Allison E. Daly1,2, George Yeh1,2, Sofia Soltero1,3 and Stephen T. Smale1,2,3 1Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, California 90095, USA; 2Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California 90095, USA; 3Howard Hughes Medical Institute, University of California, Los Angeles, Los Angeles, California 90095, USA Corresponding author: smalemednet.ucla.edu Abstract

The five NF-κB family members and three nuclear IκB proteins play important biological roles, but the mechanisms by which distinct members of these protein families contribute to selective gene transcription remain poorly understood, especially at a genome-wide scale. Using nascent transcript RNA-seq, we observed considerable overlap between p50-dependent and IκBζ-dependent genes in Toll-like receptor 4 (TLR4)-activated macrophages. Key immunoregulatory genes, including Il6, Il1b, Nos2, Lcn2, and Batf, are among the p50–IκBζ-codependent genes. IκBζ-bound genomic sites are occupied at earlier time points by NF-κB dimers. However, p50–IκBζ codependence does not coincide with preferential binding of either p50 or IκBζ, as RelA co-occupies hundreds of genomic sites with the two proteins. A common feature of p50–IκBζ-codependent genes is a nearby p50/RelA/IκBζ-cobound site exhibiting p50-dependent binding of both RelA and IκBζ. This and other results suggest that IκBζ acts in concert with RelA:p50 heterodimers. Notably, p50–IκBζ-codependent genes comprise a high percentage of genes exhibiting the greatest differential expression between TLR4-stimulated and tumor necrosis factor receptor (TNFR)-stimulated macrophages. Thus, our genome-centric analysis reveals a defined p50–IκBζ pathway that selectively activates a set of key immunoregulatory genes and serves as an important contributor to differential TNFR and TLR4 responses.

Received February 27, 2024. Accepted May 29, 2024.