Gloire Geoffrey, Horion Julie, El Mjiyad Nadia, Bex Françoise, Chariot Alain, Dejardin Emmanuel, Piette Jacques
GIGA-Research, Virology-Immunology, and Medical Chemistry Units, University of Liège, B-4000 Liège, Belgium.
J Biol Chem. 2007 Jul 20;282(29):21308-18. doi: 10.1074/jbc.M610728200. Epub 2007 May 30.
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.
IKKα在核因子-κB（NF-κB）信号通路的核阶段调节许多染色质相关事件，包括组蛋白H3的磷酸化以及从NF-κB依赖的启动子上去除共抑制因子。然而，IKKα的所有核功能尚未完全明确。在本研究中，我们利用小鼠胚胎成纤维细胞IKKα基因敲除模型，并通过逆转录病毒转导重新表达IKKα，结果表明IKKα对外源κB元件以及部分（而非全部）内源性NF-κB靶启动子上的NF-κB/p65 DNA结合活性有贡献。事实上，p65染色质免疫沉淀分析显示，IKKα对于p65结合细胞间黏附分子-1（icam-1）和单核细胞趋化蛋白-1（mcp-1）启动子的κB位点至关重要，但对IκBα启动子则不然。IKKα假定的核定位序列发生突变会阻止其核转位，或者IKKα激活环中的关键丝氨酸发生突变，都会完全抑制p65与icam-1和mcp-1启动子的结合，反而增强p65与IκBα启动子的结合。进一步的分子研究表明，染色质结合的组蛋白去乙酰化酶3（HDAC3，一种抑制p65 DNA结合的酶）的去除受到IKKα以启动子特异性方式的差异调节。实际上，IKKα缺失会导致HDAC3在icam-1启动子上的募集和抑制，而在IκBα启动子上则产生相反的效果，即p65结合更好。我们得出结论，核IKKα以基因特异性方式参与p65的DNA结合。
