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

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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.