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hSET1 Hmt Activity is Requires for Tal1-Mediated Activation of Target Genes in Hematopoiesis and Leukemogenesis

Permanent Link: http://ufdc.ufl.edu/UFE0042671/00001

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Title: hSET1 Hmt Activity is Requires for Tal1-Mediated Activation of Target Genes in Hematopoiesis and Leukemogenesis
Physical Description: 1 online resource (52 p.)
Language: english
Creator: SAENZ,RIVER URU
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

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Subjects / Keywords: Medicine -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, M.S.
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theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

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Abstract: hSET1 HISTONE METHYLTRANSFERASE ACTIVITY IS REQUIRED FOR TAL1-MEDIATED ACTIVATION OF TAL1 TARGET GENES DURING HEMATOPOIESIS By, River Saenz December 2010 Chair: Suming Huang Major: Medical Sciences--Biochemistry T-cell acute lymphocytic leukemia-1 (TAL1) also known as stem cell leukemia protein (SCL) is frequently over-expressed in T-cell acute lymphoblastic leukemia (T-ALL.) TAL1 is an important transcription factor that functions at two crucial stages of hematopoiesis. First, it is important in the expansion of early hematopoietic stem cells. Second, TAL1 is important for terminal erythroid differentiation. Deregulation of these processes is commonly involved in leukemic transformation 1. TAL1 is a member of the basic helix-loop-helix (bHLH) family of proteins. TAL1 forms heterodimers with other bHLH proteins, known as E2A proteins. TAL1 confers tissue-specific transcription and regulates target genes involved in early hematopoietic stem cell differentiation and erythroid differentiation 2. TAL1 can activate or repress transcription of target genes by recruiting transcriptional coactivators or corepressors, respectively. The results herein show that TAL1 is associated with the histone H3K4 methyltransferase hSET1 complex. More specifically, these results show that TAL1 directly associates with the ASH2L subunit of the hSET1 complex and TAL1 associates with endogenous hSET1 complex in T-ALL Jurkat cells. In addition, hSET1 is required for TAL1-mediated activation of the P4.2 target gene by changing the histone methylation status from a repressed status to active status (H3K4 trimethylation.) Furthermore, knockdown of hSET1 decreases the TAL1 mediated activation of the p4.2 promoter activity. The regulation of P4.2 in erythroid differentiation reveals a common epigenetic mechanism in which histone H3K4 methyltransferase hSET1 is involved in the TAL1-mediated transcriptional activation during hematopoiesis and possible leukemogenesis. This information will eventually help to design new therapeutic approaches to treat leukemia. Moreover, the study will shed light on the control of transcription factors on hematopoiesis.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by RIVER URU SAENZ.
Thesis: Thesis (M.S.)--University of Florida, 2011.
Local: Adviser: Huang, Suming.

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Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0042671:00001

Permanent Link: http://ufdc.ufl.edu/UFE0042671/00001

Material Information

Title: hSET1 Hmt Activity is Requires for Tal1-Mediated Activation of Target Genes in Hematopoiesis and Leukemogenesis
Physical Description: 1 online resource (52 p.)
Language: english
Creator: SAENZ,RIVER URU
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: Medicine -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: hSET1 HISTONE METHYLTRANSFERASE ACTIVITY IS REQUIRED FOR TAL1-MEDIATED ACTIVATION OF TAL1 TARGET GENES DURING HEMATOPOIESIS By, River Saenz December 2010 Chair: Suming Huang Major: Medical Sciences--Biochemistry T-cell acute lymphocytic leukemia-1 (TAL1) also known as stem cell leukemia protein (SCL) is frequently over-expressed in T-cell acute lymphoblastic leukemia (T-ALL.) TAL1 is an important transcription factor that functions at two crucial stages of hematopoiesis. First, it is important in the expansion of early hematopoietic stem cells. Second, TAL1 is important for terminal erythroid differentiation. Deregulation of these processes is commonly involved in leukemic transformation 1. TAL1 is a member of the basic helix-loop-helix (bHLH) family of proteins. TAL1 forms heterodimers with other bHLH proteins, known as E2A proteins. TAL1 confers tissue-specific transcription and regulates target genes involved in early hematopoietic stem cell differentiation and erythroid differentiation 2. TAL1 can activate or repress transcription of target genes by recruiting transcriptional coactivators or corepressors, respectively. The results herein show that TAL1 is associated with the histone H3K4 methyltransferase hSET1 complex. More specifically, these results show that TAL1 directly associates with the ASH2L subunit of the hSET1 complex and TAL1 associates with endogenous hSET1 complex in T-ALL Jurkat cells. In addition, hSET1 is required for TAL1-mediated activation of the P4.2 target gene by changing the histone methylation status from a repressed status to active status (H3K4 trimethylation.) Furthermore, knockdown of hSET1 decreases the TAL1 mediated activation of the p4.2 promoter activity. The regulation of P4.2 in erythroid differentiation reveals a common epigenetic mechanism in which histone H3K4 methyltransferase hSET1 is involved in the TAL1-mediated transcriptional activation during hematopoiesis and possible leukemogenesis. This information will eventually help to design new therapeutic approaches to treat leukemia. Moreover, the study will shed light on the control of transcription factors on hematopoiesis.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by RIVER URU SAENZ.
Thesis: Thesis (M.S.)--University of Florida, 2011.
Local: Adviser: Huang, Suming.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0042671:00001


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1 hSET1 HISTONE METHYLTRANSFERASE ACTIVITY IS REQUIRED FOR TAL1 MEDIATED ACTIVATION OF TAL1 TARGET GENES DURING HEMATOPOIESIS By RIVER SAENZ A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2011

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2 2011 River Saenz

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3 To Baron K. Faille, who has given me the opportunity to finish this

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4 ACKNOWLEDGMENTS I acknowledge my family and friends f or thei r continued encouragement in me I also acknowledge my adviser for giving me the opportunity to study in his laboratory.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 LIST OF ABBREVIATIONS ................................ ................................ ............................. 9 ABSTRACT ................................ ................................ ................................ ................... 12 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 14 Hematopoiesis ................................ ................................ ................................ ........ 14 Leukemia and Leukemogenesis (Regulation) ................................ ......................... 14 Introduction to TAL1 ................................ ................................ ............................... 15 TAL1 Is A Critical Regulator of Hematopoiesis and Leukemogenesis .................... 15 Chromatin Structure and Function ................................ ................................ .......... 17 hSET1 Is A Histone Modifying Enzyme Involved in Transcriptional Activation. ...... 18 Protein 4.2 (P4.2): Red Blood Cell Membrane Protein ................................ ........... 19 Rationale for This Study ................................ ................................ .......................... 21 2 MATERIALS AND METHODS ................................ ................................ ................ 24 Cell Culture ................................ ................................ ................................ ............. 24 Mass Spectrometry and Immunoprecipitation ................................ ......................... 24 GST Pull Down Assay ................................ ................................ ............................ 24 H3K4 Specific Methylation Assay ................................ ................................ ........... 25 Chromatin Immunoprecipitation Sequencing (ChIP seq) ................................ ........ 25 P4.2 Promoter Driven Luciferase Reporter Assay ................................ .................. 25 Cloning of the pREP4 P4.2 luc Plasmid ................................ ........................... 26 P4.2 Reporter Assay ................................ ................................ ........................ 26 siRNA Knockdown of hSET1 in the P4.2 Reporter Assay ................................ 27 3 THE ASSOCIATION OF TAL1 WITH hSET1 ................................ .......................... 29 The TAL1 Complex As sociates with the hSET1 Complex ................................ ...... 29 TAL1 Directly Interacts with the ASH2L Component. ................................ ............. 30 The TAL1 Complex Displays H3K4 Specific Histone Methyltransferase (HMT) Activity ................................ ................................ ................................ ................. 30

