GSE116284
NF-?B 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-?B 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-?B p100 protein. The antiapoptotic function of p65 and expression of the majority of TNF-a–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-a–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. Overall design: p65 ChIP-Seq in HeLa after p65 KO and reconstitution with wildtype or mutant p65, before and after TNF stimulation (1x), RNA-Seq of the same cells (2x), RNA-Seq of p65 KO MEFs reconstituted with wt and mutant p65 (1x)