Regulation of gene-specific nuclear NF-kappaB functions by cofactors and posttranslational modifications

转录因子NF-kappaB在应激诱导的基因调控中起着核心作用。对于核转录因子kappaB激活的胞浆事件的鉴定进展迅速，但对该转录因子在细胞核中的基因特异性功能知之甚少。在我们的初步工作中，我们发现了新的核辅因子(CDK6和TRIP6)以及几种核因子-kappaB p65翻译后修饰的功能和调节。上述工作构成了一个旨在更详细地阐明核因子-kappaB的核功能的两方研究计划的基础。第一部分将探讨CDK6和TRIP6基因调控功能的机制和信号通路。我们还将研究TRIP6磷酸化在糖皮质激素介导的抑制NF-kappaB依赖基因表达中的作用。全基因组分析将阐明CDK6是否是专门向核因子-kappaB传递细胞周期特异性信号，或者该激酶是否与广泛的核因子-kappaB激活刺激具有普遍相关性。第二部分将研究翻译后修饰的NF-kappaB p65和一个DNA结合突变体在全基因组范围内的招募，以了解它们在转录激活和染色质结合方面的生理功能。我们还将使用最近发现的一种构象特异性抗体来研究刺激诱导的核因子-kappaB p65构象转换的功能后果。这些实验将得出关于p65功能的翻译后修饰、上下文特定构象和DNA结合的重要性的结论。这两位申请者的重叠和互补的专业知识将显著提高对健康和疾病细胞中依赖于核因子-kappaB的基因调控可塑性的分子机制的理解。

The transcription factor NF-kappaB plays a central role for stress-induced gene regulation. The identification of the cytosolic events mediating NF-kappaB activation is rapidly progressing, whereas the gene-specific functions of this transcription factor in the nucleus are poorly understood. In our preliminary work we identified novel nuclear cofactors (CDK6 and TRIP6) and the function and regulation of several postranslational modifications of NF-kappaB p65. This preceding work forms the basis for a bipartite research program that aims to elucidate the nuclear functions of NF-kappaB in much more detail. The first part will investigate the mechanisms and signaling pathways mediating the gene regulatory functions of CDK6 and TRIP6. We will also investigate the contribution of TRIP6 phosphorylation for its function in glucocorticoid-mediated repression of NF-kappaB-dependent gene expression. Genome-wide analyses will clarify whether CDK6 is specialized to communicate cell cycle-specific signals to NF-kappaB or whether this kinase is of general relevance for a broad range of NF-kappaB activating stimuli. The second part will investigate the genome-wide recruitment of posttranslationally modified NF-kappaB p65 and a DNA-binding mutant to investigate their physiological function for transcriptional activation and chromatin association. We will also investigate the functional consequences of a stimulus-induced conformational switch of NF-kappaB p65 using a recently identified conformation-specific antibody. These experiments will allow conclusions on the significance of posttranslational modifications, context-specific conformation and DNA-binding for p65 functions. The overlapping and complementary expertise of both applicants will allow a significantly improved understanding of the molecular mechanisms underlying the plasticity of NF-kappaB-dependent gene regulation in healthy and diseased cells.