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 the 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 investigate the role of p50 and nuclear IkBs in macrophage activation, we performed chromatin-associated RNA-seq on bone marrow-derived macrophages (BMDMs) lacking each of these subunits. We stimulated wild-type, Nfkb1-/-, Nfkbiz-/-, Bcl3-/-, and Nfkbid-/- BMDMs with lipid a for 0, 0.5, 1.0, 2.0, and 6.0 hours. To evaluate possible redundancy between p50 and p52, we generated Nfkb1-/-, Nfkb2-/- macrophages and performed bulk RNA-seq on macrophages stimulated with Lipid A for 0 and 2.0 hours. To understand the selective regulation of IkBz, we generated both bulk and chromatin-associated RNA-seq from BMDMs stimulated with TNFa for 0, 0.5, 1.0, 2.0, and 6.0 hours. To evaluate if the ectopic expression of IkBz could restore the expression of IkBz-dependent genes with TNFa stimulation, we performed bulk RNA-seq on BMDMs overexpressing IkBz and stimulated with TNFa for 0, 0.5, 1.0, 2.0, and 6.0 hours. To understand if early (0.5h) p50-dependent genes are due to the role of p105 in stabilizing and upstream mediator of the MAPK/ERK pathway, we performed chromatin-associated RNA-seq on BMBMs pretreated with ERK-inhibitors and stimulated with lipid A for 0, 15, 30, and 60 mins.