Michael G. Rosenfeld - US grants

Understanding the mechanisms by which hormones regulate specific function in normal and tumor tissues is the central theme of our research efforts. We have described a novel mechanism for regulation of hormone biosynthesis during study of "switching" events in rat medullary thyroid carcinoma lines (MTCs). A single hormone-encoding gene can generate five to six structurally different mRNAs, in a tissue-specific fashion, as a consequence of alternative RNA processing events. These mRNAs encode discrete proteins productive of entirely different hormones. Thus, in the case of calcitonin gene expression, one mature mRNA produced in thyroidal "C" cells encodes two component hormones (calcitonin and a new hormone which we refer to as CCP); while a second mRNA, produced in the hypothalamus, encodes a new putative hormone, which we refer to as calcitonin gene-related peptide (CGRP). The generation of alternative RNA and protein products from a single endocrine gene is referred to as "peptide switching." Several experimental approaches will be utilized to establish whether these switching events result from bona fide RNA processing alterations, or are consequential to altered transcriptional initiation or termination or to altered genomic structure. Experiments designed to demonstrate the production of predicted peptides and to define functions of the new putative hormones will be initiated. The production of CGRP by hypothalamus and its tissue distribution will be confirmed by construction and sequencing of appropriate cDNA clones. Possible hormonal regulation of "peptide switching" events will be studied in clonal cell lines derived from MTC tumors. Additional studies will explore the possible clinical ramifications of these events in the diagnosis and biology of human cancers, particularly carcinomas of the lung. (C)

Polypeptide hormones exert rapid, specific effects on protein biosynthesis and gene expression in their target tissues, critical for normal development and homeostasis. Defining the precise biochemical mechanisms responsible for this regulation would provide a conceptual framework with which to investigate many diseases of disorders regulation. Thyrotropin releasing hormone (TRH), epidermal growth factor (EGF), bombesin, phorbol esters (TPA), and cAMP act within minutes of their addition to clonal rat pituitary cell cultures (GH4) to produce specific transcriptional stimulation of the prolactin gene; growth hormone releasing factor exerts comparable actions on the GH gene. The site of regulation of rat prolactin and growth hormone (GH) biosynthesis by polypeptide hormones is therefore at the level of gene transcription. We propose to define the precise genomic sequences in the less than 200 bp of 5' flanking information which quantitatively transfer polypeptide and steroid hormonal regulation characteristic of prolatin and GH gene regulation using a combinatorial analysis including DNA-mediated gene transfer of fusion genes containing genetically modified prolactin and growth hormone genomic fragments into homologous cell lines and into primary pituitary cell cultures, and into normal tissues using appropriate retroviral vector systems. Several approaches will be utilized to define the sequences imparting tissue-specific neuroendocrine gene expression and to identify the critical trans-acting factors. Affinity chromatography and a novel expression strategy will be adapted to isolate the transactive regulatory factors critical for hormonally- and developmentally- mediated patterns of gene expression, and to clone their encoding mRNAs. Transgenic animal approaches will be used to further define the complex patterns of sequence recognition and chromosomal position effects which adjudicate the tissue-specific developmental patterns of gene expression characteristic of the neuroendocrine system. The structure and regulatory function of two novel basic chromatin associated proteins which are rapidly phosphorylated in response to polypeptide hormones aned cAMPL will be elucidated via recombinant DNA technology. Based upon the cloning of the EGF receptor cDNA in this laboratory and the identification of polymorphic RNA products, we propose genetic evaluation of the structural components of the EGF receptor required for growth, protein synthesis, and transformation.

Project Abstract We are pleased to report that our Training Program has successfully trained graduate students who have become the next generation of creative independent investigators in the fields of gene regulation, mechanism of hormone and signal action, molecular genetics of development, homeostasis, metabolism and disease, inflammation, learning and neuronal function, global genomics and contemporary bioinformatics. This achievement reflects the strength of our training program fueled by its innovative, forward-thinking, and distinguished core faculty, who reflect the rich research environment of the La Jolla Mesa area that includes the UCSD School of Medicine, School of Biology, the Jacobs School of Engineering and the UCSD Program in Bioinformatics and Systems Biology. Notably, it also includes the Salk Institute for Biological Studies, The Scripps Research Institute, and the Sanford Burnham Prebys Medical Discovery Institute. Additionally, the recently constituted UCSD Institute for Genomic Medicine has further strengthened training in the contemporary fields of global genomics and chromatin biology. The core faculty are all internationally recognized for their research accomplishments and have strong records of training graduate students who have gone on to very productive academic and industry careers. Our program is highly interdisciplinary involving faculty from various Schools and Programs as well as the Salk Institute and The Scripps Research Institute. Graduate students are immersed in contemporary research approaches to important questions and have the full range of training in contemporary global genomics techniques, biochemistry, molecular biology, genetics, and gene regulation. Most importantly, students are expected to acquire a broad background of the scientific literature, and critical thinking, as well as the creative spirit that underlies success in these research areas. Formal coursework, hands-on bench experience, and intensive interaction and collaboration opportunities with program faculty will also prepare trainees for pursuing independent academic careers in these disciplines. We hope to be permitted the opportunity to continue this highly successful predoctoral training grant with a request for expansion for two postdoctoral trainees. Six predoctoral trainees per year are drawn from the highly competitive PhD programs. Candidates are chosen from more than 1000 applicants. Established criteria include research background, grade point average, GRE scores, and interviews with faculty and senior graduate students. All programs have a full complement of Diversity Initiatives. Trainees are directed towards careers in contemporary gene regulation, endocrinology, epigenomics, informatics, chromatin biology, metabolism and neurobiology, with a perspective towards translational application to human disease. Trainees are encouraged to develop skills in bench science, critical thinking, grantsmanship, teaching, and translational research in order to become independent investigators at leading academic institutions and make important contributions to biomedical science.

