GENOME-WIDE EPIGENETIC CONTROL OF CIRCADIAN METABOLISM BY HEME RECEPTOR REV-ERB
Mitchell A Lazar
University Of Pennsylvania, 3451 Walnut Street, Philadelphia, Pa 19104
Grant 1RC1DK086239-01 from Office Of The Director, National Institutes Of Health
Abstract: This application addresses the broad Challenge Area (08) Genomics and specific Challenge Topic 08-DK-107 Nuclear Receptor mediated assembly of functional transcriptional units. A major goal of this laboratory is to understand the transcriptional mechanisms by which nuclear receptors (NRs) regulate circadian and metabolic physiology. The heme receptor Rev-erb was identified by the PI in 1989, and has since emerged as an important repressor of key genes controlling circadian rhythm and glucose metabolism. Prior studies of Rev- erb have been based on candidate gene approaches focused on promoters. Thus, it is not surprising that only about ten Rev-erb-target genes are known, given that genome-wide location analyses for other nuclear receptors have revealed that most binding occurs outside of promoters. We hypothesize that Rev-erb functions through binding of hundreds of genes, many tissue-specifically. To address this, we will perform a genome-wide analysis of Rev-erb binding in mouse liver and white adipose tissues. Specific Aim 1 is to identify Rev-erb gene targets on a genome-wide scale in liver at different circadian times, and understand their modes of regulation. We hypothesize that circadian fluctuation of Rev-erb protein level will affect its ability to access recognition sequences in the genome, which, in turn, will have downstream effects on circadian and metabolic processes. We will test this by genome-wide location analysis using chromatin immunoprecipitation (ChIP) followed by sequencing of the isolated DNA (ChIP-seq). Specific Aim 2 is to identify Rev-erb gene targets on a genome-wide scale in liver from fed versus fasted mice and from mice under restricted feeding, and understand their modes of regulation. We hypothesize that metabolic regulation of Rev-erb activity will alter Rev-erb binding at many genes, and will test this by ChIP- seq using liver isolated from fed and fasted mice. Preliminary data reveal that Rev-erb protein level is higher in liver from fasted mice than in liver from fed mice, suggesting that Rev-erb binding activity may be modulated by metabolic signals. Specific Aim 3 is to identify Rev-erb gene targets on a genome-wide scale in white adipose tissue, and understand their modes of regulation. We hypothesize that Rev-erb mediates circadian expression of adipocyte genes and will test this by performing ChIP-seq for adipose Rev- erb at different circadian times and feeding regimens. This will expand our understanding of Rev-erb function in adipose biology and, when combined with the liver data set, it will identify tissue-specific targets and functions for Rev-erb in liver and adipose tissues. These studies can be accomplished in a two-year time frame because we have validated antibodies for ChIP of endogenous Rev-erb in metabolic tissues, and the experimental paradigms involve short-term studies. The genome-wide insights into in vivo tissue-specific regulation of circadian and metabolic genes will be enormous, and contribute greatly to our understanding of how these physiological processes are interrelated, and potentially dysregulated in obesity and diabetes, which are epidemic in the United States. Metabolic disorders such as diabetes and obesity are affected by sleep patterns and shift work. Thus, it is exciting that the focus of this proposal, the nuclear receptor Rev-erb, has emerged as a critical link between circadian and metabolic physiology. The proposed experiments aim to determine the extent that Rev-erb contributes to, or even controls, circadian and metabolic processes and the crosstalk that occurs between them. Ultimately, greater insight into the regulation of circadian rhythm and metabolism by Rev-erb will contribute greatly to our understanding of how these physiological processes are interrelated, and potentially dysregulated in obesity and diabetes, which are epidemic in the United States
Keywords: Address; Adipocytes; Adipose Cell; Adipose tissue; Affect; Antibodies; Area; Binding; Binding (Molecular Function); Biology; Body Tissues; CHIP assay; Candidate Disease Gene; Candidate Gene; Cell Communication and Signaling; Cell Signaling; ChIP (chromatin immunoprecipitation); Circadian Rhythms; Cues; DNA; Data; Data Set; Dataset; Deoxyribonucleic Acid; Diabetes Mellitus; Diurnal Rhythm; Eating; Epidemic; Epigenetic; Epigenetic Change; Epigenetic Mechanism; Epigenetic Process; Fasting; Fat Cells; Fats; Fatty Tissue; Fatty acid glycerol esters; Food Intake; GWAS; Gene Targeting; Gene Transcription; Genes; Genetic Transcription; Genome; Genomics; Goals; Intermediary Metabolism; Intracellular Communication and Signaling; Laboratories; Leptin; Link; Lipocytes; Liver; Liver Extract; Location; METBL; Mammals, Mice; Mature Lipocyte; Mature fat cell; Mediating; Metabolic; Metabolic Control; Metabolic Diseases; Metabolic Disorder; Metabolic Processes; Metabolism; Mice; Molecular; Molecular Interaction; Murine; Mus; Nuclear Receptors; Nyctohemeral Rhythm; Ob Gene Product; Ob Protein; Obese Gene Product; Obese Protein; Obesity; Organism-Level Process; Organismal Process; PAI-1; PAI1; PLANH1; Pathway interactions; Pattern; Physiologic Processes; Physiological Processes; Physiology; Plasminogen Activator Inhibitor 1; Promoter; Promoters (Genetics); Promotor; Promotor (Genetics); Proteins; Protocols, Treatment; RGM; RNA Expression; Regimen; Regulation; Role; Serine or Cysteine Proteinase Inhibitor Clade E Member 1; Signal Transduction; Signal Transduction Systems; Signaling; Sleep; Targetings, Gene; Testing; Thesaurismosis; Time; Tissues; Transcription; Transcription, Genetic; Treatment Protocols; Treatment Regimen; Treatment Schedule; Twenty-Four Hour Rhythm; Type 1 Plasminogen Activator Inhibitor; United States; adipokines; adipose; adiposity; base; biological signal transduction; body system, hepatic; chromatin immunoprecipitation; circadian; circadian process; corpulence; corpulency; corpulentia; daily biorhythm; day shift; diabetes; diurnal variation; experiment; experimental research; experimental study; fasted; fasts; feeding; gene product; genome wide association scan; genome wide association studies; genome wide association study; genome, mouse; genome-wide; genome-wide analysis; genome-wide scan; genomewide association scan; genomewide association studies; genomewide association study; genomewide scan; glucose metabolism; heme binder, membrane; heme receptor; hemopexin-heme receptor; in vivo; insight; mRNA Expression; metabolism disorder; mouse genome; night shift; night work; ob/ob mouse; obese; obese people; obese person; obese population; organ system, hepatic; pathway; public health relevance; research study; resistin; shift work; social role; white adipose tissue; whole genome association studies; whole genome association study; yellow adipose tissue
Relevance: Broad Challenge Area: (08) Genomics Specific Challenge Topic: 08-DK-107 Nuclear Receptor mediated assembly of functional transcriptional units Metabolic disorders such as diabetes and obesity are affected by sleep patterns and shift work. Thus, it is exciting that the focus of this proposal, the nuclear receptor Rev-erb¿, has emerged as a critical link between circadian and metabolic physiology. The proposed experiments aim to determine the extent that Rev-erb¿ contributes to, or even controls, circadian and metabolic processes and the crosstalk that occurs between them. Ultimately, greater insight into the regulation of circadian rhythm and metabolism by Rev-erb¿ will contribute greatly to our understanding of how these physiological processes are interrelated, and potentially dysregulated in obesity and diabetes, which are epidemic in the United States
Project start date: 2009-09-19
Project end date: 2011-08-31
Budget start date: 19-SEP-2009
Budget end date: 31-AUG-2010
PFA/PA: RFA-OD-09-003
1RC1DK086239-01 (2009): $500000
Sponsored Links Excellgen http://Excellgen.com
Grants awarded to Mitchell A Lazar
THYROID HORMONE RECEPTORS - REGULATION AND FUNCTION
Mitchell A Lazar
University Of Pennsylvania, 3451 Walnut Street, Philadelphia, Pa 19104
Grant 3R37DK043806-19S2 from National Institute Of Diabetes And Digestive And Kidney Diseases
