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. Overall design: 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.