Gerd A. Blobel - US grants

All hematopoietic cell types arise from pluripotent stem cells through multiple steps of lineage commitment and subsequent maturation. Cellular decisions regarding proliferation and differentiation are tightly controlled to ensure homeostasis of all circulating blood cells. Disruption of this control can lead to hematological aplasias or malignancies. The efforts of this laboratory are directed towards understanding the development of the erythroid cell lineage by focusing on the transcription factor GATA-1. GATA-1 participates in the regulation of virtually all erythroid-restricted genes including the globin genes. It causes cell cycle arrest and prevents apoptosis of erythroid precursor cells. This project centers around our recent observation that the transcriptional integrator CBP serves as cofactor for GATA-1 and is required for erythroid cell maturation. In addition to GATA-1, CBP regulates various transcription factors involved in cellular differentiation and is associated with translocations found in certain types of leukemias. CBP possesses histone acetylase activity towards all four core histones. Recently, we have discovered that CBP acetylates GATA-1 specifically at functionally important sites. Under Aim 1 we will determine the precise residues of GATA-1 acetylated in vitro and in vivo. Subsequently, we will monitor acetylation of GATA-1 in vivo during cell cycle progression and cellular differentiation. In Aim 2 we will analyze the functional consequences of GATA-1 acetylation through biochemical assays and through gene complementation experiments using a unique GATA-1-deficient erythroid cell line. This cell line allows the testing of GATA-1 constructs in the physiological environment of maturing erythroid cells. Studies outlined in Aim 3 are designed to delineate the transcriptional pathways by which CBP controls erythroid maturation. Using various cellular assays, we will determine which domains of CBP are required for erythroid differentiation and GATA-1 activation. Together, these studies should provide new insights into the mechanisms by which cellular differentiation and tissue-specific gene expression is accomplished and might lead to novel approaches for therapy of hematopoietic disorders.

DESCRIPTION (provided by applicant): Transcription factors control gene expression by recruiting high molecular weight protein coactivator complexes to the regulatory regions of genes. Some of these complexes contain chromatin modifying enzymes. One class of such enzymes consists of histone acetyltransferases which includes the widely expressed molecules CBP, its close relative p300, and the p300/CBP-associated factor PCAF. CBP/p300 and PCAF are critical targets of viral oncoproteins which interfere with differentiation and promote cell cycle progression. In addition, the CBP and p300 genes are rearranged in chromosomal translocations associated with certain forms of leukemia. Recent evidence suggests that CBP/p300 and PCAF are regulated by signals that control cell growth and differentiation. The goal of the proposed studies is to understand the roles of CBP and PCAF during the differentiation of hematopoietic cells. Hematopoiesis serves as an ideal model system in which to study the processes of lineage commitment, cell maturation, and cell cycle exit. The hematopoietic transcription factor NF-E2 is a key regulator of erythroid and megakaryocytic gene expression. Our preliminary studies show that NF-E2 associates with and is acetylated by CBP and PCAF. Experiments in Specific Aim 1 examine the molecular and biological consequences of NF-E2 acetylation. Our preliminary results also indicate that PCAF protein levels are differentially regulated upon differentiation of distinct hematopoietic cell lineages. Specific Aim 2 examines the role of PCAF regulation during hematopoietic cell differentiation. Furthermore, this Aim will analyze the activities and subunit compositions of the CBP and PCAF complexes during hematopoietic differentiation. Together, these studies will lead to an improved molecular understanding of acetyltransferases which stand as potential targets for pharmacological intervention in various hematological disorders.

