Amy Susan Weinmann
University Of Washington
Project start date: 2004-06-01
Project end date: 2015-01-31
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Molecular Characterization Of T-bet´s Role In Immunity
Amy Susan Weinmann
Immunologyuniversity Of Washington
office Of Sponsored Programs
seattle, Wa 981959472
Grant 5R01AI061061-05 from National Institute Of Allergy And Infectious Diseases IRG: IMB
Abstract: Lineage-restricted transcription factors play a critical role in the hematopoietic differentiation process and the deregulation of even a single factor can lead to disease states such as leukemia, autoimmunity, and allergic reactions. Part of how this occurs is that the selective loss of a factor can drastically alter the development of critical effector cell populations. Therefore, identifying the gene expression cascades influenced by these factors will allow us to address the molecular mechanisms that contribute to the disease phenotypes. The transcription factor T-bet appears to play a significant role in the development of CD4+ T helper 1 cells. Th1 cells secrete IFNgamma and are important in the development of cell-mediated immunity. A hyperactive Th1 response can contribute to autoimmune diseases such as Crohn´s disease and type 1diabetes. In addition to Th1 cells, T-bet is also expressed in multiple lineages in the immune system and is thought to contribute to cell-specific functions in each effector cell population. In the absence of T-bet, cell-specific defects occur in natural killer, dendritic, and B cells. It is also worth noting that murine disease models for Crohn´s disease, asthma, and lupus have all been established by altering T-bet levels. Therefore, one may hypothesize that T-bet plays an important role in the developing immune response. The main goal of this proposal is to address the molecular mechanisms behind T-bet´s role in the individual hematopoietic effector cell populations. Aim 1 is designed to identify direct T-bet target genes in each cell population. Is T-bet targeted to loci in a cell-specific manner or does it interact with the same genes in all cellular settings? To address this question, target gene identification strategies will be employed that utilize modifications to the chromatin immunoprecipitation procedure. These techniques will only identify genes that are directly bound by T-bet within the context of a given nuclear environment. In Aim 2, the functional consequence of T-bet´s association with select promoters will be examined to determine if this interaction is productive in the different cellular settings. The studies in Aim 1 and 2 will provide a means to address T-bet´s cell-specifications. Aims 3 and 4 will address the role T-bet plays in the transcriptional regulation of a few select target genes. Does T-bet function as a transcriptional activator or repressor? Does T-bet influence chromatin events or aid in RNA polymerase II recruitment to the promoter? Detailed transcriptional regulation studies of a few select target genes will aid in answering these questions
Keywords: cell, immunity, role Crohn`s disease, DNA directed RNA polymerase, asthma, autoimmunity, cell type, chromatin, chromatin immunoprecipitation, dendritic cell, disease /disorder model, element, environment, gene, gene expression, gene targeting, health /scientific organization, immune response, immune system, insight, insulin dependent diabetes mellitus, lead, leukemia, macrophage, phenotype, play, repression, thinking, transcription factor
Project start date: 2004-06-01
Project end date: 2010-05-31
5R01AI061061-05 (2008): $282026
Molecular Characterization Of T-bet s Role In Immunity
Amy Susan Weinmann
University Of Washington Office Of Sponsored Programs Seattle, Wa 98105
Grant 5R01AI061061-04 from National Institute Of Allergy And Infectious Diseases IRG: IMB
