Emery H. Bresnick - US grants

DESCRIPTION: (Adapted from investigator's abstract) The restriction of globin synthesis to cells of the erythroid lineage represents a fundamental problem in molecular hematology. The transcriptional activity of the beta globin locus is controlled by four erythroid specific nuclease hypersensitive sites at the 5' end of the beta globin locus on chromosome 11. This region (LCR) is critical for maintaining the globin cluster in an active chromatin conformation. The long term goal of this project is to determine how the components of the LCR interact to provide an autonomous chromosomal domain during erythropoiesis. The transcriptional activation property of the LCR can be shared by multiple promoters on a single chromosome. Two classes of models for the mechanism of the LCR are consistent with this result. The hypersensitive sites could function either independently or as an integrated unit. In this application, distinction between the two models will be made by asking if a single hypersensitive site of the LCR can activate multiple promoters on a single chromosome. Biochemical and molecular biological approaches will also be used to test the hypothesis that the hypersensitive core represents ordered nucleoprotein complexes. Elucidating the molecular mechanism of the LCR has important practical implications for expressing genes in transgenic organisms and in human gene therapy. Understanding how the LCR functions may also help in the design of synthetic LCRs that are more effective than natural ones and could lead to new approaches to the treatment of hematologic disorders.

We propose to study the involvement of coactivators in the function of the beta-globin locus control region (LCR), a genetic element that regulates the chromatin structure, transcription, and replication of the beta-globin genes. Multiple hematopoietic and ubiquitous transcription factors are important for transactivation by the LCR. A common theme among transcriptional regulatory proteins is their involvement in protein-protein interactions with coactivators . Coactivators mediate activation by covalent modification of chromatin or engagement in protein-protein interactions that facilitate assembly of RNA polymerase II preinitiation complexes. To date, no coactivators have been identified that mediate LCR function. Because the LCR disrupts chromatin over long distances (greater than 50-kb), we hypothesize that the LCR recruits chromatin modifying enzymes, specifically histone acetylases (HATs), and a ubiquitin ligase, which acts as coactivators to catalyze a chromatin changes necessary for long-range activation. In Specific Aim 1, we will assess the roles of the HATs, CBP/p300, P/CAF, and GCN5 in long- range activation by the LCR. To test the hypothesis that HATs mediate transactivation of globin genes through interactions with the LCR, the influence of wild-type HATs and acetylase-deficient mutants on LCR function will be studied in transfection assays. In Specific Aim 2, we will determine whether the ubiquitin ligase WWP1 and related WW domain proteins influence LCR function. Based on physical and functional interactions between WW domains and the LCR binding protein, NF-E2, we will test the hypothesis that such proteins are important for LCR function. In Specific Aim 3, we will test whether amino acid sequences of NF-E2 important for CBP/p300 and WW domain binding are required for long-range transactivation by the LCR. We have delineated distinct sequences within the activation domain of NF-E2 necessary for CBP/p300 and WW domain binding and will determine whether these interactions are required for NF-E2 mediated transactivation. These studies represent the first phase of our long-term goal to understand the role of coactivators in LCR function, to test whether hematopoietic signaling pathways modulate their activity, and to ascertain their role in hematopoiesis.

Description: (Investigator's abstract) We propose to study how the beta-globin locus control region (LCR) confers high-level transcriptional activity to the beta-globin genes over a long distance on a chromosome. Mechanisms of long-range transactivation are poorly understood. In Specific Aim 1, we will define protein components of the LCR in living cells and will determine if they differ during erythroid differentiation. Despite extensive in vitro studies, little is known about the composition of native LCR complexes. We hypothesize that the components of the LCR vary during erythropoiesis to accommodate differential requirements for transactivation during development. A chromatin immunoprecipitation (ChIP) assay will be used to measure the binding of candidate proteins to the LCR. In Specific Aim 2, we will define the histone acetylation and phosphorylation patterns of the beta-globin locus and will determine if the patterns change during erythroid differentiation. We hypothesize that the LCR recruits coactivators that establish a specific pattern of histone modifications throughout the beta-globin locus, which is necessary for long-range transactivation. We will define the pattern of histone modifications, will assess whether it changes during erythropoiesis, and will determine whether pharmacological inducers of fetal hemoglobin specifically modulate the pattern. We hypothesize that the histone modification pattern is established via primary and modulatory determinants. In Specific Aim 3, we will identify primary determinants of the acetylation pattern. We have shown that the histone acetylase CBP/p300 is critical for LCR-mediated transactivation. We will test whether CBP/p300 is a primary determinant and will identify CBP/p300-interacting coactivators in erythroid cells. We will also assess whether specific histone deacetylases are primary determinants. The native structure of enhancer and LCR complexes and the histone modification pattern of a domain have not been defined in any system. Since long-range mechanisms control the transcription of multiple crucial genes that regulate cell proliferation and differentiation, our studies will yield principles of broad physiological and pathophysiological relevance. The long-term objective is to therapeutically modulate beta-globin gene expression in humans with hemoglobinopathies by perturbing specific steps of the mechanism by which the LCR regulate the beta-globin genes.

