IκB Kinase α-Mediated Derepression of SMRT Potentiates Acetylation of RelA/p65 by p300

Abstract

Over the last several years, significant progress has been made in identifying chromatin-regulated events that govern NF-κB transcription. Using either laminin attachment or tumor necrosis factor alpha as a physiological stimulus of NF-κB activation, we demonstrate that IκB kinase α (IKKα) is recruited to chromatin in distinct phases. In the initial phase, IKKα is responsible for derepressing the silencing mediator for retinoic acid and thyroid hormone receptor (SMRT)-histone deacetylase 3 (HDAC3) corepressor complex from the p50 homodimer. However, in the latter phase, chromatin-bound IKKα coordinates the simultaneous phosphorylation of RelA/p65(S536) and SMRT(S2410) as detected by chromatin immunoprecipitation (ChIP) assays. Although phosphorylated SMRT remains bound to the active p50-RelA/p65 heterodimer of NF-κB, derepression of SMRT is evidenced by the loss of chromatin-associated HDAC3 activity. ChIP and re-ChIP analysis demonstrates that phosphorylation of RelA/p65(S536) and SMRT(S2410) occurs prior to acetylation of RelA/p65 at K310. Moreover, IKKα-induced phosphorylation of RelA/p65(S536) displaces corepressor activity, allowing p300-mediated acetylation of RelA/p65. Introduction of nonphosphorylatable mutants of RelA/p65 and SMRT proteins or the inhibition of IKK activity results in active repression of NF-κB promoters by tethering the SMRT-HDAC3 complex. Similar to phosphorylation within the Rel homology domain of RelA/p65, which governs an exchange of HDAC1 for CBP/p300 acetyltransferases, we demonstrate that phosphorylation within the transactivation domain of RelA/p65(S536) displaces SMRT-HDAC3 repressor activity, allowing p300 to acetylate RelA/p65.

We thank A. Sherman and H. Bateman for editorial assistance. Also, M. L. Privalsky (University of California, Davis, CA), H. Nakajima (University of Tokyo, Tokyo, Japan), D. Livingston (Dana-Farber Cancer Institute, Boston, MA), L. Schmitz (University of Bern, Bern, Switzerland), B. Ashburner (University of Toledo, Toledo, OH), and W. Toriumi (Osaka, Japan) for providing expression plasmids encoding GST-SMRT and pSP72-SMRT, Flag-SMRT, p300, Gal4-p65, pGEM-p65, and GST-p65, respectively.

This work was supported by the National Cancer Institute (R01CA104397 and R01CA095644) and a Paul Mellon Prostate Cancer Institute award to M.W.M.