Selective Regulation of a Defined Subset of Inflammatory and Immunoregulatory Genes by an NF-kB p50-IkBz Pathway

The five NF-kB family members and three nuclear IkB proteins play diverse biological roles, but the mechanisms by which distinct NF-kB and NF-kB: IkB complexes contribute to selective gene transcription remain poorly understood. Using nascent transcript RNA-seq, we observed considerable overlap between p50-dependent and IkBz-dependent genes in Toll-like receptor 4 (TLR4)-activated macrophages. Key inflammatory and immunoregulatory genes, including Il6, Il1b, Nos2, Lcn2, and Batf, were among the p50-IkBz co-dependent genes. IkBz typically bound genomic sites occupied earlier by NF-kB dimers. However, p50-IkBz co-dependence did not coincide with preferential binding of either protein, as p50, IkBz, and RelA co-occupied thousands of sites. A common feature of p50-IkBz co-dependent genes was close proximity to a p50/RelA/IkBz co-bound site exhibiting p50-dependent binding of RelA and IkBz. This result and others suggest that IkBz function is not restricted to p50 homodimers. Notably, IkBz and the p50-IkBz target genes comprise a high percentage of genes that exhibited the greatest differential expression between TLR4-stimulated and tumor necrosis factor receptor (TNFR)-stimulated macrophages, with ectopic IkBz rescuing a subset of these genes. These results reveal a defined p50-IkBz pathway that selectively activates a set of key inflammatory and immunoregulatory genes and serves as an important contributor to the differential responses to TNFR and TLR4. To understand the binding kinetics of p50 and IkBz we performed ChIP-seq experiments with Lipid A stimulation for 0, 0.5, 1.0, 2.0, and 6.0 hour timepoints. To evaluate the dependence of IkBz and RelA on p50 for chromatin interactions, we performed IkBz and RelA ChIP-seq in p50-/- BMDMs stimulated with Lipid A for 2.0 hours. To explore the binding interaction between p50, RelA, and IkBz on a global level, we employed sequential ChIP-seq. We first performed a RelA immunoprecipitation followed by sequential immunoprecipitation by either p50, IkBz, or p53.