NF-kB subunits direct kinetically distinct transcriptional cascades in antigen receptor-activated B cells [RNA-seq]

The NF-kB family of transcription factors orchestrates signal-induced gene expression in a diversity of cell types. Cellular responses to NF-kB activation are regulated at the level of cell- and signal-specificity, as well as differential use of family members (subunit specificity). Here we used time-dependent multi-omics to investigate selective functions of Rel and RelA, two closely related NF-kB proteins, in primary B lymphocytes activated via the B cell receptor. Despite large numbers of shared binding sites genome wide, Rel and RelA directed kinetically distinct cascades of gene expression in activated B cells. Single-cell RNA-Seq revealed marked heterogeneity of Rel- and RelA-specific responses and sequential binding of these factors was not a major mechanism of protracted transcription. Moreover, nuclear co-expression of Rel and RelA led to functional antagonism between the two factors. By rigorously identifying target genes of each NF-kB subunit, these studies provide insights into exclusive functions of Rel and RelA in immunity and cancer. Overall design: A total of 44 splenic B cell sample pools were isolated from 5 wildtype C57BL/6 mice (#000664, The Jackson Laboratory) (WT), 5 RelA fl/fl mice (gift from Harmel-Laws Steinbrecher) (RelA-flfl), 5 RelA cKO mice (cross between RelA fl/fl and Cd19-cre (006785, The Jackson Laboratory) (RelA cKO), or 5 Rel KO mice (gift from Hsia Cheng) (Rel KO). Each sample was treated with 10µg/ml goat anti-mouse IgM Fab`2 for 0, 1, 4, or 18 hour (N = 2 per condition). In addition, WT mice were pre-treated for 1 hour with 1uM BAY 11-7082 (BAY) and treated with 10µg/ml goat anti-mouse IgM Fab`2 for 1, 4, or 18 hours (N = 2 per condition). RNA was extracted from all samples and processed through RNA-Seq.