Christopher Glass - US grants

Macrophages and related cells of the reticuloendothelial system play essential roles in host defense mechanisms against infectious disease and neoplasia. Abnormalities in macrophage development and function are implicated in the pathogenesis of a wide variety of human disease states, including certain leukemias, autoimmune diseases and atherosclerosis. The long term objectives of this proposal are to elucidate the molecular mechanisms by which receptors for retinoic acid and Vitamin D regulate macrophage development. AIM 1 proposes to identify retinoic acid and Vitamin D-responsive genes in the human leukemia cell line, HL60, using subtractive hybridization strategies. These studies are expected to lead to the identification of genes that play important roles in the development and terminal differentiation of hematopoietic cells. Furthermore, characterization of the transcriptional control of these genes will be a prerequisite to the understanding of the mechanisms by which retinoic acid and Vitamin D exert tissue-specific effects on gene expression. Biochemical studies of HL60 cell nuclear proteins have led to the identification of two cell-specific factors that interact with the retinoic acid receptor to alter its sequence-specific DNA binding properties. The major hypothesis raised by this proposal is that these factors act to restrict and direct the actions of the retinoic acid receptor to the appropriate sets of target genes in hematopoietic cells. AIM 2 proposes to characterize and clone these factors in order to test this hypothesis. The retinoic acid receptor interacts with these cell-specific factors via amino acid sequences that overlap with domains involved in ligand binding and transcriptional activation. AIM 3 proposes to perform a mutational analysis of this protein-protein interaction interface in order to understand the relationship between these interactions and the transcriptional properties of the receptor. The proposed studies are likely to provide new insights into the mechanisms by which the retinoic acid and Vitamin D receptors influence the hierarchy of regulatory genes that act to specific cellular phenotype and control cell growth.

The development of atherosclerosis has been hypothesized to involve the uptake of modified low density lipoprotein (LDL) by macrophages and other cells within the artery wall. Recent studies have led to the identification of specific proteins that are likely to play direct roles in the modification and subsequent metabolism of LDL. These include 15 lipoxygenase, which is capable of promoting oxidative modification of LDL, and the acetyl LDL receptor, which is capable of binding and internalizing oxidized LDL. Both of these proteins are expressed in macrophages within early fatty streak lesions of the artery wall. The emphasis of the studies proposed in this Unit will be to examine the molecular mechanisms that function to regulate the expression of the acetyl LDL receptor and 15 lipoxygenase genes in macrophages. Experiments will be performed to identify hormones and other regulatory molecules that act to increase or decrease the expression of these genes. Activators of protein kinase C have recently been demonstrated to stimulate acetyl LDL receptor expression, and the mechanisms responsible for this effect will be examined in model macrophage cell lines. To further characterize the mechanisms controlling acetyl LDL receptor transcription , genomic sequences encoding the promoter and upstream regulatory elements have been cloned. Functional characterization of cis active elements will be performed by transfection analysis of wild type and mutant genomic sequences linked to suitable reporter genes. Complementary experiments will be performed in transgenic mice to verify the physiologic importance of cis elements defined In vitro. DNA binding assays will be performed using nuclear extracts prepared from cells that express the scavenger receptor to identify putative transcription factors that direct its expression. Several approaches will be used to purify these proteins, determine their functional importance, and clone their corresponding cDNAs. These studies are expected to lead to an improved understanding of the pathogenesis of atherosclerosis and may permit the development of novel strategies for prevention or treatment.

molecular biology; gene expression; biomedical facility; genetic registry /resource /referral center; chimeric proteins; synthetic nucleotide; oligonucleotides; plasmids; fusion gene; nucleic acid probes; nucleic acid sequence; nucleic acid hybridization; polymerase chain reaction; nucleic acid chemical synthesis;

Retinoic acid receptors (RARs) are ligand-dependent transcription factors that regulate diverse aspects of development and homeostasis by binding to response elements in target genes as heterodimers with retinoid X receptors (RXRs). Although several lines of evidence support a critical role of RARs in controlling the development of myeloid cells, the mechanisms by which RARs activate or repress transcription so as to regulate complex developmental programs are not understood. The objectives of this proposal are to identify and characterize proteins that mediate the transcriptional activities of RARs during myeloid development. Our recent studies of RAR/RXR heterodimers have led to the discovery that receptor-receptor and receptor-DNA interactions play important roles in regulating the transcriptional outcomes to RAR and RXR-specific ligands. The major hypothesis of this proposal is that transcriptional activation and repression by RAR is mediated through distinct co-activator and co-repressor proteins that interact with nuclear receptor heterodimers in a manner that is controlled by ligand, heterodimer composition and organization of the DNA binding Site. We have recently identified a 270 kDa protein, termed N-CoR, that mediates transcriptional repression by unliganded RAR. Several proteins have also been identified that interact with the ligand-dependent activation function of RAR and represent putative co-activator proteins. We propose to characterize these proteins and examine their roles in mediating the biological actions of normal and mutant RARs during myeloid cell development. Studies proposed in AIM 1 will examine the mechanisms by which ligand binding, dimerization and DNA binding control the interactions of RXR heterodimers with co-activators and co-repressors. In AIM 2, studies are proposed to examine potential mechanisms by which N-CoR functions as a co-repressor. The goal of AIM 3 will be to determine the role of N-CoR in mediating the actions of normal and mutant RARs during the differentiation of myeloid progenitor cells. Studies proposed in AIM 4 will be directed at the cloning and characterization of coactivator proteins that are required for positive regulation of gene expression in hematopoietic cells. Insights derived from these studies are likely to lead to an improved understanding of the mechanisms by which retinoic acid receptors positively and negatively regulate gene expression.

Monocyte-derived macrophages play a central role in the pathogenesis of atherosclerosis. They actively promote oxidation of LDL, express scavenger receptors, and secrete cytokines and growth factors which influence the migration, growth and function of arterial cells. They may also contribute to plaque instability b secretion of metalloproteinases. Our laboratory characterized transcriptional control elements of the scavenger receptor A (SR-A) gene that are required for macrophage-specific expression of this gene. We also demonstrated that regulatory elements of the SR-A gene can be used to specifically target reporter reporter gene expression in macrophage foam cells of murine atherosclerotic lesions. A major aim of this unit is to fully develop and validate a variety of vector systems for the overexpression or disruption of genes in a macrophage-specific manor. Vector based on the SR-A promoter and other macrophage-specific promoters will be developed to allow targeting of cDNAs to mature macrophages and macrophage progenitor cells. These vectors will also be used to direct expression of Cre-recombinase or a doxycycline-inducible transcriptional activator in transgenic mice. The ability of these vectors to direct efficient macrophage-specific expression of genes in forms cells of atherosclerotic lesions will be assessed in apo E- deficient, or LDL receptor-deficient (LDLR) mice, utilizing reporter genes, such as human growth hormone. A second aim will be to utilize this information to determine the impact on atherogenesis of macrophage specific overexperssion of genes that could influence atherogenesis. Overexpression of 15-lipoxygenase will serve as an example of a gene that can enhance the oxidation of LDL, and overexpression of phospholipid-hydroperoxide glutathione peroxidase as an example of a gene that should decrease oxidation of LDL. In a similar manner we will determine the consequences to the integrity of the artery wall of macrophage-specific overexpression of metalloproteinases, including the 92 kDa gelatisase, interstitial collagenase, and stromelysin, expressed individually and in combination. The effects of overexpression of these genes on the atherosclerotic process will be tested in appropriate murine models. Finally, we will determine the consequences for atherogenesis of macrophage-specific disruption of the c-jun gene. C-jun appears to regulate transcription of several important macrophase genes, including SR-A, cytokines, and metalloproteinases. Because a global disruption of c-jun results in embryonic lethality, macrophage-specific disruption will serve as a paradigm for the evaluation of gene required for normal development but which may contribute to atherogenesis. The information gained from these studies should yield fundamental insights into the role that macrophages play in atherogenesis and could be used to develop novel and effective therapeutic strategies.

gene targeting; atherosclerosis; biomedical facility; genetically modified animals; laboratory mouse;

colony stimulating factor; laboratory mouse

This training program renewal is intended to provide rigorous research training in the fields relevant to atherosclerosis and vascular biology. Emphasis is placed on training in the disciplines of molecular biology, biochemistry, bioengineering and cell biology, with opportunities to apply these basic sciences to studies in intact animals and human subjects. A group of 11 dedicated faculty provide an integrated interdisciplinary program that will equip trainees for successful, independent research careers investigating the causes, prevention and treatment of atherosclerosis. Request is made for five postdoctoral positions. This training grant faculty will tailor the educational program to the needs of each trainee and provide assessment of progress and guidance in career development. Trainees will participate in an appropriate mix of direct research involvement with preceptors, attend data clubs, journal clubs, seminars and formal courses and attend scientific meetings. The training program relates very closely and draws much of its strength from the well-established Specialized Center of Research on Molecular Medicine and Atherosclerosis and from participating faculty who are leaders in the fields of gene therapy, bioengineering,lipid biochemistry and metabolic disease.