DESCRIPTION (provided by applicant): The goal of this application is to understand how trans-acting factors and coregulators function through the beta-globin locus control region (LCR) to confer strong transcription to the beta-globin genes over a long distance on a chromosome. We discovered that RNA polymerase II (pol II) resides at the LCR in erythroid cells and formulated a new model of LCR function called long-range pol II transfer (LPT). This model proposes that LCR-bound pol II relocalizes to beta-globin promoters in a regulated fashion, thereby constituting a critical step in activation. The following Specific Aims will address how pol II is recruited to the LCR and the resulting functional consequences. Aim 1. To define the role of GATA-1 and Friend of GATA-1 (FOG-l) in pol II recruitment to the LCR. Pol II associates with the LCR in erythroid cells, and GATA-1 induces pol II recruitment. We hypothesize that GATA-1 functions with other factors to recruit pol II. GATA-I-null cells will be used to define how GATA-1 recruits pol II. Cells lacking the GATA-1 coregulator FOG-1 will be used to define if FOG-1 is required. Experiments will address whether single hypersensitive sites autonomously recruit pol II. Aim 2. To determine if pol II recruitment to the LCR is dynamic during erythropoiesis. As the concentrations and activities of erythroid-specific factors change during erythropoiesis, we hypothesize that pol II loading on the LCR is developmentally dynamic. Experiments will assess if pol II recruitment to the LCR differs between embryonic and adult erythroid cells. Aim 3. To discriminate among models for understanding the function of pol II at the LCR. Besides LPT, LCR-bound pol II might engage in transcription-dependent chromatin remodeling or the generation of regulatory transcripts. Experiments will discriminate among models to explain the function of pol II at the LCR. These studies will increase our understanding of how the LCR regulates the beta-globin genes, which should have broad relevance to mechanisms controlling diverse cellular and organismal processes. The long-term objective is to therapeutically control beta-globin genes in humans with hemoglobinopathies by regulating specific steps of the LCR mechanism.

DESCRIPTION (provided by applicant): A highly orchestrated gene expression pattern endows stem cells with the capacity to differentiate into diverse cell types. Assembly of cell type-specific chromatin domains and transcriptional networks in stem cells and multipotent progenitors is poorly understood, despite powerful experimental systems, such as mouse bone marrow hematopoietic stem cells (HSCs) and embryonic stem (ES) cell-derived hematopoietic precursors. HoxB4, Bmi 1, beta-catenin, TAL-1 and GATA-2 transcription factors control HSC self-renewal and/or function. We hypothesize that comparing transcriptional mechanisms regulating production of these factors will yield important principles vis-a-vis stem cell biology and hematopoiesis. Studies to test this hypothesis will be instituted with the following Specific Aims. As no examples exist in which the native nucleoprotein structure of an endogenous chromatin domain has been defined in HSCs, this work falls under the rubric of "pilot and feasibility" studies. Aim 1. To determine if the histone modification pattern of the GATA-2 domain is developmentally dynamic. GATA-2 is upregulated in erythroid precursors upon targeted disruption of GATA-1, and GATA-1 represses GATA-2 transcription. Using GATA-1-null G 1E cells, we showed that repression involves GATA-l-dependent displacement of GATA-2 from an upstream region (-2.8 kb) and reduced histone acetylation domain-wide. We hypothesize that GATA-2 maintains acetylation and GATA-1 abrogates autoregulation by displacing GATA-2, reconfiguring nucleoprotein complexes, and reducing acetylation. We will determine if this mechanism is operational in HSCs and progenitors and ES cell-derived hematopoietic precursors. Aim 2. To determine the importance of an upstream GATA factor binding region of the GATA-2 locus. Our studies provided correlative evidence that GATA-2 and GATA-1 binding to the -2.8 kb region confer activation and repression, respectively. We will test this model by generating ES cells with a deletion of GATA sites within the -2.8 kb region and will construct a polydactyl zinc finger protein that prevents GATA factor binding to this region. The impact of the deletion and the inhibitor on GATA-2 expression and hematopoiesis in vitro will be assessed. The studies will define the regulation and function of GATA-2 as a prelude to developing principles for how chromatin domains and transcriptional networks assemble in and control HSCs.

