The Role of TFII-I and USF in Beta-Globin Gene Regulation

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The Role of TFII-I and USF in Beta-Globin Gene Regulation
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To my parents whose support and encouragement have meant everything to me. 2


ACKNOWLEDGMENTS I would first like to thank my mentor, Dr. Jrg Bungert, for the opportunity to develop as a scientist. He provided a positive learning environment and gave constant encouragement. He always found a way to find something interesting in results, even if I thought that the experiment had failed terribly. I came out with a more positive attitude about my research after discussing it with him. Despite his busy schedule, he always found time to discuss experiments or go over presentations or papers. I am grateful for his guidance and for his unending optimism. I thank my committee members, Drs. Thomas Yang, Michael Kilberg, Philip Laipis, and Maurice Swanson. They have provided terrific feedback of my research on giving suggestions and guidance. Committee meetings were never times to dread, but rather times for me to learn from these men. Their critiques have made me more closely analyze my research and led me to paths that I might not have otherwise thought of. I am grateful for their time and support. I would also like to thank Dr. Brian Cain for critically evaluating and providing me with advice concerning my presentation at the 2006 Medical Guild Research Competition. I am grateful for all of the hard work that Dr. James B. Flanegan, the department chair, and all the administrative and secretarial staff do to make the department run smoothly. I appreciate the time that the secretarial staff has taken to arrange my travel to conferences and to register me for classes. There has never been a problem that they could not help me solve. I thank all of the past and present members of the Bungert lab. I am grateful for Sung-Hae Lee Kang’s guidance as I first entered the lab. She provided me with excellent feedback and advice as a new graduate student. I would like to thank Karen Vieira for her knowledge and friendship, also for her townhouse which I bought when she moved. Her drive to succeed in life was inspiring and I wish her all the best in her goal to retire by the age of 30. Padraic Levings was like a little, pesky brother to me. I appreciate not only the entertainment he provided in the 3


lab, but also all of his guidance and suggestions. I would especially like to thank Archana Anantharaman for all her hard work in the lab for the past two years. She has been a great help to my research. I wish her all the best in medical school. I would also like to thank all of the many other members of the lab, including undergraduate, rotation, and high school students who I have worked with and especially Felicie Andersen, Boris Thurisch, and Babak Maghimi. I wish the current graduate students all the best with their research. Finally, I would like to thank all of the lab managers who have helped my experiments to run smoothly in placing orders and keeping the lab in general order. I would like to thank my parents, Vic and Stephanie Crusselle, for their constant encouragement and support. My dad was the first to introduce me to research in my fruit fly experiment for the sixth grade science fair. I appreciate the time he takes to help me with any problems I encounter. My mom has always been there to listen to and support me in my disappointments and achievements. My parents have provided me with a strong work ethic and moral background, for which I am thankful. I have found that even in my twenties, my parents are still always right and it is best to follow their advice. I would also like to thank my extended family for their support. I would like to thank my husband, Jonathan Davis, for his love, patience, and support. I appreciate his efforts to understand my research and for listening to me daily and discuss my activities in the lab. He has supplied me with much needed laughter and distractions. Without them graduate school would have been a much more stressful time. I am grateful for his belief in me and for supporting me in my desire to succeed. 4


Lastly, I would like to thank my Heavenly Father and Savior, Jesus Christ, for providing me with guidance, comfort, and my many blessings. I know by putting my faith and trust in them that I will be happy and successful in this life. 5


TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................3 LIST OF FIGURES .........................................................................................................................8 ABSTRACT ...................................................................................................................................10 CHAPTER 1 INTRODUCTION..................................................................................................................12 Hemoglobin and -thalassemia...............................................................................................12 Overview of Thalassemia and Symptoms.......................................................................12 Current Treatments of -thalassemia...............................................................................13 Chromatin Structure and Gene Regulation.............................................................................15 Chromatin Remodeling...................................................................................................16 Histone Modification.......................................................................................................17 Chromosome Territories..................................................................................................18 The -globin Gene Locus.......................................................................................................18 Hemoglobin Switching...........................................................................................................21 Gene Competition............................................................................................................21 Autonomous Silencing....................................................................................................22 Role of Transcription Factors..........................................................................................22 Adult -Globin Gene Promoter and Regulatory Region........................................................25 TFII-I......................................................................................................................................27 Structure..........................................................................................................................27 Isoforms...........................................................................................................................28 Role in Cell Signaling.....................................................................................................29 TFII-I Regulated Genes...................................................................................................30 USF.........................................................................................................................................31 Structure..........................................................................................................................32 Regulation of Expression and Activity............................................................................32 USF Regulated Genes......................................................................................................33 Summation..............................................................................................................................35 2 MATERIALS AND METHODS...........................................................................................39 Construction of Protein Expression Vectors...........................................................................39 Cell Culture and Transfections...............................................................................................41 Chromatin Immunoprecipitation, Double-Chromatin Immunoprecipitation, and Co-immunoprecipitation...........................................................................................................42 RNA Interference....................................................................................................................44 Protein Isolation and Western Blotting...................................................................................44 RNA Isolation, Real-Time PCR, and PCR.............................................................................45 Immunofluorescence...............................................................................................................47 6


3 REGULATION OF -GLOBIN GENE EXPRESSION BY TFII-I AND USF....................48 Introduction.............................................................................................................................48 Results.....................................................................................................................................50 Discussion...............................................................................................................................55 4 RECRUITMENT OF CHROMATIN MODIFYING ENZYMES BY TFII-I AND USF TO THE -GLOBIN LOCUS.................................................................................................66 Introduction.............................................................................................................................66 Results.....................................................................................................................................69 Discussion...............................................................................................................................74 5 CALCIUM REGULATION OF -GLOBIN EXPRESSION THROUGH TFII-I AND USF.........................................................................................................................................94 Introduction.............................................................................................................................94 Results.....................................................................................................................................97 Discussion...............................................................................................................................99 6 CONCLUSIONS AND FUTURE DIRECTIONS...............................................................106 The Role of TFII-I and USF in -globin Gene Expression..................................................106 Recruitment of Chromatin Modifying Enzymes by TFII-I and USF to the -globin Locus.................................................................................................................................110 Developmental-Stage Specific Regulation of -globin Expression by TFII-I and USF......111 Calcium Regulation of -globin Expression through TFII-I and USF.................................114 Summary...............................................................................................................................116 LIST OF REFERENCES.............................................................................................................117 BIOGRAPHICAL SKETCH.......................................................................................................142 7


LIST OF FIGURES Figure page 1-1 Schematic of the organizational structure of the human and murine -globin gene loci......................................................................................................................................37 1-2 Sequence alignment of the human -globin downstream promoter region.......................38 3-1 Experimental system for expressing wild-type or dominant-negative proteins. t.............59 3-2 TFII-I function in MEL cells.............................................................................................60 3-3 p70 function in K562 cells.................................................................................................61 3-4 TFII-I function in K562 cells.............................................................................................62 3-5 USF activates -globin gene expression in MEL cells......................................................63 3-6 Interaction of USF and TFII-I with the -globin gene locus during erythroid differentiation of murine embryonic stem cells.................................................................64 3-7 Coimmunoprecipitation of USF1, USF2, and TFII-I in K562 and MEL cells..................65 4-1 TFII-I and HDAC3 interaction..........................................................................................80 4-2 TFII-I and HDAC3 are repressors of -globin gene expression in K562 cells.................81 4-3 Suz12 and H3K27me ChIP analysis of K562 and MEL cells...........................................83 4-4 Suz12 knock-down in K562 cells......................................................................................84 4-5 TFII-I and Suz12 interaction in K562 and MEL cells.......................................................85 4-6 PCAF, Set7/9, p300, and CBP localization at the -globin locus.....................................86 4-7 Interaction of USF1 with p300 and CBP...........................................................................87 4-8 USF binding to the -globin gene locus in MEL cells expressing A-USF........................88 4-9 Interactions of RNA Pol II, p300, and modified histones with the -globin gene locus in MEL cells expressing dominant-negative A-USF.........................................................89 4-10 Analysis of MEL cells transiently transfected with pITRp543f2AUSF4..........................91 4-11 Model for -globin gene regulation by helix-loop-helix proteins USF and TFII-I...........93 5-1 Bungert laboratory model of calcium regulation of -globin expression........................101 8


5-2 USF cleavage pattern in K562 and MEL cells.................................................................102 5-3. Induced MEL protein extract treated with recombinant m-calpain.................................103 5-4 Effect on -globin expression upon treatment with calcium ionophore..........................104 5-5 Intracellular localization of TFII-I...................................................................................105 9


Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy THE ROLE OF TFII-I AND USF IN BETA-GLOBIN GENE EXPRESSION By Valerie Jean Crusselle-Davis May 2007 Chair: Jrg Bungert Major: M edical Sciences--Biochemistry and Molecular Biology The human -globin locus contains five functional genes which are arranged in the order of their developmental expression. Gene proximal and distal cis-regulatory DNA elements and interacting trans-factors restrict expression of the genes to the embryonic, fetal, or adult stages of erythropoiesis. Understanding the regulation of -globin gene expression will give insight into how expression of other genes is controlled and may aid in developing therapies which could benefit those afflicted with -thalassemia or sickle-cell disease. To aid in this aim, we examined helix-loop-helix proteins TFII-I and USF which have been found to be bound to the downstream -globin promoter. We hypothesized that these proteins act to aid in the regulation of the developmental stage-specific expression of the adult -globin gene. Expression of dominant-negative proteins and over-expression assays demonstrated that TFII-I acts to repress -globin transcription while USF acts to activate it. In addition, TFII-I is found at the adult -globin promoter early in embryonic stem cell differentiation when the adult -globin gene is repressed, while USF proteins are found at the adult -globin promoter at later stages when the adult -globin gene is activated. One role DNA binding proteins play is to recruit chromatin modifying or remodeling enzymes, making regions of DNA more or less accessible for other factors to bind and for 10


transcription to occur. We have found that TFII-I interacts with HDAC3 and Suz12, a component of the polycomb repressor complex 2 (PRC2). Knocking-down each of these proteins results in an increase in -globin expression, suggesting that TFII-I recruits HDAC3 and PCR2 to the -globin promoter in an embryonic environment to aid in repression. USF proteins were found to preferentially interact with p300 and CBP in an adult environment, suggesting that USF recruits p300 and CBP to aid in the activation of -globin expression. Our data implicates a role for calcium in -globin regulation through TFII-I and USF. Phosphorylated, cytoplasmic TFII-I has been found to inhibit the entry of calcium by preventing the insertion of calcium channels into the plasma membrane, while USF is cleaved by a calcium dependent protease, m-calpain. Our data suggest that TFII-I is localized to the nucleus in embryonic erythroid cells which allows for calcium entry and activation of m-calpain, leading to cleavage of USF. A decrease in USF cleavage in adult erythroid cells, suggests that in these cells full length USF is able to activate -globin expression. 11


CHAPTER 1 INTRODUCTION Hemoglobin and -thalassemia The hemoglobin molecule found in erythrocytes is responsible for carrying oxygen from the lungs to tissues throughout the body and for carrying carbon dioxide from the tissues back to the lungs. The globin, or protein, portion of the molecule is comprised of a dimer of dimers, two pairs of unlike globin chains [1]. The majority of adult hemoglobin is comprised of two and two chains (HbA, 2 2 ); fetal hemoglobin is comprised of two and two chains (HbF, 2 2 ); and embryonic hemoglobin is comprised of two and two chains (Hbs, 2 2 ) [2, 3]. The production of these various forms is a reflection of differing oxygen concentrations at various stages of development. Fetal hemoglobin has a much higher affinity for oxygen than does adult hemoglobin to facilitate the transfer of oxygen across the placenta [4]. Each globin chain harbors a central heme group which gives blood its red color and is responsible for the reversible binding of oxygen. The heme consists of a porphyrin ring containing a central iron atom [5]. Disruption of hemoglobin structure, caused by mutations in the genes encoding and chains, results in serious medical consequences. Overview of Thalassemia and Symptoms Thalassemia is the most common monogenetic disease worldwide. It is estimated that 7% of the population are carriers and 300,000-500,000 infants are born each year with this disorder [6]. Individuals with thalassemia show increased resistance to malaria, therefore thalassemias are prevalent mainly in tropical regions, such as Africa, India, and Mediterranean countries, where there are also high incidences of malaria. Thalassemia is a group of disorders which are caused by a defective and imbalanced globin chain synthesis, resulting from mutations in the globin genes. There are over 200 known mutations of the -globin gene which cause -thalassemia [7]. 12


These mutations either reduce or eliminate -globin chain synthesis, by altering -globin mRNA transcription, interfering with mRNA processing, or translation [8]. This results in an imbalance of and chains, causing a precipitation of the unpaired chain within red blood cells and forming ragged inclusion bodies, which is the primary cause of pathophysiological changes [9]. There are three categories of -thalassemia: -thalassemia major, intermedia, and minor. Individuals afflicted with -thalassemia major suffer from direct affects of anemia, including cachexia, fatigue, and heart failure. They have skeletal abnormalities due to expansion of extramedullary hematopoiesis, splenomegaly, spinal cord compression, and growth retardation [10]. Lysis of red blood cells results in gall stones, leg ulcers, and pulmonary hypertension [11-15]. Those individuals with -thalassemia intermedia have a less severe anemia. With advancing age, they develop complications from marrow expansion, including bone abnormalities, growth retardation, infertility, and hypercoagulability. -thalassaemia minor is asymptomatic with abnormal erythrocyte morphology, but with little or no anemia, and elevated levels of the minor adult component of hemoglobin [8]. Current Treatments of -thalassemia Conventional treatment of -thalassemia is based primarily on blood transfusions. The combination of the production of nonviable red blood cells due to the thalassemia and additional blood being added to the system stimulates iron absorption. However, the capacity of transferrin to carry iron is limited and iron emerges in the plasma creating an iron overload [16-18]. Heart cells are the most vulnerable to excess iron and cardiac complications are the most common cause of mortality in thalassemic patients [19]. Therefore, chelation therapy with desferrioxamine (DFO), a medication that requires daily, prolonged infusion, is necessary. However, in many parts of the world the high cost of the drug makes it unavailable for most patients. It is estimated that DFO is prescribed for 25,000 of 72,000 patients with thalassemia 13


major who received regular transfusions [20]. Those that do receive chelation therapy are often in great pain from the treatment and daily infusions can be burdensome, resulting in non-compliance. Induction of fetal hemoglobin is another therapy investigated to treat -thalassemia. Increasing -globin is expected to decrease the imbalance of globin chains which is the major cause of the pathophysiology of the disease. Those patients with -thalassemia which have elevated levels of fetal hemoglobin have less severe complications [21]. 5-Azacytidine, an inhibitor of DNA methyltransferase, was the first drug used as an inducer of globin expression [22-24]. Due to its potential toxicity with long term use, its use was discontinued and hydroxyurea was investigated to induce fetal hemoglobin. Although it has been shown to be highly effective for patients with sickle cell disease, hydroxyurea has minimal effects for those afflicted with -thalassemia and therefore has not been approved by the FDA for the treatment of -thalassemia [25, 26]. Hematopoietic stem cell transplantation using allogenic bone marrow is a curative treatment for -thalassemia. The overall thalassemia-free survival rate is 80-90% for patients with minimally advanced disease [8]. Transplants are most successful in younger patients with the mildest organ damage, with their survival increasing to 91% [27, 28]. However, in older patients with many complications, graft rejection occurs 30% of the time and disease-free survival rates are much lower [29, 30]. Despite its curative nature, this treatment is highly dependent on finding a suitable donor and acceptance of foreign bone marrow. A permanent cure for -thalassemia would result from expressing a normal -globin gene in red blood cells. Much research is being done to create constructs which would give erythroid specific, differentiation stage specific, and high level expression. Also, it is necessary to develop 14


a transfer system which would provide therapeutic levels of transgene expression and minimal risk of insertional oncogenesis [31]. Retroviral vectors, which integrate permanently into the host genome, are being investigated for gene transfer. Lentiviruses, which have the ability to translocate into the nucleus and integrate in the absence of cell division [32, 33], were the first retroviruses used to treat mice with -thalassemia intermedia [34]. There have been rare instances of insertional oncogenesis in which the viral vector used relied on a powerful, non-tissue specific enhancer/promoter [35, 36]. Therefore, it is crucial to design vectors which are lineageand differentiation stage-restricted. The incorporation of tissue-specific promoters and enhancers in addition to genetic elements with enhancer-blocking properties will aid in reducing the toxicity and increasing the efficiency and stageand tissuespecific expression of the -globin gene [37, 38]. In addition, understanding how the -globin gene is regulated throughout development is essential for designing efficient constructs. Chromatin Structure and Gene Regulation The nucleus contains three meters of DNA, far too long to fit inside of the nucleus. DNA is compacted through several levels of hierarchical organization into chromatin. This compression is not only essential to fit DNA into the nucleus but it is also crucial for gene regulation. The basic structural unit of chromatin is the nucleosome, which consists of approximately 146 base pairs of DNA wrapped around a histone octamer comprised of two molecules of each core histone, H2A, H2B, H3, and H4 [39-41]. This structure is referred to as “beads on a string”. The second level of compaction occurs through coiling of nucleosomes into a helical structure termed the 30 nm fiber. The chain of nucleosomes is then folded into a dense, highly compact arrangement, referred to as higher-order chromatin. Regions of chromatin are either euchromatic, less compact and more permissible for proteins to bind and transcription to occur, or heterochromatic, more compact and generally not permissible for protein binding and 15


transcription. Because chromatin poses a barrier to transcription and gene expression must be tightly regulated, chromatin must be highly dynamic. At least two highly conserved chromatin remodeling activities which regulate accessibility of chromatin have been identified: adenosine triphosphate (ATP)-dependent chromatin remodeling and post-translational modifications of histones. Chromatin Remodeling Chromatin remodeling complexes contain adenosine triphosphatase (ATP-ase) activity which disrupts DNA-histone interactions. The result of remodeling is an alteration in the structure and/or position of nucleosomes through dissociation, sliding, or relocation of individual nucleosomes along the DNA. All eukaryotes contain at least five families of chromatin remodelers: SWI/SNF, ISWI, NURD/Mi-2/CHD, INO80, and SWR1. The SWI/SNF remodelers contain a bromodomain near their C-terminal tail which targets these enzymes to histones with acetylated lysine residues on their N-terminal tails [42]. They primarily act to disorder and reorganize nucleosomes to promote factor binding and transcriptional activation [43]. ISWI complexes contain SANT and SLIDE domains which help ISWI recognize histone tails and linker DNA found in between nucleosomes [44]. These complexes have central roles in the ordering and spacing of nucleosomes following DNA replication and primarily act to repress transcription [45-47]. Both SWI/SNF and ISWI complexes act to slide nucleosomes along the DNA. NURD complexes have been found to act as repressors [48]. INO80 complexes slide nucleosomes and act to evict them at sites of DNA breaks to aid in double-strand break repair [49]. SWR1 has been found to mediate the exchange of core histones with histone variants [50]. These complexes are essential for regulating factor binding and gene expression. 16


