Polycomb ( Pc ), were described in Drosophila by Lewis and colle a g u es in the 1940s. They were named after the mutation phenotype: male flies grew extra sex combs on the second and third legs, which w ere usually restricted to the first legs ( Lewis, 1978 ; Lewis, 1947 ) Further mechanistic studies revealed that the PcG mutant cells inappropriately reactivate specific Hox genes which should ha ve been repressed transforming one body segment in to another ( Jurgens, 1985 ; Struhl, 1983 ) This failure in the cellular memory system leads to the idea that PcG function s to maintain the repressed state of Hox gene ( Pirrotta, 1997 ) Although originally identified in Drosophila PcG functi on has been shown to be fairly well conserved throughout evolution : several Polycomb mutants in mice exhibit anterior posterior transfo rmations and other abnormalities of the axial skeleto n ( Akasaka et al., 1996 ; Core et al., 1997 ; del Mar Lorente et al., 2000 ) The TrxG proteins have been characterized as an antagonistic system of PcG proteins which set up an active state for the Hox gene. In the absence of Trithorax ( TRX ) the be st characterized member in TrxG, multiple homeotic genes become repressed in a PcG dependent fashion from cells where they are express ed in early stage embryos. Consequently, flies show segmental transformations, similar to the phenotypes of Hox gene muta nt s ( Breen and Harte, 1991 ; Orlando and Paro, 1995 ) Therefore, PcG and TrxG work together, through either repressive or activating mechanisms, to achieve the appropriate temporal and spatial pattern of Hox gene expression. Although PcG and TrxG proteins were firstly discovered to regulate Hox

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26 genes, further studies have identified a variety of target genes in volved in stem cell maintenance, cell cycle control, apoptosis, etc. Genetic lesions inducing inappropriate PcG and TrxG activity may perturb these fundamental biological processes, leading to pathogenesis such as tumo r ( Mills, 2010 ) Chromatin Structures and Epigenetic Regulations the heritable chan ges of gene expression pattern that are not caused by changes of primary DNA sequence. Epigenetic inheritance can be produced by distinct mechanisms, such as DNA methylation, chromatin modifications, a nd non coding RNA. Here we f ocus on the epigenetic regulati on through the modification of chromatin structures. In eukaryotic cells, DNA wraps around core histone octamers to form nucleosomes, which are further fold ed into higher order chromatin. Different chromatin conformations are usually associated with varyi ng DNA accessibility and transcriptional potential resistant to transcriptional activity. The impac t of heterochromatin configuration on gene silencing has been noticed decades ago through the study of position effect variegation (PEV), which reveals that gene activity is dependent on its position relative to a heterochromatin region on chromosome. When a Drosophila gene required for red eye pigmenta tion was placed in proximity to the pericentric heterochromatin, it became silenced in a subset of cells and resulted in a mosaic eye color ( Muller, 1932 ) The chromatin structures can be changed by histone modifications. The N terminal tails of core histones that protrude from nucleosomes are subject to a variety of post translational covalent modifications. The histone modifications provide a scaffold for the

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27 recruitment of regulatory proteins or chromatin remodeling factors, which in turn define distinct ch romatin states ( Jenuwein and Allis, 2001 ) For example, trimethylation at lysine 9 or lysine 27 on histone H3 serves as repressive histone mark for the transcriptionally silent heterochromatin, whereas trimethylation at lysine 4 on histone H3 as well a s acetylation on histone H3 has been closely linked transcriptionally active chromatin. PcG /Trx G Mediated Chromatin Modifications In flies, there are at least two types of multi protein complexes working together to conduct PcG mediated silencing. They are referred to as the Polycomb Repressive Complex 1 and 2 (PRC1 and PRC2), which function to main tain and establish the silenced chromatin st ates, respectively. The more recently characterized PhoRC mainly contains PHO/PHOL and SFMBT (Scm related gene containing four MBT domains), which may process DNA binding properties ( Schwartz and Pirrotta, 2007 ) The biochemically purified Drosophila PRC2 core complex consists of Enhancer of zeste (E(Z)), Suppressor of z este 12 (SU(Z)12) and Extra sex comb (ESC). E(Z) carries an enzymatically active SET domain, which has histone methyltransferase activity and is able to catalyze trimethylation at lysine27 of histone H3 (H3K27Me3), a repressive histone mark associated with gene silencing ( Cao et al., 2002 ; Czermin et al., 2002 ; Mller et al., 2002 ) The Drosophila PRC1 complex is more diverse and mainly contains Polycomb (PC), Posterior sex combs (PSC), Polyhomeotic (PH) and RING ( Sau rin et al., 2001 ; Shao et al., 1999 ) The Chomodomain in PC specifically recognizes the H3K27Me3 established by PRC2, and functions to stabilize the repressed status of chromatin. The core components o f mammalian PRC1 and PRC2 are very similar to

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28 those in Drosophila bu t involve more paralogs (T able 1 1 ), which may function as alternatives to target different genes or different tissues ( Levine et al., 2002 ) How PRCs modulate transcriptional silencing remains to be fully understood. The potential mechanisms include facilitating chromatin compaction, impeding RNA Pol II initiation and elongation, recruiting DN MTs (DNA methyltransferases) to target genes, as well as blocking the modulation of SWI/SNF chromatin remodeling complex ( Sparmann and van Lohuizen, 2006 ) In addition, the RING finger domain of RING protein possesses E3 ubiquit in ligase activity and induces mono ubiquitinat ion of H2AK119, which is also associated with gene silencing ( Cao et al., 2005 ; de Napoles et al., 2004 ; Wang et al., 2004 ) Similar to PcGs, TrxG pro teins also form multi component complexes. They maintain an active chromatin state through either direct histone modification or ATP dependent nucleosome remodeling ( Strahl and Allis, 2000 ; Vignali et al., 2000 ) The founding member of the Drosophila TrxG family, TRX, is a histone methyltransferase which catalyzes H3K4 trimethylation to favor transcriptional activation ( Santos Rosa et al., 2002 ) TrxG proteins also regulate chromatin dynamics through nucleosome remodeling: The SWI SNF complex c ontains ATP dependent chromatin remodeling proteins, which are able to alter the nucleosome structures to facilitate basal transcription machinery ( Smith and Peterson, 2005 ) In addition to direct methylation, both PcG and TrxG complexes can recruit other histone modifiers to ensure transcriptional repression or activation. For example, they both can rec ruit histone demethylases (HDM). The TrxG proteins can recruit HDMs (e.g. UTX) that specifically remove methyl groups from repr essive histone mark of

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29 H3K27me3. A human HDM JARI D1, has been found to demethylate H3K4me3 and associate with PcG protei ns ( Agger et al., 2007 ; Hong et al., 2007 ; Lee et al., 2007 ; Shi, 2007 ) Other histone modifiers such as histone acetyltransferase (HAT) and histone deacetylase (HDAC) are also recruited either directly or indirectly to modulate transcription ( Mills, 2010 ; Sparmann and van Lohuizen, 2006 ) (Fig 1 2 ). It is believed that the coordinated removal of repressive marks and deposition of pos itive marks (and vice versa) is important for chromatin dynamics and transcription Epigenetic R egulation of A poptosis Dysregulation of PcG Targeting Epigenetic regulation could be disturbed to promote tumorigenesis without significant changes of the global level of supp ressive histone modification For instance, the long non coding RN A HOTAIR can promote tumorigenesis through genome wide re targeting of PRC 2 to a pattern that resembles undifferentiated/lowly differentiated cells ( Gupta et al., 2010 ) Such aberrant targeting of PcG proteins is known to promote the silencing of tumor suppressor genes, which a re in a transcription ready (bivalent) state in normal stem cells ( Ohm et al., 2007 ) Altered PcG targeting can be induced by oncogenic proteins. For instance, oncogenic Ras can lead t o repression of Fas exp ression. Although at the end stage the silencing of Fas (and several other tumor suppressor genes) is manifested as both DNA hypermethylation and increased s uppressive histone modification a mechanistic analysis indicated that PcG proteins such as Bmi1 an d EzH2 are required for Ras induced silencing of Fas ( Gazin et al., 2007a ) Interestingly, a small adenoviral protein, E4 ORF6, can ac tivate a n unknown mechanism that leads to the formation of facultative heterochromatins in P53 targeted stress responsive genes ( Soria et al., 2010a ) Since

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30 E4 ORF6 lacks any enzymatic domain that can modify histone tails, it must act through a cellular pathway that can specifically silence P53 targeted stress respons ive genes. Insight into this mechanism would certai nly advance our understanding of how PcG mediated epigenetic silencing can be targeted to specific groups of genes. Epigenetic Silencing of Pro apoptotic Genes Epigenetic regulation of P53 targeted stress responsive genes is also observed during Drosophil a development. A 33 kb intergenic region located in the pro apoptotic gene cluster is responsible for mediating P53 dependent induction of reaper hid and sickle This region, I rradiation Responsive E nhancer R egion (IRER), is open in early embryonic stages when most cells are proliferating, conveying high sensitivity to DNA damage and other stresses. However, at embryonic stage 12, when most cells enter post mitotic differentiation, this region forms a heterochromatin like structure enriched for both H3K9M e3 and H3K27Me3. Consequently, the three pro apoptotic genes can no longer be induced following irradiation ( Zhang et al., 2008 ) This open to closed transition of IRER requires the function of PcG proteins such as Pc and Su(z)12, as well as HDAC and Su(var)3 9 ( Figure 1 3). Interestingly, epigenetic suppression is strictly limited to the irradiation responsive enhancer region without affecting the transcribed reg ions of the pro apoptotic genes. This limitation is important since those pro apoptotic genes are expressed in a cell lineage specific pattern in late stage embryos, and are required for lineage specific cell death ( Tan et al., 2011 ) The demarcation of epigenetic blocking is achieved by a chromatin barrier separating IRER from the promoter and transcribed regions of reaper This barrier, loc ated within a 294bp DNA fragment, appears to be highly conserved as it can block heterochromatin propagation when tested in a vertebrate system ( Lin et al., 2011 )

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31 The epigenetic blocking of IRER provided a nice system for elucidating the cellular mechanism s that regulate targeted suppressive histone modification at tumor suppressor/stress responsive genes. However, it left several fascinating questions unanswered. First, genome wide alignment revealed that IRER is highl y conserved throughout evolution: the underlying DNA sequence is even better conserved than the neighboring coding sequence for reaper suggesting the crucial regulatory function may exist in this reg ion ( Zhang et al., 2008 ) Second, a more detailed study needs to be performed to gain an insight into specific mechanisms controlling the epigenetic regulation o f IRER My dissertation research defined the functional significance of IRER in development and tumorigenesis by providing evidence that an open IRER not only convey s sensitivity to irradiation and DNA damage, but also increase s the cellular sensitivity t o overproliferation and competition induced cell death. I n addition, I developed a novel method to monitor the epigenetic status of IRER at cellular resolution in this study, which revealed a dynamic pattern of IRER accessibility which likely reflects th e stochastic nature of epigenetic regulation of stress responsive genes in a given cell type.

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32 Figure 1 1. Apoptotic pathway s in Drosophila mammals and C.elegans The key cell death regulators are conserved across different species and the functional o rthologues are highlighted in the same color. In Drosophila i n response to apoptotic stimuli, expression of the pro apoptotic genes hid reaper grim and sickle releases the initiator caspases (i.e. Dronk and Dreed) and effecter caspases (i.e. Drice and Dcp 1) from inhibition by Diap1, and induce s apoptosis. Stimulatory and inhibitory inte ractions are indicated by arrows and T bars, respectively. The extrinsic apoptotic pathway (or: death receptor pathway) is not included in this figure. IAP, inhibitor of apoptosis; Dark (also named as Dapaf 1 or HAC 1), Drosophila Apaf 1 related killer; SMAC, second mitochondria derived activator of caspases; DIABLO, direct IAP binding protein with low pI; Cyto c cytochrome c; APAF1, apoptotic protease activating factor 1; CED, cell death abnormality.

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33 Figure 1 2. PcG and TrxG proteins regulate gene expression by modulating c hromatin structure PcG and TrxG proteins directly methylate specific histone residues to establish repressive (H3K27me3) and active (H3K4me3) hi stone marks, respectively. In addition, they are able to recruit enzymes that modulate other histone modifications such as acetylation and demethylation as well as DNA methylation. PcG complexes can associate with HDACs, H3K4me3 specific HDMs and DNMTs to suppress gene expression, whereas TrxG complexes recruit HATs and H3K27me3 specific HDMs to activate gene expression.

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34 Figure 1 3. Schematic diagram summar izing previous findings regarding the IRER. Using insulator containing P element insertions, previ ous work from our lab has mapped the irradiation responsive enhancer region (IRER) to the 33kb intergenic sequence on the 3 rd chromosome. It is located between two pro apoptotic genes reaper and sickle including the putative p53 response elem ent (P53 RE) This enhancer region is subject to PcG mediated epigenetic regulation and undergoes an open to closed transition during embryonic stage 11 12. The open chromatin structure in early stage em b r yos facilitates irradiation induced transcription of reaper and hid and leads to apoptosis ; whereas the condensed chromatin in late stage d embryos precludes transcription and blocks apoptosis The facultative heterochromatin formation is restricted to IRER by the IRER left barrier (ILB) which allows the reaper promote r to remain open throughout development and accessible to regulation. The barrier activity requires binding of the Cut protein which may recruit chromatin modifying enzyme s such as CBP; mechanistically, much remains to be elucidated.

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35 Table 1 1. Many his tone modifiers are evolutionary conserved and implicated in tumorigenesis Complex Drosophila protein Human homologues Functional domains Biochemical activity PcG proteins PRC1 Polycomb (PC) CBX2, CBX4, CBX6, CBX7, CBX8 Chromodomain Binding to H3K27me3 Posterior sex comb (PSC) PCGF1(NSPc1), PCGF2(MEL18), PCGF4(BMI1) Zinc finger Cofactor for Ring Polyhomeotic (PH) PHC1, PHC2, PHC3 Zinc finger and SAM Required for silencing Sex combs extra (SCE or RING) RING1A, RING1B, RNF2 RING zinc finger H2AK119 ubi quitin ligase Sex comb on midleg (SCM) SCMH1, SCML2 SAM, MBT, Zinc finger recruitment of the PcG protein PRC2 Enhancer of zeste (E(Z)) EZH1 and EZH2 SET Histone methyltransferase, establish H3K27me3 Extra sex combs (ESC) EED WD40 repeats Co factor for E(Z) Extra sex combs like (ESCL) EED WD40 repeats Co factor for E(Z) Suppressor of zeste 12 (SU(Z)12) SUZ12 Zinc finger Co factor for E(Z) Polycomb like (PCL) PCL1(PHF1), PCL2(MTF2), PCL3(PHF19) PHD PhoRC Pleiohomeotic (PHO) YY1, YY2 Zinc finger DNA binding Pleiohomeotic like (PHOL) YY1, YY2 Zinc finger DNA binding SFMBT L3MBTL2, MBTD1 MBT, SAM Binding to mono and dimethyl H3K9, H4K20 TrxG proteins TAC1 Trithorax (TRX) MLL, MLL2, MLL3, MLL5 SET Histone methyltransferase, establishes H3K4me3 dCBP CBP KIX, IBiD, zinc finger Histone acetyltransferase dUTX UTX JmjC di and trimethylated H3K27 demethylase ASH1 ASH1L SET H3K4 and H3K36 methylase ASH2 ASH2L, WDR5 WD40 repeats Essential for H3K4me3

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36 Table 1 1. Continued Complex Drosophila protein Human homologues Functional domains Biochemical activity TrxG proteins (continued) TrxG proteins (continued) SWI SNF nucleosome remodeling complex OSA ARID1A, ARID1B BRM BRM, BRG1 SWI SNF like helicase, Bromodomain ATPase activity, bind s acetylated histones SNR1 SNF5, ARID4A, ARID4B Non catalytic core subunit Other related histone modifiers SIR2 SIRT1 Zinc finger NAD + dependent class III histone deacetylase LID JARID1 JmjC di and trimethylated H3K4 demethylase SU(VAR)3 3 L SD1 SWIRM, amine oxidase domain mono and dimethylated H3K4 demethylase PcG, polycomb group; TrxG, trithorax group; TAC, trithorax acetylation complex; SFMBT, Scm related gene containing four MBT domains; CBP, CREB binding protein; ASH, absent, small, or homeotic discs; SIR, silent information regulator; UTX, ubiquitously transcribed tetratricopeptide repeat, X chromosome; LID, little imaginal discs; CBX, chromobox homologue; PHC, polyhomeotic homologue; EZH, enhancer of zeste homologue; EED, embryonic ect oderm development; YY, Yin Yang transcription factor; MLL, mixed lineage leukemia; LSD, lysine specific demethylase; PHD, plant homeodomain; WDR5, WD repeat domain 5.