Sickle cell disease is caused by a point mutation the beta-globin gene. Under hypoxic conditions, hemoglobin[unreadable] containing mutant beta-globin protein (HbS) forms insoluble polymers, leading to defects in red cell shape,[unreadable] flexibility and adhesion. Elevated levels of y-globin reduce hemoglobin polymerization and improve the[unreadable] clinical manifestation of the disease. Therefore, treatment of patients is aimed at raising expression of the y[unreadable] globin gene, which is, normally silent in adult life. This application proposes to explore two approaches to raise[unreadable] y-globin levels. Specific Aim I focuses on the use of synthetic DNA ligands, called polyamides, to raise the y[unreadable] to beta-globin ratio. Polyamides are low molecular weight, cell permeable polymers consisting of pyrrole and[unreadable] imidazole derivatives that brad to predetermined DNA sequences with specificities and affinities approaching[unreadable] those of natural DNA binding proteins. In collaboration with Dr. Peter Dervan whose laboratory is at the[unreadable] forefront in developing polyamides, we synthesized and purified several polyamides designed to inhibit[unreadable] transcription factor binding to essential reomalatory sites in the b-globin gene promoter. We will examine these[unreadable] polyamides in primary human bone marrow and umbilical cord erythroid cells for their effectiveness in[unreadable] inhibiting beta-globin expression in vivo. In addition, we will design new polyamides with the goal to activate y[unreadable] globin gene expression directlv. Studies in Specific Aim II will test a novel viral vector for its capacity to[unreadable] express a transgene in a fashion that is resistant to gene silencing. One major limitation of viral vectors is:[unreadable] that they are often subject to gene silencing, especially in hematopoietic (HS) and embryonic stem (ES) ceils[unreadable] and their differentiated progeny. Virally induced gene silencing is associated with deacetylation of histones,[unreadable] and treatment of cells with deacetvlase inhibitors leads to reactivation of viral gene expression. We have[unreadable] produced a viral vector that contains a modified histone acetvltransferase that binds specifically to the viral[unreadable] DNA sequence. The proposed studies wil! test this vector s ability to express a transgene at high levels and in a[unreadable] fashion resistant to gene silencing in HS and ES cells. The long term goal of this Specific Aim is to use this[unreadable] vector for y-globin gene expression in HS cells of test animals and ultimately patients with sickle cell disease.

[unreadable] DESCRIPTION (provided by applicant): The transcription factor GATA-1 is essential for the development of the erythroid and megakaryocytic lineages. GATA-1 regulates the expression of all erythroid-restricted genes examined to date, including globins. Mutations in the GATA-1 gene occur in patients with anemias, thrombocytopenias, and leukemias. Here, we focus on the mechanism by which GATA-1 regulates gene is expression. Previous work funded under this grant showed that GATA-1 associates with the acetylase CBP in vivo and in vitro and that GATA-1 is acetylated by CBP at conserved, functionally important sites. Restoration of GATA-1 activity in the GATA-1-deficient cell line G1E leads to localized increases in CBP recruitment and concomitant histone acetylation at the beta-globin locus in vivo, indicating that GATA-1 acts in part by regulating chromatin structure. Specific Aim #1: Given the importance of site-specific modifications of histones, we will determine the exact sites within histones that are acetylated (or otherwise modified) in response to GATA-1 activation in vivo. This will serve in part to understand which chromatin modifiers are recruited by GATA-1 and form the basis for studies examining the molecular consequences of targeted histone modification. Specific Aim #2: The essential GATA-1 cofactor FOG-1 is required for histone modifications at certain GATA-1-dependent regulatory regions. This Aim addresses the role of FOG-1 during GATA-1-induced chromatin remodeling. Specific Aim #3: Preliminary results using microarrays suggest that GATA-1 represses nearly as many genes as it activates. Repression of some of these, which includes the cell cycle regulator c-myc, requires cooperation between GATA-1 and FOG-1. Little is known about the repressive functions of GATA-1. This Aim examines the roles of GATA-1 and FOG-1 during transcriptional repression at the c-myc locus. An important aspect of this work will be the regulation of chromatin structure that accompanies gene repression and the conditions that determine whether GATA- 1 will activate or repress gene expression. An important long-term goal of these studies is to identify chromatin remodeling enzymes that are recruited to GATA-1-dependent genes. These might serve as novel drug targets for intervention in hemoglobinopathies and other blood diseases, similar to compounds such as histone deacetylase inhibitors, which are currently in clinical trials [unreadable] [unreadable]

