IkappaB kinase alpha-mediated derepression of SMRT potentiates acetylation of RelA/p65 by p300.

Over the last several years, significant progress has been made in identifying chromatin-regulated events that govern NF-kappaB transcription. Using either laminin attachment or tumor necrosis factor alpha as a physiological stimulus of NF-kappaB activation, we demonstrate that IkappaB kinase alpha (IKKalpha) is recruited to chromatin in distinct phases. In the initial phase, IKKalpha 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 IKKalpha 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-kappaB, 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, IKKalpha-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-kappaB 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.

COMMD proteins, a novel family of structural and functional homologs of MURR1.

MURR1 is a multifunctional protein that inhibits nuclear factor kappaB (NF-kappaB), a transcription factor with pleiotropic functions affecting innate and adaptive immunity, apoptosis, cell cycle regulation, and oncogenesis. Here we report the discovery of a new family of proteins with homology to MURR1. These proteins form multimeric complexes and were identified in a biochemical screen for MURR1-associated factors. The family is defined by the presence of a conserved and unique motif termed the COMM (copper metabolism gene MURR1) domain, which functions as an interface for protein-protein interactions. Like MURR1, several of these factors also associate with and inhibit NF-kappaB. The proteins designated as COMMD or COMM domain containing 1-10 are extensively conserved in multicellular eukaryotic organisms and define a novel family of structural and functional homologs of MURR1. The prototype of this family, MURR1/COMMD1, suppresses NF-kappaB not by affecting nuclear translocation or binding of NF-kappaB to cognate motifs; rather, it functions in the nucleus by affecting the association of NF-kappaB with chromatin.

SMRT derepression by the IkappaB kinase alpha: a prerequisite to NF-kappaB transcription and survival.

Understanding how signaling cascades stimulate chromatin-remodeling events through derepression is one of the foremost questions in the transcription field. Here, we demonstrate that NF-kappaB transcription requires IKKalpha to phosphorylate SMRT on chromatin, stimulating the exchange of corepressor for coactivator complexes. IKKalpha-induced phosphorylation coincides with a loss of chromatin-associated SMRT and HDAC3 and with nuclear export of the SMRT corepressor, events required for expression of the NF-kappaB-regulated cIAP-2 and IL-8 genes. Although SMRT derepression corresponds with the recruitment of TBL1/TBLR1, this complex alone is insufficient to relieve repression. Using a nonphosphorylatable SMRT protein, we demonstrate that IKKalpha-induced phosphorylation is required to recruit 14-3-3epsilon and Ubc5 for SMRT derepression. Failure of IKKalpha to stimulate the removal of SMRT from chromatin inhibits the recruitment of NF-kappaB to promoters, blocking transcription and sensitizing cells to apoptosis. Our work provides evidence that IKKalpha orchestrates SMRT derepression, a prerequisite for NF-kappaB transcription and survival.

Modulation of NF-kappaB-dependent transcription and cell survival by the SIRT1 deacetylase.

NF-kappaB is responsible for upregulating gene products that control cell survival. In this study, we demonstrate that SIRT1, a nicotinamide adenosine dinucleotide-dependent histone deacetylase, regulates the transcriptional activity of NF-kappaB. SIRT1, the mammalian ortholog of the yeast SIR2 (Silencing Information Regulator) and a member of the Sirtuin family, has been implicated in modulating transcriptional silencing and cell survival. SIRT1 physically interacts with the RelA/p65 subunit of NF-kappaB and inhibits transcription by deacetylating RelA/p65 at lysine 310. Treatment of cells with resveratrol, a small-molecule agonist of Sirtuin activity, potentiates chromatin-associated SIRT1 protein on the cIAP-2 promoter region, an effect that correlates with a loss of NF-kappaB-regulated gene expression and sensitization of cells to TNFalpha-induced apoptosis. While SIRT1 is capable of protecting cells from p53-induced apoptosis, our work provides evidence that SIRT1 activity augments apoptosis in response to TNFalpha by the ability of the deacetylase to inhibit the transactivation potential of the RelA/p65 protein.