Ronald M. Evans - US grants

? DESCRIPTION (provided by applicant): Asthma affects more than 300 million people worldwide, but despite the current availability of multiple therapies, many patients remain refractory to standard treatments. Furthermore, specific therapies have been linked to adverse and sometimes life-threatening reactions. Thus, an urgent need for new therapies exists. Asthma is associated with chronic airway inflammation that involves both the innate and adaptive immune systems. While Th2 cells and eosinophils play key roles in the pathogenesis of the majority of asthma cases, recently Th17 cells have been implicated in a subgroup of asthmatics associated with neutrophilic airway inflammation and corticosteroid-resistance. Thus, Th2 and Th17 cells represent potential therapeutic targets in the treatment of asthma. Nuclear hormone receptors (NHRs) are validated anti-inflammatory therapeutic targets. This proposal is based on findings that the NHRs PPAR? and REV-ERB? are expressed in Th2 and Th17 cells, where they attenuate T cell effector function and suppress IL-4 and IL-17A expression, respectively. Since both Th2 and Th17 cells are associated with asthma development and pathogenesis, this proposal will investigate whether PPAR? and REV-ERB? are therapeutically accessible targets to ameliorate airway inflammation and asthma. The goal of Aim 1 is to determine the role of PPAR? in Th2 associated airway inflammation and airway hyper-reactivity (AHR), utilizing wild-type and T cell-specific PPAR? knockout (PPAR? cKO) mice in an ovalbumin-induced AHR model. Furthermore, the pharmaceutical potential of PPAR? will be determined by comparing responses of wild-type and PPAR? cKO mice to the PPAR? ligand rosiglitazone (Rosi), in the AHR model. In Aim 2, the molecular mechanisms underlying PPAR? and the corepressor SMRT functions in Th2 cells will be characterized. Global transcriptomic and cistromic analyses will be conducted in Th2 cells in the absence or presence of PPAR? ligand and these results correlated to identify direct and indirect PPAR? targets. The goal of Aim 3 is to determine the role of REV-ERB? in Th17-associated airway inflammation and AHR using novel T cell-specific REV-ERB? loss-of-function and gain-of-function mouse models, as well as a synthetic REV-ERB agonist, to test the hypothesis that activating REV-ERB? can ameliorate Th17 cell associated AHR and asthma. In Aim 4, the molecular mechanism of REV-ERB? and ROR?t interactions in Th17 cells will be dissected by mapping the ROR? and REV-ERB? cistromes to gain mechanistic insight into their global DNA binding activities. These genomic binding maps will be compared with transcriptional outcomes in wild-type and REV-ERB? knockout Th17 cells, with the goal of elucidating the REV-ERB? dependent transcriptional network. The proposed studies will advance the understanding of the molecular mechanisms of PPAR? and REV-ERB? actions in T helper cell subsets, and explore their therapeutic potential in T cell function to ameliorate airway inflammation and asthma.

The goal of this research is to understand hormonal mechanisms that control gene expression and how these molecules influence developmental and physiologic processes. Our previous studies have led to the identification of a superfamily of regulatory proteins that include receptors for steroid hormones, thyroid hormones and the vertebrate morphogen retinoic acid. The molecular interactions of the glucocorticoid receptor and thyroid hormone receptor with their binding sites will be examined using DNA- binding mutants that have altered target gene specificities or altered activational properties. The potential for functional receptor heterodimers will be examined. The DNA-binding domains of the GR and TR will be overproduced in E. coli to solve their three-dimensional structure in solution and in crystals by nuclear magnetic resonance (NMR) and X-ray crystallographic studies, respectively. Activation domains of the thyroid hormone receptors will be defined by deletion, truncation and point mutational analysis. Putative domains will be transferred to hybrid activators and potential interaction of the activator domains with the transcriptional machinery will be characterized through a transcriptional inteference analysis. Proteins interacting with the glucocorticoid and thyroid hormone receptors will be sought by biochemical approaches to identify the encoding genes. Biochemical approaches include chromatography of nuclear extracts to glucocorticoid receptor and thyroid hormone receptor affinity columns and the detection of receptor-DNA complexes with altered electrophoretic mobilities. Recent studies have shown the erbA oncogene is capable of functioning as a thyroid hormone receptor antagonist. The mechanism of this antagonism will be sought be examining the relative binding affinities of the oncogene and the thyroid hormone receptor for their response elements as well as identifying the specific mutations involved in converting the wild-type receptor to its oncogenic form. The thyroid hormone receptor repressor activity will be characterized. In summary, the proposed experiments should provide important information on basic questions such as the molecular nature of protein-DNA interactions in eukaryotic gene regulation, the interactions between activator proteins and the transcription machinery, and molecular interpretation for how steroid and thyroid hormones act to control human physiology as well as pathologic changes associated with reproduction, metabolism and cancer.