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6 4 THE EFFECT OF hSET1 ON TAL1 TARGET GENES ................................ ........... 34 TAL1 Binding Is Associated with Increased H3K4me3 in the P4.2 Promoter. ........ 34 The Effect of hSET1 on TAL1 Mediated Transcription Using a P4.2 Promoter Driven Reporter Assay. ................................ ................................ ........................ 35 5 DISCUSSION ................................ ................................ ................................ ......... 42 The Association of hSET1 and TAL1 ................................ ................................ ...... 42 The Effect of hSET1 on TAL1 Target Genes ................................ .......................... 43 How hSET1 Act ivates Transcription in Hematopoiesis. ................................ .......... 45 Future Directions ................................ ................................ ................................ .... 46 LIST OF REFERENCES ................................ ................................ ............................... 48 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 52

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7 LIST OF TABLES Tabl e page 1 1 Histone modifying enzymes required for erythropoiesis ................................ ..... 22 1 2 Conserved SET domain containing complexes ySET1 hSET1 and MLL1 complexes share components. ................................ ................................ ........... 22

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8 LIST OF FIGURE S Figure page 1 1 TAL1 is critical for differe ntiation of hematopoietic cells ................................ ..... 23 1 2 The structure of the TAL1 protein ................................ ................................ ....... 23 1 3 SET1 protein domains ................................ ................................ ........................ 23 2 1 Structure of the pREP P4.2 luc plasmid ................................ ............................. 28 3 1 Identification o f the TAL1 interacting proteins ................................ ..................... 32 3 2 TAL1 recruits endogeno us hSET1 and the H3K4 HMT in Jurkat cells. .............. 32 3 3 GST pulldown assays ................................ ................................ ......................... 33 3 4 HMT assay ................................ ................................ ................................ ......... 33 4 1 A correlation between TAL1 localization at the P4.2 promoter and H3K4 methylation ................................ ................................ ................................ ......... 37 4 2 TAL1 and H3K4me3 are correlated with expression of P4.2 upon differentiat ion. ................................ ................................ ................................ ..... 38 4 3 The experimental strategy for determining the effect of TAL1 as well as the effect of hSET1 knockdown on the P4.2 TAL1 target genes .............................. 39 4 4 The effect of TAL1 concentration on TAL1 target genes ................................ .... 40 4 5 The effect of hSET1 knockdown on TAL1 target genes ................................ ..... 41

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9 LIST OF ABBREVIATI ONS ADP Adenosine diphosphate, a phosphate donor. AML Acute myeloid leukemia ASH2L hSET1 complex component, important in the structure and catalytic activity of the hSET1 and MLL histone methyltransferase complexes. Cdk6 Cycling dependent kinase 6, a cell cycle checkpoint protein CD34 Cluster of differentiation marker for early hematopoietic stem cells ChIP seq Chromatin immunoprecipitation. Chromatin is isolated from nuclear or whole cell extract, before immunoprecipitation (see below .) followed by additi on of Solexa adaptors and massive parallel sequencing. DMSO Dimethyl sulfoxide, used to differentiate MEL cells DTT Dithiothreitol, a small molecule redox reagent known as Cleland's reagent C 4 H 10 O 2 S 2 ; its oxidized form is a disulfide bonded 6 membered rin g EDTA Ethylenediaminetetraacetic acid is a polyamino carboxylic acid and a colourless, water soluble solid. It is widely used to dissolve scale and in buffers, as a hexadentate ("six toothed") ligand and chelating agent i.e. its ability to "sequester" m etal ions such as Ca 2+ and Fe 3+ and diminish their reactivity. EPO Erythropoietin, required for differentiating CD34+ cells into CD36+ hematopoitic stem cells to erythroid progenitors. GATA1 Transcription factor involved in erythroid differentiation which binds to GATA motifs in DNA GST Gluathione S tranferase, which has an affinity for biotin. H2A Histone protein 2A H2B Histone protein 2B H3 Histone protein 3 H3K4 Lysine at the 4 th position of the N terminal tail of H3 H3K4 me3 Trimethylated H3K4

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10 H4 Histo ne protein 4 HDAC H istone deacetylases HSC Hematopoitic stem cell, the precuror to all of the blood cell types in the body. HMT Histone methyltransferase 3 H SAM Tritiated S Adenosyl Methionine, the donor of hydrogen atoms in methylation reactions, both in vivo and in vitro hSET1 Human histone methyltransferase, member of the complex which methylates histone H3 on lysine 4; required in transcriptional activation of gene promoters IP Immunoprecipitation, using antibodies to pull down proteins of interest, t he digesting the proteins and purifying the DNA which can then be polymerase chain reaction (PCR) amplified using primers specific to a gene of interest. LCR The locus control region involved in regulating globin genes, including globin. Ldb1 A transcription factor also misregulated in T ALL. Associated with TAL1 to aid in differentiation of erythroid cells. Involved in P4.2 activation. Also known as NLI. LMO2 A transcription factor also misregulated in T ALL. Associated with TAL 1 to aid in differentiation of erythroid cells.Involved in P4.2 activation. LSD1 Lysine specific demethylase 1 also known as KDM1 MEL Mouse erythroleukemia cells derived from spleens of mice susceptible to the Friend virus. MLL Mixed lineage leukemia prote in, a histone methyltransferase from the hSET1 family in complexes MLL1 4. P4.2 Red cell membrane protein, band 4.2 PMSF phenylmethanesulfonylfluoride or phenylmethylsulfonyl fluoride, a serine protease inhibitor commonly used in the preparation of cell ly sates RbBP5 hSET1 complex member, important to the structure and function of the hSET1 and MLL complexes.

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11 SDS PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis a technique widely used in biochemistry forensics genetics and molecular biolog y to separate proteins according to their electrophoretic mobility (a function of length of polypeptide chain or molecular weight .) SDS gel electrophoresis of samples have identical charge per unit mass due to binding of 1.4g SDS per g of protein, which re sults in fractionation by size alone towards a cathode. TAL1 T cell acute lymphoblastic leukemia protein number 1 T ALL T cell acute lymphoblastic leukemia Tris HCl Tris has a pKA of 8.06. Tris HCl, the acid salt, when titrated to pH = pKa with the corresp onding counterion (OH for tris HCl) it is an effective buffer similar to biological condition. It is widely used as a component of buffer solutions especially for solutions of nucleic acids WDR5 hSET1 complex component important in the structure and fun ction of hSET1 and MLL histone methyltransferase complexes. WDR82 hSET1 complex component unique to this complex, and also important in trimethylation of H3K4.

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12 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science hSET1 HISTONE METHYLTRANSFERASE ACTIVITY IS REQUIRED FOR TAL1 MEDIATED ACTIVATION OF TAL1 TARGET GENES DURING HEMATOPOIESIS By River Saenz May 2011 Chair: Suming Huang M ajor: Medical Sciences -Biochemistry T cell acute lymphocytic leuke mia 1 ( TAL1 ) also known as stem cell leukemia protein (SCL ) is frequently over expressed in T cell acute lymphoblastic leukemia (T ALL ) TAL1 is a n important transcription f actor that func tions at two crucial stages of hematopoiesis. First, it is important in the expansion of early hematopoietic stem cells. Second, TAL1 is important for terminal erythroid differentiation. Deregulation of these processes is commonly involved in leukemic tran sformation 1 TAL1 is a member of the basic helix loop helix (bHLH) family of protein s. TAL1 forms hetero dimers with other bHLH proteins, known as E2A proteins. T AL1 confers tissue specific transcription and regulates target genes involved in early hematopoietic stem cell differentiation and erythroid differentiation 2 TAL1 can activate or repress transcription of target genes by recruiting transcriptional coactivators or corepressors, respectively The results herein show that TAL1 is associated with the histone H3K4 methyltransferase hSET1 complex. More specifically, these results show that TAL1 directly associates with the ASH2L subunit of the hSET1 complex and TAL1 associates with endogenous hSET1 complex in T ALL Jurkat cells. In addition, hSET1 is required for TAL1 mediated activation of the P4.2 target gene by changing the histone methylation status from a repressed status to

