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IKKalpha regulates many chromatin events in the nuclear phase of the NF-kappaB program, including phosphorylation of histone H3 and removal of co-repressors from NF-kappaB-dependent promoters. However, all of the nuclear functions of IKKalpha are not understood. In this study, using mouse embryonic fibroblasts IKKalpha knock-out and reexpressing IKKalpha after retroviral transduction, we demonstrate that IKKalpha contributes to NF-kappaB/p65 DNA binding activity on an exogenous kappaB element and on some, but not all, endogenous NF-kappaB-target promoters. Indeed, p65 chromatin immunoprecipitation assays revealed that IKKalpha is crucial for p65 binding on kappaB sites of icam-1 and mcp-1 promoters but not on ikappabalpha promoter. The mutation of IKKalpha putative nuclear localization sequence, which prevents its nuclear translocation, or of crucial serines in the IKKalpha activation loop completely inhibits p65 binding on icam-1 and mcp-1 promoters and rather enhances p65 binding on the ikappabalpha promoter. Further molecular studies demonstrated that the removal of chromatin-bound HDAC3, a histone deacetylase inhibiting p65 DNA binding, is differentially regulated by IKKalpha in a promoter-specific manner. Indeed, whereas the absence of IKKalpha induces HDAC3 recruitment and repression on the icam-1 promoter, it has an opposite effect on the ikappabalpha promoter, where a better p65 binding occurs. We conclude that nuclear IKKalpha is required for p65 DNA binding in a gene-specific manner.

The human T cell leukemia/lymphotropic virus type 1 (HTLV-1) Tax protein strongly activates viral and cellular gene transcription. It mainly functions by interacting with cellular transcription factors and the KIX domain of the p300/CBP coactivators. Tax can also repress the transcription of cellular genes through the basic helix-loop-helix (bHLH) protein family. To investigate the molecular mechanisms of this Tax-mediated inhibition, we analyzed its effect on the transcriptional activity of the myogenic MyoD protein, which was used as a paradigm of bHLH factors. In this study, we show that overexpression of the p300 coactivator in transient transfection assays was sufficient to rescue MyoD repression by Tax. Furthermore, an N-terminal domain of p300 (amino acids 379-654) containing the region of KIX serving as the Tax binding site was found, when overexpressed, to potentiate Tax-mediated transactivation of HTLV-1 proviral as well as MyoD-dependent transcription, and to antagonize the inhibition by Tax of the transcriptional activity of MyoD. These results revealing the presence of an N-terminal MyoD binding site were confirmed by in vitro protein-protein interaction assays that demonstrate that MyoD binds to the KIX domain of p300 and that Tax competes with MyoD binding in a nonreciprocal manner. These observations provide evidence that Tax binding to the KIX domain of p300 prevents bHLH proteins from contacting this N-terminal domain of the coactivator, thus resulting in their transcriptional repression. As bHLH proteins are implicated in many developmental fate decisions, especially during thymopoiesis, Tax-mediated inhibition of their transcriptional activity may contribute to the induction of HTLV-1-linked leukemogenesis.