Klaus H. Kaestner - US grants

DESCRIPTION (taken from the application) Diabetes mellitus is a significant health problem, affecting approximately 16 million people in the United States. Loss of sufficient insulin production by the pancreatic beta cell is a hallmark of both type I and type II diabetes. The homeobox transcription factor Pdx1 (pancreatic and duodenal homeobox gene 1; also known as IPF-1, IDX-1, STF-1) is a master regulator of pancreatic development, as its homozygous deletion leads to pancreatic agenesis in mice and men. In addition, heterozygous mutation of Pdx1/IPF-1 is linked to early onset non-insulin dependent diabetes mellitus (MODY4). Recently, the winged helix transcription factor HNF (hepatocyte nuclear factor) 3Beta was shown to be an important regulator of Pdx1 transcription in the developing pancreas. Consequently, it is likely that HNF3Beta plays an important role in the control of pancreatic development and that HNF3Beta mutations can contribute to the pathogenesis of diabetes. However, gene targeting of HNF3Beta has thus far been not informative in this regard, as embryos homozygous for a null mutation die before the onset of pancreatic development. Therefore, we propose to assess the function of HNF3Beta in pancreatic development and physiology through conditional gene targeting using the loxP/Cre recombinase system. In preliminary work required for this proposal we have already generated and tested mice homozygous for a loxP-flanked HNF3Beta gene, the HNF3BetaloxP/loxP mice, and obtained Beta-cell specific Cre-recombinase transgenic mice. The specific aims of this proposal are: First, we will investigate whether HNF3Beta is required for pancreatic development through crosses of our HNF3Bet loxP/loxP mice with pancreas-specific Cre-recombinase transgenics. If our hypothesis is correct, deletion of HNF3Beta in the pancreas will lead to abnormal pancreatic development depending on the timing of deletion. Pancreatic development will be assessed by histological and immunohistochemical criteria. Second, through the use of a Beta-cell specific and steroid-inducible Cre-recombinase, we will address the question whether HNF3Beta also controls the maintenance of the specific cellular phenotype of the pancreatic Beta-cell If this is the case, induced deletion of HNF3Beta will be reflected in a perturbation of cell lineage allocation in the pancreatic islet and/or the regulation of glucose homeostasis. Together these studies will further our understanding of the transcriptional regulation of pancreatic development and the pathogenesis of diabetes. Insights gained about the role of HNF3Beta in Beta-cell function could be incorporated into future therapeutic strategies, including gene therapy.

The intestinal epithelium is one of the most rapidly renewing tissues in the body, and thus the ideal tissue to study somatic stem and progenitor cell biology. The small intestinal epithelium is composed of a single layer of cells that contains four major differentiated cell types as well as intestinal stem cells (ISCs) and progenitor or transit amplifying cells that replenish differentiated cells throughout life. While the past twenty years have seen great progress in our understanding of the signaling pathways and transcriptional regulators that control intestinal proliferation and differentiation, our understanding of the epigenetic factors that control these important processes is rather limited. Equally important is the identification of the intestinal stem cell niche, and the characterization of its function in molecular detail. To address this knowledge gap, I propose the following Specific Aims: In specific Aim 1, we will determine if Foxl1+ subepithelial telocytes are required for providing critical Wnt signals during gastrointestinal development and in R-spondin free enteroid culture. We will employ our newly developed genetic and molecular tools to determine the signaling pathways controlled by telocytes during intestinal development using mouse models. In Aim 2, we will investigate the contribution of polycomb complex mediated gene repression via histone H3K27 trimethylation on intestinal stem cell biology and regeneration. To this end, we will employ tissue and cell type specific gene ablation of two genes encoding critical H3K27me3 demethylases in the intestinal epithelium, both under homeostatic conditions and after ablation of Lgr5 stem cells. RELEVANCE (See instructions): Gastrointestinal cancer is a significant health problem, ranking fourth in incidence and second in death among cancers in the United States. Abnormal differentiation and increased proliferation of the intestinal epithelium are hallmarks of carcinogenesis. The molecular mechanisms that regulate cellular proliferation and differentiation in gastrointestinal development are far from being understood completely. Therefore, we will analyze the impact of the intestinal stem cell niche cells on intestinal growth and function, and test the contribution of histone demethylation to intestinal health.

Diabetes mellitus is a significant health problem, affecting approximately 16 million people in the United States. Loss of sufficient insulin production by the pancreatic beta cell is the hallmark of type I diabetes, while in type II diabetes there is in addition peripheral insulin resistance. Future therapeutic approaches of regeneration of beta cells both in vivo and in vitro will benefit greatly from a complete understanding of the development and differentiation of the pancreatic islets. We propose to establish a functional genomics resource that will provide detailed information about the complex patterns of gene expression that govern this process. Specific aim 1 of this proposal is to develop normalized and subtracted cDNA libraries enriched for rare transcripts expressed in the developing endocrine pancreas and to determine full length sequences of previously unknown transcripts. Aim 2 is to generate cDNA microarrays representing thousands of pancreas- specific or pancreas-enriched transcripts. This microarray facility will be established within the context of the University of Pennsylvania Diabetes Center. Clones for these microarrays will be obtained from three sources: a) existing, commercially available microarrays screened with pancreas specific probes, b) the normalized newborn islet cDNA library obtained in Aim 1, and c) subtracted cDNA libraries representing rare pancreatic transcripts. The latter will take advantage of RNA amplification from single cells; which was pioneered at Penn, to generate cDNA libraries specific for the individual endocrine cell types. Thus we will be able to generate a complex microarray of thousands of cDNAs which represent both abundant and rare pancreatic mRNAs. Specific aim 3 is to determine the expression profile of thousands of genes during pancreatic development and in mice carrying mutations that affect pancreatic development and function. We will utilize the microarrays generated in aim 2 to determine expression profiles during the development of the endocrine pancreas, both in the entire pancreas as well as in individual endocrine cell lineages. We will utilize the computer resources of the University of Pennsylvania Bioinformatics Center for the efficient analysis of the large body of data generated. This bioinformatics database, as well as the microarrays, will be made available to the general research community. Establishing these state-of-the-art technologies, and applying them to the endocrine pancreas, will enable principal investigators to formulate and test novel hypotheses that address the causes and progression of diabetes.