Recent studies of the atherogenic process in hypercholesterolemic animals suggest that the macrophage plays a role in the initiation and possibly the progression of atherosclerotic lesions. Although much is known about macrophage biology in general, very little is known about the properties of the macrophage while resident within the developing lesion other than its capacity for accumulating large amounts of lipid. The objectives of this study are to further elaborate how macrophages are involved in the atherogenic process, and specifically to determine whether the arterial macrophage is functionally and antigenically different from macrophages resident within other tissues. Initially, macrophage- derived foam cells will be isolated from atherosclerotic rabbit arteries using an enzyme digestion procedure, and purified using rate zonal gradient centrifugation, both under sterile conditions. The isolated foam cells will be placed in culture and the capacity of the cells to secrete mitogens, chemotactic factors, lipoprotein lipase, and Apo E will be assayed. In addition, the capacity of the foam cells to bind, degrade, and modify lipoproteins and proliferate will be determined. Subset of the foam cells, defined on the basis of size and buoyant density, type of lesion or location within a lesion will be purified and placed in culture. The functional assays listed above will be performed in order to determine whether there is any functional differences in the subsets of these cells. The purified foam cells, as well as crude homogenates of atherosclerotic lesions, and lipid-laden cultured macrophages will be used as immunogens to generate specific monoclonal antibodies that recognize the subsets of foam cells. In this fashion, we hope to demonstrate a correlation between the functional and antigenic properties of these cells. Knowledge of phenotypic heterogeneity of arterial macrophages should help define the natural history of the atherosclerotic lesion and aid in the design of therapeutic strategies.

Understanding the physiological mechanisms of brain development and maintenance of neuronal phenotype is central to elucidating abnormalities that may undertie severe neurological and psychiatric abnormalities. The molecular mechanisms responsible for activating gene expression responsible for establishing normal neural phenotypes and patterns of connections are unknown. Identification of the factors normally dictating these developmental events is critical for understanding abnormalities in brain development and function. We have recently identified a large family of brain-specific transcription factors containing a novel domain, referred to as the POU-domain. The identification of members of the POU-domain family expressed in discrete temporal and spatial patterns during neural development, referred to as Brn-1, Brn-2, Brn-3, Brn4, and Tst-1. Their expression in the cortex, sensory ganglia, hippocampus and hypothalamus permit a strategy to investigate transcriptional activation and regulation of genes establishing distinct neuronal phenotypes with the central nervous system. The functions of the POU-domain proteins for brain development will be investigated by targeting ectopic expression in transgenic mice, by creating POU-domain gene negative transgenic animals, and by characterizing known mutants for defects in expression of functional POU-domain proteins. A detailed analysis of the POU-domain structure and function, protein interactions, hormone-induced covalent modifications, and regulatory DNA cognate elements will be performed. Additional positive and restrictive factors required for cell-specific expression have been characterized and cloned, permitting investigation of combinatorial codes of cell-specific gene activation. This cis-active elements dictating the ontogeny of POU-domain gene activation during pituitary development will be established, the responsible trans-activating factors identified, and their encoding genes isolated. Retinoic acid receptor regulates expression of specific POU-domain genes, binding to specific cis-active DNA recognition elements. This binding appears to require interaction with another protein(s), referred to as comorphogens. A series of putative factors that can form heterodimers with the retinoic acid receptor, and that are required for binding and developmental function in neural cell lines, will be identified and structurally characterized, and their functional roles in morphogenesis will be defined. These studies should provide insights into the complex interactions of regulatory factors that control development and homeostatic control characteristic of the mammalian neuroendocrine system. These investigations will ultimately leave clear implications to problems of mental health, and these issues can be effectively explored based on the initial, proposed investigations.

Understanding the physiological mechanisms of brain development and maintenance of neuronal phenotype is central to elucidating abnormalities that may undertie severe neurological and psychiatric abnormalities. The molecular mechanisms responsible for activating gene expression responsible for establishing normal neural phenotypes and patterns of connections are unknown. Identification of the factors normally dictating these developmental events is critical for understanding abnormalities in brain development and function. We have recently identified a large family of brain-specific transcription factors containing a novel domain, referred to as the POU-domain. The identification of members of the POU-domain family expressed in discrete temporal and spatial patterns during neural development, referred to as Brn-1, Brn-2, Brn-3, Brn4, and Tst-1. Their expression in the cortex, sensory ganglia, hippocampus and hypothalamus permit a strategy to investigate transcriptional activation and regulation of genes establishing distinct neuronal phenotypes with the central nervous system. The functions of the POU-domain proteins for brain development will be investigated by targeting ectopic expression in transgenic mice, by creating POU-domain gene negative transgenic animals, and by characterizing known mutants for defects in expression of functional POU-domain proteins. A detailed analysis of the POU-domain structure and function, protein interactions, hormone-induced covalent modifications, and regulatory DNA cognate elements will be performed. Additional positive and restrictive factors required for cell-specific expression have been characterized and cloned, permitting investigation of combinatorial codes of cell-specific gene activation. This cis-active elements dictating the ontogeny of POU-domain gene activation during pituitary development will be established, the responsible trans-activating factors identified, and their encoding genes isolated. Retinoic acid receptor regulates expression of specific POU-domain genes, binding to specific cis-active DNA recognition elements. This binding appears to require interaction with another protein(s), referred to as comorphogens. A series of putative factors that can form heterodimers with the retinoic acid receptor, and that are required for binding and developmental function in neural cell lines, will be identified and structurally characterized, and their functional roles in morphogenesis will be defined. These studies should provide insights into the complex interactions of regulatory factors that control development and homeostatic control characteristic of the mammalian neuroendocrine system. These investigations will ultimately leave clear implications to problems of mental health, and these issues can be effectively explored based on the initial, proposed investigations.

morphology; biomedical facility; light microscopy; tissue /cell preparation; autoradiography; photomicrography; computer graphics /printing; immunocytochemistry; electron microscopy; image processing; in situ hybridization;

Development of neurons, such as those of the endocrine hypothalamus invoves regulation by the actions of nuclear receptors and by other classes of transcription factors, such as the Class III POU domain factors, as well as the actions of specific signaling molecules. The initial five years under this Merit Award have proven to be our most productive and innovative cycle under this Grant. We have contributed a series of discoveries concerning the molecular mechanisms by which nuclear receptors mediate gene repression and activation events and that have established a new paradigm concerning the molecular mechanisms of gene regulation. Our central theme in our laboratory under this grant has been to define the molecular mechanisms by which nuclear receptors, POU domain factors, and other classes of transcriptionfactors function via recruitment of corepressors and coactivators, and to define a role for these factors in establishing neuronal and other cellular phenotypes and in regulating function. We have identified novel molecular mechanisms that regulate tissue- and cell type-specific proliferation and activation, and defined roles for corepressors in these events. We will investigate the molecular basis of action of components of the TBLR1/TBL1/GPS2/ HDAC3 complexes, investigating the potential roles of GPS2 as an inhibitor of specific protein kinases, and the roles of site-specific phosphorylation of N-CoR/TBLR1 and TBL1 in the required recruitment of specific ubiquitin ligase 19S proteasome machinery using neuronal models. The relationship of these factors to the actin-related complexes that may detect nuclear localization and alterations in chromatin structure will be defined

