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 studied predominantly 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 find that loss of RNA Pol II pausing stalls pluripotent state transitions in the epiblast of the early mouse embryo. Using Nelfb-/- mice and a novel NELFB- degron mouse embryonic stem cells, we show that mouse ES cells (mESCs) representing the naive 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. Consistently, we find an increase in chromatin-associated NELF during pluripotency transitions. Overall, our work reveals the molecular and phenotypic roles of Pol II pausing in pluripotency and introduces Pol II pausing as a modulator of cell state transitions.

Generation of knock-in degron tags for endogenous proteins in mice using the dTAG system

“…Additionally, immunostaining revealed that, while the homozygous embryos exhibit SOX2 staining, a marker of anterior epiblast at E7.5, they lack NANOG staining on the posterior side, a marker of the posterior primed epiblast ( Figure 5 D). These defects closely resemble the null allele defects we previously reported ( Abuhashem et al., 2022a ). Note that degradation and recovery kinetics for each protein can be different, and they should be defined prior to attempting recapitulating a null defect.…”

“…In the presence of continuous degradation, the resulting tissue is expected to phenocopy a mouse model with a null allele. We recently reported that null Nelfb mouse embryos exhibit reduced growth and failure to specify the primed posterior epiblast at embryonic day (E) 6.5 ( Abuhashem et al., 2022a ). To recapitulate this defect, we crossed Nelfb dTAG/dTAG male with Nelfb dTAG/+ female.…”

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

“…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.…”

“…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 cerevisiae and Schizosaccharomyces pombe (9,10)), model plants (Arabidopsis thaliana, Oryza sativa, and Zea mays (13)(14)(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.…”

“…1A) (1)(2)(3)(4). The rate at which polymerases are "released" from a paused state into productive elongation is actively regulated by transcription factors (5), and is therefore essential for proper development in most animal species (6)(7)(8). However, unicellular model organisms, including yeast (9,10), do not have a promoter-proximal pause.…”