Abstract: Thyroid hormone receptors (TRs) are ligand-dependent, transcriptional regulators of metabolism. TRs repress gene expression in the absence of hormone, which is paradigmatic for other nuclear receptors (NRs) that function as repressors in the unliganded state. Repression is mediated by interaction with corepressors N- CoR (Nuclear Receptor Corepressor) and SMRT (Silencing Mediator of Retinoid and Thyroid receptors), which exist in stoichiometric association with the chromatin-modifiying enzyme, histone deacetylase 3 (HDAC3). HDAC3, in turn, derives its catalytic activity from interacting with N-CoR/SMRT via their unique Deacetylase Activation Domain (DAD). The DAD-dependent N-CoR/SMRT7HDAC3 complex is critical for repression by TR and other NRs in vitro, but the role of this interaction in vivo is unknown. Here we propose to use state of the art methods of gene targeting and mouse phenotyping to test, for the first time, the physiological relevance of the N-CoR7HDAC3 and SMRT7HDAC3 corepressor complexes. We hypothesize that these DAD-dependent interactions are very important, and affect distinct physiological pathways involving NRs. Specific Aim 1 is to determine the physiological function of the N-CoR DAD domain. Knockout of N-CoR is embryonic lethal; we hypothesize that interaction with HDAC3 subserves a subset of N-CoR´s developmental and physiological functions. To discover what those functions are, we have generated mice with a point mutation in the N-CoR DAD domain that prevents HDAC3 interaction. Preliminary data demonstrate that mice homozygous for this mutation are viable, with intriguing abnormalities that point to the biological importance of N-CoR7HDAC3. Specific Aim 2 is to determine the physiological function of the SMRT DAD domain. Similar to N-CoR, the physiological role of SMRT7HDAC3 is unknown. We hypothesize that SMRT subserves unique functions that are mediated by HDAC3, and may also have HDAC3-dependent functions that are redundant with those of N-CoR. We will test these hypotheses by generating knockin mice with a point mutation in the SMRT DAD domain. The N-CoR and SMRT homozygous mutant mice, and doubly homozygous mutants, will be carefully analyzed to determine the physiological function of the N-CoR/SMRT7HDAC3 interaction. Specific Aim 3 is to determine the physiological, tissue-specific functions of HDAC3. HDAC3 will be deleted in mice to test the hypothesis that losses of HDAC3 function will phenocopy the doubly homozygous N-CoR/SMRT DAD mutant mice. The HDAC3 knockout will be conditional, enabling us to investigate tissue-specific functions of HDAC3. Together, these innovative and unique studies will elucidate mechanisms regulating transcription repression by TR and other NRs in a physiological context. The insights gained from this work will shed new light on the transcriptional and epigenetic control of key biological pathways, including metabolism and inflammation. This has the potential to lead to new and deeper insights into metabolic disorders, such as obesity, diabetes, and cardiovascular disease, as well as cancer. Relevance In the past decade, corepressors have emerged as critical regulators of hormone receptors. The proposed studies will innovatively and uniquely elucidate mechanisms regulating the action of hormones and other metabolic regulators. The insights gained from this work will shed new light on key biological pathways, with the potential to lead to new and deeper insights into metabolic disorders, including obesity, diabetes, and cardiovascular disease, as well as cancer
Keywords: 21+ years old; Adult; Affect; Arts; Biological; Body Tissues; Cancers; Cardiovascular Diseases; Cell Culture Techniques; Chemotherapy-Hormones/Steroids; Chromatin; Complex; Data; Deacetylase; Development; Diabetes Mellitus; Embryo; Embryonic; Endocrine Gland Secretion; Enzymes; Epigenetic; Epigenetic Change; Epigenetic Mechanism; Epigenetic Process; Funding; Gene Down-Regulation; Gene Expression; Gene Targeting; Gene Transcription; Genetic Alteration; Genetic Change; Genetic Transcription; Genetic defect; Goals; HDAC; HDAC Proteins; HDAC3; HDAC3 enzyme; Histone Deacetylase; Hormone Receptor; Hormones; Human, Adult; INFLM; In Vitro; Inflammation; Intermediary Metabolism; Isoforms; Knock-out; Knockout; Knockout Mice; Laboratories; Lead; Ligands; Light; METBL; Malignant Neoplasms; Malignant Tumor; Mammals, Mice; Mediating; Metabolic; Metabolic Diseases; Metabolic Disorder; Metabolic Processes; Metabolism; Methods; Mice; Mice, Knock-out; Mice, Knockout; Mice, Mutant Strains; Modeling; Murine; Mus; Mutant Strains Mice; Mutation; Nuclear Receptors; Null Mouse; Obesity; Pathway interactions; Pb element; Phenocopy; Phenotype; Photoradiation; Physiologic; Physiological; Point Mutation; Property; Property, LOINC Axis 2; Protein Isoforms; RNA Expression; Regulation; Repression; Role; Silencing Mediator of Retinoid Thyroid Receptor; Specificity; Targetings, Gene; Technology; Testing; Therapeutic Hormone; Therapeutic Intervention; Thesaurismosis; Thyroid Hormone Receptor; Time; Tissues; Transcription; Transcription Repression; Transcription, Genetic; Transcriptional Repression; Transgenic Organisms; Work; adiposity; adult human (21+); base; cardiovascular disorder; corpulence; corpulency; corpulentia; diabetes; gene repression; genome mutation; heavy metal Pb; heavy metal lead; histone deacetylase 3; in vivo; innovate; innovation; innovative; insight; intervention therapy; malignancy; member; metabolism disorder; mouse mutant; mutant; neoplasm/cancer; novel; obese; obese people; obese person; obese population; pathway; prevent; preventing; receptor function; recombinase; social role; transgenic
Project start date: 2010-01-15
Project end date: 2010-03-31
Budget start date: 15-JAN-2010
Budget end date: 31-MAR-2010
PFA/PA: PA-07-070
3R37DK043806-19S2 (2010): $99500
5R37DK043806-20 (2010): $471448
5R37DK043806-19 (2009): $462341
THYROID HORMONE RECEPTORS--REGULATION AND FUNCTION
Mitchell A Lazar, Sylvan H. Eisman Professor Of Medicine &
University Of Pennsylvania 3451 Walnut Street Philadelphia, Pa 19104
Grant 5R01DK043806-07 from National Institute Of Diabetes And Digestive And Kidney Diseases IRG: END
Abstract: Thyroid hormone (T3) is crucial for normal growth, development, and adult function. Nuclear T3 receptor (TRs) regulated transcription of specific genes in a T3-dependent manner. There are three distinct TRs which bind to T3-response elements (TREs) in target genes as monomers, homodimers, and heterodimers with retinoid X receptors (RXRs) and other proteins. There are also C-terminal TR variants which do not bind T3 but are inhibitors of T3 action. It is presumed that the multiple TR isoforms and heterodimer partners are responsible for the tissue-specific and diverse effects of T3. The goal of this research is to specifically relate TR expression and biochemistry to the cellular and molecular physiological effects of T3. The first specific aim of this project is to study the hormonal regulation of TRbeta2 mRNA and protein expression in pituitary and neural cell lines. Of particular importance is the ability of RA to antagonize the down-regulation of TRbeta2 by T3. RXR- specific ligands will be used to discover the mechanism underlying this hormonal cross-talk. The second specific aim is to address the molecular physiology to T3 actio using the technique of differential display. Novel T3-responsive target genes will be identified and characterized according to how they are regulated positively or negatively; by both T3 and RA or by T3 alone; and universally or i a cell-specific manner. In this way, the rules governing positive vs. negative regulation, hormonal specificity vs. promiscuity, and cell-specificity of T3 action will be clarified. The third specific aim is to compare and contrast the TRE- binding properties of TR monomers, homodimers, and heterodimers. These TR, and correlate these biochemical properties with transcriptional function. The fourth specific aim is to understand the function and significance of C-terminal TR variants, alpha2 alpha3. The DNA-binding and transcriptional regulatory properties of these proteins will be determined and contrasted. The ability of phosphorylation to regulate the functions of TRalpha2 and alpha3 will be investigated, to address the paradox that TRalpha2 is highly expressed (especially in brain) yet binds DNA poorly in vitro and is a weak inhibitor of T3 action. Together, these studies will link the molecular biology and biochemistry of the TRs to the cellular and physiological responses to T3. This knowledge will provide insight into hormone action in general, and enhance understanding of many pathophysiological states including thyroid hormone deficiency and excess, genetic resistance to thyroid hormone, and hormone-responsive malignancies, such as breast cancer, prostate cancer, and promyelocytic leukemia.