DESCRIPTION (provided by applicant): Diabetes mellitus is a significant health problem, affecting approximately 16 million people in the United States. Future therapeutic approaches to diabetes will benefit greatly from a complete understanding of the expression profile of the beta cell under normal and pathological conditions and the functional annotation of differentially expressed genes. The goal of this application is to pool the complementary expertise available in three laboratories for mining of an exciting new resource-the more than 7,700 unique cDNAs cloned by the prior NIDDK-funded consortium on "Functional Genomics of the Developing Endocrine Pancreas". Our goals are three-fold: Aim 1 of this proposal will establish a large cDNA microarray enriched for genes expressed in the endocrine pancreas by combining the 7,700 non-redundant cDNAs described above with the 3,400 clones of our current PancChip 2.0. We will employ this microarray for the screen of six paradigms of perturbed P-cell function to identify candidate genes to be analyzed further in aims 2 and 3. Aim 2 will transfer 1,000 selected cDNA clones from our collection into the FLEXGene repository. This repository will allow for high-throughput transfer of cDNAs into multiple expression vectors. In addition, we will select antigens for the production of antisera to derive marker antibodies of beta cells and their precursors. In Aim 3 we will functionally evaluate 500 selected cDNAs for their potential role in beta-cell biology. Clones transferred into the FLEXGene repository and sequence verified (Aim 2) will be subcloned into adenovirus vectors to allow for efficient transduction of INS-1 cells. In some cases, the sequence of differentially expressed genes will be used for design of interference RNA (RNAi) oligonucleotides to allow suppression of target gene expression. The effect of modulation of target gene expression will then be tested in various models of R-cell function. Candidate cDNAs that are positive in this screen will be further evaluated in adenovirus-transduced islets and/or transgenic animals. Genes identified in this fashion may become candidate drug targets or could be useful in development of surrogate p-cells for cell-based insulin replacement therapy. This project will serve as a valuable gene discovery effort that will complement the program implemented by the NIDDK-funded beta-cell biology consortium. The new resources generated through this project will be made available to the NIDDK-funded biotechnology centers and the diabetes research community at large.

The liver constitutes one of the few, normally-quiescent tissues in the adult body that has the capacity to regenerate. As a result, it provides a unique, multi-cellular, physiologically normal system in which to study the mitogenic response of epithelial cells. Previously, we identified more than 40 novel immediate-early and delayed-early genes activated during liver regeneration. Immediate-early or primary response genes many of which encode proto-oncogenes have been shown to have important effects on cell growth and differentiation. Of the genes we identified, PRL-1 is particularly interesting, because it is the only gene that is induced at a high level in regenerating liver, but is constitutively expressed in insulin-treated rat H35 hepatoma cells which otherwise show normal regulation of immediate-early genes. It is a liver-enriched immediate- early gene because its induction is much higher in the regenerating liver than in other mitogen activated cells. We reasoned that PRL-1 could have important effects on growth of hepatic cells. Initial studies revealed that PRL-1 encodes a unique protein tyrosine phosphatase (PTPase) with no homology to other PTPases outside the active site. In cells, PRL-1 migrates as a 21 kD protein, and is located primarily in the triton- insoluble fraction of the cell nucleus. Stably transfected cells which overexpress PRL-1 demonstrate altered cellular growth and morphology, and a transformed phenotype. Other PTPases have been shown to have important effects on cellular growth control. PTPases such as cdc25 and MKP-1 have very specific intracellular substrates, cdc-2, Map kinase, respectively, thus establishing their importance in cell growth control. Thus far, Dr. Taub's laboratory has not identified specific intracellular targets for the PRL-1 phosphatase. However, like other PTPases, PRL-1 may be important in normal cellular growth in liver regeneration and other cells in which it is expressed, and could contribute to the tumorigenicity of hepatomas and other cancer cells. We have two major goals in this proposal. We will demonstrate the importance of PRL-1 in hepatic and cell growth by more carefully defining its expression patterns in animals, and modulating its expression in the 3T3 and H35 (liver) cell lines and animals. We will learn more about the specifics of PRL-1 function by characterizing interacting proteins, and identifying specific intracellular substrates. Ultimately these two goals will merge and allow us to define precisely how PRL-1 functions to regulate hepatic growth during development and regeneration, and contributes to oncogenesis in liver tumors. We will accomplish these goals by (1) more precisely defining the expression of PRL-1 in liver regeneration, fetal and adult tissues; (2) testing the specific effects of PRL-1 on cell growth and oncogenesis by carefully regulating PRL-1 expression in transfected 3T3 and H35 cells and liver cells of transgenic animals; (3) identifying the cellular substrates of PRL-1 by characterizing PRL-1 associated proteins, expression cloning and testing candidate substrates; and (4) characterizing potential members of the PRL-1 gene family through homology cloning.

DESCRIPTION (provided by applicant): Diabetes mellitus is a lifelong incapacitating disease with worldwide prevalence estimated at 150 million patients in 2000. Loss of sufficient insulin production by the pancreatic beta-cell is a hallmark of both type 1 and type 2 diabetes. The development of allogenic islet transplantation using steroid-sparing immunosuppression has raised new hope for better treatment of severe diabetes. However, the experience at multiple clinical centers over the past 4 years has shown that transplant outcome is highly variable, with insulin-independence ranging from 30% to 80% 1 year after transplant. We propose to systematically evaluate the quality of human pancreatic islets as a potentially crucial factor in transplant outcome utilizing both biochemical as well as functional genomics approaches, with the ultimate goal of developing a rapid, economic and reproducible assay of islet quality that correlates with the outcome of the transplant. We will pursue this goal in two specific aims: First, we will determine the expression profile of human islet preparations procured at the University of Pennsylvania using the human PancChip cDNA array developed in the Kaestner lab. In addition, we will measure glucose stimulated insulin secretion using islet perifusion, and evaluate islet function in vivo using minimal islet mass transplantation into diabetic NOD-Scid mice. We will then correlate the expression profile data with the in vitro and in vivo islet function tests, to determine which set of genes is most predictive of highly functional islets. Furthermore, we will investigate whether any of the 3 parameters determined in the lab correlate with clinical outcome as determined by following the decrease of insulin requirement in the transplant recipients. Second, we will apply discriminant analysis to the microarray data to identify a set of up to 96 genes that are most predictive of islet function. We will use this information to develop a TaqMan low density array that will allow for the rapid determination of the corresponding mRNA levels using real time PCR. We will test this "Array-by-Design" as a predictive tool of islet quality and function using the metrics described in Aim1. If successful, this rapid screen could be incorporated as a quality control step for human islets before transplant, and in addition as a cost-effective tool to evaluate beta-cells derived from other sources such as adult stem cells or hepatocytes.

