Transcriptional Regulation Via GATA Switches: Proteasome-Mediated Exchange of Chromatin-Bound GATA Factors.

Abstract The transcription factors GATA-1 and GATA-2 confer both shared and unique activities to regulate hematopoiesis. GATA-2 is essential for the survival and proliferation of hematopoietic stem cells and multipotent hematopoietic precursors, whereas GATA-1 is critical for erythropoiesis. GATA-1 and GATA-2 are expressed reciprocally during erythropoiesis, with GATA-2 expression preceding that of GATA-1. Erythroid maturation appears to require GATA-2 repression. We have shown that chromatin-bound GATA-2 is displaced by GATA-1, which instigates a distinct transcriptional output at the respective locus. We hypothesize that such “GATA switches” are an important transcriptional mechanism that drives erythropoiesis. Their distinct transcriptional outputs suggest that GATA-1 and GATA-2 have unique biochemical properties. However, biochemical analyses have primarily identified similarities between the factors. For instance, the two zinc fingers of GATA-1 are highly homologous to those of GATA-2, and therefore both factors interact with the cofactor FOG-1 and bind an identical WGATAR sequence with high affinity. We found that GATA-2 is considerably less stable than GATA-1. Studies using cycloheximide to block protein synthesis in erythroid precursor cell lines derived from embryonic stem cells indicate that the half-life of endogenous GATA-2 is ~45–90 min, whereas that of endogenous GATA-1 is >4 h. Biosynthetic labeling studies to measure the stabilities of endogenous GATA-1 and GATA-2 expressed in the same cell confirmed that GATA-2 is considerably less stable than GATA-1. GATA-2 levels increase in cells treated with the proteasome inhibitors MG132 or clasto-lactacystin β-lactone. Importantly, we have found that rapid GATA-2 proteolysis is essential for the proper temporal control of erythroid-specific gene transcription. Quantitative chromatin immunoprecipitation analysis revealed that proteasomal inhibition attenuated the ability of GATA-1 to displace GATA-2 from target genes, which are regulated by GATA switches. Proteasomal inhibition resulted in impaired GATA-2 displacement from GATA-2, and consequently a significantly lower degree of GATA-2 repression. Proteasomal inhibition also diminished GATA-1-mediated displacement of GATA-2 from GATA-1, α-globin, and ALAS-2, genes at which GATA switches precede transcriptional activation. We have therefore identified the 26S proteasome as a key regulator of GATA factor exchange on chromatin. Our studies indicate that rapid proteasomal degradation of GATA-2 is required for GATA-1 and GATA-2 to establish unique patterns of gene expression during erythropoiesis. Studies are underway to determine whether GATA-2 is similarly unstable in multipotent hematopoietic precursors, or if its instability is established upon commitment to the erythroid lineage.