This proposal requests funding to establish a DNA microarray core facility at the University of California, San Diego School of Medicine. Two instruments are requested that will provide state-of-the-art capabilities for the analysis of gene expression; the Beecher Instruments Model A9600 fully automated cDNA microarrayer and the Beecher Instruments Model R3000 scanning confocal laser reader. These instruments are based on prototypes built for the Laboratory of Cancer Genetics at the National Human Genome Research Institute (NHGRI). The microarray core facility will thus directly benefit from the continuous evolution of software and protocols developed at the NHGRI. The cDNA microarrayer and scanning confocal laser reader will enable users to assess changes in patterns of gene expression, investigate mechanisms of mRNA processing, detect expression polymorphisms and analyze genomic rearrangements. The ability to make custom cDNA arrays will enable investigators to exploit model organisms such as Dictyostelium and yeast, as well as to perform focused evaluations of specific genetic pathways at low cost. This instrumentation will revolutionize the analysis of gene expression by the participating users, provide an important resource for training graduate students and postdoctoral fellows, and serve as a focal point for interactions between molecular biologists, geneticists and computer scientists.

Human exposure to environmental pollutants has been proposes as a risk factor for endocrine disruption and the development of cancers of the breast and reproductive tract. Environmental pollutants are thought to exert effects on the endocrine system in part by influences the activities of nuclear hormone receptors that regulate diver aspects of growth, development and homeostasis. Some of the most common environmental pollutants found at Superfund Sites, including polychlorinated biphenyls (PCBs) and phthalates, can bind to members of the nuclear receptor superfamily and regulate their activities. These chemicals therefore have the potential for disrupting normal programs of endocrine regulation, resulting in disease. The major goals of this multi disciplinary research program are to use recombinant DNA approaches to assess the impact of exposure to Superfund chemicals on cellular and molecular events that affect the regulation of gene expression. In this project, we propose to take advantage of recent developments that affect the regulation of gene expression. In this project, we propose to take advantage of recent developments in the molecular biology of nuclear receptors to develop novel methods to assess effects of environmental pollutants on specific endocrine signaling pathways in transgenic mice. These methodologies, which can be tailored to allow evaluation of effects of environmental pollutants on human receptors in an in vivo setting, are potentially applicable to all members of the nuclear receptor super family. These methods will initially be developed to assess effects of Superfund site chemicals on estrogen and peroxisome proliferator activated receptor function. Estrogen receptors represent one of the most intensively studied targets of endocrine disruptors, allowing validation of these new methodologies by comparison to existing assay systems. The peroxisome proliferator activated receptors are much less well understood, but represent important emerging targets of endocrine disrupting compounds. The specific aims of this proposal are to: 1) Develop transgenic reporter systems that allow quantitative assessment of the transcriptional activities of estrogen and peroxisome endogenous hormones and identify physiologic target genes; and 3) Assess effects of endocrine disrupters found at Superfund Sites on the activities of estrogen and peroxisome- proliferator-activated receptors in vivo and their effects on the expression of downstream target genes.

DESCRIPTION: (Adapted from the applicant's abstract) Members of the nuclear receptor superfamily play critical roles in development and homeostasis by regulating gene expression in response to the binding of small molecular weight ligands. The central hypothesis of this proposal is that transcriptional responses to regulatory ligands are determined by the exchange of corepressor complexes for one or more coactivator complexes. Sequential or combinatorial recruitment of biochemically distinct coactivator complexes is proposed to underlie cell and gene-specific responses to a particular ligand. Aspects of this hypothesis will be tested using biochemical and cell-based assays and by determining the consequences of knocking out the NCoR and SMRT genes in mice. The biological focus of these studies will be to explore whether distinct coactivator and corepressor complexes underlie specific programs of hematopoietic differentiation that are controlled by retinoic acid receptors (RARs) and peroxisome proliferator activated receptors (PPARs). Three specific aims are proposed: The first is to test the hypothesis that transcriptional activation of RAR and PPARgamma target genes requires sequential or coordinate recruitment of p160/CBP and DRIP/TRAP/ARC coactivator complexes. These experiments will address the question of whether these complexes act sequentially or combinatorially, and whether both complexes are required on different PPARgamma and RARalpha target genes. The second specific aim is to determine the roles of NCoR and SMRT in the control of hematopoiesis. The NCoR knockout mice die around embryonic day 16 due to profound anemia. Experiments are proposed to define the molecular basis for this phenotype and determine the roles of NCoR and SMRT in regulating the differentiation of granulocytes and macrophages. The third specific aim is to test the hypothesis that ligand-dependent inhibition of NF-kB-target genes by PPARgamma other nuclear receptors involves the recruitment of inhibitory molecules to the CBP coactivator complex. PPARgamma, RARalpha and many other ligand-dependent nuclear receptors appear to exert important biological effects by inhibiting the activities of other signal-dependent transcription factors in a ligand-dependent manner. Preliminary studies suggest important roles for CBP/p300 and additional receptor-associated proteins in the transrepression process. Together, the proposed studies are intended to lead to new insights into the biochemical and biological roles of coactivators and corepressors in the regulation of gene expression by RARs and PPARs. This knowledge may suggest new approaches for the development of nuclear receptor ligands useful in the treatment of a broad spectrum of human diseases.

DESCRIPTION (provided by the applicant): Complications of atherosclerosis continue to be the major causes of death in industrialized societies. Although cholesterol-lowering drugs can significantly reduce the risk of myocardial infarction in hypercholesterolemic patients, they are not sufficient to prevent the development of atherosclerosis in all high-risk individuals and there remains an urgent need for additional therapeutic approaches. The peroxisome proliferator-activated receptors (PPARs), and liver X receptors (LXRs) are nuclear hormone receptors that have recently been shown to exert both atherogenic and anti-atherogenic effects on patterns of gene expression. Based on the development of selective estrogen receptor modulators (SERMs) that exert estrogenic effects on some genes and anti-estrogenic effects on others, it may be possible to develop analogous modulators of PPARs and LXRs that selectively exert anti-atherogenic effects. Studies are proposed in this Unit to determine the molecular mechanisms by which SERMs exert selective effects on gene expression and to characterize the roles of specific co-activators and co-repressors in mediating the transcriptional activities of PPAR and LXRs alpha and beta. Three specific aims are proposed. Specific Aim 1 will test the hypothesis that SERMs exert anti-estrogenic or estrogenic effects depending on whether or not the nuclear receptor co-repressors N-CoR or SMRT are recruited to estrogen receptor target genes. An understanding of the molecular mechanisms responsible for the selective actions of SERMs is likely to facilitate the development of selective modulators of other classes of nuclear receptors. Specific Aim 2 will investigate the roles of N-CoR and SMRT in mediating transcriptional repression and activation by LXRs. Preliminary studies suggest that activation of LXR target genes can be achieved by oxysterol-independent mechanisms that inhibit interactions between LXRbeta and N-CoR. These studies may suggest new strategies for development of selective LXR modulators. Specific Aim 3 will investigate molecular mechanisms by which PPARgamma activates transcription of anti-atherogenic and atherogenic genes, focusing on the LXRalpha and CD36 genes as models. These studies will test the hypothesis that different co-activators are used to activate different PPARgamma target genes, providing an additional avenue for the development of selective modulators. In concert with collaborative efforts outlined in Units 2, 3, 4 and 5, these studies should provide significant new information on the roles of specific nuclear receptors in the development of atherosclerosis and the molecular mechanisms responsible for their actions. Insights derived from these studies may ultimately facilitate the development of novel drugs that can selectively modulate programs of gene expression to more effectively inhibit the development of atherosclerosis.

