NF-κB phosphorylation controls enhancer selection to shape the inflammatory gene program

Inflammatory transcriptional responses mediated by NF-κB are diverse despite similar upstream activation signals. Because phosphorylation of the NF-κB subunit RelA/p65 promotes interaction with co-activators CREB-binding protein/E1A binding protein p300 (CBP/p300) and selectively affects gene expression, we asked whether this modification distinguishes regulatory architectures underlying distinct immune functions. In macrophages carrying a Rela Ser276Ala mutation, p65 phosphorylation at serine 276 (S276) was dispensable for NF-κB binding to primed enhancers but essential for recruitment to nucleosome-occluded regions lacking pre-bound transcription factors (TFs) and activating histone marks. Such latent enhancers required phosphorylated NF-κB to recruit CBP/p300, promote histone acetylation, and open chromatin. Genes linked to these enhancers, including inflammatory mediators, were selectively impaired in S276A macrophages. Although antiviral responses remained intact, Rela Ser276Ala mice resisted endotoxin shock but were susceptible to bacterial infection. Thus, NF-κB responses are functionally partitioned by enhancer architecture: phosphorylation-sensitive sites define a latent enhancer compartment with NF-κB-dominated regulatory architecture, whereas primed sites are embedded in broader networks of lineage-determining and inducible TFs, yielding distinct biological outputs.