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37 CHAPTER 2 MONITORING DNA ACCESSIBILITY AND EPIGENETIC STATUS IN INDIVIDUAL CELLS AND LIV E ANIMALS WITH A FLUORESCENT REPORTER Introduction Even though all cells share the same genome in one organism which genes are expressed and when/how they are expressed are largely determined by epigenetic regulation. This is achieved in large part by con trolling the accessibility of the DNA to the basic transcription machinery and DNA binding transcription factors ( Grewal and Moazed, 2003 ; Schwartz and Pirrotta ) The major epigenetic mechanisms that affect DNA accessibility are DNA methylat ion and histone modification However, DNA methylation in Dros ophila is restricted to early stage of embryo genesis and lacks a systematic study ( Lyko et al., 2000 ; Nanty et al., 2011 ) T he main form of epigenetic regulation in D rosophila is post trans l ational histone modification. marks lead to open chromatin structure and increased transcription histone marks are usually associated with condensed conformation s and reduced levels of gene expr ession ( Jenuwein and Allis, 2001 ) DNA accessibil ity and epigenetic modification can be mo nitored by biochemical assays such as DNase I sensitivity assay chromatin immunoprecipitation ( ChIP), etc. These methods generally require homogenizing 10 6 ~ 1 0 7 cells to produce a readout. T he obtained result actually reflects an averaged l evel of the te sted population. The methods are applicable to the cultured cell line s but may not be suitable for measuring epigen etic status in vivo given our knowledge that e pigenetic regulation could be and in many cases is ce ll specific. Cells in the same tissue m ay have totally different epigenetic profile s according to their differentiation status and many other factors Even cells of the same tissue type and differentiation status may display significant individual

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38 variation in epige netic regulation. For exa mple when the white gene is in serted in close proximity of a pericentromer ic region its expression is influenced by the centromere heterochromatin formation and variegat es among eye cells in the transgenic fly ( Karpen, 1994 ) The change of DNA accessibility can directly influence t he responsiveness of genes to environmental stimuli. It i s well known that cellular sensitivity to irradiation induced cell death can vary dramatically depending on cell ty pe and differentiation status. Using the developing Drosophila embryo as a model, w e found that the sensitive to resistant transition of the radiation responsiveness of pro apoptotic genes was mediated by an epigenetic mechanism ( Zhang et al., 20 08 ) The irradiation responsive enhancer region (IRER) is required for mediating the irradiation responsiveness of three pro apoptotic genes reaper hid and sickle accessible in proliferating and undifferentiated cells during early em bryogenesis ( 3 7hr after egg laying (AEL)). Irradiation treatment o f embryos at this stage induces a 5 10 fold increase of the pro apoptotic genes within 15~30 minutes, foll owed by wide spread apoptosis. However, this region forms a closed, DNase I resist ant str ucture in most cells during pos t mitotic differentiation (7 12 hr AEL) and renders the three pro apoptotic genes irresponsive to irradiation ( Zhang et al., 2008 ) The open to closed transition of IRER is accompanied by a dramatic increase of both trimethylated H3K27 (H3K27Me3) and trimethylated H3K9 (H3K9Me3) in the region. The epigenetic blocking of IRER requires the function of HDAC 1, Su(var)3 9 (H3K9 me thytransferase), and several key Polycomb group (PcG) proteins ( Zhang et al., 2008 )

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39 In Drosophila embryogenesis, cell nuclei undergo 13 rounds of proliferation during the blastoderm stage prior to o nset of differentiation. This unique event allowed us to monitor the epigenetic status of IRER by collect ing large amounts of pr oliferating c ells from early stage embryos and perform ing DNa se I sensitivity assay and C hIP. The relatively synchronized initiation of cellular differentiation around embryonic stage 12 was conceivably crucial in allowing us, using population based biochemical methods, to detect the change of DNA accessibility and epigenetic modification in IRER during the proliferation to differentiation transition. Intriguingly, a lthough late stage embryos are resistant to irradiation induced cell death, many cells in developing imaginal discs at larval stage become sensitive to irradiation induced pro apop totic gene expression and apoptosis suggesting they may have an open IRER The larval tissues are composed of cells from different lineages and/or at different di fferentiation st atuse s. Thus, it is impossible to discern the chromatin conformation of IRER in such a tissue using the same biochemical approaches we applied to embryos, i.e. DN ase sensitivity assay or ChIP. To solve this problem, we designed a strategy which integrated a fluorescent reporter into the epigenetically regulated IRER. Our experiment al verification indicated that the fluorescent reporter could be used to monitor cell and tissue specific epigenetic regulation and changes of DNA accessibility dur ing animal development. Materials and Methods G enerati on of IRER{ubi DsRed } Two bacterial artificial chromosome clones (BACR35F04 and BACR04F07) oordinates of Genome release 4.3 ) were obtained from the BACPAC Resources Center ( http://bacpac.chori.org/ ) The ~6.5kb targeting region is locat ed in the middle region of

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40 IRER and is highly accessible to DNase I digestion. The targeting region was cut by BamHI/XhoI, purified from agarose gel cloned into the p[Endsout2] vec tor and verified by Sanger sequencing. A ~3kb ubiquitin DsRed cassette was prepared by polymerase chain reaction (PCR) from fly genomic DNA with N d eI sites added to the PCR primers The NdeI { ubi DsRed } NedI PCR product was then sub cloned into an endogenous NdeI site (18,375,553) withi n the targeting IRER fragment in the p[Endso ut2] vector, to generate the donor construct Multiple transgenic strains carrying the donor construct were generated with the p element based integration. An inserti on on the X chromosome was selected as the donor strain for homologous recombination. The e nds out homol ogous recombination was conducte d as previously described ( Gong and Golic, 2003 ) One recombinatio n event was recovered from about 6,000 progenies based on autosomal inheritance. The successful recombination into the endogenous IRER on the 3 rd chromosome wa s verified by genetic mapping, inverse PCR, and southern blot. Fly Handling Unless otherwise mentioned, all the crosses were done at 25 degree A lleles used: Ser Gal4 (Bloomington #6791) Hdac[303], Hdac[326] and Hdac[328] (Dr. Mottus), Su(var)3 9[1] (Blo omington #6209), Su(var)3 9[2] (Bloomington #6210), UAS RNA i strains (Vienna Drosophila RNAi Center). DNase I Sensitivity Assay and Chrom atin Immunoprecipitation (ChIP) The DNase I sensitivity assay and ChIP assay on staged embryos were performed as previo usly described ( Zhang et al., 2008 )

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41 Immunofluorescence Embryo in situ hybridization was performed as stated ( Zhang et al., 2008 ) For the imagin al disc in situ hybridization: third instar larvae of appropriate genotype were briefly dissected in DEPC PBS to facilitate imaginal disc attachment to the larv al body wall. Lar vae were immediately fixed with 4% paraformaldehyde for 30 minutes, dehydrated with a graded methanol series (50% 75% 90% 100%), and stored at 20C til l use. The in situ hybridization signal was amplified and detected by TMR conjugated tyramide (TSA kit f rom PerkinElmer Life). I maginal discs were further dissected and mounte d onto glass slides. F luorescent images were taken with a Leica upright fluorescent microscope using OpenLab software Flourometric Measurement of DsRed Expression For each genotype, fi ve newly eclosed male flies (< 2 d ays) were collected into a 1.5mL micro centrifuge tube containing 250uL assay buffer (50mM NaH 2 PO 4 10mM Tris HCl Ph8.0, 200mM NaCl) on ice. The sample was homogenized by pestle, and centrifuged at 200g for 1 minute to remo ve tissue chunks. 200uL of supernatant containing DNA was transferred to a new tube. Meanwhile, a blank s ample consisting solely of 200uL of assay buffer was prepared. The samples were stained with Hoeschst33342 to a final concentration of 10 g/mL for 5 mi nutes on ice, and 40uL was immediately p ipet ted in to a 96 well plate Fluorometric measurement was done with the Perkin Elmer Victor 3 V 1420 Multiabel Counter with the settings of DsRed, excita tion 55519nm, emission 5905nm ; H33342, excitation 35519nm, em issi on 4605nm. If the blank reading wa s too high, it was subtract ed from each sample reading to correct for background fluorescence. For each sample, I d ivide d the D sRed value by the H33342 value, which reflects the expression level of DsRed relative to D NA content.

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42 The two tailed, un t test was used for statistical analysis and a p value < 0.05 was considered significant Results Inserting the ubi DsRed Reporter into IRER via Homologous Recombination The silencing of transgene s by local e pigenetic regulation has been observed in several contexts. For instance, the white gene in P element insertion s near the centromere shows variegated expression in the eye, which indicates the marker gene is silenced in some cells but not others ( Konev et al., 2003 ; Sun et al., 2004 ) This silencing is due to the spread of H3K9Me3 modification during the formatio n of the pericentromeric heterochromatin. Similarly, the UAS lacZ transgene contai ned in a P insertion in the regulatory region of bithorax(bx) can not be induced by ubiquitously expressed Gal4 in the anterior segments, because the bx locus there is epigene tically suppressed by PcG mediated trimethylat ion of H3K27 ( McCall and Bender, 1996 ) In contrast, the expression of the UAS lacZ can be induced by Gal4 in posterior segments where the regulatory region is not epigenetically suppresse d. These reported observations prompted us to postulate that the expression pattern of a repor ter gene with a constitutively active promoter inserted into IRER may reflect the epigenetic status o f IRER in any cell at any developmental point. s ( Gong and Golic, 2003 ) we first generated a transgenic strain carrying the donor construct p(LA ubi DsRed.T4 SV40PolyA RA) in the X chromoso me (Figure 2 1A ). DsRed.T4 is a modified ver sion of the original DsRed p rotein with a maturation half time of about 40 mins ( Bevis and Glick, 2002 ) LA and RA are two ~3kb consecutive segments of IRER linked by an endogenou s Nde 1 site at 18,375,553 558 on the 3 rd

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43 chromosome (the coordinates in Chapter 2 are based on genome release 4.3 unless otherw ise stated). The ubi is a 2.2 kb regulatory region extracted from the endogenous ubiquiti n p63E gene that includes the first non codin g exon and an intron. The same regulatory fragment has been used to ge nerate markers/reporters that gi ve ubiquitous expre ssion pattern throughout development ( Brow n and Castelli Gair Hombria, 2000 ; Supatto et al., 2005 ) Th e reporter flanked by the homologo us arms was released from the X chromosome with the FLP recombinase and subsequently cut by the I SceI endonuclease as described by Gong and Golic ( Gong and Golic, 2003 ) A homologous recombination event was recovered from about 6 ,000 progeny screened for autosome based inheritance of the fluorescent reporter as well as a decrease of the fluorescent signal The su ccessful recombination was then verified with genetic mapping, Inve rse PCR, and southern blot (Figure 2 1 B). The recombined strain was named as IRER{ ubi DsRed }. The DsRed mRNA, meas ured by in situ hybridization, i s ubiquitously expressed at high level s in early st age embryos which is in agreement with the fact that IRER is open and accessible in proliferating cells at this stage (Figure 2 1 C D) However, beginning with later embryogenesis, DsRed mRNA expression becomes restricted t o a small percentage of cells (F igure 2 1 E F) In newly hatched larvae, strong DsRed fluorescent si gnal can be detected from scattere d cells in the nerve cord ( Fig ure 2 2B ), a place known for continued neuroblast proliferation and apoptosis in late embryogenesis and early larval stage s ( Peterson et al., 2002 ) The pattern of cells positive for DsRed during the larval stages is very dynamic. 2 day s AEL (1 st Instar larvae ), a group of cells at the posterior epidermis develop a dis tinct signal ( Fig ure 2

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44 2 C ). In 3 rd instar larvae, most cells have little or no detectable DsRed. In addition, the restricted expression of the DsRed reporter was also observed from the arterial tip of the germarium (Figure 2 1 G I) and the polar cell s of e gg chambers in the adult ovary (Figure 2 1J). Verifying the Responsiveness of the Reporter to Changes in DNA Accessibility The restricted expression pattern of IRER{ ubi DsRed } suggests that it is sub ject to epigenetic regulation. To verify whether the DNA accessibility of the reporter is co regulated with IRER, we monitored the DNase I sensitivity of the DsRed gene before and after the ope n to closed transition of IRER (Figure 2 3A) Previously, through DNase I sensitivity assay and ChIP analysis, we have d ocumented that IRER undergo es an open to closed transition during embryonic stage 12 which is accompanied by the formation of repressive histone marks (H3K27Me3, H3K9Me3) and the binding of PcG proteins and HP1 ( Zhang et al., 2008 ) Here w e found that like the surrounding IRER, the DsRed locus became significantly more resistant to DNase I treatment following the open to closed transition of IRER. The magnitude of the change at the reporter locus ( DsRed ) was not as large a s the neighboring genomic loci. Consequently, the reporter locus was not as resistant to DNase I as t he nei ghboring IRER region (Figure 2 3 B ) This is likely due to the impact of the strong ubiquit in enhancer/promoter on the local environment of histone modif ication and DNA accessibility. However, this impact appears to be limited to the reporter locus without affecting epigenetic suppression o f IRER during the transition. The immediate neighboring loci (18,374, 377) became as resistant to DNase I as the heterochromatin control (H23), same as what was observed in wild type animals ( Zhang et al., 2008 )

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45 Even though the change of DNase sensitivity at the DsRed locus i s not prominent until the end of embryoge nesis, it appears that the enrichment of suppressive histone mark H3K27Me 3 as well as binding of Polycomb group protein PSC (posterior sex comb) is similar to the neighboring IRER loci (Figure 2 3 C ). Similarly, the accumulation of H3K9Me3 and the binding of HP1 to the reporter are also comparable to that of the neighboring loci and the H23 constitutive centromeric het erochromatin region (Figure 2 3 D ). The fo rmation of suppressive hist one marks and the binding of PSC an d HP1 appear to play a significant role in constraining t he expression of the reporter. This may be due to the fact that the accessibility to large complexes s uch as RNA Pol II can be blocked lo ng before the DNA becomes inaccessible to DNase I a phenomenon that has been reported previously and noticed by us in our early study of IRER ( Zhang et al., 2008 ) By the time animals reach 2 nd in star larval stage (~3 4 days AEL at 25 o C), few cells have strong DsRed signal comparable to early embryogenesis. To verify whether DsRed can only be expressed at a high level in cells with an open conformation of IRER, we used fluorescence activated cell sorting (FACS) to separate DsRed positive (+) and negative ( ) cells isolated from a mixture of 2 nd and 3 rd instar larvae. Using a protocol adapted from Okataba et al. ( Oktaba et al., 2008 ) we found that about 2% cells dissociated from shredded larval tissues can be r eliably separated as DsRed(+). ChIP analysis of the sorted cells indicates that both the reporter and the IRER region are enri ched for H3K27Me3 and H3K9Me3 in DsRed( ) cells There is also a significant binding of PSC and HP1 to the reporter and IRER in these cells (data not shown). In contrast, DsRed(+) cells have much lower level s of suppressive histone modificat ions

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46 which is similar to what has been observed for undifferentiated ce lls during early embryogenesis Correspondingly, DsRed(+) cells are extremely sensitive to the stress associated with tissue dissociation and FACS. Many DsRed(+) cells are dying or dea d following FACS as monitored by DAPI staining via microscopy (data not shown) The level of reaper in DsRed(+) cells is about 8 times as high as that in DsRed( ) cells (Fig ure 2 3 E ). The aforementioned evidence indicate s that IRER{ ubi DsRed } is a reliabl e reporter for the DNA accessibility of IRER. High er level s of reporter expression correlate with an open chromatin conformation in the IRER while lower levels of IRER{ ubi DsRed } expression correlate with the formation of supp ressive histone marks in IRER. Since the ubiquitin promoter is presumably a constitutively active promoter, it may interfere with the suppressive histone modification s in IRER. It has been reported that active tra nscription can disrupt Polycomb mediated silencing ( Schmitt et al., 2005 ) However, to our surprise, there is no significant delay of the open to c losed transition of IRER loci flanking the repo rter (loci 374, 377 in Figure 2 3) compared to wild type In addition, there is no detectable delay of the sensitive to resistant transition of the responsiveness of reaper and hid to irradiation. This is likely because the DsRed transcription i s term inated by the SV40 polyA and does n ot exten d into the neighboring region. The insertion of the ubi DsRed cassette has no notice able impact on animal viability or fertility and the reporter strain has been stably kept for over 5 0 generations in the lab. Monitoring Cell and Tissue Specific Ep igenetic Changes Using IRER{ubi DsRed} The IRER{ ubi DsRed } reporter allow ed us to monitor the dynamic changes of epigenetic status of IRER in individu al tissues and specific cells. While essentially all cells in the early stage embryo have strong DsRed sig nal, cells in the developing larvae

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47 disp lay different ial levels of DsRed. Some of the observed differences in DsRed expression may due to cell type and/or differentiation status. For instance, i n the transgenic fly carrying a p{ ubi GFP } insertion on the se cond chromosome, GFP is expressed ubiquit ously from the entire disc (Figure 2 4 A ). However, it is clear that cells at the D/V boundary as well as the border of the wing pouch have relatively higher level s of GFP than other cells. Compared to p{ubi GF P}, the striking feature of IRER{ ubi Ds Red } expression is the varie gated expression (Figure 2 4 B In the wing discs of IRER{ ubi DsRed }, patches of cells have DsRed levels that a re not detect able, indicating the reporter i s total ly silenced in these c ells. For cells with detectable level of DsRed, the level of expression appears significantly lower than p based, or phiC31 medi ated insertion of ubi DsRed into an euchromatin locus. The variegated expression of IRER{ ubi DsRed } i s not specific to wing disc s; all imaginal discs from the transgenic strain display varie gated expression of the reporter ( Figure 2 5 A D ). While the silenced, i.e. IRER{ ubi DsRed } negative, clones can be observed at most positions of the discs, it seems they are more frequently oc curred in regions with relatively condensed chromatin as indicated by the level of DsRed expression i n surrounding cells The occurrence of patched DsRed positive or negative clones indicates that t heir progenitor cells may contain distinct DNA accessibi lities at the IRER locus and that the epigenetic status of IRER is, to a certain extent mitotically stable during subsequent proliferation While all discs in IRER{ ubi DsRed } larvae r eared under normal condition have DsRed negative clo nes, the size of t he clones, as well as the overall area covered by th e clones varies among discs. This suggests that the epigenetic silencing of the reporter could happen within a relatively long time window of

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48 disc develo pment. We did notice some small DsRed positive clon es completely surrounded by DsRed negative cells, which seems to suggest that it is possib le to reverse from a silenced state to an open state during normal development. To verify that the variegated expression of IRER{ ubi DsRed } is due to epigenetic silen cing of the reporter, i.e. through a similar mechanism as that described for position effect variegation (PEV), we sought to knock down Su(v ar)3 9 in part of the wing disc tissues. Su(v ar)3 9 or Suppressor of v ariegation 3 9 encodes a histone methyltran sferase responsible for catalyzing suppressive heterochromatin marks H3K9Me2 and H3K9Me3 ( Ebert et al., 2004 ) We have shown previously that the function of Su(var )3 9 is required for blocking IRER accessibility during embryogenesis ( Zhang et al., 2008 ) When a double strand hairpin RNA targeting Su(v ar)3 9 mRNA i s specif ically expressed in Serrate ( Ser ) expressing cells in the wing discs using the UAS Gal4 system (Figure 2 4 C) essen tially all cells in the Ser domain have dramatically increased level of DsRed expression (Fig ure 2 4 D). In addition, there i s no DsRed negativ e clone in the Ser domain upon Su(var)3 9 kno ckdown indicating variegation is abolished via red uced level s of Su(v ar)3 9 and consequently the repressive hist on e mark H3K9Me3 To investigate whether the de repression of IRER is a cell autonomous response t o Su(var)3 9 knockdown, we simultaneously labeled the RNAi expressing cells with a GFP transgene and noticed that the IRER{ ubi DsRed } is primarily induced from GFP positive cells, suggesting a cell autonomous de repression of IRER (Figure 2 4 F To ru le out the possibility that the c hange of DsRed expression is due to abnormal activation of the ubiquitin promoter, we examined the responsiveness of p{ ubi GFP } on the 2 nd chromosome to the knockdown of Su(v ar)3 9 Interestingly,

