Hematopoietic Regulation of GATA Switches

GATA 开关的造血调节

• 批准号: 7566034
• 负责人: Emery H Bresnick
• 金额: $ 23.55万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-02-01 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7566034
• 关键词: Affect; Attenuated; Binding; Blood Cells; Cell Differentiation process; Cell Line; Cell Survival; Cells; Chromatin; Chromatin Structure; Code; Complex; Coupled; DNA; Development; Developmental Process; Elements; Family; Friends; Gene Targeting; Genes; Genetic Transcription; Genome; Genomics; Half-Life; Hematopoiesis; Hematopoietic; Histone Acetylation; Homeostasis; In Vitro; Mediating; Modeling; Multipotent Stem Cells; Mus; Nucleoproteins; Null Lymphocytes; Output; Pattern; Population; Process; Proteasome Inhibitor; RNA Polymerase II; Regulation; Relative (related person); Repression; Site; Specific qualifier value; Specificity; Staging; Stem cells; Testing; Time; Ubiquitination; chromatin immunoprecipitation; embryonic stem cell; histone modification; human GATA1 protein; in vivo; insight; progenitor; protein protein interaction; stem; transcription factor

GATA transcription factors (GATA-1-6) regulate mammalian development. GATA-2 is important for hematopoietic stem (HSC) and multipotent progenitor cell differentiation/survival. GATA-1 and GATA-2 occupy a small subset of the binding motifs in cells and can occupy the same chromatin region at distinct developmental times, but with different functional outputs. The following Aims will analyze mechanisms that regulate GATA-2 transcription, how changes in GATA-2 levels affect hematopoiesis, and mechanisms underlying GATA factor chromatin occupancy. 1. To analyze the mechanism of the GATA switch at chromatin sites during hematopoiesis. GATA-2 has a short half-life (~1 h) and is stabilized by treatment of cells with proteasome inhibitors. When GATA-2 is stabilized, GATA-1-mediated displacement of GATA-2 from chromatin is attenuated. We will test the hypothesis that ubiquitination destabilizes GATA-2, and instability is required for GATA-1 to access GATA-2-bound chromatin sites. We will also test whether an excess of GATA-1 versus GATA-2 is required for the switch. 2. To dissect the mechanism of GATA-2 transcription in vivo. GATA-2 occupies the-2.8 kband-1.8kb regions of the active GATA-2 locus, whereas GATA-1 occupies predominantly the -2.8 kb region of the inactive locus. GATA-1 binding displaces GATA-2 from both regions and is coupled to repression. We generated targeted deletions of the -2.8 kb and -1.8 kb regions to test the hypothesis that these regions confer activation and the -2.8 kb region mediates repression. We will determine if the deletions affect assembly of the histone modification pattern, RNA polymerase II recruitment, and transcription. 3. To test whether GATA-1 and GATA-2 have differentiation stage-specific target genes. Wepropose that intrinsic features of the motifs, nearby c/s-elements, protein-protein interactions and chromatin structure constitute a GATA Recognition Code (GRC) that specifies occupancy. Elucidating the GRC requires analysis of occupancy at multiple target genes. GATA factor occupancy will be defined by quantitative chromatin immunoprecipitation (ChIP) and ChIP coupled with genomic microarrays. The studies will reveal how GATA switches regulate GATA-2 transcription, how GATA-1 and GATA-2 select DMA motifs, and insights of broad relevance to diverse developmental processes.

加塔转录因子（加塔-1-6）调节哺乳动物的发育。加塔-2对于 造血干细胞（HSC）和多能祖细胞分化/存活。加塔-1和加塔-2 占据细胞中结合基序的一小部分，并且可以在不同的位置占据相同的染色质区域。 发展时间，但具有不同的功能输出。以下目标将分析机制 调节加塔-2转录，加塔-2水平的变化如何影响造血， 加塔因子染色质占据的潜在机制。1.分析其作用机理， 造血过程中染色质位点的加塔转换。加塔-2具有短半衰期（~1小时）， 通过用蛋白酶体抑制剂处理细胞来稳定。当加塔-2稳定时，加塔-1介导的 加塔-2从染色质的置换被减弱。我们将检验泛素化 使加塔-2不稳定，并且不稳定性是加塔-1接近加塔-2结合的染色质位点所必需的。 我们还将测试交换机是否需要超过加塔-1和加塔-2。2.到 探讨加塔-2在体内的转录机制。加塔-2占用-2.8 kb带宽-1.8kb 在活性加塔-2基因座的-2.8 kb区域，而加塔-1主要占据活性GATA-2基因座的-2.8 kb区域。 非活性基因座加塔-1结合从两个区域置换加塔-2并与阻遏偶联。我们 产生了-2.8kb和-1.8kb区域的靶向缺失，以检验这些区域 赋予激活，而~ 2.8kb区域介导阻遏。我们将确定删除是否会影响 组蛋白修饰模式的组装、RNA聚合酶II募集和转录。3.到 检测加塔-1和加塔-2是否具有分化阶段特异性靶基因。我们建议 基序的内在特征，附近的c/s元件，蛋白质-蛋白质相互作用和染色质 结构构成指定占用加塔识别码（GRC）。阐明GRC 需要分析多个靶基因的占有率。加塔因子占用率将由以下定义： 定量染色质免疫沉淀（ChIP）和与基因组微阵列偶联的ChIP。的 研究将揭示加塔开关如何调节加塔-2转录，加塔-1和加塔-2 选择DMA基序，和广泛的相关性，以不同的发展过程的见解。