Studies of hormonal signalling have fundamental implications for the molecular basis of organ physiology, embryonic development, cell differentiation, and human disease. The goal of this proposal is to establish a molecular basis for the interaction for steroid and related receptors with proto-oncogene signalling pathways. We have reported that AP-1 is able to block induction by the glucocorticoid and retinoic acid receptor and reciprocally, that glucocorticoids and retinoids are able to inhibit activation of promoters containing AP-1 sites. In this proposal, we will utilize the yeast two hybrid interaction screen to identify nuclear proteins that may mediate AP-1 and glucocorticoid receptor cross-talk. This will be based on a sequential screen isolating interacting clones for one protein and rescreening against the other to identify the unique subsets specific for both. Full-length cDNA clones will be isolated for dually reactive proteins and antibodies to these newly cloned factors will be prepared. These proteins will be subjected to extensive truncation and deletion analysis to map sites of interaction with the receptors utilizing co-immunoprecipitation in vitro as a rapid assay. In addition, functional studies will be initiated using co- transfection based assays. We have recently demonstrated that CBP may represent a common co-factor for both CREB and the glucocorticoid receptor. These studies will be extended to determine whether or not this may also serve as a putative co-factor mediating retinoid signalling. Assays including mobility shifts, microinjection of anti-CBP antibody into cultured cells and co- transfection will be used to characterize these regulatory interactions. Utilizing deletion and truncation mutants, the sites of interaction will be localized. As with AP-1, we have shown that p65/cREL inhibits the activity of the glucocorticoid receptor. We will characterize the functional interactions of p50/p65 (NF-kappaB) with the GR and if successful, we will extend these studies to a similar analysis with the RAR. GR interacting clones from the yeast two hybrid screen will be rescreened with NF-kappaB. Full-length clones for these co-interacting proteins will be isolated, sequenced and antibodies will be prepared. Alternatively, proteins being characterized as associated with NF-kappaB complexes and the activation of these complexes will be directly screened utilizing co- immunoprecipitation, cross-linking, and mobility shift assays for their association with the GR. In summary, the work in this proposal focuses on an alternative route for steroid hormone action mediated by the hormone dependent association of the receptors with factors or co-factors that serve as critical mediators for alternative signalling pathways. The work in this proposal will provide a molecular basis for this hormonal signalling pathway.

technology /technique development; animal tissue; lasers; neoplasm /cancer; human tissue; genetics; biotechnology; biomedical resource; biological products;

The nuclear body, POD, has been identified as the oncogenic target in acute promyelocytic leukemia. It is known that the chromosomal translocation between the PML and RARa gene occurs in this disease and the cellular distribution of PML is changed. In both the patient and the cellular model, treatment with retinoic acid results in reformation of the POD and remission in the patient. In order to obtain a better understanding of the POD and its unknown function, we wish to follow the dynamic recreation of this nuclear body in situ. To perform this study, a fusion between the PML and GFP proteins will be used to track the PML in the living cell. Secondly, optical trapping will be used to both assist in the microinjection of this marker into a suspension model cell line and also to trap the cell during visualization of this dynamic process.

The Salk Institute DNA Sequencing Facility was initiated in January 1997. The hart of the facility consists of two ABI 377 XL state-of-the-art automated sequencers and accompanying Macintosh 7200 computers. The facility also contains two Macintosh computers, centrifuges for preparation of samples, and two PCR machines. The facility is an essential, productive shared resource that is used extensively by Cancer Center members.

