RSC primes the quiescent genome for hypertranscription upon cell-cycle re-entry.

Christine E Cucinotta,Keean Ca Braceros,Rachel H Dell,Toshio Tsukiyama

doi:10.7554/elife.67033

Abstract

Quiescence is a reversible G0 state essential for differentiation, regeneration, stem-cell renewal, and immune cell activation. Necessary for long-term survival, quiescent chromatin is compact, hypoacetylated, and transcriptionally inactive. How transcription activates upon cell-cycle re-entry is undefined. Here we report robust, widespread transcription within the first minutes of quiescence exit. During quiescence, the chromatin-remodeling enzyme RSC was already bound to the genes induced upon quiescence exit. RSC depletion caused severe quiescence exit defects: a global decrease in RNA polymerase II (Pol II) loading, Pol II accumulation at transcription start sites, initiation from ectopic upstream loci, and aberrant antisense transcription. These phenomena were due to a combination of highly robust Pol II transcription and severe chromatin defects in the promoter regions and gene bodies. Together, these results uncovered multiple mechanisms by which RSC facilitates initiation and maintenance of large-scale, rapid gene expression despite a globally repressive chromatin state.

Highlights

For decades scientists have used budding yeast to uncover mechanisms of chromatin regulation of gene expression; and the vast majority of these studies were performed in exponentially growing cultures [1]
To determine the earliest time at which transcription reactivates during quiescence exit, we fed purified quiescent cells YPD medium and took time points to determine the kinetics of polymerase II (Pol II) C-terminal domain (CTD) phosphorylation by western blot analysis (Fig. 1A)
Pol II CTD phosphorylation occurred within three minutes (Fig. 1A, compare lanes 1 and 2), which was our physical limit of isolating cells during this time course

Summary

Results

To determine the earliest time at which transcription reactivates during quiescence exit, we fed purified quiescent cells YPD medium and took time points to determine the kinetics of Pol II C-terminal domain (CTD) phosphorylation by western blot analysis (Fig. 1A). In the absence of RSC at these sites, histone density is unexpectedly lower at NDRs in quiescence but does not change during quiescence exit (Fig. 5F), suggesting defective chromatin structure at and downstream of the NDR Together, these results are consistent with the notion that co-transcriptional movement of RSC facilitates passage of Pol II through nucleosomes immediately downstream of TSSs through chromatin regulation. At these sites, we observed RSC ChIP-seq signals at NDRs in quiescence and spreading during exit (Fig. 6C). It is likely that these genes have an intrinsic property to allow anti-sense transcription to occur when not properly regulated, and RSC is targeted to them to ensure sense transcription takes place through formation of proper NDRs

344 Discussion

Materials and Methods

590 Acknowledgements

Methods