CREB-Binding Protein Acetylates Hematopoietic Transcription Factor GATA-1 at Functionally Important Sites

Abstract

The transcription factor GATA-1 is a key regulator of erythroid-cell differentiation and survival. We have previously shown that the transcriptional cofactor CREB-binding protein (CBP) binds to the zinc finger domain of GATA-1, markedly stimulates the transcriptional activity of GATA-1, and is required for erythroid differentiation. Here we report that CBP, but not p/CAF, acetylates GATA-1 at two highly conserved lysine-rich motifs present at the C-terminal tails of both zinc fingers. Using [³H]acetate labelling experiments and anti-acetyl lysine immunoprecipitations, we show that GATA-1 is acetylated in vivo at the same sites acetylated by CBP in vitro. In addition, we show that CBP stimulates GATA-1 acetylation in vivo in an E1A-sensitive manner, thus establishing a correlation between acetylation and transcriptional activity of GATA-1. Acetylation in vitro did not alter the ability of GATA-1 to bind DNA, and mutations in either motif did not affect DNA binding of GATA-1 expressed in mammalian cells. Since certain functions of GATA-1 are revealed only in an erythroid environment, GATA-1 constructs were examined for their ability to trigger terminal differentiation when introduced into a GATA-1-deficient erythroid cell line. We found that mutations in either acetylation motif partially impaired the ability of GATA-1 to induce differentiation while mutations in both motifs abrogated it completely. Taken together, these data indicate that CBP is an important cofactor for GATA-1 and suggest a novel mechanism in which acetylation by CBP regulates GATA-1 activity in erythroid cells.

ACKNOWLEDGMENTS

We thank Merlin Crossley, Colyn Crane-Robinson, Warren Pear, Marc Montminy, and Michael Rosenfeld for providing plasmids, antibodies, and cell lines. We are grateful to Dawn Eastmond and Toshio Asakura for assistance with HPLC separation of GATA-1 peptides. Peptide synthesis and protein-sequencing services were provided by the Protein Chemistry Laboratory of the Medical School of the University of Pennsylvania. We thank Margaret Chou, Merlin Crossley, Stuart Orkin, and Morty Poncz for helpful suggestions and for critically reading the manuscript.

This work was supported by the Baldasare Award (G.A.B.), the American Society of Hematology Scholar Award (G.A.B.) and the Cooley’s Anemia Foundation (G.A.B.). Services provided by the Protein Chemistry Laboratory of the Medical School of the University of Pennsylvania were supported by core grants of the Diabetes and Cancer Centers (DK-19525 and CA-16520).