Enhanced transcriptional heterogeneity mediated by NF-kappa B super-enhancers

The transcription factor NF-kappa B, which plays an important role in cell fate determination, is involved in the activation of super-enhancers (SEs). However, the biological functions of the NF-kappa B SEs in gene control are not fully elucidated. We investigated the characteristics of NF-kappa B-mediated SE activity using fluorescence imaging of RelA, single-cell transcriptome and chromatin accessibility analyses in anti-IgM-stimulated B cells. The formation of cell stimulation-induced nuclear RelA foci was abolished in the presence of hexanediol, suggesting an underlying process of liquid-liquid phase separation. The gained SEs induced a switch-like expression and enhanced cell-to-cell variability in transcriptional response. These properties were correlated with the number of gained cis-regulatory interactions, while switch-like gene induction was associated with the number of NF-kappa B binding sites in SE. Our study suggests that NF-kappa B SEs have an important role in the transcriptional regulation of B cells possibly through liquid condensate formation consisting of macromolecular interactions. Author summaryNF-kappa B produces an all-or-none activation response upon the activation of B cell receptors. These dynamics modulate the amplitude and frequency of target mRNA induction in cell populations. In this research, we performed single-cell assessment of chromatin accessibility and RNA expression, coupled with fluorescence imaging to characterize the nuclear dynamics of NF-kappa B proteins in B cell upon receptor stimulation. We found that upon cellular activation, NF-kappa B-mediated long-range activation of enhancers cooperatively evoked RNA production. In addition, predicted DNA contacts brought by open chromatin led to the high heterogeneity of RNA levels in cell populations. Stimuli-dependent NF-kappa B foci formation was further inhibited by 1,6-hexanediol (liquid-liquid phase separation inhibitor) and JQ1 (coactivator protein BRD4 inhibitor). We thus propose that nuclear NF-kappa B plays an important role in the transcriptional regulation of B cell development possibly through the formation of liquid condensates.