The GATA transcription factor family (GATA-1-6) regulates critical aspects of mammalian development. Our studies revealed that GATA-1 and GATA-2 occupy GATA motifs in cells with exquisite specificity and can occupy the same chromosomal region, but with distinct functional outputs. Mechanisms underlying chromatin occupancy specificity and differential activities of GATA factors are unknown. GATA-2 is expressed in hematopoietic stem/progenitor cells and is required for hematopoiesis. The following Aims will analyze mechanisms that regulate GATA-2 transcription, how changes in GATA-2 levels affect hematopoiesis, and mechanisms underlying GATA factor target gene specificity. Aim 1. To analyze the switch in GATA factor occupancy at chromatin sites during hematopoiesis. GATA-1 and GATA-2 are reciprocally expressed during hematopoiesis, and GATA-1 represses GATA-2 transcription. We hypothesize that GATA-2 confers positive autoregulation, and that GATA-1 displaces GATA-2, reconfigures nucleoprotein complexes, and reduces acetylation, abrogating autoregulation. Studies are proposed to test this hypothesis. A key assumption of the model is that chromatin occupancy by GATA-1 and GATA-2 confer distinct functional outputs. Differential activities could result from different levels of expression or from qualitatively distinct mechanisms, and this will be tested. Aim 2. To dissect the mechanism of GATA-2 transcription in vivo, GATA-2 occupies the -2.8 kb and -1.8 kb regions of the GATA-2 locus in the active state, whereas GATA-1 binding and displacement of GATA-2 is coupled to repression. We have generated targeted deletions of the -2.8 kb and -1.8 kb regions to test whether these regions are required for assembly of the histone modification pattern, for recruiting RNA polymerase II, and for GATA-2 transcription. Aim 3. To test whether GATA-1 and GATA-2 have differentiation stage-specific target genes. We propose that a combinatorial code involving intrinsic features of GATA motifs, nearest-neighbor proteins, and chromatin structure specifies GATA factor occupancy. Elucidating this code requires systematic/analysis of GATA-1 and GATA-2 occupancy at known GATA target genes and the identification of additional direct target genes. This will be accomplished via quantitative ChIP analysis and ChIP coupled with genomic microarray chip of GATA factor occupancy in primary hematopoietic cells.

[unreadable] DESCRIPTION (provided by applicant): GATA transcription factors (GATA-1-6) regulate mammalian development. GATA-2 is important for hematopoietic stem (HSC) and multipotent progenitor cell differentiation/survival. GATA-1 and GATA-2 occupy a small subset of the binding motifs in cells and can occupy the same chromatin region at distinct developmental times, but with different functional outputs. The following Aims will analyze mechanisms that regulate GATA-2 transcription, how changes in GATA-2 levels affect hematopoiesis, and mechanisms underlying GATA factor chromatin occupancy. 1. To analyze the mechanism of the GATA switch at chromatin sites during hematopoiesis. GATA-2 has a short half-life (~1 h) and is stabilized by treatment of cells with proteasome inhibitors. When GATA-2 is stabilized, GATA-1-mediated displacement of GATA-2 from chromatin is attenuated. We will test the hypothesis that ubiquitination destabilizes GATA-2, and instability is required for GATA-1 to access GATA-2-bound chromatin sites. We will also test whether an excess of GATA-1 versus GATA-2 is required for the switch. 2. To dissect the mechanism of GATA-2 transcription in vivo. GATA-2 occupies the -2.8 kb and -1.8 kb regions of the active GATA-2 locus, whereas GATA-1 occupies predominantly the -2.8 kb region of the inactive locus. GATA-1 binding displaces GATA-2 from both regions and is coupled to repression. We generated targeted deletions of the -2.8 kb and -1.8 kb regions to test the hypothesis that these regions confer activation and the -2.8 kb region mediates repression. We will determine if the deletions affect assembly of the histone modification pattern, RNA polymerase II recruitment, and transcription. 3. To test whether GATA-1 and GATA-2 have differentiation stage-specific target genes. We propose that intrinsic features of the motifs, nearby c/s-elements, protein-protein interactions and chromatin structure constitute a GATA Recognition Code (GRC) that specifies occupancy. Elucidating the GRC requires analysis of occupancy at multiple target genes. GATA factor occupancy will be defined by quantitative chromatin immunoprecipitation (ChIP) and ChIP coupled with genomic microarrays. The studies will reveal how GATA switches regulate GATA-2 transcription, how GATA-1 and GATA-2 select DMA motifs, and insights of broad relevance to diverse developmental processes. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The goal of this application is to understand how beta-globin transcription is regulated by GATA-1 and additional factors during erythropoiesis. We discovered that GATA-1 occupancy of a small subset of the GATA-1 DNA binding motifs (WGATAR) within the endogenous beta-globin locus instigates multiple molecular events, culminating in transcriptional activation. Kinetic analyses led to the development of a novel multi-step activation model. The following aims propose to rigorously test this model: 1. To elucidate a multi-step pathway of beta-globin locus activation. We hypothesize that GATA-1- mediated early molecular events reflect direct actions of GATA-1, which are required for subsequent events. We will test mechanistic issues regarding the assembly/function of regulatory complexes at the beta-globin locus. These studies will yield comprehensive molecular snapshots of the nucleoprotein structure of the endogenous locus and fundamental mechanistic insights. 2. To define the importance of individual steps in beta-globin locus activation. Having already established the temporal regulation of certain events instigated by GATA-1 during activation, studies are proposed to molecularly modulate individual steps of the mechanism. We hypothesize that certain steps are interlinked, and therefore perturbation of a single step will disrupt a subset of the remaining steps. This hypothesis will be tested through the use of protein mutants, RNAi, and novel chemical inhibitors. 3. To analyze the molecular determinants of GATA factor chromatin occupancy. GATA-1 occupies a subset of the WGATAR motifs of the beta-globin locus and additional loci. We hypothesize that a GATA Recognition Code (GRC) exists in which parameters, including protein-protein interactions, neighboring cis-elements, and chromatin structure, determine occupancy. We propose to conduct quantitative chromatin immunoprecipitation (ChIP) analysis and ChIP coupled to microarray chip technology to comprehensively determine occupancy within and surrounding the endogenous murine and human loci. Through the assembly of a database of GRC parameters and computational/statistical analysis, hypotheses regarding determinants of GATA factor occupancy will be tested.