[unreadable] DESCRIPTION: (provided by applicant) The C57BL/6 (B6) inbred strain of mice is the preferred strain of mouse geneticists, however, high throughput gene targeting/trapping has been possible to date only with 129 strain embryonic stem (ES) cells. 129 ES lines maintain self-renewal under a wide range of conditions even in absence of mouse embryonic fibroblast (MEF) feeders if supplied with Bone Morphogenetic Protein (BMP) and Leukemia Inhibitory Factor (LIF). In contrast, self-renewal of B6 ES cells is dependent on the presence of MEFs. In preliminary data, we have shown that 129-conditioned medium dramatically improves the growth characteristics of B6 ES cells. We will exploit this discovery to make B6 cells less dependent on feeder cells and, if possible completely feeder-cell independent. Importantly, our approaches make no prior assumption of the biological nature of the difference in growth characteristics between 129 and B6 cell lines. We will pursue our goals through five specific aims: In Aim 1, we will evaluate 18 male B6 ES-cell lines and 6 male albino B6 lines for cytogenetic stability, doubling time, and germ line transmission. In Aim 2, we will optimize the growth conditions for feeder-less growth of B6 ES-cell lines using both published additives as well as novel cytokines and/or growth factors. These new additives will be identified through expression profiling of 129 vs. the B6 ES lines, because conditioned medium from 129, but not B6 ES cells promotes growth and feeder-independence of B6 ES-cells. In Aim 3 we will utilize the optimized conditions of feeder-less growth of B6 ES-cells for a final assessment of gene targeting capability by targeting five genes in each cell line. In Aim 4, we will develop a novel cost-effective high throughput test that rapidly predicts germ line transmission of ES lines and with a high degree of accuracy. We will exploit expression profiling to identify a subset of 96 genes whose expression level is most indicative of self-renewal and germ line transmission. These genes will then be used to develop a qPCR-based screen to predict germ line transmission that is more rapid, accurate and less expensive than conventional cytogenetics. In Aim 5, we will use BAG recombineering and our optimize growth conditions to derive null mutations in 50 mouse genes as selected by the KOMP steering committee. In summary, in reponse to RFA-DA-06-009 we will develop high throughput gene targeting with B6 ES-cells and derive a novel molecular assay to cost-effectively and rapidly monitor B6 ES cell lines and their targeted derivatives for germ-line competence. [unreadable] [unreadable] [unreadable] [unreadable]

Diabetes mellitus is a significant health problem, affecting over 18 million people in the United States alone. Mutations in several hepatic nuclear factors have been linked to early onset non-insulin dependent diabetes mellitus (MODY), underscoring the importance of the hepatic transcription factors for glucose homeostasis. In the previous grant cycle, we have employed tissue-specific gene ablation to demonstrate the essential function of Foxa2 (previously known as HNF3(3) in the integration of the transcriptional response of the hepatocyte to fasting. In addition, Foxa2 has been proposed as a major mediator of insulin signaling in hepatocytes. We propose the following three Aims: In Aim 1, which is the direct result of the interactions within the PO1 with Dr. Birnbaum, we will test the hypothesis that Foxa2 is the major mediator of insulin signaling via AKT2 using genetic means. We will derive mice which are deficient for both AKT2 and Foxa2 in hepatocytes to test if Foxa2 is indeed required to establish the AKT2 mutant phenotype. In Aim 2, we will collaborate with Dr. Ahima to investigate the combined role of Foxa1 AND Foxa2 in hepatic metabolism. This aim is based on our discovery that Foxal and Foxa2 act jointly to enable the hepatogenic program during fetal devlopment. We hypothesize that the two genes also cooperate in transcription in the adult hepatocyte. We will use simulatenous conditional gene ablatation for Foxa1 and Foxa2 combined with physiological and genomics approaches to test our hypothesis that the two genes open chromatin to enable binding of hormone-dependent transcription factors like CREB and GR. In Aim 3, we will address a recent controversy concerning the regulation of hepatic gluconeogenesis by cAMP. Currently, two conflicting models exist regarding the regulation of the transcription factor CREB in hepatocyte. The first proposes that PKA-dependent phosphorylation of CREB is required for recruitment of the co-activator CBP/p300 and subsequent activation of target genes, while the second invokes PKA-dependent translocation of the novel co-activator TORC2 from the cytoplasm to the nucleus as the central regulator of CREB-dependent transcription. We will address the relative importance of both pathways in hepatic glucose homeostasis by genetic means. We will develop mouse models carrying a Ser/Ala mutation in the PKA-phosphorylation site of CREB or a hepatocyte-specific ablation of TORC2, and analyze the consequences to glucose metabolism both in vivo and in isolated hepatocytes. Together, these studies will further our understanding of the transcriptional regulation of hepatic metabolism and in insulin action.

ABSTRACT NOT PROVIDED

DESCRIPTION (provided by applicant): Diabetes mellitus is a lifelong chronic disease with worldwide prevalence estimated at 180 million patients in 2007. Over the past decade, it has become clear that failure of the pancreatic ¿-cell is the final event causing the transition to overt diabetes, even though obesity and peripheral insulin resistance are the factors leading to pre-diabetes. ¿-cell failure here is defined as loss of both function, i.e. glucose stimulated insulin secretion, and inadequate ¿-cell mass, either by increased apoptosis or a failure to proliferate in response to metabolic demand. While several drugs are in use to reduce insulin resistance and increase insulin secretion, none exist that address ¿-cell failure. In fact, at present, no good targets are known that would allow such drug development. Because the ¿-cell plays such a central role in the pathogenesis of diabetes, and because epigenetic factors (i.e. long term changes in the transcriptional program reflected in chromatin status) are likely to be the predominant consequence of the life-style changes and environmental factors that lead up to diabetes, we will determine the epigenome of both healthy and type 2 diabetic human ¿-cells. This effort will lay the groundwork to defining the pathways and genes that can be targeted in the future for the development of new therapies that address ¿-cell failure. Specifically, we will determine both activating and repressing chromatin marks in ¿-cells isolated from 50 healthy and 50 type 2 diabetic organ donors using ChIP-Seq technology. Secondly, we will analyze the microRNA profile in the same ¿-cell preparations, as miRNAs are strong candidates to modify the transcriptome of the diabetic ¿-cell. Third, we will analyze a novel chromatin mark discovered at Penn that links the stress mediator AMPK to chromatin status. Fourth, we will perform a comprehensive computational biology analysis to identify the nodes and regulatory pathways that are affected in diabetic ¿-cells.