DESCRIPTION (adapted from the applicant's description): Defects in septation and placement of pulmonary artery and aorta reflect diverse defects in a multifunctional pathway that controls modulation of the great vessels. Over the past two years, the PI's laboratory, based on study of homeodomain transcription factor interactions, discovered two bicoid-related homeodomain transcription factors, referred to as Pitx1 and Pitx2 that prove to be critical regulators of these events. The distribution of Pitx1 and Pitx2 expression strongly suggested that these might mediate distinct aspects of left/right asymmetry of the cardiovascular/pulmonary system, and specific aspects of subsequent cardiac development. Generation of gene-deleted mice has documented their critical function in both events. The Pitx proteins are positive transcription factors that act synergistically with other classes of transcription factors. They hypothesize that these genes are in a regulatory pathway required for aspects of correct septation and positioning of the developing aorta and pulmonary arteries, in part, by regulation of critical neural crest components. Pitx1 and Pitx2 also exert regulation in other aspects of cardiac development. The goal of this proposal is to investigate the molecular mechanisms by which Pitx1 and Pitx2 mediate these events, using several independent genetic approaches, to define their roles in normal cardiac development, their relationships with other factors, and their potential combinatorial roles in the development of the cardiac outflow tract. They propose to identify the downstream target genes that mediate their roles in cardiovascular development, thus, identifying potentially novel regulatory molecules that underlie congenital defects in outflow tract and coronary artery vessel development. Finally, they will identify and clone factors that interact with the Pitx transcription factors in the heart, and may be critically involved in the combinatorial transcriptional regulation of target genes critical for development of the truncus arteriosus. The possible role of environmental and regulatory factors in the animal model can therefore be studied, based on the modulation or alteration of these pathways.

ABSTRACT Based on androgen-dependency for growth of prostate cancer, as well as normal prostate, anti-hormone therapy is typically initiated using selective androgen receptor modulators (SARMs). It is important to delineate new, unexpected aspects critical to AR actions, and new technologies to bear on the problem of prostate cancer. Although initially effective, resistance invariably fails as a tumor becomes androgen-independent or hormone refractory. In order to design new approaches to overcome resistance, it is necessary to understand the molecular basis of resistance. Although several models of resistance have been proposed, including mutations of the androgen receptor, these can account for only a very small percent of clinical resistance. Under the initial Grant cycle, we successfully defined a model for resistance based on macrophage:prostate cancer cell interactions which resulted in IL-1-MEKK1-TAB2-dependent derepression of AR target genes in the presence of SARMs, based on dismissal of the NCoR corepressor complex. The recruitment of TAB2 to serve as a molecular beacon for these events is specific to sex steroid receptors, reflective of evolutionarily conservation for key reproductive biological functions. However, we have also obtained data for unexpected components of a pathway that normally prevents ligand-dependent AR activation, providing new therapeutic possibilities. These unexpected mechanisms for hormone independence include failure of inhibitory histone methylation events and actions of a specific regulatory component of the NCoR complex. Our recent introduction of new technologies has revealed rapid AR-dependent changes in nuclear architecture provides an entirely new concept of androgen-dependent gene activation and new targets for preventing resistance. By developing and applying a novel genome-wide promoter location analysis method sufficiently sensitive to be suitable for use with clinical biopsy specimens, to issues of chromosomal interactions, coupled with powerful cell biological tools, we propose to delineate the molecular events underlying these additional mechanisms. Together, understanding these unexpected specific regulatory strategies should provide a series of leads for new therapeutic approaches to clinical resistance in prostate cancer. Relevance: Prostate cancer represents a major health problem in the United States and elsewhere, and is a source of major familiar and personal tragedy and societal expense. Our studies of androgen receptor and prostate cancer has direct relevance to our understanding and treatment of this disease, with implications for breast cancer as well.

DESCRIPTION (provided by applicant): The precisely-regulated pattern of recruitment of DNA binding transcription factors and associated coactivators and corepressors underlies the critical events that regulate neurodevelopment and disease. We propose to validate 3 distinct technologies that will permit genome-wide promoter location analysis for these regulatory factors-ChlP-on-chip, ChlP-DASL-chip, and ChlP-RDA-for applications to test specific hypotheses in neurodevelopment and disease. Genome-wide promoter assays will be designed, constructed and validated and we will test these technologies by determining the pattern of cofactor binding in the genome. New siRNA approaches to profiling will be applied to validate occupied promoters as functional targets. We will focus on a method that provides the optimal sensitivity and applicability to gene tiling, and will generate a genome-wide murine promoter array and will tile a series of genomic intervals that will permit investigation of specific genomic loci to uncover epigenetic strategies used in signaldependent changes in gene activation/repression programs development. These include study of the roles of CaMKII delta and SMRT in early programs of neurodevelopment. This approach will permit testing of several hypotheses and will be used to identify the promoter-specific usage of key corepressors and coactivators and location of epigenetic marks. We believe that with validation of these approaches, technologies developed here will be of widespread utility in neuroscience, particularly in studies of development and neurodegenerative disease.

DESCRIPTION (provided by applicant): Under support of this grant, our laboratory has investigated 2 bicoid-related homeodomain transcription factor, referred to as Pitx1 and Pitx2 that have proved to be critical regulators of development of the cardiac outflow tract and heart, identifying a novel Wnt/Dvl/beta-catenin-Pitx2 pathway that is required for correct development of the cardiac neural crest. Our laboratory is now focused on dissection of transcriptional events that regulate specific aspects of development of the cardiovascular system and understanding the underlying molecular mechanisms and gene ontology programs that are required for these events. Our investigation of transcriptional repression and corepressors has led to the discovery of an unexpected role of the nuclear receptor corepressor, SMRT, in heart development and the identification of an unexpected mechanism for activation by regulated DNA binding transcription factors. In this competitive renewal, we will develop new strategies for genome-wide localization of regulatory transcription factors and cofactors that, in coupled with siRNA/RNA profiling strategies, will permit elucidation of direct target genes for specific transcriptional factors and signaling pathways important in cardiovascular development. Using these technologies, we will define the gene ontology programs by which Pitx2 and SMRT regulate cardiovascular development. The discovery in cardiovascular and macrophage developmental programs of a novel strategy to enforce repression checkpoints that are critical in regulation of both macrophage, epicardium and myocardium, will be pursued to define the underlying molecular mechanisms of cofactor exchange. These studies should add to our knowledge of the molecular basis of early events critical for heart and outflow tract development, and should provide a basis for evaluating new approaches for modulating these events. These studies are likely to have direct implications for several human cardiovascular defects and diseases.