GATA transcription factors (GATA-1-6) regulate mammalian development. GATA-2 is important for hematopoietic stem (HSC) and multipotent progenitor cell differentiation/survival. GATA-1 and GATA-2 occupy a small subset of the binding motifs in cells and can occupy the same chromatin region at distinct developmental times, but with different functional outputs. The following Aims will analyze mechanisms that regulate GATA-2 transcription, how changes in GATA-2 levels affect hematopoiesis, and mechanisms underlying GATA factor chromatin occupancy. 1. To analyze the mechanism of the GATA switch at chromatin sites during hematopoiesis. GATA-2 has a short half-life (~1 h) and is stabilized by treatment of cells with proteasome inhibitors. When GATA-2 is stabilized, GATA-1-mediated displacement of GATA-2 from chromatin is attenuated. We will test the hypothesis that ubiquitination destabilizes GATA-2, and instability is required for GATA-1 to access GATA-2-bound chromatin sites. We will also test whether an excess of GATA-1 versus GATA-2 is required for the switch. 2. To dissect the mechanism of GATA-2 transcription in vivo. GATA-2 occupies the-2.8 kband-1.8kb regions of the active GATA-2 locus, whereas GATA-1 occupies predominantly the -2.8 kb region of the inactive locus. GATA-1 binding displaces GATA-2 from both regions and is coupled to repression. We generated targeted deletions of the -2.8 kb and -1.8 kb regions to test the hypothesis that these regions confer activation and the -2.8 kb region mediates repression. We will determine if the deletions affect assembly of the histone modification pattern, RNA polymerase II recruitment, and transcription. 3. To test whether GATA-1 and GATA-2 have differentiation stage-specific target genes. Wepropose that intrinsic features of the motifs, nearby c/s-elements, protein-protein interactions and chromatin structure constitute a GATA Recognition Code (GRC) that specifies occupancy. Elucidating the GRC requires analysis of occupancy at multiple target genes. GATA factor occupancy will be defined by quantitative chromatin immunoprecipitation (ChIP) and ChIP coupled with genomic microarrays. The studies will reveal how GATA switches regulate GATA-2 transcription, how GATA-1 and GATA-2 select DMA motifs, and insights of broad relevance to diverse developmental processes.

DESCRIPTION (provided by applicant): The development of hallmark features of endothelial cells relies upon endothelial cell-specific transcriptional mechanisms. Multiple factors, including GATA-2, establish endothelial cell-specific transcription. Human GATA2 polymorphisms correlate with coronary artery disease, GATA-2 expression is linked to arteriosclerosis, and GATA-2 regulates genes encoding the vascular molecules endothelin-1 and vascular adhesion molecule-1. However, mechanisms underlying GATA-2 function and regulation in endothelium are unknown. Using our work on mouse Gata2 in hematopoietic cells as a foundation, studies are proposed to elucidate how GATA-2 functions in vascular endothelium. The Gata2 locus contains five "GATA switch sites" that are occupied by GATA-2 and GATA-1 at the active and inactive loci, respectively, in erythroid precursor cells. GATA-1-mediated displacement of GATA-2 from these sites is coupled to repression. The +9.5 site functions autonomously to activate a LacZ transgene in endothelium of mouse embryos and human endothelial cells, and DNA motifs that bind GATA factors are required for this activity. The following aims will test hypotheses regarding GATA-2 function and regulation in vascular endothelium: Aim 1 - To elucidate a novel GATA factor-dependent transcriptional mechanism in vascular endothelium. We will test whether the +9.5 site enhancer in endothelium uniquely requires a GATA factor- dependent core module and additional regulatory modules. Chromatin immunoprecipitation (ChIP) and ChIP coupled to microarray chip (ChIP-chip) assays will be conducted to determine which GATA factor(s) occupy GATA2 in endothelial cells. Mutant transgenes will be analyzed to determine whether vascular and hematopoietic specificities can be dissociated and whether the +9.5 site can be reprogrammed to yield novel specificities. Chromosome conformation capture analysis will be used to determine whether GATA2 adopts a unique conformation in endothelial vs. hematopoietic cells. Mutant mice lacking the +9.5 site will be generated to determine whether it functions nonredundantly. Aim 2 - To identify GATA-2 target sites on human endothelial cell chromosomes. Only a small fraction of GATA motifs are occupied in erythroid cells. The specificity of transcription factor occupancy has not been studied in endothelial cells. We will test the hypothesis that endothelial cells resemble hematopoietic cells in that the vast majority of GATA motifs are not occupied. GATA-2 occupancy will be measured throughout human chromosome 3 in endothelial cells. Bioinformatics analysis will test whether E-boxes and other motifs are enriched at occupied versus nonoccupied GATA motifs and will identify an ensemble of genes as prospective components of circuitry underlying GATA-2 function. Project Narrative This project focuses on understanding how GATA-2, a nuclear protein expressed in endothelial cells and in certain blood cells, functions and is regulated. As GATA-2 is implicated in the development of atherosclerosis and coronary artery disease, the proposed studies are expected to provide important insights into mechanisms underlying these human disorders. Furthermore, GATA-2 is crucial to maintain hematopoietic stem cells and therefore uncovering mechanisms underlying GATA-2 function in any system has outstanding potential to further knowledge on adult stem cell biology.