A major goal in efforts to understand the mechanisms by which signal transduction pathways regulate programs of gene expression is to identify their target genes and to determine the specific components of the transcriptional machinery that are recruited to these genes in response to hormonal signals. The DERC Genomics Core Facility will provide two complementary services to advance these efforts; conventional cDNA microarray analysis and recently developed promoter microarray analysis. Conventional microarray analysis will utilize glass slides spotted with PCR products corresponding to specific genes, allowing large-scale assessment of relative levels of gene expression. These arrays are intended to complement the use of commercially available microarrays (e.g., Affymetrix microarrays). For example, microarrays containing a few hundred to a few thousand cDNA targets of particular interest can be printed at relatively low cost, allowing multiple experimental conditions to be examined that would be prohibitively expensive using Affymetrix arrays. Microarrays for several organisms are currently available, including human and murine gene microarrays. Recent progress in combining the use of chromatin immunoprecipitation (CHIP) assays with DNA microarrays has recently allowed genome-wide analysis of transcription factor localization to specific promoter sequences in living cells. The DERC Genomics Core Facility will fabricate a human and murine promoter microarrays to allow genome-side location analysis of transcription factors such as nuclear hormone receptors and signal-dependent transcription factors. The fabrication of a murine promoter array will be a unique resource, and allow full exploitation of genetically engineered mouse models and cell lines.

peroxisome proliferator activated receptor

functional /structural genomics; cell biology; macrophage; biomedical facility; lipid metabolism; reagent /indicator; cooperative study; gene expression; cell line; genetically modified animals; laboratory mouse;

A major goal in efforts to understand the mechanisms by which signal transduction pathways regulate programs of gene expression is to identify their target genes and to determine the specific components of the transcriptional machinery that are recruited to these genes in response to hormonal signals. The DERC Genomics Core Facility will provide two complementary services to advance these efforts; conventional cDNA microarray analysis and recently developed promoter microarray analysis. Conventional microarray analysis will utilize glass slides spotted with PCR products corresponding to specific genes, allowing large-scale assessment of relative levels of gene expression. These arrays are intended to complement the use of commercially available microarrays (e.g., Affymetrix microarrays). For example, microarrays containing a few hundred to a few thousand cDNA targets of particular interest can be printed at relatively low cost, allowing multiple experimental conditions to be examined that would be prohibitively expensive using Affymetrix arrays. Microarrays for several organisms are currently available, including human and murine gene microarrays. Recent progress in combining the use of chromatin immunoprecipitation (CHIP) assays with DNA microarrays has recently allowed genome-wide analysis of transcription factor localization to specific promoter sequences in living cells. The DERC Genomics Core Facility will fabricate a human and murine promoter microarrays to allow genome-side location analysis of transcription factors such as nuclear hormone receptors and signal-dependent transcription factors. The fabrication of a murine promoter array will be a unique resource, and allow full exploitation of genetically engineered mouse models and cell lines.

[unreadable] DESCRIPTION (provided by applicant): Many nuclear receptors act in trans to repress transcriptional responses to signaling pathways as a central aspect of their biological functions, but the underlying mechanisms remain an important and unresolved question in development and homeostasis. Studies performed during the current funding period of this grant demonstrated that the glucocorticoid receptor (GR), the peroxisome proliferator-activated receptor y (PPAR?) and liver X receptors (LXRs) repress responses to toll-like receptor activation in a promoter-, and nuclear receptor-specific manner. The current proposal seeks to define the underlying mechanisms responsible for these specific programs of repression on a genome-wide scale. Preliminary studies indicate that PPAR? represses a subset of inflammatory response genes in macrophages by preventing the signal- dependent dissociation of NCoR co-repressor complexes. Surprisingly, this mechanism involves ligand- dependent sumoylation of the PPAR? ligand binding domain, which targets PPAR? to NCoR/HDACs co- repressor complexes on inflammatory gene promoters. The interaction of sumoylated PPAR? with NCoR/HDAC3 complexes prevents the recruitment of ubiquitylation/igS proteosome machinery that normally mediates their signal-dependent removal. Studies in Drosophila Schneider cells suggests that this sumoylation-dependent transrepression pathway is evolutionarily conserved and is utilized by both orphan and ligand-dependent nuclear receptors. Based on these observations, we propose to explore the roles of sumoylation and NCoR co-repressor complexes in nuclear receptor-dependent transrepression of innate immune responses on a genome-wide scale. Three Specific Aims are proposed. Specific Aim i will test the hypothesis that sumoylation of nuclear receptors is a broadly used mechanism in transrepression of inflammatory programs of gene expression. Specific Aim 2 will test the hypothesis that sumoylation of nuclear receptors mediates repression of inflammatory programs of gene expression by preventing co- repressor/co-activator exchange. Specific Aim 3 will test the hypothesis that the function of NCoR co- repressor complexes is modulated by the histone methyltransferase SMYDs. The results of these studies are likely to lead to new insights into the mechanisms underlying transrepression of inflammatory responses that can be exploited for development of novel therapeutic approaches. [unreadable] [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): In this application we propose to investigate molecular mechanisms by which PPARs counter-regulate innate immune responses and influence the ability of macrophages to contribute to atherosclerotic vascular disease. There is now extensive evidence that PPARa, PPAR?, and PPARd ligands inhibit inflammatory processes in macrophages and other cells within the artery wall that are linked to the progression of atherosclerosis and its clinical complications, but the mechanisms that are responsible for these effects remain poorly understood. We recently identified an NCoR/SUMOylation-dependent pathway by which PPAR? ligands inhibit specific subsets of toll-like receptor (TLR) 4-responsive genes. This mechanism involves ligand-dependent sumoylation of PPAR?, which later targets it to NCoR corepressor complexes on the promoters of inflammatory response genes. This in turn prevents the signal-dependent removal of NCoR complexes that is normally a prerequisite for transcriptional activation. Three specific aims are proposed to explore the significance of these findings with respect to the control of inflammatory programs of gene expression that underlie innate immune responses and the development of atherosclerosis. Specific Aim 1 will utilize a combination of molecular, cellular and genomics approaches to test the hypothesis that NCoR, and the highly related corepressor SMRT, are differentially required to maintain inflammatory response genes silent under quiescent conditions. We will test the hypothesis that the NCoR/SMRT repression checkpoint is inappropriately relieved in macrophage foam cells in vivo, resulting in a partially activated phenotype. Specific Aim 2 will test the hypothesis that the NCoR/SMRT/SUMOylation dependent pathway is a quantitatively important mechanism mediating anti-inflammatory actions of PPARy and PPARd agonists in macrophages. We will define the genome-wide profiles of PPAR? and PPARd repression of genes activated by the TLR2 agonist Pam3 in NCoR-/- and SMRT-/- macrophages. We will determine the ability of PPAR? and PPARd -specific agonists to exert anti-inflammatory effects in macrophages lacking the SUMO E3 ligase PIAS1. Specific Aim 3 will test the hypothesis that the NCoR/SMRT/SUMOylation-dependent pathway plays a quantitatively important role in mediating ant-atherogenic effects of PPAR? agonists in vivo. We will use a combination of selective PPAR? modulators and PPAR? mutants to define the relative importance of ligand- dependent activation and ligand-dependent repression in inhibiting the development of atherosclerosis in LDL receptor-deficient (LDLR-/-) mice. The results of these studies are likely to contribute to an improved understanding of the inflammatory component of atherosclerosis and the anti-atherogenic actions of PPAR? agonists. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): A new PPG application is proposed to identify and characterize gene networks in adipocytes and macrophages that influence insulin action. A combination of molecular, cellular, genetic and bioinformatics approaches will be used to define the components and behaviors of these networks at a genome-wide scale. The proposed studies will test the hypothesis that macrophage/adipocyte interactions result in altered programs of inflammatory gene expression in both cell types that contribute to insulin resistance. We will further test the hypothesis that PPARy agonists exert insulin-sensitizing effects by counter-regulating feed forward mechanisms that amplify inflammation within obese adipose tissue. Microarray and genome-wide location analysis will be performed to define the roles of NCoR/SMRT corepressor complexes as transcriptional checkpoints in PPARy-, NF-KB-, and AP-1-dependent gene expression and determine the importance of these complexes in mediating anti-inflammatory actions of PPARy agonists. Candidate genes identified by microarray studies and associated bioinformatics approaches will be tested for their pathophysiological roles in high fat diet-induced insulin resistance in mouse models. The results of these studies are likely to lead to new insights into the mechanisms underlying obesity-associated insulin resistance that can be exploited for development of novel therapeutic approaches.