Abstract: Lineage-restricted transcription factors play a critical role in the hematopoietic differentiation process and the deregulation of even a single factor can lead to disease states such as leukemia, autoimmunity, and allergic reactions. Part of how this occurs is that the selective loss of a factor can drastically alter the development of critical effector cell populations. Therefore, identifying the gene expression cascades influenced by these factors will allow us to address the molecular mechanisms that contribute to the disease phenotypes. The transcription factor T-bet appears to play a significant role in the development of CD4+ T helper 1 cells. Th1 cells secrete IFNgamma and are important in the development of cell-mediated immunity. A hyperactive Th1 response can contribute to autoimmune diseases such as Crohn s disease and type 1diabetes. In addition to Th1 cells, T-bet is also expressed in multiple lineages in the immune system and is thought to contribute to cell-specific functions in each effector cell population. In the absence of T-bet, cell-specific defects occur in natural killer, dendritic, and B cells. It is also worth noting that murine disease models for Crohn s disease, asthma, and lupus have all been established by altering T-bet levels. Therefore, one may hypothesize that T-bet plays an important role in the developing immune response. The main goal of this proposal is to address the molecular mechanisms behind T-bet s role in the individual hematopoietic effector cell populations. Aim 1 is designed to identify direct T-bet target genes in each cell population. Is T-bet targeted to loci in a cell-specific manner or does it interact with the same genes in all cellular settings? To address this question, target gene identification strategies will be employed that utilize modifications to the chromatin immunoprecipitation procedure. These techniques will only identify genes that are directly bound by T-bet within the context of a given nuclear environment. In Aim 2, the functional consequence of T-bet s association with select promoters will be examined to determine if this interaction is productive in the different cellular settings. The studies in Aim 1 and 2 will provide a means to address T-bet s cell-specifications. Aims 3 and 4 will address the role T-bet plays in the transcriptional regulation of a few select target genes. Does T-bet function as a transcriptional activator or repressor? Does T-bet influence chromatin events or aid in RNA polymerase II recruitment to the promoter? Detailed transcriptional regulation studies of a few select target genes will aid in answering these questions.
Project start date: 2004-06-01
Project end date: 2009-05-31
5R01AI061061-04 (2007): $287488
Grants awarded to Amy Susan Weinmann
TRANSCRIPTIONAL CONTROL OF INTERFERON-GAMMA IN T AND NK CELLS
Amy Susan Weinmann, Assistant Professor
University Of Washington, Office Of Sponsored Programs, Seattle, Wa 98195-9472
Grant 5R01AI071272-10 from National Institute Of Allergy And Infectious Diseases
Abstract: IFN-?, a cytokine produced primarily by NK cells and by Th1 CD4 and CD8 T cells, plays a central role in the immune response to infection with intracellular pathogens. Conversely, protection against extracellular metazoans is dependent on Th2 CD4 T cells and the cytokines they produce, which include IL-4, IL-13 and IL-5. Expression of IFN-? and of Th2 cytokines is primarily regulated at the level of transcription. While transcription factors that govern IFN-? expression have in recent years been elucidated in considerable detail, very little is known regarding where and how they act. By contrast to IFN-?, regulation of the Th2 cytokines IL-4, IL-13 and IL-5, which are clustered into a single ~150 kb locus in mammals, is now understood in considerable molecular detail. In this application, we propose to gain a similar level of knowledge regarding IFN-? by comprehensively identifying the regulatory elements governing IFN-? expression and the functional boundaries of the Ifng locus through 3 Aims Aim 1 Identify regulatory elements governing the expression of IFN-? through comprehensive chromatin profiling of the murine Ifng locus. Hypothesis DNase hypersensitive sites and regions enriched in transcriptionally favorable histone modifications extend ~50 kb upstream and downstream of the murine Ifng gene and identify regulatory elements governing IFN-? expression. Aim 