DESCRIPTION (provided by applicant): While erythropoietin (Epo) is commonly used to treat anemia in patients with cancer and kidney disease, recent studies have raised major safety concerns. There is great interest in developing new strategies to modulate erythropoiesis, and in this regard, we have been analyzing GATA transcription factor function. GATA-2 regulates the proliferation/survival of hematopoietic stem cells (HSCs), whereas GATA-1 promotes the differentiation of erythroid, megakaryocyte, eosinophil, and mast cells. Analysis of GATA-1 function in GATA-1-null erythroid precursor cells revealed a novel target gene, mFam55B. GATA-1 directly activates mFam55B transcription, and mFam55B expression is highly restricted to definitive erythroblasts in murine fetal liver and adult bone marrow. Although mFam55B has minor sequence similarity to Intercellular Cell Adhesion Molecule-4 (ICAM4), which is implicated in formation of functionally important erythroblastic islands, the bulk of mFam55B sequence does not resemble ICAM4 or other known proteins. mFam55B appears to be a Type II membrane protein, whereas ICAMs are Type I membrane proteins. We discovered four mammalian mFam55B-related genes, which have not been described, but only mFam55B is GATA-1-regulated. mFam55B is predicted to have a transmembrane helix and two extracellular immunoglobulin (Ig)-like repeats. mFam55B and its paralogs define a new protein family, and we hypothesize that mFam55B is an erythroid precursor membrane protein with important functions in erythroid cell biology. The following aims describe pilot/exploratory studies to develop reagents/models to define mFam55B function and to investigate the role of mFam55B in the formation of erythroblastic islands. Aim 1 - To determine whether mFam55B is a GATA-1-regulated transmembrane protein in erythroid precursor cells. We hypothesize that mFam55B is a transmembrane protein in erythroid precursor cells that functions via intercellular signaling. We will test whether GATA-1 regulates endogenous mFam55B levels, whether mFam55B exhibits predominant plasma membrane localization, and whether it resides in erythroblastic islands. Aim 2 - To investigate biological functions of mFam55B via loss-of-function studies in zebrafish and mice. We identified five novel zebrafish genes with significant sequence similarity to mFam55B. Knocking down one of these genes yielded severe anemia. We will knockdown the mFam55B-related genes to determine if they regulate erythropoiesis, will define their expression patterns, and will test whether their expression is GATA-1-dependent. We will also generate a mFam55B-null mouse to begin to dissect the role of mFam55B in erythroid cell function and/or erythropoiesis. PUBLIC HEALTH RELEVANCE: This project focuses on analyzing a novel GATA-1 regulated protein, mFam55B, that we discovered, which is predicted to be a single-pass transmembrane protein on erythroid precursor cells and to represent the founding member of a new protein family. As GATA-1 is a fundamental regulator of erythropoiesis, we hypothesize that mFam55B has important functions to regulate red blood cell development and/or erythroid precursor cell function. The proposed pilot/exploratory studies therefore have potential to yield high impact findings of relevance to novel strategies to stimulate red blood cell development, to elucidating how erythroid precursor cell function is altered in pathophysiological states, and to unraveling fundamental aspects of protein structure/function.