PROJECT SUMMARY Core C: Administrative core An Administrative Core is proposed to support the administrative needs of the Program Project Grant. The major functions of the Core will be to provide a structure for decision making, organization of regular PPG meetings, mechanisms for internal and external advice and review, and effective utilization of resources. This Core will be Co-Directed by Drs. Glass and Witztum. Dr. Glass, Dr. Witztum, Dr. Evans, Dr. Loomba and Dr. Benner (PIs of Projects 1, 3, 2, and Core B respectively) will form an executive committee for routine decision making. An internal advisory committee consisting of Dr. Nicholas Schork, Dr. Alan Saltiel and Dr. Tatiana Kisseleva will provide both administrative and scientific advice. An external advisory group will be constituted to review the scientific program on an annual basis. The external advisory group will be selected to include expertise in NASH, atherosclerosis, inflammation, gene expression and statistics. The Administrative Core will be responsible for ensuring that the PPG conforms to the Resource Sharing Plan, the Sharing of Model Organisms and the Data Sharing Plan. The Administrative Core will also be responsible for preparation of the Annual Non Competing renewal.

A major goal in efforts to understand the mechanisms by which signal transduction pathways regulate programs of gene expression is to identify their direct target genes and to determine the specific components of the transcriptional machinery that are recruited to these genes in response to regulatory signals. To support these goals, the Transcriptional Genomics Core will provide three complementary services to PPG investigators; conventional gene expression (Chip) microarray analysis, recently developed genomic (ChIPChip) microarray analysis, and associated Bioinformatics support for experimental design oversight and data analysis. Conventional expression analysis will utilize commercially available microarrays (e.g., Affymetrix, Agilent and Illumina microarrays). Recent progress in combining the use of chromatin immunoprecipitation (ChIP) assays with DMA microarrays has allowed genome-wide analysis of transcription factor localization to specific promoter sequences in living cells. The PPG Transcriptional Genomics Core will fabricate murine intergenic/promoter microarrays to allow genome-wide location analysis of PPARs, NCoR, SMRT, and other transcription factors of relevance to this application. Effective utilization of genome-wide approaches requires an understanding of the strengths and limitations of these technologies, particularly with respect to sources of error and the number of experimental replicates that are required to develop gene lists at defined and acceptable false positive and false negative rates. Personnel within the PPG Transcriptional Genomics Core will interact with scientists within each of the Projects to provide experimental design oversight focused on these issues. Once microarray experiments are performed and raw data is collected, the Transcriptional Genomics Core will utilize standard tools to develop gene lists at specified levels of confidence and perform secondary analysis (e.g., Gene Ontology analysis, mapping to KEGG pathways, etc.). The Transcriptional Genomics Core will provide a database infrastructure for data storage and retrieval to allow integration of data collected across the PPG and the application of more sophisticated bioinformatics approaches outlined in each of the Projects.

In this Project, we will investigate molecular mechanisms by which PPARs counter-regulate innate immune responses and influence the ability of macrophages to contribute to atherosclerotic vascular disease. There is now extensive evidence that PPARa, PPARy and PPARd ligands inhibit inflammatory processes in macrophages and other cells within the artery wall that are linked to the progression of atherosclerosis and its clinical complications, but the mechanisms that are responsible for these effects remain poorly understood. We recently identified an NCoR/SUMOylation-dependent pathway by which PPARy ligands inhibit specific subsets of toll-like receptor (TLR) 4-responsive genes. This mechanism involves ligand-dependent sumoylation of PPARy, which targets it to NCoR corepressor complexes on the promoters of inflammatory response genes. This in turn prevents the signal-dependent removal of NCoR complexes that is normally a prerequisite for transcriptional activation. Three Specific Aims are proposed to explore the significance of these findings with respect to the control of inflammatory programs of gene expression that underlie innate immune responses and the development of atherosclerosis.: Specific Aim 1 will utilize a combination of molecular, cellular and genomics approaches to test the hypothesis that NCoR, and the highly related corepressor SMRT, are differentially required to maintain inflammatory response genes silent under quiescent conditions. In collaboration with Project 3, we will test the hypothesis that NCoR and SMRT are actively removed from these promoters upon stimulation of macrophages and B1 cells by modified LDL and specific TLR2 and TLR4 agonists. We will test the hypothesis that the NCoR/SMRT repression checkpoint is inappropriately relieved in macrophage foam cells in vivo, resulting in a partially activated phenotype. Specific Aim 2 will test the hypothesis that the NCoR/SMRT/SUMOylation dependent pathway is a quantitatively important mechanism mediating anti-inflammatory actions of PPARy and PPARd agonists in macrophages. In collaboration with Project 2, we will define the genome-wide profiles of PPARy and PPARS repression of genes activated by the TLR2 agonist Pam3 in NCoR[-/-] and SMRT[-/-] macrophages. We will determine the ability of PPARy and PPARd-specific agonists to exert anti-inflammatory effects in macrophages lacking the SUMO E3 ligase PIAS1. Specific Aim 3 will test the hypothesis that the NCoR/SMRT/SUMOylation-dependent pathway plays a quantitatively important role in mediating antiatherogenic effects of PPARy agonists in vivo. In collaboration with Project 4, we will use a combination of selective PPARy modulators and PPARy mutants to define the relative importance of ligand-dependent activation and ligand-dependent repression in inhibiting the development of atherosclerosis in LDL receptordeficient (LDLR[-/-]) mice. The results of these studies are likely to contribute to an improved understanding of the inflammatory component of atherosclerosis and the anti-atherogenic actions of PPARy agonists.

Macrophages and related cell types play critical roles in many aspects of immunity and homeostasis and contribute to the pathogenesis of human diseases that involve chronic inflammatory responses, including atherosclerosis and diabetes mellitus. Lipid metabolism in the macrophage is subject to extreme physiological and pathophysiological programs of regulation, providing a powerful model system to explore lipidomics. Core D will contribute to each of the integrated LIPID MAPS Specific Aims as follows: Specific Aim 1: Employ lipidomics to advance mechanistic understanding of metabolism: Analyses of lipid metabolites in mouse macrophages coupled with perturbations of these cells and the mice from which they are derived will advance our mechanistic understanding of biochemical pathways. Core D will serve as a central source of macrophages for Specific Aim 1 centrally planned perturbation experiments performed by all of the Lipidomics Cores and will provide macrophages for Core-specific projects as needed. This structure will maximize uniformity of cell preparations across the Lipidomics Cores. Core D will also perform corresponding transcriptome analysis for all centrally planned lipidomics experiments to correlate changes in lipids with changes in gene expression. Specific Aim 2: Employ lipidomics to investigate macrophages and tissues under pathological conditions as disease models: Analyses of lipid metabolites in mouse cells and tissues coupled with perturbations of these cells and the mice from which they are derived will advance our understanding of interacting lipid pathways under normal and pathological conditions. Core D will serve as a central source of macrophages for the disease model studies that will be centrally planned and performed by the Lipidomics Cores. Macrophages and tissues will also be supplied for Core-specific projects as needed. Core D will perform corresponding transcriptome analysis for all centrally planned lipidomics experiments to correlate changes in lipids with changes in gene expression. Specific Aim 3: Develop lipid networks and maps from lipidomics data analysis Using transcriptomic data generated in the first two specific aims, we will contribute to new and comprehensive informatic approaches to create networks and maps of biochemical pathways and to determine the fluxes of metabolites through these pathways. Macrophages play essential roles in normal immunity, but also contribute to the development of heart disease and diabetes. The procedures and experiments performed by this Core will enable the LIPID MAPS consortium to better understand how different classes of lipids, including fat and cholesterol, are metabolized by macrophages. Knowledge gained from these studies may lead to the development of new strategies for the prevention and treatment of these diseases.