Keywords: gene expression, genetic regulatory element, hormone receptor, hormone regulation /control mechanism, receptor expression, transcription factor, triiodothyronine, DNA binding protein, chemical binding, conformation, genetic regulation, messenger RNA, phosphorylation, protein isoform, protein structure /function, retinoate, retinoid binding protein, vitamin receptor, DNA footprinting, gel mobility shift assay, northern blotting, polymerase chain reaction, reporter gene, site directed mutagenesis, tissue /cell culture, western blotting
Project start date: 1991-06-01
Project end date: 1998-05-31
5R01DK043806-07 (1997): $271197
5R01DK043806-05 (1995): $242306
2R01DK043806-04 (1994): $233416
5R01DK043806-03 (1993): $215648
5R01DK043806-02 (1992): $204738
GENOME-WIDE EPIGENETIC CONTROL OF CIRCADIAN METABOLISM BY HEME RECEPTOR REV-ERB
Mitchell A Lazar
University Of Pennsylvania, 3451 Walnut Street, Philadelphia, Pa 19104
Grant 5RC1DK086239-02 from Office Of The Director, National Institutes Of Health
Abstract: This application addresses the broad Challenge Area (08) Genomics and specific Challenge Topic 08-DK-107 Nuclear Receptor mediated assembly of functional transcriptional units. A major goal of this laboratory is to understand the transcriptional mechanisms by which nuclear receptors (NRs) regulate circadian and metabolic physiology. The heme receptor Rev-erb was identified by the PI in 1989, and has since emerged as an important repressor of key genes controlling circadian rhythm and glucose metabolism. Prior studies of Rev- erb have been based on candidate gene approaches focused on promoters. Thus, it is not surprising that only about ten Rev-erb-target genes are known, given that genome-wide location analyses for other nuclear receptors have revealed that most binding occurs outside of promoters. We hypothesize that Rev-erb functions through binding of hundreds of genes, many tissue-specifically. To address this, we will perform a genome-wide analysis of Rev-erb binding in mouse liver and white adipose tissues. Specific Aim 1 is to identify Rev-erb gene targets on a genome-wide scale in liver at different circadian times, and understand their modes of regulation. We hypothesize that circadian fluctuation of Rev-erb protein level will affect its ability to access recognition sequences in the genome, which, in turn, will have downstream effects on circadian and metabolic processes. We will test this by genome-wide location analysis using chromatin immunoprecipitation (ChIP) followed by sequencing of the isolated DNA (ChIP-seq). Specific Aim 2 is to identify Rev-erb gene targets on a genome-wide scale in liver from fed versus fasted mice and from mice under restricted feeding, and understand their modes of regulation. We hypothesize that metabolic regulation of Rev-erb activity will alter Rev-erb binding at many genes, and will test this by ChIP- seq using liver isolated from fed and fasted mice. Preliminary data reveal that Rev-erb protein level is higher in liver from fasted mice than in liver from fed mice, suggesting that Rev-erb binding activity may be modulated by metabolic signals. Specific Aim 3 is to identify Rev-erb gene targets on a genome-wide scale in white adipose tissue, and understand their modes of regulation. We hypothesize that Rev-erb mediates circadian expression of adipocyte genes and will test this by performing ChIP-seq for adipose Rev- erb at different circadian times and feeding regimens. This will expand our understanding of Rev-erb function in adipose biology and, when combined with the liver data set, it will identify tissue-specific targets and functions for Rev-erb in liver and adipose tissues. These studies can be accomplished in a two-year time frame because we have validated antibodies for ChIP of endogenous Rev-erb in metabolic tissues, and the experimental paradigms involve short-term studies. The genome-wide insights into in vivo tissue-specific regulation of circadian and metabolic genes will be enormous, and contribute greatly to our understanding of how these physiological processes are interrelated, and potentially dysregulated in obesity and diabetes, which are epidemic in the United States. Metabolic disorders such as diabetes and obesity are affected by sleep patterns and shift work. Thus, it is exciting that the focus of this proposal, the nuclear receptor Rev-erb, has emerged as a critical link between circadian and metabolic physiology. The proposed experiments aim to determine the extent that Rev-erb contributes to, or even controls, circadian and metabolic processes and the crosstalk that occurs between them. Ultimately, greater insight into the regulation of circadian rhythm and metabolism by Rev-erb will contribute greatly to our understanding of how these physiological processes are interrelated, and potentially dysregulated in obesity and diabetes, which are epidemic in the United States
Keywords: Address; Adipocytes; Adipose Cell; Adipose tissue; Affect; Antibodies; Area; Binding; Binding (Molecular Function); Biology; Body Tissues; CHIP assay; Candidate Disease Gene; Candidate Gene; Cell Communication and Signaling; Cell Signaling; ChIP (chromatin immunoprecipitation); Circadian Rhythms; Cues; DNA; Data; Data Set; Dataset; Deoxyribonucleic Acid; Diabetes Mellitus; Diurnal Rhythm; Eating; Epidemic; Epigenetic; Epigenetic Change; Epigenetic Mechanism; Epigenetic Process; Fasting; Fat Cells; Fats; Fatty Tissue; Fatty acid glycerol esters; Food Intake; GWAS; Gene Targeting; Gene Transcription; Genes; Genetic Transcription; Genome; Genomics; Goals; Intermediary Metabolism; Intracellular Communication and Signaling; Laboratories; Leptin; Link; Lipocytes; Liver; Liver Extract; Location; METBL; Mammals, Mice; Mature Lipocyte; Mature fat cell; Mediating; Metabolic; Metabolic Control; Metabolic Diseases; Metabolic Disorder; Metabolic Processes; Metabolism; Mice; Molecular; Molecular Interaction; Murine; Mus; Nuclear Receptors; Nyctohemeral Rhythm; Ob Gene Product; Ob Protein; Obese Gene Product; Obese Protein; Obesity; Organism-Level Process; Organismal Process; PAI-1; PAI1; PLANH1; Pathway interactions; Pattern; Physiologic Processes; Physiological Processes; Physiology; Plasminogen Activator Inhibitor 1; Promoter; Promoters (Genetics); Promotor; Promotor (Genetics); Proteins; RNA Expression; Regimen; Regulation; Role; Serine or Cysteine Proteinase Inhibitor Clade E Member 1; Signal Transduction; Signal Transduction Systems; Signaling; Sleep; Targetings, Gene; Testing; Thesaurismosis; Time; Tissues; Transcription; Transcription, Genetic; Twenty-Four Hour Rhythm; Type 1 Plasminogen Activator Inhibitor; United States; adipokines; adipose; adiposity; base; biological signal transduction; body system, hepatic; chromatin immunoprecipitation; circadian; circadian process; corpulence; corpulency; corpulentia; daily biorhythm; day shift; diabetes; diurnal variation; experiment; experimental research; experimental study; fasted; fasts; feeding; gene product; genome wide association scan; genome wide association studies; genome wide association study; genome-wide; genome-wide analysis; genome-wide scan; genomewide association scan; genomewide association studies; genomewide association study; genomewide scan; glucose metabolism; heme binder, membrane; heme receptor; hemopexin-heme receptor; in vivo; insight; mRNA Expression; metabolism disorder; mouse genome; night shift; night work; ob/ob mouse; obese; obese people; obese person; obese population; organ system, hepatic; pathway; research study; resistin; shift work; social role; white adipose tissue; whole genome association studies; whole genome association study; yellow adipose tissue