DESCRIPTION (provided by applicant): Abstract Funds are requested for an Illumina Genome Analyzer IIX and associated Cluster Station to support the ongoing NIH-funded PI-initiated research programs in the Functional Genomics Core of the Institute for Diabetes, Obesity, and Metabolism (IDOM) as well as the new Epigenomics Program at the University of Pennsylvania School of Medicine. These are very exciting programs, linked by the recognition that environmental factors, including diet and lifestyle, influence the development of diabetes and obesity phenotypes via epigenetic regulation of gene expression. The Functional Genomics Core has already established an ultra- high throughput sequencing capability, supported by institutional funds. With the success and productivity of the nine major investigators focused on epigenomics and gene expression, including the PI and new senior recruits to the University of Pennsylvania, the demand on the current instrumentation is so great that users must wait 4-6 weeks for their samples to be analyzed, even with the instrument running non-stop. The current and future studies of genome-wide transcription factor binding, epigenetic mapping, microRNA discovery, and re- sequencing of candidate human disease regions are critical for the rapid progress we are making, and the proposed acquisition of a second Illumina Genome Analyzer IIX will allow us keep up with the increased demand. The University of Pennsylvania School of Medicine has made a major financial commitment towards this effort, including the funding of equipment and service contract costs not covered by this proposal as well as the staff of the Functional Genomics Core, which is itself a key component of Penn's NIDDK-funded Diabetes and Endocrinology Research Center. In addition, the institution has allocated dedicated space to house the instrument and its associated hardware in the new Fisher Translational Research Building, where the Functional Genomics Core, IDOM, and Epigenomics Program will be co- located. The School of Medicine has also provided a large server room and the required ultra- high speed connections for data storage and access. Scientific oversight will be accomplished by a committee comprised of the PI of the proposal, the IDOM director, and two faculty representatives from the user group. Thus, the Illumina Genome Analyzer will be highly utilized by outstanding, well-funded investigators focused on epigenomic and genomic research with the potential to have a critical impact on major diseases, including diabetes and obesity.

DESCRIPTION (provided by applicant): Several different sources of cells for the generation of insulin producing cells beta-cells for the treatment of diabetes can be envisioned. These include pluripotent stem cells as well as adult endoderm derivatives such as liver cells. The current focus in producing beta-cells from pluripotent precursors is on the use of extrinsic factors, whereas the generation of beta-cell from hepatic cells involves genetic reprogramming using gene transfer strategies. This project brings together investigators working on these different approaches for the purpose of integrating relevant information and using it to guide us towards the most efficient way of producing transplantable beta-cell equivalents. Specifically, epigenetic analysis of differentiation intermediates from all projects will be used to inform rational decisions about the extrinsic/genetic manipulations required in each system to achieve the final goal of therapeutically useful cells. Because in vivo reprogramming of liver cells requires the use of adenoviral vectors, the role of this virus in reprogramming must be elucidated. Heterogeneity of the in vitro products is expected in each system and hence reagents to purify more epigenetically homogeneous populations are needed. We propose further development of surface reactive monoclonal antibodies for this purpose. Finally, functional validation of the differentiation products requires in vivo testing upon transplantation into animal models. PUBLIC HEALTH RELEVANCE: Transplantation of islets from cadaveric donors has shown that type 1 diabetes can be successfully treated by cell therapy. However, high quality cadaveric islet donors are rare and new sources of transplantable beta-cells must be found in order for this approach to realize its clinical potential. This proejct will produce patient-matched autologous beta-cells for the treatment of type 1 diabetes. Successful execution will impact large numbers of patients world-wide.

DESCRIPTION (provided by applicant): Diabetes mellitus is a lifelong chronic disease with worldwide prevalence estimated at 180 million patients in 2007. Over the past decade, it has become clear that failure of the pancreatic (-cell is the final event causing the transition to overt diabetes, even though obesity and peripheral insulin resistance are the factors leading to pre-diabetes. (-cell failure here is defined as loss of both function, i.e. glucose stimulated insulin secretion, and inadequate (-cell mass, either by increased apoptosis or a failure to proliferate in response to metabolic demand. While several drugs are in use to reduce insulin resistance and increase insulin secretion, none exist that address (-cell expansion and regeneration. In fact, at present, no good targets are known that would allow such drug development. There is a dramatic decline in the proliferative potential of the (-cell with age, both in humans and in rodents. The epigenetic events that underlie this change have been only partially explored. We hypothesize that a full understanding of the epigenetic changes that correlate with the regenerative capacity of the (-cell can be utilized to enable aged (-cells to proliferate. Specifically, we will determine both activating and repressing chromatin marks as well as expression profiles of (-cells isolated from young and aged mice by ultra-high throughput sequencing technology. We will also investigate the epigenetic changes that occur when a recently divided (-cell enters a refractory period. Secondly, we utilize the epigenetic profile of the young (-cell obtained in AIM 1 in a low-throughput screen of conditions aimed at reversing the aged phenotype of human (-cells. Third, we will evaluate the ubiquitin ligase adaptor PCIF1, shown to be an important regulator of the chromatin modifier BMI1, as a target for reversal of the epigenetic phenotype of the aged human (-cell. In summary, we will utilize genome-wide approaches and computational biology tools to transform our knowledge of how the epigenome reflects the aging of the (-cell and its loss of proliferative potential. These data will serve two purposes: to guide the development of interventions that aim to rejuvenate human islets, and to evaluate how their epigenome can be manipulated to favor (-cell replication. Therefore, our proposal has the potential to transform diabetes research and treatment in the future. PUBLIC HEALTH RELEVANCE: Health outcomes for both type I and type II diabetics could be improved if we could develop drugs to stimulate the proliferation of the beta-cell, the insulin producing cell in the pancreas. Likewise, more type I diabetics could be treated with islet transplantation if we could expand beta-cell mass in vitro before transplantation. While beta-cells in young children have very high proliferative capacity, this declines dramatically as we age. We hypothesize that epigenetic changes are responsible for this decline in proliferative capacity. Therefore, we will determine the epigenome of both young and aged beta-cells, define their differences, and use these as a tool to investigate treatments that can reverse the aged phenotype of the human beta-cell. If successful, this novel approach holds great promise for the development of innovative treatments for diabetes.