The surprising relationship between macrophages and insulin resistance provides a promising interface in which to apply our emerging understanding of the molecular mechanisms of nuclear receptor actions and recent progress in defining the underlying strategies of transrepression. In Project 2, we will focus on NCoR/SMRT corepressor complexes as transcriptional checkpoints controlling both ligand-dependent regulation of gene expression by nuclear receptors and the activities of signal-dependent transcription factors that drive inflammatory programs of gene expression. We will better define the molecular mechanisms and roles of the TBLi, TBLRi and GPS2 components of N-CoR corepressor complexes in the regulation of AP1/NF-kB target genes and we will use genome-wide location analyses (GWLA) to investigate the roles of these proteins in positive and negative regulation of macrophage and adipocyte gene expression. Three Specific Aims are proposed. Specific Aim i will test the hypothesis that N-CoR/SMRT complexes regulate inflammatory responses that contribute to insulin resistance and are targets of anti-diabetic actions of PPARy agonists. These studies will be performed in collaboration with Units i and 3 using mice reconstituted with N-CoR-/- or SMRT-/- fetal liver hematopoietic progenitor cells. Specific Aim 2 will investigate the hypothesis that the TBLi/TBLRi exchange complex is regulated by signal-specific phosphorylation of TBLRi/TBLi. We will investigate the protein kinase control of corepressor complex dismissal from AP-1 and NF-kB target genes, and the role of these events in PPARy-mediated activation of positively regulated genes and transrepression of inflammatory response genes. Specific Aim 3 will explore the role of GPS2 and KIAA1787 in JNK-dependent gene activation/repression events and to test the hypothesis that GPS2 is required for normal insulin sensitivity based on observations that JNK-expression and activity are consistently elevated in diet-induced obesity models and that AP-1 activity is constitutively increased on a subset of gene targets in N-CoR-/- macrophages. These studies will utilize a combination of single cell nuclear microinjection of siRNAs, an ultra-sensitive, multiplexed RNA quantification method (RASL) and ChlP-DASL to define roles of GPS2 in signal-dependent activation of inflammatory response genes.

2,4-Thiazolidinedione, 5-004-02-((((methyl-2-pyridinylamino)ethoxy)phenyl)methyl)-; 5-((4-(2-methyl-2-(pyridinylamino)ethoxy)phenyl)methyl)-2,4-thiazolidinedione-2-butenedioate; Adhesions; Adipocytes; Adipose Cell; Adipose tissue; Affinity; Animals; Avandia; Binding; Binding (Molecular Function); Blood monocyte; Body Tissues; Bone Marrow; Bone Marrow Transplant; Bone Marrow Transplantation; Candidate Disease Gene; Candidate Gene; Causality; Cells; Chemotaxis; Chronic; Co-culture; Cocultivation; Coculture; Coculture Techniques; Collaborations; Communicating Junction; Condition; Data; Diabetes Mellitus, Adult-Onset; Diabetes Mellitus, Ketosis-Resistant; Diabetes Mellitus, Non-Insulin-Dependent; Diabetes Mellitus, Noninsulin Dependent; Diabetes Mellitus, Slow-Onset; Diabetes Mellitus, Stable; Diabetes Mellitus, Type 2; Diabetes Mellitus, Type II; Epidemic; Etiology; Fat Cells; Fatty Tissue; Gap Junctions; Gene Expression Monitoring; Gene Expression Pattern Analysis; Gene Expression Profiling; Gene Targeting; Gene Transfer Techniques; Generations; Genes; Glaxo Wellcome brand of rosiglitazone maleate; GlaxoSmithKline brand of rosiglitazone maleate; Glucose Intolerance; Grafting, Bone Marrow; Human; Human, General; Humulin R; Hyperinsulinemia; Hyperinsulinism; INFLM; In Vitro; Inflammation; Inflammatory; Insulin; Insulin (ox), 8A-L-threonine-10A-L-isoleucine-30B-L-threonine-; Insulin Resistance; Insulin, Regular; KIAA1047; Knock-out; Knockout; Knockout Mice; Lentivirinae; Lentivirus; Ligands; Lipocytes; Low-resistance Junction; MODY; Macrophage Activation; Mammals, Mice; Man (Taxonomy); Man, Modern; Marrow Transplantation; Marrow monocyte; Mature Lipocyte; Mature fat cell; Maturity-Onset Diabetes Mellitus; Mediating; Metabolic; Metabolic syndrome; Methods; Mice; Mice, Knock-out; Mice, Knockout; Mice, Transgenic; Modeling; Molecular; Molecular Interaction; Mother Cells; Murine; Mus; N-CoR; N-CoR1; NCOR1; NCOR1 protein, human; NIDDM; Nexus; Nexus (resin cement); Nexus Junction; Non-Insulin Dependent Diabetes; Non-Insulin-Dependent Diabetes Mellitus; Novolin R; Nuclear Receptor Co-Repressor 1; Nuclear Receptor Corepressor; Nuclear Receptors; Null Mouse; Numbers; Obesity; Pathogenesis; Pathway interactions; Phenotype; Play; Process; Production; Profilings, Gene Expression; Progenitor Cells; Promoter; Promoters (Genetics); Promotor; Promotor (Genetics); Receptor Protein; Recruitment Activity; Repression; Reticuloendothelial System, Bone Marrow; Role; Series; Site; SmithKline Beecham brand of rosiglitazone maleate; Specific qualifier value; Specified; Stem cells; Subfamily lentivirinae; Syndrome; System; System, LOINC Axis 4; T2D; T2DM; TRAC1; Targetings, Gene; Technology; Testing; Thyroid Hormone- and Retinoic Acid Receptor-Associated Corepressor 1; Tissues; Trans-Activation (Genetics); Transactivation; Transcript Expression Analyses; Transcript Expression Analysis; Transgenesis; Transgenic Mice; Transplantation; Type 2 diabetes; Type II diabetes; United States; Virus-Lenti; adipose; adiposity; adult onset diabetes; cell type; combinatorial; concept; corpulence; corpulency; corpulentia; cytokine; desire; disease causation; disease etiology; disease/disorder etiology; disorder etiology; experiment; experimental research; experimental study; hCIT529I10; hN-CoR; human NCOR1 protein; in vitro Assay; in vivo; insight; insulin resistant; insulin sensitivity; ketosis resistant diabetes; knock-down; knockout animal; lentiviral-mediated; macrophage; maturity onset diabetes; migration; monocyte; mutant; novel; obese; obese people; obese person; obese population; pathway; receptor; recruit; research study; rosiglitazone; shRNA; short hairpin RNA; small hairpin RNA; social role; transplant; vector; white adipose tissue; yellow adipose tissue