DESCRIPTION (provided by the applicant): This proposal will focus on biochemical, molecular and cellular aspects of atherogenesis and inflammation, especially as it relates to signaling by the nuclear hormone receptors, PPARg, PPARd and LXR. A major hypothesis of this proposal is that PPAR/LXR target genes in the monocyte/macrophage are transcriptionally by ligands present in the oxLDL particle as well as by drugs such as the TZDs. We have previously identified the scavenger receptor CD36, LXRa and the reverse cholesterol transporter ABCA 1 as either direct or indirect PPAR target genes. Through these genes PPARg has the potential to both promote (CD36) or inhibit (LXR/ABCA1 atherogenic function. The goal of Specific Aim I is to investigate whether the anti-atherogenic effects of TZDs are dependent upon PPAR expression in macrophages. Currently, it is simply unknown whether TZDs principle action is in peripheral tissue or in the lesion. This will be approached by using PPARg null bone marrow in transplantation studies in LDLR-/- male mice. Coupled to this goal will be the use of cDNA microarrays to identify the critical set of target genes that respond to PPARg, both in vitro as well as in the lesion. In Specific Aim 2, we have a unique opportunity to characterize the role of PPARd in macrophage differentiation and inflammation. This is possible because we have recently generated a line of PPARd null mice and in addition, have been able to derive PPARd null ES cells. We will analyze the role of this receptor in macrophage differentiation and particular, we will explore the possibility that the anti-inflammatory effects of TZDs may be exerted through their ability to activate this receptor. In Specific Aim 3, we will expand on these observations by characterizing in detail the role of PPARd in lipid metabolism, macrophage gene expression and atherogenesis. By transplanting PPARd null bone marrow into the LDLR-/- mice, we will determine the specific contribution of PPARd expression in macrophage to the atherosclerotic process. Again, as PPARd is a ligand activated transcription factor this provides an ideal situation to characterize the set of genes that serve as targets for this receptor and its ligands. Together these studies will provide valuable new insight into the biochemical and molecular mechanisms that underlie broad aspects of macrophage function and in particular, the role of oxLDL, inflammation, and anti-diabetic drugs in both the promotion, maintenance, and potential treatment of the atherogenic lesion.

hormone regulation /control mechanism; biomedical resource; biological signal transduction; methylation;

DESCRIPTION (provided by applicant): This UCSD-NHLBI Translational Cardiovascular Science and Medicine Training Program is designed to develop highly qualified Ph.D./Postdoctoral Fellows, and Physician/Scientists that have been training at the forefront of cardiovascular science and medicine. The Program incorporates several of the top molecular scientists in the La Jolla scientific community at our three neighboring Institutions, including The Salk Institute, Scripps Research Foundation, and the Burnham Institute. In every case, these scientists hold cross-appointments with UCSD, have a history of active participating in the IMM program and are currently collaborating with investigators at UCSD toward scientific and technological advances that will have a direct impact on our understanding of cardiovascular diseases. Thus, the Program will promote the cross-fertilization between Institutions and fields, while maintaining a strict focus on cardiovascular biology and medicine. In this manner, the Program will eventually allow future opportunities to translate new developments in molecular cardiovascular sciences into therapeutic and prognostic capabilities in clinical cardiology. Accordingly, the objectives are: 1) To identify, recruit, and training highly qualified, accomplished, and committed Ph.D. postdoctoral fellows, and M.D and combined degree M.D./Ph.D. fellows with a sincere interest in translational cardiovascular science and medicine; 2) To harness the strengths of the most advanced technologies to understand the mechanistic basis of key aspects of cardiovascular biology and disease; 3) To translate these fundamental findings into new therapeutic, diagnostic, and prognostic advances in the mainstream of clinical cardiology, by creating direct collaborations amongst clinical cardiovascular scientists, basic cardiovascular scientists, and existing scientific strengths in genetics, molecular/cellular biology, and bioengineering on the UCSD campus and within the La Jolla scientific community.

DESCRIPTION (provided by applicant): Xenobiotic nuclear receptors, PXR and CAR, are activated by numerous chemicals and induce the Phase I, II and III clearance pathways through their association with specific transcriptional coactivators. Although some environmental contaminants including those found in the Superfund sites are known to activate PXR, CAR or both in a species-specific manner, the effects of the receptor activation on the toxicity are not clear. The studies proposed in this project are aimed at defining, in molecular terms, how the receptors and their xenobiotic and endobiotic ligands influence physiology by controlling gene expression patterns. The hypothesis is that certain toxic environmental agents activate either PXR and/or CAR, leading to both acute and chronic perturbations of the enterohepatic clearance system, production of supertoxic metabolites and endocrine disruption. To test these ideas, the investigators will combine genomic, molecular genetic, and biochemical approaches to 1) identify environmental toxins that activate PXR and/or CAR using a high-throughput cell-based reporter systems and an in vitro coactivator recruitment assay; 2) create a transgenic mouse line that replaces endogenous PXR with recombinant human PXR in both the liver and intestine to humanize the xenobiotic response over the entire enterohepatic system; 3) determine the contribution of PXR and CAR towards the long-term and acute toxicity of chemicals using knockout and transgenic humanized animals; and 4) identify PXR and CAR target genes that contribute to the toxicogenomic response using DNA oligonucleotide microarray analysis. Taken together, the proposed experiments will uncover details of the molecular mechanisms by which environmental substances found at Superfund sites exert adverse effects and will also provide unique and quantitative tools to predict toxicity.