The objective of the Functional Genomics Core is to provide state of the art experiment planning, sample preparation, quality assessment, microarray, DNA sequencing, and data analysis services to DRC members.

Targeting hepatic CREB to reduce glucose output as a treatment option for diabetes is a highly controversial notion. Inhibition of CREB activity in the liver using a dominant-negative approach caused increased hepatic triglyceride content, suggesting that CREB is a poor target. In contrast, a recent report using antisense oligonucleotides (ASO) suggested that CREB inhibition prevented hepatic steatosis and insulin resistance However, given the systemic application of the ASO, and the repression of CREB in other metabolic tissues such as adipose tissue, this report did not directly address the function of CREB in the liver. During the past grant cycle, we have completed a systematic, genome-wide evaluation of CREB binding in the mammalian liver using ChlP-Seq analysis. Strikingly, four genes central to circadian regulation are direct CREB targets, suggesting a novel role for CREB in the maintenance of the peripheral clock in the liver. This is relevant, because night-shift work and other disruptions of circadian rhythms increase the risk of metabolic syndrome, including obesity, insulin resistance, and dyslipidemia in humans. Based on the above, we propose three specific aims. In AIM 1, we will determine if CREB ablation in hepatocytes can be used to alleviate hepatic insulin resistance and steatosis. We have derived two new models that will enable us to analyze CREB function in a precise, cell-type specific manner, a conditional null allele for CREB (CREB[loxp), and a conditional CREB Ser133 to Ala133 mutation (CREB][loxp](S133A)). These two alleles allow us to dissociate the contribution of CREB phosphorylation by protein kinase A versus all CREB functions. In AIM 2, we will determine the contribution of CREB to the control of the peripheral clock in the liver. Mice will also be subjected to restricted feeding to study the contribution of CREB to shifting part of the liver's circadian clock in this paradigm. In AIM 3, we will determine the role of CREB in nutritional control of the neuroendocrine axis. We will ablate CREB in the hypothalamus to assess its contribution to the regulation of TRH, and its role in feeding-dependent thermogenesis

The function of the Gene Targeting Core (Core C) is to facilitate gene-targeting and BAC- transgenic projects for all progrann investigators. The Gene Targeting Core will continue to perform all aspects of mouse embryonic stem cell culture and gene targeting, including the preparation of feeder layers, the culture and electroporation of ES cells (both 129 and C57/BL6 derived), the isolation of DNA, the expansion of targeted ES cell clones, and the preparation of targeted ES cells for blastocyst injection. We will also maintain Cre-lines for program investigators, and assist with the construction of new alleles or transgenes via BAC recombineering. In addition, we will carry out a systematic evaluation of available Cre lines for gene ablation in adipose tissue for the P01 investigators.

DESCRIPTION (provided by applicant: Diabetes is a disease of multiple organs responding to complex genetic and environmental factors. A complete understanding of insulin resistance and type 2 diabetes mellitus (T2DM) requires an integrative approach that asks how different cell types influence each other through hormonal, neural, and metabolic signals all in the context of extra-organismal stresses including overnutrition and disruptions in normal circadian rhythms. A goal of all studies proposed in this application is to explain normal and pathological metabolism in molecular terms, emphasizing both cell autonomous processes as well as those that depend on organismal integration. The Program Project brings together five outstanding investigators, each with considerable past success as an independent investigator, but each also with a genuine belief in the value of scientific collaboration. Each PI focuses on a specific organ system and how it interacts with other tissues and external stresses, and works in close communication other PIs who study related problems. In Project 1, Lazar addresses how resistin coordinates the multi-organ response to nutritional overload, in which an inflammatory response leads to adverse consequences in insulin target tissues and the cardiovascular system. In Project 2, Stoffers focuses on the response of the beta cell to the stress of peripheral insulin resistance, testing an intriguing model that connects transcriptional regulation to endoplasmic reticulum biosynthesis and cell growth. In Project 3, Ahima uses a mouse feeding entrainment model that mimics circadian disruption in humans to examine how the central nervous system influences hepatic metabolism. In Project 4, Kaestner also studies the response to a perturbed central clock, but in the context of how the hormonal milieu influences the transcriptional control of liver glucose metabolism. Lastly, in Project 5, Birnbaum also considers how hepatic metabolism responds to the stress of obesity, but also asks how it is normalized by the antidiabetic drug metformin. The projects are supported by three Cores that provide histochemical analysis, generation of genetically modified mice, and their metabolic phenotyping.

DESCRIPTION (provided by applicant): Maintenance of glucose homeostasis is central to our health, and its failure results in severe debilitating diseases including diabetes and familial hyperinsulinism. Diabetes mellitus is a metabolic disorder that affects over 285 million people worldwide and is a leading cause of death in many countries. The disease is characterized by either absolute insulin deficiency due to the autoimmune destruction of pancreatic insulin-producing ?-cells [Type 1 diabetes mellitus (T1DM)], or relative insulin deficiency due to defective insulin secretion or insulin sensitivity [Type 2 diabetes mellitus (T2DM)]. The resulting elevated blood glucose levels eventually lead to an impairment of the microvasculature followed by kidney failure, blindness, neuropathy and heart disease. Consequently, diabetes is currently the sixth leading cause of death in the United States (CDC). During the past grant cycle, we have made the exciting discovery that the imprinted MEG3 locus is strongly down-regulated in islets from type 2 diabetics. The MEG3 locus is of particular interest in that it encodes a cluster of 54 microRNAs, which we have found to target anti-apoptotic genes, suggesting that dysregulation of this locus contributes to ?-cell failure in type 2 diabetes. Here, we propose to investigate both epigenetic regulation and biological function of this locus in mouse and human ?-cells. In Aim 1, we will investigate the molecular mechanism that causes dysregulation of the MEG3 locus in diabetes. Specifically, we will test the hypothesis that hyper-methylation of a differentially methylated region (DMR) near the promoter of the MEG3 gene causes loss of binding of ?-cell specific transcription factors in an enhancer in the MEG3 gene. In Aim 2, we will determine the specific function of the MEG3 locus in ?-cell physiology and survival using both mouse genetic and innovative human epigenetic inactivation. Multiple biochemical and molecular assays will be performed on MEG3 deficient ?-cells. Together, these experiments will provide important new insights into the molecular etiology of ?-cell failure in type 2 diabetes.