2 Define the functional boundaries of the Ifng locus using BAG transgenes to complement the defect in IFN-? knockout (GKO) mice. Hypothesis BAG transgenes containing the murine Ifng gene and extending >= 50 kb 5´ and 3´ will reconstitute proper IFN-? expression and thereby complement the immunological defect in GKO mice. Aim 3 Identify the mechanisms bv which the transcriptional regulatory elements within the Ifng locus influence IFN-? expression and the key transcription factors that act at these sites. Hypotheses T-bet, STAT4, NFAT, NF-?B and AP-1 bind not only to the Ifng promoter but to additional upstream and downstream regulatory elements, thereby affecting IFN-? expression; Ifng5´CNS 2 and 3 and/or Ifng3´CNS3 help to insulate the Ifng locus from the influence of surrounding genes
Keywords: AP-1; AP-1 Enhancer-Binding Protein; AP1; AP1 protein; Activator Protein-1; Address; Adopted; Affect; B Cell Differentiation Factor I; B cell activating factor; B cell growth factor; B cell growth factor 2; B-Cell Differentiation Factor-1; B-Cell Growth Factor-1; B-Cell Growth Factor-I; B-Cell Growth Factor-II; B-Cell Proliferating Factor; B-Cell Stimulating Factor; B-Cell Stimulating Factor-1; B-Cell Stimulation Factor-1; B-Cell Stimulatory Factor-1; BAF; BCDF-1; BCGF; BCGF-1; BCGF-II; BCGF2; BCGF2 (B cell growth factor 2); BCSF 1; BSF-1; BSF1; BSF1 (B cell stimulating factor 1); Binding; Binding (Molecular Function); Binetrakin; CD4 Positive T Lymphocytes; CD4 T cells; CD4 lymphocyte; CD4+ T cell; CD4+ T-Lymphocyte; CD4-Positive Lymphocytes; CD8; CD8B; CD8B1; CD8B1 gene; Cell Function; Cell Process; Cell physiology; Cells; Cells, CD4; Cellular Function; Cellular Physiology; Cellular Process; Chromatin; Complement; Complement Proteins; Cytokine Gene; Cytotoxic cell; DNA Methylation; DNase; Defect; Deoxyribonucleases; Distal; EDF; Enhancer-Binding Protein AP1; Eo-CSF; Eosinophil Differentiation Factor; Epigenetic; Epigenetic Change; Epigenetic Mechanism; Epigenetic Process; Gamma interferon; Gene Transcription; Genes; Genes, Regulator; Genetic Transcription; IFN; IFN-Gamma; IFN-g; IFNG; IL-13; IL-4; IL-5; IL13; IL4; IL4 Protein; IL5; IgA enhancing factor; Immune response; In Vitro; Infection; Interferon Gamma; Interferon Type II; Interferon gamma (human lymphocyte protein moiety reduced); Interferon, Immune; Interferon-gamma; Interferons; Interleukin 5 (Colony-Stimulating Factor, Eosinophil); Interleukin 5 Precursor; Interleukin-13; Interleukin-4; Interleukin-4 Precursor; Interleukin-5; K lymphocyte; Knock-out; Knockout; Knowledge; LYT3; Lymphocyte Stimulatory Factor 1; MCGF-2; Maintenance; Maintenances; Mammalia; Mammals; Mammals, General; Mammals, Mice; Mast Cell Growth Factor-2; Mediating; Mice; Molecular; Molecular Interaction; Murine; Mus; NK Cells; Natural Killer Cells; Nucleases, DNA; Nucleic Acid Regulatory Sequences; Play; Production; Programs (PT); Programs [Publication Type]; Promoter; Promoters (Genetics); Promotor; Promotor (Genetics); RNA Expression; Regulation; Regulator Genes; Regulator Regions, Nucleic Acid; Regulatory Element; Regulatory Regions; Regulatory Regions, Nucleic Acid (Genetics); Regulatory Sequences, Nucleic Acid; RegulatoryElement; Relative; Relative (related person); Repression; Reticuloendothelial System, Thymus; Role; STAT4; STAT4 gene; Site; Subcellular Process; T cell replacing factor; T-Cell Growth Factor 2; T-Cell Replacing Factor; T-Cells; T-Lymphocyte; T4 Cells; T4 Lymphocytes; Thymus; Thymus Gland; Thymus Proper; Thymus-Dependent Lymphocytes; To specify; Transcription; Transcription Factor AP-1; Transcription Regulation; Transcription, Genetic; Transcriptional Control; Transcriptional Regulation; Transcriptional Regulatory Elements; Transgenes; cytokine; extracellular; genetic regulatory element; helper T cell; histone modification; host response; immunoresponse; in vitro Model; in vivo; lFN-Gamma; pathogen; programs; reconstitute; reconstitution; regulatory gene; social role; thymus derived lymphocyte; trans acting element; transcription factor
Project start date: 2000-05-01
Project end date: 2011-06-30
Budget start date: 1-JUL-2010