Histone Modification Histones contain long, flexible amino-terminal tails which protrude from the nucleosome. There are many residues which can be post-translationally modified along these tails. Such modifications include: methylation, acetylation, phosphorylation, ubiquitination, sumoylation, and ADP-ribosylation. Depending on the modification and the residue modified, an addition to the N-terminal tail can have various effects on the state of surrounding chromatin and recruitment of chromatin-associated factors. The various combinations of histone modifications which can elicit unique cellular responses has been termed the “histone code” [51]. It has been found that mono-, di-, or tri-methyl marks can be placed on histone 3 (H3) lysine (K) residues 4, 9, 27, 36 and 79 and histone 4 (H4) K20 by histone methyltransferases (HMTs). Methylation of H3K9, K27, and H4K20 have been found to be generally associated with heterochromatin and transcriptional repression. However, H3K9me has also been found to be associated with the transcribed region of active genes [52]. Methylation of H3K4, 36, and 79 have been found associated with euchromatin and active genes. Methylation of histone residues exert their effect by recruiting proteins with chromodomains, tudor domains, or WD40-repeat domains, which interact with specific methylated lysine residues [53-55]. These proteins can act themselves or recruit additional enhancer or repressor proteins to stimulate or repress transcription. It was once believed that methylation of histones was a stable mark, not able to be removed. However, in 2004 it was demonstrated that LSD1 can demethylate monoand dimethylated H3K4 in an amine oxidase reaction [56, 57]. Since that time, several Jumonji C (JmjC)-domain-containing proteins which demethylate diand tri-methylation of histones have been discovered [58-61]. Histone acetylation has been the most thoroughly analyzed modification. It has been found that histone 3 is acetylated at lysine residues 9, 14, 18, and 23 while histone 4 lysine residues 5, 17


8, 12, and 16 are acetylated. Histone lysines undergo acetylation-deacetylation switches dependent upon physiological conditions [62]. The balance between these states is dependent upon histone acetyltransferase (HAT) and histone deacetylase (HDAC) enzymes. HAT enzymes catalyze the transfer of an acetyl group from acetyl-CoA molecules to the lysine group, while the HDAC proteins remove it. Two functions of histone acetylation explain how it is able to aid in transcriptional activation. Acetylation of histone tails neutralizes their charge and directly inhibits chromatin condensation by disrupting normal core histone tail domain function [63]. In addition, it is a signal for binding of trans-acting factors through their bromodomains [64]. Chromosome Territories An additional level which regulates gene expression is that of the spatial location of the gene in the nucleus. Chromosomes are organized as chromosome territories (CTs), built up from a hierarchy of chromatin domains, separated from each other by interchromosome domains (ICDs) [65]. Active genes have been found at the periphery of CTs and also at infoldings in the interior portion of the CTs which creates an inter-chromatin compartment which allow for their interaction with factors necessary for transcription and processing of mRNA [66-68]. Silenced genes are located in the interior of the CTs, inhibiting any interaction with machinery necessary for transcription. The -globin Gene Locus The human -globin gene locus spans 80 kb on the short arm of chromosome 11 (11p15.15) and is embedded within one of the multiple olfactory receptor gene arrays [69]. It is comprised of five functional genes (5’--G-A---3’), which are arranged in the order of their developmental expression (Figure 1-1), [70]. The embryonic -globin gene is expressed in the embryonic yolk sac. After six to eight weeks of gestation, the site of hematopoiesis switches to 18


the fetal liver, the -globin gene is repressed, and the -globin genes are expressed. In a second switch, which is completed shortly after birth, the major site of hematopoiesis is found in the bone marrow, with minor production in the spleen. The -globin genes are silenced and the adult -globin gene is activated. The -globin gene is also activated, but is only expressed at levels less than 5% of the -globin gene due to a mutation in its promoter. The mouse -globin locus is located on chromosome 7 and is highly homologous to the human locus. It is also comprised of a series of developmentally regulated genes (5’--h1-maj-min-3’) (Figure 1-1). and h1 are expressed in the embryonic yolk sac, while maj and min are expressed in the fetal liver and in the adult bone marrow. Approximately six to twenty-two kilobases upstream of the embryonic gene in both human and mouse resides a regulatory region crucial to the high level and erythroid specific expression of the -globin genes, termed the locus control region (LCR) [71, 72]. LCRs are able to enhance the expression of linked genes in a tissue specific, copy number dependent, and position independent manner [73]. The human -globin LCR is comprised of 5 domains highly sensitive to DNase I (hypersensitive sites, or HSs) in erythroid cells [74, 75]. The HSs consist of a core region 200-400 bp and are flanked by several kb between them [76-78]. HSs 1 to 4 are only formed in erythroid cells, while HS5 is found in multiple cell lineages [79]. HS2 behaves as a classical enhancer [80], while enhancer activity in HS3 and 4 can only be detected when they are integrated into chromatin [81]. HS3 contains the main chromatin opening activity of the LCR [82]; HS5 functions as a chromatin insulator [83-85]; the function of HS1 is still undefined. Each LCR core element is comprised of many potential binding sites for ubiquitous and erythroid-specific transcription factors. 19


The LCR has been found to directly interact with the active -globin genes, while the inactive genes loop out [86, 87]. This spatial unit of regulatory DNA elements has been termed the active chromatin hub (ACH) [88]. The ACH incorporates the locus flanking 3’ HS1, 5’ HS-60/-62, and also HS1-5 of the LCR. The globin genes switch their association depending on the developmental stage. Several models have been proposed of how these loop structures are formed: random collision, tracking, and linking [89]. The random collision model proposes that by Brownian motion transcription factors act as a protein bridge, mediating the interaction between an enhancer and a promoter [90]. The tracking model suggests that the LCR moves along the chromatin fiber of the -globin locus until it reaches and binds to a promoter. The linking model proposes that the LCR promotes the formation of a chain of facilitator proteins that extend along the chromatin fiber between the LCR and the globin gene. The LCR then tracks along the protein complexes until it reaches a promoter [89, 91]. Histone acetylation has been postulated to play a role in these interactions. The higher the histone acetylation levels are the more flexible the chromatin is, thus making looping more plausible. The LCR has been found to be highly acetylated at all stages of development, while elevated acetylation levels at the globin genes are associated with gene activation [92, 93]. Histones in the -globin region are hypoacetylated in the fetal stage, therefore the chromatin flexibility is low and favors contacts between the LCR and the hyperacetylated -globin promoter. In the adult bone marrow, the region between the and -globin genes is hypoactylated which forces a large loop to form between the LCR and the hyperactylated -globin gene [94]. 20


Hemoglobin Switching One of the more interesting and most studied events of the -globin locus is Hb switching, e.g. the repression of the -globin gene accompanied by the expression of the previously silent -globin gene [95]. The first evidence for developmental changes in hemoglobin was observed in the 19 th century when it was found that newborn hemoglobin is alkali-resistant, while adult hemoglobin is alkali-sensitive [96]. This difference in sensitivity is due to a difference in oxygen affinity of the hemoglobins. As previously mentioned, fetal hemoglobin has a higher affinity for oxygen than does adult in order to more efficiently extract oxygen from the maternal placenta [4]. The creation of different developmental forms of hemoglobin is accomplished by the stage-specific expression of the -globin genes. The switch from to -globin gene expression is controlled exclusively at the transcriptional level and two different modes of regulation are known to control this switch: gene competition and autonomous silencing [96]. Gene Competition Competition of the -globin genes for interaction with the LCR was first seen when the -globin or -globin gene alone was linked to the LCR in transgenic mice [97, 98]. In both instances, the genes failed to display proper developmental regulation. The -globin gene was expressed in both the embryo and the fetus and also in the adult, although at a much lower level. The -globin gene was active at all stages of development and expression was as high in the embryo and fetus as it was in the adult. However, when and -globin genes were linked together with the LCR, developmental regulation was restored. The deletion of the -globin promoter has been shown to result in higher frequency of association of the -globin gene with the active chromatin hub (ACH) and higher level of expression [99]. Additional experimental evidence shows that competition by and -globin genes silences the -globin gene during early 21


stages of development [100]. These observations suggest that during the fetal stage of development there is a preferential interaction of the LCR with the -globin gene and during the adult stage the LCR prefers to interact with the -globin gene. It has also been shown that the relative distance and order with respect to the LCR plays a role in -globin gene expression. Reducing the relative distance between the genes and the LCR reduces the competitive advantage of the proximal gene [101]. In experiments where the five -globin genes are inverted in transgenic mice, the -globin gene is inappropriately expressed in the embryonic yolk sac and fetal liver [102]. Autonomous Silencing When the endogenous -globin LCR is deleted from the locus in embryonic stem cells and somatic cell lines, -like globin transcripts are reduced but the developmental expression pattern remains normal suggesting that other factors play a role in the regulation of -globin gene expression [103]. It has been found that elements contained in or adjacent to the gene are responsible for autonomous silencing. When the -globin gene alone is linked to the LCR, its developmental expression remains normal [104]. Sequences within both the distal and proximal promoter have been found to be responsible for its silencing [105-107]. It has been postulated that the factors bound to the -globin promoter turn off its expression by interfering with the interaction between -globin and the LCR [96]. Sequences responsible for -globin silencing have been located to the -378 to -730 region of the upstream -globin promoter, which contains a GATA site [108]. Role of Transcription Factors Many transcription factors have been identified which regulate the stage-specific expression of the -globin genes. EKLF (erythroid krupple-like factor), an erythroid-specific transcription factor, has been found to serve as an adult switching factor [109]. It binds to the 22


CACCC site with higher affinity than to the slightly different -globin CACCC box and preferentially activates the -globin gene [95, 110, 111]. Over-expression of EKLF has been found to lead to an earlier switch in globin expression [112]. It acts by recruiting a chromatin-remodeling complex, SWI/SNF, to the -globin promoter, which leads to a local change in nucleosome organization [113]. EKLF has also been found to bind to HS3 of the LCR and may act to increase the interaction of the -globin gene promoter and the LCR [114]. In addition, it is required for the formation/stabilization of the ACH [115]. It is unlikely that EKLF is the main or only factor responsible for -globin transcription because it is essential for -globin expression even in the absence of gene competition and EKLF’s expression remains the same throughout all stages of development [116]. EKLF is also involved in -globin expression. It recruits the repressor complex, mSIN3a/HDAC to the -globin region and may be involved in remodeling the embryonic chromatin into a repressed state [117]. FKLF (fetal krupple-like factor) has been found to activate the and -globin genes with it being more active on the -globin promoter [118]. It also activates -globin transcription, but to a lesser extent. FKLF-2, a related fetal transcription factor, has been found to activate -globin expression. It increases expression of the -globin more than forty fold compared to the six fold activation of FKLF [119]. FKLF-2 also activates other erythroid promoters, suggesting that it may play a role in erythroid differentiation. GATA-1 also is an erythroid specific transcription factor and has binding sites in the globin gene promoters and in the HSs of the LCR. It acts as a repressor or activator depending on the context of its binding site and interacting proteins. GATA-1 activates -globin expression, but when bound to the -globin silencer in the presence of YY1 it acts as a repressor [105, 120]. It acts as an activator when bound to the -globin promoter or HS 1-5 [121]. GATA-1 has been 23


found to interact with histone acetyltransferase CREB-binding protein (CBP), suggesting that the recruitment of CBP by GATA-1 will help lead to gene activation [121]. As EKLF, it is necessary for the formation/stabilization of the ACH [122]. SSP (stage selector protein), a heterodimer comprised of ubiquitous CAAT binding protein CP2 and erythroid-specific NF-E4, has been found to bind to the SSE (stage selector element) of the -globin promoter [123-125]. The NF-E4 subunit confers SSE binding specificity and the preferential activation of the over the -globin promoter. Deleting the SSE element in transgenic mice leads to a delay in Hb switching. Introducing the SSE into the -globin promoter leads to fetal stage expression of the -globin gene [126]. NF-E2, a heterodimer between the NF-E2 subunit (p45) and a member of the small Maf family of proteins (p18), has been found to act as a transcriptional activator. Expression of p45 is restricted to erythroid cells and megakaryocytes, while p18 expression is ubiquitous [127-129]. The p45 subunit activates transcription, while p18 confers DNA binding specificity. NF-E2 binding sites, termed MAREs, Maf recognition elements, have been found in HSs 2, 3, and 4 of the LCR and also the -globin promoter [130-133]. Binding within the LCR is essential for its erythroid specific and high level enhancer activity. It has been found to facilitate the transfer of RNA polymerase II from the LCR to the -globin promoter [134]. Also, in addition to GATA-1 it aids in histone acetylation of the LCR and -globin gene by recruiting CBP [135]. COUP-TFII, a retinoic acid orphan receptor that corresponds to the erythroid specific binding activity of NF-E3 [136, 137], binds to the and -globin CAAT elements and acts as a repressor [106, 138]. Its expression peaks at the to -globin switch [106]. Polypyrimidine (PYR), an erythroid specific and stage-specific chromatin remodeling complex [139], is an adult specific factor which binds to a pyrimidine-rich DNA stretch between 24


the and -globin genes [140, 141]. Deletion of the PYR element leads to delayed switching. Individuals which have the 7.2 kb of the intergenic gamma-delta region upstream of the delta gene deleted, have between 88% and 90% fetal hemoglobin, only mild anemia, and no transfusion requirements, suggesting that their -globin gene is active and able to compensate for the lack of -globin expression [142]. Adult -Globin Gene Promoter and Regulatory Region The core -globin promoter is comprised of a TATA-like element (CATAAA), a CCAAT box at approximately -75, and a CACC box at approximately -90 [143-145]. The CACC box has been shown to be bound by Sp1, whose recruitment is stabilized and promoted by the LCR [146]. In addition, a direct repeat motif (DRE) is located between the CCAAT and TATA elements (at positions -53 to -32). The murine DRE consists of an imperfect direct repeat of a 10-bp motif, GGCAGGAGCCAGGGCAGAGC. It is the only core element found exclusively at the adult globin promoter. DRF, -globin direct repeat factor, was found to bind to this region and stimulates transcription of -globin [147]. The combination of these elements is essential for formation of the pre-initiation complex and for transcription to occur. A pyrimidine rich, functional initiator (Inr) element is found at +1 of the -globin promoter [148]. Several models have been proposed of how proteins initiate transcription through Inr elements. One suggests that factors bound to the Inr element are contained in the TFIID complex [149]. Another proposes that initiator-bound proteins such as YY1, TFII-I, and USF stimulate transcription [150-152]. Both TFII-I and USF proteins have been found to be bound to the -globin Inr [153]. Additionally, recognition of the Inr by RNA polymerase II may serve as a nucleation event [154]. 25


A 3’ enhancer of the -globin gene confers high level expression. It is located 500 to 850 bp downstream of the polyadenylation site of the -globin gene. Containing four binding sites, each is bound by GATA-1 in addition to one other protein [155]. The 3' enhancer is erythroid-specific but not developmental stageor gene-specific [156, 157]. Several silencer regions have been identified in the proximal -globin promoter. Two silencer regions upstream of the -globin gene have been denoted as binding sites for a common protein, termed the beta protein 1 (BP1). The first BP1 binding site identified coincides with silencer I located between nucleotides -546 to -521 relative to the initiation site, the other is between -302 and -294 bp. This AT rich sequence consists of two (AC) dinucleotides, followed by seven (AT) dinucleotides and seven T-nucleotides [(AC)2(AT)7(T)7]. A second protein, BP2, has been shown to bind to -275 to -263. Binding of BP1 and BP2 to these sequences have been shown to have a negative effect on -globin expression [158, 159]. Several elements downstream of the transcription initiation start site have been identified. TAF1/TAF(II)250, of the TFIID complex, makes sequence specific contacts to a downstream core element (DCE) and its binding is necessary for DCE function [160]. The DCE contains three subelements approximately residing at +6 to +11 (CTTC), +16 to +21 (GTGT) , and +30 to +34 (AGC) [161]. Additionally, a MARE/AP1-like element at +24 is bound by NF-E2 [130]. The downstream promoter element also contains two conserved E-boxes, whose motif is CANNTG (Figure 1-2). One overlaps with the initiator while the other is 60 bp downstream of the transcription initiation start site [153]. The upstream E-box is bound by helix-loop-helix proteins TFII-I and USF, while the downstream E-box is bound by USF1 and USF2. The downstream E-box is essential for high level transcription of the -globin gene. The function of TFII-I and USF proteins at the -globin promoter is unknown, therefore it is the goal of this 26


work to determine if they play a role in the developmental stage-specific expression of the adult -globin gene. TFII-I TFII-I is a ubiquitously expressed, multifunctional transcription factor that is activated by a variety of extracellular signals. Individuals with Williams-Beuren syndrome (WBS) have a hemizygous deletion of TFII-I [162]. The neurodevelopmental disorder involves supravalvar aortic stenosis, hypercalcemia in infancy, mild to moderate mental retardation, cognitive defects, and characteristic facial features [163, 164]. The frequency of this disease is estimated to be 1 in 20,000 live births [162]. The genetic material from the region q11.2 of chromosome 7 is deleted in WBS patients, including more than 20 genes. TFII-I was originally discovered to act as a basal transcription factor that binds and functions through the initiator (Inr) element in promoters [151]. Subsequently it was found to bind to E-boxes, which are known to be recognized by families of helix-loop-helix proteins. These findings raised the possibility that TFII-I acts as both a basal transcription factor and as an activator, thus bridging the basal machinery at the promoter and activator complexes at upstream regulatory sites [151]. Structure TFII-I is divided into two domains, a 70 kDa N-terminal domain which is responsible for DNA binding and a 43 kDa C-terminal domain which is necessary but not sufficient for activation function [165]. TFII-I is comprised of six 90 amino acid direct reiterated I-repeats, R1-R6, each containing a putative HLH motif and each representing a potential protein-protein interaction surface. It, however, only contains one basic region (BR) just before R2 (amino acids 301-306) [166]. The BR in traditional HLH proteins has been shown to be a sequence-specific DNA binding domain [167]. In fact, the BR in TFII-I has been found to be necessary for its DNA 27


binding properties. However, deletion of the N-terminal 90 amino acids, including a putative leucine zipper, leads to a loss of binding despite the BR being intact [168]. Therefore, there may be other DNA recognition surfaces present in TFII-I. Isoforms TFII-I has multiple isoforms, (977 aa), (978 aa), (998 aa), and (957 aa), generated by alternative splicing [162, 169, 170]. The difference between these isoforms lies in additional exons after the first I-repeat. The -isoform does not contain any additional exons, while -isoform contains exon A (encoding 20 amino acids), contains exon B (encoding 21 amino acids), and contains exons A and B. Tissue and species distribution of these isoforms suggest that they do not have overlapping functions. The -isoform is found predominantly, if not exclusively, in neuronal cells [162]. The -isoform is expressed higher in murine cells than in human cells [169] and the -isoform seems to be absent in murine cells [171]. Functions of the isoforms may be regulated by their mutual interactions. TFII-I isoforms primarily interact with each other or themselves through the leucine zipper domain and additional interactions are mediated by the I-repeats [168]. Homomeric and heteromeric interactions lead to their preferential nuclear localization. A nuclear localization deficient mutant of the -isoform, which remains exclusively in the cytoplasm, is found to be in the nucleus when co-expressed with any of the other TFII-I isoforms [169]. Also, various homoor heterodimers of TFII-I may regulate basalvs. signal-induced transcription functions and serve different transcription functions on different promoters. Recently it has been found that and -isoforms in murine fibroblasts have distinct subcellular localizations and mutually exclusive transcription functions on the c-fos gene in the context of growth factor signaling. In the resting state, the -isoform is found in the nucleus and recruited to 28