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13 active status (H3K4 trimethylation ) Furthermore, knockdown of hSET1 decreases the T AL1 mediated activation of the P 4.2 promoter activity. The regulation of P4.2 in erythroid differentiation reveal s a common epigenetic mechanism in which histone H3K4 methyltransferase hSET1 is involved in the TAL1 mediated transcriptional activation during hematopoiesis and possible leukemogenesis. This information will eventually help to design new therapeutic appr oaches to treat leukemia. Moreover, the study will shed light on the control of transcription factors on hematopoiesis

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14 CHAPTER 1 INTRODUCTION Hematopoiesis Hematopoiesis is characterized by the development of distinct cell types through the regulated dif ferentiation and proliferation of pluripotent hematopoietic stem cells ( HSC ) and oligo potent progenitors 3 The two common oligopotent hematopoietic progenitor cell lineages, are lymphoid and erythroid/myeloid. The lymphoid progenitor cells have the ability to differentiate into all of the white blood cell types that exist in the body, including B cells and T cells. The erythroid/myeloid progenitor cells have the ability to differentiate into erythrocytes as well as megakaryocytic a nd granulocytic cells ( Figure 1 1) Transcription factors specific to each cell type as well as cytokine s play key roles in the process 4 Gene targeting studies in mice have identified a series of regulators that function non redundantly at various stages of hematopoietic cell differentiation and in sp ecific blood cell lineages 5 Thus a carefully orchestrated binding of transcription factors is key in proper development of hematopoietic cells and the deregulati on of this process leads to leukemogenesis 6 Leukemia and Leukemogenesis (Regulation) The study of transcriptional regulation of hematopoiesis provide s crucial insight into how aberrant r egul ation of gene expression leads to leukemogenesis. Many genes that encode crucial hematopoietic transcription factors are also deregulated in human leukemias. Fusions as well as deletions of these transcription factors resulted from chromosome transloca tion events are frequently involved in leukemogenesis 3 For example, a fusion of SET domain containing MLL protein, which is also a histone methyltransferase, and of the same family as the SET1 protein, is involved in acute

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15 myeloid and lymphoblastic leukemia 7 Further, Ldb1 and LMO2, which are hematopoietic transcription factors and also specifically involved in activation of the erythroid target gene P4.2 are also involved in translocations lead ing to leukemogenesis as is TAL1 1 8 9 Introduction to TAL1 TAL1 was originally identified due to a (1:14) translocation in human T cell acute l ymphoblastic leukemia (T ALL) patients, which involves deletion of a 90 bp regulatory segment placing the TAL1 gene under the control of a T cell receptor promoter which is active in T cell development. This leads to th e ectopic expression of TAL1 in T cells In fact, TAL1 is ectopically expressed in 60 % of all T cell acute leukemias 3 It is a member of the basic helix loop helix (bHLH) family of transcription factors and is required for the development of all hematopoietic cell lineages 10 11 TAL1 forms heterodimers with the products of the ubiquitously expressed bHLH genes, E2A or HEB, and binds a hexanucleotide sequence known as an E box, CANNTG, to regula te transcription 12 ( Figure 1 2 ) The DNA binding function of TAL1 is dispensable in the early expansion of hematopoietic cells yet crucial in terminal eryt hroid diffe rentiation 13 In addition to direct effec ts as a transcription factor, TAL1 also exerts influence on target genes by recruiting histone methyltransferases, histone acetyltransferases, histone demethylases, histone deacetylases and transcription factors which change the conformation of chromatin t o allow transcription of the target genes at the appropriate stages of development for specific mature hematopoietic cell types TAL1 Is A Critical Regulator o f Hematopoiesis and Leukemogenesis Duri ng the process of hematopoiesis genes critical for differ enti ation of early hematopoietic stem cells into specific lineages of oligopotent progenitors, then finally

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16 into mature blood cells are actively transcribed. At the same time, t ranscription of cell proliferation genes are turned off, in part by epigenetic mechanisms 1 Deletion of Tal 1 in mice leads to embryonic lethality 8.5 days post conception (E8.5) due to complete loss of yolk sac hematopoietic cells 14 Further, Tal1 null embryonic stem (ES) cells are unable to generate both primitive and definitive erythroid cells in vitro and do not contribute to hematopoiesis in vivo i n chimeric mice suggesting a key role of TAL1 in erythropoiesis 15 In addition, studies indicated that TAL1 is also required for proper B and T lineage development 15 16 ( Figure 1 1 ) During hematopoiesis, TAL1 can function as repressor or activator of transcription dependin g on the sequence context and differentiation stage of the cells. TAL1 recruits mSin3a and histone deacetylase 1/2 (HDAC1/2) associated corepressor complexes, which mediate its transcription repressive activity in certain stages of erythroid differentiati on. While TAL1 activity is required throughout erythroid differentiation, the TAL1 complexes containing HDACs and deacetylase activity are markedly decreased during differentiation 17 Consistent with the role of histone methylation in TAL1 mediated transcriptional repression, our lab has recently discovered that TAL1 interacts with the histone lysine specific demethylase 1 (LSD1 or KDM1 ) in T ALL J urkat and erythroleukemia cells 18 LSD1 removes methyl groups from histone H3 lysine 4 (H3K4) residues. This H3K4 trimethyl modification is found in transc riptionally active or competent chromatin 19 In proliferating erythroleukemia cells, TAL1 represses target gene expression in part by recruiting LSD1 to demethylate H3K4 dimethylatio n marks at promoter regions 18 However, despite the role of TAL1 in transcriptional repression, i t has also been shown that TAL1 interacts with co activating histone m odifying enzymes

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17 such as p300 and the p300/CBP associated factor (PCAF) in hema topoietic cells as well as in T cell leukemia 17 20 These data also support that despite its known status as a transcriptional repressor, TAL1 can also positively regulate transcription. Chromatin Structure and F unction Within eukaryotic nuclei, DNA wraps around histone octamers, consistin g of two copies each of H2A, H2B, H3 and H4, to form repetitive units known as nucleosomes. The N terminal tails of core histones protrude out from the nucleosome and are dynamically regulated by covalent post translational modifications. Enzymatic modific ations of the histone N terminal tails have been implicated in the modulation of chromatin structure and gene expression. Like DNA methylation, histone modification patterns, associated with specific gene loci, are often maintained during replication and t ransmitted to daughter cells 21 Histone modifications include phosphorylation, acetylation, methylation, ubiquitination, sumolation, adenosine di phosphate (ADP) rib osylation and proline isomerization 22 Among these modifications, histone acetylation and methylation occurring at lysine (K) residues on histone H3 and H4 tails have been recognized as an important epigenetic mechanism with links to transcriptional activation and repression 22 23 These epigenetic modifications are critical for maintaining normal gene expression patterns in developmental proce sses such as hematopoiesis. Further, hematopoietic cell differentiation is often perturbed in malignancies such as leukemia 4 24 25 Throughout hematopoietic lineage development, histone acetylation and methylation patterns play critical roles in converting cell fate decisions into epigenetic information that determine the gene expression patterns into specific mature blood cell types 16 This process is initiated by the binding of sequence specific transcription