Relevance: Broad Challenge Area: (08) Genomics Specific Challenge Topic: 08-DK-107 Nuclear Receptor mediated assembly of functional transcriptional units Metabolic disorders such as diabetes and obesity are affected by sleep patterns and shift work. Thus, it is exciting that the focus of this proposal, the nuclear receptor Rev-erb¿, has emerged as a critical link between circadian and metabolic physiology. The proposed experiments aim to determine the extent that Rev-erb¿ contributes to, or even controls, circadian and metabolic processes and the crosstalk that occurs between them. Ultimately, greater insight into the regulation of circadian rhythm and metabolism by Rev-erb¿ will contribute greatly to our understanding of how these physiological processes are interrelated, and potentially dysregulated in obesity and diabetes, which are epidemic in the United States
Project start date: 2009-09-19
Project end date: 2011-08-31
Budget start date: 1-SEP-2010
Budget end date: 31-AUG-2011
PFA/PA: RFA-OD-09-003
5RC1DK086239-02 (2010): $500000
Sponsored Links Excellgen http://Excellgen.com
DIABETES ENDOCRINOLOGY RESEARCH CENTER
Mitchell A Lazar, Sylvan H. Eisman Professor Of Medicine &
Biochemistry And Biophysicsuniversity Of Pennsylvania
3451 Walnut Street
philadelphia, Pa 19104
Grant 3P30DK019525-25S1 from National Institute Of Diabetes And Digestive And Kidney Diseases IRG: ZDK1
Abstract: The Philadelphia DERC participates in the nationwide interdisciplinary program established two decades ago by the NIDDK to foster research and training in the area of diabetes and related endocrine and metabolic disorders. The Philadelphia DERC, located at and administered by the University of Pennsylvania, serves 87 participants, including technical staff, from several institutions including Hershey, Jefferson, Penn and Temple. This highly interactive team represents 21 departments and institutes. Investigators of the Biomedical Research Component of the Center are organized in the following research focus groups Beta-Cell Physiology and Pathology; Immunology and Transplantation; Insulin and Growth Factors; Intracellular Signaling; Genetics of Diabetes; Nutrition, Metabolism and Obesity; Complications of Diabetes. Eight Core facilities are designed to facilitate the interdisciplinary investigations of these scientists (Administrative Core; Electron Microscopy/Morphology Core;Genetic Analysis Core; Islet Cell Core; Protein Chemistry Core; Radioimmunoassay Core; Transgenic Animal Core; Viral Vector Core). A Pilot and Feasibility Grant Program serves to foster new initiatives in diabetes research primarily of junior faculty. An intensive academic enrichment program which organizes seminars and various symposia is designed to keep Center investigators abreast of the latest discoveries and to maintain the research program at this center at the forefront of biomedical science. The present Center grant (NIDDK 19525) remains the foundation of interdisciplinary diabetes research in the Philadelphia Metropolitan Area
Keywords: biomedical facility, diabetes mellitus
Project start date: 1977-03-01
Project end date: 2002-03-31
3P30DK019525-25S1 (2001): $99000
3P30DK019525-25S2 (2001): $158500
3P30DK019525-25S3 (2001): $159310
3P30DK019525-25S4 (2001): $183676
3P30DK019525-24S1 (2000): $50000
5P30DK019525-25 (2001): $1188750
5P30DK019525-24 (2000): $1188741
Univ Of Pennsylvania Diabetes Endocrinology Res CTR
Mitchell A Lazar, Sylvan H. Eisman Professor Of Medicine &
University Of Pennsylvania 3451 Walnut Street Philadelphia, Pa 19104
Grant 3P30DK019525-30S1 from National Institute Of Diabetes And Digestive And Kidney Diseases IRG: ZDK1
Abstract: The Penn Diabetes and Endocrinology Research Center (DERC) participates in the nationwide inter-disciplinary program established over two decades ago by the NIDDK to foster research and training in the areas of diabetes and related endocrine and metabolic disorders. The Penn DERC is located at and administered by the University of Pennsylvania. It serves 65 diabetes-oriented investigators primarily from the University of Pennsylvania School of Medicine, but also from other Schools within the University of Pennsylvania as well as additional Philadelphia institutions including Jefferson and Temple. The Penn DERC is highly interactive and interdisciplinary, representing many basic science and clinical departments at Penn and the other institutions. The Research Base of the Penn DERC includes investigators organized in the following 4 focus groups Beta-cell Physiology and Pathology; Signaling by Insulin and Other Hormones; Genetics and Complications of Diabetes; and Obesity, Metabolism, and Nutrition. The Penn DERC facilitates and supports research in diabetes and endocrinology in a variety of ways. Seven Biomedical Research Core facilities are designed to facilitate the inter-disciplinary investigations of Penn DERC scientists Biomedical Imaging Core; Functional Genomics Core; Islet Cell Biology Core, Mouse Phenotyping, Metabolism, and Physiology Core; Radioimmunoassay Core; Transgenic and Chimeric Animal Core, and Viral Vector Core. A Pilot and Feasibility Grant Program that has been extremely successful for over two decades serves to nurture new investigators in diabetes and to foster new initiatives in diabetes. An intensive Academic Enrichment Program organizes Diabetes and Endocrinology Research seminars and an annual Diabetes Day symposium as well as other activities that facilitate communication and collaboration of Penn DERC investigators while keeping them abreast of the latest discoveries in diabetes and endocrinology. Penn DERC investigators mentor trainees at every level (undergraduate, predoctoral, and post-doctoral Ph.D., M.D., and combined M.D./Ph.D.), and the DERC academic activities provide a superb environment for training in diabetes research. An Administrative Component coordinates and publicizes the Biomedical Cores, Pilot and Feasibility Grant Program, and Academic Enrichment Program. These functions of the Penn DERC maintain the diabetes-related research program at this Center at the forefront of biomedical science. Moreover, the present Center grant (NIDDK19525) remains the foundation of inter-disciplinary diabetes research in Philadelphia.
