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. ? ? ? ? ?
| Linnemann, Amelia K; O'Geen, Henriette; Keles, Sunduz et al. (2011) Genetic framework for GATA factor function in vascular biology. Proc Natl Acad Sci U S A 108:13641-6 |
