NF‐κB p65 dimerization and DNA‐binding is important for inflammatory gene expression

Increasing evidence shows that many transcription factors execute important biologic functions independent from their DNA‐binding capacity. The NF‐κB p65 (RELA) subunit is a central regulator of innate immunity. Here, we investigated the relative functional contribution of p65 DNA‐binding and dimerization in p65‐deficient human and murine cells reconstituted with single amino acid mutants preventing either DNA‐binding (p65 E/I) or dimerization (p65 FL/DD). DNA‐binding of p65 was required for RelB‐dependent stabilization of the NF‐κB p10O protein. The antiapoptotic function of p65 and expression of the majority of TNF‐α–induced genes were dependent on p65′s ability to bind DNA and to dimerize. Chromatin immunoprecipitation with massively parallel DNA sequencing experiments revealed that impaired DNA‐binding and dimerization strongly diminish the chromatin association of p65. However, there were also p65‐independent TNF‐α‐inducible genes and a subgroup of p65 binding sites still allowed some residual chromatin association of the mutants. These sites were enriched in activator protein 1 (AP‐1) binding motifs and showed increased chromatin accessibility and basal transcription. This suggests a mechanism of assisted p65 chromatin association that can be in part facilitated by chromatin priming and cooperativity with other transcription factors such as AP‐1.—Riedlinger, T., Liefke, R., Meier‐Soelch, J., Jurida, L., Nist, A., Stiewe, T., Kracht, M., Schmitz, M. L. NF‐κB p65 dimerization and DNA‐binding is important for inflammatory gene expression. FASEB J. 33, 4188–4202 (2019). www.fasebj.org