therapy design /development

[unreadable] DESCRIPTION (provided by applicant): Our work focuses on the mechanisms by which transcription factors regulate the development of diverse hematopoietic lineages, and how these factors orchestrate terminal differentiation with cell cycle arrest. These functions require key nuclear factors to be able to both activate and repress transcription. GATA-1 activates as many genes as it represses during terminal erythroid differentiation in a manner largely dependent on the interaction with its co-factor FOG-1. We recently identified two novel mechanisms by which GATA-1 and FOG-1 regulate transcription. First, we purified a FOG-1-associated co-represser complex NuRD (nucleosome remodeling and deacetylase) that is required for GATA-1/FOG-1-mediated transcriptional repression. Second, using chromosome conformation capture (3C) we showed that GATA-1 and FOG-1 are required for the formation of long-range chromatin loops at the p-globin locus. In Aim I we propose to study the function of the FOG-1-NuRD complex at the molecular and cellular level as well as in murine models. This includes the identification of the modules that mediate NuRD binding by FOG-1, determining their atomic structure, testing the function of NuRD subunits in erythroid cell lines, generating mice with mutations that disrupt the FOG-1-NuRD interaction, and the conditional knock out of the NuRD core subunit Mi-2p\ Since FOG-1 can function as co-activator and co-repressor for GATA-1, Aim II examines the mechanisms by which FOG-1 switches between these opposing functions. These studies include the analysis by CHIP and siRNA of NuRD components at activated and repressed genes. This Aim will also investigate the role of posttranslational modifications of FOG-1 and NuRD with regard to the switch in activity. Based on encouraging preliminary results, in Aim III we will examine by 3C whether GATA-1 and FOG-1 actively form chromatin loops during the repression of the GATA-2 and c-kit genes. This requires a thorough prior characterization of the GATA-2 and c-kit loci with regard to GATA-1 binding and histone acetylation. The role of loop formation will be directly addressed in functional studies. We hope that together these studies will help to explain how key transcriptional regulators can switch between activating and repressive functions. This knowledge directly impacts on how nuclear proteins promote the formation of one lineage at the expense of another, and how they activate genes associated with the mature phenotype while simultaneously inhibiting a program that maintains the immature proliferative state. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): We propose to manipulate cellular gene expression using a novel strategy involving the generation of chromatin loops, focusing on the [unreadable]-globin locus in developing erythrocytes. The locus control region (LCR) of the [unreadable]-globin locus is required for the expression of all [unreadable]-like globin genes and functions over great distances. Throughout development, the LCR interacts dynamically with the active globin gene promoters via the formation of chromatin loops. Here we will examine the cause-effect relationships between loop formation and gene transcription by establishing chromatin loops at endogenous cellular genes in vivo. Specifically, we will examine 1) whether forced loop formation between the LCR and a designated ?-globin gene leads to transcriptional activation in immature erythroid cells, 2) whether inducing the LCR to loop to a developmentally silenced globin gene can lead to its reactivation, 3) whether a silent non-globin gene can be activated by bringing the LCR into close proximity by forced loop formation. Our approach makes use of artificial zinc finger proteins designed to bind to unique, predetermined sequences at the endogenous LCR and the target gene promoters. This will be accomplished by an already established collaboration with Sangamo Biosciences. This company has in its possession a large archive of zinc finger proteins and the required bioinformatics tools to assemble polydactyl zinc finger proteins to bind to the desired target sequences with high selectivity and affinity. These zinc finger proteins will be fused to protein moieties that allow drug-induced heterodimerization such as FKBP and FRB from the Argent(tm) system (ARIAD Pharmaceuticals). This system, which we have acquired, is well suited to generate high affinity interactions between FKBP and FRB bearing proteins upon exposure to the cell permeable, non-toxic drug AP21967. Alternative approaches to manipulate cellular gene expression typically involve the introduction of transcriptional activators, repressors or molecules that interfere with such transcription factors. A potential advantage of the system proposed here is that it does not rely on the effects of a single factor but harnesses the regulatory potential of an entire enhancer or LCR. The long-term goal of this study is to explore whether this strategy is suitable to activate the ?-globin gene in human primary adult erythroid cells. If successful, this might provide a novel tool for the treatment of sickle cell disease and the [unreadable]-thalassemias. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Our work focuses on the mechanisms by which transcription factors regulate the development of diverse hematopoietic lineages, and how these factors orchestrate terminal differentiation with cell cycle arrest. These functions require key nuclear factors to be able to both activate and repress transcription. GATA-1 activates as many genes as it represses during terminal erythroid differentiation in a manner largely dependent on the interaction with its co-factor FOG-1. We recently identified two novel mechanisms by which GATA-1 and FOG-1 regulate transcription. First, we purified a FOG-1-associated co-represser complex NuRD (nucleosome remodeling and deacetylase) that is required for GATA-1/FOG-1-mediated transcriptional repression. Second, using chromosome conformation capture (3C) we showed that GATA-1 and FOG-1 are required for the formation of long-range chromatin loops at the p-globin locus. In Aim I we propose to study the function of the FOG-1-NuRD complex at the molecular and cellular level as well as in murine models. This includes the identification of the modules that mediate NuRD binding by FOG-1, determining their atomic structure, testing the function of NuRD subunits in erythroid cell lines, generating mice with mutations that disrupt the FOG-1-NuRD interaction, and the conditional knock out of the NuRD core subunit Mi-2p\ Since FOG-1 can function as co-activator and co-repressor for GATA-1, Aim II examines the mechanisms by which FOG-1 switches between these opposing functions. These studies include the analysis by CHIP and siRNA of NuRD components at activated and repressed genes. This Aim will also investigate the role of posttranslational modifications of FOG-1 and NuRD with regard to the switch in activity. Based on encouraging preliminary results, in Aim III we will examine by 3C whether GATA-1 and FOG-1 actively form chromatin loops during the repression of the GATA-2 and c-kit genes. This requires a thorough prior characterization of the GATA-2 and c-kit loci with regard to GATA-1 binding and histone acetylation. The role of loop formation will be directly addressed in functional studies. We hope that together these studies will help to explain how key transcriptional regulators can switch between activating and repressive functions. This knowledge directly impacts on how nuclear proteins promote the formation of one lineage at the expense of another, and how they activate genes associated with the mature phenotype while simultaneously inhibiting a program that maintains the immature proliferative state.