The nuclear receptor family is made up of ligand-activated transcriptional regulators that have been implicated as key regulators of development and metabolic homostatsis of the human body. Many common diseases such as atherosclerosis, hepatic and pulmonary fibrosis, cancer and chronic inflammation have their origins in loss metabolic or developmental control. We will focus on exploring the mRNA expression profile of nuclear hormone receptors in human diseases including but not limited to nonalcoholic steatohepatitis (NASH), atherosclerosis and diabetes, skin diseases (eg psoriasis), brain tumors and myeloid leukemia. In addition, selective mouse models will be used to examine the expression pattern of mRNA NR family during early and late phases of progression of metabolic and inflammation diseases such as atherosclerosis. Profiling will be achieved through the quantitative PCR NR NURSA platform which provides a sensitive and highly quantitative method for analyzing NR mRNA levels. The data collected from these studies will be contained within a shared Bioinformatics Resource for data mining by the wider scientific community. In addition one of the major goals of this project is to develop of new analytical tools for determining and altering the expression of all members of the nuclear receptor family in specific cells and tissues. In addition, we will design, develop and validate a comprehensive lentiviral shRNA knockdown library that targets the entire NR family. Finally, we will use the library to interrogate cell lines and animal models of metabolic, inflammatory and cancer states in which a receptor has been shown to have a dynamic and/or dominant expression profile. The NR lentiviral shRNA knockdown library will be made available other NURSA members with the eventual goal of supplying the research community with a powerful new tools for analyzing NR function.

DESCRIPTION (provided by applicant): Xenobiotic nuclear receptors, PXR and CAR, are activated by numerous chemicals and induce the Phase I, II and III clearance pathways through their association with specific transcriptional coactivators. Although some environmental contaminants including those found in the Superfund sites are known to activate PXR, CAR or both in a species-specific manner, the effects of the receptor activation on the toxicity are not clear. The studies proposed in this project are aimed at defining, in molecular terms, how the receptors and their xenobiotic and endobiotic ligands influence physiology by controlling gene expression patterns. The hypothesis is that certain toxic environmental agents activate either PXR and/or CAR, leading to both acute and chronic perturbations of the enterohepatic clearance system, production of supertoxic metabolites and endocrine disruption. To test these ideas, the investigators will combine genomic, molecular genetic, and biochemical approaches to 1) identify environmental toxins that activate PXR and/or CAR using a high-throughput cell-based reporter systems and an in vitro coactivator recruitment assay; 2) create a transgenic mouse line that replaces endogenous PXR with recombinant human PXR in both the liver and intestine to humanize the xenobiotic response over the entire enterohepatic system; 3) determine the contribution of PXR and CAR towards the long-term and acute toxicity of chemicals using knockout and transgenic humanized animals; and 4) identify PXR and CAR target genes that contribute to the toxicogenomic response using DNA oligonucleotide microarray analysis. Taken together, the proposed experiments will uncover details of the molecular mechanisms by which environmental substances found at Superfund sites exert adverse effects and will also provide unique and quantitative tools to predict toxicity.

The rapid accumulation of large data sets from genomic sequencing, coupled to DNA expression and RNA profiling, provides a unique opportunity to map the integrated function of genes that serve as targets for nuclear hormone receptors (NHRs). The availability of oligonucleotide microarrays like the Affymetric chip to simultaneously interrogate the expression of thousands of genes at a time provides a powerful tool for examining the roles of specific receptors in regulating complex programs of gene expression. Alternatively a specific set of targets such as the NHRs and associated cofactors and key target genes can also be measured in a high-throughput format by quantitative PCR (QPCR) techniques in combination with robotics. The Gene Profiling Resource (GPR) will process RNA samples provided by the RAG (Receptor Atlas Group) members and interrogate with the use of customized Affymetrix microarrays. In addition through an established robotic HT-QPCR platform RNA samples provided by the RAG members will be profiled quantitatively for the expression pattern of specific genes or gene families like the NHR family. Data collected by GPR will be distributed to the submitting RAG laboratory, as well as deposited in the public NURSA bioinformatics relational database for analysis, comparison, data mining for the scientific community.