DESCRIPTION (provided by applicant): Elucidating mechanisms that regulate erythroid cell maturation is critical for devising targeted therapies for red cell disorders including thalassemias, polycythemia vera and anemias, and for developing strategies to generate red blood cells for clinical transfusion. The PI3-kinase-AKT pathway promotes and suppresses erythroid maturation. The inhibitory mechanism involves AKT-mediated phosphorylation of FoxO3, which leads to its sequestration in the cytoplasm. FoxO3 is upregulated/activated during erythroid maturation and by oxidative stress and controls the erythroid cell cycle and maturation, red blood cell lifespan, and protects against oxidative stress. We developed evidence that the serine/threonine kinase mTOR engages in crosstalk with FoxO3 to control erythroid maturation. Mechanisms underlying FoxO3- mTOR crosstalk in any context are poorly understood. FoxO3 and mTOR were known to have critical independent functions to control autophagy in non-erythroid cells. Autophagy mediates the consumption of damaged cellular components and differentiation-associated cellular remodeling, including mitochondria disposal as a key step in erythrocyte development. We discovered that FoxO3 facilitates GATA-1-instigated autophagy gene activation and accumulation of the autophagosome during erythroid maturation. GATA-1 also activates autophagy by directly inducing FoxO3 expression. We hypothesize that GATA-1-FoxO3 cooperativity controls autophagy and is modulated by mTOR signaling, which would constitute a new paradigm with considerable potential for therapeutic modulation of erythropoiesis and understanding erythroid pathophysiologies. The Bresnick and Ghaffari groups will collectively test this hypothesis and elucidate mechanisms. Aim 1 - To elucidate mechanisms underlying FoxO3 regulation of terminal erythroid maturation. We will test the hypothesis that FoxO3 has a critical function to promote autophagy in late erythroid maturation. We will determine whether FoxO3 stimulates autophagy in steady-state and stress erythropoiesis contexts, if oxidative stress influences erythroid maturation by controlling autophagy, and if mTOR-FoxO3 crosstalk regulates autophagy. As inhibition of mTOR signaling improves anemia in a b-thalassemia model, we will address the potential function of autophagy in b-thalassemia. Aim 2 - To discriminate among models to explain how GATA-1 and FoxO3 cooperatively control autophagy and erythroid maturation. We hypothesize that GATA-1-FoxO3 cooperativity represents a physiological mechanism to control erythroid maturation. We will distinguish between models to understand the cooperativity, which conforms to a type I coherent feed-forward loop, and will determine how it contributes to establishment/maintenance of the erythroid genetic network. Given the importance of GATA and FoxO factors for regulating diverse processes, the studies will yield broad mechanistic and biological insights.

DESCRIPTION (provided by applicant): The ENCODE projects have generated a wealth of high-quality genomic datasets with the applications of high- throughput next generation sequencing (NGS) to create a catalog of functional elements in the human and model organism genomes. Although the NGS technologies, embraced by ENCODE, are enabling interrogation of genomes in an unbiased manner, the data analysis efforts of the ENCODE projects have thus far focused on mappable regions of the genomes and thereby have not fully leveraged these data to their full advantage. A major bottleneck to a comprehensive understanding of data from the ENCODE projects is the lack of statistical and computational methods that can identify functional elements in repetitive regions. We will address this critical impediment in four specifi aims by building on our expertise in ChIP-seq and RNA-seq analysis. In Aim 1, we will develop probabilistic models and accompanying software for utilizing reads that map to multiple locations on the genome (multi-reads) from multiple types of *-seq datasets (ChIP-, DNase-, MeDIP-, and FAIRE-seq). This will enable cataloging of regulatory elements in repetitive regions. In Aim 2, we will improve the specificity of the discoveries in repetitive regions from ou probabilistic models by utilizing multiple related *- seq datasets simultaneously. Specifically, we will devise methods to supervise analysis of ChIP- and RNA-seq datasets by external ChIP-seq datasets. This will facilitate accurate inference for repetitive elements with near identical sequences, e.g., segmental duplications, long interspersed nuclear elements, and boost accuracy of gene and isoform quantification with RNA-seq. In Aim 3, we will focus on identifying co-occupied/enriched regions to infer cell-specific modules of regions/genes and their regulatory profiles. We will also develop a formal differential co-enrichment framework to study cell-specific wiring and interactions of regulatory factors. This will elucidate how interactions among regulatory factors vary across cells/tissues/conditions. Aim 4, we will apply our methods from Aims 1-3 to relevant ENCODE data to understand GATA factor functions in hematopoiesis and vascular biology. The GATA system in human and mouse will serve as a training and validation platform for our methods. Statistical and computational resources generated from the project, which will be disseminated as modular and robust software, will help to enhance and maximize the impact of ENCODE-derived data on the biomedical research community. PUBLIC HEALTH RELEVANCE: The ENCODE projects have generated a wealth of high-quality functional genomic datasets with the applications of high-throughput next generation sequencing (NGS) to create a catalog of functional elements in the human and model organism genomes. A central limitation to a comprehensive understanding of these ENCODE data from the point of development, differentiation, and disease is the lack of statistical and computational methods that can identify functional elements in repetitive regions of the genomes. In this proposal, we will develop statistical and computational methods that can fully leverage ENCODE-derived data to their full advantage and catalog functional repetitive regions of the genomes.