the c-fos promoter and maintains basal level transcription. The -isoform in this condition is largely cytoplasmic. Upon growth factor signaling, the -isoform translocates to the nucleus and is recruited to the c-fos promoter where it activates the gene. The -isoform, on the other hand, is exported from the nucleus [172]. The function of TFII-I isoforms is sure to be cell-type and gene specific. Role in Cell Signaling TFII-I has been shown to be basally phosphorylated at serine and tyrosine residues. Phosphorylation of TFII-I is dispensable for its specific DNA-binding activity however, at least phosphorylation of Y248, which is enhanced through extracellular signaling, is required for its transcriptional activity [173]. TFII-I is tyrosine-phosphorylated by Bruton’s tyrosine kinase (Btk), which is a member of the Tec family of Src-like tyrosine kinases characterized by the presence of a pleckstrin homology (PH) domain. The non-receptor Btk is preferentially expressed in hematopoietic cells of B and myeloid lineages [174]. TFII-I is tethered to Btk in the cytoplasm primarily through Btk’s PH domain and the first ninety amino acids of TFII-I, which includes the putative leucine zipper domain [175]. This interaction prevents dimerization of TFII-I which is necessary for its translocation into the nucleus. Upon B cell antigen receptor engagement, Btk phosphorylates TFII-I and the subsequent release allows for nuclear import of TFII-I [176]. TFII-I also undergoes cell signaling mediated phosphorylation in non-lymphoid cells. TFII-I is tyrosine phosphorylated in response to pervanadate treatment and PDGF and epidermal growth factor (EGF) stimulation in fibroblasts and epithelial cells. This leads to TFII-I mediated transcriptional activation of c-fos through interaction with the serum response element (SRE) and c-sis/PDGF-inducible factor element (SIE) of the c-fos promoter [173, 177]. Several tyrosine 29


kinases have been identified to act on TFII-I in non-lymphoid cells, such as JAK2 and c-Src [178, 179]. The TFII-I D-box motif, a consensus MAP kinase binding domain found at amino acids 282 to 293, is bound by the ERK1 MAP kinase in a signal-transduction dependent manner. Only through the phosphorylation of TFII-I by JAK2 is ERK able to associate with TFII-I and thus to activate c-fos [179]. A mechanism similar to that of Btk has been identified in fibroblasts in which TFII-I is sequestered in the cytoplasm. TFII-I is bound by 190 RhoGAP, via its FF domains, in the cytoplasm. Upon PDGF receptor-mediated phosphorylation of the FF domain, TFII-I is released from p190 and translocates to the nucleus where it activates transcription of serum-inducible genes including c-fos [180]. Phosphorylation of TFII-I by multiple tyrosine phosphorylases at different sites implies that TFII-I is regulated by multiple independent pathways. A further level of control is through the regulation of the nuclear translocation of TFII-I by distinct signaling mechanisms that involve cytoplasmic sequestering and phosphorylation. The role of serine phosphorylation is less clear and it is not clear whether it is dependent upon growth-factor or serum signaling. It has been found that serine 633 is phosphorylated by MAPK and mutation of this residue decreases TFII-I dependent activation of the c-fos promoter [177]. TFII-I Regulated Genes TFII-I, in addition to the activation of c-fos, regulates a number of growth-signal dependent genes. Under normal growth conditions, TFII-I is recruited to the cyclin D1 promoter and transcriptionally activates the gene [181]. TFII-I binds to both the Inr and to three regulatory E boxes in the human vascular endothelial growth factor receptor-2 (VEGFR-2) promoter and acts to enhance its transcription [182]. The transcription of goosecoid (Gsc), a homeodomain-containing transcription factor known to regulate formation and patterning of embryos, is 30


enhanced upon growth factor beta (TGFbeta)/activin stimulation. TFII-I is recruited to the distal element (DE) of the Gsc promoter, and activates Gsc transcription [183]. TFII-I has been shown to act as a repressor of a subset of estrogen-responsive genes, only those containing Inr elements, by recruiting estrogen receptor (ER) alpha and corepressors to these promoters. Such genes include pS2, cyclin D1, GREB1 and amphiregulin [184]. TFII-I has also been implicated as a regulatory component of the endoplasmic reticulum (ER) stress pathway. It acts by binding to the ER stress element, which is highly conserved in promoters of ER stress-inducible genes such as Grp78/BiP and ERp72 [185, 186]. An inducer of the GRP stress response, through depletion of the ER Ca(2+) store, elevates the level of TFII-I transcript and protein level in the nuclei of the stressed cells [186]. TFII-I has been found to activate a number of viral genes. TFII-I is necessary for the induction of latent HIV-1 in response to T-cell activation signals. It interacts with USF1 and USF2 to form the complex RBF-2 (Ras response element binding factor 2), which binds to the upstream RBEIII of the HIV-1 promoter [162]. TFII-I is bound to and essential for transcriptional activity of the the Rous sarcoma virus (RSV) long terminal repeat (LTR), which contains an enhancer and core promoter composed of a TATA box and an Inr-like sequence [187]. USF The members of the upstream stimulatory factor (USF) family, USF1 and USF2, are part of the basic-helix-loop-helix-leucine zipper transcription factor family and also the Myc family of regulatory proteins. The USF1 gene is found on chromosome 1 and USF2 on chromosome 19 in humans [188, 189]. The ubiquitously expressed proteins bind with high affinity to E-box regulatory elements, which are largely represented across the genome. USF proteins mediate the 31


recruitment of chromatin remodeling enzymes and interact with co-activators and members of the transcription pre-initiation complex to activate or repress transcription. Structure USF1 and USF2 have a high degree of homology at their C-terminal DNA binding domain (>70% identical residues) and consequently display identical dimerization and DNA binding specificities [190]. The C-terminal domain includes a DNA binding basic region followed by helix-loop-helix (HLH) and leucine zipper (LZ) motifs, which are involved in dimerization. USF1 and USF2 share a small but extremely conserved domain called the USF-specific region (USR), located just upstream of the basic region. This domain is implicated in nuclear localization and specific activities of promoters containing both a TATA box and an Inr element [191]. USF1 and USF2 differ in their N-terminal domains, which includes at least one additional transcription activation domain. It is therefore possible for USF1 and USF2 to control different sets of genes by interacting with other proteins [191, 192]. Accordingly, USF1 and USF2 knockout mice display distinct phenotypes. USF1 -/mice are viable and fertile, with only slight behavioral abnormalities. These mice contain elevated levels of USF2, which may compensate for the absence of USF1. In contrast, USF2-null mice contain reduced levels of USF1 and display an obvious growth defect. The double knockouts are embryonic lethal [193]. The predominant form of USF is the USF1USF2 heterodimer. USF1 homodimers are less abundant and USF2 homodimers are rarely found [190]. USF dimers bind DNA as a four helix bundle, with the basic domain from each monomer contacting half of the DNA binding site [194-196]. Regulation of Expression and Activity Expression of USF1 and USF2 is controlled by alternative splicing. The 38 kDa USF2b variant lacks exon 4, which contains an additional positive-regulatory domain, and acts as a dominant-negative regulator of USF mediated activation [197]. USF2aH, although containing 32


an intact leucine zipper domain, lacks the second helix of the HLH motif due to a 24-bp in-frame deletion occurring by the use of a cryptic splice site within the ninth exon [188]. However, this isoform is very rare in humans and its biological relevance is still to be determined. Excision of part of exon 4 of USF1 results in the deletion of the N-terminal LZ domain, creating isoform USF1/BD. USF1/BD acts as a modulator of USF1 to control the expression of target genes [198]. The DNA-binding activity of USF proteins is modulated by multiple signal transduction pathways. Phosphorylation of the p38 family of kinases through the stress-activated signaling cascade leads to phosphorylation of USF1 by p38 [199]. USF1 was also found to be phosphorylated by protein kinase C (PKC) and cAMP-dependent protein kinase (PKA) [200]. Phosphorylation of USF1 by the cdk1 pathway suggests that USF plays a role in the progression of the cell cycle [201]. The phosphatidylinositol 3-kinase (PI3K) pathway also is shown to phosphorylate USF1 [202]. Deletion studies of USF-1 suggest that amino acids 143-197, which comprise the USR, regulate DNA-binding activity in a phosphorylation-dependent manner [201]. Phosphorylation increases USF1 binding affinity, except for the PI3Kinase-regulated form of USF which does not bind to DNA. USF Regulated Genes As mentioned previously, USF proteins are involved in the stress response pathway. USF1 is involved in the tanning response to UV light by activating UV responsive genes such as, POMC, MC1R, Tyrosinase, TYRP-1, and Dct [199, 203]. USF1 is involved in both the humoral, B-cell and cell mediated, T-cell immune response. The enhancers and promoters of the Ig chain genes contain critical E-box elements [204]. Thus, USF1 has been shown to bind to and promote the transcription of the 2-Ig light chain gene [205]. In addition, USF is crucial for efficient J chain transcription and polymeric Ig receptors (pIgRs) in activated B-cells [206, 207]. USF 33


activates the C4 complement gene, which plays a crucial role in the complement pathway and induced in response to acute inflammation or tissue injury [208]. It also regulates 2-microglobulin activation, which plays a role in antigen presentation and Ig transport [197], and CTIIA activation, which is required by MHC class II gene induction [209]. USF proteins play a role in G1/S and G2/M cell cycle progressions by regulating the expression of cyclin and Cdk genes. The CDK4 gene, which is involved in the G1/S transition, is activated by USF2 [210]. USF1 has been found to be an active regulator of cyclin B1 and cdc2 gene expression which are involved in the G2/M transition [211, 212]. USF proteins have been found to have anti-proliferative properties and thus involved in the regulation of tumor suppressor genes. USF1 and USF2 proteins are critical for the expression of the adenomatous polyposis coli (APC) gene, whose loss of function is associated with the development of colorectal carcinogenesis [213]. USF1 is essential for transcriptional activation of the estrogen receptor alpha (ERalpha) gene, whose expression correlates with breast cancer pathophysiology [214]. USF proteins also activate BRCA2 which is a hereditary breast cancer susceptibility gene that may be involved in the cellular response to DNA damage [215]. The p53 tumor suppressor gene which plays an important role in the regulation of cellular proliferation and differentiation is also activated by USF proteins [216]. USF activity, therefore, plays a large role in inhibiting tumor formation and progression. USF seems to play an opposite role in lung cancer. USF2 has been found to regulate transcription of arginine vasopressin, which can act as a growth factor for lung cancer tumors. In non-small cell lung cancer, endogenous USF-2 expression is low, and this basal level appears to be insufficient to activate transcription of arginine vasopressin, thus possibly inhibiting growth 34


of the tumor. Whereas in small cell lung cancer, USF2 expression is high, vasopressin is activated, thus aiding in the growth of the tumor [217]. USF proteins also function in glucose and lipid metabolism. USF participates in the regulation of genes involved in hepatic glucose sensing such as: the insulin gene [218], the insulin growth factor-binding protein 1 gene [219], the glucagon receptor gene [220], the islet-specific glucose-6-phosphatase catalytic subunit related gene [207], and the glucokinase gene [221]. Glucose response elements, containing E-boxes, have been characterized for the L-pyruvate kinase gene, which is required for glycolysis, and the S14 gene, whose expression is induced by lipogenic stimuli. USF is involved in the activation of these genes through the glucose response pathway [222]. USF also regulates fatty acid synthase, apolipoprotein, hepatic lipase, acetyl-CoA, and carboxylase genes, which are all involved in lipogenesis [202, 223-225]. Familial combined hyperlipidaemia (FCHL), which is characterized by elevated levels of total cholesterol and/or triglycerides, was recently linked and associated to the USF1 gene [226]. USF1 has also been associated with type 2 diabetes and its variation was found to be the influencing feature of both glucose and lipid homeostasis [227, 228]. Summation The -globin locus is one of the most studied eukaryotic loci due to its role in -thalassemia, one of the most common monogenetic diseases world-wide. In order to develop effective treatments and/or cures for -thalassemia, such as gene therapy, it is crucial to understand how the -globin genes are regulated. Determining the mechanism of hemoglobin switching, the silencing of the fetal -globin gene accompanied by the activation of the adult -globin gene, would greatly aid in this understanding. In addition, the -globin locus serves as a model of gene regulation which can elucidate the control of expression of other genes. It is known that the LCR and also trans-acting factors aid in this switch of gene expression. It is our 35


goal to contribute further knowledge of trans-acting factors which regulates the adult -globin gene. It has been previously been shown by our laboratory that TFII-I and USF proteins are bound to the downstream promoter of the -globin gene [153] . TFII-I and USF proteins have been found to have various roles in gene regulation dependent upon interacting proteins and on the sequence context of the promoter region. Therefore, it was the goal of this work to understand the role of these proteins on -globin expression both in vitro and in vivo. It has been seen that transcription factors often exert their effect by recruiting coactivators which modify histones or mobilize nucleosomes at regulatory sites. Therefore, determining the mechanism by which TFII-I and USF regulate the -globin gene was the next goal of this work. Finally, recent reports implicate a role of TFII-I in calcium entry [229] . Therefore, the final goal of this work was to determine if calcium entry, regulated by TFII-I, plays a role in -globin expression. 36


A Human: G A LCR HS 5 4 3 2 ~15 kb ~60 Globin gene expression embryonic adult fetal liver during human development yolk sac bone marrow B Mouse: h maj min LCR HS 6 5 4 3 2 1 Globin gene expression during mouse development embryonic fetal liver & yolk sac adult bone marrow Figure 1-1. Schematic of the organizational structure of the human and murine -globin gene loci. A) The human -globin locus consists of five genes which are expressed in a developmental stage-specific manner in erythroid cells as outlined. The expression of the genes is regulated by a locus control region composed of five DNase I HS and located about 15 to 27 kbp upstream of the embryonic -globin gene. B) The murine -globin gene locus consists of four genes which are expressed either in erythroid cells of the embryonic yolk sac (EY or H1) or in definitive erythroid cells derived from fetal liver or bone marrow hematopoiesis (maj and min). The murine LCR also contains multiple HS required for high-level globin gene expression. 37


Figure 1-2. Sequence alignment of the human -globin downstream promoter region. Shown are three sequences of the adult -globin downstream promoter region from human (H), mouse (M) and rabbit (R). Shaded boxes highlight the position of E-box motifs (CANNTG). Two of these E-boxes, the one overlapping the initiator and the distal E-box, are conserved in all three species, whereas the E-box located at +20 is only present in the human and rabbit genes. The open box delineates the position of the MARE-like sequence. 38


CHAPTER 2 MATERIALS AND METHODS Construction of Protein Expression Vectors The plasmid pCMV/A-USF was obtained from Charles Vinson (NIH) and has been described previously [230]. The A-USF open reading frame, including an N-terminal hemagglutinin (HA) tag, was amplified by PCR. The reaction was carried out using Elongase (Invitrogen), 0.4 mM forward primer (5'-GCGC CTTAA GCTCCACC ACCATGGCGTATCC-3') containing an AflII restriction site (underlined), 0.4 mM reverse primer (5'-GCGC TCTAGA AGAAGCTTTTAGTTGCTGTCATTC-3') containing a XbaI restriction site (underlined), 1.5 mM Mg 2+ , and 5 ng pCMV/A-USF as a template in a final volume of 50 l. PCR conditions were as follows: 94C for 30 s, 35 cycles of 94C for 30 s, 61C for 30 s, and 68C for 30 s, with a final extension at 68C for 2 min. The amplified product and pcDNA4/TO (Invitrogen) were digested with AflII and XbaI and subsequently ligated to create pTO/A-USF. pET11d/USF1 was digested with BamHI and XbaI to isolate the USF1 open reading frame containing an N-terminal Flag tag. The fragment was subcloned into pcDNA4/TO and then properly oriented by digestion with PmeI and ligation back into pcDNA4/TO to create pTO/USF1. pET11d/p70 and pET11d/TFII-I were digested with NcoI and SpeI to isolate the open reading frames. The fragments were subcloned into the pLitmus 29 vector (New England Biolabs). A subsequent digest with BamHI and SpeI isolated the open reading frames, which were subcloned into the pcDNA 3.1(+) vector (Invitrogen). The open reading frames were properly oriented by digestion with PmeI and ligation back into pcDNA 3.1(+). The open reading frames were isolated from pcDNA 3.1(+) by digestion with AflII and XbaI and subcloned into pcDNA4/TO, thus creating pTO/p70 and pTO/TFII-I. pTO/HA-p70 and pTO/HA-TFII-I 39


expressing an HA-tagged p70 mutant and HA-tagged TFII-I and containing a neomycin resistance gene, was created by amplifying the p70 and TFII-I open reading frame by PCR using pTO/p70 and pTO/TFII-I as a template and the forward primer containing a BamHI site (5'-GCGC GGATCC CATGGCCCAAGTTGCAATGTCC-3') and the reverse primer containing an XbaI site (5'-GCGC TCTAGA GTTCAGGTTTTTTAACAACGAAC-3'). The PCR product and pTO/A-USF were digested with BamHI and XbaI and subsequently ligated, creating pcDNA4/TO/HA-p70 and pcDNA4/pTO/HA-TFII-I. These constructs were digested with XbaI and BstZ171 to remove the Zeocin resistance gene. pCMV-566 (obtained from Charles Vinson) was digested with PciI, filled in using a Klenow reaction, and then digested with XbaI to isolate a fragment containing the neomycin resistance gene. These two fragments were then ligated to create pTO/HA-p70 and pTO/HA-TFII-I. pTO containing the neo resistance gene, the empty control vector for pTO/HA-p70 and pTO/HA-TFII-I, was created by digesting pcDNA4/TO with XbaI and BstZ171. pCMV-566 was digested with PciI, filled in using a Klenow reaction, and digested with XbaI. The fragments were then ligated. The pITRp543f2AUSF4 construct was created by digesting pITRp543f2beta4 with NcoI and PmeI, which removed the -globin gene, the 3’ enhancer, and the 3’ chicken HS4. The AUSF coding region was isolated from the pCMV/AUSF plasmid by digesting with NcoI and PmeI. The two fragments were then ligated creating pITRp543f2AUSF. The 3’enhancer and 3’chicken HS4 elements were amplified using pITRp543f2beta4 as template and with primers containing PmeI sites and cloned into the pTOPO vector (Invitrogen). The pTOPO/3’enhancer, 3’cHS4 and pITRp543f2AUSF were subsequently digested with PmeI and then ligated to form pITRp543f2AUSF4. 40


The inserts of all generated plasmids were sequenced to verify that no mutations were introduced and to ensure that coding sequences were in the correct reading frames. Cell Culture and Transfections The K562 cells used in this study were obtained from Ann Dean (NIH) and grown in RPMI 1640 medium supplemented with 15% fetal bovine serum and 5% penicillin-streptomycin. Murine erythroleukemia (MEL) cells were grown in RPMI containing 10% fetal bovine serum and 5% antibiotic-antimycotic. Cells were grown in 5% CO 2 at 37C and maintained at a density between 1 x 10 5 cells/ml and 5 x 10 5 cells/ml. In DMSO induction studies, MEL cells were incubated with 2% DMSO for 48 hours. Stable MEL cell lines were created following the protocol for FuGENE 6 transfection reagent (Roche Molecular Biochemicals). Stable pTO/A-USF-pcDNA6/TR (pTR) (Invitrogen) and pTO-pTR MEL cell lines were created by cotransfecting pTO/A-USF or pTO (control vector) with pTR. Stable pTO/USF1-pTR and pTO-pTR (control vector) MEL cell lines were created by transfecting MEL pTR cells with either pTO/USF1 or pTO. Stable pTO/p70-pTR, pTO/TFII-I-pTR, and pTO-pTR (control vector) MEL cell lines were created by transfecting stable MEL pTR cells (previously transfected with 3 g of pTR) with 1 g of pTO/p70, pTO/TFII-I, or pTO. Cells were allowed to recover for 48 h before selection with 5 g/ml blasticidin and 300 g/ml Zeocin. For K562 transfections, 10 6 cells were resuspended in 100 l of cell line Nucleofector solution V (Amaxa GmbH) and nucleofected with 1 g of pTO/HA-p70 or pTO/HA-TFII-I or pTO and 3 g of pcDNA6/TR, using the provided nucleofection program for K562 cells (T-03). Cells were allowed to recover for 3 days before selection with 4 g/ml blasticidin and 300 g/ml G418. 41


