RNA Pol II pausing facilitates phased pluripotency transitions by buffering transcription

Abstract

Promoter-proximal RNA Pol II pausing is a critical step in transcriptional control. Pol II pausing has been predominantly studied in tissue culture systems. While Pol II pausing has been shown to be required for mammalian development, the phenotypic and mechanistic details of this requirement are unknown. Here, we found that loss of Pol II pausing stalls pluripotent state transitions within the epiblast of the early mouse embryo. Using Nelfb−/− mice and a NELFB degron mouse pluripotent stem cell model, we show that embryonic stem cells (ESCs) representing the naïve state of pluripotency successfully initiate a transition program but fail to balance levels of induced and repressed genes and enhancers in the absence of NELF. We found an increase in chromatin-associated NELF during transition from the naïve to later pluripotent states. Overall, our work defines the acute and long-term molecular consequences of NELF loss and reveals a role for Pol II pausing in the pluripotency continuum as a modulator of cell state transitions.

Evolution of promoter-proximal pausing enabled a new layer of transcription control

“…After the depletion of NELF-B, we observed paused Pol II “creeping” across the first couple of kilobases of the gene body ( fig. S10 ), similar to observations made in S. pombe , which has no NELF ( 7, 10 ). As a result, Pol II which needs to be released from pause by p-TEFb is no longer in a fixed location, in proximity to promoter-bound transcription factors.…”

“…We also included a number of metazoan organisms representing major taxa, including the cnidarian ( Nematostella vectensis ), the sea urchin ( Strongylocentrotus purpuratus ), the water flea ( Daphnia pulex ), the butterfly ( Dryas iulia ), and the cyclostome ( Petromyzon marinus ). Finally, we have augmented our PRO-seq atlas by integrating published data from a fly ( Drosophyla melanogaster (11) ), a nematode ( Caenorhabditis elegans (12) ), yeast (Saccharomyces cerevisia e and Schizosaccharomyces pombe ( 9, 10 )), model plants ( Arabidopsis thaliana, Oryza sativa , and Zea mays ( 13–15 )), and mammals ( Homo sapiens and Mus musculus ( 7, 16 )). These species occupy key positions along the phylogenetic tree, allowing us to investigate most major transitions in the animal lineage.…”

“…Our analysis of NELF evolution shows that the core NELF subunits, NELF-B and -C/D, evolved earlier than the ancillary subunits, NELF-A and -E. To test the functional impact of core and ancillary subunits in mammalian cells, we generated FKBP12-homozygously tagged mouse embryonic stem cell (mESC) lines that rapidly degrade either NELF-B (13) or NELF-E after treatment with the small molecule dTAG-13 ( Fig. 3A-B; ( 7, 31 )). The NELF-B dTAG was reported and validated in a recent paper ( 7 ), while the NELF-E dTAG cell line is novel here.…”

“…3A-B; ( 7, 31 )). The NELF-B dTAG was reported and validated in a recent paper ( 7 ), while the NELF-E dTAG cell line is novel here. We verified that the FKBP12-tagged NELF-E protein was properly localized and that NELF-E was nearly undetectable within 30 min after the addition of 500 nM dTAG ( fig.…”

“…To rule out the possibility that our observed differences in HSF1-dependent gene activation were driven by changes in gene expression following dTAG-13 ( 7 ), including the accumulation of Pol II trickling into the gene body ( fig. S10 ), we focused our analysis on the gene body downstream from NELF-induced Pol II trickling regions.…”