DESCRIPTION (provided by applicant): The prevalence of Diabetes Mellitus has reached epidemic proportions world-wide and is predicted to increase rapidly in the years to come, putting a tremendous strain on health care budgets in both developed and developing countries. There are two major forms of diabetes and both are associated with decreased beta-cell mass. No treatments have been devised that increase beta-cell mass in vivo in humans, and transplantation of beta-cells is extremely limited due to lack of appropriate donors. For these reasons, increasing functional beta-cell mass in vitro, or in vivo prior to or after transplantatio, has become a Holy Grail of diabetes research. Our previous studies clearly show that adult human beta-cells can be induced to replicate, and - importantly - that cells can maintain normal glucose responsiveness after cell division. However, the replication rate achieved was still low, likely due in part to the known age-related decline in the ability of the beta-cell to replicate. W propose to build on our previous findings and to develop more efficacious methods to increase functional beta-cell mass by inducing replication of adult beta-cells, and by restoring juvenile functional properties to aged beta-cells. We will focus on mechanisms derived from studies of non- neoplastic human disease as well as age-related phenotypic changes in human beta-cells. In Aim 1, we will target the genes altered in patients with marked beta-cell hyperplasia, such as those suffering from Focal Hyperinsulinism of Infancy, Beckwith-Wiedemann Syndrome or Multiple Endocrine Neoplasia. Expression of these genes will be altered in human beta-cells via shRNA-mediated gene suppression and locus-specific epigenetic targeting. Success will be assessed in transplanted human islets by determination of beta-cell replication rate and retention of function. In Aim 2, we will determine the mechanisms of age-related decline in beta-cell function and replicative capacity, by mapping the changes in the beta-cell epigenome that occur with age. Selected genes will then be targeted as in Aim 1 to improve human beta-cell function, as assessed by glucose responsiveness. To accomplish these aims, we will use cutting-edge and emerging technologies that are already established or are being developed in our laboratories. The research team combines clinical experience with expertise in molecular biology and extensive experience in genomic modification aimed at enhancing beta-cell replication. By basing interventions on changes found in human disease and normal aging, this approach will increase the chances that discoveries made can be translated more rapidly into clinically relevant protocols.

? DESCRIPTION (provided by applicant): A better understanding of the liver's response to toxic injury, which includes hepatocyte proliferation, activation and differentiation of facultative hepaic stem cells (oval cells), and - unfortunately - an increased risk for hepatocellular carcinoma, is a prerequisite for the development of novel clinical treatments for chronic liver disease and improved cancer prevention. Likewise, cell replacement therapy, either through direct hepatocyte transplantation or in bio-artificial liver devices, needs to be improved in order to become a reliable alternative to liver transplantation. To date, investigations of hepatocyte proliferation have frequently focused on the partial hepatectomy paradigm, a noninjury model that is not reflective of liver injury in humans and which has therefore failed to identify specifi targets for either improved regeneration following toxic injury or for limiting proliferation in HC in humans. In Specific Aim 1, we will determine which genes and gene combinations promote or repress hepatocyte repopulation following toxic liver injury using an innovative genetic approach. In Specific Aim 2, we will employ expression of key hepatic transcription factors to improve the differentiation of hepatic progenitor cells to functional hepatocytes. Together, these approaches will provide an improved understanding of the liver's response to toxic injury, and facilitate the discovery of new cell replacement therapies to treat chronic liver disease and liver failure.

Penn Human Pancreas Procurement and Analysis Program Abstract Utilizing our existing infrastructure and scientific collaborations, we have assembled 6 cores with expertise ranging from pancreas procurement and islet isolation to data integration for a comprehensive and integrated Human Pancreas Procurement and Analysis Program based at the University of Pennsylvania. Core A will procure a spectrum of human pancreata and detailed donor medical history; perform high resolution HLA typing by next generation sequencing; isolate islets; and distribute islets and tissues to the other Cores for further analysis or processing. Core B will perform physiological phenotyping on the isolated islets. Core C will quantify and characterize memory T cell subsets by flow cytometry and single cell qPCR analysis; characterize suppressive activity of Tregs and the ability of related effector cells to be suppressed; B cell phenotyping; and generate chromatin accessibility maps of enhancers in pathogenic cell types. Core D will perform multiple advanced modalities for the molecular profiling of isolated islets including RNAseq and microRNAseq of sorted islet cell populations; mass cytometry for single cell quantification of more than 20 cell surface and intracellular markers; and single cell RNAseq. Core E will process tissues using multiple modalities that will allow for analysis using advanced technologies such as multiplexed immunoflourescent staining, combinatorial barcoded FISH (combFISH), whole slide imaging, and quantitative image analysis of protein markers and immune cell infiltrates. This Core will adapt 2-dimensional mass cytometry to pancreatic sections utilizing multiplexed ion beam imaging (MIBI) technology. This Core will also archive tissues as well as DNA and blood, and facilitate sample distribution to HPPAP approved researchers. Finally, Core F will assemble, annotate and maintain an open access database for the Program and its member-researchers, and collaborate with the HIRN in the sharing of data from both programs. The entire Program will be executed by an Administrative Core consisting of the PIs, with assistance from an Executive Committee consisting of the core leaders. The Administrative Core will interface with an external committee to review applications for HPPAP biosample use, and will collaborate with the HIRN. The Program will also interact with the HPPAP member/PANC DB user community to provide a richly annotated source of physiologic, genomic and immunologic data on the tissue-based landscape governing T1D.