To induce protein expression, stable MEL and K562 cell lines were treated with 1 g/ml doxycycline for 36 hours at a cell concentration of approximately 2x10 5 cells/ml. Murine embryonic stem cells were cultured and differentiated into erythroid cells as previously described by Levings et al [231]. Briefly, ESD3 cells (ATCC, CRL-1934) were seeded onto 0.2% gelatin at a density of 10 5 cells/25 cm 2 in ES media (DMEM, 4.5g/l glucose, 1.5g/l sodium bicarbonate, 15% FBS, 0.1 mM 2-mercaptoethanol and 10 6 U/ml LIF), grown for two days, and then passaged (1:6) and grown for another day. An aliquot of the cells (3-4x107) was taken at this time (Day 0) and subjected to ChIP analysis. The remaining day 0 cells were then seeded onto confluent OP9 stromal cells in OP9 media (-MEM with ribonucleosides and deoxyribonucleosides; 20% FBS) in the absence of LIF at a density of 2x10 4 cells/well in 6 well tissue culture dishes. At day 3 Epo was added (2U/ml and 50ng/ml, respectively) for the remainder of the course of induction. On day five of induction, cells were collected for ChIP and the remaining cells were passaged and reseeded onto fresh OP9 cultures at a density of 10 5 cells/well. On day 8 cells were collected and subjected to ChIP analysis. Chromatin Immunoprecipitation, Double-Chromatin Immunoprecipitation, and Co-immunoprecipitation Chromatin immunoprecipitation (ChIP) was performed as previously described by Leach et al. [153]. Double ChIP for K562 was carried out following the same ChIP procedure with the addition of dialysis of the elutant from the first immunoprecipitation against 200 ml of dilution buffer with complete mini-protease inhibitors (Roche) using Slide-A-Lyse mini-dialysis units (Pierce) at 4C with constant stirring for 2 h. Samples were removed from dialysis units, and 500 l dilution buffer was added to each. The second immunoprecipitation was performed, again following the previously referenced ChIP protocol. 42


The immunoprecipitation of TFII-I from K562 cells, examining looking for HDAC3 and USF2 interactions, was carried out following the ChIP protocol as previously described by Leach et al. [153], with the addition of protease inhibitors in all buffers for co-immunoprecipitation assay. All other immunoprecipitations were performed using extract from 1 x 10 8 K562 or MEL cells lysed with 1 ml of NP-40 buffer (150 mM NaCl, 1% NP-40, 50 mM Tris, pH 8.0). Extract was precleared with 100 l of anti-rabbit immunoglobulin G (IgG) beads (eBioscience) for 30 min. Precipitation was carried out by incubating extracts with antibody for 2.5 h. Complexes were captured by incubating extracts with 100 l of anti-rabbit IgG beads for 2 h. All incubations were performed on a spinning wheel at 4C. Samples were washed three times with NP-40 buffer and eluted with Laemmli buffer (Bio-Rad) at 95C for 10 min. The eluted samples were loaded onto a 5%, 7.5%, or 10% Ready gel (Bio-Rad) and analyzed by Western blotting. Antibodies used in the experiments are as follows: acetyl-histone H3 06-599, RNA polymerase II clone CTD4H8 05-623, monomethyl-Histone H3 Lys27 07-448, dimethyl-Histone H3 Lys4 07-030, dimethyl-Histone H3 Lys9 07-521, anti-HA tag 05-904, and Suz12 07-379 (purchased from Upstate Biotechnology, Inc.); USF1 (H-86) sc-8983, USF1 (C-20) sc-229, USF2 (N-18) sc-861, USF2 (C-20) sc-862, p300 (N-15) sc-584, CBP (A-22) sc-369, GAPDH (FL-335) sc-25778, HDAC3 (H-99) sc-11417 and PCAF (H-369) sc-8999 (purchased from Santa Cruz Biotechnology); IgG C6409 (anti-chicken IgG; Sigma); anti-TFII-I (a gift from R. G. Roeder, Rockefeller University); anti-TFII-I (a gift from A. Roy, Tufts University); anti-HDAC3 (a gift from E. Seto, University of South Florida); SET 7/9 IMG-587 (purchased from Imgenex); Histone H3 tri methyl K27 ab6002, TFII-I ab10464, Suz12 ab12073 (purchased from abcam); GATA-1 (16012644) (purchased from Geneka Biotechnology); and anti-calpain II AB81013 (purchased from Chemicon International). 43


RNA Interference siGENOME SMARTpool reagents for human TFII-I (M-013638-0), HDAC3 (M-003496-00), USF2 (M-003618-00), Suz12 (D-001810-10-05), CAPN2 (L-005804-00-0005), siCONTROL nontargeting pool (D-001206-13-05), and siGLO RISC-free (D-00160-01-05) were obtained from Dharmacon. A total of 10 6 K562 cells were nucleofected with 0.5 g of short interfering RNA (siRNA) or mock transfected following the protocol for K562 cells (Amaxa GmbH). RNA and protein from cells were collected after 48, 72, and 96 h. Transfection efficiency was determined by first fixing and then DAPI (4,6-diamidino-2-phenylindole) staining K562 siGLO cells. Pictures were taken using an Axioplan 2 microscope (Zeiss) under 40 power. Protein Isolation and Western Blotting Whole cell extracts were obtained by incubating cells in RIPA buffer (50 mM Tris-HCl pH 7.4, 50 mM NaCl, 10 mM EDTA, 0.25% Na-Deoxycholate, 1% NP-40, 0.1% SDS, and 1 complete protease inhibitor tablet (Amersham)) at 4C for 20 minutes, spinning. The lysate was centrifuged and the pellet discarded. Nuclear and cytoplasmic extracts were prepared as in Bungert et al. [232]. A total of 20 g of protein, unless otherwise noted, was run on Ready gels (Bio-Rad). The detection of proteins on membranes was performed with an ECL Plus system according to the manufacturer's instructions (Amersham Pharmacia Biotech). The primary antibodies used were the same as those used for ChIP, in addition to anti-HA tag (a gift from Michael Kilberg, University of Florida) and anti-TFII-I (a gift from Ananda Roy, Tufts University). The secondary antibodies used were goat anti-rabbit IgG-horseradish peroxidase (HRP) (Kirkegaard & Perry Laboratories and Santa Cruz Biotechnology; sc-2004), goat anti-rabbit IgG-HRP sc-2004, goat anti-mouse IgG-HRP sc-2005 (Santa Cruz Biotechnology), and 44


anti-rabbit IgG TrueBlot (eBiosciences) for coimmunoprecipitation. The concentration of antibodies used followed the manufacturers' guidelines. RNA Isolation, Real-Time PCR, and PCR RNA was isolated using the guanidine thiocyanate method [233] and reverse transcribed as described previously [234]. RNA for TFII-I and HDAC3 RNAi experiments was isolated using the Aurum total RNA kit (Bio-Rad) and reverse transcribed using the iScript cDNA synthesis kit (Bio-Rad). Quantitative PCR (qPCR) was performed using the Opticon 2 (MJ Research) or the MyiQ (Bio-Rad). The reactions were carried out using either DyNAmo HS SYBR green master mix (MJ Research) or iQ SYBR green super mix (Bio-Rad). Real-time PCR conditions were as follows: 95C for 15 min, 40 cycles of 94C for 10 s, 59C for 20 s, and 72C for 30 s (the plate was read after each extension step), final extension at 72C for 5 min, a melting curve from 55C to 95C (reading every 0.5C), and reannealing at 72C for 5 min. The 20-l reaction mixtures contained 20 ng of cDNA from dominant-negative and overexpression assays, a 1:40 dilution of cDNA from RNAi assays, or 2 l of DNA from ChIP assays, 0.3 M of forward and reverse primer, and 10 l of the 2 SYBR green master mix. All primers for expression analysis span introns to ensure exclusive amplification of cDNA. Amplicons ranged in size from 110 to 120 bp. All reactions were carried out in duplicate with a no-template control. Standard curves were generated using 10-fold serial dilutions of cDNA or input DNA for ChIP assay from the appropriate cell line. Final quantification analysis was performed using the relative standard curve method, and for reverse transcription (RT)-qPCR, results were reported as the relative expression of the control after normalization of the transcript to the endogenous control, which was either -actin (data not shown) or GAPDH. PCR for semiquantitative ChIP samples was performed using the following conditions: 2 l DNA, 1 M each forward and reverse primer, and 8 l of 2.5 master mix (Eppendorf) in a 45


20-l reaction mixture. PCR cycle parameters were as follows: 95C for 5 min, 30 cycles of 95C for 95 s, 60C for 95 s, and 72C for 95 s, and a final extension at 72C for 2 min. PCR products were run on Criterion 5% polyacrylamide gels (Bio-Rad), stained with SyBr Green, and scanned using a storm scanner. Real-time RT primers used were as follows: mouse maj -globin, upstream (US) 5-CTGTGGGGAAAGGTGAACTC-3 and downstream (DS) 5-GCAGAGGCAGAGGATA GGTC-3; mouse -actin, US, 5-ACTGCTCTGGCTCCTAGCAC-3, and DS, 5-ACATCTGCT GGAAGGTGGAC-3; mouse GAPDH, US, 5-CCAAGGTCATCCATGACAACT-3, and DS, 5-ATCACGCCACAGCTTTCC-3; human -globin, US, 5-GCACGTGGATCCTGAGAACT-3, and DS, 5-GCCACCACTTTCTGA TAGGC-3; human -globin, US, 5-CTGAGTGAGCT GCACTGTGA-3, and DS, 5-TGCACTTCAGGGGTGAACTC-3; human -globin, US, 5’-TGAATGTCCAAGATGCTGGA-3’, and DS, 5’-CATGATGGCAGAGGCAGAG-3’; human -actin, US, 5-CCAACCGCGAGAAG ATGAC-3, and DS, 5-ACGATGCCAGTGGTACGG-3; and human GAPDH US, 5-GAAGGTGAAGGTCGGAGTCA-3, and DS, 5-GAGGTCAATG AAGGGGTCAT-3. Primers for ChIP samples used are as follows: mouse maj-globin promoter, US, 5-AAGCCTGATTCCGTAGAGCCACAC-3, and DS, 5-CCCACAGGCAAGAGACAG CAGC-3; mouse HS2, US, 5-TGCAGTACCACTGTCCAAGG-3, and DS, 5-ATCTGGCC ACACACCCTAAG-3; mouse -globin promoter, US, 5’-ATGACCTGGCTCCACCCAT-3’, and DS, 5’-TCTTTGAGCCATTGGTCAGC-3’; mouse GAPDH promoter, US, 5-GATGATG GAGGACGTGATGG-3, and DS, 5-GGCTGCAGGAGAAGAAAATG-3; human HS2, US, 5’-CGCCTTCTGGTTCTGTGTAA-3’, DS, 5’-GAGAACATCTGGGCACACAC-3’; human -globin promoter, US, 5’-TGCACATACATGAGGAGCCA-3’, DS, 5’-GTCAGCAGTGATGG 46


ATGGAC-3’; human -globin promoter, US, 5’-CCTTCAGCAGTTCCACACAC-3’, DS, 5’-CTCCTCTGTGAAATGACCCA-3’; and human -globin promoter, US, 5’-GTCAGGGCAG AGCCATCTAT-3’, DS, 5’-AACGGCAGACTTCTCCTCAG-3’. Primers for double ChIP analysis are as follows: human -globin, US, 5-GCTCCTTTA TATGAGGCTTTCTTGG-3, and DS, 5-AATGCACCATGATGCCAGG-3; human -globin, US, 5-TATCTTAGAGGGAGGGCTGAGGGTTTG-3, and DS, 5-CCAACTTCATCCACGTT CACCTTGC-3 and human HS2/-3 linker, US, 5-TGGGGACTCG AAAATCAAAG-3, and DS, 5-AGTAAGAAGCAAGGGCCACA-3. Primers used for pTO/TFII-I RT-PCR are as follows: US, 5-CCCGGAGTTCTTGTA TGTGG-3, and DS (bovine growth hormone, BGH), 5-TAGAAGGCACAGTCGAGG-3. Primers used for pITRp543f2AUSF4 transgenic mice to detect A-USF are: US, 5’-TGACG AAGAAGAACTCGAGGA-3’, and DS, 5’-ACGACCTCTAATCCGTGGTG-3’. Immunofluorescence Cells were washed in 1x PBS and resuspended to 5x10 6 cells/ml in 1x PBS. Twenty l of cells was dropped onto a slide and incubated in a humid chamber for 20 minutes. Cells were fixed in fixation solution for 18 minutes and then washed in PBS. Cells were hybridized with anti-HA tag in 1/100 dilution in PBS for 2 hours and then washed in PBS. Cells were then hybridized with the secondary antibody (FITC or Texas Red) in 1/300 dilution in PBS for 1 hour and then washed in PBS. A drop of Vectashield-DAPI was placed on each cell drop and overlaid with a cover slip. Pictures were taken under 450x using the florescent cube for DAPI and the appropriate cube for the secondary antibody. 47


CHAPTER 3 REGULATION OF -GLOBIN GENE EXPRESSION BY TFII-I AND USF Introduction The mammalian -globin genes are expressed exclusively in erythroid cells and organized along the chromosome according to the timing of expression during development [70, 89]. The embryonic genes are located at the 5 end of the -globin gene locus, and the adult genes are located at the 3 end. Tissueand developmental stage-specific expression of the genes is regulated by complex mechanisms involving gene-proximal and -distal DNA regulatory elements [96]. High-level transcription of -globin genes is mediated by the locus control region (LCR), a powerful regulatory DNA element located far upstream of the -globin gene [75, 235]. The LCR is composed of several DNase I-hypersensitive sites (HS) that function together to regulate chromatin structure and globin gene expression [234, 236-240]. The arrangement of the genes with respect to the LCR is important for correct developmental expression of the -globin genes [101, 241, 242]. Inversion of the genes relative to the LCR activates the adult -globin gene in embryonic cells and represses -globin gene expression throughout development [102]. The -globin genes have also been shown to compete for interaction with the LCR [97, 98]. Those genes which are in close proximity to and interact with the LCR have been shown to be transcriptionally active [86, 87]. A number of trans-acting factors have been found to aid in the developmental regulation -globin expression by binding to the individual gene promoter regions. A well characterized example of this is EKLF which activates the adult -globin gene during definitive erythropoiesis [109]. The promoter of the human adult -globin gene is characterized by a TATA-like element, CATAAA, 25-30 bp upstream of the transcription initiation start site [143]. Deviation from consensus TATA sequences often weakens promoters and requires additional elements for the 48


efficient recruitment and stabilization of transcription complexes. In accordance, the -globin promoter also contains an initiator sequence, which overlaps with the transcription initiation start site. Both the initiator and TATA-like element interact with the TFII-D protein complex [148, 160]. In addition to TAF250 and TAF150, components of the TFII-D complex, a number of additional proteins have been shown to interact with initiator elements. Notable among these are helix-loop-helix proteins TFII-I and USF which have been implicated in the recruitment of transcription complexes to TATA-less promoters and in the stabilization of transcription complexes in TATA-box containing promoters [151, 232, 243]. The downstream promoter region has been found to be comprised of a MARE/AP1-like element, which is bound by NF-E2, and two conserved E-boxes, one overlapping the initiator element and the other sixty bases downstream of the transcription initiation start site [153]. TFII-I and USF proteins have also been found to interact with the E-box elements. TFII-I and USF1 are found at the initiator/E-box, while USF1 and USF2 interact with the +60 E-box. Mutations of the E-box at +60 reduced transcription to a level comparable to that observed after mutation of the initiator sequence [153]. This suggests that the E-box and initiator significantly contribute to the formation and/or the stabilization of transcription complexes and that all three elements, the TATA-like element, initiator, and +60 E-box, act together to establish functional transcription complexes. TFII-I and USF are ubiquitously expressed helix-loop-helix proteins that are involved in the transcriptional regulation of genes containing E-box or initiator sequences. The USF family is comprised of USF1 and USF2 which are highly related proteins that interact with DNA either as homoor heterodimers [188]. They often bind E-box motifs in the vicinity, sometimes downstream, of transcription start sites and have been shown to aid in the formation of 49


transcription complexes [232, 243]. Interestingly, a recent genomic survey revealed that many Drosophila genes harbor E-box elements located about 60 bp downstream of the transcription start site [244]. Likewise, TFII-I has been implicated in the recruitment of transcription complexes to promoters containing an initiator sequence overlapping the transcription start site [151]. USF and TFII-I can act as activators or repressors of transcription, depending on interacting proteins and the sequence context of the promoter region [166, 184, 245-247]. Initial studies suggest that TFII-I is not necessary for transcription of the -globin gene. Antibodies against TFII-I were not able to inhibit transcription of the -globin gene in vivo and crosslinking of TFII-I to the -globin gene in adult erythroid cells is inefficient compared to USF1 and USF2 [153]. Mutation of the +60 E-box, which diminishes -globin transcription, suggests that USF proteins are necessary for transcription. Here in we show that reducing the activity of TFII-I, by expressing a dominant-negative form of TFII-I, leads to the derepression of the -globin gene in erythroid cells. Whereas, reduction of USF activity, through expression of a dominant-negative protein, results in a decrease of -globin expression. The results presented here support the hypothesis that antagonistic activities exerted by TFII-I and USF during embryonic and adult erythroid development contribute to the stage-specific expression of the -globin gene. Results We previously demonstrated that the helix-loop-helix proteins USF and TFII-I interact with sequences located at or downstream of the transcription start site of the adult -globin gene [153]. The interaction of TFII-I was more pronounced in cells not expressing the -globin gene, suggesting that it may be involved in repressing -globin gene expression. In contrast, USF1 and USF2 efficiently interact with the -globin gene in cells that express the gene. In this study, we examined the consequence of reducing or increasing the activities of TFII-I and USF on -globin 50


gene expression in human K562 erythroleukemia cells, in which the -globin gene is repressed, and in MEL cells, in which the adult -globin gene is expressed. In this study, we over-expressed wild-type or mutant TFII-I proteins in erythroid cell lines. The erythroid cell lines utilized were K562 cells, human erythroleukemia cells, which repress the adult -globin gene and express the embryonic and fetal -globin genes, and MEL cells, murine erythroleukemia cells, in which the adult -globin genes are expressed and the embryonic and fetal genes are silenced [248-251]. These cell lines have been extensively characterized and frequently utilized to understand regulation of the -globin locus. The effect of expressing these proteins on -globin gene expression was monitored by quantitative RT-PCR. We related -globin gene expression to the expression of two internal control genes, -actin (data not shown) and GAPDH, and obtained similar results. We used a dominant-negative mutant of TFII-I (p70) that lacks the C-terminal acidic activation domain as described previously to interfere with the function of wild type TFII-I [165]. p70 forms dimers with TFII-I but does not allow for interactions with coregulators. The sequence encoding full-length wild-type TFII-I or p70 was cloned into an expression vector and placed under the control of the cytomegalovirus (CMV) promoter and the Tet operator. K562 and MEL cells were stably co-transfected with constructs expressing either wild-type TFII-I or the p70 mutant and with a Tet repressor expression construct. The expression of these proteins was induced by incubating the cells with doxycycline (Figure 3-1). The expression of TFII-I and p70 was confirmed by RT-PCR and/or Western blotting (Figure 3-2A, C, Figure 3-3A, and Figure 3-4A). Over-expression of TFII-I in MEL cells was difficult to assess from the Western blotting experiment, but the message was detected by RT-PCR using primers specific for TFII-I expressed from the transfected plasmid (Figure 3-2C). 51


