Chromatin Domain Regulation in Stem Cells

描述(申请人提供)：GATA转录因子(GATA-1-6)调节哺乳动物的发育。GATA-2对造血干细胞(HSC)和多能祖细胞的分化/存活具有重要作用。GATA-1和GATA-2在细胞中占据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。以下目的将分析调节GATA-2转录的机制，GATA-2水平的变化如何影响造血，以及GATA因子染色质占据的潜在机制。1.分析造血过程中染色质部位GATA转换的机制。GATA-2的半衰期很短(~1h)，并通过蛋白酶体抑制剂处理细胞而稳定下来。当GATA-2稳定时，GATA-1介导的GATA-2从染色质上的置换被减弱。我们将检验这样的假设，即泛素化破坏GATA-2的稳定性，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.剖析GATA-2在体内转录的机制。GATA-2占据了GATA-2活性位点的-2.8kb和-1.8kb区域，而GATA-1主要占据了非活性位点的-2.8kb区域。GATA-1结合取代了这两个区域的GATA-2，并与抑制物偶联。我们产生了有针对性的-2.8kb和-1.8kb区域的缺失，以检验这些区域提供激活和-2.8kb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的招募和转录。3.检测GATA-1和GATA-2是否具有分化阶段特异性靶基因。我们认为，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有率的GATA识别码。阐明GRC需要分析多个靶基因的占位情况。GATA因子占有率将通过定量染色质免疫沉淀(CHIP)和芯片与基因组微阵列相结合来定义。这些研究将揭示GATA开关如何调控GATA-2转录，GATA-1和GATA-2如何选择DMA基序，以及与不同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.