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49 ectopic GFP signal is indu ced from wing discs, with an induction pattern totally different from that of IRER{ ubi DsRed } and seems not to overlap with the Ser domain. (Figure 2 5) The induction of p{ ubi GFP }, unlike the induction of IRER{ ubi DsRed }, probably reflects an independent response to Su(var)3 9 knockdown. This further indicates that the induced DsRed expression in Su(var)3 9 knockdown cells is mostly attributable to de repression of IRER. Moreover, IRER{ubi DsRed} expression is als o significantly induced when d sRNA targetin g HDAC3 is expressed in the same experimental setup, suggesting the epigenetic silencing of IRER requires multiple his t one modifiers ( Figure 2 7 ). As we have shown previously, an open IRER is responsible for mediating immediate (within 15 30 minutes) in duction of reaper and hid in early stage embryos following gam m a irradiation ( Zhang et al., 2008 ) Following development stage 12, reaper and hid become irrespon sive to irradiation in most cells due to the formation of heterochr omatin like structu re in IRER. Intriguingly, reaper mRNA is readily detected from Su(var)3 9 RNAi discs via in situ hybridizatio n. This suggests that when the DNA acc essibility of IRER is i ntensely increased, it is sufficient to drive cells to undergo apoptosis. Yet another possibility is that cells cannot survive when the level of a fundamental his t one modifier such as Su(var)3 9 is down regulated or totally abolished Further studies are r equired to differentiate the two possibilities. However, they are not mutually exclusive with each other because IRER may serve as a key regulatory region to define the cell fate by mediating its epigenetic status. Monitoring Epigenetic Status of IRER at O rganismal Level As afore mentioned, the IRER{ ubi DsRed } reporter allowed us to monitor the epigenetic status of IRER from individual tissues and cells. However, this requires a

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50 considerable amount of tissue dissection work. Since the DNA accessibility of I RER is accessibility of IRER by directly measuring the fluorescent intensity of DsRed from the whole animal. T he reporter strain can be used to screen for histone modifiers required for the appropriate epigenetic regulation of IRER. In the previous study, we have identified several histone modifiers including Hdac1(rpd3), Su(var)3 9, and Pc which are required for the epigenetic silencing of IRER during embryogenesis ( Zhang et al., 2008 ) To test whether they still perform such a role in adults, we crossed their mutant strains individually with the IRER reporter strain and measure d the DsRed expression from the ir progeny Among all the tested mutants, three alleles of Hdac1 mutant ([303], [326], and [328]), one Pc mutant (Pc3), and two Su(var)3 9 mutant s ([1] and [2]) consistently showed a modest but significant increase of DsRed si gnal from IRER{ ubi DsRed }/Mutant fly when compared t o the IRER{ ubi DsRed }/+ control (Figure 2 8A) Therefore, we concluded that these histone modifiers are required for epigenetic silencing of IRER, similar to what has been observed in embryos. We hypothe size that the homozygous mutant s should further release the condensed chromatin and induce still higher levels of DsRed. However, none of th e histone modifier mutants tested is homozygous viable, presumably due to their essential roles of conducting proper epigenetic regulation during normal development This may explain the vigorous induction of DsRed from the Su(var)3 9 RNAi wing discs but not from the wing discs heterozygous for Su(var)3 9 (data not shown). Nonetheless, the stable increase of DsRed obse rved from the histone modifier mutants is statistically sig nificant. T he

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51 IRER{ubi DsRed} reporter can also be used as a convenient tool to measure the DNA accessibility of IRER at the organism al level and screen for any environmental factors or drug trea tments sufficient to change the epigenetic status of IRER One unique feature of epigenetic regulation is that it is dynamic, responsive to environmental and dietary factors, and under many circumstances, reversible. Interestingly, our preliminary data sho wed that dietary restriction (DR), a reduction in food intake without malnutrition, significantly induced DsRed expression (Fig 2 8B ), suggesting that the epigenetically regulated IRER is de rep ressed at the organism al level Recen t studies have provided extensive evidence to show that DR can extend life span of diverse organisms, including y east, flies, rodents, and monkeys ( Anderson et al., 2009 ) Accum ating evidence suggests that the biological effects of DR are closely related to chromatin function, although the underl ying mechanisms are still unclear ( Heydari et al., 2007 ) Here we linked the DR to an epigenetically regulated enhancer region that is required for s tress response. This work will help us to better understand how epigenetic regulation is responsible for the effects on lifespan by dietary restriction Discussions Histone modifications, by controlling the expression profiles of different sets of genes, p lay a pivotal role in defining distinct cellular propert ies over the otherwise identical genome. Not only are specific histone modifications important for maintaining the differentiation potential of stem cells, but also the dynamic change of epigenetic st a tus of specific sets of genes is one of the main mechanism s of cellular differentiation. Mis r egulation of epigenetic status has been implicated in the etiology of a variety of diseases such as mental disorders and cancers ( Isles and Wilkinson, 2008 ; Johnstone

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52 and Baylin, 2010 ) Traditional biochemical methods of monitoring epigenetic status rely on h omogenizin g millions of cells. In this work, we showed that a fluorescent reporter could be applied to monitor the epigenetic status of a given locus in individual cells. This not only allowed us to monitor the cellular specificity of epigenetic regulation, but als o enabled us to assess the DNA accessibility of the targeted locus without tissue dissection or sacrificing the animal Epigenetic Regulation with Cellular Resolution In any given tissue, the epigenetic statu s of a particular gene could vary significantl y depending on the cell typ e and /or differentiation status. For the development of certain diseases, such as cancer, it is conceivable that the causative epigenetic change may be initially restricted to a very small number of cells or even a single cell. Monitoring epigenetic status at the cellular level is thus essential for us to under stand the mechanisms of how mis regulation of epigenetic status may arise as a consequence of development and aging or following environmental stresses. Cell specific epi genetic regulation was first observed with the variegated expression of the white gene when it was transposed close to the centromere ( Henikoff, 1992 ; Karpen, 1994 ) A UAS lacZ reporter inserted into the regulatory region of bithorax was used successfully to show that this region became inaccessible to Gal4 and the transcription apparatus in cells in the anter ior segments, where it is silenced by PcG mediated silencing, but remained open in cells at posterior segments ( McCall and Bender, 1996 ) In canonic regulation of homeotic genes such as the bithorax the formation of facultative heter ochromatin is determined by the position (and id entit y) of the cell and is largely irre versible in later development. However, there are man y loci whose epigenetic status depend s on the differentiation status of the cell, or is controlled

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53 by a particular s ignal transduction pathway, or changes in r esponse to particular stimuli. For instance, PcG mediated silencing/repression of differentiation regulators in stem cells could be relieved in differentiating cells ( Reik, 2007 ) And the expression of oncogenic Ras could specifically induce the silencing of pro apoptotic genes such as Fas ( Gazin et al., 20 07b ) Our previous analysis indicated that, unlike canonic PcG mediated suppression of homeotic genes, epigenetic regulation of IRER is not cell lineage dependent ( Zhang et al., 2008 ) The IRER{ ubi DsRed } reporter allowed us to show that its regulation is rather dynamic during development and is also responsive to environmental factors. The cellular resolution offered by this fluorescent reporter based strategy also highlighted the stochastic nature of epigenetic regulation, i.e. cells of the same tissue and similar differentiation status may display different DNA accessibility at a particular locus. This feature has been very well documented before with variega ted expression of the white gene, and henc e variegated eye pigmentation. However, fluorescent reporter such as IRER{ ubi DsRed } allowed this stochastic fea ture to be monitored not just in the eye, b ut in essentially all tissues. This should provide a rich o pportunity for us to investigate t he mechanism s that control/a ffect the stochastic regulation of epigenetic status. Limitations of the Strategy There are certainly limitation s to this strategy that need to be ad dressed in future studies First, the ubiquit in promoter we used in the reporter line is not Even in the attB{ubi DsRed} control line, t he expression levels of DsRed are not totally even in every cell in the imaginal discs. The unevenness of the reporter system in different cells will compound the association between the expression level of the

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54 reporter and the epigenetic status. This limitation could be partially addressed by using a better ubiquitously expressed driver We are in the process of generating another reporter line us ing the tubulin regulatory region as a driver, which has been reported to direct ubiquitous transcription and is sensitive to chromosomal position effects ( O'Donnell et al., 1994 ) In the new version of reporter, GFP is used to replace the DsRed a s a reporter gene, in case DsRed itself may have a cell specific expression. If we can observe a similar expression pattern of the reporter signal from the IRER{tub GFP} line, it will be a strong and independent verification of our stra tegy. More importantly, the current method relies on monitoring the fluorescent reporter signal from dissected tissues or homogenized animals In order to follow the epigenetic changes in normal development, we need to access the reporter signal in liv e an imals without disturbing normal development. It is possible to quantify the DNA accessibility of the targeted region in live animals with the adaptation of a quantitative fluorescence measurement technolog y We have collaborated with the research group of Dr. Huabei Jiang in the department of biomedical engineering, University of Florida, and developed a method to quantitatively measure the DNA accessibility in live animals via fluorescence molecular tomography (FMT) (data not shown)

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55 Figure 2 1. Generat ion of IRER{ubi DsRed}. A) Schematic drawing of the strategy for inserting the ubi the targeting construct P(LA ubi DsRed.T4 SV40PolyA RA) on X chromosome was firstly generated. LA and RA were two ~3kb homologous sequence of IRER(coordinates based on genome release 4.3). B) Southern blot verification of the recombination. Probe was corresponding to the red bar in A. Hybridization of the p32 labeled DNA probe to the Bgl II digested genomic DNA specifically recognized the 8kb recombined fragment from the IRER{ubi DsRed} strain, confirming the successful recombination. BglII site s were labeled as arrows in A. C F) Monitor the DsRed expression in embryos by RNA in situ hybri dization with a DsRed probe. C) DsRed was expressed ubiquitously at high level in stage 11 embryos, which was in agreement with the fact that IRER is open and accessible in prol iferating cells at this stage. D) Stage 11 embryos from the wild type control d id not show any signal, indic a ting the high s pecificity of the DsRed probe. E) Starting from stage 13, when IRER has formed a condensed chromatin structure, the overall DsRed level was dramatically decreased. Not ice that cells in the midline were still lab eled (red arrow head) F) By the end of embryogenesis, the general DsRed mRNA level was steadily at a reduced level; salivary gland showed strong hyb ridization signal (red arrow). G J) T he restricted DsRed expression in adult ov ary by fluorescent microscop y. G) GFP was ubiquitously expressed in every single cell in the control ovary: w[1118];P{w[ +mC]=Ubi GFP(S65T)nls}2R/CyO. H) In the IRER{ubi DsRed} ovary, the DsRed expression (red) was restricted to only a few locations. Nuclear DNA was c ounter stained b y DAPI (blue). I) Higher magnification picture of the ovary germarium, showing bright DsR ed signal at the anterior tip. J) Higher magnification picture of the stage 10B egg chamber, showing DsRed was specifically expressed from the polar cells (white arrow head), with TOTO 3 DNA counter stain (green). Scale Bars, 25m. The southern blot in B) was performed by Dr. Nianwei Lin (former graduate student in the Zhou laboratory); the egg chamber staining in J) was provided by Bingqing Zhang and Dr. Brian Calvi in Department of Biology at the Indiana University.

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56 Figure 2 2. The expression of IRER{ubi DsRed} from embryo s and larvae. A) In early embryos, the reporter was expressed ubiquitously at high levels. B) In newly hatched larvae, the strongest reporter expr ession was detected from nerve cord cells (blue arrow). C) In day2 larvae, a group of cells at the posterior epidermis have distinct DsRed signal (blue arrowhead). The larvae were counter stained with DAPI.

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57 Figure 2 3 Verification of the IRER{ubi DsRed } reporter for DNA accessibility and epigenetic status of IRER. A) Schematic representation of the IRER locus and DsRed reporter. The positions of DNA amplicons for quantification of DNase I sensitivity and ChIP assay were shown below the IRER map. B) DNas e I sensitivity in IRER and the reporter in early and late embryogenesis, when the reaper gene induction is sensitive or resistant to DNA damage, respectively. Both IRER and DsRed loci showed high sensitivity to DNase I at the sensitive stage, and reduced sensitivity in the resistant stage. Note that the insertion of the ubi DsRed reporter did not disrupt the closed conformation of its surrounding environment (i.e. that of 374 & 377) at late em bryogenesis (resistant stage). C) Enrichment of H3K27Me3 and PSC binding in the reporter locus in late stage embryos. ChIP data was normalized against the recovery rate of t he positive control locus bxd. D) Enrichment of H3K9Me3 and HP1 binding at the reporter locus in later stage embryos. ChIP data was normalized agai nst the positive control locus H23 (centromeric heterochromatin). E) DsRed (+) cells were very sensitive to the stress associated with tissue disassociation and FACS. The level of reaper presented as RNA/DNA ratio, was much higher in DsRed (+) cells than that in ( ) cells. Experiments in D E) were performed by the Dr. Nianwei Lin.

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58 Figure 2 4 Monitor ing the change of ep igenetic status of IRER in wing discs. A the control wing discs: w[1118];P{w[+mC]=Ubi GFP(S65T)nls}2R/ CyO, G FP was ubiquitousl y expressed. B from IRER{ubi DsRed} wing disc. The expression of DsRed was under the control of not only ubi promoter, but also the local environment of IRER, and exhi bited a variegated expression. C D) Cell spec ific knock down Su(var)3 9 in the Ser expressing domain in wing discs led to de repression of IRER, shown by intensely increased DsRed signal and abolished mosaic pattern, verifying that the variegated expression of IRER{ubi DsRed} is due to epigene tic sil encing of the reporter. E) Upon Su(var)3 9 knock down, the pro apoptotic gene reaper was induced from the Ser domain, shown by in situ hybridization using a reaper probe. F in Su(var)3 9 RNAi cells (see the overlapping between DsRed (red) and Ser>GFP (green)).

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59 Figure 2 5 The expression pattern of the ubi DsRed reporter is largely controlled by the local chromosome environment. When inserted into the epigenetically regulated IRER, t he reporter sho ws a variegated expre ssion from A) wing discs, B) eye discs and C) haltere discs in third instar larvae. D) Th e variegated expression pattern is also observed from imaginal discs at earlier developmental stage. When the ubi DsR ed construct was inserted into a relatively open l ocus on 2 nd chromosome, the DsRed singal is fairly ubiquitous from all examined tissues E) wing discs; F) eye discs; G) haltere discs

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60 Figure 2 6 Su(var)3 9 knockdown induced DsRed expression is not due to abnormal activation of the ubiquitin promote r. Using a Ser Gal4 driver, the construct of UAS Su(var)3 9 dsRNA was expressed from either IRER{ubi DsRed} wing d isc or Ctr{ubi GFP} wing disc. A) Ser expression pattern in win g disc was labeled by UAS GFP. B) Upon Su(var)3 9 knockdown, IRER{ubi DsRed} wa s primarily induced from Ser expressing cells. Compare the DsRed signal in B with the GFP signal in A. C) Interestingly, ubi GFP was also induced by Su(var)3 9 knockdown The induced GFP signal wa s mainly distributed in the center of the wing porch and hin ge but not from Ser expres sing cells. This suggests a non cell autonomous induction of ubi GFP following Su(var)3 9 RNAi.

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61 Figure 2 7 The expression of IRER{ubi DsRed} is responsible to HDAC. We tested a list of RNAi strains for their effect on altern ating DsRed expression, which reflects the role on epigenetic regulation of IRER. Among the h istone modifiers we have tested B) Su(var)3 9 and C) HDAC3 RNAi caused induction of DsRed signal, suggesting that they are required for epigenetic silencing of IRER during development. We are stil l in the process of testing additional histone modifiers with more RNAi constructs for their potential role i n regulating local DNA accessibility of IRER.

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62 Figure 2 8 Monit oring the epigenetic status of IRER in differ ent genetic background s and in respons e to dietary restriction (DR). A) Flourometric measu rement of DsRed was performed on males eclosed within 1 day. The DsRed value from the mutant strains IRER{ubi DsRed}/mutant was normalized to that from the control st rain IRER{ubi DsRed}/w[1118]. Interestingly, DsRed was increased from several histone modifier mutants, including HDAC1, Pc, and Su(var)3 9, indicating their requirement for the reporter gene silencing. p<0.05 from unpaired student t test B) The IRER{ u bi DsRed} was induced by dietar y restriction (DR) in both males(blue) and females(red). DsRed was measured from adults at different time points (0/3/6/9 day) after feeding with either DR food(10S0Y) or control food(SY). The DR induced DsRed change was calc ulated by dividing the DsRed value from 10S0Y feeding animal by that of a SY feeding animal. Solid line, IRER{ubi DsRed}, carrying an ubi DsRed within IRER; Dash line, attB{ubi DsRed}, carrying an ubi DsRed in a different genomic locus. The DR induced DsRe d expression was only observed from IRER{ubi DsRed} but not attB{ubi DsRed}, indicating the DsRed induction requires the function of IRER.

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63 Table 2 1. List of RNAi lines used for the screen of epigenetic modifiers of IRER RNAi target gene VERC stock # Win g morphology IRER{ubi DsRed} signal in wing disc dHDAC3 20814 Small curl ed wing Moderate increase dHDAC1 30600 Small curl a little Moderate increase E(z) 107072 Normal No change Lid 103830 normal size, curl a little No change Hpo 104169 Severely cu rled No change UTX 105986 Normal No change Su(var)3 9 39378 Lethal Strong increase Su(z)12 42423 Normal No change CBP 104064 Normal No change UAS GFP (Ctr) Normal No change In order to identify the factors required for establishing and/or maintain ing the epigenetic status of IRER, individual UAS RNAi lines were crossed with Ser Gal4; IRER{ubi DsRed} to knock down target genes from Ser expressing cells in wing discs. The wing disc DsRed signal and wing morphology of the progenies were compared with that in the control (Ser Gal4>UAS GFP). Through this round of screen ing Su(var)3 9. HDAC1 and HDAC3 were identified as potential key repressors for IRER.