ABSTRACT Under this Grant, we have investigated brain-specific and hormone specific strategies of transcriptional regulation, focusing on the hypothalamic: pituitary axis, repression complexes, and the neural stem cell state. These studies have elucidated roles of the NCoR/SMRT corepressor complexes, and associated factors, including TBL1/TBLR1, HDACs and TAB2 as components of the machinery that maintain a repression checkpoint, as well as the enzymatic machinery that regulated the exchange of corepressor:coactivation complexes. Genetic approaches have elucidated the functional distinctions between NCoR and SMRT in differentiation of NSCs along a neuronal pathway, in part based on roles of SMRT in repressing the RAR-dependent induction of H3 K27 Me3 demethylation factors, revealing an additional regulatory pathway in maintaining the NSC state. By elucidating the in vivo role of the histone demethylase LSD1 and the role of methyltransferases in preventing constitutive activation by nuclear receptors. We have elucidated roles for these members of these parameters in regulation of specific gene cohorts. We have provided initial evidence of ligand-dependent interchromosomal interactions dependent on ligand- binding of nuclear receptors, and apparently requiring a motor system. These events are hypothesized to exert critical functional roles in ligand-dependent gene activation events. The central Specific Aims in this competitive renewal are to investigate the molecular mechanisms by which signals and ligands modulate long-distance, interchromosomal networks of gene interactions, and their possible actions dependent upon the presence of DNA binding site in specific classes of DNA repeats. We have developed several technologies to investigate these transcriptional strategies in a genome-wide fashion, permitting us for the first time to directly address the broad roles of DNA repeats and ncRNAs in regulated interchromosomal interactions. These will permit us to access potential roles of these events in gene regulatory programs chromosomal translocation events in tumors, signal pathways and genotoxic stress. We will investigate function connections between these signaling events by histone demethylase and roles of receptor bound enhancers in promoting interactions with interchromatin granules, linking elongation and RNA processing events to transcriptional initiation. We further propose to investigate the hypothesis that noncoding RNA transcripts and specific subclasses of DNA repeats that bind specific nuclear receptors exert roles as sensors of genetic stress and in control of nuclear architecture, using neuronal cells and neuronal stem cells as models. We suggest that these mechanisms are likely to provide important insights, and possibly new intervention strategies, into many human diseases, including neurodegeneration, defects of development, diabetes and cancer.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The intracellular domain of APP is the center of a protein network involved in Alzheimer disease. We are interested in the dynamic of this complex during cellular stress.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Histones do change the interacting partners upon disease, cellular differentiation and during stress. Histone modifications determine the expression level of different genes in spatial and temporal manner. We are intersed in finding out the modifications and interacting partners of histones during such conditions.

DESCRIPTION (provided by applicant): Understanding the transcriptional programs that define cell type and regulate the inflammatory responses in macrophages and vascular endothelium is of central interest in understanding/preventing prevalent cardiovascular diseases. The molecular strategies that dictate these critical transcriptional programs reflect, in large part, the actions of dedicated repression "checkpoints" and the functions of enhancers that modulate cell type-specific gene expression programs. How the programs dictated by an enhancer "code" underlying such programs remains a fundamental question in regulatory and cardiovascular biology. Our studies under this Grant have linked integration of inflammatory and anti-inflammatory signaling pathways and inhibiting atherosclerosis, uncovering functionally-distinct pathways utilized by PPAR3 and LXRs, and provided initial insights into the large programs of transrepression critical for blocking inflammatory pathways. Here, we will use genetic and epigenetic approaches to uncover the in vivo roles of dedicated enhancer networks in cardiovascular disease. We propose to focus on the molecular determinants of cell type-specific gene enhancer programs and three-dimensional genomic interaction networks that underlie developmental and newly discovered regulatory programs in the cardiovascular system and macrophages. We will link these programs to two important myocardial infarction susceptibility loci, based on results from genome-wide association studies (GWAS), providing an unprecedented opportunity, based on human genetic models, to further define and delineate the role of three-dimensional "enhancer networks" and epigenetic strategies in development and disease of the cardiovascular system. These studies should provide a general approach to investigating disease susceptibility loci for many classes of disease. PUBLIC HEALTH RELEVANCE: Coronary artery disease, the predominant cause of myocardial infarction, is caused by coronary artery atherosclerosis, with an estimated 1.5 million Americans experiencing a coronary attack/infarction in 2009. Because atherosclerosis reflects both inflammatory and lipid metabolism disorder in which monocytes/macrophages play a central role in all phases of atherosclerosis, our proposal to link the discovery of the key regulated "enhancer codes" in tissues that underlie cardiovascular disease and regulation of their nuclear architecture to genome-wide association studies of susceptibility to coronary artery disease can provide new insights and approaches to the prevention and treatment of this major disease of the cardiovascular system.

DESCRIPTION (provided by applicant): This Merit Award has permitted the opportunity to focus over the past decade on the molecular/epigenetic strategies that combinatorially regulate programs of gene transcription, that include investigation of histone demethylases, the actions of non-coding RNAs in recruiting regulatory protein complexes in control of transcriptional programs, the role of regulated nuclear architecture in transcriptional regulation and tumor translocation events, the linkage of transcription and DNA damage/repair, the role of specific phosphatases in gene regulation, regulated apoptosis/survival, and the strategies of exchanging corepressor coactivators. Under the first Specific Aim, and based on extensive preliminary data, new histone demethylases affecting cell cycle regulation will be explored and various new aspects of enhancer programming and regulation will be investigated. Under the second Specific Aim, new strategies to investigate tumor translocation by sex steroid receptors, the connection between specific demethylases and septic shock will be explored, and a new strategy for screening chemical libraries is being developed. The recent introduction of powerful new technologies, including next-generation sequencing, has permitted us to generate preliminary data designed to uncover previously- unsuspected aspects of epigenetic regulation of transcription, with particular relevance to translational aspects of common disease including breast and prostate cancer and cytokine storm syndromes, translational areas that represent important research objectives in this Competitive Renewal. We propose specific areas of investigation based on several novel and promising new technologies we are developing under this Grant that will be of broad utility to the scientific community, centered on key, unanswered questions concerning gene regulation by nuclear receptors. We propose to vigorously pursue these fundamental issues in order to accelerate the discovery of as yet unknown putative tumor translocation events in breast cancer, and to complete the development of new, powerful strategies including a new multiplexed screen to identify new approaches to intervene in these regulatory and pathological events.

