Transcriptional shut-off of MAP kinase signaling enables pluripotency maintenance during diapause [TT-seq]

Exposure of unicellular or multicellular organisms to adverse environmental conditions, including nutrient deprivation, may induce a state of suspended animation or diapause. We here report that broad repression of RNA Pol II-driven gene expression by inhibiting the BET family's bromodomain-containing proteins (BET) triggers diapause in mouse embryonic stem (ES) cells. The diapause ES cells upregulate a functionally linked group of genes encoding negative regulators of MAP kinase signaling (NRMKS), which play a crucial role in ES cell differentiation. The elevated NRMKS expression is a hallmark of cells exposed to distinct diapause-inducing conditions, including mTOR inhibition, and are required for the pluripotency maintenance during diapause. Mechanistically, inhibition of mTORC1/2 leads to rapid decline of the Capicua transcriptional repressor (CIC) at the NRMKS gene promoters, followed by rapid transcriptional NRMKS gene upregulation. The mTOR and BET-dependent transcriptional switch supporting the undifferentiated state of the diapause ES cells suggests a broader usage of this mechanism in maintaining the undifferentiated state of metabolically dormant stem- or stem-like cells in different tissues. Overall design: Our study here based on bulk RNA-seq with time course dependent samples suggested the rapid gene expression changes occurring in ES cells upon mTOR inhibition by Torin1. To further investigate the rapid gene expression changes in ES cells upon mTOR inhibition by Torin1, we performed TT-seq by using 4-thiouridine (4sU) labeled nascent RNA samples derived from time course dependent assays with Torin1 treatment including 15min, 30min and 60min. Three biological replicates were included in each condition of the assay.