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64 CHAPTER 3 AN EPIGENETICALLY REGU LATED INTERGENIC REGION CONTROLS ORGAN SIZE AND MEDIATES DMYC INDU C ED APOPTOSIS I N DROSOPHILA Introduction Cell autonomous apoptosis following Myc over expression has been regarded as a major tumor suppression mechanism (reviewed by ( Meyer et al., 2006 ) ). Suppression of apoptosis, by means such as overexpression of Bcl 2, is required for unleashing the tumorigenic potential of Myc in mammalian cells ( Letai et al., 2004 ; Pelengaris et al., 2002 ; Strasser et al., 1990 ) Whether cells with elevated level of Myc undergo apoptosis depends on the availability of growth factors such as IGF ( insulin like growth factors ) ( Harrington et al., 1994 ) It has been post ulated that Myc induced cell autonomous apoptosis reflects a fundamental mechanism that maintains tissue homeostasis by inducing apoptosis when overproliferation is sensed. However, the mechanistic details of this mechanism remain enigmatic. One extensivel y studied pathway implicated in Myc induced cell autonomous apoptosis is the P53 mediated activation of pro apoptotic genes and/or suppression of anti apoptotic genes (reviewed by ( Hoffman and Liebermann, 2008 ) ). Many important pro apoptotic genes, such as apaf 1 and caspase 9 have been implicated in Myc induced apo ptosis in cell culture systems. However, study with animal models suggested that Myc induced cell death can proceed in the absence of apaf 1 or caspase 9 ( Scott et al., 2004 ) It is also clear that at least under some circumstances, Myc induced cell death can proceed without the participation of P53. Despite its enigmatic nature, the mechanism of Myc induced apoptosis appears to be highly conserv ed. Overexpression of dMyc, the only Myc ortholog in Drosophila also induces cell autonomous apoptosis ( de la Cova et al., 2004 ; Montero et al., 2008 )

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65 Intriguingly, while the level of dP53 ( Drosophila ortholog of mammalian tumor suppresser P53) mRNA is significantly increased following dMyc expression, the function of dP53 appears to be dispensable for dMyc induced cell death in Drosophila ( Montero et al., 2008 ) Ectopic expression of dMyc leads to increased cell size but fails to result in significant hyperplasia on its own ( Johnston et al., 1999 ) Moderate tissue overgrowth was only observed after dMyc induced apoptosis was blocked by co expression of the viral caspase inhibitor P35 ( Montero et al., 2008 ) This indicates that, similar to what was observed with mammalian tumorigenesis models, blockage of a poptosis is required for dMyc induced hyperplasia in Drosophila In this study, we showed that the induction of apoptosis following dMyc induced overproliferation requires a highly conserved intergenic regulatory control region in the RHG ( reaper hid a nd grim ) genomic block. The ~ 33kb intergenic region was originally named as IRER ( i rradiation r esponsive e nhancer r egion) since it was found to be required for mediating the induction of reaper hid and sickle following ionizing irradiation of the embryo ( Zhang et al., 2008 ) This region contains a previously identified response element for Drosophila P53 ( Brodsky et al., 2000 ) Interestingly, the epigenetic status of this region undergoes a dramatic change at embryonic development stage 12, when most cells enter into post mitotic differentiation. During the transitio n, this region becomes enriched for H3K27me3, H3K9me3, and bound by Polycomb group (PcG) proteins as well as HP1 (Heterochromatin Protein 1). Consequently, the DNA in this region becomes as inaccessible to DNase I as the pericentromeric heterochromatin reg ion. This epigenetic blocking of IRER specifically renders the pro apoptotic genes irresponsive to ionizing

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66 irradiation while other branches of the DNA damage response, such as the DNA repair pathway, remain active ( Zhang et al., 2008 ) In this work, we found that the IRER is required for mediating overproliferation induced apoptosis and plays a pivotal role in maintaining tissue h omeostasis during development. A nimals lacking IRER are viable but display tissue hyperplasia in several organs, indicating it is required for controlling cell numbers during development. The functional significance of this regulatory control region is heightened in the context of oncoge nic stress. IRER is required for the induction of apoptosis associated with oncogene induced overproliferation. While overexpression of dMyc alone failed to induce hyperplasia, it led to significant overproliferation in cells lacking IRER. Materials and Me thods Drosophila Strains and Culture Flies were maintained on a standard corn agar medium at 25C, except otherwise mentioned. The strains used in this study were described in supplementary table I. Irradiation was performed as described previously ( Zhang et al., 2008 ) Clone Induction The IRER mutant clones were generated with either the standard FLP/FRT system or the MARCM system ( Morata and Ripoll, 1975 ; Wodarz, 2000 ) Hsp flp and ey flp were used to induce clones from imaginal discs and adult eyes, respectively. Clone size was determined by measuring the 2 dimensional area of clones at 200X magnification. Immunohistochemistry and Microscopy Imag inal discs were dissected from third ins tar larvae in 1XPBS, fixed in 4% paraformaldehyde for 25 min at room temperature washed three times with 1XPBT for 5min each, counter stained with DAPI ( 1ug/mL ) and mounted on slides with Vectashield

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67 HardSet mounting medium (Vector laboratories). To detect the apoptotic cells, fixed discs were incubated with blocking buffer ( 5% norma l goat serum in PBT ) for 30min ~1hr at room temperature, followed by incubation with the anticleaved Caspase 3 antibody (Cell signaling, 1:200) for 2hr at roo m temperature or overnight at 4 C and incubation with the Alexa Fluor 488 /568 Goat anti Rabbit IgG secondary antibody (Molecular Probes, 1:400) for 2hr at room temperature or overnight at 4 C In situ hybridization was carried out as described ( Zhang et al., 20 08 ) FISH signal was detected by HRP conjugated anti DIG antibody (1:500, Roche) and subsequently amplified by the Tyramid Signal Amplification Kit (PerkinElmer, Waltham, MA, USA). Fluorescent images were taken with a Zeiss Axioplan imaging 2 microscope or a Lei ca SP5 Confocal Microscope The Open lab software was used to acquire the images. Clone size was m easured at 200X magnification in Photoshop (Adobe). Scanning electron microscopy (SEM) was performed as described ( Sullivan et al., 2000 ) I mages were taken at 1 000 magnification. Wing Size Measurement F lies were r aised at a standard density (~20 flies per vial) and three replicate vials were established for each genotype Both wings were dissected from adult flies and mounted onto slid es in Permount mounting medium (Fisher Scientific); only one wing from each fly was used for size measurement and the other one served as back up. Wing images were acquired using the Leica DMLB light microscope and w ing area was deter mined using methods as described ( Gilchrist and Partridge, 1999 ) Gene Expression Analysis RNA was extracted from ~ 20 wing discs or ~ 10 larvae of the desired genotype s using the RNeasy Minikit (Qiagen). cDNA was synthesized with the high capacity cDNA

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68 archive kit (Applied Biosystems). Quantitative PCR was run using the 7500 fast real time PCR system (Applied Biosystems) and signal was d etected based on SYBR Green intensity (SYBR Green Master Mix from Applied Biosystems). 10ng cDNA was used as template for each quantitative PCR reaction in duplicates. Statistics Wilcoxon rank sum test was used t o compare the difference between clone size and twin spots. In other cases, the data are subject to normal distribution and the tests were used to determine the statistical significance. Bioinformatics Hi C chromatin interaction : The Hi C data for D.melanogaster embryos (16 18hr) was take n from a recent genome wide study of three dimensional chromatin interactions ( Sexton et al., 2012 ) The data in the 600kb region (Ch3L: 18,000,001 18,600,000) with the resolution of 10kb bin was used to generate the heat map. The contact enrichment was calculated in the same way as described in Sexton et al. And the heat map was generated with R (R version 2.9.2, R Dev elopment Core Team, 2009). dMyc and P53 binding sites : Due to the unavailability of the Myc and P53 binding motifs in Drosophila we took the motifs for cMyc and P53 in vertebrates from TransFac dataset (Release 2012.1) ( Issigonis et al., 2009 ) We applied MATCH algorithm ( Kel et al., 2003 ) with the minFP (minimal false positive) setting to identify the potential binding sites for each motif in the RHG region. Then the identified b inding sites were pooled together for Myc (V$CMYC_01, V$CMYC_02) and P53 (V$P53_01, V$P53_02, V$P53_03, V$P53_04, V$P53_05).

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69 Results IRER Mediates DNA damage induced Pro apoptotic Gene Expression in Post embryonic Tissues Our previous work revealed that IR ER is required for mediating irradiation induced reaper hid and sickle expression in embryos before stage 12 ( Zhang et al., 2008 ) (Fig. 3 1A) In embryos defi cient for this intergenic region, i.e. homozygotes of Df(IRER), the transcriptional response of the three pro apoptotic genes to DNA damage is totally blocked. While the irradiation responsiveness of these three pro apoptotic genes is robust in stage 9 11 embryos, it is diminished in most cells in embryos past developmental stage 12. This sensitive to resistant transition is due to a targeted epigenetic regulation that requires the function of Polycomb group (PcG) proteins and histone deacetylase (HDAC) ( Zhang et al., 2008 ) To test whether IRER is also required for DNA damage induced cell death in post embryonic tissues, we measured irradiation induced caspase a ctivation and pro apoptotic gene expression in the developing imaginal discs of Df(IRER) It has been well documented that exposure of Drosophila larvae to irradiation induces a rapid and wide spread apoptosis in imaginal discs ( Brodsky et al., 2000 ; Ollmann et al., 2000 ) Specifically, it has been shown that the significant increase of apoptotic cells in t he wing disc at 4 hours following irradiation is dependent on the function of dP53 ( Wichmann et al., 2006 ) W e subjected third instar larvae to ray. Indeed, we found that at 4 hours post irradiation, there is a substantial increase in the amount of apoptosis in the wild type wing dis cs, preferentially at the wing margin (Fig. 3 1C vs. B ). However, in sharp contrast, there is little detectable increase of caspase activation in discs from animals homozygous to Df(IRER) (Fig. 3 1E vs. D ).

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70 W e then measured the mRNA level s of the RHG genes by quantitative PCR. In a time course analysis, we found that the induction of reaper and hid was highest between 1 and 2 hours fo llowing irradiation (Fig. 3 1F). At this time interval, irradiation induced expression of reaper and hid was significantly l ower in wing discs homozygous to Df(IRER), when compared to the wild type control (Fig. 3 1G). The levels of sickle and grim mRNAs in the wing discs were rather low and barely detectable even after irradiation. We noticed that, unlike the embryo, the induc tion of reaper and hid was not completely blocked in the wing discs. It has been observed that a P53 indpendent mechanism also contributes to irradiation/DNA damage induced cell death in the wing disc. In animals mutated for dp53 or chk2 such a mechanism could eventually compensate for the loss of dP53 or Chk2 function and induce apoptosis at a much later (12 16hr post IR) time point ( Wichmann et al., 2006 ) It is possible that the incomplete block of reaper / hid induction in the wing discs reflects the existence of such a mechanism in wing disc cells. However, it is c lear that the significant suppression of ray induced reaper / hid expression in Df(IRER) was sufficient to result in a significant decrease of apoptotic cells, i.e. cells with activated caspase 3, at 4 hours post irradiation. Ther efore, we conclude that IRER also mediates DNA damage induced pro apoptotic gene e xpression and cell death in po st embryonic tissue The cis Regulatory Function of IRER is Required for Tissue Homeostasis and Organ Size Control ray induced cell death in the developing imaginal discs, we n oticed that discs from homozygous Df(IRER) animal s are conspicuously larger than those with wild type IRER or heterozygous t o the deficiency ( Fig. 3 2 A B & D E). This is accompanied by a decreased level of apoptotic cells in the

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71 wing disc even without any irradiation treatment. A detailed statistical analysis revealed that both eye and wing imaginal discs from homozygous Df(IRER) larvae are significantly larger than those from their heterozygous l ittermates (Fig. 3 2 C&F). Close inspection of the discs indic ated that the size of the cell in the Df(IRER) discs, reflected both as the distance between DAPI stained nuclei as well as density of cells within a given area, was indistinguishable from that of the heterozygous discs. Thus it appears that there are mor e cells in the developing discs lacking the function of IRER. To make sure that the observed increase in imaginal disc size was not due to changes in the timing of proliferation or developmental cell death, we further evaluat ed the size of the adult wing. About 10 20% homozygous Df(IRER) animals survive to adulthood. The wings of these Df(IRER) homozygous flies are significantly larger than those of wild type animals raised in parallel or heterozygous animals develope d in the same vial (Fig. 3 3 When di fferent compartments of the wing were monitored, it appear ed that both anterior and posterior compartments of the wing overgrew with the change being relatively more pronounced in the posterior co mpartments ( Fig. 3 4 A B) The increased size of the wing wa s not due to increased cellular size but purely due to the increased number of cells that contributed to the adult wing. When the densities of the trichome (one from each wing cell) were measured, to our surprise, the density was slightly higher in Df(IRER ) wing than those of wild type or heterozygous to Df(IRER). The difference was statistically significant for the posterior compartments (Fig. 3 3 B ). When corrected for this difference in cell d ensity, wings from homozygous Df(IRER) animals have about 10% m ore cells than t he wings of wild type animals.

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72 To rule out the possibility that the phenotype was due to background mutation on the Df(IRER) chromosome, complementation test was performed with well defined deficiencies of this region (Fig. 3 3 C) Both Df(E D224) and Df(ED225) were generated by the DrosDel project with defined break points and in isogenic background ( Ryder et al., 2004 ) While both deficiencies are homozygous lethal, the transheterozygous with Df(IRER) are viable. The size of the wings of Df(IRER)/Df(ED225) animal is about the same as Df(IRER) homozygous. In contrast, the overgrown phenotype was partially complemented by the Df(ED224), which lacks t he transcribed regions of reaper grim and hid but has the IRER region intact (Fig. 3 1A). This seems to suggest that either the expression of sickle can contribute to the size control or that IRER can act in trans to regulate the expression of reaper and /or hid Since the genetic background of Df(ED225) is independent of that of Df(IRER), we conclude that the observed overgrown phenotype is due to the lack of the genomic region of IRER. The abnormality of organ size control is due to the cis regulatory fu nction of IRER. There is no predicted coding or non coding gene in the IRER genomic region (Flybase.org). In addition, no transcript with coding potential has been detected in this region by any RNA sequencing projects (based on data collected by Flybase o r ModENCODE). To rule out the possibility that the phenotypes associated with Df (IRER) might be due to a low level of non coding transcripts from IRER, we cloned the genomic region encompassing the IRER as a potential rescue construct (Fig 3 1A). A BAC cl one containing the genomic region Chr 3L: 18391335 18432601, corresponding to the span from just before the start of Reaper ORF to the stop codon of Sickle (Fig. 3 1A), was used to make a transgenic fly via phiC31 integrase mediated transformation Animals

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73 carrying the 2 nd chromosome tha t contains this reaper sickle interval fragment appear to be normal without any noticeable change in viability or organ size. When this chromosome was recombined with the Df(IRER) chromosome it failed to rescue the partial lethality and increased wing size associated with homozygous Df(IRER) (Fig. 3 3 D ). Homozygous Df(IRER) flies carrying this rescue construct have enlarged wings indistinguishable from those without the rescue construct. From these evidences, we concluded th at the cis regulatory function of IRER is required for controlling tissue homeostasis. To monitor how the lack of IRER cis regulatory function affects pro apoptotic gene expression in development, w e compared the expression of the four pro apoptotic gene s, reaper hid grim and sickle between wild type and homozygous Df(IRER) larvae. We found that the level of reaper mRNA was significant ly lower ( 50% ) in Df(IRER) mutant larvae. The level of sickle was reduced by about 60%, while the levels of hid and grim were n ot significantly changed (Fig. 3 3 E). The level of reaper mRNA in Df(IRER) wing discs was reduced to similar extent as that in the whole la rvae (Fig. 3 3 F). However, the level of sickle in the wing discs was too low to be reliably measured with QPCR. Similarly, the level of hid mRNA in the wing discs was not significantly changed. The level of grim was also too low to be detectable from the win g discs. The measurement of RHG mRNA levels was normalized against housekeeping genes rp49 or GAPDH. The trends as well as the magnitude of changes do not seem to be significantly affected by th e choice of standard genes. To observe whether IRER is als o responsible for mediating developmental cell death in other tissues, we monitored developmental cell death in the embryonic CNS.

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74 We noticed that about 10% of the homozygous embryos have an enlarged nervous sys tem ( Fig.3 5 A B). Comparing the nervous syst em phenotype of Df(IRER) with that of H99 suggests that most RHG mediated developmental cell death likely proceeds normally without the function of IRER ( White e t al., 1994 ; Zhou et al., 1995 ) Within the embryonic system, the programmed cell death of the midline glia l cells has been shown to be dependent on the EGF signals provided by the axons ( Bergmann et al., 2002 ) Using the slit 1.0 lacZ reporter that labels the ensheathing midline glia we noticed that in stage 16 17 embryos homozygous to Df(IRER), there were extra slit 1.0 lacZ positive cells present in abdominal segments ( Fig. 3 5 C D) This suggests that IRER function is also required for mediating this EGF dependent c ell death of the midline glia. The pattern of cells expressing reaper and sickle in develo ping tissues is dynamic, which is partly due to the fact that cells expressing these genes are quickly removed via apoptosis. The expression pattern of these two genes, especially reaper seems to be sporadic in many developing tissues such as the wing dis c. Since it is impossible to predict which cells in the developing wing will express reaper we turned to the embryo. We noticed that in places where the expression of reaper is cell lineage specific, such as in neuroblasts in post stage 14/15 embryos, th ere is no detectable difference in Df(IRER) homozygous embryos. In contrast, the segmentally repeated expression of reaper at the epidermis was significantly weakened in animals lacking IRER (Fig. 3 6 ). demonstrated that the number of epidermal cells that can survive in the embryonic epidermis is strictly dependent on the EGF signaling ( Parker, 2006 )

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75 Taken together, our observations indicate that the intergenic region of IRER plays a fundamental role in regulating the appropriate number of cells in s everal tissue compartments. This function is likely achieved by activating/increasing the expression of pro apoptotic genes in response to increased cell number and consequently decreased availability of growth factors. The impact of losing IRER appears to be more significant in tissues that have development al cell death regulated by growth hormones as opposed to tissues where developmental cell death is largely cell lineage specific IRER is Required f or the Induction of Apoptosis following dMyc i nduced Overproliferation The fact that IRER is required for mediating appropriate cell number during tissue development prompted us to see whether IRER is also involved in mediating the pro apoptotic response f ollowing dMyc induced overproliferation. Similar to what has been observed for c Myc in mammalian systems, overexpression of dMyc in developing Drosophila tissues leads to increased metabolism, increased cell size, and overproliferation (reviewed in ( Gallant, 2009 ) ). The dMyc induced overp roliferation is quickly followed by induction of apoptosis which essentially cancels out the impact on final number of cells. For instance, when dMyc was expressed in differentiating eye cells driven by GMR Gal4, there was a significant increase of cell pr oliferation as documented by incorporation of BrdU ( Montero et al., 2008 ) However, the overproliferation was quickly followed by increased TUNEL signals among dMyc expressing cells. Overall, GMR driven dMyc (GMM) has little noticeable effect on the final number of cells in the eye. These reported observations were reconfirmed when we established the system wit h GMR Gal4 and UAS dMyc (Fig. 3 7 A&H).