The peroxisome proliferator-activated receptor y (PPAR) regulates adipocyte differentiation and glucose homeostasis and is the molecular target of thiazolidinediones that act as insulin-sensitizers in patients with type 2 diabetes. PPAR? is also expressed in macrophages and negatively regulates the program of macrophage activation by repressing a subset of AP-i and NK-KB-dependent genes. The recent discovery that macrophages accumulate in obese adipose tissue and are a major source of inflammatory mediators that are linked to insulin resistance raises the possibility that the macrophage is a key target of the anti-diabetic actions of TZDs. Consistent with this, selective deletion of the PPAR? gene from macrophages results in mild insulin resistance in lean animals and a more severe degree of insulin resistance in diet-induced obesity. Studies performed in collaboration with the Rosenfeld laboratory have uncovered a mechanistic pathway by which PPAR? inhibits inflammatory gene expression in activated macrophages that involves the nuclear receptor co-repressor, N-CoR. This Project will test the overall hypothesis that adipocyte-macrophage interactions drive inflammatory programs of gene expression that contribute to insulin resistance and that TZDs act as insulin sensitizers in part by counter-regulating these responses in macrophages. Four Specific Aims are proposed: Specific Aim i will define PPAR? and N-CoR-dependent gene networks in macrophages using a combination of gene expression profiling and ChlP-Chip technologies. These studies will test the hypothesis that N-CoR co-repressor complexes are required for PPAR?-mediated repression of broad sets of inflammatory response genes and will complement studies in Project 2 examining glucose homeostasis in mice reconstituted with N-CoR-deficient macrophages. Specific Aim 2 will investigate the basis for insulinresistance in mice selectively lacking PPAR? expression in macrophages. In collaboration with Project i, these studies will evaluate macrophage accumulation and gene expression in adipose tissue, skeletal muscle and livers of wild type mice and mice lacking PPAR? expression in macrophages. Specific Aim 3 will test the hypothesis that PPAR? counter-regulates pro-inflammatory programs of gene expression induced by free fatty acids. This will be accomplished by determining the effects of free fatty acids on macrophage gene expression in control and PPAR?-deficient macrophages and by evaluating the ability of free fatty acids to stimulate the transrepression activities of PPAR?. Specific Aim 4 will utilize selective modulators of PPAR? to test the hypothesis that insulin-sensitizing activities of PPAR? can be achieved through either activation of direct target genes in adipocytes or through transrepression of inflammatory response genes in macrophages. These studies have implications for novel approaches to treatment of obesity-induced diabetes.

DESCRIPTION (provided by applicant): We propose an ambitious program of collaborative research to identify and characterize molecular mechanisms responsible for altered programs of inflammatory gene expression that contribute to insulin resistance. We will capitalize on our recent discoveries of unexpected roles of GPS2 and the nuclear receptor co-repressors NCoR and SMRT in regulation of inflammatory signaling pathways in macrophages and adipocytes. We will extend studies of novel mechanisms mediating the recruitment of pro-inflammatory macrophages into adipose tissue that have clear translational potential. We will utilize newly developed technologies to characterize the genomic locations and functions of PPARy in adipose tissue macrophages in vivo to determine mechanisms by which macrophage PPARy contributes to insulin-sensitizing functions of thiazolidinediones. We will investigate the roles o 3 dimensional chromatin interactions and non-coding RNAs in positive and negative regulation of inflammatory gene expression. Overall, the proposed studies are expected to lead to new insights into mechanisms underlying obesity-associated insulin resistance that will facilitate development of new approaches for the prevention and treatment of type 2 diabetes.

DESCRIPTION (provided by applicant): We propose to establish the feasibility of a potentially transformative approach, applicable to a wide variety of human diseases, which we refer to as Enhancer Therapy. This approach is based on the discovery of the functional importance of eRNAS; non-coding RNAs that are transcribed from tissue and disease-specific enhancers. Remarkably, we find that knockdown of these eRNAs using derivatized anti-sense oligonucleotides (ASOs) reduces expression of nearby target genes in macrophages and breast cancer cells. These findings, in concert with the clinical development of ASO technology by Isis Pharmaceuticals, open a pathway for the development of ASOs that result in tissue specific inhibition of pathogenic gene expression in humans. We therefore propose to investigate the feasibility of 'Enhancer Therapy' using inflammation and breast cancers as initial models, with the following approach: (i) We will use established and novel genome-wide methods to generate atlases of enhancers and enhancer RNAs in mouse and human tissues and cell types that are relevant to both pathological conditions and normal tissue homeostasis; (ii) Using these atlases, we apply GRO-seq and our recently developed 3D-DSL methodology to generate high-resolution maps of the interconnections of specific enhancers of interest with their target genes; (iii) We will next select cell-specific enhancers that express eRNAs and interact with disease-relevant genes to use as models for eRNA targeting. In collaboration with ISIS Pharmaceuticals, we will develop corresponding ASOs that specifically reduce expression of target eRNAs in primary mouse macrophages and human breast cancers suitable for use in vivo. The functional consequences of eRNA knockdown will be ascertained by appropriate secondary assays, e.g., suppression of inflammatory gene expression in macrophages and proliferation/metastasis of breast cancer cells. (iv) Using the results of these in vitro studies, we will proceed to test ASOs for their ability to reduce expression of selected target genes in cells and in vivo in a cell/tisse specific manner and explore appropriate disease models. Our goal is to directly test the novel idea that inhibition of eRNA expression can result in a therapeutic outcome, thereby establishing a transformative approach to treatment of human disease. PUBLIC HEALTH RELEVANCE: We will focus on macrophages and breast cancer cells as the primary target cells for studies of feasibility of Enhancer Therapy. These two cell types provide models that are relevant to a broad range of inflammatory diseases, such as atherosclerosis and type 2 diabetes, and cancer, which collectively account for a substantial fraction of overall morbidity and mortality in industrialized societies.

Project 3 will investigate transcriptional networks in macrophages that influence Insulin resistance. Our proposed studies will primarily focus on understanding unexpected physiological and cellular consequences of deletion of the NCoR co-repressor in macrophages and on deflning the molecular mechanisms by which macrophage PPARy contributes to normal glucose homeostasis and insulin sensitizing effects of thiazolidinediones (TZDs). These lines of investigation will complement studies performed in Projects 1 and 2 to improve our understanding of central pathogenic mechanisms that drive the development of insulin resistance. Speciflc Aim 1 will test the hypothesis that macrophage-speciflc disruption of NCor results in enhanced insulin sensitivity due to de-repression of LXR and/or PPARy target genes that drive production of anti-inflammatory fatty acids. These studies have the potential to identify a fundamentally new pathway by which macrophages influence insulin resistance that may be amendable to therapeutic intervention. Specific Aim 2 will investigate mechanisms by which macrophage PPARy contributes to normal glucose homeostasis and anti-diabetic effects of TZDs. We will test the hyothesis that the genome-wide locations and functions of PPARy are compromised in adipose tissue macrophages of obese adipose tissue and are restored by insulin-sensitizing PPARy ligands. These studies will make use of new in vivo approaches for determining macrophage-specific PPAR location and function in adipose tissue that do not require extensive purification methods. Studies in Specific Aim 3 will be performed in collaboration with Project 2 to test the hypothesis that alternative macrophage activation alters the chromatin interactome so as to facilitate PPARy-dependent gene expression and antagonize TLR4-dependent gene expression. These studies will test a new concept for understanding how anti-inflammatory and pro-inflammatory signals are integrated at a 3 dimensional level in the nucleus. RELEVANCE (See instmctions): The proposed studies will be of signiflcance in improving our understanding of central pathogenic mechanisms that drive the development of insulin resistance and in shaping future therapeutic approaches to prevent and treat type 2 diabetes.

A major goal in diabetes research is to understand how alterafions in the epigenome and subsequent responses in gene expression impact disease phenotype and treatment regimens. The Epigenefic and Genomics Core (EGC) will provide access to microarray platforms, massively parallel sequencing instrumentafion and computafional infrastructure to advance the diabetes and metabolism research goals of DRC investigators. The EGC will provide the following services: 1. Expression microarray technology; Affymetrix, Agilent, Codelink, NimbleGen, and lllumina platforms will be provided for microarray-based mRNA expression profiling. Affymetrix, Life Technologies and Exiqon platforms will be provide for miRNA profiling. 2. Technical support will be provided for high-throughput sequencing assays on lllumina HiSeq 2000 platforms, including RNA sequencing (RNAseq), microRNA sequencing (mlRNAseq), global run-on sequencing (GRO-Seq), ribosome profiling and deep sequencing (Ribo-Seq), chromafin immunoprecipitafion linked to massively parallel sequencing (ChlP-Seq) and MethylC-sequencing. 3. Bioinformatics support will be provided for assistance with experimental design, choice of technological platform, data analysis and data quality control. Implementafion of new data management and analysis pipelines will facilitate effective data mining. 4. Training of students, postdoctoral fellows, invesfigators and technical staff in the application of highthroughput sequencing methodologies and data analysis 5. High-performance computing resources, systems administrators, and data storage/backup systems will enable users to efficiently access and analyze their data. A major emphasis in the configurafion of the ECG will be implementafion of new Core services to reduce barriers to entry to new investigators. This will be achieved by providing direct Core support at three of the crifical steps required to take advantage of sequencing-based technologies; 1) Preparafion of sequencing libraries, 2) provision of computafional resources, and 3) assistance with data analysis through both training and provision of informatics services.