The Analytical Core will provide a variety of specialized analytical services for the investigators of the Program Project to assist in the conduct of the molecular, cellular and in vivo studies. The Analytical Core will be a collaborative effort between units at the Salk Institute under the direction of Dr. Ronald Evans, and one located at the University of California, San Diego under Dr. Joseph Witztum. The overall goal of the Analytical Core is to take advantage of specialized resources and expertise to provide investigators with selected core services that will assist in the research mission of each Project. The Aims of this Core are: Specific Aim 1: To provide high throughput quantitative PCR analysis (HT-QPCR) of selected target genes, such as nuclear receptors or target genes that impact inflammation and atherosclerosis. This platform will allow rapid exploratory mechanism of impact of various perturbations on nuclear hormone receptor expression in macrophages and B-1 cells, as well as in other cells or tissue. Specific Aim 2: To provide high throughput and highly sensitive analysis of cytokines and chemokines in small sample volumes, such as plasma or culture fluids, through use of Bioplex suspension array proteomic profiling. Specific Aim 3: To provide immunological services including the generation of antigen-specific antisera and monoclonal antibodies, preparation of reagents and performance of a variety of immunological assays, as for example, antibody levels in plasma of murine models under study. Specific Aim 4: To provide quantitative and qualitative analysis of lipids and lipoproteins, to prepare isolated lipoprotein fractions needed, such as LDL, as well as provision of standardized modified lipoproteins, such as oxidized LDL and mmLDL for use by all investigators.

Atherosclerosis is now widely recognized as a chronic inflammatory disease involving a complex interplay between resident vascular endothelial and smooth muscle cells and infiltrating immune cells, particularly macrophages. An underlying hypothesis of this work is that inflammation can be modulated at the transcriptional level by nuclear receptors and their co-repressors. Previous studies in our laboratory identified PPARd as an "inflammatory switch" within the macrophage, in which ligands act to control the concentrations of free and nuclear receptor-bound fractions of the co-repressors BCL-6 and SMRT. Biochemical, molecular, genetic, and physiologic approaches will be used to uncover the roles for these factors in inflammation and atherosclerosis. Finally, we will examine functions for PPARs throughout the artery wall and atherosclerotic lesion by utilizing conditional knockout models of PPARg and PPARd in the vascular endothelium. Studies will include both molecular and cell biology experiments and in vivo experiments, taking advantage of unique nuclear receptor knockout and conditional knockout-derived cells and mouse models using newly developed orally active PPARd-specific drugs along with the available class of PPARg therapeutics

Cancer Center Support Grant; programs

DESCRIPTION (provided by applicant): Over the past decade the field of NR signaling has generated a large volume of global datasets that collectively describe sequences of NR and coregulator genes (genomics); the regulation by NRs and coregulators of gene networks in specific target tissues (transcriptomics); protein-protein interactions required for the efficient function of NRs and coregulators (proteomics); specific sites of action of NRs in target gene promoters (cistromics); covalent modification of chromatin (epigenomics); and, more recently, specific functional endpoints in the form of regulation of cellular metabolic pathways (metabolomics). In order to effectively leverage these resources, we propose the continued development of the Nuclear Receptor Signaling Atlas (NURSA) web resource that will assemble and integrate these datasets, build user-friendly data analysis tools and present these to the community for hypothesis generation and validation. To fully engage the research community, we propose to administer a program of NURSA Data Source Projects (NDSPs) that will generate new global scale datasets that will be submitted to the website and integrated with existing datasets. We will also engage in outreach efforts that will offer members of the research community to participate in NURSA as Affiliate members, as well as in testing software tools during their development. With the involvement of the community, we anticipate that NURSA will be an important research resource for this field.