Project Summary/Abstract Genomic technologies have revealed extensive transcription through ?non-coding? regions of genomes to yield regulatory RNAs that control development and physiology. A host of cellular constituents implicated in regulating RNAs function in broadly expressed multimeric complexes. Many questions remain unanswered regarding how these complexes control differentiation, proliferation and survival, and how they function in cell type-specific contexts, e.g. in diverse cells of the hematopoietic system. We discovered that the RNA- regulatory exosome complex (exosome) opposes primary erythroid cell maturation, and the erythroid transcription factor GATA-1 represses genes encoding exosome subunits. Downregulating exosome subunits abrogates intra-complex protein-protein interactions and promotes erythroid maturation. Our progress revealed the exosome confers expression of the vital receptor tyrosine kinase c-Kit that mediates Stem Cell Factor (SCF) pro-proliferation signaling in erythroid precursors, while opposing pro-differentiation erythropoietin (Epo) signals. These results establish a paradigm in which an RNA-regulatory complex orchestrates a developmental signaling transition to balance proliferation and differentiation. This paradigm has considerable importance for understanding mechanisms governing erythrocyte genesis in physiological and pathological states and provides a foundation for devising strategies to control this process independent of Epo-dependent interventions. In one aim, as specified by SHINE II, we propose mechanistic/biological analyses to elucidate how the exosome regulates c-Kit expression/function in primary mouse and human erythroid cells. In Aim 1, we will elucidate how the exosome confers SCF/c-Kit signaling. Exosome dismantling decreases Kit mRNA and primary transcript levels, the opposite response predicted from exosome function to degrade RNAs. As the exosome occupies insulators and promoters, is implicated in superenhancer function and chromatin modification, and suppresses promoter upstream transcripts that regulate genes, we predict it directly confers Kit transcription (Model 1). Since the exosome degrades regulatory RNAs, exosome dismantling might elevate regulatory RNA(s) that act in trans to repress Kit (Model 2). These equally important mechanisms represent a new dimension on how cells mount Stem Cell Factor (SCF) signaling. In Aim 1a, we will test the hypothesis that direct exosome function at Kit regulates transcription. In Aim 1b, we will test whether exosome dismantling selectively or broadly expels Pol II and/or its functionally distinct isoforms. In Aim 1c, we will test whether the mechanism operates in primary human erythroid cells, which is supported by compelling initial data. These studies will unravel a mechanism governing acquisition of SCF/c-Kit signaling and how the exosome controls erythroid maturation. Given the crucial c-Kit functions in normal and malignant hematology, regenerative biology and more broadly, mechanistic analyses of this innovative paradigm will yield findings of high fundamental and translational significance.

UWCCC Cancer Genetic and Epigenetic Mechanisms (GEM) Program Summary Co-Leaders: Emery Bresnick and Michael Newton PROJECT SUMMARY/ABSTRACT As genetic and epigenetic mechanisms underlie human cancer initiation and progression, components of these mechanisms represent extremely promising targets for cancer prevention, diagnosis, and therapy. Considering the multitude of regulatory factors involved, and the many unanswered mechanistic questions, our vision is that much of the mechanistic knowledge and druggable space remain to be discovered. Thus, from both fundamental and clinical/translational perspectives, elucidating cancer genetic and epigenetic mechanisms continues to hold great promise. The UWCCC Cancer Genetic and Epigenetic Mechanisms Program (GEM) consists of 27 highly collaborative members spanning 13 departments and 6 schools at UW-Madison. The program members include basic and translational scientists conducting multidisciplinary research, with 3 members directing clinical cancer research programs and engaged in patient care. Senior members actively mentor junior faculty, and additional faculty recruitments are ongoing. During the prior funding period, GEM members published 453 papers, many of which appeared in high-impact journals including Cancer Cell, Mol. Cell, Science, Science Trans. Med., Nature Chem. Biol., J. Clin. Invest., and Genome Res. The 27 GEM members brought in a total of $9.98 M in direct cost cancer-relevant funding for which they are the PI (NIH total, $5.79 M, of which NCI, $1.25 M). GEM Thematic Aims are: 1) Discover and elucidate cancer genetic mechanisms; and 2) Discover and elucidate cancer epigenetic mechanisms. Multidisciplinary GEM teams are analyzing genetic and epigenetic mechanisms in breast, prostate, sarcoma, myeloid leukemia, and other cancers. The discoveries are used to develop new paradigms in cancer biology, and via new intra- and inter- programmatic collaborations, are applied toward clinical trials to improve cancer prevention, diagnosis, and therapy. GEM-derived methodological/technological innovations continue to dramatically enable GEM and broader UWCCC cancer research. An overarching theme is our invention and deployment of powerful strategies to discover and elucidate cancer mechanisms and enable diagnostic and therapeutic modalities unlikely to emerge from existing paradigms.