The Genomics and Bioinformatics Core will provide resources and technical and bioinformatics support for the application of massively parallel sequencing-based technologies to the understanding of regulated gene expression in macrophages, T cells, B cells and endothelial cells. The Genomics and Bioinformatics Core will thereby enable the acquisition and sophisticated analysis of data generated by ChlP-Seq, RNA-Seq, GRO-Seq and Ribo-Seq experiments. These methods provide extremely powerful approaches to addressing key mechanistic and pathophysiologic questions by each ofthe four projects ofthe PPG.

Project 1 will investigate the regulation of inflammation and lipid homeostasis in the context of macrophage foam cell formation and the pathogenesis of atherosclerosis. These studies will leverage our recent discovery that desmosterol, an intermediate in the cholesterol biosynthetic pathway, unexpectedly plays a key role in integration of the homeostatic response to excess cholesterol by activating LXR target genes required for cholesterol efflux, suppressing SREBP target genes involved in cholesterol and fatty acid biosynthesis, and inhibiting inflammatory responses by LXR-dependent and independent mechanisms. These findings have a number of unanticipated implications for understanding the pathophysiology of atherosclerosis and suggest new therapeutic approaches. In Specific Aim 1, we will test the hypothesis that de-compensation of cholesterol homeostasis and 'expression of genes that amplify inflammatory responses in macrophage foam cells results from extrinsic, pro-inflammatory signals that are generated in the artery wall and induce TLR signaling. In Specific Aim 2, we will test the hypothesis that in addition to serving as TLR-induced transcriptional activators of inflammatory-response genes, NFKB and IRF3 mediate TLR? dependent suppression of LXRs. Delineation of the mechanisms by which NFKB and IRF3 inhibit the lipid homeostatic and anti-inflammatory activities of LXRs may suggest new approaches for therapeutic intervention. In Specific Aim 3, we will test the hypothesis that increasing endogenous levels of desmosterol or mimicking its activities will promote sterol excretion and inhibit the development of atherosclerosis. Conventional LXR ligands promote cholesterol efflux and inhibit atherosclerosis in mice, but are not useful drugs because they induce the expression of SREBP1c and cause marked hypertriglyceridemia. In contrast, desmosterol activates LXRs but suppresses SREBP processing. In this aim, we will investigate whether treatment with a putative desmosterol mimetic or raising endogenous desmosterol levels can be used as approaches to restore cholesterol homeostasis in lesion macrophages without causing adverse effects on circulating lipoprotein levels.

PROJECT 1 ? SPATIOTEMPORAL ARCHITECTURE OF THE GENOME SUMMARY A central challenge of molecular biology is to understand how transcriptional regulatory elements are selected from the genome thereby specifying cellular identity and cell-specific responses. In this project, we will use systems biology approaches and the maps-to-model paradigm to gain insights into general mechanisms responsible for the selection and function of cis-regulatory elements necessary for transcriptional responses to pathogens. These activities consist of the following four Specific Aims. First, we will generate a map and use this to model the selection of cell-specific enhancer landscapes. This aim is based on the hypothesis that relatively simple combinations of lineage-determining transcription factors (LDTFs) play dominant roles in selecting a large fraction of cell-specific enhancers. We will develop and test a mechanistic network model that begins with genome-wide predictions of binding sites for macrophage LDTFs and progresses to predict the genome-wide binding locations of other collaborating transcription factors that contribute to enhancer selection. Second, we will map and model the role of signal-dependent transcription factors (SDTFs) in regulating the enhancer landscape. This aim is based on the hypothesis that an essential feature of a functional enhancer is that it is actively transcribed. In this aim, we will extend the mechanistic network model developed in Aim 1 to predict binding of SDTFs and subsequent transcriptional activation of enhancers following lipopolysaccharide exposure or adenoviral infection. The predictive value of the model will be tested by loss-of-function studies of the SDTFs and by evaluation of effects of natural genetic variation. Third, we will try to predict transcriptional activity as a function of enhancer interactions. We propose extending the mechanistic network model achieved in Aim 2 to consider co-regulated transcriptional start sites in the context of spatial co-localization. This aspect of the model will be tested by evaluating loss-of-function mutants and the impact of natural genetic variation on enhancer- promoter interactions using a modified version of the Hi-C assay that focuses sequencing power on interactions involving promoters. Finally, we will map and model the 3D virus-host genome interaction hubs and transcriptional networks that determine the outcome of infection across tissues and species. We hypothesize that viral genomes target and subvert the 3D organization and interactions of the host genome to activate different viral and host transcriptional programs in the time course of infection. The proposed studies will map and model 3D genome interactions and transcriptional programs within different tissue types that determine viral tropism and replication. These studies are of particular interest because, while the `early' program of human adenovirus infection is intact in mouse cells, their productive lytic replication/expression is blocked `late' through mechanisms that are poorly understood. A molecular understanding of these mechanisms would be highly significant at both conceptual and practical levels.

Project 3 will investigate transcriptional networks in macrophages that influence Insulin resistance. Our proposed studies will primarily focus on understanding unexpected physiological and cellular consequences of deletion of the NCoR co-repressor in macrophages and on deflning the molecular mechanisms by which macrophage PPARy contributes to normal glucose homeostasis and insulin sensitizing effects of thiazolidinediones (TZDs). These lines of investigation will complement studies performed in Projects 1 and 2 to improve our understanding of central pathogenic mechanisms that drive the development of insulin resistance. Speciflc Aim 1 will test the hypothesis that macrophage-speciflc disruption of NCor results in enhanced insulin sensitivity due to de-repression of LXR and/or PPARy target genes that drive production of anti-inflammatory fatty acids. These studies have the potential to identify a fundamentally new pathway by which macrophages influence insulin resistance that may be amendable to therapeutic intervention. Specific Aim 2 will investigate mechanisms by which macrophage PPARy contributes to normal glucose homeostasis and anti-diabetic effects of TZDs. We will test the hyothesis that the genome-wide locations and functions of PPARy are compromised in adipose tissue macrophages of obese adipose tissue and are restored by insulin-sensitizing PPARy ligands. These studies will make use of new in vivo approaches for determining macrophage-specific PPAR location and function in adipose tissue that do not require extensive purification methods. Studies in Specific Aim 3 will be performed in collaboration with Project 2 to test the hypothesis that alternative macrophage activation alters the chromatin interactome so as to facilitate PPARy-dependent gene expression and antagonize TLR4-dependent gene expression. These studies will test a new concept for understanding how anti-inflammatory and pro-inflammatory signals are integrated at a 3 dimensional level in the nucleus. RELEVANCE (See instmctions): The proposed studies will be of signiflcance in improving our understanding of central pathogenic mechanisms that drive the development of insulin resistance and in shaping future therapeutic approaches to prevent and treat type 2 diabetes.

? DESCRIPTION (provided by applicant): Microglia are tissue macrophages that reside in the central nervous system (CNS) and perform unique and critical auxiliary functions important to CNS development, homeostasis, immunity and repair. Despite their importance, remarkably little is known about the mechanisms that control microglia development and function, particularly in humans. Here we propose to leverage recent technical and computational breakthroughs to investigate how interactions between lineage-determining and signal-dependent transcription factors regulate gene expression in human and mouse microglia. Specific Aim 1 will test the hypothesis that the human brain microenvironment is an important determinant of microglia gene expression. Microglia transcriptomes will be obtained immediately upon isolation and after one week in a tissue culture environment supplemented with putative regulators of microglia phenotypes. Importantly, all samples will be analyzed in the context of full genomic sequence. These studies will provide qualitatively new information on core transcriptional signatures of microglia, the influence of the brain microenvironment, and the relationships of genotype to gene expression patterns. Specific Aim 2 will test the hypothesis that PU.1 and a limited set of alternative lineage determining factors drive the selection of the majority of enhancers that determine microglia identity and regulatory potential. We will use ChIP-Seq and ATAC-Seq methods to define enhancer atlases in freshly isolated human microglia. This information will suggest key microglia lineage determining factors and identify microglia super enhancers. We will exploit the combination of ChIP sequencing and natural genetic variation to identify core transcription factor combinations needed for the selection and activation of microglia-specific enhancers. These findings will provide qualitatively new insights into molecular mechanisms that underlie human microglia development and function and will inform efforts to more faithfully reprogram human iPS cells to microglia phenotypes. Specific Aim 3 will test the hypothesis that environmental factors and transcriptional circuits required for microglia-specific gene expression are conserved between mouse and human. Mouse microglia transcriptomes and enhancer atlases will be obtained under the same conditions as described for human microglia in Specific Aims 1 and 2. Different strains of mice will be used as a source of natural genetic variation to define core transcription factor combinations needed for enhancer selection and activation. Roles of specific transcription factors predicted to have conserved roles in regulating human and mouse microglia development and function will be assessed by loss of function experiments. These experiments will provide important information on the extent to which mouse microglia can be used to model human microglia and establish the functional importance of conserved transcription factors.