3. Function of Core A This PPG proposes to use deep sequencing-based approaches to define transcriptional mechanisms that regulate inflammation and insulin resistance. An essential aspect of all three projects will be to define the consequences of gain and loss of function of regulatory proteins on global programs of macrophage and adipocyte gene expression using massively parallel RNA sequencing technologies. All three projects will also employ ChiP-Sequencing technologies to define the cistromes of wild type and mutant PPARy and components of NCoR/SMRT co-repressor complexes. Finally, Projects 2 and 3 will use recently developed assays of three-dimensional genomic interactions (3D-DSL) to interrogate the relationship between nuclear architecture and gene expression. The PPG Deep Sequencing Core will provide two complementary services to advance these efforts; technical support for preparation and sequencing of the specific libraries required for each assay and bioinformatics/computational support to enable effective effective experimental design and utilization of the resulting data.

PROJECT ABSTRACT Many genetic disorders are characterized by defects in neuronal function and cognitive ability. To gain key insights into new ways of approaching these diseases, it is critical to understand the molecular basis for regulating neuronal transcriptional programs and take advantage of emerging global genomic technologies, which provide deep insights into the molecular basis for these disorders. Here, we focus on a severe neurological disorder that is caused by mutations of the methyl-CpG-binding protein, MeCP2, and how it causes alterations in gene expression programs required for neuronal function. We propose to investigate this perplexing syndrome, which can be categorized under the umbrella of ?autism spectrum? disorders, to test a previously unappreciated mechanism for regulating neuronal transcription programs based on chromosomal architecture. Specifically, we will determine the effects of MeCP2 loss on chromatin architecture and its relation to changes in gene transcription, as well as determine the functional role of MeCP2 on the nuclear matrix-dependent architectural network. We will utilize several mouse genetic models to target several important brain cell types and apply genome wide methodologies to elucidate the contribution of each of these to the observed brain phenotype. We expect to expand our understanding of MeCP2-dependent transcriptional de- regulation due to structural effects on chromosome architecture, and uncover the role of MeCP2 in regulation of chromosomal boundary/ sub-nuclear architectural interactions as an underlying mechanism in Rett syndrome. This will enable new understanding of the molecular mechanisms of this devastating neurological disease, and lay the groundwork for potential new classes of potential therapeutic avenues.  

PROJECT ABSTRACT Alzheimer?s disease (AD) is conventionally characterized by specific neuropathological features, including the appearance of extracellular amyloid deposits and the accumulation of intracellular neurofibrillary tangles. While several gene mutations are clearly associated with early onset Alzheimer?s disease, the large number of individuals exhibiting delayed onset, aging-associated AD, are likely to harbor many alterations in linked modifier genes that predispose to AD susceptibility. Genetic and genome wide association studies (GWAS) have identified numerous genes and risk alleles that indicate both cell autonomous and non-cell autonomous mechanisms contributing to loss of neurons and cognitive decline. In this regard, the majority of risk variants identified by GWAS reside in non-coding regions of the genome, implying that they act in part to alter gene expression. This proposal responds to the RFA indicating a particular need for approaches designed to delineate the transcriptional and cellular consequences of combinations of SNPs in the risk alleles by generating new cell line reagents to help unravel the question of the causative SNPs and their target genes in specific neurons derived from iPS cells of AD individuals. There are two features of sporadic AD that require molecular explanation- the potential role of aging in AD susceptibility, and the striking gender disparity, with the incidence of AD being exaggerated in females. These issues can only now be addressed based on new technologies and the availability of patient-derived samples. Our proposed research plan takes advantage of the invaluable samples stored at the brain bank of the Shiley-Marcos Alzheimer's Disease Research Center (ADRC) at UCSD, and the iPSC-derived neurons (Salk). This approach will interrogate the effects of different genetic variants with other risk factors (e.g. age, sex), and assess their effects on cell type-specific enhancer landscapes. By merging these data, we can begin to identify the potential causative SNPs that result in altered function of cell-type specific enhancers. We propose using a high throughput 4C screening approach (UMI-4C), and Hi-ChIP, to identify the most likely causative, enhancer-associated SNPs for functionally-implicated coding target genes. Exploiting the power of contemporary gene editing approaches in control or patient-derived iPS cells to specific neuronal cell types, and to astroglia, we can assess the transcriptional phenotypes and functional behaviors of neurons harboring different combinations of risk alleles, both in the isolated cell lines alone and in combination with coculture experiments with astroglia and microglia, as effects of these SNPs may be manifest only with astroglial:neuronal interactions. Together these studies will use powerful contemporary global genomic approaches to determine the coding transcriptional targets of several of the most significant SNPs in enhancers, and the link to roles of estrogen receptor in the gender disparity for AD.

ABSTRACT We propose a study of functional interactions between nuclear receptors in breast cancer cells as a project under this USA-NFSC initiative, with great scientific and intellectual benefit, based on the efforts of two closely- collaborating investigators with shared research interests, but with disparate areas of expertise. Drs. Michael Rosenfeld and Wen Liu have an extensive history of effective collaborations, as evidenced in a number of fundamental discoveries over the past ten years, including nine co-authored papers. This project, involving a sustained, close scientific interaction and collaboration is based on their complementary expertise, focus and resources. We propose to establish an unappreciated, but critical, molecular strategy that serves as the basis for large enhancer-dependent programs of coding target gene transcriptional repression important in breast cancer. This program depends on the fact that ER? is recruited in trans to the basally active enhancers that mediate the repressive transcriptional program, with trans-bound ER? receptor recruiting a demethylase, based on the availability of its DNA binding domain, which in turn recruits machinery leading to dismissal of Pol II from these basally highly active enhancers, causing their repression. This mechanism therefore represents a previously unappreciated type of repressive strategy, and involves a gene set that serves as a powerful prognostic indicator of a ten-year metastasis-free survival. This would uncover a set of largely overlooked prognostic biomarkers for breast cancer patients, perhaps ultimately providing a potential target for treatment or prevention of aggressive breast cancers. A strategy is proposed to underlie the ability of liganded glucocorticoid receptor to inhibit the ER?-activated regulatory enhancers in breast cancer cells, based on competition between different members of the nuclear receptor family.

PROJECT ABSTRACT A fundamental question in regulatory biology is whether enhancers regulate only the coding target genes with spatial proximity, or whether they regulate chromosomal architecture, exerting transcriptional effects on genes far removed in a specific chromosome. Thus, in addition to exhibiting regulated interactions with its cognate promoter, and perhaps with other enhancers in a single TAD, it is important to solve whether signal-induced proximity of specific robustly-activated regulatory enhancers, separated by vast linear distances within a chromosome, constitute a ?first tier? network that alters the transcription of specific, interacting component enhancers. We hypothesize that this ?first tier? network, while not impacting the ability of each component enhancer to loop to and activate its cognate target coding gene promoter, nucleates formation of an architectural ?structure? that provides the machinery that licenses the robustness of the transcriptional response imparted by these individual enhancers. We will use both GRO-seq and assays of 3D architecture to test whether conceptualize a ligand-dependent distributive superenhancer connectome dictates chromosomal architecture. This would represent an entirely new perspective on enhancer networks, revealing interactions of E2-regulated enhancers that modulate whole chromosome architecture and ensemble chromosomal structures result in an unexpected integrated transcriptional response network based on actions of single, robust ?first tier? enhancers.  