PROJECT SUMMARY (See instructions): Superfund site xenobiotics and other environmental toxicants are human health hazards whose toxicity is, in part, associated with altered patterns of gene expression. The goal of this project is to provide molecular mechanisms and models for exposure, focusing on the classic xenobiotic receptors (XenRs) PXR and CAR, and their induction of gene networks encoding the Phase I, II and III clearance pathways. Accordingly, to define the chemical space of XenRs in response to environmental toxins, in Aim II we will initiate a comparative chemical library screen using high throughput (HT) cell based luciferase reporter assays. Recently, we have determined that the nuclear receptor ERalpha is capable of responding to anticoagulants, antibacterial and anti-inflammatory drugs thus identifying it as a candidate xenobiotic sensor. Therefore as part of this Aim we will include ERalpha in the above XenRs screen. Some of our HT screens will include extracts gathered from Superfund sites by the Research Translation Core. Comparative gene expression studies will be conducted in Aim II to establish the overlap of ERalpha dependent gene regulation with known PXR and CAR target genes. The in vivo relevance will be established using a humanized hPXR/hCAR reporter mouse. In Aim III we will determine how XenRs control the xenobiotic response at the genome-wide level. Chromatin immunoprecipitation coupled with massively parallel deep sequencing (ChlPSeq) will be used to identify PXR, CAR and ERalpha cistromes, before and after treatment with high affinity agonists to reveal unique and common (core) xenobiotic networks. The aggregate binding sites will comprise a xenobiotic cistrome. Finally, in Aim I, we describe a new HT screening platform called NHR Transcriptional Promoter Ontology which allows us to explore xenobiotic regulation by all human NHRs (+/- ligands) by screening against a panel of ~300 drug metabolism reporter constructs comprised of P450 and conjugation enzyme and transporter sets. This is a unique opportunity to redefine the molecular basis of NHR-xenobiotic regulation and will provide a new roadmap for future study. We will collaborate with the Research Translation Core and Community Engagement Core to share this work with our SRP tribal science partners, industry, EPA, and ATSDR.

Atherosclerosis is a chronic inflammatory disease involving a complex interplay between resident vascular wall cells and infiltrating immune cells. Loss of T cell homeostasis contributes to both plaque development and inflammation. Regulatory T cells (Tregs) inhibit atherosclerotic development and progression through suppression of immune responses, including the actions of pro-inflammatory T helper 17 (Th17) cells. We identified the expression of the nuclear receptors PPARy and REVERBa/B in thoracic periaortic Tregs and Th17 cells, respectively. Our underlying hypothesis is that the development of the atherogenic lesion is driven by a pro-inflammatory gene network that can be modulated at the transcriptional level by PPARy and REVERBa/B. Our approach is to employ complementary loss-of-function and drug? mediated gain-of-function mouse studies, as well as genome-wide transcriptomic and cistromic studies, to define the molecular mechanisms of PPARy and REVERBs actions in atherogenesis. In Aim 1, we will employ a novel Treg-specific PPARy knockout mouse in combination with PPARy agonists, to understand the role of PPARy-dependent signaling in visceral adipose tissue-resident Tregs during the development of atherosclerosis. In Aim 2 we will determine PPARy's genome-wide chromatin binding sites, define the PPARy-dependent transcriptome, and map the translatome (Ribo-seq) of peri-aortic fat Tregs in order to determine the signaling pathways and molecular mechanisms of PPARy action in Tregs. In Aim 3 we will examine the role of REVERBa/B in attenuating pro-Inflammatory Th17 cells in the context of atherogenesis. These REVERBs naturally oppose RORy, the Th17 lineage specifier. Our approach is to utilize mice with selective deletion of both REVERBa and REVERBB in T cells, in combination with the biologically-available REVERB agonist, SR9009, to determine the consequences of REVERB signaling on the differentiation of Th17 cells and the resultant effects on the initiation and progression of atherosclerosis. Similarly, in Aim 4 we will identify direct and indirect target genes of REVERBa and REVERBB by comparing their genome? wide chromatin binding sites to the REVERB-dependent transcriptomes in Th17 cells, thereby shedding light on the mechanisms of REVERB-mediated repression. These highly integrated Aims employ biochemical, molecular, genetic, pharmacologic and physiologic approaches to clarify the competing roles of the immuno? suppressive Tregs and the pro-inflammatory Th17 cells in this disease. Characterization of the roles of these therapeutically-accessible nuclear receptors in these immune cell populations may lead to the development of novel small molecule drugs for the prevention and treatment of atherosclerosis.

