Inferring gene regulation from stochastic transcriptional variation across single cells at steady-state

Regulatory relationships between transcription factors (TFs) and their target genes lie at the heart of cellular identity and function; however, uncovering these relationships is often labor-intensive and requires perturbations. Here, we propose a principled framework to systematically infer gene regulation for all TFs simultaneously in cells at steady-state by leveraging the intrinsic variation in the transcriptional abundance across single cells. Through modeling and simulations, we characterize how transcriptional bursts of a TF gene are propagated to its target genes, including the expected ranges of time delay and magnitude of maximum covariation. We distinguish these temporal trends from the time-invariant covariation arising from cell states, and we delineate the experimental and technical requirements for leveraging these small but meaningful co-fluctuations in the presence of measurement noise. While current technology does not yet allow adequate power for definitively detecting regulatory relationships for all TFs simultaneously in cells at steady-state, we investigate a small-scale dataset to inform future experimental design. This study supports the potential value of mapping regulatory connections through stochastic variation, and it motivates further technological development to achieve its full potential. Overall design: For 4-thiouridine (4sU) experiments, cells were plated in 6-well plates ~12h before the start the experiment at a density of 5x 10^5 – 8 x 10^5 cells/mL in 5mL of fresh media. 4sU (Sigma, T4509-25MG) in DMSO was added to culture wells at a final concentration of 100µM for 24h, with renewal every 6-8h. Between the pulse and chase phases, the cells were washed twice to remove any residual traces of 4sU. Subsequently, media with saturating concentrations of uridine (Sigma, U6381) at 10mM was added. Cells were collected at 0h, 2h, 4h, 6h, 8h, and 10h. All the samples from each pulse-chase experiment were processed the same day to minimize batch effects. To control for genes induced by the long exposure to 4sU and to uridine, two control samples with chase only or DMSO only were included.