The expression of p70 in MEL cells led to a threeto four-fold increase in maj-globin gene expression and a three-fold increase in -globin expression in K562 cells (Figure 3-2B and 3-3B). There is a minimal effect on -globin expression in K562 cells compared to the effect on -globin expression, suggesting that this effect is specific for the adult -globin gene (Figure 3-3C). The p70 mutant protein is expressed in transfected cells even in the absence of doxycycline, demonstrating that there is leaky expression from the CMV/tetracycline-responsive element promoter. Over-expression of TFII-I in MEL and K562 cells decreased maj-globin/-globin gene expression by about 70% (Figure 3-2D and 3-4B). There was a very minimal effect on -globin expression in K562 cells, again suggesting that TFII-I is specific for the adult -globin gene (Figure 3-4C). We observed higher expression of p70 and TFII-I in the absence of doxycycline in MEL cells relative to p70 and TFII-I expression in K562 cells; however, the expression of the proteins in the untreated MEL cells had effects on globin gene expression similar to those in Dox-treated cells. The MEL cells used in these experiments express the maj-globin gene in the absence of dimethyl sulfoxide (DMSO) induction. Incubating the cells in the presence of 3% DMSO leads to a 5to 10-fold increase in maj-globin gene expression (data not shown). It is interesting that the DMSO-mediated increase is similar to that with the up-regulation of maj-globin gene expression by p70. Taken together, the data demonstrate that TFII-I functions as a repressor of -globin gene expression in both MEL and human K562 cells. Many studies have shown that USF proteins can function as either activators or repressors of transcription. We previously demonstrated that USF1 and USF2 interact prominently with the adult -globin gene promoter in MEL cells [153]. In addition, in vitro transcription of the -globin gene was impaired in promoter constructs containing mutations in the USF binding site. 52


To analyze the role of USF in a cellular context, we expressed a dominant-negative mutant of USF, A-USF, in MEL cells. The expression construct was kindly provided by Charles Vinson (NIH). A-USF lacks the basic DNA binding region, which has been replaced by an acidic domain, and also the N-terminal activation domain. It has been found to dimerize with both USF1 and USF2 and thus reduce total USF activity [230]. We generated an A-USF expression construct in which the coding region is under control of the CMV promoter and the Tet operator. This construct was cotransfected into MEL cells with a Tet repressor expression plasmid. A-USF was expressed as an HA-tagged protein, thus the induction of A-USF expression by doxycycline was monitored by Western blotting experiments using antibodies against the HA tag (Figure 3-5A). In these experiments, we again observed some expression of A-USF in the absence of doxycycline, demonstrating that the Tet repressor does not effectively repress the expression of A-USF. However, after the induction with doxycycline, we do detect a higher level of expression of A-USF. The expression of A-USF led to a five-fold decrease in maj-globin gene expression in induced MEL cells transfected with pTO/A-USF and only a 1.5-fold decrease in uninduced cells transfected with pTO/A-USF (Figure 3-5B). We also over-expressed USF1 in MEL cells and found that it increased maj-globin gene expression threeto fourfold (Figure 3-5D). The inducible system again revealed some leakiness, as we detected a significant increase in USF1 expression in both the presence and the absence of doxycycline (Figure 3-5C). However, the expression of USF1 in the untreated MEL cells had effects on globin gene expression similar to those in Dox-treated cells. Nevertheless, the combined data demonstrate that USF is an activator of -globin gene expression in MEL cells. The association of RNA polymerase II and transcription factors with the murine -globin gene locus has previously been analyzed during the erythroid differentiation of murine 53


embryonic stem cells [231]. Differentiation is induced by erythropoietin and is accompanied by the expression of the embryonic globin genes at day 5 and the activation of the adult globin genes at day 8. Zhuo Zhou, a current graduate student in the Bungert laboratory, utilized this differentiation system to examine the interaction patterns of USF and TFII-I with locus control region element HS2 and the maj-globin gene promoter by ChIP assay (Figure 3-6). As a control, we examined the association of histone H3 dimethylated at lysine 4 (H3K4 dimethyl), which is a mark for transcriptionally competent chromatin. Dimethylated H3K4 is detectable in LCR HS2 at all stages but reduced at the -globin promoter at day 0, which is consistent with our previous findings [231]. USF2 is detectable at the LCR and the maj-globin gene promoter in undifferentiated cells at day 0 and continues to interact with these elements throughout differentiation. TFII-I is detectable at HS2 and the -globin promoter at day 5, while USF1 is found only at HS2 at this stage. At day 8, USF1 interacts with the -globin promoter and HS2. Interestingly, TFII-I is no longer detectable at HS2 or the -globin promoter at day 8, when -globin gene expression is activated. These results are consistent with the conclusion that TFII-I is a repressor of -globin gene expression, while USF is an activator. Furthermore, these results, together with those of other interaction studies, strongly suggest that USF and TFII-I directly act on the globin gene locus. Previous studies have shown that TFII-I interacts cooperatively with USF1 and stimulates the binding of USF1 at Inr and E-box elements [151, 166]. We analyzed protein-protein interactions involving USF1 by co-immunoprecipitation in K562 and MEL cells (Figure 3-7). The results demonstrate that USF1 interacts strongly with TFII-I in K562 cells but not as strongly in MEL cells. USF1 and USF2 are seen to interact efficiently in MEL cells and less efficiently in K562 cells. We observed the same results in experiments using USF2 antibodies 54


for immunoprecipitation and USF1 antibodies for Western blotting. TFII-I, USF1, and USF2 are expressed at similar levels in K562 and MEL cells as previously shown by Leach et al. [153]. This data suggests that in an embryonic environment TFII-I and USF1 interact and bind the Inr/E-box element while in an adult environment USF1 preferentially interacts with USF2 and binds to the +60 E-box. Discussion The mechanisms which control the correct developmental expression of the -globin genes involve both distal and proximal cis-elements. Throughout development, the LCR aids in the correct timing of -globin gene expression through interaction with the proper -globin gene. The active gene is in close proximity to the LCR whereas inactive genes are distant. Relative order and distance to the LCR also plays a role in the correct developmental expression [101, 102]. Proximal promoter elements have also been found to be crucial in the correct timing of -globin gene expression. Many different transcription factors have been found to bind to these elements and either activate or repress transcription. Such proteins which are known to mediate stage-specific expression of the -globin genes are EKLF, FKLF, and NF-E2. EKLF and NF-E2 preferentially activate the adult -globin gene over that of the -globin genes, whereas FKLF promotes and -globin gene transcription [95, 110, 134]. Our previous studies demonstrated that -globin promoter activity is regulated by DNA sequences located downstream of the transcription start site [153]. One of these elements is an E-box motif located 60 bp downstream of the transcription initiation site. This element interacts with USF proteins in vitro and is precipitated by USF antibodies in ChIP experiments in cells expressing the -globin gene. Another element located at and downstream of the transcription start site is composed of overlapping initiator and E-box elements and interacts with TFII-I and USF proteins. Other studies have shown that USF interacts with a functionally important E-box 55


motif in LCR element HS2 [252]. In this study, we have analyzed the contribution of helix-loop-helix proteins TFII-I and USF to -globin gene expression in K562 and MEL cells. The human erythroleukemia cell line K562 exhibits an embryonic-cell-like phenotype and predominantly expresses the and -globin genes. In contrast, MEL cells express the adult majand min -globin genes but not the embryonic yolk and h1-globin genes. The results of the present study show that the inhibition of TFII-I activity through the expression of a dominant-negative mutant upregulates adult -globin gene expression in K562 and MEL cells. In addition, over-expression of TFII-I in both cell lines results in a decrease of -globin expression. Further evidence was derived from erythroid differentiation studies. TFII-I was found to crosslink to the adult -globin promoter during early stages of erythroid differentiation in murine embryonic stem cells when the -globin gene is silenced and not at later stages when the -globin gene is active. This collective evidence suggests that TFII-I acts as a repressor of adult -globin gene expression. It is likely that the ubiquitously expressed transcription factor TFII-I functions in conjunction with stageand erythroid-cell-specific activities to repress the expression of the -globin gene. Repressor complexes have previously been characterized that mediate stage-specific repression of the -globin gene in adult cells [253]. USF proteins have previously been shown to interact with HS2 of the -globin locus control region, which contains conserved E-box motifs [252, 254]. In addition, previous studies have shown that USF also interacts with the -globin gene promoter [153]. Mutations in the downstream promoter E-box motif decreased in vitro transcription efficiency of the -globin gene. Our findings of reduced -globin gene transcription in MEL cells expressing a dominant-negative mutant and increased expression in MEL cells over-expressing USF1 are consistent with the in vitro data. Additionally, USF1 was found to crosslink to the -globin promoter in 56


later stages of erythroid differentiation in murine embryonic stem cells which express the -globin gene and not in earlier stages where -globin is preferentially expressed. These data suggest that USF proteins act to enhance -globin gene expression. It has previously been shown that both TFII-I and USF1 bind to the initiator element and the E-box of the adenovirus major late promoter and HIV-1 LTR and interact cooperatively [151, 243]. TFII-I increases the affinity of USF1 for the HIV-1 LTR [229] and to initiator elements [166]. Their interaction is necessary for the transcription of these genes. In contrast, we have found that TFII-I and USF1 interact preferentially in K562 cells, where the adult -globin gene is silenced. Transcriptional regulation by TFII-I and USF is context dependent in that activation may require expression of specific co-activators, or cooperative interactions with other transcription factors. Therefore, TFII-I and USF1 may interact with additional co-factors which leads to the repression of the -globin gene. In MEL cells but not K562 cells, we have found that USF1 and USF2 preferentially interact. These data support a model in which TFII-I and USF1 interact and bind to the Inr/E-box of the -globin promoter, repressing transcription, in an embryonic environment. In an adult environment, USF1 and USF2 interact and bind to the +60 E-box aiding in transcription of the -globin gene. To understand the mechanism of TFII-I and USF regulation of -globin gene expression, it will be crucial to determine how these ubiquitously expressed proteins have tissueand stage-specific functions. DNA binding proteins are known to recruit co-activators which modify and mobilize nucleosomes leading to the activation or repression of transcription. Therefore, TFII-I and USF could possibly recruit tissueand stage-specific co-activators to the -globin locus. It is also known that TFII-I and USF are phosphorylated through cell signaling pathways. 57


Modifications of these proteins may target them or inhibit them from binding to the -globin promoter. 58


Figure 3-1. The experimental system for expressing wild-type or dominant-negative proteins. Coding sequences for TFII-I, p70, USF1, and A-USF are under the control of the CMV enhancer/promoter. A Tet operator sequence interacts with the Tet repressor, for which an expression construct is cotransfected into these cells. Doxycycline interacts with the Tet repressor and relieves repression. TRE, tetracycline-responsive element. TFII-I, p70, USF1, or AUSF TetR CMV/TRE TFII-I, p70, USF1, or AUSF + Doxycycline TetR 59


0123456p70 + doxp70pTO +/doxRelative Beta-Major Expression + doxTFII-IpTO +/doxRelative Beta-Major Expression A B C D Figure 3-2. TFII-I function in MEL cells. The expression of the dominant-negative mutant p70 (A and B) or wild-type TFII-I (C and D) was induced in stably transfected MEL cells lines by using 1 g doxycycline per ml culture medium for 36 h. The expression of TFII-I or p70 was analyzed by Western blotting and/or RT-PCR analysis as indicated. For RT-PCR analysis of TFII-I expression, RNA was isolated from induced MEL cells harboring expression constructs for TFII-I or the empty vector pTO and converted to cDNA. The cDNA was analyzed by PCR with forward primers corresponding to the 3' region of the TFII-I-coding region and the reverse bovine growth hormone (BGH) primer hybridizing to the pTO vector. For gene expression analysis, RNA was isolated from transfected cell lines, reverse transcribed, and analyzed by quantitative PCR for the expression of the maj-globin gene. maj-globin expression is presented as the ratio of expression in pTO/p70 or pTO/TFII-I cells relative to the expression in pTO cells using GAPDH as the internal reference. +/–, pTO + dox cells served as control for doxycycline-treated cells, and pTO dox served as control for untreated cells. 60


A pTO + dox p70 + dox pTO p70 p70 00.511.522.533.54p70 + doxp70 doxpTO +/doxRelative Beta-globin Expression 00.511.522.533.54p70 + doxp70 doxpTO +/doxRelative Epsilon-globin Expression B C Figure 3-3. p70 function in K562 cells. The expression of dominant-negative mutant p70 was induced in stably transfected K562 cells by using 1 g doxycycline per ml culture medium for 12 h. The expression of p70 was analyzed by Western blotting using an HA-tag antibody (A). RNA was isolated from transfected cell lines, reverse transcribed, and analyzed by quantitative PCR for expression of the -globin (B) and -globin (C) genes. and -globin gene expression is presented as the ratio of expression in pTO/p70 relative to expression of cells transfected with the pTO vector using GAPDH as the internal reference. +/–, pTO + dox cells served as control for doxycycline-treated cells, and pTO dox served as control for untreated cells. 61


Figure 3-4. TFII-I function in K562 cells. The over-expression of TFII-I was induced in stably transfected K562 cells by using 1 g doxycycline per ml culture medium for 36 h. The expression of TFII-I was analyzed by Western blotting using an HA-tag antibody (A). RNA was isolated from transfected cell lines, reverse transcribed, and analyzed by quantitative PCR for expression of the -globin (B) and -globin (C) genes. and -globin gene expression is presented as the ratio of expression in pTO/TFII-I relative to expression of cells transfected with the pTO vector using GAPDH as the internal reference. +/–, pTO + dox cells served as control for doxycycline-treated cells, and pTO dox served as control for untreated cells. 62

PAGE 63 + doxAUSF doxpTO +/doxRelative Beta-Major Expression 00.511.522.533.544.5USF1 + doxUSF1 doxpTO +/doxRelative Beta-Major Expression A B C D Figure 3-5. USF activates -globin gene expression in MEL cells. Stable MEL cell lines were created containing either A-USF (pTO/A-USF) or USF1 (pTO/USF1) and the Tet repressor (TR). The expression of A-USF and USF1 was induced by incubating the cells with doxycycline and analyzed by Western-blotting experiments (A and C). A-USF was detected using an antibody against the HA-tag, while US1 was detected with a USF1 specific antibody. maj-globin gene expression in transfected cell lines was analyzed by quantitative RT-PCR, using the expression of GAPDH as an internal reference, and compared to the expression in cells transfected with the empty vector (pTO), whose expression level was set to 1 (B and D). +/–, pTO + dox cells served as control for doxycycline-treated cells, and pTO dox served as control for untreated cells. 63


Figure 3-6. Interaction of USF and TFII-I with the -globin gene locus during erythroid differentiation of murine embryonic stem cells. Murine embryonic stem cells were cultured and induced to differentiate as previously described by Levings et al. At day 5, after the addition of erythropoietin, cells were collected and subjected to ChIP analysis using antibodies against dimethylated histone H3K4 (-dimethyl H3K4), USF1, USF2, and TFII-I. IgG antibodies were used as negative controls in these experiments. The DNA was isolated from the precipitate and analyzed by PCR using sets of primers specific for LCR element HS2 and the maj-globin gene promoter. 64


Figure 3-7. Coimmunoprecipitation of USF1, USF2, and TFII-I in K562 and MEL cells. K562 or MEL cell extract was precleared with anti ()-rabbit IgG beads and precipitated with -USF1, and complexes were captured by incubation with anti-rabbit IgG beads. Complexes were eluted off the beads with Laemmli buffer and incubation at 95C for 10 min and loaded onto 10% Ready gels (Bio-Rad). After transfer, the membrane was cut into strips to probe with antibodies against either USF1, USF2, or TFII-I. The strips were then reassembled for phosphorimaging. 65


CHAPTER 4 RECRUITMENT OF CHROMATIN MODIFYING ENZYMES BY TFII-I AND USF TO THE -GLOBIN LOCUS Introduction The activation of gene expression is controlled at different levels, including positioning genes to transcriptionally active domains in the nucleus, increasing chromatin accessibility, recruitment of transcription complexes, transcription elongation, and posttranscriptional mechanisms [255, 256]. It is becoming increasingly clear that one role DNA binding proteins play is to recruit co-activators that modify histones or mobilize nucleosomes at regulatory sites. It is believed that the modification and mobilization of nucleosomes render DNA accessible for interactions with transcription complexes. Recent reports support the view that chromatin-remodeling activities act locally at promoter and perhaps enhancer regions to allow the subsequent assembly of transcription or enhanceosome complexes [257]. These activities, which are often part of large protein complexes, must be recruited to the site of action by proteins that recognize specific DNA motifs. EKLF represents a well-described example of a protein that recruits chromatin-remodeling activities to regulatory sites in the -globin gene locus. EKLF interacts with LCR elements HS2 and HS3, as well as with the adult -globin gene, and recruits the chromatin-remodeling complex SWI/SNF, resulting in a local change in nucleosome organization [141, 258]. The ablation of EKLF in mice leads to a reduction of DNase I HS formation in HS3 and in the -globin gene promoter [259]. EKLF is particularly important for -globin gene expression and does not significantly affect the expression of other genes in the locus [259]. The LCR of the -globin locus is known to mediate high-level and erythroid specific expression of the -globin genes. Recent models propose that macromolecular protein complexes involved in modulating chromatin structure and transcription of the -globin genes are first 66


recruited to the LCR [260, 261]. These activities are used to establish stage-specific accessible chromatin domains, and transcription complexes are subsequently transferred from the LCR to the globin genes. Stage-specific modification of chromatin structure would provide a means to restrict the transfer of macromolecular complexes from the LCR to individual globin genes [262]. We have previously shown that helix-loop-helix proteins TFII-I and USF antagonistically regulate -globin expression. TFII-I represses while USF proteins activate -globin transcription. As these proteins are ubiquitously expressed, it is most likely that they interact with co-activators to exert stage-specific control. Histone deacetylase 3 (HDAC3) is one of the four members of the human class I histone deacetylases which are implicated in transcriptional repression through deacetylation of amino-terminal tails of core histones. It is present in a unique large multi-subunit protein complex, is essential for cell viability, and is localized both in the nucleus and the cytoplasm of cells [263-265]. TFII-I has been found to physically and functionally interact with HDAC3 [247, 266]. Residues 373-401 of HDAC3 have been found to be crucial for HDAC3-TFII-I association, while residues 363-606, which include I-repeats R2-R4, of TFII-I are required [247]. It is currently unclear exactly how these proteins interact, since HDAC3 does not contain a HLH domain. Nevertheless, this interaction leads to a possible mechanism of gene repression by TFII-I. Polycomb group proteins (PcG) play key roles in developmental regulation. These complexes have been found to regulate the correct expression of the segmental homeo box (Hox) genes by maintaining a repressive state [267]. Initital repression of these genes is mediated by DNA binding transcriptional repressors and then PcG proteins modify chromatin to maintain a 67


repressive state [268]. The PcG proteins are conserved in mammals where they have also been implicated in control of cell proliferation and oncogenesis [269]. PcG proteins function in two distinct complexes, Polycomb Repressive Complex 1 (PRC1) and PRC2. The core of the PRC2 complex consists of EZH2, the WD-repeat protein EED, the zinc-finger protein Suz12, and histone-binding protein RbAp48 [270, 271]. EZH2 is a H3K27 methyltransferase and Suz12 is required for this activity [272]. H3K27 methylation is required for PRC2-mediated gene silencing which is thought to provide a binding surface for PRC1. PRC1 facilitates oligomerization, condensation of chromosomes, and inhibition of chromatin remodeling activity in order to maintain silencing[273]. Components of PRC2 are expressed at high levels in embryonic tissues and are essential for the earliest stages of vertebrate development. They also have been found to occupy a special set of developmental genes in embryonic stem (ES) cells that must be repressed to maintain pluripotency and that are poised for activation during ES cell differentiation [274]. As the adult -globin gene is repressed during the early stages of development and is poised for activation, it is possible that PCR2 regulates -globin expression at the embryonic and fetal stage of development. A recent report demonstrated that USF is a critical component of the boundary function of the chicken HS4 (cHS4) insulator [275]. cHS4 establishes a boundary between open and closed chromatin in the chicken -globin gene locus. It was shown that USF interacts with cHS4 in vivo and recruits histone-modifying activities that may be involved in separating inactive from active chromatin domains. Thus, part of the in vivo activating function of USF may be due to the recruitment of chromatin-modifying activities to DNA regulatory elements. USF has also been shown to interact with a conserved E-box located in LCR element HS2 [252, 254]. A mutation of this E-box impairs the enhancer function of HS2. 68