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76 The cis regulatory function of IRER is required for overproliferation induced apoptosis in the GMM model. Under normal conditions, there is no detectable defect in the eyes of surviving homozygous Df(IRER) adults (Fi g. 3 7 B), suggesting that either there is a minimal level of developmental cell death in the eye, or the developmental cell death can proceed in the absence of the regulatory function of IRER. As previously reported ( Montero et al., 2008 ) we found that co expressing P35, the viral caspase inhibitor, marginally enhanced the hyperplasia induced by GMM (Fig. 3F). However, when dMyc was over expressed in eye discs lacking IRER, there was a massive increase of the size o f the adult eye (Fig. 3 7 C & I). The superfluous ommatidia caused disorganization of eye, which was never seen with GMM alone. This enhancer effect w as noticeable even in the animals heterozygous to Df(IRER) ( Fig. 3 7 D). The dominant e nhancing effect of Df(IRER) was similar to that observed for heterozygous Df(ED225) (Fig 3 7 E), a ~450kb deletion that covers the transcribed regions of grim reaper sickle and more than 10 other genes (Fig. 3 1A). The se observations strongly suggest that IRER plays a central role in regulating the expression of pro apoptotic genes in the context of GMM induced overproli feration. It has been shown cell autonomous apo ptosis following dMyc induced overproliferation is accompanied by the induction of reaper and sickle ( Montero et al., 2008 ) Using fluorescent in situ hybridization (FISH), we found that reaper is signific antly induced in GMM eye disc. Cells with significantly elevated reaper mRNA are posterior to the morphogenetic furrow (Fig. 3 7 K), consistent with previously reported observation of apoptotic cells in this model. This dMyc induced cell

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77 autonomous expression of reaper was totally absent in discs ho mozygous to Df(IRER) (Fig. 3 7 L). It has been documented that constitutively elevated level of dMyc introduced by t ubulin > d M yc ( Tub dMyc ) leads to an increased size of the wing ( de la Cova et al., 2004 ) mostly due to the increased cellular size instead of cell numbers. When T ub dMyc was crossed into the Df(IRER) background, we found that the wing sizes were significantly larger than either Df(IRER) or Tub dMyc alone (Fig. 3 7 M). Significantly more cells survived when IRER is absent in dMyc overexpressi ng wings. To make sure that the observed effect of Df(IRER) on dMyc induced cell death is indeed cell autonomous, we generated clones of cells deficient for IRER and simultaneously over expressing dMyc via the MARCM strategy ( Morata and Ripoll, 1975 ) (Fig. 3 8 A). Clones of Act Gal4>UAS dMyc;IR ER( / )Gal80( / ) cells (Fig. 3 8 D E) were significantly larger than that of Act Gal4>UAS d Myc;IRER(+/+)Gal80(+/+) (Fig. 3 8 B C). Taken together, our observations indicate that IRER is required for the induction of apoptosis, and consequently the suppression of hyperplasia, following dMyc induced overproliferation. This is not limited to a pa rticular tissue, but appears to be true for a variety of tissues exami ned in this study. Cells with Relatively Open IRER are More Sensitive to dMyc induced Cell Death Our previous analyses have shown that th e accessibility of IRER in developing embryos is subject to epigenetic regulation ( Zhang et al., 2008 ) In cells with open IRER, reaper sickle and hid can be dramatically induced within 15 30 minutes followin g ionizing irradia tion. In contrast, none of these three genes can be induced when IRER forms a heterochromatin like structure enriched w ith both H3K27me3 and H3K9me3. To monitor the accessibility of IRER in individual cells, an ubiquitin DsRed

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78 reporter w as inserted into IRER through homologous recombination (Fig. 3 9 A). This reporter, IRER{ ubi DsRed }, gives relatively low and variegated expression i n devel oping wing discs (Fig. 3 10 A). When Su(var)3 9, which is required for epigenetic blocking of IRER dur ing embryogenesis, was knocked down via RNAi, the reporter was dramati ca lly de repressed (Fig. 3 10 B). The expression of IRER{ ubi DsRed } is also sensitive to environmental stress suc h as irradiation (Fig. 3 11 ) and nutrition. It has been shown that dMyc dr iven by dpp Gal4 at the anterior/posterior boundary of the wing discs induces apoptosis within the dMyc expressing zone as well as the zone immediately anterior to the dMyc expressing zone ( de la Cova et al., 2004 ) We reproduced the system in the presence of IRER{ ubi DsRed }. We noticed that, as reported, the sizes of cells with dMyc overexpression are noticeably larger than the neighboring cells, which is reflected by the dilut ed DAPI signal intensity (Fig. 3 9 C D). Surprisingly, there is a conspicuous lack of DsRed positive cells in the dMyc expression zone as well as the zone immediately anterior to it. In contrast, the variegated patterns of DsRed in oth er regions of the dpp>dMyc wing discs are indistinguishable from those in discs without dMyc overexpression (Fig. 3 IRER{ ubi DsRed } expressing cells is not specific to dpp>dMyc Similar phenomenon was observed when clones of dMyc expressing cells were generated with Act >y>Gal4; UAS dMyc (Fig. 3 9 E To verify that cells with higher levels of DsRed, and presumably more accessible IRER, are preferentially eliminated by dMyc induced cell autonomous cell death, we c o expressed Diap1 in those cells that have dpp Gal4 expression The variegated pattern of DsRed signal in the dpp zone was largely restored by co expressing of Diap1(Fig. 3 9

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79 D positive cells in this zone without Diap1 was due to preferential elimination of cells with relatively open IRER. This observation support s the hypothesis that epigenetic regulation of IRER plays an important role in controlling cellular sensitivity to overprol iferation induced cell death. Cells Lacking the cis Regulatory Function of IRER have the Propensity to Overgrow and are Resistant to Stress induced Cell Death To mimic the behavior of cells with completely blocked IRER in proliferating tissues, we generated mosaic clones bearing IRE R deletions in the developing imaginal discs. The heat shock induced FLP system was titrated to generate about 1 clone per disc, which allowed us great confidence in identifying and measuring the size of the clones and the simultaneously generated twin sp ot s. As shown in Figure 3 12 control (wild type) clones and the ir twin spots grow to similar size in either the wing or the eye discs ( Fig 3 12 A & C). In contrast, clones lacking either the left section of IRER (Df(IRER_left)) (Fig. 3 12 B & D) or the en tire IRER (Df(IRER)), have a great propensity to significantly overgrow the ir respective twin spots. Although there is considerable variation in how much the mutant clones overgrow their twin spots, the overall differences are statistically significant ( op en bars in Fig. 3 12 E & F; F ig. 3 13 ). The propensity of IRER mutant clones to overgrow (wild type counterparts) was significantly heightened when cytotoxic stress was present. When growing animals were subjected to a non lethal dose of IR (10Gy) at 24 hrs following clone induction, the ratio between the size of the IRER deficient clones and their respective twin spots was greatly increased, both in the wing discs (Fig. 3 12E & G) and the eye discs (Fig. 3 12F & Fig. 3 13 A ). Under this heightened stress con dition, we noticed that in about 15% of the cases, the wild type twin spots had all been eliminated leaving only the IRER

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80 mutant clones at the end of larval development. Although these instances were not included in calculating the ratio of sizes between mutant clone vs. twin spot, the overgrown propensity of both Df(IRER_left) and Df(IRER) clones was still statistically signific ant (Fig. 3 12 E F, closed bars). The overgrown propensity of cells lacking the cis regulatory function of IRER is accompanied by resistance to stress induced cell death. When irradiation induced cell ray treatment (40Gy), it is clear that, while there are abundant apoptotic cells in the twin spots or the hetero zygous tissues, there is a conspicuous lack of activated caspase 3 signal in the clones deficient for IRER (or IRER_left) (Fig. 3 12 H). The RHG Genomic Regulatory Block Contains Consensus Myc Binding S ites The RHG genes reside in a ~250kb synteny that is m arked by unusually long intergenic regions surrounding reaper and grim as well as an unusually high level of conservation of nucleotide sequences within these intergenic regions ( Lin et al., 2009 ) It is interesting to note that when sequences from distantly related Drosophila species are considered, nucleotide sequences within IRER are better conserv ed than those underlying the reaper coding region ( Zhang et al., 2008 ) IRER is enriched for highly conserved non coding e lements (HCNEs) ( Engstrom et al., 2007 ; Lin et al., 2009 ) Functional studies in both vertebrates/mammals and insects have shown that HCNEs ofte n function as long range enhancers to regulate complex temporal and spatial expression patterns of target genes ( Pennacchio et al., 2006 ; Woolfe et al., 2005 ) Direct evidence that the genomic region encompassing the RHG genes forms a regulatory block was provided by recent whole genome Hi C analysis ( Sexton et al., 2012 ) By analyzing the data set generated by Sexton et al, it became clear that the

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81 previously observed synteny correlates very well with a domain defi ned by chromosomal contacts (Fig. 3 14 A). Extensive chromosomal contacts exist within this region, which for simplicity, we will refer to as the RHG genomic regulator block. Independent of this whole genome Hi C analysis, 3C analysis conducted by us also r evealed that there are extensive interactions between the IRER and the RHG gene promoters. However, what remains to be addressed is whether the pattern of chromosomal interactions within the RHG genomic regulatory block is the same among different cells, o r likely to be dynamic depending on the cell type or differentiation status. Bioinformatics analysis also revealed that there are binding sites for Myc and P53 in IRER (Fig. 3 14 B). Unfortunately, we were not able to experimentally verify the predicted bi nding sites due to the unavailability the dMyc antibody suitable for ChIP. The mechanism of interaction between dMyc and IRER (e.g. direct or indirect interaction) needs to be explored in further study. Discussions Our study provided evidences indicating t hat the regulatory region IRER in the RHG genomic regulatory block plays a pivotal role in controlling apoptosis to maintain tissue homeostasis. Animals lacking this regulatory region are viable but have overgrown organs, suggesting cells lacking IRER have decreased sensitivity to growth factor levels or other intrinsic cell number controlling mechanisms. The regulatory function of this region is also required for mediating the induction of apoptosis, and suppression of hyperplasia, following dMyc overexpre ssion in a variety of tissues. The fact that the accessibility of IRER is subject to epigenetic control implies that cellular sensitivity to proliferation induce apoptosis, and consequently the balance between

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82 proliferation and cell death, is subject to ep igenetic mechanisms which in turn are regulated by differentiation status and/or environmental factors. IRER Serves as the Gatekeeper for Overproliferation i nduced Apoptosis A tenacious link between proliferation and apoptosis has long been observed in bot h developmental and pathological (tumorigenesis) settings (Reviewed in ( Hipfner and Cohen, 2004 ) ). While there is little dispute on the importance of this mechanism in maintaining tissue homeostasis and preventing hyperplasia, the mechanistic details remain enigmatic. Our data indicate tha t in Drosophila IRER serves as the converging point for induction of apoptos is following overproliferation. First, we showed that the cis regulatory function of IRER is required for controlling appropriate cell numbers in the developing tissues. The conse quence of lacking IRER seems to be more significant in tissues where cell survival is largely dependent on the availability of growth factors. While the overall impact of Df(IRER) on the size of the embryonic CNS is marginal, the survival of midline glial cells, which have been shown to be dependent on the EGF signal provided by the neuronal axons ( Bergmann et al., 2002 ) are significantly impacted. In post emb ryonic tissues, cell death in the wing disc has been shown to be strongly influenced by the BMP/dpp signaling ( Gibson and Perrimon, 2005 ; Martin Castellanos and Edgar, 2002 ; Moreno and Basler, 2004 ; Moreno et al., 2002a ; Shen and Dahmann, 2005 ) Interestingly, wing discs are very sensitive to ionizing radiation and heat shock induced apoptosis ( Perez Garijo et al., 2004 ; Ryoo et al., 2004 ) This sensitivity is at least in part mediated through regulatory elements reside in IRER, since irradiation induced cell death is significantly inhibited in discs lacking this regulatory region (Fig. 3 1). It has been estimated that during normal development, at any given time point, about 1.4% cells in the larval developing wing

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83 disc are TUNEL positive ( Milan et al., 1997 ) Since ap optotic cells are quickly removed by phagocytosis, the overall number of cells that undergo apoptosis during the whole development period of the wing disc is certainl y to be much higher. The consequence of a reduced level of cell death caused by the absenc e of IRER is significantly different from that of blockage of cell death by Bac ulovirus caspase inhibitor P35. Inhibiting the final demise of cells with P35 leads to distorted formation in the wing disc and the adult wing ( Perez Garijo et al., 2004 ; Ryoo et al., 2004 ) Apparently cells with higher levels of RHG genes are not only on the path to final dem ise, but also secret growth signals to induce compensatory proliferation of neighboring cells ( Perez Garijo et al., 2004 ; Ryoo et al., 2004 ) In contrast, although animals lacking IRER have about 10% more cells, the structure of the wing is essentially normal. While there is a perceivable decrease of apoptotic cells in Df(IRER) discs (Fig. 3 1D), the ove rall pattern of apoptotic cells was not significantly different from that of wild type discs. This suggests that lacking IRER tunes down the sensitivity of wing disc cells to environmental constraints, such as growth factor availability, without fully bloc king cell death regulation. The fact that about a 50% 60% reduction of selected RHG genes can have a significant impact on the number of cells forming the wing is rather interesting. It should be noted that the overgrowth phenotype we observed was not repo rted for animals lacking the transcribed region of reaper ( Moon et al., 2008 ) or grim ( Wu et al., 2010 ) or sickle ( Tan et al., 2011 ) alone. This is strikingly reminiscent of what was observed for development cell death of the embryonic neuroblasts, in which the deletion of a central regulatory region (NBRR) gave a mo re prominent phenotype than deleting individual

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84 RHG gene ( Tan et al., 2011 ) Large deletions that delete multiple RHG genes, such as H99, ar e embryonic lethal, preventing the analysis of tissue growth at later developmental stages. The relatively similar wing disc overgrown phenotype between Df(IRER) homozygous and the Df( IRER )/Df(ED225) transheterozygous strongly suggest that IRER plays a piv otal role in regulating RHG gene expression in respo nse to increased cell numbers. In addition to its role in limiting cell number during development, we showed that IRER is also required for mediating the induction of apoptosis following d Myc induced over proliferation. IRER is certainly required for the cell autonomous apoptosis following dMyc overexpression, as have been demonstrated with the overgrown phenotype of d Myc;IRER( / ) clones (Fig. 3 8 ). However, we cannot exclude that potential inhibition of s uper competition induced cell death in Df(IRER) might also contributed to the hyperplasia observed for GMM in Df(IRER) animal. Indeed, additional analysis indicates that IRER is required for competition induced elimination of weaker cells. However, the mechanism seems to be different and involves mainly the role of IRER in regulating hid expression. In the context of dMyc induced cell autonomous cell death, ( Montero et al., 2008 ) indicate that reaper and sickle are likely responsible. The levels of these two genes, but not that of hid were also significantly decreased in Df(IRER) larvae that have overgrown tissue. Thus, it appears that overproliferation induced apoptosis is achieved through IRER mediated induction of reaper and/or sickle It remains to be seen as to which enhancer is responsible for mediating dMyc in duced cell autonomous expression of reaper (and sickle ), and whether the same

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85 enhancer(s) is(are) responsible for regulating these two genes in developing tissues. Although there are consensus dMyc/cMyc binding sites within IRER, the lack of a suitable ant ibody prevented us from verifying whether any of these sites indeed binds with dMyc. Whole genome dMyc binding in the cultured Kc167 cells was previously revealed via the DamID method ( Orian et al., 2003 ) the published list did not contain any binding site in the RHG genomic block. However, since the IRER is enriched for suppressive histone modifications and has very low accessibility in the Kc167 cells, it very likely does not reflect cells in vivo that have relatively open IRER. Functional Significance of E pigenetic Regulation of IRER in Development and in Tumorigenesis Epigenetic regulation of IRER during normal development likely plays an important role in ad justing cellular sensitivity to apoptosis inducing signals in accordance with cell type and differentiation/proliferation status. During embryogenesis, IRER is open in rapidly proliferating cells prior to stage 12, conferring hypersensitivity to environme ntal stress ( Zhang et al., 2008 ) It likely also confers sensitivity to growth factor signaling, based on our observation that reaper expression in the developin g epidermis is significantly impeded in Df(IRER) embryos ( Fig. 3 6 A). Although it remains to be seen whether the expression of reaper at the epidermis is directly regulated by the EGF pathway, it has been shown that apoptosis in the epidermis, and the numb er of cells surviving at each segment, is dependent on the EGF signaling ( Parker, 2006 ) In addition, overexpression of a Ras gain of function mutant has been found to suppress hid expression in embryos specifically around this stage (5 7 hr post eg g laying) ( Kurada and White, 1998 ) In post stage 12 embryos, when most cells enter into post mitotic differentiation, IRER is closed and renders cells resista nt to stress such as

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86 irradiation. The formation of heterochromatin in IRER never reaches the proximal enhancer and promoter regions of reaper which is due to the presence of about a 200bp DNA fragment that functions as a chromatin barrier ( Lin et al., 2011 ) This IRER specific epigenetic blocking specifically tunes down the sensitivity of RHG genes to stress, while still allowing them to be expressed in cell linea ge specific patterns. For instance, r eaper (and sickle ) is expressed in neuroblasts in stage 14 16 embryos to mediate lineage specific apoptosis ( Peterson et a l., 2002 ; Tan et al., 2011 ) Using the IRER{ ubi DsRed } reporter, we were able to observe that the accessibility of IRER varies significantly even among neighboring cells in developing wing or eye discs cells. The variegated pattern of IRER{ ubi DsRed } in developing tissues suggests that there is a stochastic component of epigenetic regulation of IRER. A direct consequence of this stochastic regulation is that there is a distribution of cells with varying degree of IRER accessibility in a given tissue compartment. Our results with dpp>dMyc indicate that cells with relatively open IRER are preferentially removed when over proliferation is sensed among the population of cells. A lasting puzzle of apoptosis re gulation in development has been that how can some cells chosen to commit suicide while many of their sister cells remain live. Our analysis indicated that stochastic epigenetic regulation, and the resulting varying degree of sensitivity, may be an importa nt mechanism to tier apoptotic response to lowering growth fac tors or environmental stimuli. Abnormal chromatin regulation has been hypothesized as a major oncogenic mechanism (reviewed in ( Baylin and Jones, 2011 ) In this work, we found that the epigenetically regulated IRER is required for the induction of apoptos is following dMyc

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87 induced overproliferation. It has been elegantly demonstrated in mammalian models that the tumorigenic potential of Myc is only fully manifested when apoptosis is blocked by ectopic expression of key anti apoptotic regulators such as Bcl 2 (reviewed in ( Meyer et al., 2006 ) ). In this study, we showed that higher levels of dMyc can lead to dramatic tissue hyperplasia in Df(IRER ) animals, where the sensitivity to stress induced apoptosis was impeded due to the absence of the regulatory region. The accessibility of IRER is subject to epigenetic modification, and formation of heterochromatin in IRER is capable of blocking this reg ion to transcription factors ( Zhang et al., 2008 ) Our observations strongly suggest that over expression of dMyc, coupled with epigenetic blocking of IRER, coul d lead to tumorigenesis in the absence of any other genetic mutation. Such a mechanism appears to be also present in mammals. It has long been observed that Bmi 1 the mammalian ortholog of PcG protein PSC, corroborates with cMyc in promoting tumorigenesis but suppressing apoptosis ( Jacobs et al., 1999 ) It has also been hypothesized that epigenetic silencing of pro apoptotic genes by latent EBV infection may be the underlying mechanism of endemic which is strongly associated with EBV infection and characterized by ectopic expression of Myc ( Allday, 2009 ) In addition to complete silencing of pro apoptotic genes or regulatory regions, it is also possible that disturbance of the stochastic distribut ion of epigenetic statuses may lead to increased heterogeneity observed for cancerous cells ( Pujadas and Feinberg, 2012 ) T he mechanism that underlies the epigenetic regulation of IRER r emains to be fully elucidated. Interestingly, the chromatin barrier (IRER left barrier or ILB) that prevents the spreading of suppressive histone modification from IRER to the promoter

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88 of reape r also functions as a potent silencing barrier when tested in a vertebrate system ( Lin et al., 2011 ) ing the formation of facultative heterochromatin in IRER. There is no discernable PRE (Polycomb response element) in IRER and the formation of heterochromatin in IRER seems to be independent of anterior/ posterior position of the cell. Rather, observing the expression of IRER{ ubi DsRed } throughout the development of various tissues suggests that there is no simple cause effect relationship between cellular differentiation and/or proliferation status and the accessibility of IRER. It appears that epigenetic r egulation of IRER is dynamic during development, stochastic in tissue compartments, and responsive to environmental conditions. This regulation may very well present as a unique system for us to further explore how epigenetic mechanism controls the fundame ntal balance between proliferation and apoptosis, and how dysregulation of this mechanism can contribute to tumorigenesis.