Budget end date: 30-JUN-2011
5R01AI071272-10 (2010): $331801
MOLECULAR CHARACTERIZATION OF T-BET´S ROLE IN IMMUNITY
Amy Susan Weinmann, Assistant Professor
University Of Washington, Office Of Sponsored Programs, Seattle, Wa 98195-9472
Grant 2R01AI061061-06A1 from National Institute Of Allergy And Infectious Diseases
Abstract: At cell fate decision checkpoints, a precise series of events occur to establish developmentally appropriate gene expression profiles. At the molecular level, lineage-determinant transcription factors are required for the simultaneous activation and repression of genes that define the fate of a cell. Currently, it is unclear how these factors mechanistically achieve this precise control on a global level. In the immune system, naive CD4+ T helper cells begin with the potential to become a number of phenotypically distinct lineages, with key transcription factors committing them to a defined fate that is appropriate for a pathogenic insult. The T-box transcription factor T-bet is responsible for the differentiation of the Th1 cell lineage. Previous work has shown that T-bet positively regulates the effector cytokines and chemokine receptors that are the prototypic genes in Th1 cellular differentiation. To activate these select target genes, T-bet participates in at least three physically separable activities 1) H3K27-demethylation, 2) H3K4-methylation, and 3) transactivation events that occur independent from the chromatin environment. It is currently unclear, however, whether these separable functional activities are required at all target promoters, or rather they are selectively utilized in a context-specific manner. In addition, the mechanism by which T-bet represses the gene expression profiles for the alternative helper T cell lineages and whether T-bet´s ability to negatively regulate these genes requires the same or distinct activities is unknown. We will examine these questions on both a global and select target gene level. To this end, we will utilize mutant constructs deficient in each of T-bet´s defined functional activities and assess their ability to regulate the global T-bet-dependent gene expression patterns in Th1 cell differentiation. We also will create mice deficient in defined interacting proteins to determine their biological relevance in regulating T-bet-dependent gene expression profiles in Type 1 immune responses. We will utilize this knowledge to model T-bet-dependent target gene networks that are based upon the mode of regulation. Together, these studies will allow us to define the mechanisms that are needed for the precise regulation of Th1 differentiation. A new and exciting possibility in medical research is to selectively target therapeutic interventions for individual pathways that are pathogenically altered in human disease. This will allow for the greatest therapeutic benefit with the least number of unintentional detrimental side effects. In order to accomplish this goal in molecular medicine, we must precisely define the pathways that are regulated by the individual activities of the key factors involved in maintaining a healthy state. Our studies will address this from the standpoint of a key factor that regulates immune responses, which when altered, has been shown to be associated with diseases such as type 1 diabetes, ulcerative colitis, multiple sclerosis, cancer metastasis, and increased susceptibility to infectious disease
Keywords: 21+ years old; Address; Adult; Adverse effects; Autoimmune; Autoimmune Process; Biological; Body Tissues; Boxing; CD4 Positive T Lymphocytes; CD4 T cells; CD4 lymphocyte; CD4+ T cell; CD4+ T-Lymphocyte; CD4-Positive Lymphocytes; Cardiac Abnormalities; Cardiac Defects, Congenital; Cardiac Malformation; Cardiac defect; Cell Differentiation; Cell Differentiation process; Cell Lineage; Cells; Cells, CD4; Chromatin; Classification; Cleft Palate; Commit; Communicable Diseases; Congenital Heart Defects; Development; Developmental Gene; Diabetes Mellitus, Brittle; Diabetes Mellitus, Insulin-Dependent; Diabetes Mellitus, Juvenile-Onset; Diabetes Mellitus, Ketosis-Prone; Diabetes