To understand the regulation of -globin gene expression by TFII-I and USF, we investigated whether these proteins recruit chromatin modifying enzymes to aid in repression or activation of transcription. We show here that TFII-I is complexed with HDAC3 and that both proteins interact together at the -globin core promoter in embryonic erythroid cells. We also show that Suz12 of the PRC2 complex is involved in repression of -globin transcription in an embryonic environment. In addition, USF is seen to interact with histone acetyltransferase proteins (HATs) and a decrease in USF activity decreases their recruitment as well as RNA Polymerase (Pol) II recruitment to the -globin locus. These results support the hypothesis that the recruitment of chromatin modifying enzymes to the -globin locus by TFII-I and USF contribute to the developmental regulation of the -globin gene. Results Previous studies have shown that TFII-I physically and functionally interacts with HDAC3 [247, 266]. We therefore sought to determine if TFII-I recruits HDAC3 to the adult -globin promoter in erythroid cells. We first analyzed their interaction in K562 cells by coimmunoprecipitation (Figure 4-1A). The data show that TFII-I but not USF2 interacts with HDAC3. We next compared their interaction in an embryonic environment where the -globin gene is repressed (K562 cells) and in an adult environment where is the adult -globin gene is expressed (MEL cells) (Figure 4-1B). TFII-I and HDAC3 are seen to exclusively interact in K562 cells. We then examined whether TFII-I and HDAC3 simultaneously interact with the -globin gene promoter in K562 cells. Karen Vieira, a previous graduate student in the Bungert laboratory, using double-chromatin immunoprecipitation assay, first precipitated cross-linked and sheared chromatin with TFII-I antibodies. The precipitate was subsequently immunoprecipitated with HDAC3 antibodies. The DNA purified from this precipitate was analyzed by PCR. The results show that HDAC3 interacts with the -globin promoter in TFII-I69


selected chromatin fragments (Figure 4-1C). We did not detect interactions of TFII-I or HDAC3 with the embryonic -globin gene promoter or with a region between LCR HS2 and HS3. Together, the data suggest that TFII-I recruits HDAC3 to the -globin gene promoter in an embryonic environment to repress transcription. To further determine that TFII-I and HDAC3 act as repressors of -globin gene transcription, we reduced the expression of TFII-I, HDAC3, and USF2 by RNA interference in K562 cells (Figure 4-2). Knock-down of USF2 was used as a control as it is not expected to play a role in -globin expression in an embryonic environment. We transfected K562 cells with siGENEOME SMARTpool reagents (Dharmacon) using a Nucleofector and observed close to 100% transfection efficiency in these cells (Figure 4-2A). Western blotting experiments revealed specific reductions in the expression of TFII-I, HDAC3, and USF2 compared to that in cells transfected with a non-targeting (neg) siRNA or mock transfected cells (Figure 4-2B). Reducing the protein levels of either TFII-I or HDAC3 resulted in a significant increase in -globin gene transcription (Figure 4-2C). The increase of -globin gene expression in cells transfected with TFII-I-directed siRNA was about 2.5-fold. Cells transfected with siRNA directed against HDAC3 revealed a threefold increase in -globin gene expression. The expression of -globin was not affected, demonstrating that TFII-I and HDAC3 specifically act on the -globin gene. A reduction of USF2 expression had no effect on the expression of either the -globin or the -globin gene in K562 cells. The data strongly suggest that a protein complex containing the DNA binding protein TFII-I and the coregulator HDAC3 interacts with the -globin gene promoter in embryonic erythroid cells and represses transcription by rendering the chromatin structure inaccessible for the transcription complex. 70


Polycomb repressor complex 2 (PRC2) has been found to repress genes during early stages of development which is necessary to maintain pluripotency. These genes are poised for activation during cell differentiation. Since the adult -globin gene is repressed during early stages of development and is activated during erythroid differentiation, we looked for PRC2 presence at the -globin locus using ChIP (Figure 4-3A). We determined that Suz12, a component of the PRC2 complex, is bound at the repressed -globin promoter in K562 cells. It was not seen at the active -globin promoter. In MEL cells, Suz12 was not observed at the repressed -globin promoter or at the active maj-globin promoter, suggesting that Suz12 aids in the specific repression of the -globin gene in an embryonic environment. The repressive function of PRC2 is mediated by its methylation of H3K27. H3K27 can be mono-, di-, or trimethylated. No H3K27me3 marks are seen at the promoters of the globin genes in either K562 or MEL cells (Figure 4-3A). However, the promoters of the globin genes in K562 and MEL cells are monomethylated at H3K27, which corresponds with recent data shown by the Blobel group (Figure 4-3B) [276]. H3K27me1 levels at the globin gene promoters inversely correspond to the expression of the globin genes. The repressed adult -globin gene has high levels of H3K27me1 at its promoter, H3K27me1 levels are decreased at the expressed -globin gene promoter compared to the -globin promoter, and the highly expressed -globin gene has the lowest H3K27me1 levels at its promoter. This suggests that H3K27me1 marks aid in the control of globin gene expression. To determine if Suz12 plays a role in the repression of -globin gene expression, we transfected K562 cells with siRNAs against Suz12 to knock-down Suz12 protein levels. A similar approach was taken as with the TFII-I and HDAC3 RNAi experiments. Specific Suz12 protein knock-down was seen as compared to K562 cells nucleofected with non-targeting (neg) 71


siRNA or mock transfected cells (Figure 4-4A). -globin expression was seen to increase while knock-down of Suz12 had less of an effect on -globin expression and only a slight effect on -globin expression. -globin expression increased 3 to 4.5 fold, while -globin expression was seen to increase 2 to 2.5 fold, and -globin expression increased 1.5 fold over that of the non-targeting and mock controls (Figure 4-4B). This effect correlates to the expression of the globin genes. The expression of the repressed -globin gene increases the greatest when Suz12 is knocked-down, while the expression of the highly expressed -globin gene is least effected. These results correlate with H3K27me1 levels at the globin promoters and suggest that Suz12 aids in the repression of the globin genes. We have shown that TFII-I plays a repressive role in -globin transcription partly by recruiting HDAC3. TFII-I has multiple protein interaction domains and thus it is possible that TFII-I could recruit more than one repressive protein/complex to the -globin promoter. Thus, we analyzed the interaction of Suz12 and TFII-I in both K562 and MEL cells. Through co-immunoprecipitation, Suz12 and TFII-I are seen to interact in both cell lines (Figure 4-5). These data suggest that TFII-I recruits the PRC2 complex to the -globin promoter which aids in repression of the -globin genes through mono-methylation of H3K27. It has been reported that USF1 is found at the chicken HS4 boundary element and recruits histone modifying enzymes such as: the H3K4-specific methyltransferase SET7/9, H3-specific acetyltransferase PCAF, and histone acetyltransferases p300 and CBP. We therefore investigated whether these proteins are present at the -globin locus in K562 and MEL cells (Figure 4-6). PCAF and SET7/9 were not observed at any of the globin promoters or HS2 in either cell line. However, both p300 and CBP are present at the active maj-globin promoter and HS2 in MEL cells. In K562 cells, these proteins were just observed at HS2. Since USF1 has been shown to 72


associate with the active -globin promoter and also HS2, these data suggest that p300 and CBP are recruited to these regions by USF1 and aid in the activation of the adult -globin gene. To determine whether USF1 physically interacts with p300 and CBP we performed co-immunoprecipitations using both K562 and MEL cell extracts (Figure 4-7). Results suggest that p300 and CBP are expressed at about equal levels between K562 and MEL cells. p300 and CBP are seen to interact with each other in both K562 and MEL cells. Both p300 and CBP were seen to strongly interact with USF1 in MEL cells. No interaction was observed in K562 cells, suggesting that USF1 preferentially interacts with p300 and CBP in an adult erythroid environment. To analyze whether USF is involved in the recruitment of transcription complexes to the globin gene locus, we performed ChIP experiments in MEL cells transfected either with the dominant-negative USF, A-USF, expression construct or an empty control vector (pTO). We first determined whether the expression of A-USF affects the interaction of USF1 and USF2 with the -globin gene locus (Figure 4-8). We and others previously demonstrated that USF interacts with LCR element HS2 and the -globin gene promoter [153, 252, 254]. The expression of A-USF inhibits the binding of USF1 to both HS2 and the maj-globin gene promoter. There is less of an effect on the interaction pattern of USF2. This result suggests that A-USF preferentially inhibits USF1/USF2 heterodimers and USF1/USF1 homodimers. We next analyzed the interaction of RNA Pol II and the co-regulators p300 and CBP as well as the association of acetylated histone H3 (AcH3) with LCR element HS2 and the maj-globin promoter by ChIP. The results show that the interactions of essentially all of the factors with the -globin gene locus were reduced in cells expressing A-USF compared to that in cells harboring the control vector (Figure 4-9A). A reduction in factor binding was not only seen at 73


the -globin promoter region but also occurred at LCR element HS2, which contains several E-box elements. The decrease in factor binding was accompanied by reductions in the association of AcH3 with the globin locus. There was very little change in chromatin marks or factor association with the control GAPDH promoter (Figure 4-9B). Overall, the results suggest that USF proteins not only regulate the recruitment of transcription complexes but also play roles in establishing accessible chromatin domains in the globin gene locus by recruiting chromatin modifying enzymes. To further understand the role USF and TFII-I of in -globin gene regulation it will be crucial to look at their actions in vivo. We have created a construct in which the expression of A-USF is under the control of the -globin promoter and HS3 and HS2 and flanked by chicken HS4 elements for insulation, pITRp543f2AUSF4 (Figure 4-10A). Thus, A-USF should be exclusively expressed in erythroid cells. To first to ensure that A-USF will be expressed, we transiently transfected MEL cells. A-USF expression is seen after 48 hours transfected with either 1, 3, or 6 g of plasmid (Figure 4-10B). In addition, a 40-50% decrease in maj-globin expression is seen in these cells (Figure 4-10C). These results suggest that A-USF is expressed from this construct and is functional. We then created transgenic mice using pITRp543f2AUSF4 and have preliminarily verified integration by PCR using primers for A-USF, human HS3, and the flanking region of human HS3 and HS2 (Figure 4-10D). Further analysis of these mice is necessary. This approach will also be used to create transgenic mice expressing the dominant-negative TFII-I, p70, exclusively in erythroid cells. Discussion The mechanisms governing the correct expression of -globin genes during development involve DNA binding proteins that interact with specific regulatory elements in the globin locus in a developmental stage-specific manner and aid in the modification of chromatin structure 74


and/or in the recruitment of transcription complexes [96]. Relative gene order and activity of stage-specific DNA binding proteins regulate chromatin accessibility and ultimately the recruitment of active transcription complexes to the genes. Among the proteins known to mediate stage-specific expression of globin genes is EKLF, which activates the adult -globin gene but not the embryonic globin genes. EKLF recruits ATP-dependent chromatin-remodeling activities to the -globin gene promoter [141]. It is becoming increasingly clear that the local remodeling of nucleosomes in the promoter region is an important step in the activation of gene transcription [257]. The results of the present study show that TFII-I through the recruitment of HDAC3 and Suz12 aids in the repression of the -globin gene. We have shown that the inhibition of either TFII-I or HDAC3 activity upregulates -globin gene expression in K562 cells. Furthermore, we show that TFII-I and HDAC3 interact with each other exclusively in K562 cells and not in MEL cells. Also, TFII-I and HDAC3 interact at the -globin gene promoter in K562 cells. It has been observed that TFII-I and HDAC3 have very similar expression patterns in the developing mouse embryo [266]. Therefore, the results presented here along with the previous studies suggest that the transcription activity of TFII-I may be controlled by HDAC3 during early development. Our data demonstrate that TFII-I functions as a repressor of -globin gene expression by recruiting histone deacetylase activity to the promoter. This is consistent with observations that acetylation of the human -globin promoter region is reduced in erythroid cells with an embryonic phenotype [277]. Polycomb group complex (PRC) 2 has been implicated in the repression of genes required for pluripotency and those that are poised for expression during differentiation. We therefore examined the presence of Suz12, a component of PRC2, at the -globin locus. Suz12 was 75


observed at the promoter of the repressed -globin gene but not the promoter of the active -globin gene in K562 cells. Also, Suz12 is not seen at the -globin locus in MEL cells, suggesting that Suz12 acts at the adult -globin promoter in an embryonic environment. However, knockdown of Suz12 results in an increase in both and -globin expression with minimal effect on -globin expression. The function of PRC2 is to methylate H3K27 residues, which leads to transcriptional repression. We did not detect H3K27me3 at the -globin locus in either K562 or MEL cells but did observe H3K27me1 at the promoters of the -globin genes. The extent of this modification at the specific globin promoter seems to be dependent on the state of activation of the globin gene. Low levels of H3K27me1 are observed at -globin gene which is highly expressed in K562 cells, with moderate levels at -globin which is expressed at lower levels, and highest levels are seen at the -globin gene which is repressed. Therefore, it is possible that the PRC2 complex acts globally at the -globin promoters. In addition, we have shown that TFII-I interacts with Suz12 in both K562 and MEL cells. There is only a slight increase in the Suz12 IP compared to our negative control in K562 cells suggesting that this may be a transient interaction. Our results suggest that Suz12 and possibly the PRC2 complex is actively recruited to the -globin promoter to aid in repression of the and -globin genes through the mono-methylation of H3K27. It has been shown that H3K27me1 levels are not affected in Suz12 knock-out mice [278]. However, Eed is required not only for diand tri-methylation, but also mono-methylation. Thus, it is possible that Eed in a complex with Suz12 actually is responsible for the modification of H3K27 at the -globin genes promoters. It is likely that the ubiquitously expressed transcription factor TFII-I functions in conjunction with stageand erythroid-cell-specific activities to repress the expression of the 76


globin gene. Repressor complexes have previously been characterized that mediate stage-specific repression of the -globin gene in adult cells [253]. Alternatively, activities that counteract the establishment of inaccessible chromatin mediated by TFII-I, HDAC3, and Suz12 are present in adult erythroid cells but may not be expressed or active at the embryonic stage. The reversal of gene order in the globin locus with respect to the LCR leads to the expression of the adult -globin gene in embryonic cells [102]. This result shows that proximity to the LCR can override activities involved in repressing -globin gene expression at the embryonic stage. In the normal configuration, an open and accessible chromatin domain extends over the LCR and the embryonic genes, while TFII-I and other proteins recruit histone-modifying activities to the -globin gene to establish an inaccessible chromatin domain. In the “genes-inverted” orientation, the -globin gene becomes part of an environment in embryonic cells that is dominated by activities recruited to and possibly spreading from the LCR. These activities override the stage-specific silencing of the -globin gene. It is possible that in the wild-type configuration, stage-specific insulators between the embryonic and adult domains prevent the spread of chromatin opening into the region containing the adult genes. Inversion of the genes with respect to the LCR would place the same insulators between the LCR and the embryonic genes. This may explain why the embryonic -globin gene is not expressed in a transgenic -globin locus in which the genes are inverted relative to the LCR [102]. The Felsenfeld group recently showed that USF interacts with a chromatin boundary element in the chicken -globin gene locus and recruits the coregulators p300, CBP, SET7/9, and PCAF [275]. It was discussed that the chromatin-modifying activities recruited by USF could establish an open, accessible chromatin region, counteracting the spread of heterochromatin. USF could play a similar role in establishing accessible chromatin in the LCR and the adult 77


globin gene promoter. We have shown that p300 and CBP, but not PCAF and SET7/9, are located at the promoter of the active adult -globin gene and also HS2. USF1 is observed to interact with p300 and CBP exclusively in an embryonic environment. These data suggest that USF1 recruits p300 and CBP to the promoter of the active -globin gene to aid in transcriptional activation. The ChIP analysis clearly shows that a reduction of USF activity not only reduces the modification of histone tails that are associated with accessible chromatin domains but also affects the recruitment of Pol II to the globin locus. Our data show that the association of p300 and CBP with LCR element HS2 and the maj-globin promoter, both of which associate with USF, is reduced in cells expressing A-USF. In addition, we have previously shown that RNA polymerase II dissociates from the LCR and globin genes during replication in synchronized K562 cells [277]. In these studies, we found that USF interacts with the LCR and globin genes before RNA polymerase II reassociates with the -globin gene locus. This result supports the hypothesis that USF recruits chromatin-modifying activities that confer or maintain an open chromatin conformation in specific regions of the -globin gene locus. The ablation of the genes encoding either USF1 or USF2 does not significantly affect the function of the hematopoietic system [193]. The ablation of both genes leads to an embryonic lethal phenotype, suggesting that the proteins can compensate for one another during embryonic development. The dominant-negative mutant protein that we used in this study inactivates USF1 and, to a somewhat lesser extent, USF2, which could explain the more dramatic effect on -globin gene expression. In summary, our data strongly suggest that the ubiquitously expressed helix-loop-helix proteins TFII-I and USF participate in restricting -globin gene expression to the adult stage of erythropoiesis (Figure 4-11). We propose that the primary function of these proteins is to recruit 78


protein complexes that regulate chromatin accessibility in the -globin gene locus. Tissueand stage-specific regulation of the -globin gene locus involves complex mechanisms, and to understand this process, it is important to determine the function of not only erythroid cell-specific activities but also ubiquitously acting factors that function together with cell type-specific proteins. 79


Figure 4-1. TFII-I and HDAC3 interaction. A) Coimmunoprecipitation experiment demonstrating interactions between TFII-I and HDAC3. K562 cells were lysed and subjected to immunoprecipitation (IP) with antibodies directed at either TFII-I or USF2. The precipitate was loaded onto a 7.5% denaturing polyacrylamide gel, and the gel was subjected to Western blotting with antibodies against HDAC3. The lane labeled no Ab represents the no-antibody control. B) K562 or MEL cell extract was precleared with anti ()-rabbit IgG beads and precipitated with -HDAC3 or IgG, and complexes were captured by incubation with anti-rabbit IgG beads. Complexes were eluted off the beads with Laemmli buffer and incubation at 95C for 10 min and loaded onto a 10% Ready gel (Bio-Rad). The membrane was probed with -TFII-I and then stripped and probed with -HDAC3 as a positive control. C) Double-ChIP experiment demonstrating simultaneous associations of TFII-I and HDAC3 with the -globin gene promoter in K562 cells. K562 cells were subjected to ChIP using antibodies against TFII-I. The precipitate was subsequently immunoprecipitated with antibodies against HDAC3. The DNA in the second precipitate was purified and analyzed by PCR using primers specific for the human or -globin genes or for a region between LCR HS2 and -3. The lanes labeled no Ab2 represent samples from the no-antibody control experiments. 80


A B Figure 4-2. TFII-I and HDAC3 are repressors of -globin gene expression in K562 cells. (A) To determine transfection efficiency, K562 cells were nucleofected with 0.5 g of siGLO RISC-free. Cells were collected on days 2 and 4, fixed, and stained with DAPI for nuclear visualization; siGLO is seen in red. (B) Knockdown of TFII-I, HDAC3, and USF2 proteins in K562 cells. K562 cells were nucleofected with 0.5 g of siGENOME SMARTpool targeting either TFII-I, HDAC3, USF2, or 0.5 g of siCONTROL nontargeting pool (Neg.) or mock transfected. A total of 20 g of protein was run on gels from cells collected on day 2 for TFII-I and HDAC3 Western blots, and 10 g of protein was loaded from cells collected on day 3 for the USF2 Western blot. Blots were probed with anti ()-TFII-I (upper left panel), -HDAC3 (upper middle panel), and -USF2 (upper right panel). Blots were stripped and reprobed with -GAPDH for loading control (bottom panels). (C) Relative and -globin expression of TFII-I, HDAC3, or USF2 knockdown K562 cells. On day 4, RNA was collected from K562 cells transfected as described above, reverse transcribed, and analyzed by real-time PCR. Expression is set relative to either non-targeting siRNA (Neg.) or mock transfected cells, with GAPDH as the internal reference. Error bars indicate standard error of the means. 81