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89 Table 3 1. Drosophila s trains used in this study strain source note Df(IRER) & Df(IRER_left) ( Zhang et al., 2008 ) IRER deletion Df(IRER),FRT80B & Df(IRER_left), FRT80B Generated in this work Df(3L)ED224 Bloomington #8080 Chromosomal deletion: 75B1 -75C6 Df(3L)ED225 Bloomington #8081 Chr omosomal deletion: 75C1 -75D4 Bac IRER Generated in this work Targeting site: 51D UAS p35 Bloomington #6298 IRER{ubi DsRed} 5 Generated in this work dpp Gal4,UAS GFP A gift from Laura Johnston Act Gal4,UAS GFP Generated in this work tub>dMyc>G al4 A gift from Eduardo Moreno UAS Su(var)3 9 RNAi VDRC #39378

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90 Figure 3 1. IRER mediates DNA damage induced reaper and hid expression in the developing wing discs. A) The four pro apoptotic RHG genes ( reaper hid grim and sickle ) are clustered i n a ~ 280 kb genomic region. The four genes are transcribed to the same direction and the two intergenic regions surrounding reaper are unusually long (99 kb and 40kb). Regulatory regions, such as NBRR (Neuroblast Regulatory Region) ( Tan et al., 2011 ) or IRER ( Zhang et al., 2008 ) controls the expres sion of multiple RHG genes during development or in response to stress. The regions deleted in deficiency strains used in this study are indicated by dashed lines. The region cloned into the BAC IRER rescue construct is indicated by the red bar on top of t he DNA. The figure is not drawn to scale due to space limitation. Nucleotide coordinates are relative to the reaper TSS and based on Drosophila genome release 5.42. B E) Caspase 3 staining of wing discs without IR (B and D), or at 4 hours after 40Gy of IR (C and E). Irradiation induced a widespread of apoptosis in wild type wing discs (compare C with B) but not in course of irradiation induced reaper expression from whole larvae. Early third instar larvae were irradiated with 40Gy of IR and collected for quantitative RT PCR (qRT PCR) at various times afte r IR. The induction level of reaper, i.e. the ratio of IR vs. control (NT), appeared to be the highest between 1 and 2 hour after IR in both wild type and Df(IRER) larvae. G) Both reaper and hid transcripts are greatly induced from wild type wing discs at 1.5~2 hours following IR as shown by qRT PCR. The induction magnitude is much smaller in Df(IRER) wing discs. For all the qRT PCR data, means + s.d. are shown (n=3).

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91 Figure 3 2 IRER is required for the control of imaginal disc size. A B) The represent ative images of wing imaginal discs from third insta r larvae either heterozygous or ho mozygous to IRER deletion. C) The size measurements of wing discs of 3 rd in star larvae we re summarized in the whiskers box plot. The top and bottom bars of the box plot corresponded to the max the min values, respectively. The homozygous Df(IRER) larvae had enlarged wing discs when compared to their heterozygous littermates (p = 0.0024, Student t test; n(IRER +/ )=20, n(IRER / )=32 ). D E) The representative images of third instar eye imaginal discs from the labeled genotypes. F) Similar to what was observed with the wing discs, the size of eye discs from homozygous Df(IRER) larvae was also significantly larger than that fr om heterozygous Df(IRER) larvae (p = 1.9e 5, Student t test; n(IRER +/ )=23, n(IRER / )=27)

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92 Figure 3 3. The cis regulatory function of IRER is required for organ size control A) A representative image of the wild type wing, with five longitudinal veins labeled as L1 through L5. The squares indicate the areas sampled to measure hair number Wings from IRER / adult flies (red) are larger than those from wild type flies (black). Bar=10 B) T he density of cells was estimated by counting the number of trichomes from fixed area s on the w ing blade. Four 75px 2 areas (squares in A) were selected for trichome counting (L1 L2, L2 L3, L4 L5 and L5). In the posterior compartment, i.e. L4 5 and L5, t he cell density is higher i n IRER / wings ( p= 0.0022 and 0.0002, respectively ; n=12 ). There is no significant difference in cell density between the two genotypes in the anterior compartment. ( p= 0.0566 and 0.0783, for L1 2 and L2 3, respectively ; n=12). C) Quanti fication of adult wing sizes As shown in the wild type controls ( p = 2.6e 5 ) A similar wing size increase is observed with the transheterozygous Df(ED225)/Df(IRER) (p< 0.0001 between ED225 /IRER (n=20) and +/+ (n=25)), p= 0.0581 between ED225 /IRER and IRER / (n=26)). In contrast,

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93 the wing ove rgrown phenotype was partially rescued by Df(ED224) (p< 0.0001 between ED224 /IRER (n=27) and +/+, p= 0.0236 between ED224 /IRER and IRER / ). The deletion regions in ED224 and ED225 are indicated in Fig 1A. D ) Introducing the BAC IRER to the second chromo some failed to rescue the increased wing size of IRER / (p< 0.0001 between Bac IRER, IRER / (n=30) and +/+, p= 0.2669 between Bac IRER, IRER / and IRER / E ) The basal levels expression of reaper and sickle are significantly reduced in IRER / l arvae a s measured by qRT PCR. F) The expression of reaper is also decreased in the IRER / wing discs. The unpaired student t test was used to calculate p value.

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94 Figure 3 4 Differentially increased compartment size in Df(IRER) wings A) Schematic figure showe d the A P compartment of the Drosophila wing. Compartment size was measured as the number of pixels within the contour lines B) The size of A/P compartment was measured separately for both wild type and Df(IRER) wings. When comparing to the corresponding compartment in wild type wings, A compartment of Df(IRER) wing s showed a ~10% size increase, and P compartment of Df(IRER) wing s showed a ~19% size increase. p= 0.0151 between A (+/+) and A (IRER / ), p= 0.0012 between P (+/+) and P (IRER / ), student t te st; n=7 for each genotype.

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95 Figure 3 5 Homozygous Df(IRER) embryos have enlarged central nervous systems. A B) About 10% of the embryos homozygous to IRER deletion contained an enlarged central nervous system (CNS) at the end of embryogenesis (st. 17). Anti Elav staining was used to label differentiated neurons. C D) Anti galactosidase staining was performed to st.17 embryos bearing the P[ slit 1.0 lacZ reporter to label the midline glia cells. The heterozygous Df(IRER) embryos have approximately three l abeled cells per segment, whereas the homozygous Df(IRER) embryos have 2~5 extra cells/segment.

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96 F igure 3 6 Decreased expression level s of reaper i n homozygous Df(IRER) embryos. A) We analyzed the transcriptional regulation of the pro apoptotic gene rea per by performing whole mount in situ hybridization (ISH) in both wild type and IRER deficient embryos Compared to the wild type embryos, IRER deficient embryos at stage 10 11 have much less ISH signal. There was no difference at later stage when reaper is mainly expressed in the neuroblasts. B ) To quantify the reaper transcription level from staged embryos, st.9 12 embryos were collected and qRT PCR was performed. In consistence with the ISH results, decreased levels of reaper mRNA was observed for both Df(IRER) and Df(IRER_left) embryos. The experiments in this figure were performed by Dr. Nianwei Lin.

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97 Figure 3 7. IRER is required for dMyc induced cell death. A) Consistent with previously reported results ( Montero et al., 2008 ) expression of dMyc under the control of the GMR Gal4 driver (GMM) induced both overproliferation and apoptosis and had little impa ct on the size of the adult eye. B) There is no significant over grow of eyes in IRER / adults. C) Expression of dMyc in the homoz ygous Df(IRER) animals results in significant overgrown phenotype with some eyes containing protruding cell mass (black arrow ). D) The enhancer effect of Df(IRER) on GMM induced hyperplasia is detectable in anim als heterozygous to Df(IRER) E F) A similar enhancing effect was observed from fli es heterozygous to Df(ED225) or flies over expressing the viral caspase inhibitor P35 G I) The hyperplasia of GMM,IRER / was evident

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98 when observed with scanning electron micrographs (SEM). Note that the ommatidia from GMM,IRER / were disorganized. J L) Fluorescent in situ hybridization (FISH) was performed to the third instar eye discs to detect reaper transcripts. Corresponding with the eye phenotype, we found that reaper was significantly induced by GMM in the GMR d omain in wild type eye discs and this induction was absent in IRER / eye discs. M) Either lack of IRER (IRER / ) or over expression of dMyc (provided by tub>dmyc) alone is sufficient to increase wing size (p < 0.0001 between +/+ (n=30) and IRER / (n=26), p< 0.0001 between +/+ and tub>dmyc (n=20), p=0.8818 between IRER / and tub>dmyc). When combined together (tub>dmyc,IRER / )(n=19), the wing size is increased even further, suggesting the synergy between Df(IRER) and dMyc overexpression (p = 0.0012 between IRER / and tub>dmyc,IRER / p=0.0027 between tub>dmyc and tub>dmyc,IRER / ). N) The representative wing images of tub>dm yc (upper panel) and tub>dmyc,IRER / (lower panel, merged layer of tub>dmyc,IRER / and tub>dmyc).

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99 Figure 3 8. Cell autonomous role of IRER regu lates dMyc induced overgrowth. A) Schematic representation of the MARCM strategy used to induce dMyc expres sion from IRER mutant clones. Compared to wild ty pe clones with dMyc expression ( B & C mutant clones ( D expression are significantly larger in size. Clones were monitored at 72hours after heat shock. The percentage occupan cy of clones was examined by calculating the ratio between the GFP positive area and the entire disc area. The dMyc,IRER / clones occupy a much larger area than dMyc expressing clones in both eye disc F) and wing disc G).

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100 Figure 3 9. Cells with an open IRER are more sensitive to dMyc induced cell death A) An ubiquitin DsRed reporter was inserted, via homologous recombination, into the middle of IRER, a locus that showed strongest resistance to DNase I in late stage embryos ( Zhang et al., 2008 ) ubi DsRed served as a reporter for the epigenetic status of IRER. The IRER{ ubi DsRed } reporter signal is variegated in the d eveloping wing discs. B (green) in the dpp domain does not change the variegated expression pattern of IRER{ ubi DsRed } C dMyc was co expressed with GFP there was a conspicuous lack of DsRed positive cells in the dpp zon e (yellow arrows). D When cell death in the dpp zone was blocked by co expressing UAS diap1, many DsRed positive cells were rescued (yellow arrows). E ) dMyc overexpression clones, marked by GFP (dashed lines), were generated by the Act>y>Gal4 fli p out in conjugation with UAS GFP and UAS dMyc transgene expression. Note cells in the dMyc overexpressing clones have lower levels of IRER{ubi DsRed} signal comparing to neighboring cells. Figure E Dr. Sergio Casas Tint ( Cajal Instit ute, Spanish Research Council (CSIC), Madrid, Spain) and Dr. Eduardo Moreno (Molecular Oncology Program, Spanish National Cancer Centre (CNIO), Madrid, Spain).

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101 Figure 3 10. Su(var)3 9 is required for the epigenetic silencing of IRER in wing discs. A) Th e expression of the reporter IRER{ubi DsRed} was variegated in the developing wing discs (red) B) The reporter activity can be significantly de repressed when Su(var)3 9 was knocked down from Ser expressing cells. Cross: Ser Gal4; IRER{ubi DsRed} X Su(var )3 9 RNAi. The expression pattern of Ser Gal4 was visualized using a nuclear localized GFP (green GFP; blue DAPI ). Cross: Ser Gal4 X UAS nlsGFP. The de repression is not exclusively cell autonomous, potentially due to secondary stress to neighboring cell s.

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102 Figure 3 11 Epigenetic silencing of IRER is responsive to stress. A Low and variegated expression of IRER{ubi DsRed} in the wing disc. B ) & C C Irra diation led to a n increase of the reporter signal Wing discs were dissected from third in star larvae at either 4hrs or 24hrs after a 40Gy x ray treatment.

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103 Figure 3 12. Cells lacking IRER have the propensity to overgrown and are resistant to stress induced cell death A D) Clones we re induced using the FLP/FRT mediated mitotic recombinatio n and labeled by the absence of GFP (white lines). The sibling wild type twin spots are marked by 2XGFP (brighter green). W hile neutral clones in either wing or eye imaginal discs generally grow to similar size as the simultaneously induced twin spots, h omozygous IRER deficient clones tends to overgrow (A & B, wing disc; C & D, eye disc, 72 hr following clone induction). E F) While there is a considerable variation in how much the Df(IRER) clone overgrows, the overgrown phenotype is significant (open bars in E & F. wing disc: p=0.017 between Ctr and Df(IRER left ), p<0.001 between Ctr and Df(IRER); eye disc: p<0.001 between Ctr and Df(IRER left), p=0 .002 between Ctr and Df(IRER)). Wilcoxon rank sum test was used to for p value calculation. When animals are subject to a sub lethal dose (10Gy) of x ray at 24 hours following clone induction, the size difference between Df(IRER) clones and the twin spots is significantly increased (closed bars in E & F. wing disc: p=0.021 between Ctr and Df(IRER left), p=0.002

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104 b etween Ctr and Df(IRER); eye disc: p=0.027 between Ctr and Df(IRER left), p=0.002 between Ctr and Df(IRER)). Size was measured at 72~ 96 hr after clone induction and the ratio of clone size vs. twin spot size was calculated for different groups G) Scatter p lot of clone size and twin spot size measured at 72~96hr after clone induction. Red dots and green dots represent the clones induced wit h or without IR, respectively. H) Animals were irradiated (40Gy) at 48 hr post clone induction. Apoptosis was measured b y Caspase 3 staining at 4hr following IR. There is a lack (or reduced level) of apoptotic cells in the Df(IRER) clones. The size difference between the mutant clone and the twin spot in this setting is not significant due to the relatively short time follo wing clone induction and x ray treatment.

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105 Figure 3 13. Df(IRER) clones overgrow their twin spot s in the eye discs. A) The clone size from eye discs was summarized in the 2D plot B D) Mitotic clones wer e induced in adult eye s (clone red; twin spot white ). Both Df(IR ER left) and Df(IRER) clones gre w larger than their twin spots.

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106 Figure 3 14. High order chromatin organization brings IRER in to the proximity of multiple RHG gene promoters. A) Hi C data for D.melanogaster embryos was taken from a recent g enome wide study ( Sexton et al., 2012 ) The region demarcated by the black lines indicates the predicted physical interaction domain, which is corresponding to the synteny block and enriched for HCNE (highly conserved noncoding elements). The HCNE data is derived from Ancora ( Lin et al., 2009 ) The region contai ning IRER is enlarged in panel B). There are multiple predicted binding sites for both cMyc/dMyc and P53 within IRER. The bioinformatics was done by Guangyao Li.

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107 Figure 3 15. Diagram summarizing the mechanism of IRER. It has been known that ectopic expression of dMyc leads to overproliferation and oncogenic potential, which is quickly followed by reaper / sickle dependent apoptosis to remove the oncogenic cells. We here describe t he regulatory elements within IRER functio n as the central regulatory element for the induction of apoptosis fo llowing overproliferation It is still not clear whether dP53 is involved in the process and how dMyc induces apoptosis through IRER (e.g. direct binding or indirect targeting into IRER) and requires further studies. In addition, e pigenetic regulation of IRER defines cellular sensitivity to developmental constraints and oncogenic stress. In general, cells with an open IRER are more sensitive to stress induced apoptosis and have less potential to over proliferate; whereas cells with a closed IRER are more resistant to stress and consequently, easier to undergo over proliferation when they are confronted with over expression of oncogene such as Myc.

