Repression of GCN5 Histone Acetyltransferase Activity via Bromodomain-Mediated Binding and Phosphorylation by the Ku–DNA-Dependent Protein Kinase Complex

ABSTRACT

GCN5, a putative transcriptional adapter in humans and yeast, possesses histone acetyltransferase (HAT) activity which has been linked to GCN5’s role in transcriptional activation in yeast. In this report, we demonstrate a functional interaction between human GCN5 (hGCN5) and the DNA-dependent protein kinase (DNA-PK) holoenzyme. Yeast two-hybrid screening detected an interaction between the bromodomain of hGCN5 and the p70 subunit of the human Ku heterodimer (p70-p80), which is the DNA-binding component of DNA-PK. Interaction between intact hGCN5 and Ku70 was shown biochemically using recombinant proteins and by coimmunoprecipitation of endogenous proteins following chromatography of HeLa nuclear extracts. We demonstrate that the catalytic subunit of DNA-PK phosphorylates hGCN5 both in vivo and in vitro and, moreover, that the phosphorylation inhibits the HAT activity of hGCN5. These findings suggest a possible regulatory mechanism of HAT activity.

ACKNOWLEDGMENTS

We thank T. Carter for DNA-PKcs monoclonal antibodies and for help in purification of Ku70/80 DNA-PKcs; W. H. Reeves for α-Ku monoclonal antibodies; D. Jensen for help in the two-hybrid screen; N. Malik for help in purification of Ku and DNA-PKcs; and T. Carter, P. Lieberman, G. Moore, X. Nui, J. Ozer, F. J. Rauscher III, D. Reinberg, and R. Sheikhatter for valuable discussions and critical reading of the manuscript.

The work was supported by grants from the NSF and The Council for Tobacco Research to S.L.B.; grants from the NIGMS and the NSF to J.L.W.; and a Cancer Core grant from the NIH and a grant from the Pew Charitable Trust to The Wistar Institute. T.O.-H. was the recipient of an EMBO long-term fellowship; S.L.B. is the recipient of an ACS Junior Faculty Research Award, and J.L.W. is a Leukemia Society Scholar.