C Figure 4-2. Continued. 82


MEL00.00050.0010.00150.0020.00250.003IgGSuz12 H3K27me3Fraction of Input -major -globin K562 00.00050.0010.00150.0020.00250.003IgGSuz12 H3K27me3 Fraction of Input -globin -globin A B K56200.0010.0020.0030.0040.005-globin-globin-globinHS2Fraction of Input MEL00.00040.00080.00120.00160.002-major-globinHS2Fraction of Input IgG H3K27me1 Figure 4-3. Suz12 and H3K27me ChIP analysis of K562 and MEL cells. A) Antibodies against Suz12 and H3K27me3 and non-specific IgG were used in a ChIP assay with K562 and MEL cells. Quantitative PCR (qPCR) was performed with primers which amplified the promoters of the genes as indicated. B) Antibodies against H3K27me1 and non-specific IgG were used for ChIP assay with K562 and MEL cells. qPCR was performed with primers which amplified the promoters of the genes as indicated and also HS2 for MEL cells. 83


Figure 4-4. Suz12 knock-down in K562 cells. K562 cells were nucleofected with Suz12 siRNA, non-targeting siRNA (neg), or mock transfected. A) Protein was collected after two days and ran on gels for Western blotting. Blots were probed with -Suz12 in the upper panel and then stripped and reprobed with -GAPDH for a loading control. B) Relative -, and -globin expression in Suz12 knock-down cells. RNA was collected, reverse transcribed, and analyzed by qPCR. Expression is set relative to either non-targeting siRNA (neg) samples or mock transfected cells, with GAPDH as the internal reference. 84


Figure 4-5. TFII-I and Suz12 interaction in K562 and MEL cells. K562 and MEL extracts were precleared with anti-rabbit IgG beads and precipitated with 2.5 g of -Suz12 for K562 and 5 g of -Suz12 for MEL and 2.5 g of -GATA1 (as negative control). The complexes were captured by incubation with anti-rabbit IgG beads. Complexes were eluted off the beads with Laemmli buffer and incubation at 95C for 10 min and loaded onto a 10% Ready gel (Bio-Rad). The membrane was probed with -TFII-I. 85


MEL00.0010.0020.0030.0040.0050.006IgGp300CBPFraction of Input -globin -major HS2 K56200.00050.0010.00150.0020.00250.003IgG p300 CBPFraction of Input -globin -globin -globin HS2 MEL00.00050.0010.00150.0020.00250.003IgG PCAF SET7/9 Fraction of Input -globin -major HS2 K56200.00050.0010.00150.0020.00250.003IgG PCAF SET7/9 Fraction of Input -globin -globin -globin HS2 A B Figure 4-6. PCAF, Set7/9, p300, and CBP localization at the -globin locus. K562 and MEL cells were subjected to ChIP analysis with antibodies against PCAF, Set7/9 (A) and p300, CBP (B). Purified DNA was analyzed by qPCR with primers as indicated. 86


Figure 4-7. Interaction of USF1 with p300 and CBP. K562 and MEL cell extract was precleared with anti-rabbit IgG beads and precipitated with -USF1, -p300, -CBP, or -HDAC3 (as negative control), and complexes were captured by incubation with anti-rabbit IgG beads. Complexes were eluted off the beads with Laemmli buffer and incubation at 95C for 10 min and loaded onto a 5% Ready gel (Bio-Rad). The membrane was probed with -p300 and then stripped and probed with -CBP. 87


Figure 4-8. USF binding to the -globin gene locus in MEL cells expressing A-USF. pTO/A-USF, TR (A-USF), and pTO, TR (control [Ctrl.]) MEL cells were induced with doxycycline. Cells were subjected to ChIP, immunoprecipitating with -USF1, -USF2, or no antibody (No Ab). Semiquantitative PCR was performed on samples, including input, using primers specific for the -major promoter, HS2, or the promoter. 88


Figure 4-9. Interactions of RNA Pol II, p300, and modified histones with the -globin gene locus in MEL cells expressing dominant-negative A-USF. MEL cells transfected with the A-USF expression vector (pTO/A-USF) or empty vector pTO were subjected to ChIP analysis with antibodies against H3K4me2, AcH3, RNA Pol II, p300, or CBP. DNA was purified from the immunoprecipitate and analyzed by qPCR with primers specific for the maj-globin promoter and LCR element HS2 (A) or the GAPDH promoter (B). The error bars represent the results from two independent experiments. Ctrl represents either no antibody or nonspecific IgG antibody. 89


Figure 4-9. Continued. 90


Figure 4-10. Analysis of MEL cells transiently transfected with pITRp543f2AUSF4. A) Diagram of pITRp543f2AUSF4 construct. B, C) MEL cells were transiently transfected with 1, 3, or 6 g of pITRp543f2AUSF4. Protein and RNA was collected after 48 hours. B) Expression of A-USF was determined by Western blotting, using -USF1 (C-terminal) antibodies, which detects both endogenous USF1 and A-USF. C) RNA was reverse transcribed and analyzed by quantitative PCR. GAPDH was used as the internal reference. D) Purified DNA from pITRp543f2AUSF4 transgenic mice (lanes 1, 2, and 3) and pITRp543f2-globin4 (lanes 4 and 5, negative controls for A-USF primers and positive controls for HS3 and HS3/2 primers) transgenic mice was used as template for PCR reactions using primers against A-USF, human HS3, or the flanking region of human HS3 and HS2. 91


Figure 4-10. Continued. 92


Figure 4-11. Model for -globin gene regulation by helix-loop-helix proteins USF and TFII-I. The -globin promoter consists of a TATA-like sequence (CATA) located 25 bp upstream of the transcription start site, an initiator with an overlapping E-box, and a downstream E-box element located at +60. We propose that a protein complex containing TFII-I, HDAC3, PRC2, and possibly other proteins interacts with the -globin promoter in embryonic and fetal erythroid cells. The modification of histones by HDAC3 and PRC2 confers or maintains inaccessibility of the globin gene to the transcription complex. In adult cells, USF interacts with the -globin promoter and recruits coactivator complexes that modify the chromatin structure to increase accessibility for transcription complexes. 93


CHAPTER 5 CALCIUM REGULATION OF -GLOBIN EXPRESSION THROUGH TFII-I AND USF Introduction Calcium plays a vital role in the anatomy , physiology and biochemistry of organisms and of the cell , particularly in signal transduction pathways. Ca 2+ ions are one of the most widespread second messengers used in signal transduction. Calcium enters the cytoplasm either through the cell membrane via calcium channels or from internal calcium storages . There, calcium can have a number of various actions depending on the cell type: secretory cells release vesicles , muscle cells contract, neurons release synaptic vesicles . Mouse erythroleukemia (MEL) cells provide an excellent model for studying the molecular mechanisms controlling terminal differentiation. Treatment with reagents such as dimethyl sulfoxide (DMSO), hexamethylene, bisacetamide, X-irradition, and hypoxanthine results in expression of erythroid-specific genes and loss of cellular immortality [279]. However, their mechanism is not known. The inducing agents trigger the pathways of differentiation, therefore continual exposure to the inducer is not necessary. Following exposure to the inducer, a latent period of eight to twelve hours occurs before the cells begin to differentiate. Changes in cytosolic calcium concentration have been suggested to play a role in inducing these early changes. In MEL cells induced with DMSO, EGTA (a calcium chelator) blocks the commitment to differentiate. The addition of excess calcium to the media results in a reverse of this block [280]. Additionally, A23187, a calcium ionophore which increases the permeability of membranes with high selectivity for calcium ions, abolishes the latent period which occurs with DMSO treatment and also induces the commitment to differentiate [280, 281]. However, despite these colonies being completely hemoglobinized, they do not express elevated levels of -globin or Band3 whose expression is characteristic of differentiated erythroid cells [281]. Only after the cells are 94


placed in medium free of the ionophore does -globin and Band3 transcripts accumulate. In contrast to these studies, Arrow and Macara have demonstrated that DMSO decreases cellular calcium levels in MEL cells [282]. In addition, intracellular calcium concentrations were measured in precursor erythroid cells at various stages (proerythroblast, basophil erythroblast, and normoblast erythroblast) and also red blood cells. Calcium concentration was seen to increase in cells from zero to 24 hours and then begin to decrease at 48 hours until it reaches the lowest point in red blood cells [283]. It is possible that calcium plays a different role in in vitro differentiation compared to in vivo. Therefore, despite a large number of studies the precise effect of calcium on erythroid differentiation is undetermined. It has recently been reported that TFII-I, which we have previously shown act as a repressor of -globin gene transcription, plays a role outside of the nucleus in calcium entry. Receptor tyrosine kinases and G-protein coupled receptors initiate intracellular calcium signaling by activating or -isoforms of phospholipase C (PLC) [284]. PLC catalyzes the generation of inositol-1,4,5-triphosphate (IP3) which triggers intracellular calcium release . PLCpromotes calcium entry through transient receptor potential (TRP) channels, such as TRPC3, by stimulating their surface expression [285]. A partial pleckstrin homology (PH) domain of PLCbinds to a complementary PH-like domain in TRPC3 and results in an increase of TRPC3 insertion into the plasma membrane [286]. This interaction is disrupted by TFII-I. When phosphorylated by Btk, TFII-I binds to a Src homology 2 (SH2) domain on PLC[229]. In addition, TFII-I binds to PLCthrough partial PH domains found in both TFII-I and PLC-. Thus, TFII-I competes with TRPC3 for binding to PLC-. The interaction of TFII-I and PLCtherefore results in a decrease of TRPC3 insertion into the plasma membrane, thus inhibiting calcium entry. 95


Members of the calpain family, a heterogeneous group of cysteine proteases, are involved in a variety of calcium-regulated processes, such as signal transduction, cell proliferation and differentiation, apoptosis, membrane fusion, and platelet activation [287]. The proteolytic domain of calpains configures to form an active catalytic pocket only when calcium is present which is bound by the EF-hand domain. The ubiquitous, classic calpains, -calpain and m-calpain, are heterodimers consisting of an 80 kDa subunit, which shares 55-65% sequence homology between the two proteases, and an identical 28 kDa regulatory subunit. These two calpains differ in the concentration of calcium required for activation; -calpain requirements in the micromolar range and m-calpain in the millimolar range [288]. Upon exposure to calcium, both the 80 and 28 kDa subunits undergo an autoproteolysis and express catalytic activity at calcium concentrations close to the physiological range. A large number of proteins have been found to be cleaved by calpains, such as cytoskeletal proteins, kinases, phosphatases, membrane-associated proteins, and some transcription factors. Unlike most proteases which cause extensive degradation of proteins, cleavage by calpain usually produces large, limited proteolytic fragments cleaved in the vicinity of the boundary of two domains. A protein destabilizing sequence, enriched in proline, glutamic acid/aspartic acid, and serine/threonine, in combination with structural features may be involved in substrate recognition by calpain [289]. Human erythrocytes have been found to contain both and m-calpain [290]. -calpain knock-out mice have been found to have hematopoietic homeostasis alterations, specifically defects in platelet aggregation and clot retraction [291]. USF proteins, which we have previously shown to activate -globin gene transcription, are cleaved by m-calpain [292]. Three major peptides with molecular masses of 18.5, 16.5, and 14.5 kDa result from the incubation of recombinant USF with m-calpain in the presence of calcium. 96


The DNA binding domain is suggested to reside within the 18.5 and 16.5 kDa peptides. Other proteases, such as V8 protease, papain, and trypsin, have also been shown to cleave USF but creating products of molecular masses different from that of m-calpain. Also, unlike m-calpain, longer digestion with these proteases leads to the complete disappearance of the cleavage products. The product from proteolysis of USF retains specific DNA-binding ability but is not able to stimulate transcription. It has been suggested that limited proteolysis may be involved in the activity of USF. M-calpain has been found in the nucleus and thus may play a role in USF regulation or turnover. We here propose that calcium concentration regulates -globin gene expression (Figure 5-1). The cellular localization of TFII-I has been found to be regulated through phosphorylation. We therefore propose that in embryonic erythroid cells, TFII-I is located to the nucleus and acts as a repressor of -globin transcription. Since TFII-I would not be found in the cytoplasm, calcium entry through the plasma membrane would not be inhibited. Higher calcium levels would ensure that m-calpain is active which would result in the cleavage of USF. USF then would not be able to act as an activator of -globin gene transcription in embryonic erythroid cells. In adult erythroid cells, we propose that TFII-I is localized in the cytoplasm and not acting to repress -globin gene expression. It is instead inhibiting the entry of calcium. M-calpain would not be active, thus USF, in its complete form, would be able to act as an activator at the -globin promoter. We here provide evidence for the above proposed model of calcium regulation of -globin expression. Results USF has been found to be cleaved into three peptides of molecular weights between 19 and 14 kDa by m-calpain. If m-calpain is active in erythroid cells we expect to detect a similar pattern. We examined the molecular weight patterns of USF in K562 cells (embryonic erythroid 97


cells), MEL cells (adult erythroid cells), and MEL cells induced with DMSO (Figure 5-2). Three bands are observed in K562 cells between 15 and 20 kDa when probed with antibody against USF2 and bands of similar size when probed with antibody against USF1. However, in MEL cells only the smallest band of approximately 15 kDa is observed when probed with -USF2. In induced MEL cells, in which we observe a fifteen fold increase in maj-globin expression (data not shown), no smaller products are observed when probed with -USF2 and only the higher molecular weight band when probed for -USF1. In fact, an increase in full length USF2 is observed in the induced MEL cells compared to uninduced MEL cells and K562 cells. This data suggests that m-calpain is active in K562 cells and partially in MEL cells and acts to cleave USF proteins. In MEL cells, which are induced to express maj-globin, m-calpain is not active and USF proteins are intact. To determine if the small cleavage products that we observed are actually USF peptides formed by m-calpain cleavage, we treated induced MEL extract with recombinant m-calpain (Figure 5-3). The same bands between 15 and 20 kDa are seen with extract treated with m-calpain in the presence of calcium and magnesium. In the reactions with no calcium or magnesium, cleavage products are not observed suggesting that m-calpain is only catalytically active in the presence of these ions and able to cleave USF. A23187, an ionophore which increases intracellular calcium levels, has been shown to induce erythroid cell differentiation [281]. Therefore, Babak Moghimi treated MEL cells, which were induced with DMSO, with A23187 to observe there is a change in -globin expression (Figure5-4). A significant decrease in -globin expression is observed in the A23187 treated MEL cells compared to the untreated cells. The decrease in -globin expression does appear to be A23187 concentration dependent. MEL cells treated with 0.45 g/ml A23187 have a seven98


fold decrease in expression while cells treated with 0.9 g/ml have a seventy-fold decrease. A smaller decrease in -globin expression is observed in uninduced MEL cells, although the effect again is concentration dependent. There is only a slight decrease with 0.45 g/ml A23187 treatment but a four-fold decrease with 0.9 g/ml. These data suggest that an increase in intracellular calcium levels in MEL cells results in the decrease of -globin gene expression. According to our model, TFII-I is predominately localized in the nucleus in K562 cells acting to repress -globin gene transcription. In MEL cells, we hypothesize that TFII-I is localized predominately in the cytoplasm where it acts to inhibit calcium entry. To test this theory, Babak Moghimi visualized HA-tagged TFII-I in K562 cells through immunofluorescence (Figure 5-5A). TFII-I is seen to be strongly localized to the nucleus with little seen in the cytoplasm. There are a number of cells in which TFII-I was not visualized. This is most likely due to cells losing the HA-TFII-I expression vector after stable selection or incomplete induction of HA-TFII-I expression with doxycycline treatment. In addition, we collected nuclear extracts from K562, MEL, and induced MEL cells to observe TFII-I localization (Figure 5-5B). TFII-I is seen in the nucleus of all three cell types, but two bands are observed in K562 cells, suggesting there is an additional TFII-I isoform in the nucleus in these cells which may have a repressive role. In MEL cells, this isoform should be localized to the cytoplasm where it may act to inhibit calcium entry. The TFII-I isoform seen in all three cell types may not act at the -globin promoter or act to maintain a basal level of transcription. Discussion Calcium is a ubiquitous secondary messenger that regulates a wide range of activities in every cell type. It acts both in the cytoplasm and the nucleus to activate signaling pathways [293]. In many cases these signaling cascades result in the activation of transcription factors and kinases which regulate gene expression [294]. For example, the CRE binding protein (CREB) 99

PAGE 100

binds to the cAMP responsive element (CRE) of the c-fos gene upon calcium signaling and stimulates gene expression [293]. We have here presented evidence that calcium is able to regulate -globin expression not through the activation of a transcription factor but through the degradation of transcription factor USF. USF, an activator of -globin transcription, has been shown to be cleaved into three small peptides by m-calpain, a calcium dependent protease, thus inactivating the protein. Our evidence supports the hypothesis that USF cleavage is regulated by the ability of TFII-I to modulate calcium entry. Findings of high levels of USF cleavage products in K562 cells and nuclear localization of TFII-I suggest that the absence of cytoplasmic TFII-I allows for the entry of calcium and the activation of m-calpain. USF is thus not able enhance -globin transcription. Two of the USF peptides contain the DNA-binding domain, thus these peptides can still bind to the -globin promoter but they lack activity due to the loss of the transcriptional activation domain. These peptides may act as dominant-negative proteins. Only one isoform of TFII-I is found in the nucleus of induced MEL cells, where the adult -globin gene is expressed. The other isoform is therefore most likely localized to the cytoplasm where it inhibits the entry of calcium. Inducing MEL cells to differentiate results in the absence of USF cleavage and a significant increase in maj-globin expression. Treatment of induced MEL cells with a calcium ionophore results in the appearance of USF cleavage products and a decrease in -globin expression. These results suggest that in induced MEL cells cytoplasmic TFII-I blocks calcium entry, which inactivates m-calpain, and thus USF, in its full form, is able to activate -globin transcription. 100

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Figure 5-1. Bungert laboratory model of calcium regulation of -globin expression. A) In embryonic erythroid cells, TFII-I is located to the nucleus and acts as a repressor of -globin transcription. Calcium entry through the plasma membrane is not inhibited and higher calcium levels would ensure that m-calpain is active which would result in the cleavage of USF. B) In adult erythroid cells, TFII-I is localized in the cytoplasm, inhibiting the entry of calcium. M-calpain is not active, thus USF, in its complete form, acts as an activator at the -globin promoter. 101

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Figure 5-2. USF cleavage pattern in K562 and MEL cells. Protein extracts from K562 cells, MEL cells, and MEL cells induced with 2% DMSO for 48 hours were ran on a 12% gel for Western blotting. The membrane was probed with -USF2 (C-terminal) and then stripped and re-probed with -USF1 (C-terminal). 102

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Figure 5-3. Induced MEL protein extract treated with recombinant m-calpain. MEL cells were induced with 2% DMSO for 48 hours. Protein extract was collected and 20 g was incubated with 0.1 or 1 unit of rat recombinant calpain-2 (208718, Calbiochem), with or without 5 mM CaCl 2 and 5 mM MgCl 2 at 30C for 10 minutes. Mixture was boiled in Laemmeli buffer and ran on a 12% gel for Western blotting. The membrane was probed with -USF1 (C-terminal) and -USF2 (C-terminal). 103

