GATA Factor Function in Vascular Endothelium

描述(由申请人提供)：这项建议侧重于阐明GATA因子在血管系统中作用的分子机制，作为一种合理和创新的手段来识别内皮中新的GATA因子靶基因。这一点很重要，因为GATA因子失调与冠状动脉疾病和动脉粥样硬化等各种血管病理生理机制之间存在着多种联系，但人们对其在血管中的正常功能知之甚少。要阐明GATA因子功能改变在血管病理生理学发展中所起的确切作用(S)，需要更多地阐明它们在内皮细胞中作用模式的分子机制。此外，扩大我们对下游GATA因子靶基因和血管中靶基因网络的了解，将有助于我们深入了解GATA因子在这一背景下调控的生理过程。最近，我们发现了一个Gata2内含子增强子元件(位于Gata2 1S启动子下游9.5kb)，它自主地激活了小鼠胚胎血管内皮细胞和心内膜中的LacZ转基因基因，这是导致Gata2在血管内皮细胞表达的第一个分子决定簇。9.5位在胎肝(一个主要的造血点)和培养的红系前体细胞中也很活跃。相反，其他GATA2调节区(-77和-3.9kb)在造血祖细胞中具有GATA依赖的增强子活性，在体内缺乏内皮增强子功能。9.5位的内皮细胞增强子活性需要E-box-GATA复合元件，通过改变其插入序列的间距来使其失活。虽然-77位点也包含保守的E-box和GATA基序，但它们的间距和方向与9.5位点不同，这表明E-box-GATA复合元件在内皮细胞中有严格的结构限制。这是这一提议的中心假设，以下目标将定义调控内皮细胞中E-box-GATA复合元件功能的结构规则，并利用这些规则在血管内皮细胞中发现新的依赖于GATA基序和E-box的靶基因。 目的1.确定血管中GATA因子依赖的增强子活性的分子机制 内皮细胞。目的：寻找新的依赖GATA因子、血管内皮细胞特异的转录调控元件和靶基因。

DESCRIPTION (provided by applicant): This proposal focuses on elucidating the molecular mechanisms underlying GATA factor function in the vasculature as a rational and innovative means of identifying novel GATA factor target genes in endothelium. This is important since there are several links between GATA factor dysregulation and various vascular pathophysiologies such as coronary artery disease and atherosclerosis, but little is known about their normal functions in blood vessels. To elucidate the precise role(s) that altered GATA factor function plays in the development of vascular pathophysiologies demands greater clarification of the molecular mechanisms underlying their modes of action in endothelium. Furthermore, expanding our knowledge of downstream GATA factor target genes and target gene networks in the vasculature will yield insights into the physiologic processes regulated by GATA factors in this context. Recently, we discovered a Gata2 intronic enhancer element (located +9.5 kb downstream of the Gata2 1S promoter) which functions autonomously to activate a LacZ transgene in the vascular endothelium and endocardium of mouse embryos, representing the first molecular determinant implicated in establishing Gata2 expression in endothelium. The +9.5 site is also active in a subset of cells in the fetal liver (a major site of hematopoiesis) and in cultured erythroid precursors. By contrast, other Gata2 regulatory regions (-77 and -3.9 kb) with GATA-dependent enhancer activity in hematopoietic precursors lack endothelial enhancer function in vivo. Endothelial cell enhancer activity of the +9.5 site requires an E-box-GATA composite element, which is inactivated by altering the spacing of its intervening sequence. While the -77 site also contains conserved E-box and GATA motifs in close proximity, their spacing and orientation differ from that of the +9.5 site, suggesting that E-box-GATA composite elements have strict architectural constraints in endothelium. This is the central hypothesis of this proposal and the following aims will define the architectural rules governing the function of E-box-GATA composite elements in endothelial cells and exploit these rules to discover novel GATA motif- and E-box-dependent target genes in vascular endothelium. Aim 1. To define molecular mechanisms underlying GATA factor-dependent enhancer activity in vascular endothelium. Aim 2. To identify novel GATA factor-dependent, vascular endothelial cell-specific transcriptional regulatory elements and target genes.