Chromatin Domain Regulation in Stem Cells

描述（由申请人提供）：GATA转录因子（GATA-1-6）调节哺乳动物的发育。GATA-2对于造血干细胞（HSC）和多能祖细胞的分化/存活非常重要。GATA-1和GATA-2占据细胞中结合基序的一小部分，在不同的发育时期可以占据相同的染色质区域，但具有不同的功能输出。以下Aims将分析调节GATA-2转录的机制，GATA-2水平的变化如何影响造血，以及GATA因子染色质占用的机制。1. 分析造血过程中染色质位点GATA开关的机制。GATA-2半衰期短（约1小时），用蛋白酶体抑制剂处理细胞后稳定。当GATA-2稳定时，gata -1介导的GATA-2从染色质上的位移减弱。我们将验证泛素化使GATA-2不稳定的假设，而GATA-1进入GATA-2结合的染色质位点需要不稳定。我们还将测试交换机是否需要额外的GATA-1和GATA-2。2. 探讨GATA-2在体内的转录机制。GATA-2占据活性GATA-2位点的-2.8 kb和-1.8 kb区域，而GATA-1主要占据非活性GATA-2位点的-2.8 kb区域。GATA-1结合将GATA-2从两个区域置换，并与抑制相结合。我们产生了-2.8 kb和-1.8 kb区域的靶向缺失，以验证这些区域赋予激活而-2.8 kb区域介导抑制的假设。我们将确定这些缺失是否会影响组蛋白修饰模式的组装、RNA聚合酶II的募集和转录。3. 检测GATA-1和GATA-2是否存在分化阶段特异性靶基因。我们提出，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了一个指定占用的GATA识别代码（GRC）。阐明GRC需要分析多个靶基因的占用情况。GATA因子占用率将通过定量染色质免疫沉淀（ChIP）和ChIP结合基因组微阵列来定义。这些研究将揭示GATA开关如何调节GATA-2转录，GATA-1和GATA-2如何选择DMA基序，以及与多种发育过程广泛相关的见解。

DESCRIPTION (provided by applicant): 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 kb and -1.8 kb 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. We propose 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.