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A 00.511.522.5InducedInduced + 0.45g/ml A23187Induced + 0.9g/ml A23187Non-inducedNon-induced +0.45 g/mlA23187Non-induced +0.9 g/mlA23187Ratio of Beta-globin expression Figure 5-4. Effect on -globin expression upon treatment with calcium ionophore. RNA from MEL cells induced with DMSO cells treated with A23187 and also RNA from uninduced MEL cells treated with A23187 was extracted, reverse transcribed, and analyzed by qPCR. Values are relative to -actin. 104

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Figure 5-5. Intracellular localization of TFII-I. A) Stable K562 pTO/HA-TFII-I cells were induced with 1 g/ml doxycyline for 36 hours. Cells were fixed, hybridized with -HA tag, and then the labeled secondary antibody. A drop of Vectashield-DAPI was placed on the cells and then visualized under a fluorescence microscope. B) Nuclear protein extracts were collected from K562, MEL, and MEL cells induced with 2% DMSO for 48 hours and ran on a 5% gel for Western blotting. Membrane was probed with -TFII-I. 105

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CHAPTER 6 CONCLUSIONS AND FUTURE DIRECTIONS The Role of TFII-I and USF in -globin Gene Expression Expression of -globin genes is regulated at multiple levels. The LCR is known to play a crucial role in both the high-level and erythroid specific expression of these genes. Rearranging the genes relative to the LCR disrupts their normal restricted developmental expression. However, is has been found that the developmental expression of the -globin genes remains uninterrupted in the absence of the LCR. Thus, proximal cis-elements which are bound by trans-acting factors are also crucial to the regulation of the expression of these genes. Understanding how transcription factors influence -globin expression is crucial in the understanding of hemoglobin switching. I have determined the function of ubiquitously expressed, helix-loop-helix proteins TFII-I and USF in -globin expression. Over-expressing TFII-I decreases, while reducing the level of TFII-I increases -globin transcription. In embryonic stem cell erythroid differentiation assays, TFII-I is found to associate with the adult -globin promoter early, suggesting that it acts as a repressor. Over-expressing USF increases, while reducing the function of USF1 and USF2 decreases -globin transcription. In addition, USF1 proteins associate with the adult -globin promoter later in embryonic stem cell differentiation, suggesting that it acts an activator of -globin expression. TFII-I and USF1 are seen to associate in embryonic erythroid cells while USF1 and USF2 associate in adult erythroid cells. These proteins may differentially associate for the proper regulation of -globin expression. As the erythroleukemia cell lines used in these studies do not accurately reflect the identities of erythroid cells that differentiate during human or mouse development, it will be important to determine the functions of TFII-I and USF during the development and 106

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differentiation of erythroid cells in an animal model. We have created transgenic mice which express the dominant-negative USF (A-USF) under control of the LCR and the -globin promoter. A-USF, thus, should be expressed exclusively in erythroid cells. While USF1 knock-out mice are viable and fertile and USF2 knock-out mice have growth defects, knocking-out both USF1 and USF2 has shown to be embryonic lethal [193]. Thus, it is essential that this dominant-negative USF, which affects the function both USF1 and USF2, only be expressed in erythroid cells. This will also allow us to determine the direct effect of A-USF on -globin expression without interfering with USF function in other cell types. Also, by using a dominant-negative protein which affects both USF1 and USF2 function we eliminate the possibility that reducing expression of one protein will upregulate the expression of the other. Elevated levels of USF2 are seen in USF1 knockout mice and are expected to offset the lack of USF1, thus a fairly normal phenotype is observed [193]. Both USF1 and USF2 play a role in the regulation of -globin expression, thus inhibiting the activity of both is crucial in understanding their in vivo function. We would expect these A-USF transgenic mice to be anemic, however early results suggest that heterozygous mice are phenotypically normal. It will be necessary to create homozygous mice to increase expression levels of A-USF. Also, phenylhydrazine (PHZ) injections will be utilized to induce hemolytic anemia and to convert the spleen primarily to the site of erythropoiesis. The spleen will be collected and -globin levels will be compared to wild type mice. We would expect there to be a decrease in -globin gene expression in the transgenic mice. We are also in the process of creating a construct similar to that used for the A-USF transgenic mice to create transgenic mice expressing a dominant-negative TFII-I. In place of A-USF will be the construct for p70, a dominant-negative TFII-I. Thus p70 will also be exclusively 107

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expressed in erythroid cells. No TFII-I knockout mice have been generated, so it is not known what effect eliminating the activity of this protein would have on the system as a whole. Expressing p70 in erythroid cells, we would expect to see an effect early in erythroid development. RNA interference (RNAi) is a tremendous tool to assay the activity of a specific protein. It has many benefits when used in vivo compared to gene ablation experiments. Creating a targted gene deletion can be quite time-consuming. Also, eliminating gene expression completely may be deleterious to the organism, but reducing it to a certain degree may allow effects to be seen. In addition, a classical targeted deletion, even if inducible, is irreversible, whereas an inducible siRNA mediated “knock-down” approach is not. An inducible RNAi system allows researchers to reduce expression of a target gene at different stages of development and in a tissue specific manner. This approach may also be used to understand the in vivo role of TFII-I and USF in the regulation of -globin expression. We have transgenic mice which express the tetracycline repressor (tetR) protein. When bred with mice containing a construct with a shRNA (short hairpin RNA) under the control of a tetracycline operator (tetO) in a modified U6 promoter, the tetR will silence the shRNA construct. Ohkawa and Taira first reported the successful regulation of expression by this system [295] and many researchers have used it to express a shRNA to date [296-298]. Addition of tet or doxycycline inhibits tetR binding to tetO, allowing transcription. The level of doxycycline can be titrated to allow various levels of expression. Doxycycline can be administered to the mother of the transgenic mice while they are still in the fetus and will cross the placental barrier. Therefore, expression of TFII-I or USF can be regulated throughout development in these animals and the role of these proteins play in the -globin expression at various developmental stages can be determined. 108

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I have generated mouse TFII-I shRNA constructs, however no knock-down of TFII-I was observed in stable MEL cells. There has been much research done in order to create algorithms for functional siRNA sequences. However, even if the sequence does fit the algorithm, there is no guarantee that the siRNA will efficiently reduce expression of the protein of choice. In order to guarantee success, it will be best to find siRNA sequences that have previously been used. The chromosome conformation capture (3C) assay allows for the examination of long range interactions between genes and regulatory regions. It has been utilized to determine the interaction of distant elements of the -globin locus. In non-expressing tissue, the -globin locus adopts a linear conformation. In erythroid cells, the hypersensitive sites of the locus cluster together, forming an active chromatin hub (ACH). The active globin gene is found to be within close proximity to the ACH with the intervening chromatin, containing the inactive globin genes, looping out [87]. Deletion of the -globin promoter leads to a reduction of interaction with the ACH. However, a substantial interaction does remain suggesting that multiple interactions between elements in the ACH and the -globin gene are required [99]. In agreement with the competitive interaction of the -globin genes with the LCR, this deletion results in an increase of transcription of the and -globin genes. It has been found that trans-factors are crucial for the formation of these interactions. EKLF, GATA1, and FOG-1 are essential for the interaction between the -globin gene and the LCR [115, 122]. Using the 3C method, we would be able to determine if TFII-I or USF play a role in the formation of the ACH. By either over-expressing or expressing a dominant-negative TFII-I or USF, the impact on the interaction between the -globin gene and the LCR could be observed in both embryonic and adult erythroid cells. USF proteins may in fact aid in the interaction between HS2 and the -globin promoter. Hydrodynamic measurements of USF protein-DNA complexes 109

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have shown that USF factors can exist as a bivalent homotetramer [167]. USF homotetramers could potentially interact with two DNA recognition sites, such as HS2 and the -globin promoter, which would allow for DNA looping [299]. In an embryonic environment, TFII-I may aid in inhibition of the of the -globin promoter coming in close proximity to the LCR by recruiting factors which impede this interaction. Recruitment of Chromatin Modifying Enzymes by TFII-I and USF to the -globin Locus It is becoming increasingly evident that one way transcription factors aid in the repression or activation of gene expression is by the recruitment of co-factors which modify histones or mobilize nucleosomes at promoters. In order to understand how TFII-I and USF proteins are able to regulate -globin gene expression, I investigated if TFII-I and USF interacted with chromatin modifying enzymes. TFII-I is found to interact with HDAC3 preferentially in embryonic erythroid cells and both TFII-I and HDAC3 are found at the -globin promoter in an embryonic environment. TFII-I also interacts with Suz12 of the PRC2 complex in both embryonic and adult erythroid cells. USF proteins are found to interact with p300 and CBP preferentially in adult erythroid cells. USF function is required for the recruitment of p300 and CBP to HS2 and the adult -globin promoter. By recruiting these specific chromatin modifying enzymes, TFII-I is able to repress while USF is able to activate -globin expression. The dominant-negative protein used to investigate the function of TFII-I (p70) lacks the C-terminal activation domain, consisting of R5 and R6, and is still able to bind to DNA. It has been determined that HDAC3 binds to the R3 and R4 domains, thus this interaction is not disrupted in cell lines expressing p70 [247]. Therefore, it would be interesting to determine if there is a larger effect on -globin expression by expressing a dominant-negative TFII-I which lacks the DNA binding domain in K562 cells. TFII-I may interact with additional proteins at R5 and R6, such as 110

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Suz12, which aid in the repression of -globin expression. Thus, we would expect to see a much more dramatic effect with a total loss of function of TFII-I. Another method to investigate TFII-I function would be to look at the difference in proteins bound or chromatin marks at the -globin locus, using ChIP, in cells where TFII-I is knocked-down by RNAi or expressing the dominant-negative DNA binding mutant TFII-I. Our current method of RNAi does not allow for the collection of a large number of cells, which is needed for ChIP, because of its transient nature. Stable cell lines which carry a construct containing a shRNA against TFII-I would be needed. In these knock-down or dominant-negative cell lines, it would be interesting to see if there is an increase in histone acetylation or decrease in H3K27me1 levels at the -globin promoter. In order to further investigate the role of the PRC2 complex on -globin gene regulation, it will be important to determine the exact mechanism of its repression. PRC2 repression is mediated by methylation of H3K27. We and others have found a lack of di-, and trimethylation of H3K27 at the -globin locus but monomethylated H3K27 marks are present. Suz12 and EZH2 are not needed for H3K27me1 but EED is required [278, 300]. Therefore, it will be important to determine if EED is present at the -globin locus and if its knock-down results in an increase of -globin expression and a decrease in H3K27me1. Because we have observed Suz12 at the -globin promoter, we would hypothesize that Suz12 is present in the complex with EED which monomethylates H3K27 but may not be required for activity. Since PRC2 histone modifications are thought to be docking sites for the PRC1 complex, it will be important to see if this complex is located at the -globin promoter as well. Developmental-Stage Specific Regulation of -globin Expression by TFII-I and USF TFII-I and USF are ubiquitously expressed proteins, therefore it will be essential to determine how they are able to regulate -globin expression in a stage specific manner. It is 111

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known that phosphorylation of transcription factors is an effective mechanism by which DNA binding and transcriptional activity can be modulated. Both TFII-I and USF are regulated by signal transduction pathways. Phosphorylation of TFII-I and USF as a result of these cascades may regulate their developmental stage-specific function at the -globin promoter. TFII-I is sequestered in the cytoplasm until it is phosphorylated. Upon phosphorylation, it is able to dimerize and translocate into the nucleus. TFII-I has been found to be regulated by various tyrosine kinases: Btk in hematopoietic cells of B and myeloid lineages and JAK2, c-Src, and 190 RhoGAP in non-lymphoid cells. Phosphorylated USF1 has an increased binding affinity to DNA. Protein kinases such as p38, PKC, PKA, and phophatydylinositol-3 kinase (PI3K), have been found to phosphorylate USF1. Hematopoiesis is controlled by intricately regulated signal transduction cascades that are mediated by cytokines and their receptors. It has been determined that JAKs, mitogen-activated protein (MAP) kinases (including the p38, ERK, and c-Jun families), PI3K, and the Src family of kinases are activated by a variety of hematopoietic growth factors and their cytokines [301]. These kinases have various functions in the differentiation process. Therefore, it is possible that developmental-specific signal transduction pathways which activates any one or a combination of these kinases results in the regulation of TFII-I and USF function. According to our model, we would hypothesize that in an embryonic environment TFII-I would be phosphorylated resulting in its translocation to the nucleus where it represses -globin transcription. Conversely, in an adult environment, we would hypothesize that USF1 is phosphorylated which increases its affinity to bind to the -globin promoter and HS2 where it enhances -globin transcription. To test these hypotheses, phospho-specific antibodies against TFII-I and USF could be used to determine if these modified proteins are present in K562 or 112

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MEL cells. ChIP assays could be used to determine if the phosphorylated protein is bound to the -globin promoter. In addition, immunofluorescence assays could be done to determine the localization of these phosphorylated proteins. A mutant TFII-I or USF, in which the phosphorylation site is mutated, could be over-expressed in K562 and MEL cells and the effect on -globin expression could be determined. ChIP and immunofluorescence assays could also be utilized to determine the effect of these mutant proteins. It would also be important to determine which kinase acts to phosphorylate TFII-I or USF in this developmental stage-specific manner. Reducing expression levels of suspected kinases by RNAi and observing the effect on the phosphorylation status of TFII-I or USF would help to elucidate this question. It may also be possible that interaction with developmental stage-specific proteins regulates TFII-I and USF effect on -globin expression. Currently Babak Moghimi is creating K562 and MEL cell lines which over-express a HA-, Flag-tagged TFII-I. By using pull down assays and mass spectrometry, he will be able to determine TFII-I interacting proteins. It will be especially interesting to see if there is a difference in protein interaction in the two different cell lines. The interacting proteins may regulate TFII-I so it is able to repress -globin transcription in embryonic cells but not adult. A similar approach can be taken to determine what proteins interact with USF in K562 vs. MEL cells. Isoforms of TFII-I may also aid in TFII-I’s stage-specific regulation of -globin expression. Recently is has been determined that the and -isoforms in murine fibroblasts have distinct subcellular localizations and mutually exclusive transcription functions on the c-fos gene in the context of growth factor signaling [172]. The -isoform was found to regulate c-fos in the resting state, while the -isoform translocates into the nucleus upon signaling and activates c-fos. Thus, it is apparent that the different TFII-I isoforms can be specifically activated and have 113

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their own specific function. Our preliminary data suggests that both the and -isoforms are present in the nucleus in K562 cells, but only the -isoform in MEL cells. This suggests that the -isoform acts to repress -globin expression in embryonic erythroid cells and is exported to the cytoplasm in adult cells. To test this further, isoform-specific antibodies could be used in ChIP and immunofluorescence assays. Calcium Regulation of -globin Expression through TFII-I and USF Calcium has been suggested to play a role in the process of erythroid cell differentiation. Increased calcium levels have shown to effect early cellular events which lead to differentiation. However, a calcium ionophore, A23187, has been shown to inhibit transcription of -globin [281]. Calcium has also been found to be affected by TFII-I and to affect USF. Recent reports indicate that cytoplasmic TFII-I inhibits the entry of calcium through the plasma membrane, while USF is cleaved by a calcium dependent protease, m-calpain. We have shown that USF cleavage products are abundant in K562 cells, with less in MEL cells, and very little in induced MEL cells. Treatment of induced MEL cells with a calcium ionophore results in the cleavage of USF and decrease of adult -globin expression. TFII-I is found to localize to the nucleus in K562 cells. These results support our model that in embryonic erythroid cells, TFII-I is located to the nucleus which allows the entry of calcium, thus activating m-calpain leading to the cleavage of USF. Thus, -globin expression is repressed. In adult erythroid cells, TFII-I is located to the cytoplasm, inhibiting calcium entry, which inhibits m-calpain activity leading to USF activation of -globin expression. A TFII-I mutant lacking the nuclear localization signal (NLS) is being created by Babak Moghimi in order to determine the effect of TFII-I in the cytoplasm in K562 cells or uninduced MEL cells. We would expect that increased cytoplasmic TFII-I would decrease calcium levels, decrease USF cleavage, and thus increase -globin expression. 114

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M-calpain activity is not only regulated by calcium levels, but also by the ERK/MAP kinase pathway [281]. Growth factor induced phosphorylation of serine residue 50 of m-calpain by ERK leads to its activation. ERK activation of m-calpain was found in the absence of excess calcium suggesting that activation of m-calpain by phosphorylation could be an alternative to, or coordinate with, activation by calcium fluxes. In addition, TFII-I has been found to be phosphorylated by and interact with ERK [302]. The interaction between TFII-I and ERK results in their translocation from the cytoplasm to the nucleus. Therefore, it will be important to determine what function ERK plays in the regulation of -globin expression. This can be accomplished by reducing expression levels of ERK through RNAi in both K562 and MEL cells. The effect on -globin expression, USF cleavage, and TFII-I localization should be determined. Over-expression of a mutant m-calpain containing a mutation at serine residue 50 would determine if cleavage of USF and corresponding repression of -globin expression is dependent upon ERK phosphorylation or an increase in calcium levels. Determining the intracellular calcium levels in K562 and MEL cells would shed light on whether calcium is essential for m-calpain activation. We would expect the calcium concentration to be higher in K562 cells than in MEL cells. We have seen no effect on USF cleavage products or -globin expression when m-calpain expression is decreased by siRNA treatment. Because this is a transient assay, m-calpain expression may not be reduced long enough to see an effect. The USF cleavage products may be very stable and thus a decrease of m-calpain for three or four days does not effect their accumulation. Also, if the cleavage products act as dominant-negative proteins, a small amount may be enough to disrupt the function of USF and thus no change in -globin expression would be seen. Also, m-calpain is not completely eliminated in these cells, a small amount still remains. 115

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This may be enough m-calpain to cleave USF. Therefore, to test directly whether cleavage of USF by m-calpain represses -globin transcription, calpastatin, an inhibitor of m-calpain, could be over-expressed in K562 cells or the cells could be treated with a calpain inhibitor. Additionally, co-immunoprecipitation assays could be performed to see if m-calpain does interact with USF in K562 cells and not in induced MEL cells. Because calcium plays so many roles in various pathways, it will be crucial to determine that the effect we are seeing in our experiments is not due to off-target effects. Summary The work presented here elucidates the role of factors, TFII-I and USF, in the developmental stage-specific expression of -globin gene through the recruitment of histone modifying enzymes and the regulation of intracellular calcium levels. This knowledge can be applied to understand the regulation of other genes that are controlled by these proteins. This work also will aid in developing gene therapy constructs which express -globin at proper levels. 116

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BIOGRAPHICAL SKETCH Valerie Jean Crusselle-Davis was born on July 2, 1979 in San Diego, California, to Vic and Stephanie Crusselle of Cocoa Beach, Florida and Oasis, Utah, respectively. During elementary and middle school her family moved quite frequently. Valerie attended Suffield High School, in Suffield, Connecticut, graduating eighth in her class in 1997. She then attended the University of Utah and graduated in May of 2001 with a B.S. degree in biology with a minor in chemistry. Valerie was awarded an internship at Pacific Northwest National Laboratory, in Richland, Washington, the summer of 2001. She stayed on for a year before starting in the Interdisciplinary Program in Biomedical Sciences in the College of Medicine at the University of Florida in the fall of 2002. Valerie was awarded a Grinter Fellowship and a University of Florida Alumni Fellowship upon entering the IDP. During her time at the University of Florida, Valerie won first place in the Medical Guild research competition and the Howard Hughes Medical Institute Science for Life Graduate Student award. She graduated with her PhD from the Department of Biochemistry and Molecular Biology in May 2007. Upon graduating, Valerie will be working in Dr. Trevor Archer’s laboratory at the NIEHS as a post-doctoral fellow investigating the role of chromatin structure on gene expression and its impact on disease. Long term, she would like to teach at the collegiate level or work in biotechnology. 142