DESCRIPTION (provided by applicant): Proper regulation of gene expression is essential to the normal development and health of organisms, whereas aberrant gene regulation is known to cause many genetic diseases, including some inherited anemias, and it is thought to be a major contributor to complex phenotypes such as susceptibility to common diseases. Understanding the molecular mechanisms of gene regulation may provide novel candidates for therapeutic interventions. Our studies aim for a deeper molecular understanding of global aspects of gene regulation in an important biological process, the maturation of erythroid precursor cells to become red blood cells. Building on our progress using patterns in sequence alignments to predict cis-regulatory modules for erythroid genes and deciphering functional correlations of their evolutionary history, we propose to acquire genome-wide information on biochemical features associated with regulation to reach a more complete understanding of gene regulation in erythroid cells. Specifically, we propose to use high throughput biochemical assays such as chromatin immunoprecipitation followed by hybridization to microarrays and deep re-sequencing to acquire data on genomic DNA sequences (Aim 1) occupied in vivo by critical tissue-specific transcription factors, (Aim 2) bound by histones with modifications associated with gene activation or repression, (Aim 3) in chromatin with an altered structure, and (Aim 4) transcribed in a mouse erythroid cell model that undergoes maturation upon restoration of the critical transcription factor GATA-1. Then we will (Aim 5) apply existing software and develop new data-processing algorithms to determine peaks of signals that are likely to represent the locations of the features targeted in aims 1-4. Aim 6 will mine the peak-calling results, along with raw data, multiple sequence alignments and other information to investigate their covariation structure and integrate them to predict cis-regulatory modules, classify the modules by function, identify motifs associated with specific protein occupancy, and deduce the phylogenetic depth of preservation of critical motifs in the regulatory modules. Aim 7 will experimentally test biological hypotheses that arise from the analyses in Aims 6 and 7, determining the extent to which we can validate the locations of protein occupancy and transcripts, the predictions of both positive and negative cis-regulatory modules by gain-of-function cell transfection assays, and the role of motifs implicated in occupancy by directed mutagenesis and in vivo binding assays. We will test whether the motif- constraint hypothesis for protein-occupied DNA segments involved in enhancement applies to transcription factors in addition to GATA-1, and we will conduct additional experiments probing deeper biological issues. This research will provide not only global insights into mechanisms and effects of gene regulation during erythroid maturation, but the techniques and analytical tools developed here can be applied to better understand the development and differentiation of any tissue.

DESCRIPTION (provided by applicant): The study of the epigenetic mechanisms that ensure the correct propagation of transcriptional states throughout the cell cycle is important for understanding lineage commitment and cellular reprogramming. The hematopoietic transcription factor GATA1, an acetylated protein, is essential for the differentiation and commitment of several hematopoietic lineages. We recently found that GATA1 interacts with the bromodomain protein Brd3 in an acetylation-dependent manner in vitro and in vivo. Anti-Brd3 ChIP-seq analysis showed that GATA1 is a key determinant of Brd3 recruitment in interphase cells in vivo. Notably, we found that Brd3, like some other Brd family proteins, remains bound to chromatin during mitosis when the vast majority of nuclear factors are removed from chromatin. Brd3 binding might thus provide an epigenetic memory function to bookmark genes in a manner that facilitates the appropriated re-assembly transcription factor complexes at the correct sites upon re-entry into the G1 phase of the cell cycle. In Aim 1, we will study the function of Brd3 in hematopoietic development, and biochemically and structurally dissect the Brd3-GATA1 interaction. We will determine the in vivo acetylation sites of GATA1 and monitor acetylation dynamics during the cell cycle and at varying stages of cell growth and differentiation. In Aim 2 we will examine the genome-wide occupancy pattern of Brd3 in mitosis and define the mechanisms of mitotic and interphase Brd3 recruitment. We will examine whether Brd3 serves a bookmarking function of hematopoietic genes and study how Brd3 transmits transcriptional information through mitosis. This will involve the testing of Brd3-occupied elements in gain-of- function assays at the single allele level in living cells and the targeted destruction of Brd3 specifically during mitosis. In Aim 3 the in vivo function of Brd3 will be assessed by conditional deletion in mice. Together, the proposed studies are designed to elucidate the transcriptional and epigenetic machinery that underlies the establishment and stable propagation of gene expression patterns in the hematopoietic system.

