TNF stimulation primarily modulates transcriptional burst size of NF-κB-regulated genes.

Cell-to-cell heterogeneity is a feature of the tumor necrosis factor (TNF)-stimulated inflammatory response mediated by the transcription factor NF-κB, motivating an exploration of the underlying sources of this noise. Here, we combined single-transcript measurements with computational models to study transcriptional noise at six NF-κB-regulated inflammatory genes. In the basal state, NF-κB-target genes displayed an inverse correlation between mean and noise characteristic of transcriptional bursting. By analyzing transcript distributions with a bursting model, we found that TNF primarily activated transcription by increasing burst size while maintaining burst frequency for gene promoters with relatively high basal histone 3 acetylation (AcH3) that marks open chromatin environments. For promoters with lower basal AcH3 or when AcH3 was decreased with a small molecule drug, the contribution of burst frequency to TNF activation increased. Finally, we used a mathematical model to show that TNF positive feedback amplified gene expression noise resulting from burst size-mediated transcription, leading to a subset of cells with high TNF protein expression. Our results reveal potential sources of noise underlying intercellular heterogeneity in the TNF-mediated inflammatory response.

NF-κB-Chromatin Interactions Drive Diverse Phenotypes by Modulating Transcriptional Noise.

Noisy gene expression generates diverse phenotypes, but little is known about mechanisms that modulate noise. Combining experiments and modeling, we studied how tumor necrosis factor (TNF) initiates noisy expression of latent HIV via the transcription factor nuclear factor κB (NF-κB) and how the HIV genomic integration site modulates noise to generate divergent (low-versus-high) phenotypes of viral activation. We show that TNF-induced transcriptional noise varies more than mean transcript number and that amplification of this noise explains low-versus-high viral activation. For a given integration site, live-cell imaging shows that NF-κB activation correlates with viral activation, but across integration sites, NF-κB activation cannot account for differences in transcriptional noise and phenotypes. Instead, differences in transcriptional noise are associated with differences in chromatin state and RNA polymerase II regulation. We conclude that, whereas NF-κB regulates transcript abundance in each cell, the chromatin environment modulates noise in the population to support diverse HIV activation in response to TNF.