Contact PD/PI: ROSENFELD, MICHAEL G PROJECT ABSTRACT Alzheimer?s disease (AD) is conventionally characterized by specific neuropathological features, including the appearance of extracellular amyloid deposits and the accumulation of intracellular neurofibrillary tangles. While several gene mutations are clearly associated with early onset Alzheimer?s disease, the large number of individuals exhibiting delayed onset, aging-associated AD, are likely to harbor many alterations in linked modifier genes that predispose to AD susceptibility. Genetic and genome wide association studies (GWAS) have identified numerous genes and risk alleles that indicate both cell autonomous and non-cell autonomous mechanisms contributing to loss of neurons and cognitive decline. In this regard, the majority of risk variants identified by GWAS reside in non-coding regions of the genome, implying that they act in part to alter gene expression. This proposal responds to the RFA indicating a particular need for approaches designed to delineate the transcriptional and cellular consequences of combinations of SNPs in the risk alleles by generating new cell line reagents to help unravel the question of the causative SNPs and their target genes in specific neurons derived from iPS cells of AD individuals. There are two features of sporadic AD that require molecular explanation- the potential role of aging in AD susceptibility, and the striking gender disparity, with the incidence of AD being exaggerated in females. These issues can only now be addressed based on new technologies and the availability of patient-derived samples. Our proposed research plan takes advantage of the invaluable samples stored at the brain bank of the Shiley-Marcos Alzheimer's Disease Research Center (ADRC) at UCSD, and the iPSC-derived neurons (Salk). This approach will interrogate the effects of different genetic variants with other risk factors (e.g. age, sex), and assess their effects on cell type-specific enhancer landscapes. By merging these data, we can begin to identify the potential causative SNPs that result in altered function of cell-type specific enhancers. We propose using a high throughput 4C screening approach (UMI-4C), and Hi-ChIP, to identify the most likely causative, enhancer-associated SNPs for functionally-implicated coding target genes. Exploiting the power of contemporary gene editing approaches in control or patient-derived iPS cells to specific neuronal cell types, and to astroglia, we can assess the transcriptional phenotypes and functional behaviors of neurons harboring different combinations of risk alleles, both in the isolated cell lines alone and in combination with coculture experiments with astroglia and microglia, as effects of these SNPs may be manifest only with astroglial:neuronal interactions. Together these studies will use powerful contemporary global genomic approaches to determine the coding transcriptional targets of several of the most significant SNPs in enhancers, and the link to roles of estrogen receptor in the gender disparity for AD. Project Summary/Abstract Page 7

Project Summary Genetic and genome wide association studies (GWAS) have identified numerous genes and risk alleles that indicate both cell autonomous and non-cell autonomous mechanisms contributing to Alzheimer's Disease (AD). In addition to genes expressed by neurons, the observation that several risk alleles, such as TREM2, are exclusively or mainly expressed in microglia, has led to increased efforts to understand the roles of microglia in AD pathology. Importantly, the majority of risk variants identified by GWAS reside in non-coding regions of the genome, implying that some act to alter gene expression. Our recent comparisons of neurons derived by trans-differentiation of fibroblasts from AD subjects and age matched controls demonstrate marked changes in gene expression in AD neurons. In parallel, our recent ability to globally analyze the transcriptomes and enhancer atlases of human microglia demonstrated marked individual variation in expression of immune genes associated with AD risk alleles. Collectively, these findings suggest widespread alterations in the expression of genes that may contribute to susceptibility of AD independent of the generation of ?amyloid. Enhancers have emerged as major points of integration of intra and extra-cellular signals associated with development, homeostasis and disease, resulting in context-specific transcriptional outputs. By defining a cell's enhancer landscape, it is possible to both infer the environmental signals the cell is receiving and explain its consequent program of gene expression. In this application, we propose to define the `Enhancer codes of Alzheimer's Disease' to qualitatively advance our understanding of cell autonomous and non-cell autonomous factors that drive pathogenic programs of gene expression. In Specific Aim 1, we will define transcriptomes and enhancer landscapes of nuclei isolated from neurons and microglia derived from sporadic and genetic AD brains and brains from age and sex-matched controls. These studies will enable an unprecedented analysis of the regulatory landscapes of neurons and microglia in the intact aging and AD brain. In Specific Aim 2, we will validate and explain AD-specific enhancer codes of neurons by direct reprogramming of fibroblasts from sporadic and genetic AD patients and age/sex-matched control subjects. In Specific aim 3, we will define cell autonomous AD-specific enhancer codes of microglia obtained by reprograming of iPSCs and monocytes from control and AD subjects. These studies will build upon our recent characterization of human microglia transcriptomes and enhancer landscapes that demonstrate striking levels of individual variation in the expression of genes linked to risk of AD. In Specific Aim 4, we will define consequences of neuron-microglia interactions on the transcriptomes and epigenomes of each cell type. By leveraging existing resources and data sets, these studies will define transcriptional networks that are dysregulated in neurons and microglia in AD, provide proof of concept for defining the mechanistic basis of inherited forms of AD, and nominate additional pathways for further investigation.