Keywords: biomedical facility, diabetes mellitus, endocrinology, glucose metabolism, insulin, clinical research
Project start date: 1977-05-01
Project end date: 2007-03-31
3P30DK019525-30S1 (2006): $142930
5P30DK019525-30 (2006): $1522074
5P30DK019525-29 (2005): $1558635
Sponsored Links Excellgen http://Excellgen.com
5P30DK019525-28 (2004): $1579665
5P30DK019525-27 (2003): $1565040
2P30DK019525-26 (2002): $1530920
Nuclear Hormone Receptors In Adipocyte Differentiation
Mitchell A Lazar, Sylvan H. Eisman Professor Of Medicine &
University Of Pennsylvania 3451 Walnut Street Philadelphia, Pa 19104
Grant 5R01DK049780-13 from National Institute Of Diabetes And Digestive And Kidney Diseases IRG: END
Abstract: The nuclear hormone receptor PPAR_/is a critical regulator of adipocyte differentiation and glucose metabolism, and hence a potential link between obesity and diabetes. PPARgamma, activity is regulated allosterically by activating ligands such as thiazolidinedione (TZD) antidiabetic drugs as well as by phosphorylation, which inhibits PPARgamma, activity. A major goal of this laboratory is to understand the relationships between regulation of PPARgamma, activity, adipogenesis, and insulin resistance. Specific Aim 1 is to determine the physiological role of PPARgamma phosphorylation. We hypothesize that phosphorylation regulates cell- and tissue-specificity of PPARgamma activity. Preliminary evidence indicates that mice whose PPARgamma prevent cannot be phosphorylated (S 112A) are protected from diet-induced insulin resistance. The metabolic effects of PPARgamma phosphorylation will be further dissected by comparing S112A mice with mice whose PPARgamma is mutated to mimic the less active, phosphorylated state (S 112D) in different genetic backgrounds and on TZD therapy. Specific Aim 2 is to determine the effects of PPARgamma phosphorylation on adipogenesis. We will test the hypothesis that phosphorylation of PPARgamma by plays a physiological role in adipogenesis by studying fibroblasts from S112A and S 112D mutant mice. These models are ideal for exploring the putative role of PPARgamma phosphorylation in the regulation of adipogenesis by cytokines and growth factors. We are also interested in adipocyte target genes that contribute to insulin sensitization by PPARgamma ligands. We have identified glycerol kinase (GyK) as a novel adipocyte target of PPARgamma ligands. In Specific Aim 3, we will determine the metabolic consequences of GyK induction in adipose tissue. We hypothesize that induction of GyK contributes to reduction in serum FFA levels and thereby to insulin sensitivity caused by TZD treatment. This will be directly tested by expressing GyK in adipose tissue of transgenic mice, whose glucose and lipid homeostasis will characterized. Finally, in Specific Aim 4, in which we will determine mechanisms by which PPARgamma activation selectively modulates adipoeytes gene expression. GyK induction is TZD-dependent, whereas the PPARgamma target aP2 is induced in the same adipocytes without endogenous ligand. We will use molecular and cellular techniques to test the hypothesis that the difference between adipocyte aP2 and GyK regulation is due to the PPARgamma binding sites, leading to differential cofactor recruitment or histone modification. These studies have important implications for designing selective PPARgamma, modulators (SPPARMs) that improve insulin sensitivity with fewer side effects. Together, this proposal addresses major questions about the molecular mechanisms underlying PPARgamma regulation of gene expression and metabolism, and will provide new understanding of obesity and type 2 diabetes, which are epidemic in our society.
Keywords: adipocyte, cell differentiation, hormone receptor, hormone regulation /control mechanism, insulin, lipid biosynthesis, receptor expression, biological signal transduction, body weight, diabetes mellitus, gene expression, genetic transcription, glucose metabolism, insulin sensitivity /resistance, intermolecular interaction, obesity, phosphorylation, protein structure function, gene targeting, genetically modified animal, laboratory mouse, photon absorptiometry, tissue /cell culture
Project start date: 1995-07-01
Project end date: 2008-06-30
5R01DK049780-13 (2007): $451532
5R01DK049780-12 (2006): $451399
5R01DK049780-11 (2005): $448842
2R01DK049780-10 (2004): $436246
Proteomic Profiling Corepressors
Mitchell A Lazar, Sylvan H. Eisman Professor Of Medicine &
University Of Pennsylvania 3451 Walnut Street Philadelphia, Pa 19104
Grant 1U19DK062434-010005 from National Institute Of Diabetes And Digestive And Kidney Diseases IRG: ZDK1
Abstract: The functional atlas of nuclear receptors (NRs) sets out to obtain novel insights into the regulation of the transcriptional activity of NRs in their unliganded and liganded states. Our laboratory has been especially focused on the mechanism of repression by unliganded NRs, using as models the thyroid hormone receptor, the retinoic acid receptor, and the orphan receptor RevErb. We have been extremely interested in protein interactions made by N-CoR and the related silencing mediator of retinoid and thyroid receptors (SMRT). Our work, and that of others, has suggested that numerous proteins interact with N-CoR and SMRT, including a variety of histone deacetylases (HDACs) and other potential transcriptional mediators including mSin3, SUN-CoR, ETO, TBL1. We hypothesize that critical NR repression complexes vary as a function of cell type and environment. The goal of the present project is to utilize a proteomic approach to characterize these potential NR repression complexes, and to utilizes genomic, bioinformatics, and ultimately proteomics to determine their target genes in various cell types under different conditions. Specific Aim 1 is to create and characterize stable cell lines over-expressing N-CoR and SMRT. We and others have found numerous other proteins with the potential to interact with N- CoR/SMRT. To determine the importance of these other interactions in different cell types, we will stably over-express epitope tagged-versions of SMRT and N-CoR in multiple cell types, including HeLa cells, human embryonic kidney cells, myoblasts, preadipocytes, hematopoietic, and teratoma cells. Complexes will be purified biochemically and evaluated proteomically. Specific Aim 2 is to create and characterize stable cell lines over-expressing co-repressing co-reppressor interacting proteins. N- CoR and SMRT interact with a variety of histone deacetylases (HDACs) and other potential transcriptional mediators that may have important functions in co-repressor complexes, and these proteins will be systematically epitope tagged and stably expressed in the same cell type as in Aim 1, and their biochemical complexes will be evaluated proteomically. Specific Aim 3 is to delineate co-repressor gene targets in living cells. Co-repressors do not directly interact with target genes, but are recruited by NRs. To begin to catalog and understand their range of target genes, chromatin immunoprecipitation (ChIP) of N-CoR and SMRT from wild type cells as well as the stable lines will be performed, and libraries of associated gene fragments will be created, sequenced, and annotated. Bioinformatic analysis of such data from multiple cell types will provide great insight into the commonalities and differences of gene targets for these NR co-repressors. Together, these studies will provide critical information about NR co-repressor complexes and target in genes in various cell types, that be of great interest to the field and comprise an important component of the functional atlas of nuclear receptors.
Keywords: biological signal transduction, nuclear receptor, proteomics, DNA binding protein, amidohydrolase, cooperative study, genetic library, genetic transcription, hormone receptor, protein protein interaction, retinoate, thyroid hormone, HeLa cell, SDS polyacrylamide gel electrophoresis, immunoprecipitation, mass spectrometry, polymerase chain reaction, protein sequence, tissue /cell culture
Project start date: 2002-08-15
Project end date: 2007-07-31
PROGRAMS THAT DIFFERENTIATE PRESTRUCTURAL TISSUES
Mitchell A Lazar, Sylvan H. Eisman Professor Of Medicine &
Medicineuniversity Of Pennsylvania
3451 Walnut Street
philadelphia, Pa 19104
Grant 3P01DK049210-05S1 from National Institute Of Diabetes And Digestive And Kidney Diseases IRG: SRC
Abstract: The development of complex organs from pre-structural tissues is a mysterious process. Transdifferentiation and organ specialization depend on an interaction of biologic events which move rapidly in the assembly of structure-function relationships in the growing embryo. Cell differentiation is an active process and this suggests an on-line role for gene modulation. We believe that organ development depends on the participation and continuity of sequential sets of transcriptional proteins responding to morphogenic cues. Such developmental programs are restrictive, decisional, and usually tissue-specific. Very few pieces of this regulatory puzzle are known, and we believe a program project such as ours will cultivate new interdisciplinary research in this area. The projects in our program portfolio collectively bridge the disciplines of genetics and biochemistry with basic pathophysiology in support of six established research laboratories with common interest in the developmental biology of amniotes. Each of the project leaders come from diverse career pathways and bring special interactive strengths to the larger group. All of our projects are initially focused on rodents. Project 1 will identify new Kruppel-type zinc fingers expressed in the early stages of kidney development; Project 2 will focus on the comparison of genetic programs regulating hepatic development and regulation; Project 3 will investigate the effects of perturbations of Connexin 43 (Cx43) on the expression of gap junction proteins during the differentiation of nephrons; Project 4 will evaluate the role of forkhead domains in transcription factors that are active in early nephrogenesis; Project 5 will identify the genes important in the differentiation of adipose tissue using retinoic acid as a morphogen to probe the conversion of preadipocytes; and Project 6 will examine the molecular actions of caudal- related homeodomain proteins in the endoderm of developing intestine. Core units for project administration, molecular biology, and embryologic histochemistry will provide necessary and appropriate infrastructure. It is our hope and expectation that our work will successfully identify the positional coordinates for some important regulatory events by beginning an intensive study of significant genes in early organ development