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108 CHAPTER 4 A STRESS RESPO NSIVE REGULATORY REGION IS REQUIRED FOR COMPETITION INDUCED APOPTOSIS Introduction In multicellular organisms, cellular communication plays an es sential role in determining cel l fates and growth decisions, e. g. whether a cell can survive to contribute to the adult tissue. Cell competition is one of the sophistic ated cellular phenomenon that was originally identified in Drosophila in the 1970s, when fly geneticists were studying a class of mutations of genes that encod e ribosomal proteins ( Morata and Ripoll, 1975 ) These dominant mutations, named Minutes are homozygous lethal to cells but are able to survive in heterozygotes (M+/ ), althou gh it takes much longer for the organism to grow and reach normal size. Surprisingly, when clones of M+/ cells are induced from the wing discs containing otherwise wild type cells, they are actively eliminated and are not recovered in the adult wing tissue. Therefore, the intrinsically via ble cells could be eliminated when surrounded by cells with higher fitness, a phenomenon called cell competition ( Simpson, 1979 ; Simpson and Morata, 1981 ) Subsequent studies have show n that c ell competition is not restricted to the Minute condition and can be triggered by a variety of other genes One of the decisive regulator s in cell competition is t he transcription factor dMyc, which is the single Drosophila orthologue of the mammalian proto oncogene c Myc ( Johnston et al., 1999 ) Through a still un clear mechanism, cells can compare dMyc level s with their neighbors in the sa me tissue compart ment and establish a win/lose status: cells with relatively lower levels of dMyc will be removed while cells with relatively higher levels of dMyc will expand at the expense of the loser cells, through compensatory proliferation ( de la Cova et al., 2004 ; Moreno and Basler, 2004 ) Since both dMyc and Minute mutations

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109 affect ribosomal biogenesis and protein synthesis, it is likely that the local differences of ribosome activity are the factors leading to cell competition in this scenario ( de Beco et al., 2012 ) Interestingly, recent studies h ave highlighted the role of a group of polarity genes in the process of cell competition. This group of polarity genes, including scribble ( scrib ) lethal giant larvae ( lgl ) and disc large ( dlg ) are evolutionarily conserved neoplastic tumor suppressors. Th ey play an important role in apical basal polarity control and are vital for epithelial integrity ( Bilder, 2004 ; Hariharan and Bilder, 2006 ) Flies homozygous for scrib mutation develop normally till the end of larvae stage, when the maternally deposited scrib protein has been exhausted at that point The ir imaginal discs keep proliferating without termin al differentiation, and the animals eventually die as a giant larvae with multilayered epithelial structure s ( Wodarz, 2000 ) Interestingly, when clones of scrib mutant cells a re surrounded by wild type tissues, they are actively eliminated through cell competition instead of overgrow ing their neighbors, like one might expect ( Brumby an d Richardson, 2003 ; Pagliarini and Xu, 2003 ) Th eir elimination requires the existence of the neighboring w ild type cells, which is a hall mark of cell competition. Since cell competition leads t o and relies on the removal of suboptimal cells it is essential to fully elucidate the molecular mechanisms used to conduct their death Here we used both dMyc and scrib i n duced cell competition models to show tha t an epigenetically regulated, stress res ponsive region -IRER, is required for the induction of pro apoptotic genes in response to competition stress. Loss of IRER leads to inhibition of pro apo ptotic gene expression and increased survival of disadvantaged cells Furthermore, the epigenetic stat us of IRER can be regulated by competition stress,

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110 which can possibly change the susceptibility of cells to apoptosis. It seems to be worthwhile to better recognize the consequences of epigenetic regulation in cell competition. Materials and Methods Droso phila Strains and Culture All crosses were done at 25C and maintained on a standard corn agar medium unless otherwise mentioned. Canton S Drosophila strain was used as wild type control in this study. Defined deletion s of IRER were generated previously as described ( Zhang et al., 2008 ) Clone Induction Wild type clones in a tub>dMyc background were induced from larvae of genotype hsp70 flp ; tub>dmyc>Gal4 ; UAS G FP as previously described ( Moreno and Basler, 2004 ) Wild type clones in the tub>dMyc,Df(IRER) background were induced from larvae of genotype hsp70 flp; tub> dMyc>Gal4/UAS GFP; Df(IRER) upon the same condition. Microscopy and Immunohistochemistry Imaginal disc dissection and immunostaining were performed as described in C hapter 3. Gene Expression Analysis RNA was extracted from 8 10 larvae of the desired geno type using the RNeasy Minikit (Qiagen). cDNA synthesis and quantitative PCR were performed as described in C hapter 3.

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111 Results IRER is Required for Super Competition induced Elimination of Loser C ells In the fly wing imaginal discs, the dMyc induced cell c ompetition is extraordinarily influenced by the neighboring cells. F or example, the wild type cells are recognized as winner cells when surrounded by the dMyc mutant cells, but become losers when facing the challenge from dMyc overexpressing cells, e.g., d riven by the tubulin promoter. Here we used the previously reported tubulin > dMyc > Gal4 system to study the role of IRER in mediating super competition induced apoptosis ( Moreno and Basler, 2004 ) In this system, all cells have elevated levels of dMyc due to the ubiquitous and strong tubulin promoter. However, FLP mediated excision of FRT flanked dMyc will generate clones of cells that have wild ty pe levels of dM yc protein. The cells with lo wer levels of dMyc compared to surrounding cells, marked by GFP, will eventually be eliminated through cell competition induced apoptosis. When this system was recombined with Df(IRER) or Df(IRER_Left), we noticed that the eli mination of wild type clones was significant ly inhibited. While most GFP positive clones were eliminated at 72 hours post clone induction in wild type discs (Fig. 4 1 A), many clones survived with larger size in the wing discs that lacked an IRER (or IRE R_left) (Fig. 4 1B). IRER is Required for Super Competition induced hid Expression in Loser C ells When the level of RHG genes was monitored, we noticed a dramatic induction of hid between 24 48hrs following the generation of wild type clones by heat shock (Fig. 4 2A). Parallel heat shock experiments conducted with animals that have h s FLP but lack tubulin > dMyc > Gal4 indicated that there was no detectable difference of hid mRNA levels in heat shocked or mocked animals between 24 48 hours following the treatm ent

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112 (data not shown). This strongly suggests that the induction of hid observed in hs FLP ; tubulin > dMyc > Gal4 animal s was due to competition induced cell death in this system. To our surprise, this induction of hid following wild type clone induction in tubulin > dMyc > Gal4 animals was totally blocked in animals lacking IRER (Fig. 4 2A). Even more intriguingly, we found that although reaper (and sickle ) was not usually induced in this competition induced apoptosis system, the level of reaper mRNA was noneth eless significantly increased in Df(IRER) animals (Fig. 4 2B). The induction of reaper and sickle may be due to the accumulation of these cell death genes in the are event ually eliminated in the Df(IRER) animal. Taken together, our observations indicate that IRER is required for competition induced cell death in a dMyc overexpression system, by mediating the pro apoptotic hid expression. IRER is Required for the Eliminati on of scrib Knockdown Cells Cell competition induced apoptosis has been ob served for cells mutated for cell polarity genes. Cells mutated for scrib fail to exit cell cycle; and under many circumstances the mutant cells are eliminated by JNK mediated apopt osis ( Bilder et al., 2000 ; Brumby and Richardson, 2003 ; Pagliarini and Xu, 2003 ) Since both scrib (97B 97C) and IRER (75C) are located on the 3 rd chromosome, it is very difficult to generate clones deficient for both scrib and IRER. To overcome this obstacle, we decided to perform tissue specific RNA interference (RNAi) to knock down scrib expression in eye progenitor cells using the shRNA strategy ( Ni et al., 2011 ) In the subsequent experiments, ey1x Gal4 ( Bloomington 8228) was used to drive the expression of both the GFP reporter gene and the shRNA s targeting scrib The

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113 expression profile of the GFP signal indicated that the ey1x Gal4 was active in eye disc cells starting from late embryogenesis. In third i n star larvae, the GFP signal was mostly restricted to cells behind the morphorgenic furrow (Fig. 4 3A When scrib was depleted by RNAi, a subset of scrib knockdown cells in the eye discs failed to complete differentiation as indicated by Elav stainin g ( Fig. 4 4 B ). The adult eyes of ey scrib RNAi were smaller in size and had rough surface. Interestingly, for some eyes there were necrotic spots in the middle of the eye (Fig. 4 3C1 C2). Similar phenotypes were observed for the two shRNA strains, JF0 3229 and HMS 01490 with t he later resulting a relatively more severe phenotype than the former ( Fig. 4 5 ). When ey scrib RNAi was introduced to the IRER heterozygous background, more GFP positive cells were detectable in the eye discs in third instar larvae and some were even observed migrating towards the antenna disc ( Fig. 4 4 C C ). However, no GFP positive cell could be observed in the antenna disc a nd adult eyes display ed a very similar small & rough phenotype as the ey scrib RNAi flies in wild type b ackground (data not shown ). Strikingly, when ey scrib RNAi was introduced to the homozygous Df(IRER) genetic background, GFP positive cells were frequently found in the antenna disc in third instar larvae (Fig. 4 ogy of the discs and t he monolayered epithelial organization was disrupted ( Fig. 4 3 D). Some adults of ey scrib RNAi in Df(IRER) background (~30%for HMS 01490 ) had ectopic pigment cells in the head (Fig. 4 3 D1). About 70% of adults had extra antennae like structures mixed with an undifferentiated tissue mass (Fig. 4 3 D2). The majority of cells in the undifferentiated cell mass in the head were GFP positive (Fig. 4

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114 these cells, whereas it was turned off in most differentiated cells. When the expression of reaper and hid was monitored by fluorescent in situ hybridization (FISH) in the ey scrib RNAi discs the mRNA levels of both were elevated (Fig 4 6 A this elevatio n of reaper and hid in the ey scrib RNAi discs was absent in Df(IRER) animals (Fig. 4 6 B Disruption of Cell Polarity led to de Repression of IRER Previous studies have found that the DNA accessibility of IRER is subject to PcG mediated epig enetic regulation. The e pigenetic status of IRER dictates cellular sensitivity to developmental constraints and multiple stresses. In general, cells with an open IRER are more sensitive to stress induced apoptosis and tend to be preferentially eliminated f rom the tissue ( Zhang et al., 2008 ) Using a ubi DsRed construct inserted into IRER as a reporter, we were able to monitor the epigenetic status of IRER with a c ellular resolution. Interestingly, we found that when Scrib was knocked down in Ser expressing cells of the wing disc, there was a significant induction of the IRER{ubi DsRed} signal specifically in the Ser domain. Therefore, disruption of cell polarity, v ia down regulation of the polarity control gene scrib leads to derepression of IRER and thus increased sensitivity to stress induced cell death. The increased DNA accessibilit y of IRER in scrib knockdown cells may contribute to their elimination from the tissue, a hypothesis which requires further evidence. Nonetheless, the epigenetic response of IRER to decreased scrib activity suggests a link between epigenetic regulation and polarity control.

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115 Discussion s Myc induced Cell Autonomous Apoptosis vs. Cell N o n autonomous Apoptosis It has been long known that ectopic express ion of dMyc in the GMM context is able to induce apoptosis in a cell autonomous manner ( Montero et al., 2008 ) In C hapter 3, we identified IRER as a central regulatory region for dMyc induced apoptosis in the GMM. The fact that IRER is also required for competition induced cell deat h in the tub>dM yc>Gal4 system wa s quite a surprise to us It should be noted that IRER i s not required for competition induced cell death in the Minute +/ system ( Li and Baker, 2007 ) (personal communication with Dr. Nicolas Baker). The main commona lity between dMyc induced autonomous cell death and competiti ve cell death is that both are affected by the availability of growth factors. Howeve r, detailed analysis indicated that the two processes, although both requiring cis regulatory functions that reside in the same intergenic region of IRER, likely involve different mechanisms. First, it has been shown that dMyc induced cell autonomous apo ptosis is accompanied by an increased expression of reaper and sickle but not hid ( Montero et al., 2008 ) Th is wa s also confirmed by us in C hapter 3. And it appears t hat the effect of IRER deletion on dMyc induced cell autonomous cell death is through reducing the responsiveness of reaper ( and sickle ) In contrast, we found that hid is the only RHG gene strongl y induced in the tub>dMyc>Gal4 system between 24 48 hours following clone induction, a period when a majority of the elimination of w ild type clones (with lower levels of dMyc) occurs ( Moreno and Basler, 2004 ) This correlates with previous finding s that the function of hid is required for dMyc super competitor induced cell death ( de la Cova et al., 2004 )

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116 Secondly, we found that although the induction of hid in the tub>dMyc>Gal4 system was completely blocked in animals lacking IRER, the level of reaper mRNA, which usually is not induced in this system, was then f ound to be significantly increased (Fig. 4 2B). This phenomenon is rather intriguing and is to some exten t reminiscent of what was observed in an embryonic midline system. In the CNS midline glial cells that are destined to die, usually the expression of r eaper is not detectable while the expression of hid is prominent. However, in the hid mutant background, there is a dramatic accumulation of reaper ( Zhou et al., 19 97 ) This could be due to the fact that delayed or inhibited cell death in the absence of hid induction allows the accumulation of reaper mRNA, which would usually be too low to be detected. This is unlikely to be the case in the tub>dMyc>Gal4 larvae sin ce reaper level s in the larvae were reliably detectable. It may also reflect a redundant mechanism built into the coordinated regulation of RHG genes, which allows induction of alternative genes when the first preference is not a vailable or fails to respon d. The nature of such a mechanism, as well as the identification of distinctive elements required for cell autonomous vs. competition induced RHG genes expression, will have to await further mechanistic analysis of the IRER. Epigenetic Regulation and Cel l Competition Since cell competition leads to and relies on t he elimination of loser cells via apoptosis, a fundamental question that remains to be answered is how the differences of cellular fitness are recognized and what mechanisms are used to kill the loser cells. Recent studies using gene expression arrays have brought some exciting insights in to the process of specification of winner /loser cells. I t appears that this distinction involves the differential display of the integral membrane protein Flowe r (Fwe), which is highly

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117 conserved in multi cellular organisms ( Rhiner et al., 2010 ) Fwe is alternatively spliced in disadvantaged cells, and has been propose d to be the code that separate s them from better adapted cells However, h ow the F w e code transmits the death signal as well as which genes are involved in the process, is not yet clear In Chapter 4 we report that the stress responsive region IRER is re quired for cell competition induced elimination of loser cells, presumably by mediating the transcriptional induction of hid upon competitive stress. Intriguingly I RER is epigenetically regulated and t he DNA accessibility within this region defines c ellul ar sensitivity to stress induced apoptosis ( Zhang et al., 2008 ) The finding that IRER is derepressed in cells with lower levels of scrib is rather interestin g a nd link s epigenetic regulation and cell competition. One potential hypothesis is that decreased levels of scrib leads to higher DNA accessibility of IRER, which allows transcription of pro apoptotic gene hid and activation of cell death from scrib RNAi cell s. Further experiments would need to be done to study whether indeed the scrib RNAi cells with open IRER undergo apoptosis, e.g. by staining the wing discs of Ser>Scrib RNAi, IRER{ubi DsRed} with cleaved Caspase 3 antibo dy If this proves to be true, it wou ld be interesting to further investigate the relationship between Fwe and IRER. For example, does derepre ssion of IRER in loser cells happen before, at the same time, or after the sp l icing of the F w e (lose) isoform. Furthermore, it is worth to test whether a similar epigenetic response of IRER also exists in cell competition induced by other genes (e.g. dMyc or Minute ). Interestingly, recent work from Bondar and Medshitov presented that cell competition also ex ists between mammalian hematopoietic stem cell s and is mediated

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118 by the relative activity of P53: cells with higher levels of P 53 activity were outcompeted by cells with lower levels of P 53 activ ity. Since P 53 activation is a critical cellular response to DNA damage, the P 53 dependent cell competition l ikely reflect s a fail safe mechanism to select the progenitor cel ls with less damage ( Bondar and Medzhitov, 2010 ) Intriguing ly, there are several putative P 53 binding sites within IRER, including the previously identified dP53 response element (P53RE). The dP53 induced apoptotic response following DNA damage is totally abolished when IRER is epigenetically blocked in late stage embryos, suggesting the essential role of IRER in dP53 induced cell death. The involvement of both P53 and IRER in cell competition highlights the connection of cell competition, stress response and homeostatic maintenance. Current knowledge indicates that dMyc induced cell compet ition rem oves the loser cells via hid dependent apoptosis and likely the activation of JNK ( de la Cova et al., 2004 ; Moreno and Basler, 2004 ) A s ubstantial amount of experimental data strongly suggest s that the Eiger/JNK induced apoptosis plays a key role in eliminating the polarity deficient clones of scrib mutant cells. The stress responsive JNK signal is upregu lated from loser cells and triggers the ultimate execution of their apoptosis ( Brumby and Richardson, 2003 ; de la Cova et al., 2004 ; Igaki et al., 2006 ; Moreno and Basler, 2004 ; Pagliarini and Xu, 2003 ) Eiger, the sole Drosophila tumor necrosis factor ( TNF), has been identified as an upstream regulator of JNK activity ( Igaki et al., 2002 ; Moreno et al., 2002b ) Eiger is taken up by loser cells through endocytosis, which in turn activates downstream JNK signaling and induces apoptosis from the scrib mutant clones. Indeed, either abolishing Eiger ex pression or down regulating the endocytosis

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119 activity is sufficient to lead to tumor progression from epithelium containing polarity deficient clones ( Igaki et al., 2009 ) It is still unclear how the activated JNK pathway leads to the induction of pro apoptotic genes as well as the execution of cell death in out competed cells. Further experiments need to be done to investigate the role of IRER in mediating JNK in duced apoptosis. The results from this study will hopefully set up a link between JNK activation and apoptotic consequence in the loser cells.

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120 Figure 4 1. IRER is required for super competition induced elimination of loser cells In transgenic flies car rying tubulin>dMyc >Gal, tub>Gal4 clones are generated by inducing expression of hs FLP These t ub>Gal4 clones (marked by GFP) have wild type levels of dMyc and are eliminated through cell competition induced apoptosis because they are surrounded by cells expressing high er levels of dMyc ( Moreno and Basler, 2004 ) A) By 72 hours after clone induction, majority of tub>Gal4 clones have been eliminated B) However, the elimination of Tub>Gal4 clones is strongly inhibit ed in Df(IRER) discs The data was provided by Dr. Sergio Casas Tint ( Cajal Institute, Spanish Research Council (CSIC), Madrid, Spain) and Dr. Eduardo Moreno (Molecular Oncology Program, Spanish Natio nal Cancer Centre (CNIO), Madrid, Spain).

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121 Figure 4 2. IRER is required for super competition induced hid expression in loser cells. A) The pro apoptotic gene hid is significantly induced by cell competition from the larvae at 24~48 hours after heat sh ock. However, the hid induction is almost totally blo cked in IRER deficient larvae. B) Another pro apoptoti c gene reaper is not responsive to cell competition in the presence of IRER, but is highly induced by cell competition in homozygous Df(IRER).