DESCRIPTION (provided by applicant): Sickle cell anemia is a serious disorder caused by a point mutation in the adult 2-globin gene. Patients with linked mutations that result in elevated levels of 3-globin, which is normally expressed only during fetal development, experience a more benign clinical course of the disease. This observation provided the initial impetus to better understand the mechanisms of 3-globin gene silencing with the goal to develop new treatments that would raise 3-globin levels. Based on recent findings obtained in our laboratory, we propose to develop a high-throughput screen to identify compounds that interfere with the function of a transcription factor complex that is involved in the silencing of 3-globin expression. The screen will be validated in 384- or 1536-well format in preparation for high-throughput screening (HTS) in the Molecular Libraries Probe Production Centers Network (MLPCN). The assay will be based on the repressive function of a transcription co-factor called FOG-1, which in turn binds to a co-repressor complex called NuRD (nucleosome remodeling and deacetylase). We defined a highly conserved module consisting of just 12 amino acids at the extreme N-terminus of FOG-1 that is required for NuRD binding. Disruption of just one amino acid within this motif leads to a marked reduction in NuRD binding and loss of repressor activity of FOG-1. This indicates that the contact surface between FOG-1 and NuRD is small. Therefore, we believe that it is possible to disrupt this interaction with small molecules. If successful, these compounds will be evaluated for their ability to reactivate 3-globin in an established transgenic mouse model and in human erythroid cells. Compounds that disrupt the FOG-1/NuRD interaction and raise the expression of 3-globin could provide a targeted treatment for sickle cell anemia and 2-thalassemia. Current treatment protocols include histone deacetylase inhibitors and DNA demethylating agents, which give rise to side effects resulting from very broad and rather unpredictable effects on cellular gene expression. Inhibitors of the FOG-1/NuRD interaction would be extremely useful chemical probes to dissect the transcriptional pathways employed by these nuclear factors and to further understanding of the mechanisms of hemoglobin switching. Moreover, since other important transcription repressors share this NuRD-binding module, chemicals that interfere with their function would be powerful tools to dissect transcriptional pathways during mammalian development. PUBLIC HEALTH RELEVANCE: Despite decades of intense studies, the mechanisms of hemoglobin switching are still unclear. Recent findings in our laboratory revealed a critical role for the FOG-1/NuRD transcription factor complex in the silencing of 3-globin expression. Compounds that target the FOG-1/NuRD interaction would be extremely useful tools to dissect the transcriptional pathways employed by these nuclear factors. Raising 3-globin levels by disruption of the FOG-1/NuRD complex may provide a novel, targeted treatment for sickle cell anemia and 2-thalassemia.