Keywords: cell differentiation, developmental genetics, histogenesis, mammalian embryology
Project start date: 1995-09-01
Project end date: 2001-08-31
3P01DK049210-05S1 (2000): $151207
5P01DK049210-05 (1999): $784535
Sponsored Links Excellgen http://Excellgen.com
5P01DK049210-04 (1998): $757350
THYROID HORMONE RECEPTORS - REGULATION AND FUNCTION
Mitchell A Lazar
University Of Pennsylvania, 3451 Walnut Street, Philadelphia, Pa 19104
Grant 3R37DK043806-19S1 from National Institute Of Diabetes And Digestive And Kidney Diseases
Abstract: Studies funded by DK43806 have led to discoveries that have illuminated how the unliganded thyroid hormone receptor represses basal transcription. We have determined the role of corepressors NCoR (Nuclear Receptor Corepressor) and SMRT (Silencing Mediator of Retinoid and Thyroid receptors) for TRs and other NRs, discovered general mechanisms as well as specificity factors governing corepressor interactions, and elucidated the core NCoR/SMRT corepressor complex, which contains the chromatin-modifying enzyme, histone deacetylase 3 (HDAC3), in stoichiometric amounts. HDAC3 derives its catalytic activity from its interaction with NCoR and/or SMRT, via the Deacetylase Activation Domain (DAD) that is unique to NCoR/SMRT. With strong evidence for the function of the DAD-dependent NCoR/SMRTHDAC3 complex in cell culture models, we are excited that state of the art methods of gene targeting and mouse phenotyping allow us to test for the first time the physiological relevance of the NCoRHDAC3 and SMRTHDAC3 corepressor complexes, which is the main objective of the parent grant. The main objective of the parent grant activities is to determine the physiological relevance of the NCoR-HDAC3 and SMRT-HDAC3 corepressor complexes. This is being accomplished through 3 specific aims 1) Determine the physiological function of the NCoR DAD domain; 2) Determine the physiological function of the SMRT DAD domain; and 3) Determine the physiological, tissue-specific functions of HDAC3. Here we request to expand these studies to include an additional aim that will be accomplished in two years and will greatly enhance the importance and impact of this work Determine and characterize the genomic locations of HDAC3 corepressor complexes. These studies will identify the regions of the genome where HDAC3 is located and potentially functioning with NCoR and SMRT to regulate gene expression through interaction with various transcription factors. Once genomic regions of association have been identified, motif analysis tools will be utilized to identify the transcription factors potentially cooperating with HDAC3 at those locations. We will also evaluate the effect of tissue specific knockout of HDAC3 (generated in Aim 3) on genome-wide histone acetylation. Relational analysis of the identified HDAC3-associated regions with epigenetic changes in chromatin modification and HDAC3-dependent gene expression will provide novel insight into the mechanism of how HDAC3-NCoR/SMRT complexes regulate gene transcription. The goals of this aim are achievable within a two year period because the liver, macrophage, and adipocyte-specific HDAC3 mice are available in the laboratory, and the gene expression profiling and genome-wide ChIP-seq analysis are up and running in the laboratory. Thus this is an ideal addition to a productive project that illuminates relationships between nuclear receptor coregulators, epigenetic changes, gene expression, and metabolic physiology. not required for this submission. No change from original proposal
Keywords: Address; Adipocytes; Adipose Cell; Adipose tissue; Arts; Body Tissues; CHIP assay; Cell Culture Techniques; ChIP (chromatin immunoprecipitation); Chromatin; Complement; Complement Proteins; Complex; Coupled; Deacetylase; Enzymes; Epigenetic; Epigenetic Change; Epigenetic Mechanism; Epigenetic Process; Fat Cells; Fatty Tissue; Funding; Gene Expression; Gene Expression Monitoring; Gene Expression Pattern Analysis; Gene Expression Profiling; Gene Targeting; Gene Transcription; Genetic Transcription; Genome; Genomics; Goals; HDAC3; HDAC3 enzyme; Hdac3 protein, mouse; Histone Acetylation; Intermediary Metabolism; Investigators; Knock-out; Knockout; Kupffer Cells; Laboratories; Lipocytes; Liver; Location; METBL; Mammals, Mice; Mature Lipocyte; Mature fat cell; Metabolic; Metabolic Processes; Metabolism; Methods; Mice; Modeling; Murine; Mus; Nuclear Receptors; Phenotype; Physiologic; Physiological; Physiology; Profilings, Gene Expression; Property; Property, LOINC Axis 2; RNA Expression; Regulation; Repression; Research Personnel; Researchers; Role; Running; Silencing Mediator of Retinoid Thyroid Receptor; Specificity; Stellate Sinusoidal Macrophage; Targetings, Gene; Testing; Thyroid Hormone Receptor; Time; Tissues; Transcript Expression Analyses; Transcript Expression Analysis; Transcription; Transcription Regulation; Transcription, Genetic; Transcriptional Control; Transcriptional Regulation; Work; adipose; body system, hepatic; chromatin immunoprecipitation; chromatin modification; genome-wide; histone deacetylase 3; histone deacetylase 3, mouse; insight; interest; liver macrophage; macrophage; member; mouse Hdac3 protein; novel; organ system, hepatic; parent grant; public health relevance; social role; tool; transcription factor; white adipose tissue; yellow adipose tissue
Relevance: not required for this submission. No change from original proposal
Project start date: 1991-06-01
Project end date: 2011-08-31
Budget start date: 29-SEP-2009
Budget end date: 31-AUG-2011
PFA/PA: PA-07-070
3R37DK043806-19S1 (2009): $359936
BIOLOGY OF THE ORPHAN RECEPTOR REV/ERB
Mitchell A Lazar, Sylvan H. Eisman Professor Of Medicine &
University Of Pennsylvania 3451 Walnut Street Philadelphia, Pa 19104
Grant 2R01DK045586-05A1 from National Institute Of Diabetes And Digestive And Kidney Diseases IRG: BCE
Abstract: Rev-Erb is a fascinating orphan nuclear receptor encoded on the opposite strand of a thyroid hormone receptor gene. Rev-Erb belongs to a subset of the thyroid/retinoid receptor superfamily that includes ROR-alpha, which is crucial for cerebellar development. Rev-Erb and ROR-alpha are distinguished from other nuclear receptors because both bind DNA as monomers, requiring a receptor half-site flanked 5 prime by an extended A/T rich region. Rev-Erb and ROR-alpha are functionally antagonistic; Rev-Erb is a repressor whereas ROR-alpha is a transcriptional activator. Rev-Erb only represses transcription as a homodimer on a direct repeat of the monomer binding site spaced by 2 bp, to which it binds cooperatively. The first specific aim of this proposal is to understand the determinants of DNA binding by Rev-Erb and ROR-alpha. The roles of the extended DNA-binding domain will be delineated biochemically and by crystallographic analysis of purfied Rev-Erb bound to monomer and dimer sites. The molecular basis for homodimerization and lack of heterodimerization with RXR will be determined by analysis of receptor chimera and mutations. The second specific aim is to compare and contrast the transcriptional regulatory properties of ROR-alpha and Rev-Erb. Preliminary results suggest that transactivation by ROR-alpha requires dimerization, and the hypothesis that DNA-binding regulates interaction between ROR- alpha and transcriptional cofactors will be tested. In addition, an inherent, cell-specific repression domain in ROR-alpha will be characterized. The third specific aim is to characterize the mechanism and biological function of Rev-Erb and ROR-alpha interactions with the nuclear hormone receptor corepressors N- CoR and SMRT. Preliminary results indicate that N-CoR interacts differentially with Rev-Erb on different binding sites. Furthermore, different regions of Rev-Erb govern binding to N- CoR versus SMRT. ROR-alpha also interacts with N-CoR and SMRT. The molecular basis and function of these findings will be determined. The fourth specific aim is to characterize the mechanism and function of a novel Rev-Erb corepressor that we have isolated using the yeast two-hybrid system. The molecular basis of the interaction will be determined, and the biological relevance of the novel corepressor will be explored. Together, these studies will provide insight into the molecular mechanisms by which members of the Rev-Erb and ROR-alpha interact with target genes and coregulator proteins to both positively and negatively regulate transcription. These studies are required to understand the function of Rev-Erb and ROR-alpha and, more generally, will define novel mechanisms underlying specificity of gene regulation by the nuclear hormone receptor superfamily.
