The number of transcription factor molecules loading at an enhancer determines gene expression quantity
NF-kB is a transcription factor whose activity is controlled by intracellular signaling events and subsequent nuclear translocation. NF-kB potentially induces both threshold and graded responses of target genes via super enhancers (SEs) and typical enhancers (TEs). However, the mechanism by which NF-kB establishes quantitative gene expression using different types of the enhancers remains to be elucidated. To clarify this issue, we performed an integrated analysis of chromatin accessibility, NF-kB (RelA)-DNA binding and single cell mRNA sequence data obtained from anti-IgM-stimulated mouse primary B lymphocytes and analyzed the input-output relationship of nuclear NF-kB and target gene expression using a kinetic model. Here we show that SE activity, defined by acetylated histone H3 lysine 27 (H3K27ac) modification, was simultaneously associated with chromatin opening and enriched RelA binding, resulting in a threshold target gene response, while smaller changes in these signatures controlled TEs, resulting in a graded response. Our analysis showed that the number of RelA peaks at a SE is larger on average than at a TE. Mathematical analysis indicated that SE-mediated threshold gene expression is explained by the existence of cooperativity in multiple NF-kB binding on DNA for promoter activation. Analysis of single cell transcriptome data using a mathematical model further revealed that the SE-mediated gene expression results in a larger distribution of mRNA levels in a cell population than that by TE, indicating a biological function of SEs in B cells for divergent clonal responses.