DESCRIPTION (provided by applicant): This proposal investigates epigenetic mechanisms by which transcriptional programs are established and maintained to specify hematopoietic gene expression. Studies in Aim 1 will examine how the interaction between the GATA1-cofactor FOG1 and the NuRD histone deacetylase complex is controlled during hematopoietic gene expression. Using a mouse model in which the FOG1-NuRD interaction is disrupted, we observed that in developing erythroid cells and megakaryocytes NuRD is required for both repression and activation of specific genes. The latter was unexpected in light of the established role of NuRD as co-repressor. Here we will explore mechanisms that discern active from repressive FOG1/NuRD complexes by studying posttranslational regulation of FOG1 and NuRD. The mechanisms that govern distinct activities of the FOG1-NuRD complex are deemed critical for lineage choice and normal cellular maturation. Experiments in Aim 2 explore the mechanisms of epigenetic inheritance of transcriptional programs in hematopoiesis. We will study how specific gene expression patterns are restored after mitosis when transcription is disrupted globally. We will examine the kinetics of disassembly and re- assembly during mitosis of key erythroid transcription factors and their co-factors, such as NuRD, using chromatin immunoprecipitation of synchronized cell populations and live cell imaging. Further, we will study the dynamics of histone modifications at hematopoietic regulatory modules throughout the cell cycle. Mechanistic experiments will explore how mitotic bookmarks are established and interpreted to ensure faithful transmission of transcription patterns throughout the cell cycle. In Aim 3 we will study the higher order chromatin organization of key hematopoietic genes. Previous work under this grant identified functionally important dynamic long-range chromatin loops between hematopoietic regulatory elements. We will extend these studies by using high- throughput technology called 5C to examine in an unbiased fashion chromatin folding at developmentally controlled genes under distinct conditions and cellular contexts. Moreover, we will explore chromatin loops as potential conveyors of epigenetic information through mitosis. All Aims benefit from a unique combination of powerful cellular systems and well-characterized gene loci to tackle these fundamental problems in developmental molecular genetics. The role of epigenetic regulation of tissue-specific gene expression is timely as it pertains to mechanisms of lineage fidelity and cellular reprogramming. PUBLIC HEALTH RELEVANCE: The proposed studies are aimed to better understand the mechanisms underlying the formation of the blood lineages and their associated disorders. We examine how nuclear factors and their chemical modifications control the genes that govern the specification and differentiation of blood cells. Moreover, we study mechanisms by which cells remember their identity throughout the cell cycle. Progress is this area promises novel approaches to treat blood-related disorders including leukemias and anemias.

DESCRIPTION (provided by applicant): The eukaryotic genome is non-randomly organized in the interphase nucleus. Critical control elements can physically contact each other to form chromatin loops. Looped configurations of the chromatin fiber have been described at numerous gene loci, however, the mechanism by which these structures are established and their functional relationship with gene expression have remained unclear. The transcription co-factor Ldb1 is critical for establishing looped chromatin interactions at the murine ?-globin locus. Specifically, we showed that tethering Ldb1 to the locus via artificial zinc finger proteins in immature erythroid cells is sufficient to promote a looped interaction between the ?-globin enhancer and promoter and potently activate transcription. This suggested for the first time that chromatin looping causally underlies gene expression and raised the possibility that forced chromatin looping might be employed to effectively manipulate gene expression. This proposal builds on these findings by investigating in Specific Aim 1 the mechanisms of Ldb1 function and its broad role in genome organization. In Specific Aim 2 we will further develop the approach of forced chromatin looping to enhance and broaden its usefulness. In Specific Aim 3 we will examine forced chromatin looping as an approach to developmentally reprogram the murine and human ?-globin locus. In Specific Aim 4 proof-of-concept studies will examine whether reactivation of fetal hemoglobin via chromatin looping can ameliorate sickle cell anemia in a humanized mouse model. To our knowledge, the manipulation of higher order chromatin structure for the purpose of regulating gene expression is unique and novel in its design. The juxtaposition of complex regulatory elements promises more dramatic changes in gene activation when compared to conventional approaches, and might also be exploited to repress gene transcription for exploratory or therapeutic purposes.

DESCRIPTION (provided by applicant): Defining the regulatory architecture of hematopoietic cells to elucidate lineage determination and differentiation can produce insights into developmental biology and can help identify targets with potential application to human diseases such as leukemias and anemias. Mouse hematopoiesis is a versatile system for studying gene regulation during differentiation because we can purify populations of progenitor and differentiated cells for genome-wide mapping of transcripts and regulatory sequences, and we can genetically manipulate critical proteins and cis-regulatory modules (CRMs) to study mechanisms of regulation. This application is for a renewal of a long-standing, productive collaboration among multiple investigators with complementary expertise in hematopoietic cell differentiation, gene regulation, genomics, bioinformatics and statistics. Our previous work laid a foundation of genome-wide data sets for transcriptomes, transcription factor occupancy and chromatin states in a cultured cell model for erythroid differentiation and in maturing primary cells in the erythroid and megakaryocytic lineages, which led to key new insights about regulation. We now propose to (Aim 1) generate genome-wide data on transcriptomes and informative epigenetic features in purified cells from each stage of differentiation from mouse hematopoietic stem cells to mature cells of the erythroid and myeloid lineages. For all cell types, including multilineage progenitor cells available only in small numbers, we propose to determine transcriptomes, DNA methylation, and chromatin accessibility (using a new method based on in vitro transposition). In more abundant cell types, we will use ChIP-seq to map transcription factors and histone modifications and also the chromosome conformation capture method Hi-C to build an interaction map of distal regulatory regions with target genes. We will then (Aim 2) conduct integrative, quantitative modeling to find genes differentially expressed and with different transcription factor binding patterns in the distinct lineages; within this set are candidates for genes involved in choice of cell lineage. A hypothesis-driven Bayesian network model will learn quantitative relationships between features, including expression level, and make predictions about how the system would behave after perturbation of both transcription factors and CRMs. We will then (Aim 3) conduct genetic manipulations to test hypotheses arising from integrative analysis in Aim 2. Specific hypotheses about genes involved in lineage choice will be tested by transduction of interfering or forced expression constructs into mouse fetal liver progenitor cells and bipotential cells in culture. Hypotheses from the quantitative modeling of determinants of levels of expression will be tested, targeting specific proteins (using transfections of cells with or withou GATA1) and CRMs (by Cas9-CRISPR-guided genome editing). The result of this proposed work will be deep, widely disseminated data on the regulatory landscape in multiple hematopoietic lineages and keener insights into how changes in regulatory proteins and chromatin lead to lineage choice and progressive differentiation.