The goal of this supplement request is to continue support for NURSA biocuration and analysis took development as they relate to NIDDK's mission. NURSA biocurators will carry out detailed, systematic curation of datasets using consistent structured vocabularies and constrained unique identifiers to provide for more accurate data retrieval. NURSA-curated datasets will be citable in full concordance with the FAIR principles. Datasets will be exposed for discovery through two major dataset search engines, NIH bioCADDIE DataMed and Thomson Reuters Web Of Science. Fourthly, journal staff in two major publishers in the field of mammalian cellular signal transduction and metabolic disease, Elsevier and Public Library of Science, will facilitate interactions between NURSA staff and authors of accepted articles to gain access to datasets. The web development team will integrate transcriptomics, ChIP-Seq, protein-protein interactions, and protein translational modifications in a single searchable resource. To ensure that we deliver to the community the curated content of most immediate use and impact, we will prioritize our curational efforts in descending order of dataset date of publication (newer > older), dataset throughput (discovery-scale vs focused hypothesis-drven) and signaling pathway level of interest (greater > lesser).

Over the past decade the field of NR signaling has generated a large volume of global datasets that collectively describe sequences of NR and coregulator genes (genomics); the regulation by NRs and coregulators of gene networks in specific target tissues (transcriptomics); protein-protein interactions required for the efficient function of NRs and coregtjiators (proteomics); specific sites of action of NRs in target gene promoters (cistromics); covalent modification of chromatin (epigenomics); and, more recently, specific functional endpoints in the form of regulation of cellular metabolic pathways (metabolomics). In order to effectively leverage these resources, we propose the continued development ofthe Nuclear Receptor Signaling Atlas (NURSA) web resource that will assemble and integrate these datasets, build user-friendly data analysis tools and present these to the community for hypothesis generation and validation. To fully engage the research community, we propose to administer a program of NURSA Data Source Projects (NDSPs) that will generate new global scale datasets that will be submitted to the website and integrated with existing datasets. We will also engage in outreach efforts that will offer members of the research community to participate in NURSA as Affiliate members, as well as in testing software tools during their development. With the involvement of the community, we anticipate that NURSA will be an important research resource for this field. RELEVANCE (See instructions): Nuclear receptors (NRs) and coregulators are important therapeutic targets in many different disease states including cancer, obesity, diabetes, inflammation, neurological disorders and senescent diseases. This application proposes a web resource for information and data analysis tools for the NR research community, as well as a community research grant program that will generate global-scale 'omics datasets for distribution via the web resource. These initiatives will have tangible benefits for the progress of research in the field towards developing novel NR- and coregulator-based therapeutics.

Project Summary/Abstract: Superfund site toxicants pose a significant hazard to human health, in part through their ability to alter patterns of gene expression. One of the most prevalent diseases, toxicant-associated steatohepatitis (TASH), is phenotypically similar to nonalcoholic steatohepatitis (NASH) without the underlying obesity. Building on this similarity, this project will provide mechanistic insight into TASH-induced liver damage and models of exposure. With a focus on the epigenetic and immune responses, the overall goal of the project is to identify potential intervention or prevention therapies and/or therapeutic targets. In Aim 1 we will explore the role of regulatory enhancers, key genomic mediators of tissue fibrosis, in mouse models of TASH by mapping injury-induced epigenetic changes in stellate cells, hepatocytes, and immune cells. In parallel studies, the therapeutic efficacy of epigenetically-targeted small molecule modulators will be determined in both intervention and prevention models of TASH. In Aim 2 we will profile the immune response to toxicant-induced liver damage in mice during the initial injury, damage/repair, and resolution stages. Based on these findings, potential immune-targeted therapies for the treatment TAFLD and TASH will be explored. In Aim 3, the epigenetic and immunological consequences of chronic toxicant exposure will be explored in a novel genetic mouse model of TAFLD (SMRTRID mice). In summary, this project will provide valuable insight on the molecular mechanisms underlying TAFLD and TASH as well as a roadmap for potential new therapies.