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18 factors to the regulatory elements of target genes in hematopoietic cells. These transcription factors then recruit coregulatory enzymes, many of which possess intrinsic histone modification activities to augment or inhibit target gene transcription 1 17 20 26 27 For example, erythroid specific transcription factors, such as TAL1, GATA1, EKLF and NF E2 1 28 29 form complexes and differentially recruit histone acetyltransferases (HATs) which correlate with actively transcribed genes 20 30 or histone deacetylases (HDACs) which repress transcription 2 17 to modulate expression of erythroid specific target genes (Table 1 1 .) Aberrant regulation of hematopoietic specific transcription factors and coregulators often leads to the development of specific forms of leukemia s 2 16 24 29 31 33 Changes in epigenetic marks due to misregulation of histone modifying enzymes may alter the function of TAL1, resulting in aberrant transcription during hematopoietic development which results in malignant hematopoiesis. h SET1 Is A His tone Modifying Enzyme Involved i n Transcriptional Activation The human SET1 complex (hSET1) is a SET domain containing H3K4 transcription 34 The hSET1 complex contains SET1, in which the SET domain confers H3K4 specific histone methyltransferase (HMT) activity 35 (Table 1 2 Figure 1 3 ) H3K4me2/3 is essential for recruitment of RNA polymerase II (RNAPII) and transcription machinery to the promoter of the target gene 22 35 There are several SET domain containing protein complexes (hSET1, and MLL1 4) and they are highly conserved from yeast to man (Table 1 2 ) Though yeast has only one SET1 complex, man has several homologs 34 36 All of the SET family histone methyltransferase complexes contain ASH2L, W DR5, RbBP5 and HCFC1 (Table1 2.) Though b oth MLL1 and hSET1 have H3K4 specific HMT activity, the hSET1 complex has a broader activity than the MLL

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19 complexes, which have more specific target genes 34 Further, the hSET1 complex is known to bind to the serine 5 phosphorylated, large subunit of RNAPII, Rtf in yeast This is at the C terminal domain of the elongating p olymerase. When serine 5 of Rtf is phosphor ylated, transcription is active; 37 t his provides an additional link between hSET1 and activation of genes. The proteins within the SET1 complex have the following functions: ASH2L is required for mole cular regulation of H3K4 trimethylation, but not di or mono methylation in yeast 36 Therefore, it is likely t hat knockdown of this component will only affect trimethylation levels. However, the data herein show that TAL1 binds directly to ASH2L. Thus knockdown of ASH2L may affect transcriptional activation of the TAL1 target gene due to failure to recruit RNAPII. The hSET1 complex components WDR5 and WDR82 are also both important for th e stability of the SET1 complex 38 Further, WDR82 may be unique to the hSET1 complex as is hSET1 itself, and thus knockd own of this component and hSET1 will only affect hSET1 mediated H3K4 methylation at promoters and hSET1 target gene expression 38 It is likely that TAL1 recruits hSET1 H3K4 methyltransferase to r egulate target gene expression. One example suggesting this is the following: in addition to TAL1 binding at the P4.2 promoter, an increase in H3K4 di methylation and trimethylation at the P4.2 promoter is also observed during DMSO induced differentiation o f MEL cells. Since H3K4 trimethylation is associated with active genes, this could be the signal to activate target genes at the correct developmental stage in the cell. Protein 4.2 (P4.2) : Red Blood Cell Membrane Protein Along with globin, the expression of protein 4.2 ( P 4.2 ) is a mark of erythroid differentiation, used in previous studies because P4.2 mRNA is expressed in early

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20 erythroblasts. P4.2 is an important component of mature erythrocyte membrane skeletal networks which reg ulates the stability and flexibility of the mature red blood cells 39 Deficiency of P4.2 leads to hemolytic anemia with various penetrance levels, especially pr evalent in Japanese populations 40 There are 7 isoforms of P4.2, t he largest of which encodes a 72 kDA membrane protein. The expression of this gene has bee n studied utilizing erythroblasts from bone marrow and erythroid cells cultured by the two phase liquid culture method from burst forming unit erythroid ( BFU E ) cells in peripheral blood in mice 41 Transcription of the mouse P4.2 gene initiates at multiple sites, with the major initiation site mapped at 174 nucleotides upstream of the ATG s tart codon. The mouse P4.2 promoter is TATA less and contains multiple potential binding sites for erythroid transcription factors GATA 1, NF E2, EKLF, and tal 1/SCL. Transient transfection experiments demonstrated that a 1.7 kb mouse P4.2 promoter fused w ith the luciferase coding regions was induced in DMSO treated MEL cells. TAL1 is known to associate with many binding partners including E2A, GATA1 LMO2, LDB1, and ETO2, all of which are involved in modulating hematopoietic cell growth and differentiation and expression of P4.2 8 42 43 Deletion analysis showed that the GATA binding site at position 29 t o 24 (upstream, relative to the promoter) is required for p4.2 expression after induction in differentiated MEL cells 39 Fu rther studies show that GATA1 bound to the dual GATA sites in the promoter form a bridge with TAL1/E protein heterodimers linked by LMO2 and Ldb1 to initiate transcription of the p4.2 gene 43 GATA1, Ldb1, LMO2 and TAL1 are among transcription factors whose functi on is critical in hematopoiesis 1 8 43

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21 Additionally, in the case of P4.2 the ETO2 protein binds to the dual GATA sites to prevent premature activation of the gene by this TAL1/Ldb1/LMO2 bridge 42 Previous research regarding the P4.2 promoter shows that LSD1 is recruited to restrict the ability of progenitor cells response to cellular differentiation programs 8 12 32 42 44 Also, after diff erentiation, LSD1 is switched to repress TAL1 target genes that promote cellular proliferation such as P4.2. In supporting this view, LSD1 disappeared from the p4.2 promoter which became H3K4 hypermethylated and histone hyperacetylated during differentiati on 6 18 Rationale f or This Study We undertook the biochemical purification of TAL1 associated nuclear protein complexes from T ALL leukemia cells and showed that TAL1 copurifies with the hSET1complex suggesting that hSET1 may confer positive effects on TAL1 mediated transcription. My hypothesis is that recruitment of hSET1 plays an important role in activation of the TAL1 t arget gene P4.2 in erythropoiesis. Colocalization of TAL1 and increased H3K4 methylation at the p4.2 promoter during transcriptional activation of p 4.2 gene and the hSET1 is present in the TAL1 associated protein complex support the hypothesis that recruit ment of hSET1 by TAL1 and subsequent H3K4 trimethylation of target gene promoters plays an important function on TAL1 mediated transcriptional regulation and hematopoiesis. Thus in addition to associating with the LSD1 complex, TAL1 also associates with th e hSET1 complex, perhaps acting as a molecular switch to activate hematopoietic specific gene expression patterns during hematopoiesis.

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22 Table 1 1 Histone modifying enzymes required for erythropoiesis TFs Coactivators Corepressors References TAL1/SCL P3 00, PCAF hSET1 mSin3A, HDACs, Brg 1, LSD1 Huang, at al. 2000; Huang, et al. 1999; Huang & Brandt, 2000a; Schuh et al., 2005; Goardon et al., 2006; Hu et al., 2009 GATA 1 CBP NURD, Mi 2, HDACs Rodriguez et al., 2005; Hung et al., 1999; Hong et al., 2005; Blobel et al., 1998 EKLF CBP/p300, Brg 1 mSin3A, HDACs Chen & Bieker, 2001; Zhang et al., 2001; Armstrong et al., 1998 NF E2 CBP/p300 Hu a ng, et al. 1999 *added because of t h is study Table 1 2. Conserved SET domain containing complexes ySET1, hSET1 a nd MLL1 complexes share components. WDR82 is unique to SET1. ASH2L is required for H3K4 trimethylation activity.

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23 Figure 1 1 TAL1 is critical for differentiation of hematopoietic cells. TAL1 is important for early HSC/HPC differentiation and later in erythroid differentiation (black arrows.) In Jurkat cells, T AL1 is ectopically expressed, preventing differentiation into lymphoid lineage cell types Figure 1 2 The structure of the TAL1 protein: Shown is the N terminus (Nt) the basic DNA binding region (b) the helix loop helix region (HLH) (the bHLH domain is made up of aa 187 243) and the C terminus (Ct .) The region within aa 143 185 is required to bind protein partners such as LSD1 13 Figure 1 3 SET1 protein domains 45 SET1a forms a HMT complex that associates with TAL1. The SET domain is the catalytic unit of the protein. There is al so an RNA Recognition motif (RRM .)

