GATA Factor Regulation of Vascular Endothelium

描述(由申请人提供)：内皮细胞标志特征的发展依赖于内皮细胞特有的转录机制。包括GATA-2在内的多种因子建立内皮细胞特异性转录。人类GATA2基因多态性与冠状动脉疾病相关，GATA-2的表达与动脉硬化有关，GATA-2调控编码血管分子内皮素-1和血管黏附分子-1的基因。然而，GATA-2在内皮细胞中的功能和调节机制尚不清楚。以我们在造血细胞中对小鼠GATA-2的研究为基础，我们提出了一些研究，以阐明GATA-2在血管内皮细胞中的作用。GATA2基因包含5个“GATA开关位点”，分别由GATA-2和GATA-1在红系前体细胞的活跃和非活跃基因座上占据。GATA-1介导的GATA-2从这些位点的置换与抑制相耦合。在小鼠胚胎和人内皮细胞中，+9.5位点自主地激活LacZ转基因基因，而这一活性需要结合GATA因子的DNA基序。以下目的将检验关于GATA-2在血管内皮细胞中的功能和调节的假说：目的1-阐明一种新的GATA因子依赖的血管内皮细胞转录机制。我们将测试内皮细胞中+9.5位点增强子是否独特地需要GATA因子依赖的核心模块和额外的调节模块。将通过染色质免疫沉淀(CHIP)和芯片-微阵列芯片(CHIP-CHIP)分析来确定哪种GATA因子(S)占据内皮细胞中的GATA2。将对突变的转基因进行分析，以确定血管和造血特异性是否可以分离，以及+9.5位点是否可以重新编程以产生新的特异性。染色体构象捕获分析将用于确定GATA2是否在内皮细胞和造血细胞中采用独特的构象。将产生缺乏+9.5位点的突变小鼠，以确定其是否具有非冗余功能。目的2-确定人内皮细胞染色体上的GATA-2靶点。只有一小部分GATA基序在红系细胞中被占据。转录因子在血管内皮细胞中的特异性尚未得到研究。我们将测试内皮细胞类似造血细胞的假设，因为绝大多数GATA基序没有被占据。GATA-2的占有率将在整个人类3号染色体内皮细胞中进行测量。生物信息学分析将测试E-box和其他基序是否在被占用的GATA基序和未被占用的GATA基序上得到丰富，并将确定一组基因是潜在的GATA-2功能电路的预期组件。项目简介这个项目的重点是了解GATA-2，一种在内皮细胞和某些血细胞中表达的核蛋白，如何发挥作用和受到调控。由于GATA-2与动脉粥样硬化和冠状动脉疾病的发展有关，拟议的研究有望为这些人类疾病的潜在机制提供重要的见解。此外，GATA-2对维持造血干细胞至关重要，因此，揭示GATA-2在任何系统中潜在的功能机制，对于进一步了解成人干细胞生物学具有突出的潜力。

DESCRIPTION (provided by applicant): The development of hallmark features of endothelial cells relies upon endothelial cell-specific transcriptional mechanisms. Multiple factors, including GATA-2, establish endothelial cell-specific transcription. Human GATA2 polymorphisms correlate with coronary artery disease, GATA-2 expression is linked to arteriosclerosis, and GATA-2 regulates genes encoding the vascular molecules endothelin-1 and vascular adhesion molecule-1. However, mechanisms underlying GATA-2 function and regulation in endothelium are unknown. Using our work on mouse Gata2 in hematopoietic cells as a foundation, studies are proposed to elucidate how GATA-2 functions in vascular endothelium. The Gata2 locus contains five "GATA switch sites" that are occupied by GATA-2 and GATA-1 at the active and inactive loci, respectively, in erythroid precursor cells. GATA-1-mediated displacement of GATA-2 from these sites is coupled to repression. The +9.5 site functions autonomously to activate a LacZ transgene in endothelium of mouse embryos and human endothelial cells, and DNA motifs that bind GATA factors are required for this activity. The following aims will test hypotheses regarding GATA-2 function and regulation in vascular endothelium: Aim 1 - To elucidate a novel GATA factor-dependent transcriptional mechanism in vascular endothelium. We will test whether the +9.5 site enhancer in endothelium uniquely requires a GATA factor- dependent core module and additional regulatory modules. Chromatin immunoprecipitation (ChIP) and ChIP coupled to microarray chip (ChIP-chip) assays will be conducted to determine which GATA factor(s) occupy GATA2 in endothelial cells. Mutant transgenes will be analyzed to determine whether vascular and hematopoietic specificities can be dissociated and whether the +9.5 site can be reprogrammed to yield novel specificities. Chromosome conformation capture analysis will be used to determine whether GATA2 adopts a unique conformation in endothelial vs. hematopoietic cells. Mutant mice lacking the +9.5 site will be generated to determine whether it functions nonredundantly. Aim 2 - To identify GATA-2 target sites on human endothelial cell chromosomes. Only a small fraction of GATA motifs are occupied in erythroid cells. The specificity of transcription factor occupancy has not been studied in endothelial cells. We will test the hypothesis that endothelial cells resemble hematopoietic cells in that the vast majority of GATA motifs are not occupied. GATA-2 occupancy will be measured throughout human chromosome 3 in endothelial cells. Bioinformatics analysis will test whether E-boxes and other motifs are enriched at occupied versus nonoccupied GATA motifs and will identify an ensemble of genes as prospective components of circuitry underlying GATA-2 function. Project Narrative This project focuses on understanding how GATA-2, a nuclear protein expressed in endothelial cells and in certain blood cells, functions and is regulated. As GATA-2 is implicated in the development of atherosclerosis and coronary artery disease, the proposed studies are expected to provide important insights into mechanisms underlying these human disorders. Furthermore, GATA-2 is crucial to maintain hematopoietic stem cells and therefore uncovering mechanisms underlying GATA-2 function in any system has outstanding potential to further knowledge on adult stem cell biology.