Abstract Critical unanswered questions in the field of genome biology are how the dynamics of chromatin folding shape gene expression patterns. Our knowledge of the dynamics of higher-order 3-D folding of chromatin is severely limited, largely due to the lack of technologies to precisely image, engineer and monitor looping in a precise spatiotemporal manner across a population of cells. Here we propose to address these limitations by developing tools to dynamically alter chromatin folding in a synchronous manner across populations of cells as well as individual cells, and measure chromatin looping and its relationship to transcription at high spatial resolution in single cells. In Specific Aim 1 we will design tools to control looping dynamics. We will modify factors that fold chromatin at various levels, such as Ldb1 and CTCF by fusion to a moiety whose stability can be controlled by diffusible ligands. In combination with hi resolution 5C and single molecule imaging these tools are expected to generate fundamental insights into the relationship of nuclear architecture and gene expression mechanisms. In Specific Aim 2 we plan to engineer light-inducible systems for the precise control of looping dynamics. Using light activated dimerization domains that can be used in conjunction with designer DNA binding proteins we attempt to engineer factors used to rapidly promote or disrupt chromatin looping at various scales. This technology should enable studies not only in populations but also at the single cell level. In Specific Aim 3: we will develop reagents to study the transcriptional dynamics in relation to looping at the single cell level. We will combine RNA FISH with super-resolution imaging to develop a methodology for exploring the spatial and temporal structure of nascent transcription at high resolution. Combined with high-throughput image acquisition, we will discriminate the temporal dynamics of transcription by measuring the relative intensities arising from the different parts of the transcript. We will employ super-resolution imaging (STORM) to measure the spatial structure of transcription sites. These experiments are expected to reveal the impact of forced chromatin looping on distinct stages of the transcription cycle and elucidate the relationship between transcriptional burst kinetics and physical gene structure.

Project Summary VISION: ValIdated Systematic IntegratiON of hematopoietic epigenomes Technological advances enabling the production of large numbers of rich, genome-wide, sequence-based datasets have transformed biology. However, the volume of data is overwhelming for most investigators. Also, we do not know the mechanisms by which the vast majority of epigenetic features regulate normal differentiation or lead to aberrant function in disease. We have formed an interdisciplinary, collaborative team of investigators to address the problem of how to effectively utilize the enormous amount of epigenetic data both for basic research and precision medicine. At this point, acquisition of data is no longer the major barrier to understanding mechanisms of gene regulation during normal and pathological tissue development. The chief challenges are how to: (i) integrate epigenetic data in terms that are accessible and understandable to a broad community of researchers, (ii) build validated quantitative models explaining how the dynamics of gene expression relates to epigenetic features, and (iii) translate information effectively from mouse models to potential applications in human health. These needs are addressed by the proposed ValIdated Systematic IntegratiON (VISION) of epigenetic data to analyze mouse and human hematopoiesis, a tractable system with clear clinical significance and importance to NIDDK. By pursuing the following Specific Aims, the interdisciplinary collaboration will deliver comprehensive catalogs of cis regulatory modules (CRMs), extensive chromatin interaction maps and deduced regulatory domains, validated quantitative models for gene regulation, and a guide for investigators to translate insights from mouse models to human clinical studies. These deliverables will be provided to the community in readily accessible, web-based platforms including customized genome browsers, databases with facile query interfaces, and data-driven on-line tools. Specifically, the proposed work in Aim 1 will build comprehensive, integrative catalogs of hematopoietic CRMs and transcriptomes by compiling and determining informative epigenetic features and transcript levels in hematopoietic stem and progenitor cells and in mature cells. CRMs will be predicted using the novel IDEAS (Integrative and Discriminative Epigenome Annotation System) method. Work proposed in Aim 2 will build and validate quantitative models for gene regulation informed by chromatin interaction maps and epigenetic data. Compiling and determining chromosome interaction frequencies will predict likely target genes for CRMs. Gene regulatory models will be built that predict the contributions of CRMs and specific proteins to regulated expression; these models will be validated by extensive testing using genome-editing in ten reference loci. Finally, work in Aim 3 will produce a guide for investigators to translate insights from mouse models to human clinical studies. This effort will include categorizing orthologous mouse and human genes by conservation versus divergence of expression patterns, assigning CRMs to informative categories of epigenomic evolution, and testing the interspecies functional maps experimentally by genome-editing.