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24 CHAPTER 2 MATERIALS AND METHOD S Cell Culture Murine erythroleukemia (MEL) cells were grown at 37C with 5% CO 2 and maintained in M fetal bovine serum (FBS) a nd 1% penicillin/streptomycin (P/S .) I nduction of erythroid differentiation was performed by incubating cells in medium containing 1. 8 % dimethylsulfoxide (DMSO) for 24 h ours Mass S pect rometry and Immunoprecipitation The TAL1 associated complexes were puri fied from nuclear extract (NE) of the Jurkat T ALL cell line transduced with flag tagged TAL1 (Flag TAL1) and identified by m ass spectrometry at the T aplin facility in Harvard medical school. To prepare the nuclear extract, cells were lysed using the proto col developed by Nakatani and colleag ues 46 To confirm whether TAL1 associates with hSET1 in T ALL, TAL1 associated complexes were immunoprecipitated from Jurka t flag antibody (Sigma F3290) and then purified using an TAL1 antibody ( sc 12984 .) The resulting complex was resolved by SDS PAGE and analyzed by western blotting using SET1 antibody (Bethyl labs A300 289A, formerly BL1192) tar geting the catalytic component of the hSET1 complex. GST Pull Down Assay Both i n vitro transcribed and translated 35 S Met labeled hSET1 complex components as well as bacterially expressed GST TAL1 were incubated in bindi ng buffer (50 mM Tris HCl [ pH 8.0 ] ; 2 mM EDTA; 150 mM NaCl; 0.1% NP 40; 20 mM ZnC l 2 ; 10 mM MgCl 2 ; 1 mM DTT, 1 mM PMSF) containing 0.1 mg/mL bovine serum

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25 albumin (BSA) for 1 h our at room temperature and then 1 h our at 4 C. This incubation was followed by washing with the same binding buffer t o remove all protei n not bound to GST or GST TAL1. H3K4 Specific Methylation A ssay N uclear extracts were generated from both induced and uninduced MEL cells overexpress ing FH TAL1 before and after 24 h of DMSO induced differentiation (1.8% DMSO for 24 h) in the standard procedure used in our lab developed by Nakatani and colleague s 46 Flag TAL1 antibody. This complex was then incubated with 3 H Ado Met and comm ercially available H3 peptides, or fully H3K4 trimethylated pep tides in a buffer favorable to histone methylation ( 20 mM Tris HCl, PH 8.0; 4 mM EDTA; 1mM PMSF, 500 uM DTT ) for 1 h at 30 C The products of this reaction were then run on a 10% SDS PAGE gel. The presence of a band on an autor adiograph of this gel (in cu ba ted for 24 h at 80 C) indicates the specificity of the methylation activity of the TAL1 associated hSET1 complex for K4 residues in the histone H3 tails Chromatin Immunoprecipitation Sequencing (ChIP seq) ChIP seq data was provided by our collaborators i n the laboratory of Dr. Keji Zhao. Nucleosomes were isolated from CD34+ cells and cells differentiated to CD36+ with erythropoietin (Epo .) The DNA was purified and Solexa adaptors added before massively parallel sequencing of the P 4.2 promoter region. P4.2 P romoter D riven L uciferase R eporter A ssay To test if hSET1 is important for TAL1 transactivation, an assay containing a P4.2 driven luc if erase reporter gene was used. This pREP 4.2 luc contains a P4.2 promoter,

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26 upstream of the luciferase reporter gene. Th e pREP reporter plasmid is unique in that it can be transfected into mammalian cells and will replicate as an episome, in which the DNA and histones form the chromatin structure as it does within chromosomes. Cloning of the pREP4 P4.2 luc P lasmid The clon ing of this plasmid involved taking a pREP4 plasmid, in which there is a multi cloning site. The particular one used in this study already contained an insert with a long terminal repeat (LTR) sequence, which contains triple E box motifs that can potential ly also bind to TAL1. The P4.2 promoter, which contains dual GATA motifs and dual E boxes, was cut out of a pCDNA3.1 vector backbone and inserted into this pREP4 episomal plasmid ( Figure 2 1) An episomal plasmid replicates as a miniature chromosome within the cell in culture and forms chromatin as a normal chromosome. The P4.2 promoter was inserted with a single restriction site XhoI, into the pREP4 plasmid backbone and confirmed to be the correct orientation by restriction digest with XhoI and NotI, which is w/in the promoter clone inside the pREP4 backbone (data not shown .) P4.2 Reporter Assay Since t he P4.2 linked luciferase promoter used in this study expre sses luciferase upon activation the first step was to determine if P4.2 promoter driven lucife rase was expressed in mouse ery throleukemia (MEL) cells upon DMSO induction. First, the MEL cells were co transfected with pREP 4.2 luc and a plasmid encoding Tal1 cDNA (pCDNA TAL1) us ing the Lipofectamine 200 kit performed in 24 well plates 24 h ours later, t he cells were induced to differentiate by adding 1.5% DMSO and additional Optimem media for a total of 1mL per transfection in a 24 well plate The resulting luciferase expression was measured at 72 h ours after DMSO induction by correlating

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27 bioluminescenc e produced with expression of the protein using a luciferase reporter kit (Promega .) Therefore, this assay measured TAL mediated activation of the P4.2 driven luciferase reporter gene as a function of the amount of pCNDA3.1 Tal1 co transfected into the pro moter during erythroid differentiation The resulting luciferase expression was measured using 100 uL per well in 96 well plates with a spectrophot ometer siRNA K nockdown of hSET1 in the P4.2 Reporter Assay Further, siRNA targeting hSET1 was co transfecte d in an otherwise identical experiment, to determine the effect of hSET1 presence on TAL1 mediated activation. The hSET1 k nock d own (kd) studies were done using 100 ng of TAL1 and 10 ng of si hSET1 co transfected into MEL cells in the presence of the pREP4 P4.2 luc reporter Lipofectamine was also used in this transfection, and the siRNA was added at the same time as the pCDNA3.1 Tal1 and pREP4 P4.2 luc plasmids, 24 h ours before DMSO and additional Optimem media was added.

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28 Figure 2 1 Structure of the pREP P4.2 luc plasmid: Contains the P4.2 promoter driving the luciferase gene, which contains dual E boxes and dual GATA sites. Further, long terminal repeats from the HIV virus which contain triple E box motifs are also contained in this plasmid.