Project Summary/Abstract Bioanalytical tools to study long noncoding RNAs (lncRNAs) and their protein interactors are desperately needed. lncRNAs are critical elements in the transcriptional regulation of gene expression. They have been shown to function through several different mechanisms. They may serve directly as gene-regulatory factors, produced by transcription at the genomic site where they act. Alternatively, lncRNAs interact with proteins to control gene expression. For example, lncRNAs can act as a ?molecular sink? for proteins that interact with DNA or RNA; in this case, binding of the protein to the lncRNA competes with its interaction with its primary target. lncRNAs can also guide proteins to their DNA targets to either repress or activate transcription. They can act not only in cis, or near their site of transcription, but also in trans, at multiple genomic sites. Finally, lncRNAs can act as platforms upon which molecules can convene to perform a function as a team at a specific location and time (e.g. histone modification complexes). Each of these mechanisms requires the interaction of lncRNAs with a diverse protein cohort. Knowledge of the proteins bound to specific lncRNAs is thus essential information needed to understand mechanisms by which lncRNAs control gene expression. Unraveling this tremendous diversity of interactions demands tools capable of rapid identification and quantification of lncRNA- associated proteins. Despite the great importance of lncRNAs and the proteins that interact with them, there are significant limitations in the tools available to study them. We propose to develop and validate a suite of powerful new tools for the discovery, identification, and quantification of lncRNAs and for the comprehensive proteomic analysis of their protein interactomes. It is known that lncRNAs direct gene expression to modulate cell fate in the hematopoietic system, enabling diversification of gene programming during development. Disrupted lncRNA function can also contribute to malignant transformation in specific myeloid and lymphoid cancers. GATA factor-regulated lncRNAs, discovered in powerful genetic systems (wild type and Gata2 enhancer-mutant mice, GATA-1 genetic complementation system and GATA factor knockdowns in primary human erythroid precursor cells), control human erythroid precursor cell function and erythrocyte development. In initial profiling studies, we have identified 74 GATA factor-regulated lncRNAs. The performance of the new tools developed here will be thoroughly tested on a subset of these 74 lncRNAs to discover GATA factor-dependent regulatory circuits and networks that control hematopoiesis. The proposed research will drive state-of-the-art lncRNA identification and proteomic analysis of the lncRNA interactome, applied to one of the most important regulatory networks in hematopoiesis. We will bring a new and unprecedented level of visibility to the definition of the GATA factor-relevant lncRNA interactome, while developing powerful open-source software tools and detailed experimental protocols.

PROJECT SUMMARY Hemoglobin synthesis and erythrocyte development are often studied independently, yet their mechanisms are inextricably linked. Differentiation defects yield immature precursors, and impaired hemoglobin synthesis causes ineffective erythropoiesis. A common thread of these mechanisms is GATA transcription factor involvement. Many questions remain regarding how GATA factor networks instruct progenitors to generate vast numbers of erythrocytes, which broadly informs molecular/cellular biology and hematology. We discovered: 1) locus-specific coregulator utilization by GATA1 to control differentiation; 2) GATA factor/regeneration-activated enhancer confers expression of an unstudied sterile alpha motif domain protein that controls erythrocyte regeneration; 3) GATA factor-regulated zinc transporter switch governs differentiation; 4) mechanism of heme targeting chromatin genome-wide; 5) GATA factor-regulated solute carrier protein (SLC) cohort transports diverse small molecules to control erythropoiesis. Our multi-omic work supports the aims to analyze how GATA factors establish small molecule ensembles that target the genome and regulate the GATA factor to ensure differentiation. Aim 1 will dissect a multi-component mechanism by which GATA1 and heme control genome function and erythrocyte development. GATA1 activates genes mediating heme biosynthesis, heme facilitates or restricts GATA1 function and heme downregulates GATA1. Heme regulates transcription by downregulating the repressor Bach1, and we discovered a Bach1-independent heme-regulated mechanism. We hypothesize that Bach1-dependent and -independent mechanisms establish critical erythroid functions, and these mechanisms provide translational opportunities. Using all heme target genes and a gene-specific approach, we will establish the mechanisms. Aim 2 will elucidate a GATA factor-dependent small molecule transporter axis that regulates erythroid differentiation. We hypothesize that diverse small molecules function in GATA factor mechanisms and discovering GATA factor-regulated solute carrier (Slc) transporters will unveil new dimensions to these mechanisms. We defined a GATA1/2-regulated Slc cohort that transports diverse small molecules. We prioritized a subset with GATA factor-occupied predicted enhancers and will elucidate mechanisms that link GATA factors with small molecule ensembles and differentiation. Aim 3 will test models for how GATA1 instigates a sphingolipid-dependent regulatory network. GATA1-regulated Slcs included sphingolipid transporters. Lipidomics revealed GATA1-induced sphingolipid remodeling. Ceramide synthase inhibition blocks GATA1-mediated GATA2 downregulation, ?-globin induction and erythroid maturation. Sphingolipid signaling controls apoptosis, proliferation and migration, high S1P is deleterious in sickle cell disease, and human ceramide deficiency involves disrupted erythropoiesis. We hypothesize that sphingolipidome regulation by GATA1 is vital in biology and pathology. We will develop basic and translational insights into GATA factor mechanisms governing small molecules that control GATA factors, globin synthesis and differentiation.