GATA Factor Function in Vascular Endothelium

描述（由申请人提供）： 本研究旨在阐明加塔因子在血管系统中发挥作用的分子机制，为内皮细胞中新的加塔因子靶基因的鉴定提供一种合理和创新的方法。这是重要的，因为加塔因子失调和各种血管病理生理学如冠状动脉疾病和动脉粥样硬化之间存在几种联系，但对它们在血管中的正常功能知之甚少。为了阐明改变的加塔因子功能在血管病理生理学发展中所起的确切作用，需要进一步阐明其在内皮中作用模式的分子机制。此外，扩大我们的下游加塔因子靶基因和靶基因网络的知识，在血管系统中将产生的生理过程的见解调节加塔因素在这种情况下。最近，我们发现了一个Gata 2内含子增强子元件（位于Gata 2 1 S启动子下游+9.5 kb），其自主激活小鼠胚胎血管内皮和内皮细胞中的LacZ转基因，代表了在内皮细胞中建立Gata 2表达的第一个分子决定簇。+9.5位点在胎儿肝脏（造血的主要部位）和培养的红系前体细胞亚群中也有活性。相比之下，在造血前体中具有GATA依赖性增强子活性的其他Gata 2调节区（~ 77和~ 3.9kb）在体内缺乏内皮增强子功能。+9.5位点的内皮细胞增强子活性需要E-box-加塔复合元件，其通过改变其间插序列的间隔而失活。虽然-77位点也包含保守的E-box和加塔基序，但它们的间距和方向与+9.5位点不同，表明E-box-加塔复合元件在内皮中具有严格的结构限制。这是该提议的中心假设，并且以下目标将定义支配内皮细胞中的E-box-加塔复合元件的功能的结构规则，并且利用这些规则来发现血管内皮中的新的加塔基序和E-box依赖性靶基因。 目标1。为了明确血管内皮细胞中加塔因子依赖性增强子活性的分子机制， 内皮细胞目标二。鉴定新的加塔因子依赖性血管内皮细胞特异性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.