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29 CHAPTE R 3 THE ASSOCIATION OF TAL1 WITH h SET1 In this project, I focus ed on investigating how the opposite histone modifying enzymes, LSD 1 and hSET1 complexes, which have been identified as TAL1 ass ociated cofactors regulate the function of TAL1 in hematopoiesis and leukemogenesis. The specific aims of this research proposal are: 1 ) Analyze the role of histone methyltransferase hSET1 complex in TAL1 mediated transcriptional regulation dur i ng erythroid differentiation; 2 ) Understand the molecular basis of histon e modifying enzy mes in TAL1 induced normal. The first aim was accomplished by characterizing the association of the TAL1 associated hSET1 complex with the TAL1 protein, and the H3K4 specific methylation of the complex. The second aim examined the effect of hSET1 onTAL1 mediated transcriptional regulation in hematopoiesis as well as the hSET1 mediat ed epigenetic modification, H3K4 trimethylation, which coincides with activation of the P4.2 gene, a marker for erythropoiesis. The TAL1 Complex A ssociate s w ith t he hSET1 C omplex Previous studies in our lab have indicated that TAL1 activation or repression is determined by combinatorial association with coactivators versus corepressors. Therefore, it was important to first purify TAL1 associated complexes. The poly peptide signatures found in this analys is are listed in the end of this section ( Figure 3 1 ) These data provide evidence that the hSET1 HMT complex associates with the TAL1 complex in a T ALL leukemia cell line. Data gathered from mass spectrometry analys is shows that TAL1 containing complexes in Jurkat cells include several transcriptional corepressor and coactivator proteins ( Figure 3 1 ) They are: 1) one complex containing corepresssors such as L SD1 and Co REST and HDAC1, and 2) another complex

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30 containi ng coactivators such as hSET1, and components of the hSET1 complex (ASH2L, WDR5) suggesting that TAL1 can activate or repress target genes by interacting with coregulatory enzymes and fun ctions like a molecular switch. The TAL1 complex is associated with h SET1 in the Jurkat T ALL cell line. Jurkat leukemia cells are T cell precursors in which TAL1 is ectopically expressed, a cell culture model for T ALL leukemia. In this T ALL cell line, TAL1 associates with several components of the hSET1 complex, includin g: hSET 1, ASH2L, HCF1, and RBBP5 ( Figure 3 1 ) To confirm whether TAL1 associates with hSET1 in T ALL, TAL1 TAL1 antibody (Santa Cruz Biotechnology, sc 12984) and western blotted (WB) with hSET1 antibody ( Bethyl L abs A3 00 289A .) These data confirm that endogenous TAL1 interacts with hSET1 ( Figure 3 2 ) These results support the idea that hSET1 associates with TAL1 in normal hematopoiesis and in leukemogenesis. TAL1 Directly Interacts with the ASH2L Component In additio n to determining the association of hSET1 and TAL1, I determined which component of the hSET1 complex directly associates with TAL1 using an in vitro GST pulldown assay ( Figure 3 3 A ) These data show that TAL1 interacts with ASH2L, a component required fo r trimethylation of H3K4 36 Further, the results show that there is no direct interaction of TAL1 with the othe r hSET1 complex components tested, including RbBP5, WDR5 or the SET1 catalytic unit itself ( Figure 3 3 B ) The TAL1 Complex Displays H3K4 Specific Histone Methyltransferase ( HMT ) A ctivity To determine whether the TAL1 complex exhibits HMT activity througho ut differentiation, nuclear extracts from FH TAL1 over expressed MEL cells, before and

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31 afte r DMSO induced differentiation were prepared. The TAL1 complex was purified using an Flag immunoaffinity column, then the resulting purified complex was then use d in an in vitro methylation assay. These results indicate that the TAL1 complex not only has HMT activity in general but also that this HMT activity is H3K4 specific. This is be cause the fully trimethylated H3K4 histones were not methyl ated by the complex ( Figure 3 4 lanes 2 and 4) whereas H3 alone was methylated (lanes 1 and 3 )

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32 Figure 3 1 Identification of the TAL1 interacting proteins, including hSET1 components as well as corepressors. A partial list of polypeptides identified by mass spectrometry above is grouped by biological function. TAL1 associating polypeptides were purified from the nuclear extract of 1 10 9 Flag tagged TAL1 transduced and mock transduced Jurkat ce Flag antibody conjugated column, eluting polypeptides containing the Flag peptide. The resulting flag TAL1 complexes were resolved by SDS PAGE and t he proteins visualized with C oomassie blue staining and analyzed by mass spectrometry. F igure 3 2 TAL1 recruits endogenous hSET1 and the H3K4 HMT in Jurkat cells. The Jurkat nuclear extracts were immunoprecipitated with TAL1 antibody. The immuno complexes were resolved by SDS PAGE, and analyzed with western blot using hSET1 antibody Flag Tal1 MOCK Flag Tal1 KD 191 97 64 51 39 28 1 2 3 4 5 6 hSET1 Complex 2A hSET1 (7) 4E ASH2L (2) 2C HCFC1 (16) Transcription Factors Corepressor Complexes 3D LSD1 (11) 3F TIF1 4B MTA1/2 (15/13) 4N E2A (11) 5A HDAC1 (16) 6C LDB1 (2) 6K actin (10) 6 TAL1 (6) 4M HEB (19) 5I CoREST (10) 5J DMAP1 (4) 2B Mi 2 5B HDAC2 (2) 6L SSBP3 (3) 4L ETO 2 (3) Protein ID (No of Peptides) Protein ID ( No of Peptides) Protein ID (No of Peptides) A B Flag Tal1 191 97 64 51 39 28 1 2 3 4 5 6 hSET1 Complex 2A hSET1 (7) 4E ASH2L (2) 2C HCFC1 (16) Transcription Factors Corepressor Complexes 3D LSD1 (11) 3F TIF1 4B MTA1/2 (15/13) 4N E2A (11) 5A HDAC1 (16) 6C LDB1 (2) 6K actin (10) 6 TAL1 (6) 4M HEB (19) 5I CoREST (10) 5J DMAP1 (4) 2B Mi 2 5B HDAC2 (2) 6L SSBP3 (3) 4L ETO 2 (3) Protein ID (No of Peptides) Protein ID (No of Pepti des) Protein ID (No of Peptides) A B

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33 Figur e 3 3 GST pulldown assays. A) E xperimental scheme of GST pulldown assays. B) Radioactively labeled protein input (positive control) GST (negative control) and GST TAL1 (full length .) Figure 3 4 HMT assay. The FH TAL1 MEL nuclear extract before and aft er treatment w/ 1. 8% DMSO for 1 day was immunopre Flag antibody; the resulting complex was the flag immunoaffinity column. This complex was then incubated with 3H Ado Met and commercially available H3 peptides. In lanes 2 and 4, completely tri methylated H3K4 me3 was also used to determine the specificity of the methylation activity of the TAL1 containing complex. The presence of a band on the autoradiograph of the gel indicates methylation. A B.

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34 CHAPTER 4 THE EFFECT OF h SET1 ON TAL1 TARGET GENES After confirming the association of the two complexes, the next logical step was to confirm that these complexes exert a discernable effect of the transcription of TAL1 target genes, such as P4.2 in erythroid development. To ascertain this, two different approaches were unde rtaken. First of all chromatin immunoprecipitation is the standard biochemical method for determining colocalization of complexes at a promoter as well as any changes in chromatin structure discernable via histone modifications. Our collabo rator Keji Zhao contributed the ChIP seq studies of the colocalization of TAL1 and H3K4 trimethylation at the P4.2 promoter Therefore, another approach using a reporter assay was also employed to determine the direct effect on th e protein expression of the P4.2 gene itself. TAL1 Binding Is Associated with Increased H3K4me3 in the P4.2 Promoter If TAL1 associates with SET1, it is likely that hSET1 HMT activity plays a role in activation of TAL1 target genes during normal hematopoi esis. P4.2 is an erythroid cell membrane protein and a marker for red blood cell de velop ment1. ChIP seq data ( Figure s 4 1 and 4 2) from our collaboration with the laboratory of Dr. Keji Zhao suggest a correlation between TAL1 occupancy of the P4.2 promoter and incre ased H3K4me3 in ES cells ( Figure 4 1 ) Further, in CD34+ hematopoietic stem cells (HSC) where the P4.2 gene is silent, there is no H3K4 methylation detec ted at the P4.2 promoter ( Figure 4 2 ) After differentiation from CD34+ HSCs into the CD36+ lineage, H3K4me3 levels increase (data not shown )