Abstract Based on the importance of defining new insights into cellular senescence, we initiated studies to investigate whether there might be a specific enhancer activation ?code? that underlies cellular senescence for identifying the responsible DNA binding transcription factors. While there is rapidly-emerging, and now unassailable evidence, on the role of the 40-70,000 enhancers in each cell type in development, homeostasis and, often, pathological events, their role in cellular senescence remains undefined. Furthermore, while cellular senescence represents a fundamental process of aging and a known driver of pathologies, the causative role of newly activated enhancer cohorts underlying progression of senescence remain poorly understood. Therefore, the goal of this proposal, supported by extensive preliminary data, is to test a novel hypothesis that the de novo appearance of two specific cohorts of enhancers sets into motion a progressive, functionally- important, alteration in gene transcription programs. Based on our study of the altered enhancer and chromosomal landscape during replicative senescence, we have begun to establish that the geroprotective mTOR inhibitor, Rapamycin, markedly delays all aspects of cellular senescence, including the appearance of new, functional, enhancers. Our focus is to elucidate the functional importance of a gained enhancer program underlying cellular senescence, and identify the critical DNA binding transcription factors underlying the transcriptional programs that are determinants of replicative senescence, based on the complementary expertise of the Suh and Rosenfeld laboratories. Specifically: i) We will use unbiased screens to document that at least two distinct activated enhancer networks independently regulate the proliferation arrest and SASP aspects of replicative senescence, respectively. ii) We will identify combinatorial factors synergizing with the previously-unrecognized transcription factors, NFI-A, NFI-C, to regulate the gained enhancers underling proliferation arrest, and those that, with SMAD2/3 and NFkB, to regulate the SASP program. In parallel, we can implicate the underlying signaling pathways. iii) We will identify previously unrecognized histone modification signatures of, and their functional importance in replicative senescence . iv) We will Identify Activin and Tgf?2 as inhibitors of the proliferation and SASP enhancer programs, respectively. Our proposal promises to provide transformative insights into molecular events that initiate and perpetuate the senescent cell phenotypes, and help elucidate potential novel therapeutic modalities against the deleterious SASP program.

ABSTRACT Here we propose a Supplement to the research proposed in the parent grant Combinatorial regulation of the enhancer codes in senescence to perform a logical extension of our initial Specific Aims regarding cellular aging/senescence to achieve initial insights into the distinction between these events and distinct pathological causal features in each CNS cell type that may represent the underlying mechanisms underlying sporadic Alzheimer?s Disease (AD). The Specific Aims of the initial grant employed and developed biostatistical tools relevant to cellular aging and replicative senescence, including an examination of underlying epigenomic alterations and enhancer activation codes ultimately leading to cellular senescence approaches and proposed application of single cells approaches. Appling these specific aims/approaches is particularly suitable for enhancing our understanding of the potential initial causal events that eventuate in clinical sporadic AD, an aging- associated disease affecting both men and, to a greater extent, women. In concert with the original Aim of understanding the molecular basis for enhancer-mediated programs of cellular aging and senescence, in this Supplement, we propose to extend our original Specific Aims to uncover the enhancer program underlying the aging events in each CNS cell type, to permit examination of the central question whether the altered enhancer and transcriptome changes in AD in each cell type represent a trajectory distinct from the normal aging-related alterations in these cell types. We hypothesize that, while each cell type will, of course, exhibit specific features of enhancer activation characteristic of aging and even cellular senescence, as we have uncovered in the parent grant, AD represents a distinct trajectory for these cell types. This Supplement is licensed by our ability by our development of the technology to perform simultaneous quantitation of single nucleus (sn) RNA-seq and snATAC-seq using archival samples stored at the brain bank of the Shiley-Marcos Alzheimer's Disease Research Center (ADRC) at UCSD. We have carefully piloted this approach to ensure that we are technically able to obtain high quality data and that all of the proposed informatic pipelines and our ability to successfully aggregate such massive data sets is fully established. The Supplement would license our ability to scale the analysis to obtain data sets capable of generating statistically significant results and, therefore, informative conclusions, which can be ultimately be further validated by imputation from available data bases and using hiPSCs to generate specific cell types for validating transcriptional analyses. Our overarching goal in this Supplement is to apply the described technologies and new informatic approaches to a sufficient number of archived specimens to permit formulating the actual transcription factors and pathways that distinguish initiation of the AD process in specific CNS cell types.

ABSTRACT The intersection of genotoxic stress and altered transcriptional programs impacts numerous disease and disease risk events, but surprisingly little is mechanistically known in this regard, particularly with respect to risk alleles for myocardial infarction. Based on our initial data, we can confirm that a single base alteration in a CTCF site in the SIRT1 promoter can increase the risk of MI. We will investigate the hypothesis that this is based on the functional importance of induced binding of CTCF to in response to hypoxia or genotoxic stress to the cognate site in the SIRT1 promoter, licensing a promoter pause release that results in an acute stimulation of SIRT1 transcription. The proposed research is directed at revealing a previously overlooked strategy for signal-dependent transcriptional regulation based on redistribution of the critical chromosomal architectural protein - CTCF- in activation of a large promoter pause release program with important biological consequences, including the gene encoding critical regulator - SIRT1. We will investigate the hypothesis that stress-induced increased transcription of SIRT1 and a global genomic program of response to oxidative stress in cardiomyocytes is mediated by CTCF promoter recruitment and, in part, by long distance interactions based on liquid-liquid phase separation of CTCF and lncRNAs in cardiomyocytes, which is lost in MI risk allele carriers. We will investigate the stress-induced transcriptional program in iPSC-derived, genome-sequenced cardiac myocytes and the test the hypothesis by assessing effects of introducing the risk allele in mice. With the availability of iPSCs harboring the causative SNP for the SIRT1 risk allele, we are now in a position to delve into the precise mechanism of these events, and assess the possibility that there is a large cardiomyocyte pause-release program important with respect to cardiac response to acute insults. The idea that a key arbiter of chromosome architecture is regulated by phosphorylation of a key architectural protein, binding to a set of promoters harboring weak sites for CTCF and licensing increased transcription based of promoter pause release events reveals an unappreciated signal-dependent mechanism for controlling important biological program.

Mammalian cells exhibit a precise gene regulation process, during which enhancers play critical roles in mediating rapid gene activation in response to different signals. Indeed, much of our knowledge about gene transcriptional control comes from the long-standing investigation of the actions of nuclear receptors, well exemplified by estrogen 17?-estradiol (E2)-dependent activation of transcriptional programs. This program is controlled virtually entirely by activation of a cohort of ~1000 robustly activated ER?-bound enhancers. Under physiological conditions, many signals are pulsatile, including ligands for nuclear receptors such as ER?, representing a continuum from transient, acute stimulation to chronic stimulation. We have found that the acute 17?-estradiol (E2)-dependent activation of functional enhancers requires assembly of an eRNA-dependent ribonucleoprotein (eRNP) complex, referred to as the MegaTrans complex. A transformative, newly emerging concept, to which we are pleased to have contributed, is that acute signal of ligand-dependent activation of target enhancers causes them to form an RNA-protein condensate, with features of phase separation, that results in cooperative activation of other homotypic enhancers separated by multiple TADs and, even in other chromosomes. In contrast, chronic signal/ligand activation results in loss of the dynamic RNP condensate at the enhancers, loss of induced proximity of homotypic enhancers observed with acute activation, with enhancer activation function now confined to the nearby cognate target gene promoters. We will use global genomic, proteomic, and real time, single nucleus approaches, with appropriate informatics, to examine the patterns of enhancer activation, potential interactions in acute vs chronic ligand-dependent activation, relationship to localization in phase-separated subnuclear architectural structures and patterns of movement by single molecule imaging. Our goal is to provide a paradigm-shifting insight into ligand/hormone-regulated transcriptional programs, based on this multidisciplinary approach.

