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.