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35 The Effect of hSET 1 o n TAL1 Mediated Transcription Using a P4 .2 Promoter Driven Reporter Assay After determining the association of the TAL1 and hSET1 complexes, and the presence of histone methyltransfer ase activity of the TAL1 associated hSET1 complex, a more direct in vivo approach was used to determine the effect of hSET1 on TAL1 mediated target gene P4.2 during erythroid differentiation ( Figure 4 3 ) The in vitro model system used does lead to P4.2 pro moter driven luciferase expression upon DMSO induction ( Figure 4 4 ) Unexpectedly, the level of pCDNA3.1 TAL1 required for maximum activation of the reporter construct varied between endogenous levels and an addition of 100 ng of pCDNA TAL1 For example, co transfection with pREP4 P4.2 luc with higher concentrations of p CDNA3.1 Tal1 (150ng to 200 ng) and thus overexpression of TAL1, may result in repression of P4.2 ( data not shown ) This is likely due to varying transfection efficiency. This is not actuall y surprising since very little TAL1 binds DNA and is required in activation of the P4.2 gene. In any case, TAL1 is shown to be required for P4.2 expression in previous studies 8 39 To address the effect of hSET1 on TAL1 transactivation, DMSO treated MEL cells were then co transfected with siRNA targeting hSET1 to disrupt the function of hSET1 on TAL1 mediated activat ion of P4.2. It was expected that cotransfection of siRNA targeting the hSET1 complex would strongly decrease the TAL1 transactivation of the P4.2 driven luciferase reporter ( Figure 4 5 ) 100ng was the optimum concentration of co transfected pCDNA TAL1 for activation of the reporter gene in siRNA cotransfection studies. Repression of the gene activation did occur upon transient hSET1 knockdown. Unexpectedly, derepression also occurred in cells transfected with higher then endogenous levels of pCDNA TAL1 co transfected with si hSET1 ( Figure 4 5 ) In

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36 addition to the transfection efficiency issues it would make sense that knocking down hSET1 may not be completely abolishing hSET1 histone methyltransferase activity and overexpression of TAL1 via cotransfection wi th higher concentration of pCDNA TAL1 may compensate for this. Nonetheless, t hese results do indicate that hSET1 is involved in TAL1 mediated P4.2 activation in differentiating erythrocytes, but that there is a specific level required.

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37 Figure 4 1. A c orrelation between TAL1 localization at the P4.2 promoter and H3K4 methylation ( ChIP seq data on Bernstein ES cells over the P4.2 promoter provided by Dr. Keji Zhao, NIH .)

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38 Figure 4 2 TAL1 and H3K4me3 are correlated with expression o f P4.2 upon differe ntiation. ChIP seq data indicating a correlation between H3K4 methylation at the promoter of P4.2 in CD34+ cells upon differentiation with erythropoietin (EPO ) (data provided by Dr. Keji Zhao, NIH .)

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39 Figure 4 3 T he experimental strategy for determini ng the effect of TAL1 as well as the effect of hSET1 knockdown on the P4.2 TAL1 target genes: A) MEL cells will be induced to differentiate after being co transfected with pREP 4.2luc, pcDNA Ta1l. HCF1 siRNA into these MEL cells, thus disrupting the hSET1 complex will be compared to the result with the wild type hSET1 complex.

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40 Figure 4 4 The effect of TAL1 concentration on TAL1 target gen es. MEL cells were DMSO induced to differentiate after being co transfected with pREP 4.2luc, pcDNA Ta1l and the respective luciferase activity was quantitated spectrophotometricall y, indicating the activation level of the P4.2 promoter driving the lucife rase construct. T tests show that this is a significant difference = 0.05 n=6 (data not shown) 0 2 4 6 8 10 12 14 16 18 control 100 control 100 DMSO + DMSO Effect of siRNA kd of hSET1 on TAL1 activation of p4.2 reporter

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41 Figure 4 5 T he effect of hSET1 knockdown on TAL1 target genes. MEL cells were DMSO induced to differentiate after being co transfected with pREP 4.2luc, pcDNA Ta1l. Then co transfected with HCF1 siRNA into these MEL cells, thus disrupting the hSET1 complex will be compared to the result with the wild type hSET1 complex T tests show that this is a significant difference =0.05 n=2 (data not shown )

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42 CHAPTER 5 DISCUSSION During hematopoiesis, TAL 1 is essential for both early stem cell development and in erythropoiesis and its activity is subject to regulation. The specific function that TAL1 serves in regulating genes involved in differentiation towards either the erythroid or the megakaryocytic l ineage depends on complex combinatorial effects of additional protein complexes in conjunction with TAL1/E protein heterodimers. This suggests that TAL1 does in fact act as a molecular switch controlling cell fate by activating genes at the correct develop mental stage It makes sense that there is a histone methyltransferase associated with the coactivator complexes associated with TAL1 similar the association with LSD1 demethylation enzyme in TAL1 associated repressor complexes. Further studies are neces sary to elucidate how these proteins act in tandem to regulate genes involved in hematopoiesis. It is likely that TAL1 activates other erythroid specific genes via the same mechanism Further, given that hSET1 in conjunction with TAL1/E2A heterodimers is i nvolved in activating TAL1 target genes in early hematopoietic expansion as well as in activating misregulated genes in T ALL leukemogenesis, these studies will provide insight into the mechanisms by which the misregulation of this transcription factor con tributes to leukemogenesis. The Association of hSET 1 a nd TAL1 The data presented in this thesis support the proposed role of hSET1 in the TAL1 mediated transcriptional activation of erythroid genes in erythroid progenitor cells and is accompanied by tempor al changes in promoter chromatin status throughout hematopoietic cell development The biological action of hSET1 in hema topoiesis was previously unknown. Further, these results show that in fact there is endogenous

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43 association between TAL1 and hSET1 in he matopoietic cells, which colocalizes at the P4.2 promoter and has histone methyltransferase activity specific to histone H3K4 residue. Further these studies show that TAL1 associates with the hSET1 complex via the ASH2L subunit specifically. It remains to be shown which domain interacts with ASH2L however, and if this is the sole interaction between the two. The Effect of hSET1 on TAL1 Target Genes Further, the association of hSET1 and TAL1 can lead to activation with the correct concentration of transcript ion factors. However, the orchestration of this activation process is not simply linear. The concentration of TAL1 and how it interacts with other motifs nearby adds an additional layer of complexity to the puzzle of how TAL1 target genes and genes in gene ral are activated at the proper developmental stage. These studies contribute novel information on how this histone methyltransferase ( HMT ) tic activities, and the effect of these epigenetic modifications on hematopoietic differentiation. However, the data show n here has the limitation of only showing with certainty that TAL1 recruits hSET1 to the promoter and mediates H3K4 trimethylation and activation of the P4.2 gene in this in vitro model system One further limitation of this study is the fact that the pREP4 P4.2 luc plasmid also contains a segment with long terminal repeats (LTR) which contain E boxes. These E boxes which TAL1 heterodime rs bind to may interfere with the GATA1 Ldb1, LMO2 bridge required for activating the gene. It is possible that if the threshold level of TAL1 is crossed, ectopic TAL1 blocks expression by sequestering transcription factors or by blocking binding of the GA TA1, Lmo2, Ldb1, complexes that assist in bringing

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44 enhancers to promoters via a looping mechani sm as described in Wadman et al 43 Conversely, the E boxes contained within the long terminal repeats (LTRs) that exist in the plasmid used in this study could also activate TAL1 in lieu of the enhancers or in addition to the enhancers that act in vivo within the P4.2 promoter. Another direction for future studies on the ef fect of hSET1 on TAL1 mediated gene activation is determining that TAL1 and hSET1 colocalize at the promoter of target genes such as P4.2. The ChIP seq data generously contributed by our collaborator Keji H3K4 trimethylation are co localized. However, this H3K4 trimethylation is most likely due to hSET1 activity, because of the association of the complexes shown. Chromatin immunoprecipitation (ChIP) studies examining the binding of hSET1 at the P4.2 promote r as well as at the promoters of additional TAL1 target genes will more clearly show if there is indeed a pattern where hSET1 is the histone methyltransferase required for TAL1 activation of target genes. None theless, t he data herein suggests that TAL1 d oes in fact act as a molecular switch controlling cell fate by activating genes at the correct developmental stage. It makes sense that there is a histone methyltransferase associated with the coactivator complexes associated with TAL1 similar the associat ion with LSD1 demethylation enzyme in TAL1 associated repressor complexes. Previous r esearch on the androgen receptor shows that other transcription factors also interact dynamically with both coactivators and corepressors to regulate cell fate decisions 18 The refore, dynamic changes of the TAL1 i nteraction with LSD1 and hSET1 dictate the TAL1 function. In fact, both coregulators are involved in regulated erythroid spe cific p4.2 gene. TAL1