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122 Fig ure 4 3. IRER is required for the elimination of scrib knockdown cells. A & A1 A2) Eye antenna discs containing GFP expression driven by ey1x Gal4. The animals develop norma l eye, and antenna B B ) & B1 B2) Expression of ey1x>GFP in the homozygous Df( IRER) animals. The se animals also develop normal eye and antenna, suggesting that the loss of the regulatory function of IRER by itself does not affect the eye antenna development. C ) When scrib is knocked down from ey1 expressing cells (marked by GFP) in eye discs, the affected disc cells lose the mono layered organization and become folded as revealed by DAPI stai Some scrib RNAi cells are misplac ed and undergo local migr ation C1) The eyes of ey1x> scrib RNAi adults are small in size and have rough surface, with so me eyes showing necrotic spots C2) A ntenna development i s not affected. D ) When inducing scrib RNAi in IRER deficient background the ey1x> scrib RNAi cells (marked by GFP ) a re detectable from a ntenna discs D1 D2) Development of eye and antenna is severely affected: extra eye s (or eye like tissues) and antenna e (or antenna like tissues ) are frequen tly observed The GFP labeled scrib RNAi cells can survive to adult stage and aremainly detected in the undifferentiated tissues in th e head nnel of D1 and D2, respectively

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123 Figure 4 4. S crib knockdown leads to differentiation defects of photoreceptor cells. A ) Wild type e ye discs with ey1x>GFP expression were stained with anti Elav antibody to label the differentiated photoreceptor cells. All the cells behind the morphogenetic furrow (MF) were differentiated and positive for both GFP expression and Elav staining. B Upon scrib knockdown, some cells posterior to the MF were absent for Elav stainin g suggesting their differentiation was affected C RNAi cells failed to undergo terminal differentiation in heterozygous Df(IRER) background. Furthermore, some scrib RNAi IRER +/ cells tend to migrate within eye disc

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124 Figure 4 5. Scrib knockdown phenotype in eyes. A) Flies carrying UAS scrib RNAi construct s have normal eye development. B C) In order to knock down the scrib expression from developing eyes, two different UAS scrib RNAi lines ordered from TRiP were use d: the Long Hairpin JF03229 and the Short Hairpin HMS01490 They both cause a small, rough eye phenotype with frequently obversed necrotic spots in the eye. For the rest of the experiments, we chose the short hairpin HMS01490 for scri b knock down because it appeared to have higher efficacy of gene targeting and induce d a stronger phenotype

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125 Figure 4 6. IRER is required for scrib RNAi induced reaper and hid expression from eye discs. The transcription levels of reaper and hid mRNA were monitored by FISH. Indeed, ey1x>scrib RNAi led to a strong induction of both reaper and hid in the wild type eye discs A ) & C the homozygous Df(IRER) discs B ) & D

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126 Figure 4 7. Disruption of cell polarity le a d s to de repression of IRER. A Low and variegated expression of IRER{ubi DsRed} in the wing disc. B down scrib (cross: S er Gal4 ; IRER{ubi DsRed} X UAS scrib RNAi) led to de repression of IRER{ubi DsRed} f rom the Ser expression domain (Check Fig. 2 4C for the expression pattern of Ser Gal4 in wing discs)

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127 CHAPTER 5 DISCUSSION AND PERSPECTIVE Coordinated Transcriptional Regulation of the RHG Genes by Locus Control R egions The RHG genes as a whole are required for almost all development cell death during embryogenesis in Drosophila Melanogaster Embryos homozygous for the H99 deletion, which removes hid grim and reaper fail to hatch and have a CNS that contains about 3 times more cells than that in wild type embryos ( White et al., 1994 ) However, how individual cells are chosen to die during embryogenesis, or in later developmental pe riod, is far from clear. With the exception of hid the other three genes ( reaper grim and sickle ) seem to be exclusively expressed in cells that are to be eliminated shortly after one or a multiple of these genes is expressed ( Jiang et al., 1997 ; Lohmann et al., 2002 ; Steller, 2008 ; Zhou et al., 1995 ) Transcriptional regulation of these genes thus plays a pivotal role in determining the life/death decision for an individual cell. Centralized regulation, i.e. one regulatory region which coordinates t he expression of multiple genes, appears to be the general theme of transcriptional regulation of the RHG genes. Previously, we have found that the intergenic region between reaper and sickle (i.e. IRER) is responsible for mediating radiation induced expr ession of not one, but three pro apoptotic genes ( reaper hid and sickle ) ( Zhang et al., 2008 ) Data from shown that a ~22kb intergeni c region between grim and reaper (i.e. neuroblast regulatory region or NBRR) is required for appropriate developmental expression of reaper grim and sickle in neuroblasts at the end of embryogenesis ( Tan et al., 2011 ) While IRER is required for stress (DNA damage) induced expression of RHG genes, it is not required for the expression of RHG genes in

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128 neuroblasts during late embryogenesis. R ather, it appears that cell lineage specific expression of these genes in neuroblasts is controlled by the NBRR. Although it remains to be seen as to whether this observed separation of task will hold true for cell death regulation in other cell lineages or in respons e to different kinds of stress it is now clear that transcriptional regulations of the RHG pro apoptotic genes are coordinated by locus control regions such as the NBRR and IRER. Both IRER and NBRR, situated upstream and downstream of reaper respectively, are highly conserved intergenic regions enriched for HCNEs (Fig. 3 14A). Characterized HCNEs in both vertebrates and insects genomes often regulate target genes over long distance to guide tissue or developmental stage specific expression p atterns ( Kikuta et al., 2007 ) The extensive chromosomal interactions revealed by the whole genome Hi C analysis within this regio n (Fig. 3 14A) strongly sugges t that regions such as NBRR and IRER can coordinate the expression of RHG genes through long range chromosomal interactions. However, the detailed interaction map of the RHG genomic regulatory block, as well as the potential dynamics of the chromosomal int eractions, remains to be fully understood. Epigenetic Regulation and C ancer Traditionally cancer has been considered as a genetic disease, which can be dated back to the observation of aneuploidy associated with cancer cells. I t becomes more clear nowadays that epigenetic mechanisms play an important role in cancer development ( Jones and Baylin, 2002 ) Recently, s everal large scale transcriptome / exome studies aimed at genomic analysis of tumor genetic abnormalities have again revealed the importance of chromatin regulation in tumorigenesis ( Dalgliesh et al., 2010 ; Gui et al., 2011 ; van Haaften et al., 2009 ) For example, h istone modifiers, such as the

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129 histone H3K27 demethylase UTX, were rep eatedly identified to be mutated in a variety of cancers Besides protein complexes that directly modify histones, other protein complexes, such as chromatin barrier s also play a role in affecting chromatin status and oncogenesis. It has been reported tha t the loss of CTCF binding which is an insulator protein, will lead to the spreading of facultative heterochromatin into the promoters and/or transcribed regions of tumor suppressor genes p16 ( Witcher and Emerson, 2009 ) and p53 ( Soto Reyes and Recillas Targa, 2010 ) resulting in the ectopic silencing of these tumor suppressors. The dysregu l ation of chromatin modification associated with tumorigenesis could manifest as a global change in a particular modification such as the glob al reduction of H4K16 acetylation and H4K20 trimethylation observed in a skin carcinogenesis model ( Fraga et al., 2005 ) However, it is more common for epigenet ic silencing to be restrained to specific groups of genes, while the overall levels of su ppressive histone modification do not significantly change. For example, our data suggests that epigenetic silencing of a group of pro apoptotic genes promotes dMyc ov erexpression induced hyperplasia as well as scrib RNAi induced neoplastic growth. This finding is fairly interesting to our understanding of cancer progression. Traditionally, cancer development is considered as a multi hit process, which requires accumulat ion of multiple mutations to create the tumor potential. It has been well demonstrated that transgenic mice expressing oncegenic Myc fail to rapidly develop tumor s unless a secondary mutation impeding apoptosis (e.g. mutation of P53, or overexpression of B CL 2) is introduced to the animals. Our data suggests that in the p resence of the primary mutation e.g., oncogene overexpression or loss of a tumor suppressor gene,

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130 aberrant regulation of epigenetic machinery is somewhat sufficient to promote tumor progre ssion without secondary mutations. Is Cell Competition Relevant to Cancer? Although many efforts have been made to understand the process of cell competition, its impact on normal development and disease condition still remains to be further elucidated C ell competition has been implicated as an intrinsic mechanism to control orga n size De la Cova et al. showed that competition between dMyc expressing cells and wild type cells is essential to achieve the proper wing size, because dMyc induced overgrowth c an be compensated by elimination of the wild type cells. In fact, expression of dMyc from every single cell leads to an enlarged wing. When introducing wild type clones to the disc to induce cell competition, the wing is re stored to normal size ( de la Cova et al., 2004 ) Moreover, the recent finding of competition in the Drosophila stem cell niche suggests its role in influencing progenitor cell selection ( Issigonis et al., 2009 ) Therefore, it is becoming more popular to consider cell competition as a surveillance machinery that optimize s the fitness of progenit or cells during normal development. From an evolutionary angle, the animals benefit from select ion of the most adaptive cells to maximize the fitness of the organism. However, the continuous selection for super competitors (e.g. cells with higher levels o f dMyc) may promote tumor g rowth and has been postulated to be the initial stage of carcinogenesis ( Baker and Li, 2008 ; Moreno, 2008 ) There are several reasons for implicating cell competition in tumor formation. First, many genes causing cell competition are well defined oncegenes (e.g. dMyc ) or tumor suppressor genes (e.g. scrib, Hp o Sal Wts ) These genes ar e highly conserved from flies to mammals and their mammalian orthologues are intensively studied in cancer biology. Additionally, tumor

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131 development is a process of accumulating mutations that favor tumor growth or inhibit developmental constraints, includi ng both genetic mutations and epigenetic aberrances. The selection process between cells with different genotypes (mutations) may be conducted through cell competition. As aforementioned, competition between dMyc super competitor and normal cells results i n a local expansion of super competitors without affecting organ size or epithelial morphology. The phenomenon is strikingly similar to a clinical term called field cancerization, which describes the clonal expansion of pre tumorous cells in a particular f ield in the early stage of tumor development ( Dakubo et al., 2007 ) Additional genetic and epigenetic alterations of these cells may transform them in to malignant tumor s On e major difficulty of cancer therapy is lack of efficient detection of early stage tumors, due to their normal morphology and relatively small size. If cell competition is really involved in the initial steps of tumor progression, understanding the underly ing mechanisms may benefit early disease prognosis. It has to be noted that not all the genes involved in cell competition can transform cells into super competitors or contribute to tumor pro gression. For example, increasing copy numbers of the Minute gen e does not help to kill the surrounding wild type cells ( Moreno et al., 2002a ; Simpson, 1979 ) On the o ther hand, recent studies of cell competition induced by loss of neoplastic tumor suppressors (e.g. scrib) strongly indicate its role in tumor suppression. The precancerous scrib mutant cells are eliminated by cell competition, which may otherwise maldiffe rentiate into neoplastic tumor. So far there is no hard evidence of whether cell competition is a cancer promoting or cancer inhibiting mechanism It is possible that different types of com petitions contribute distinct ly to carcinogenesis.

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132 These puzzles w ill become easier to decipher as more knowledge of cell competition is uncovered. Unraveling Epigenetic Regulation and T umorigenesis in Drosophila Although fruit flies have long serv ed as a great model for genetic studies, they seem to not attract much at tention as a model for cancer research. One major reason is the fact that flies rarely develop cancer in nature. Because cancer is a multi hit process and requires accumulation of mutations during cell proliferation, the relative short lifespan of flies do es not allow for this to occur However, I personally consider Drosophila as a beautiful model to study cancer due to the following reasons. First, ir genome with the powerful genetic tools available specifically for flies to induce cancer. For example, by mutating the neoplastic tumor suppressor genes in the scrib lgl dlg gene family, the imaginal discs undergo neoplastic overgrowth in larvae ( Bilder et al., 2000 ) Furthermore, the imaginal discs in Drosophila provide a perfect system to study cancers of epithelial cells or carcinomas, which are the most common ly observed cancer. The imaginal discs are a sac of epithelial cells that undergo continuous proliferation in larvae stage and eventually differentiate into most of the adult tissues. Most of the tumor suppressor genes identified in flies were screened with imaginal tissues. Finally, many of t he intensively studied genes are conserved between flies and humans. Indeed, many key discoveries of cancer research were made with Drosophila such as the identification and characterization of Hippo pathway to control organ size, and the socialized cellu lar interactions such as compensatory proliferation and cell competition. Using Drosophila as a model, we and others also unravel ed the role of epigenetic regulation in tumorigenesis. For example, It has been noticed for some time that clones

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133 of cells mut ated for PRC 1 components Psc Su(z)2 or Polyhomeotic ( Ph ) in the developing wing disc and eye disc display a tumor like overgrow th phenotype, by either up regulating the cell cycle mediator CycB ( Oktaba et al., 2008 ) or abnormal ly activating the JAK STAT pathway ( Classen et al., 2009 ) or th e Notch pathway ( Martinez et al., 2009 ) The phenotype observed for mutant clones lacking PcG genes varies and largely depends on the targeting genes and af fected cell/tissue types. In some other cases, there is evidence that enhancement of PcG mediated silencing may lead to tumor like hyperplasia in Drosophila For example, clones of cells mutated for dUTX have increased level s of H3K27Me3 and significantly overgrow compared to their sister clones ( Herz et al., 2010 ) The overgrowth phenotype of these clones is significantly reduced or bl ocked in animals heterozygou s for either Pc or E(Z) mutation s indicating that it is, at least partially, due to increased silencing of PcG target ed genes ( Herz et al., 2010 ) Interestingly in addition to homeotic genes, several Notch pathway genes had increased H3K27Me3 modifications and reduced mRNA levels in dUTX heterozygous animals. The interaction between dUTX and the Notch signaling pathway was also genetically verified. Intriguingly several other histone demethylases, such as dLSD1 and Lid (litte r imaginal discs) also interact with the Notch signaling pathway ( Di Stefano et al., 2011 ; Mulligan et al., 2011 ) suggesting histone regulations play an important role in defining the pleiotropic Notch pathway. In this thesis study, we have reported the role of epig enetic regulation of a poptosis i n tumorigenesis. The epigenetically regulated IRER functio ns as the central regulatory region for the induction of apoptosis fo llowing overproliferation and cell competition The

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134 e pigenetic status of IRER defines cellular sensitivity to oncogenic and competitive stress. Cells with an accessible IRER have higher susceptibility to stress and are preferentially eliminated; whereas cells with a condensed chromatin structure of IRER are more resistant to stress and have higher possibilities to overgrow Our work highlights the importance of epigenetic mechanisms in mediating cellular sensitivity to oncogenic stress and indicates th at dysregulation of epigenetic status may contribute to tumorigenesis The utility of Drosophila as a model for unraveling t he role of epigenetic regulation in tumorigenesis is e merging. There is no doubt that mechanistic studies in fruit fly will continually provide insight on the fundamental mechanisms of chromatin regulation. In addition, the fruit fly could also serve as v aluable systems for addressing key questions related to tumorigenesis, such as how PcG mediated suppression is targeted to specific genes and what pathway(s) controls epigenetic regulation of P53 targeted tumor suppressor genes. Perspectives In my dissert ation study, I used Drosophila as a model to investigate the role of epigenetics, by transcriptional regulation of a group of pro apoptotic genes, in normal development and tumorigenesis. Our data suggests that an evolutionarily conserved IRER is required to control the appropriate levels of apoptosis, which in turn mediate s the cell number in a given tissue and control s organ size during normal development. In the context of tumor progression, because of, for example, overexpression of oncogene dMyc or los s of tumor suppressor gene scrib, IRER mediated apoptosis plays a key role in keeping the cancerous cells in check. Furthermore, IRER is subject to epigenetic regulation, which is dynamic durin g development and is responsive to environmental

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135 factors or gen omic alterations. The dynamic and rever sible epigenetic regulation provide s more plasticity to the static genome. More efforts need to be put forward to understand the molecular mechanisms of IRER. What Mechanism (S) Controls the Epigenetic Regulation of IR ER? The ChIP assay performed with late stage embryos detected a substantial enrichment of repressive histone mark H3K27me3 in IRER, suggesting the epigenetic blocking of IRER is mediated by PcG mediated silencing. Canonical epigenetic silencing of homeotic genes is mediated by the Polycomb Responsive Element (PRE). However, we were not able to identify any PRE in IRER with the most updated PRE prediction software ( Fiedler and Rehmsmeier, 2006 ) Instead, there are two non coding RNAs within IRER predicted by EST ( Zhang et al., 2008 ) Interestingly, one exciting breakthrough made over the past few years is to uncover long noncoding RNAs (lncRNAs) (e.g. HOTAIR and XIST) associated with PRC bi nding and heterochromatin silencing ( Rinn and Chang, 2012 ) Thus, it is totally possible that the epigenetic silencing of IRER is regulated by non coding RNA. A quick and rough way to test is to knock down the two non coding RNAs respec tively and examine its impact on the epigenetic blocking of IRER in late stage embryos. However, it is also possible that the epigenetic silencing is directed by other cis regulatory elements or even the lncRNAs function in trans, which requires an unbiase d screen to identify. Which Signaling Pathways Regulate the Function IRER? Our study showed that IRER is responsible for apoptosis following several stress conditions including radiation and cell competition. However, little is kn own about the signaling pa thway that function s upstream of IRER, or the transcription factors that response to upstream signal and bind to IRER to mediate RHG gene transcription. It

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136 has been well demonstrated that the immediate apoptotic response to irradiation is P53 dependent ( Brodsky et al., 2004 ) whereas activation of JNK plays a dom inant role in inducing apoptosis in the context of cell competition ( Brumby and Richardson, 2003 ; de la Cova et al., 2004 ; Igaki et al., 2006 ; Moreno and Basler, 2004 ; Pagliarini and Xu, 2003 ) Both P53 and JNK are well studied stress responsive pathways that are highly conserved from flies to humans. Therefore, it is necessary to investigate the relationship between IRER and P53/JNK, for example, by examining the P53/JNK induced cell death in IRER deficient background. In addition, we noticed that the absence of IRER seems to have a more pro minent effect on the cells whose survival is growth factor sensitive instead of cell lineage dependent (Chapter 3). It is also worthwhile to test the role of IRER on growth factor withdrawal induced cell death. What is the Relevance of our Study to Human Research? It is important to transform and apply the knowledge we obtained in Drosophila to humans. Up to now, we have not identified a sequence homologous sequence of IRER in human s However, the epigenetic mechanism established from the study on IRER cou ld be conserved and ap plicable to human research. In C hapter 3, our data indicates a synergy between epigenetic blocking of IRER and overexpression of dMyc induced tumorigenesis. A similar mechanism has long been identified in mammalian cancer research. It has been known that the mammalian ortholog of PcG protein PSC, Bmi 1, correlates with c Myc in promoting tumorigenesis by suppressing apoptosis ( Jacobs et al., 1 999 ) Moreover, it has been shown that Adenovirus is able to specifically silence P53 targeting genes to block the host response to viral infection ( Soria et al ., 2010b ) These observations all support the mechanistic themes of epigenetic regulation on stress response. After we have further dissected the cis regulatory elements

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137 responsible for the silencing machinery of IRER, we could attempt to search for the ir homologous sequence in the human genome. In addition, it will be interesting to examine whether a similar locus control r egion exists in human, which can cooperatively mediate the expression of a group of related genes, or which is subject to ep igenetic regulation

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154 BIOGRAPHICAL SKETCH Can was born in Jining, S handong province, P.R. China in 1983. She recei ved her Bachelor of Science in biological s ciences from the Ocean University of China (Qingdao) in 2005 and immediately started the Ph.D. study in marine b iology also from the Ocean University of China. One ye ar later, her husband Bing Yao was admitted to the Ph.D. program at the University of Florida in US Can made a lifetime decision of giving up her Ph.D. career in China and traveling to US to join her husband. After she came to the US, Can volunteered in D r. Lei Z gain ed some valuable research experience To pursue further education, she started her Ph.D. study in the interdisciplinary program in b iomedi cal s ciences at the University of Florida in 2007. She then officially j oined Zhou laboratory in 2008 and passed her qualifying examination in October 2009. She received her Ph.D. degree in August 2012. After graduation, Can will continue her research career as a post doctoral fellow in Dr. E mory University Outside of the science life Can enjoys watching soccer games with her husband, and relaxes through reading, sleeping, and traveling