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 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.

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

A major goal in diabetes research is to understand how alterations in the epigenome and subsequent responses in gene expression impact disease phenotype and treatment regimens. The objective of the Epigenetic and Genomics Core (EGC) is to provide cutting-edge, reliable and innovative genomic technologies to support the diabetes and related endocrinology and metabolism research goals of DRC investigators. It also offers training, education and consultation in genomics technologies in order to enhance the ability of DRC Investigators to implement these technologies in their research. The EGC will provide the following services: Technical Support For Sequencing-Based Assays: The Core will provide technical support for high-throughput sequencing assays on Illumina sequencing platforms (MiSeq, HiSeq2500, HiSeq4000), enabling RNA sequencing (RNA-seq), microRNA sequencing (miRNAseq), Global Run- On sequencing (GRO-Seq), ribosome profiling and deep sequencing (Ribo-Seq), Chromatin Immunoprecipitation linked to massively parallel sequencing (ChIP-Seq), Assay for Transposase- Accessible Chromatin with high throughput sequencing (ATAC-Seq), MethylC-sequencing, metagenomic assays, and sequencing of CRISPR Screens. Low Input and Single Cell Sequencing Assays: The Core will provide technical support for low input and single cell sequencing assays, including development of low input Standard Operating Procedures (SOPs), use of the Fluidigm C1 System and the 10X Genomics Chromium Single Cell Solution technologies, and implementation of new technologies as they arise. Training and Consultation: The Core will provide consultation and training of students, postdoctoral fellows, investigators and technical staff regarding high-throughput sequencing methodologies and data analysis. These functions will be overseen by the Functional Genomics Specialist. Bioinformatics Support: The Core will provide bioinformatics support for assistance with experimental design, choice of technological platform, data analysis and data quality control.

PROJECT SUMMARY Project 1. Macrophage-specific targeting of LXRs in CVD and NASH Fatty liver diseases account for rapidly growing morbidity in the United States, where it is estimated that 80 to 100 million individuals have non-alcoholic fatty liver disease (NAFLD) and 6 to 16 million have the more severe liver disease, nonalcoholic steatohepatitis (NASH). NAFLD is a spectrum of liver conditions strongly coupled with obesity, insulin resistance, CVD, and type-2 diabetes mellitus. Long term prospective studies indicate that the presence and severity of NAFLD independently predicts fatal and nonfatal CVD events. The development of NAFLD and CVD is influenced by combinations of genetic and environmental factors, some of which are disease-specific and others that affect both disease processes. In this Project, we will investigate the central hypothesis that impaired function of liver X receptors in Kupffer cells in the liver and macrophages within the artery wall represent a common underlying mechanism that contributes to both NAFLD and atherosclerosis, and that this mechanism can be reversed by treatment with desmosterol mimetics. A major limitation in targeting LXRs for treatment of atherosclerosis is that most synthetic agonists cause marked hypertriglyceridemia by inducing the expression of SREBP1c in hepatocytes. Our studies of macrophage foam cells led to the finding that desmosterol, an intermediate in the cholesterol biosynthetic pathway, is the most abundant endogenous LXR agonist. Unlike conventional agonists that selectively bind to LXRs, desmosterol also binds to SCAP, thereby inhibiting processing of SREBP1 and SREBP2. Unexpectedly, we recently discovered that desmosterol and synthetic desmosterol mimetics do not activate LXR or suppress SREBP target genes in hepatocytes. In vivo studies with a synthetic desmosterol mimetic further demonstrated activation of LXR target genes in Kupffer cells but not in the liver as a whole. Our findings reveal cell-specific differences in LXR responses to natural and synthetic ligands in macrophages and hepatocytes that provide a conceptually new basis for prevention of NASH and atherosclerosis. Three Specific Aims are proposed. Specific Aim 1 will test the hypothesis that LXR activity in Kupffer cells is required for normal liver homeostasis and that Kupffer cell-specific deletion of LXRs results in exaggerated NASH and atherosclerosis. These studies will exploit new mouse models allowing Kupffer cell-specific deletion of LXRs. Specific Aim 2 will use a combination of pharmacologic and genetic approaches to test the hypothesis that selective activation of LXRs in Kupffer cells with synthetic desmosterol mimetics protects mice from NASH and atherosclerosis independent of effects of these ligands within the artery wall. Specific Aim 3, performed in collaboration with Project 4, will test the hypothesis that monocyte gene expression signatures and epigenetic landscapes in peripheral blood monocytes correlate with CVD phenotypes and the extent of fibrosis in human subjects. The proposed studies may result in qualitative advances in understanding roles of LXRs in regulation of NASH and atherosclerosis and establish the potential of desmosterol mimetics to be advanced for clinical investigation.

PROJECT SUMMARY Overall Despite the development of increasingly effective therapies to reduce elevated levels of atherogenic lipoproteins, cardiovascular disease (CVD) complications are projected to rise worldwide due in part to the increasing incidence of obesity and insulin resistance. An emergent question is the extent to which non-alcoholic fatty liver disease (NAFLD), which is a spectrum ranging from fatty liver to non-alcoholic steatohepatitis (NASH) to cirrhosis, contributes to CVD risk. Among patients with NAFLD, the leading cause of death is CVD, estimated to account for 31% of total mortality. The development of NAFLD and cardiovascular disease is influenced by combinations of genetic and environmental factors, some of which are disease-specific and others that affect both disease processes. The overall hypotheses of our PPG are that liver fat and fibrosis predict CVD risk and that interventions targeting Liver X receptors (LXRs) in macrophages, the farnesyl X receptor (FXR) in the gut, and oxidation specific epitopes (OSEs) in the liver and artery wall will reveal common mechanisms that contribute to the clinical association between NASH and CVD. Importantly, each of these interventions make use of representative small molecules or antibodies that have the potential to be advanced for clinical trials. Identifying mechanisms by which known and unknown risk factors promote both NASH and CVD would be of great significance, especially if targeting one or more of these mechanisms would produce beneficial effects on both diseases. To achieve this goal, we propose a PPG consisting of four highly inter-related projects and three cores. Project 1, led by Dr. Christopher Glass, will test the hypothesis that selective activation of LXRs in macrophages and Kupffer cells with desmosterol mimetics will result in reductions of atherosclerosis and NASH without causing steatosis or hypertriglyceridemia. Project 2, led by Dr. Ronald Evans, will investigate the hypothesis that selective activation of FXR in the gut or liver will result in reductions in atherosclerosis and NASH. Project 3, led by Dr. Joseph Witztum, will test the hypothesis that antibody-mediated reductions in OSEs will coordinately reduce both atherosclerosis and NASH. Project 4, led by Dr. Rohit Loomba, will investigate the relationships of liver fat content and fibrosis with cardiovascular risk in human subjects and enable translational extension of mechanistic findings made in Projects 1, 2 and 3. A Phenotyping Core will enable Projects 1, 2 and 3 to quantitatively evaluate extent of atherosclerosis and NASH in mouse models, and enable all projects to obtain targeted lipidomic profiles and cytokine levels from relevant samples. A Genomics and Bioinformatics Core will support the application of massively parallel sequencing-based assays, such as RNA Seq, by Projects 1, 2 and 3 and provide a shared resource for bioinformatics and statistical analysis. An Administrative Core will support the overall administrative and scientific needs of the PPG.