Hematopoietic Regulation of GATA Switches

描述（申请人提供）：加塔转录因子（加塔-1-6）调节哺乳动物发育。加塔-2对于造血干细胞（HSC）和多能祖细胞分化/存活是重要的。加塔-1和加塔-2占据细胞中结合基序的一小部分，并且可以在不同的发育时间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加塔-2转录的机制，加塔-2水平的变化如何影响造血，以及加塔因子染色质占据的潜在机制。1.分析造血过程中染色质位点加塔转换的机制。加塔-2的半衰期较短（约1 h），通过蛋白酶体抑制剂处理细胞可使其稳定。当加塔-2稳定时，加塔-1介导的加塔-2从染色质的置换减弱。我们将检验这样的假设，即泛素化使加塔-2不稳定，而不稳定是加塔-1进入加塔-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.探讨加塔-2在体内的转录机制。加塔-2占据活性加塔-2基因座的-2.8 kb和-1.8 kb区域，而加塔-1主要占据非活性基因座的-2.8 kb区域。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-2.8 kb和-1.8 kb区域的靶向缺失，以测试这些区域赋予激活而-2.8 kb区域介导阻遏的假设。我们将确定缺失是否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，图案的内在特征，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加塔识别码（GRC），指定占用。阐明GRC需要分析多个靶基因的占有率。将通过定量染色质免疫沉淀（ChIP）和ChIP与基因组微阵列偶联来定义加塔因子占有率。这些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.