Keywords: gene induction /repression, genetic regulation, hormone receptor, protein structure, receptor expression, transcription factor, DNA binding protein, chimeric protein, dimer, receptor binding, structural biology, 3T3 cell, crystallization, restriction mapping, transfection, yeast two hybrid system
Project start date: 1992-09-30
Project end date: 2002-03-31
2R01DK045586-05A1 (1997): $247283
5R01DK045586-09 (2001): $263845
5R01DK045586-08 (2000): $264213
5R01DK045586-07 (1999): $258624
5R01DK045586-06 (1998): $251398
NUCLEAR HORMONE RECEPTORS IN ADIPOCYTE DIFFERENTIATION
Mitchell A Lazar, Sylvan H. Eisman Professor Of Medicine &
University Of Pennsylvania 3451 Walnut Street Philadelphia, Pa 19104
Grant 3R01DK049780-09S1 from National Institute Of Diabetes And Digestive And Kidney Diseases IRG: ZRG1
Abstract: Obesity and diabetes are leading causes of morbidity and death in the United States. Recently a nuclear hormone receptor, PPARgamma, has emerged as a critical regulator of both adipocyte differentiation and glucose metabolism, and hence a potential link between obesity and diabetes. PPARgamma is induced during adipocyte differentiation, and specific PPARgamma ligands such as the exciting new class of anti-diabetic drugs called thiazolidinediones (TZDs) not only promote adipogenesis but also enhance insulin action. PPARgamma activity is regulated allosterically by lipid soluble ligand activators as well as by MAP kinase-dependent phosphorylation which reduces PPARgamma activity by an unknown mechanism. This allows crosstalk between extracellular and intracellular signals, converging on PPARgamma. A major goal of this laboratory is to understand the relationships between regulation of PPARgamma activity, adipogenesis, and insulin resistance. We hypothesize that phosphorylation of PPARgamma contributes to cell-specific of PPARgamma activity by altering interactions with different ligands and transcriptional co- regulators. This hypothesis will be directly tested in Specific Aim 1, which is to understand the role of the N-terminus in ligand- dependent transcription by PPARgamma. Preliminary evidence indicates that phosphorylation of PPARgamma A/B domain results in reduced potency but not reduced efficacy of TZDs by lowering the ligand binding affinity of PPARgamma. This will be further evaluated by studying the effects of phosphorylation on the affinities of different ligands, and recruitment of co-activators and co-repressors. We also hypothesize the regulation of PPARgamma activity by phosphorylation plays a physiological role in maintenance of fuel metabolism. This hypothesis will be directly tested in Specific Aim 2, which is to determine the physiological and pathophysiological implications of PPARgamma phosphorylation. Here, the physiology of transgenic animals expressing PPARgamma mutants in adipose tissue will be evaluated in basal and stressed conditions, to determine the effects of dysregulation on homeostatic mechanisms regulating body weight and blood glucose levels. Finally, in Specific Aim 3, we will determine the mechanisms by which TZDs act on PPARgamma to promote insulin action in peripheral tissues. This is a major roadblock in the understanding of the role of PPARgamma activation in diabetes treatment. Given the abundance of PPARgamma in adipocytes, we hypothesize that PPARgamma ligands act on fat cells to alter expression of genes which signal muscle to respond more effectively to insulin. These signals could act directly or indirectly, and the effect of PPARgamma could be to increase or decrease their expression. We will isolate and characterize genes whose expression is regulated by PPARgamma ligands, especially TZD anti-diabetic drugs, and characterize their identity, regulation, and potential role in the mechanism of TZD action. Together, the proposed studies will address the major questions about PPARgamma and its role in body weight and glucose metabolism, which have profound implications for our society where diabetes and obesity are rampant.
Keywords: adipocyte, cell differentiation, hormone receptor, hormone regulation /control mechanism, insulin, lipid biosynthesis, receptor expression, biological signal transduction, body weight, diabetes mellitus, gene expression, genetic transcription, glucose metabolism, insulin sensitivity /resistance, intermolecular interaction, obesity, phosphorylation, protein structure function, troglitazone, embryonic stem cell, gene targeting, laboratory mouse, mutant, tissue /cell culture, transgenic animal
Project start date: 1995-07-01
Project end date: 2004-06-30
3R01DK049780-09S1 (2003): $45995
5R01DK049780-09 (2003): $348164
3R01DK049780-07S1 (2002): $34474
Sponsored Links Excellgen http://Excellgen.com
3R01DK049780-08S1 (2002): $45965
5R01DK049780-08 (2002): $339529
5R01DK049780-07 (2001): $331186
5R01DK049780-06 (2000): $323064
2R01DK049780-05 (1999): $314804
BIOLOGY OF THE ORPHAN RECEPTOR REV-ERBALPHA
Mitchell A Lazar
University Of Pennsylvania, 3451 Walnut Street, Philadelphia, Pa 19104
Grant 5R01DK045586-18 from National Institute Of Diabetes And Digestive And Kidney Diseases
Abstract: Nuclear receptors (NRs) are transcriptional regulators of metabolism. A major goal of this laboratory is to understand the biological importance of transcriptional repression by unliganded NRs. The orphan receptor Rev-erba has been particularly enlightening because it lacks an activation domain and is a constitutive represser. Transcriptional repression by Rev-erba is a key molecular component of the negative limb of the cell autonomous circadian clock. Normal circadian rhythms are disrupted both by absence of Rev-erba, and by unregulated persistence of its function. In the prior period, we showed that Rev-erba recruitment of the N-CoR/HDAC3 corepressor complex is critical for repression of circadian genes, and that modulation of Rev- erba protein stability initiates and synchronizes cellular clocks. Here we will determine the cellular mechanisms regulating Rev-erba activity, and explore the role of Rev-erba in regulating circadian activity and metabolic functions in liver and adipocytes. Specific Aim 1 is to determine molecular mechanisms regulating Rev-erba activity. Rev-erba protein is stabilized by GSKSp-dependent phosphorylation, and we hypothesize that this modification regulates association with a specific E3 ligase. Biochemical and molecular approaches will be taken to identify and validate the role of specific components of this complex, and determine their involvement in normal clock function. Preliminary data indicate that Rev-erba activity is also regulated by heme binding. We will test the hypothesis that this plays a critical role in regulating the normal role of Rev-erba in the cell autonomous clock. Specific Aim 2 is to determine the role of mechanisms regulating Rev-erba activity on circadian and metabolic physiology. We hypothesize that the mechanisms regulating cellular Rev-erba activity have important effects on circadian and metabolic processes. This will be tested by expressing informative Rev-erba mutants in liver, where Rev-erba normally regulates circadian gene expression and metabolism. Specific Aim 3 is to understand the role of Rev- erba in adipocyte biology. Rev-erba is induced during adipogenesis, and we hypothesize that it regulates not only adipocyte differentiation, but also the physiological functions of the mature adipocyte, including adipokine production and lipolysis, both of which display circadian rhythms in vivo. We will explore the role of Rev-erba activity in the basal and circadian regulation of these adipocyte genes as well as adipocyte metabolic functions. Insights gained from this work will shed new light on the transcriptional and post- transcriptional control of circadian and metabolic physiology, as well as interrelationships between circadian rhythms and metabolism. This has the potential to lead to new understanding of circadian and metabolic disorders, including obesity, diabetes, and cardiovascular disease