UC4 APPLICATION: DRIVERS AND CONSEQUENCES OF b-CELL DNA DAMAGE IN DIABETES Abstract: The prevalence of Diabetes Mellitus has reached epidemic proportions world-wide, and is predicted to increase rapidly in the years to come, putting a tremendous strain on health care budgets in both developed and developing countries. There are two major forms of diabetes and both are associated with decreased beta-cell mass. Exciting recent data have provided evidence that metabolic stress is associated with DNA double strand breaks in multiple models of impaired glycemia. Based on our exciting preliminary data, we propose to develop novel technologies further to specifically determine the accumulated mutation load, as well as physiological responses to genomic stress in human beta- cells, all at the single cell level. In Aim 1, we will determine the molecular mechanisms that cause b- cell DNA damage in diabetes, based on our hypothesis that metabolic and/or inflammatory insults and abortive replication attempts result in formation of double-strand breaks in b-cells, potentially in discrete locations. To this end, we will perform genome-wide CHIP-Seq experiments with antibodies against the DNA damage response proteins 53BP1 and gH2AX, as well as the BLISS method to reveal the location of actual DNA breaks at a single-base resolution in healthy and metabolically- stressed b-cells from mice and humans. In Aim 2, we will analyze the cumulative mutation load of human b-cells in diabetes, based on our hypothesis that metabolic insults and abortive replication attempts result in the accumulation of somatic mutations in b-cells, contributing to their loss of function and possibly to immunogenicity in diabetes. To accomplish this goal, we will determine the cumulative mutation load of stressed b-cells using single cell exome-, RNA-, and ATACseq analysis. To accomplish these aims, we will use cutting-edge and emerging technologies that are already established in our laboratories. We have assembled an outstanding team of scientist with complementary expertise, ranging from human islet transplantation to computational biology, to assemble an atlas of the human endocrine pancreas in health and disease at the single cell level. These datasets and technologies promise to greatly increase our understanding of the human beta- cell in health and disease.

PROJECT SUMMARY (OVERVIEW) The NIDDK P30 Digestive Diseases Research Core Center at the University of Pennsylvania is called the Center for Molecular Studies in Digestive and Liver Diseases (CMSDLD). Constituted in 1997 and funded continuously since then (with highly successful competitive renewals in 2002, 2007 and 2012), the CMSDLD provides an exceptional platform for basic and translational research in digestive, liver and pancreatic diseases with a vision of understanding human health and ameliorating the public health burden associated with these diseases. This overarching vision is executed through the interrelated missions or Specific Aims of the CMSDLD: (1) to support impactful interdisciplinary and collaborative digestive, liver and pancreatic research through its Members/Associate Members, who span 4 Schools at the University of Pennsylvania, multiple Departments/Centers/Institutes, importantly, Children's Hospital of Philadelphia, as well as institutions within and surrounding Philadelphia; (2) to foster the academic and professional development of its Associate Members; (3) to provide state-of-the art services and technologies through its scientific core facilities (with quality and cost-effectiveness), which in turn support the Members and Associate Members; (4) to oversee innovative enrichment (inclusive of education and mentorship) programs and a highly successful pilot and feasibility grant program; (5) to promote gender, diversity and inclusion as part of our CREED (Clinical care, Research, Education, Encouragement and Diversity) and (6) to collaborate with other Penn Centers/Institutes as well as national universities, academic medical centers, other DDRCCs and the NIH/NIDDK, and conduct outreach with the lay public. Rigor, reproducibility and transparency are also critical aspects of the CMSDLD. The CMSDLD has an administrative structure, or Core, which is overseen by the Dean, and guidance from highly interactive Penn/CHOP leaders, and internal and external advisory boards. The CMSDLD may be viewed as a stem cell, which self-renews and concurrently gives rise to differentiated entities on campus that in turn continue to interact with the CMSDLD: (A) Members who apply for and receive new interdisciplinary grants spawned by the CMSDLD; (B) Penn-CHOP Joint Center in Transitional Medicine (adolescence to adulthood) in digestive, liver and pancreatic medicine; (C) Penn-CHOP Microbiome Program. The Perelman School of Medicine and Children's Hospital of Philadelphia have invested heavily in the CMSDLD and are committed to continue to do so in the next funding cycle. The CMSDLD is positioned to build upon its successes and be a vanguard of research, education and translational clinical care in the future.

A better understanding of the liver?s response to toxic injury, which includes hepatocyte proliferation, and ? unfortunately ? an increased risk for hepatocellular carcinoma (HCC), is a prerequisite for the development of novel clinical treatments for chronic liver disease and improved cancer prevention. Existing drug therapies for HCC such as sorafenib extend patient survival by only three months. We recently developed a massively parallel in vivo screening platform to test the impact of genetic factors such as full-length cDNAs or miRNAs on liver repopulation and tumorigenesis. We have used this screening technology to build a map of all miRNAs active in liver regeneration. Here, we propose to exploit this innovative paradigm to conduct a comprehensive evaluation of the effects of the 135 most abundant but evolutionarily conserved hepatic miRNAs on the processes of recovery from toxic liver injury and HCC tumorigenesis. In Specific Aim 1, we will determine the combined benefits of three miRNAs identified in our prior screen on liver repopulation following toxic injuries, as a step toward using miRNA-mimetic drug therapies for liver diseases. This will be accomplished through delivery of miRNA-encoding plasmids or nanoparticles singly and in all combination. In Specific Aim 2, we will determine the impact of hepatic miRNAs and miRNA combinations on HCC tumor development in vivo. To this end, we have developed two models of rapid HCC development in mice, in which we will screen our library of 135 ?tough decoys? (?TuD?s?), or inhibitors of miRNA action, on tumor formation. We will quantify the abundance of all TuD?s using high throughput sequencing in the tumor-loaded liver compared to the input library. TuD?s enriched in after tumor formation target miRNAs that normally limit tumor growth, and those found less abundant target miRNAs that promote tumorigenesis. We will then test the combinations of the most potent miRNA effectors on tumor formation following systemic delivery. In Specific Aim 3 we will perform a conditional screen of miRNAs that impact Sorafenib resistance to identify novel combination treatments for the prevention or treatment of HCC. Together, our powerful genetic screens promise to identify miRNA effectors that can be employed for the treatment of acute liver injury and, in combination with Sorafenib, as a more effective treatment to prevent HCC initiation and progression.

