NF-kB subunits direct kinetically distinct transcriptional cascades in antigen receptor-activated B cells [ChIP-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. A total of 22 B cell samples from WT C57BL/6 mice (#000664, The Jackson Laboratory) were treated with 10μg/ml goat anti-mouse IgM Fab`2 for 1, 4, or 18 hours respectively, or left untreated. ChIP-Seq was performed using anti-RelA antibody (sc-372, Santa Cruz; N=2 for each of the 0, 1, and 4 hour time points), anti-Rel antibody (sc-71, Santa Cruz; N=2 for each of the 0, 1, and 18 hour time points), anti-H3K27ac antibody (ab4729, Abcam; N=2 for the 0 hour time point), anti-H3K4me1 antibody (ab8895, Abcam; N=2 for the 0 hour time-point) and anti-H3K4me3 antibody (ab8580, Abcam; N=2 for each of the 0, 1, and 18 hour time points).