Keywords: Adipocytes; Adipose Cell; Adipose tissue; Animals; Avian Erythroblastosis Virus Oncogene A; Binding; Binding (Molecular Function); Biochemical; Biological; Biology; CUL-4A; CUL4A; Cardiovascular Diseases; Cell Function; Cell Process; Cell physiology; Cells; Cellular Function; Cellular Physiology; Cellular Process; Circadian Rhythms; Complex; Cullin Homolog 4A; Cultured Cells; Data; Diabetes Mellitus; Diurnal Rhythm; Drosophila; Drosophila genus; E3 Ligase; E3 Ubiquitin Ligase; Erythroblast Transforming Gene A; Extremities; Fat Cells; Fatty Tissue; Ferrate(2-), (7, 12-diethenyl-3, 8, 13, 17-tetramethyl-21H, 23H-porphine-2, 18-dipropanoato(4-)-N21, N22, N23, N24)-, dihydrogen, (SP-4-2)-; Ferroprotoporphyrin; Fruit Fly, Drosophila; Funding; Gases; Gene Down-Regulation; Gene Expression; Gene Transcription; GeneHomolog; Genes; Genes, erbA; Genetic Transcription; Goals; Heme; Heme b; Hepatic; Homolog; Homologous Gene; Homologue; Intermediary Metabolism; Investigators; KIAA1047; Laboratories; Lead; Leptin; Light; Limb structure; Limbs; Lipocytes; Lipolysis; Liver; METBL; Mature Lipocyte; Mature fat cell; Metabolic; Metabolic Diseases; Metabolic Disorder; Metabolic Processes; Metabolism; Modification; Molecular; Molecular Interaction; N-CoR; N-CoR1; NCOR1; NCOR1 protein, human; Non-Trunk; Nuclear Receptor Co-Repressor 1; Nuclear Receptor Corepressor; Nuclear Receptors; Nyctohemeral Rhythm; Ob Gene Product; Ob Protein; Obese Gene Product; Obese Protein; Obesity; Oncogene ERB A; Organism-Level Process; Organismal Process; Orphan; PAI-1; PAI1; PLANH1; Pb element; Phosphorylation; Photoradiation; Physiologic; Physiologic Processes; Physiological; Physiological Processes; Physiology; Plasminogen Activator Inhibitor 1; Play; Post-Transcriptional Control; Post-Transcriptional Regulation; Post-Transcriptional Regulation Process; Production; Programs (PT); Programs [Publication Type]; Protein Phosphorylation; Proteins; Protoheme; Protoheme IX; RNA Expression; Receptor Protein; Recruitment Activity; Regulation; Repression; Research Personnel; Researchers; Role; Serine or Cysteine Proteinase Inhibitor Clade E Member 1; Staging; Subcellular Process; TRAC1; Testing; Thesaurismosis; Thyroid Hormone Receptor Alpha-1, Oncogene; Thyroid Hormone- and Retinoic Acid Receptor-Associated Corepressor 1; Transcription; Transcription Repression; Transcription, Genetic; Transcriptional Repression; Twenty-Four Hour Rhythm; Type 1 Plasminogen Activator Inhibitor; Ubiquitin-Protein Ligase E3; Work; adipocyte biology; adipocyte development; adipocyte differentiation; adipogenesis; adipokines; adipose; adiposity; blood glucose regulation; body system, hepatic; cardiovascular disorder; circadian; circadian clock; circadian pacemaker; circadian process; corpulence; corpulency; corpulentia; cullin 4A; daily biorhythm; diabetes; diurnal variation; erbA Genes; ferroheme; fruit fly; gene product; gene repression; glucose control; glucose homeostasis; glucose regulation; hCIT529I10; hN-CoR; heavy metal Pb; heavy metal lead; heme a; heme-binding protein; hemin-binding proteins; human NCOR1 protein; in vivo; insight; lipid biosynthesis; lipogenesis; metabolism disorder; mutant; ob/ob mouse; obese; obese people; obese person; obese population; organ system, hepatic; programs; receptor; recruit; resistin; social role; ubiquitin-protein ligase; white adipose tissue; yellow adipose tissue
Project start date: 1992-09-30
Project end date: 2011-03-31
Budget start date: 1-APR-2010
Budget end date: 31-MAR-2011
5R01DK045586-18 (2010): $390140
5R01DK045586-17 (2009): $385926
2R01DK045586-15 (2007): $377693
Corepressors For The Orphan Receptor RevErb
Mitchell A Lazar, Sylvan H. Eisman Professor Of Medicine &
University Of Pennsylvania 3451 Walnut Street Philadelphia, Pa 19104
Grant 5R01DK045586-14 from National Institute Of Diabetes And Digestive And Kidney Diseases IRG: BCE
Abstract: Nuclear hormone receptors (NRs) are key transcriptional regulators of cellular growth, differentiation, and metabolism. The NR superfamily include receptors for hormones, vitamins, metabolites, and xenobiotics, as well as receptors without known ligands, termed orphan receptors, NRs repress transcription in the absence of ligand by recruiting co-repressors N-CoR and SMRT. Hormone action is largely the result of dissociation of co-repressor, accompanied by the recruitment of co-activator molecules. A major goal of this laboratory is to understand transcriptional repression from the interaction of NRs with the co-repressors to the molecular mediators of the repressive signal. The orphan receptor RevErb has been a superb of NRs with co-repressors to the molecular mediators of the repressive signal. The orphan receptor RevErb has been a superb model for learning about activation helix, and that RevErb homodimers recruit N-CoR to specific DNA binding sites. We hypothesize that each subunit of the DNA-bound RevErb homodimer binds to a specific DNA binding sites. We hypothesize that each subunit of the DNA-bound RevErb homodimer binds to a different CoRNR motif in N-CoR. This hypothesis will be directed tested in Specific Aim 1, which will determine the molecular mechanism by which RevErb homodimers interact with co-repressor using mutagenesis, chimeric receptors and co-repressors, and proteolytic mapping. We have also identified multiple histone deacetylases (HDACs) and other proteins that interact with N-CoR/SMRT and are potential mediators of the repressive signal. We hypothesize that different co-repressors and co-repressor associated proteins are recruited by RevErb and other NRs in a receptor- and cell-specific manner. Specific Aim 2 is to determine the co-repressors and co-repressor-associated proteins involved in repression by RevErb and other NRs in vivo, using chromatin immunoprecipitation and RNA interference. We have also shown that N-CoR and SMRT act as activ ating co-factors for the HDAC3 enzyme. Specific Aim 3 is to determine the mechanism of this phenomenon, and to understand its applicability to other co-repressors and other HDACs. We hypothesize that SMRT and N-CoR have others functions related to chromatin modification and transcriptional repression. Preliminary data indicate that one such function is histone binding. Specific Aim 4 is to understand the chromatin-related function of the co-repressors. The mechanism and function of histone binding will be elucidated. Together, these studies will elucidate basic mechanisms underlying the regulation of NR interactions with co-repressors, and co-repressor regulation of transcription. The insights gained from this work will expand our understanding of the mechanisms of hormone action, and has potential to lead to novel approaches to diseases associated with NR function, including obesity, diabetes, and leukemia.
Keywords: gene induction /repression, genetic regulation, genetic regulatory element, hormone receptor, hormone regulation /control mechanism, intermolecular interaction, protein structure function, receptor expression, transcription factor, amidohydrolase, chromatin, dimer, histone, molecular site, nucleic acid structure, posttranslational modification, protein localization, gel mobility shift assay, immunoprecipitation, peptide chemical synthesis, site directed mutagenesis, tissue /cell culture
Project start date: 1992-09-30
Project end date: 2007-03-31
5R01DK045586-14 (2006): $386938
5R01DK045586-13 (2005): $396250
Sponsored Links Excellgen http://Excellgen.com