Functional Interrogation of T2D-associated genes in human stem cell-derived models and mice Type 2 Diabetes (T2D) is one of the fastest-growing diseases and a leading cause of death throughout the world. A better understanding of the disease process, including characterization of both the genetic etiology and the contribution of different cell types to disease initiation, progression and heterogeneity promises to reveal new therapeutic targets. Large-scale genome-wide association studies (GWAS) of this common complex trait have driven the rapid identification of hundreds of T2D-associated loci. However, the mechanism(s) through which most of these loci influence disease susceptibility remain poorly understood. Our interdisciplinary team at Penn brings together experts in population genetics, T2D GWAS, biostatistics, metabolic tissue biology, human cellular disease modeling and T2D pathophysiology to tackle this critical knowledge gap. In collaboration with other Consortium groups, we aim to accomplish the following goals. (1) Provide the diabetes research community with a robust pipeline for mapping T2D GWAS variants to effector genes and target tissues. (2) Identify new genes and biological pathways that modulate susceptibility to T2D. (3) Define gene regulatory networks relevant to T2D with the goal of uncovering therapeutic ?entry points? for developing new treatments. For (1), we will prioritize ?candidate effector transcripts? for downstream functional analyses by integrating multiple sources of data to gain a ?confluence of evidence? as to their disease relevance and tissue of action. These sources include publically available datasets, a unique collection of internal resources from the Million Veteran Program, and our own functional genomics (RNA-seq, ATAC-seq, chromatin conformation capture etc.) data generated from stem cell-derived T2D relevant cell types. For (2), we will examine the biological function of prioritized T2D-effector transcripts in human cell models of T2D-relevant tissue types using gain- and loss-of- function methods combined with a battery of physiological, metabolic, molecular phenotyping and genomic approaches. These studies include the use of induced pluripotent stem cell (iPSC) models for pancreatic b cells, hepatocytes, adipocytes and skeletal muscle cells, enabling precise genetic engineering and establishment of multiple cell types in the same genetic background. Through this process, we will identify 10 high priority candidate effector genes, which we will advance for comprehensive in vivo analyses in conditional mutant mouse models. For (3), we will perform network analyses through the integration of our multiple data sources to identify molecular memberships in broader pathways and search for pathway components that are potentially amenable for therapeutic targeting.

Penn Human Pancreas Analysis Program ? T2D Abstract Building on our existing infrastructure and scientific collaborations, and the expertise gained during the first two years of the HPAP effort for Type 1 Diabetes, we have assembled five cores with expertise ranging from pancreas procurement and islet isolation to data integration. These Cores form a for a comprehensive and integrated Penn Human Pancreas Analysis Program ? T2D based entirely at the University of Pennsylvania. Core A will procure a spectrum of human pancreata and detailed donor medical history; perform high resolution HLA typing by next generation sequencing; isolate islets; and distribute islets and tissues to the other Cores for further analysis or processing. Core B will perform physiological phenotyping on the isolated islets. Core C will perform multiple advanced modalities for the molecular profiling of isolated islets including RNAseq, ATACseq and DNA methylome analysis of sorted islet cell populations; single cell ATACseq and RNAseq, and mass cytometry for single cell quantification of more than 20 cell surface and intracellular markers. Core D will process tissues using multiple modalities that will allow for analysis using advanced technologies such as multiplexed immunoflourescent staining, whole slide imaging, and imaging mass cytometry. This Core will also archive tissues, DNA and blood, as well as other T2D-relevant organs such as skeletal muscle, intestine, adipose tissue and liver for future use by other NIDDK-approved consortia. Finally, Core E will assemble, annotate and maintain an open-access database for the Program and its member-researchers, and collaborate with the HIRN in the sharing of data from both programs. The entire program will directed by an Executive Committee consisting of the core leaders and the PI, who will be the interface with HIRN and NIDDK leadership. HPAP-T2D will provide physiologic, genomic, genetic, and histological analysis of the pancreas in type 2 diabetes at unprecedented detail, share the rich data with researchers world-wide before publication, and thus enable breakthrough discoveries in our understanding of this disease that has reached epidemic levels world-wide.

The Human Pancreas Analysis Program for Type 1 Diabetes (HPAP-T1D) Abstract In 2016, the NIH NIDDK selected a multi-disciplinary team of investigators from three institutions (UPENN, Vanderbilt, University of Florida) to establish the pilot phase of the Human Pancreas Analysis Program (HPAP). Over the past three years, type 1 diabetes (T1D)-relevant tissues from more than 50 organ donors were profiled at the anatomic, physiologic, metabolic, immunologic, genomic and epigenomic levels. The resulting data were compiled and organized into the publicly accessible PANC-DB database and website. Here, we propose not only to continue, but to expand our efforts to apply and develop state-of-the-art technologies designed to phenotype and molecularly profile human tissues relevant to the etiology of T1D through a series of innovative efforts by six Cores. Core A (Pancreas Procurement and Islet Isolation) will procure/process pancreatic islets, pancreas and lymphoid organs, expand donor outreach (in collaboration with the well-established nPOD program) and increase the collection of non-pancreatic tissues. Core B (PhysiologicalPhenotyping)will provide a comprehensive metabolic profile and probe the key regulatory steps that govern hormone secretion from the major pancreatic endocrine cell types. Core C (Immunobiology) will develop an immune atlas of peripancreatic lymphoid populations, obtain transcriptomic profiles of the T1D- specific T cells, and perform immune repertoire profiling of B and T cells in association with single cell and antigen-specific cell approaches. Core D (Advanced Molecular Profiling) will perform RNAseq, ATACseq and DNA methylome analysis on sorted alpha-cell, beta-cell and exocrine cell population as well as scRNAseq and scATACseq and carry out whole genome sequencing. In addition, islet endocrine and major lymphocyte populations will be quantified precisely using flow CyTOF. Core E (Tissue Analysis & Biobanking) will analyze pancreatic tissue architecture and immune cell/epithelial cell interactions using multiple modalities including imaging mass cytometry, multi-spectral imaging and CODEX. Complete image data will be made available via PancreatlasTM and PANC-DB. Finally, Core F (PANC-DB, Data Analysis and Integration) will expand the PANC-DB resource by adding new features that will make the public web page even more useful, as well as add a Computational Biology and Data Science Unit for applying state-of-the-art analytical tools, allowing for the integration and visualization of generated datasets using different experimental modalities such as multi-spectral imaging and omics technologies. In addition, Core F will continue to expand its outreach activities, exemplified via the deposition of transcriptome and epigenome data into the Diabetes Epigenome Atlas (DGA). HPAP-T1D will be directed by an experienced, collaborative multi-PI team that confers weekly and will meet in-person on a biannual basis in coordination with NIDDK leadership to review the progress of the entire program.