Dissecting the Mechanism of Endogenous GATA-2 Transcription: GATA Factor Interplay at Multiple Upstream Regulatory Regions Establishes Long-Range Transcriptional Control.

Given the simplicity of the DNA sequence that mediates binding of GATA transcription factors, GATA motifs reside throughout chromosomal DNA. However, analyses of GATA-1 occupancy by chromatin immunoprecipitation (ChIP) revealed that GATA-1 discriminates exquisitely among these sites. GATA-2 selectively occupies the −2.8 kb region of the GATA-2 locus in the active state, despite there being numerous GATA motifs throughout the locus. The GATA-1-mediated displacement of GATA-2 is tightly coupled to repression of GATA-2 transcription. We have used high-resolution ChIP to show that GATA-1 and GATA-2 occupy two additional regions, −3.9 kb and −1.8 kb of the GATA-2 locus. GATA-1 and GATA-2 had distinct preferences for occupancy at these regions, with GATA-1 and GATA-2 occupancy highest at the −3.9 kb and −1.8 kb regions, respectively. Activation of an estrogen receptor fusion to GATA-1 (ER-GATA-1) induced similar kinetics of ER-GATA-1 occupancy and loss of GATA-2 occupancy at the sites. As the distinct preferences for GATA factor occupancy were not evident from electrophoretic mobility shift assay analysis in vitro, establishment of the preferences requires the native nucleoprotein structure of the GATA-2 locus. In the transcriptionally active state, the −3.9 kb and −1.8 kb regions formed strong DNaseI hypersensitive sites (HSs), and the −2.8 kb region formed a weak HS. Whereas ER-GATA-1-instigated repression abolished the −1.8 kb HS, the −3.9 kb HS persisted in the inactive state. Transient transfection analysis provided evidence that the −3.9 kb region functions distinctly from the −2.8 kb and −1.8 kb regions. Molecular studies, including capturing chromosome conformation (3C) analysis, are underway to dissect the structure/function of the upstream regulatory regions. Based on the high sequence conservation and occupancy by GATA factors in vivo, targeted deletions of the −2.8 kb and −1.8 kb regions were generated. Mutant mice are being developed, and functional analysis is being conducted via in vitro differentiation of homozygous mutant ES cells. Our results support a model in which dynamic changes in GATA factor occupancy and chromatin structure at the −3.9 kb, −2.8 kb, and −1.8 kb regions are intimately controlled by Friend of GATA-1 and additional coregulators. This GATA factor interplay is essential for GATA-2 transcriptional regulation and therefore the control of hematopoiesis.