Small transcriptional differences among cell clones lead to distinct NF-?B dynamics

Transcription factor dynamics is fundamental to determine the activation of accurate transcriptional programs and yet is heterogeneous at single-cell level, even between very similar cells. We asked how such heterogeneity emerges for the nuclear factor ?B (NF-?B), whose dynamics have been reported to cover a wide spectrum of behaviors, including persistent, oscillatory and weak activation. We found that clonal populations of immortalized fibroblasts derived from a single mouse embryo display robustly distinct dynamics upon tumor necrosis factor a (TNF-a) stimulation, which give rise to differences in the transcription of NF-?B targets. Notably, standard transcriptomic analyses indicate that the clones differ mostly in transcriptional programs related with development, but not in TNF-a signaling. However, by combining transcriptomics data and simulations we show how the expression levels of genes coding for proteins of the signaling cascade determine the differences in early NF-?B activation; differences in the expression of I?Ba determine differences in its persistence and oscillatory behavior. The same analysis predicts inter-clonal differences in the NF-?B response to IL-1ß. We propose that small (less than twofold) differences at transcript level can lead to distinct transcription factor dynamics in cells within homogeneous cell populations, and all the more so among different cell types. Overall design: To determine the mechanisms responsible for NF-kB transcriptional dynamical variations, we isolated several clones from the original cell population of GFP-MEFs and made RNA-Seq of the original populiation (pool) as control and three clonal populations sleected based on the NF-kB dynamics, in five replicates.