Hematopoietic Regulation of GATA Switches

GATA转录因子(GATA-1-6)调控哺乳动物的发育。GATA-2对于 造血干细胞(HSC)与多能祖细胞分化/存活。GATA-1和GATA-2 在细胞中占据结合基序的一小部分，并可以在不同的 发育时间不同，但功能输出不同。以下目标将分析机制 调节GATA-2转录，GATA-2水平的变化如何影响造血，以及 GATA因子染色质占位的机制。1.分析其形成的机制。 在造血过程中，GATA在染色质部位发生切换。GATA-2的半衰期很短(~1小时)， 通过用蛋白酶体抑制剂处理细胞而稳定的。当GATA-2稳定时，GATA-1介导的 GATA-2从染色质上的位移被减弱。我们将检验泛素化的假设 破坏GATA-2的稳定，而GATA-1需要不稳定才能访问GATA-2结合的染色质位点。 我们还将测试交换机是否需要超过GATA-1和GATA-2。2.至 剖析GATA-2在体内转录的机制。GATA-2占用-2.8kb频段-1.8kb 活性GATA-2基因座的区域，而GATA-1主要占据-2.8kb的区域 不活跃的轨迹。GATA-1结合取代了这两个区域的GATA-2，并与抑制物偶联。我们 生成了-2.8kb和-1.8kb区域的有针对性的缺失，以测试这些区域的假设 授予激活和-2.8kb区域介导抑制。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的招募和转录。3.至 检测GATA-1和GATA-2是否具有分化阶段特异性靶基因。我们建议 基序、邻近c/S元件、蛋白质-蛋白质相互作用和染色质的内在特征 结构构成指定占有率的GATA识别码(GRC)。阐明GRC 需要分析多个靶基因的占位情况。GATA要素占有率将通过以下方式定义 染色质定量免疫沉淀(CHIP)及其与基因组微阵列的联用。这个 研究将揭示GATA开关如何调控GATA-2转录，GATA-1和GATA-2如何 精选DMA主题，以及与不同发展过程广泛相关的见解。

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.