ABSTRACT Pancreatic islet transplantation offers long-term treatment for Type 1 diabetes, however the shortage of donors and the need for immunosuppressive drugs restrict its therapeutic utility. Islet-like organoids generated from human pluripotent stem cells (PSCs) are an attractive alternative. Moreover, given the increasing appreciation for the role of multi-cellular organization in the functional maturation and maintenance of organs, islet-like organoids have the potential for superior functionality compared to ? cells alone. The goal of this project is to develop the next generation of human islet-like organoids (HILOs) from stem cells for efficient and immune evasive transplantation. The underlying hypothesis is that a combination of a novel 3D differentiation protocol, modulation of cell surface signaling, and anti-inflammation transcriptional machinery will enable HILOs to survive long-term and function in an immune competent environment in vivo. To achieve this goal, Aim 1 proposes to establish the long-term efficacy and safety profile of HILOs, of which the function has been validated extensively in vitro. Aim 2 proposes to develop immune-tolerant HILOs by engineering the expression of PD-L1 and demonstrating the efficacy in humanized immune-competent diabetic mice. To further extend protection of transplanted HILOs, Aim 3 proposes to apply pharmacological activation of vitamin D signaling to alleviate cytokine stress on transplanted HILOs. The goal is to determine whether the incorporation of these strategies to improve survival as well as minimize allo-rejection of transplanted HILOs will result in an unlimited supply of therapeutically viable engineered islets for treating diabetes.

Functional Genomics and Genome Sequencing Core Shared Resource - Project Summary/Abstract The mission of the Functional Genomics and Genome Sequencing Core is to facilitate cutting-edge genomics research at the Salk Institute by providing: 1) cost-effective and rapid high-throughput sequencing services, 2) expert assistance in experimental design, and 3) the development of novel methods to enable cutting edge sequencing technologies. In addition, the Core prepares sequencing libraries and performs quality control to ensure that high-quality data are extracted from each experimental sample. In the near future, the Core will acquire and implement a single cell sequencing platform. In support of Cancer Center research endeavors at the Salk, the Core specifically aims to provide: 1) access to state-of-the-art instrumentation for next-generation sequencing projects, 2) assistance with experimental design when Salk Cancer Center members are seeking to perform experiments that involve sequencing technologies, 3) novel methods and strategies for implementing cutting-edge sequencing technologies, 4) preparation and quality control of sequencing libraries, and 5) training services for Cancer Center members in library preparation or other methods required for next- generation sequencing projects.

PROJECT SUMMARY Project 2. Tissue-specific roles of FXR in CVD and NASH Hyperlipidemia and insulin resistance are commonly associated with both cardiovascular and liver diseases, however causal relationships between atherosclerosis and NASH are not well established. The farnesoid X receptor (FXR) is a regulator of systemic sterol and glucose homeostasis, and is known to contribute to the initiation and progression of both cardiovascular and liver diseases. Loss of hepatic FXR, but not intestinal FXR, results in elevated circulating and hepatic cholesterol and triglyceride levels. In contrast, both hepatic and intestinal FXR enhance hepatic repair and cholesterol excretory activities. Consistent with this, FXR agonists reliably reduce atherosclerosis and have been reported to show promising effects in liver disease models. Thus, the goal of Project 2 is to dissect the tissue-specific activities of FXR in the context of integrative hepatovascular pathophysiology. Specifically, we will explore the notion that FXR drives distinct protective programs in cardiovascular and liver diseases. The macrophage is central in the development of atherosclerosis and steatotic hepatitis through the deposition of vascular fatty lesions, as well as driving or resolving liver damage. As a key regulator of inflammation and multiple steps in the reverse cholesterol transport and excretion pathways, FXR is an established target for mitigating the development of foam cells that underlie vascular plaque deposition. Our preliminary findings indicate that plaque deposition and facets of liver disease are divisible with tissue-specific modulation of FXR activities. This project will explore the hypothesis that tissue-specific modulation of FXR will affect the progression of CVD and NASH. To achieve this goal, proprietary FXR agonists that target either the liver (hepFexD) or gut (intFexD) will be used in combination with ldlr-/- mice lacking FXR expression in either the liver (hepFXRko) or the intestine (intFXRko) to dissect the association of fatty liver disease and CVD risk. In Aim 1, the impact of hepatic FXR on atherosclerosis and liver disease will be explored, including the contribution from FXR- regulated crosstalk between parenchymal and non-parenchymal cells in collaboration with Project 1, and the role for hepatic FXR in OSE levels or clearance in collaboration with Project 3. In Aim 2, the ability of systemic signaling from intestinal FXR to affect macrophage cholesterol homeostasis will be determined in collaboration with Project 1. Finally, the relevance of our pre-clinical findings studies to human disease will be determine by interrogating curated clinical samples for biomarkers of FXR activity in collaboration with Project 4. The proposed comprehensive genetic, pharmacologic and comparative biological approach will provide a better understanding of the physiology and mechanisms by which tissue-specific FXR crosstalk impacts atherosclerosis and steatohepatitis.