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. 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.