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45 recruits LSD1 to inhibit p4.2 transcription in undifferentiated stem or progenitor cells 18 Upon induction of differentiation, disassociation of LSD1 and Recruitment of hSET1 complex (your data) appear to function as molecular switch to turn o n the p4.2 expression. Since TAL 1 only activates the p4.2 promoter in DMSO induced MEL cells and siRNA knockdown ofhSET1 abolishes the TAL1 mediated transcrip tional activation in differentiated erythrocytes support the role of hSET1 in TAL1 mediated transcriptional activation during hematopoiesis. Further studies are necessary to elucidate how these proteins act in tandem to regulate genes involved in hematopoi esis. It is likely that TAL1 activates other erythroid specific genes via the same mechanism Further, given that hSET1 in conjunction with TAL1/E2A heterodimers is involved in activating TAL1 target genes in early hematopoietic expansion as well as in act ivating misregulated genes in T ALL leukemogenesis, these studies will provide insight into the mechanisms by which the misregulation of this transcription factor contributes to leukemogenesis. How h SET 1 Activates Transcription i n Hematopoiesis These stu dies show that hSET1 is involved in TAL1 mediated transcriptional activation at the P4.2 promoter. The evidence for this is the fact that hSET1 is recruited to erythroid specific promoters by tissu e specific transcription factors such as TAL1. Further, unl ike the MLL catalytic subunit, intrinsic DNA binding domain; therefore it doe The specificity of this binding is therefore conferred by the tissue specific transcription factor, TAL1. Furthermore, it is known that H3K4 mono and di methylation both ma rk the enhancers of active genes, while H3K4 trimethylation is enriched in the promoters of all active genes 22 47 The se data show that hSET1, a H3K4 methyltransferase as well as H3K4

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46 trimethylation are both highly enriched in active p4.2 promoter in mature erythrocytes. Further linking this process to transcription is the fact that H3K4 trimethylation catalyzed by hSET1 and hSET1 can directly interact with TAF7, an important c omponent of TFIID/PolII complex 37 Future Directi ons First of all, the results of the GST pull down experiments presented here have indicated that ASH2L directly interacts with TAL1 This suggests that ASH2L bridges the TAL1 and hSET1 interaction. ASH2L is important component of hSET1 complex. It is requ ired for the integrity and enzymatic activity of whole histone methyltransferase complex. It will be interest ing to map the domain that medi ates TAL1 and ASH2L interaction in future experiments to determine the way in which hSET1 affects TAL1 mediated acti vation of target genes. Discovery of any differential regulation between different hematopoietic genes and leukemogenic genes is also key in determining the way that TAL1 mediated gene activation in tandem with hSET1. As mentioned previously, t he most impo rtant futures studies in this area should focus on confirming colocalization of hSET1 and TAL1 at target genes. The studies herein do suggest such an association due to the colocalization of H3K4 trimethylation and TAL1 at the P4.2 promoter. However, Chrom atin immunoprecipitation of TAL1 and hSET1 binding at the P4.2 promoter and other TAL1 target genes in conditions that lead to activation of the gene should also be done to confirm this. Another future direction of this work is to determine if hSET1 acti vates TAL1 mediated genes involved in leukemogenesis in the same way that it has been shown to activate P4.2, for TAL1 not only affects recruiting histone modifying enzymes and chromatin remodelin g proteins, but may also activate genes affecting cell cy cle arrest.

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47 This is substantiated by the fact that TAL1 knockdown studies show an effect on cdk6 expression 5 Further, the Runx transcription factor is also impo rtant in hematopoiesis of myeloid lineage and Runx genes are known downstream targets of TAL1 48 Finally complexes containing RBP2 associating w/ pRb has been sh own to be involved in regulating cell cycle genes 45 49 TAL1 has also been shown to interact with pRb in Mass spectrometry data from this lab (unpublished.) Discovery of the TAL1 target genes involved in cell cycle control as well as apoptosis may suggest potential new treatments for leukemia.

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48 LIST OF REFERENCES 1. Ferrando AA, Herblot S, Pa lomero T, et al. Biallelic transcriptional activation of oncogenic transcription factors in T cell acute lymphoblastic leukemia. Blood. 2004;103:1909 1911. 2. Chen Q, Cheng JT, Tasi LH, et al. The tal gene undergoes chromosome translocation in T cell leuke mia and potentially encodes a helix loop helix protein. Embo J. 1990;9:415 424. 3. Look AT. Oncogenic transcription factors in the human acute leukemias. Science. 1997;278:1059 1064. 4. Orkin SH, Zon LI. Hematopoiesis: an evolving paradigm for stem cell bi ology. Cell. 2008;132:631 644. 5. Palomero T, Odom DT, O'Neil J, et al. Transcriptional regulatory networks downstream of TAL1/SCL in T cell acute lymphoblastic leukemia. Blood. 2006;108:986 992. 6. Hu X, Ybarra R, Qiu Y, Bungert J, Huang S. Transcriptiona l regulation by TAL1: a link between epigenetic modifications and erythropoiesis. Epigenetics. 2009;4:357 361. 7. Wiederschain D, Kawai H, Shilatifard A, Yuan ZM. Multiple mixed lineage leukemia (MLL) fusion proteins suppress p53 mediated response to DNA d amage. J Biol Chem. 2005;280:24315 24321. 8. Xu Z, Huang S, Chang LS, Agulnick AD, Brandt SJ. Identification of a TAL1 target gene reveals a positive role for the LIM domain binding protein Ldb1 in erythroid gene expression and differentiation. Mol Cell Bi ol. 2003;23:7585 7599. 9. Lecuyer E, Herblot S, Saint Denis M, et al. The SCL complex regulates c kit expression in hematopoietic cells through functional interaction with Sp1. Blood. 2002;100:2430 2440. 10. Begley CG, Green AR. The SCL gene: from case rep ort to critical hematopoietic regulator. Blood. 1999;93:2760 2770. 11. Begley CG, Robb L, Rockman S, et al. Structure of the gene encoding the murine SCL protein. Gene. 1994;138:93 99. 12. Valge Archer VE, Osada H, Warren AJ, et al. The LIM protein RBTN2 a nd the basic helix loop helix protein TAL1 are present in a complex in erythroid cells. Proc Natl Acad Sci U S A. 1994;91:8617 8621. 13. Kassouf MT, Chagraoui H, Vyas P, Porcher C. Differential use of SCL/TAL 1 DNA binding domain in developmental hematopoi esis. Blood. 2008;112:1056 1067.

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52 BIOGRAPHICAL SKETCH River Saenz (formerly River Ybarra) received a B achelor of Science degree in 2007 from California State University in Sacramento with a major in molecular biology. During her undergraduate training, she prepared an honors thesis project entitled She was also selected as a Louis Stokes A lliance for Minority Participation (LSAMP) Scholar as well as a McNair Scholar, two competitive programs d esigned to bring educationally and economically disadvantaged students from underrepresented groups into grad uate programs in the sciences. In 2007, she was accepted to graduate school in the terdisciplinary Program in Biomedical Sciences (IDP) and joined the laboratory of Dr. Suming Huang. During her graduate studies, she received a res earch supplement from the National Heart Lung and Blood Institute (NHLBI )