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. ? ? ?
| Wozniak, Ryan J; Keles, Sunduz; Lugus, Jesse J et al. (2008) Molecular hallmarks of endogenous chromatin complexes containing master regulators of hematopoiesis. Mol Cell Biol 28:6681-94 |