Mellitus, Sudden-Onset; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type I; Disease; Disorder; Embryo Development; Embryogenesis; Embryonic Development; Environment; Epigenetic; Epigenetic Change; Epigenetic Mechanism; Epigenetic Process; Event; Expression Profiling; Expression Signature; Extremities; Family; Family member; Gene Down-Regulation; Gene Expression; Gene Targeting; Gene Transcription; Gene, Developmental; Generations; Genes; Genetic; Genetic Condition; Genetic Diseases; Genetic Transcription; Genetics, Human; Goals; Heart Abnormalities; Heart Defects, Congenital; Heart Malformation; Helper Cells; Helper T-Cells; Helper-Inducer T-Lymphocyte; Hereditary Disease; Histones; Human Genetics; Human, Adult; Hypophysis; Hypophysis Cerebri; IDD; IDDM; Immune response; Immune system; Immunity; Immunodeficiency and Cancer; Incidence; Individual; Inducer Cells; Infectious Disease Pathway; Infectious Diseases; Infectious Diseases / Laboratory; Infectious Diseases Research; Infectious Diseases and Manifestations; Infectious Disorder; Insulin-Dependent Diabetes Mellitus; Knowledge; Limb structure; Limbs; Logic; MS (Multiple Sclerosis); Mammals, Mice; Medical Research; Metastasis; Metastasize; Metastatic Neoplasm; Metastatic Tumor; Methylation; Mice; Modeling; Molecular; Molecular Disease; Molecular Fingerprinting; Molecular Medicine; Molecular Profiling; Multiple Sclerosis; Murine; Mus; Neoplasm Metastasis; Nervous System, Pituitary; Network-based; Non-Trunk; Pathway interactions; Pattern; Pituitary; Pituitary Gland; Play; Predisposition; Programs (PT); Programs [Publication Type]; Promoter; Promoters (Genetics); Promotor; Promotor (Genetics); Protein Methylation; Proteins; RNA Expression; Recruitment Activity; Regulation; Research, Medical; Role; Sclerosis, Disseminated; Secondary Neoplasm; Secondary Tumor; Series; Susceptibility; System; System, LOINC Axis 4; Systematics; T-Cells; T-Cells, Helper-Inducer; T-Lymphocyte; T-Lymphocytes, Helper; T-Lymphocytes, Inducer; T-bet protein; T-bet transcription factor; T1 diabetes; T1D; T1DM; T4 Cells; T4 Lymphocytes; Targetings, Gene; Th-1 Cell; Th1 Cells; Therapeutic; Therapeutic Intervention; Thymus-Dependent Lymphocytes; Tissues; Trans-Activation (Genetics); Transactivation; Transcription; Transcription Repression; Transcription, Genetic; Transcriptional Repression; Treatment Side Effects; Tumor Cell Migration; Type 1 Helper Cell; Type 1 diabetes; Ulcerated Colitis; Ulcerative Colitis; Work; adult human (21+); body system, allergic/immunologic; cancer metastasis; chemokine receptor; cytokine; demethylation; disease/disorder; gene product; gene repression; genetic disorder; heart defect; helper T cell; hereditary disorder; host response; human disease; immunoresponse; in vivo; insular sclerosis; insulin dependent diabetes; intervention therapy; juvenile diabetes; juvenile diabetes mellitus; ketosis prone diabetes; molecuar profile; molecular signature; mouse model; mutant; organ system, allergic/immunologic; pathway; programs; public health relevance; recruit; side effect; social role; therapy adverse effect; thymus derived lymphocyte; transcription factor; treatment adverse effect; type I diabetes
Relevance: A new and exciting possibility in medical research is to selectively target therapeutic interventions for individual pathways that are pathogenically altered in human disease. This will allow for the greatest therapeutic benefit with the least number of unintentional detrimental side effects. In order to accomplish this goal in molecular medicine, we must precisely define the pathways that are regulated by the individual activities of the key factors involved in maintaining a healthy state. Our studies will address this from the standpoint of a key factor that regulates immune responses, which when altered, has been shown to be associated with diseases such as type 1 diabetes, ulcerative colitis, multiple sclerosis, cancer metastasis, and increased susceptibility to infectious disease
Project start date: 2004-06-01
Project end date: 2015-01-31
Budget start date: 15-FEB-2010
Budget end date: 31-JAN-2011
PFA/PA: PA-07-070
2R01AI061061-06A1 (2010): $146250