Abstract Sickle cell disease (SCD) and some types of b-thalassemia that are caused by defects in the adult form of hemoglobin manifest shortly after birth, when the switch from the fetal to the adult form of hemoglobin is complete. Even a partial reversal of this switch is associated with an improved course of these diseases. We employed a newly improved CRISPR-Cas9 platform to carry out a kinase domain-focused genetic screen to identify potentially druggable molecules that repress fetal hemoglobin (HbF) production. This screen uncovered HRI (also known as EIF2AK1), an erythroid-specific protein kinase that regulates protein translation. Depletion of HRI elevates HbF levels in human erythroid cells with few additional perturbations. HRI loss reduces the expression of the major HbF repressor BCL11A, and restoration of BCL11A expression partially restores HbF repression. Moreover, HRI depletion reduces sickling of SCD-derived human erythroid cells in culture. In Aim 1 we will comprehensively dissect HRI function by assessing the transcriptome and proteome of HRI-depleted cells. The goals of Aim 2 are to study the mechanism by which HRI regulates BCL11A, identify additional HRI regulated HbF repressors, and examine the global impact of HRI on protein translational control in primary human erythroid cells. Aim 3 will explore synergies with previously known HbF inducers both using a candidate approach, and by unbiased genetic screens for novel synergies. In Aim 4 we will examine the effects of HRI loss on SCD by generating HRI-deficient humanized SCD mouse models. In sum, these studies explore the role of HRI in human red cell biology and examine HRI as target for pharmacologic HbF induction alone or in combination with mechanistically distinct HbF inducers.

The mammalian genome folds into tens of thousands of long-range looping interactions. A critical unknown is whether and how chromatin loops control gene expression, and a major unresolved question is how the temporal progression of loops relates to transcription dynamics. One major barrier to answering this question is that loops change on a range of timescales, necessitating the use of tools and model systems amenable to tracking and engineering loops longitudinally and in real time on both short and long timing. Here, we propose to develop and apply new engineering and imaging tools to measure, induce, and perturb loops with precise temporal control in three different biological systems spanning minutes, hours, and weeks. At the shortest timescale (minutes, Aim 1), we will examine loop dynamics in human induced pluripotent stem cell-derived neurons in response to electrical stimulation, revealing how interaction frequency is functionally connected to transcriptional bursting of immediate early and secondary response genes. On the timescale of hours (Aim 3), we will elucidate how the architectural protein YY1 connects enhancer-promoter loops that re-assemble upon the exit from mitosis by erythroid cells. On the timescale of weeks (Aim 2), we will use a cellular ?Time Machine? to longitudinally track the rare cells that undergo cellular reprogramming, allowing us to dissect the functionality of loop formation and dissolution with single-cell and subcellular resolution during the reprogramming of somatic cells to pluripotency and transition of melanoma cancer cells to a resistant phenotype. Our team consists of a highly productive and collaborative set of junior and senior investigators with complementary expertise and overlapping interests, including Dr. Gerd Blobel (epigenetics, mitosis, loop engineering), Dr. Eric Joyce (Oligopaints imaging), Dr. Bomyi Lim (nascent transcript live cell imaging), Dr. Jennifer Phillips-Cremins (chromatin architecture, loop engineering, neurobiology), Dr. Stanley Qi (CRISPR genome engineering, live cell imaging), and Dr. Arjun Raj (single cell genomics, RNA imaging, reprogramming). We will develop and apply live and fixed cell imaging techniques for chromatin contacts, and in the same cells image nascent transcription. We will build a cadre of synthetic architectural proteins to engineer loops in a time-dependent inducible manner. Successful application of our engineering and imaging tools across biological systems will yield a comprehensive and rigorous assessment of the cause-and-effect relationship between loops and distinct biological phenotypes across timescales.