Reproductive aging is a major health, personal and societal issue, but ovarian aging has received limited scientific attention, even in large genomic survey projects. Ovarian aging influences diverse health outcomes in women including lifespan, cardiovascular disease, metabolic syndromes, neurodegenerative disorders and various types of cancer. Yet the molecular mechanisms underlying ovarian aging, timing of menopause and inter-organ feedback loops remain elusive. As one of the most dynamic organs in the human body, the ovary undergoes significant remodeling across the entire reproductive period. The dynamic transcriptional regulation of and interactions between oocytes and their surrounding cells during aging remain unknown. The objective of this proposal is to understand the regulatory landscapes underlying the complex interplay among the different cell types in the ovary and to investigate the molecular mechanisms that regulate the remarkably complex processes of reproductive aging. We will apply powerful single-cell (sc) RNA-seq and scATAC-seq analysis to define specific transcriptional programs and regulated enhancer networks that are altered in distinct ovarian cell types or subtypes during aging. By defining the roles of specific enhancers in specific cell types, and how these change with aging, we aim to understand the identities of the regulatory factors and environmental signals that impact aging in each ovarian cell type. Genetic variation affecting enhancer selection and function is a major determinant of differences in cell-specific gene expression between individuals. To investigate the mechanisms by which altered regulatory enhancer landscapes contribute to ovarian aging by licensing changes in transcriptional programs, we will investigate the roles of genetic variants associated with age at menopause, detected by genome-wide association studies (GWAS), in modulating transcription programs during ovarian aging. In particular, we hope to provide mechanistic insights into genetic modulation of transcriptional regulation of critical homeostatic and inflammatory pathological functions in the granulosa cells (GC), the supporting cell type immediately surrounding the oocyte, by modeling the causal regulatory variants in human GC models that are differentiated from human ESCs engineered to carry causal variants by CRISPR gene editing.

ABSTRACT Herpes Simplex Virus 1 (HSV1) can establish both lytic and latent infections in a cell type-specific fashion with known and emerging neuropathological ramifications, respectively. Provocative data now link reactivation of latent HSV1 infection to Alzheimer?s disease (AD), the etiological basis of which remains incompletely defined. Here we propose to employ powerful new genomic technologies to identify and characterize the actual cell types that harbor latent and reactivated HSV1, extending recent findings that have revealed an increased abundance of herpes virus transcripts in affected regions of human AD brains. Using a modified single-nucleus sequencing approach, which allows for DNA accessibility and global transcription to be assessed in the same nucleus, we will interrogate human control and AD brain samples as well a HSV1-infected brain organoids and mouse models of acute and progressive HSV1-induced neurotoxicity. These studies promise to reveal cell type-specific enhancer landscapes and transcriptional profiles consequent to lytic, latent, or reactivated HSV1 in the brain while also providing insights into the cell autonomous versus non-cell autonomous effects of its presence. In addition, we propose to elucidate a novel innate immune pathway by which HSV1 lytic transcripts trigger the sentinel kinase PKR to initiate a cascade of nuclear events that include the secondary activation of the transcriptional regulator PARP1 and culminate in a NF-kB-dependent inflammatory gene expression program, potentially providing a molecular mechanism by which occasional HSV1 reactivation in the brain could contribute to an inflammatory milieu that promotes the pathogenesis of AD. Furthermore, this molecular pathway may underlie diverse microbial and possibly non-microbial inflammatory triggers in the brain that have been implicated in AD. We also hypothesize that HSV1 latency-associated transcripts (LATs) have distinct and opposing genomic functions as well as non-genomic actions in host neurons and possibly non-neuronal brain cells, the balance of which preserves neuronal cell integrity but may facilitate low-grade, chronic inflammation in the context of latent infection irrespective of viral reactivation. Based on enticing preliminary evidence, we propose to investigate the idea that the sense (S) and antisense (AS) LATs impact transcription in a partially dichotomous fashion by associating with specific regulatory elements in the HSV1 and host genomes in collaboration with the KRAB zinc-finger protein (KZFP) co-regulator KAP1. We hypothesize that these genomic events influence the AD process by affecting neuronal function through modulation of KZFP-mediated regulation of human endogenous retrovirus (HERV) repeats. We further hypothesize that the LATs have a complementary non- genomic role that mitigates the innate immune response and suppresses cell death programs, at least in part, by inhibition of PKR. Finally, we propose to exploit these protective properties of the HSV1 LATs as a unique prophylactic strategy for AD.

Abstract Type 1 diabetes (T1D) is an organ-specific autoimmune disease, whereby immune cell-mediated and inflammatory cytokines lead to loss of the insulin-producing ? cells in the pancreas. Genome-wide association studies (GWAS) have identified ~60 genomic regions associated with T1D risk. The vast majority of GWAS risk variants associated with T1D reside in non-coding regions, particularly enhancers, suggesting that gene regulatory changes substantially contribute to inter-individual differences in susceptibility to T1D. Driven by our T1D GWAS annotation data, highlighting the importance of a systematic analysis of both immune and ? cell systems of both T1D patients and controls, we propose to identify causal enhancer variants and causal target genes using contemporary computational methods and cutting-edge global genomics. We will perform our PRO-cap and PRO-seq assays to comprehensively identify active enhancers harboring T1D-associated variants in T cells, monocytes and stem cell derived ? cells (sc-? cells) from T1D patients and controls, followed by validation of active enhancers harboring T1D-associated variants using our eSTARR-seq assays (Aim 1). We will perform our Tri-HiC assays to profile the enhancer-promoter interactomes at unprecedented high resolution in primary T cells, primary monocytes and sc-? cells from T1D patients and controls as well as pancreatic islets to comprehensively identify target genes of T1D-associated variants, and refine targets of T1D-associated variants with T1D-sepcific alteration in target gene expression in each cell type using our coupled single cell nucleus (sn) RNA-seq and snATAC-seq assays (Aim 2). We will validate targets of T1D-associated variants in T cells, monocytes and ? cells using CRISPR/Cas9 endogenous enhancer mutational strategies in the context of the endogenous chromatin landscape in each cell type. Our analytical and experimental framework represents an exciting new paradigm for studying T1D, and the subsequent clinical research based on our results has the potential to develop preventive and therapeutic strategies against T1D. The data sets generated by these studies will represent important, but currently lacking, resources for the T1D research community.