Pausing of RNA Polymerase II Regulates Mammalian Developmental Potential through Control of Signaling Networks

Abstract

SUMMARY The remarkable capacity for pluripotency and self-renewal in embryonic stem cells (ESCs) requires a finely-tuned transcriptional circuitry wherein the pathways and genes that initiate differentiation are suppressed, but poised to respond rapidly to developmental signals. To elucidate transcriptional control in mouse ESCs in the naïve, ground state, we defined the distribution of engaged RNA polymerase II (Pol II) at high-resolution. We find that promoter-proximal pausing of Pol II is most enriched at genes regulating cell cycle and signal transduction, and not, as expected, at developmental or bivalent genes. Accordingly, ablation of the primary pause-inducing factor NELF does not increase expression of lineage markers, but instead causes proliferation defects, embryonic lethality and dysregulation of ESC signaling pathways. Indeed, ESCs lacking NELF have dramatically attenuated FGF/ERK activity, rendering them resistant to differentiation. This work thus uncovers a key role for NELF-mediated pausing in establishing the responsiveness of stem cells to developmental cues.

The positive transcriptional elongation factor (P-TEFb) is required for neural crest specification

“…6 bottom route). Recently it has been shown that regulation of transcriptional elongation to be important for genes regulating cell cycle and signal transduction (Williams et al, 2015). c-Myc is an important regulator for such processes and as we show here it is clearly important in the regulation of the neural crest.…”

The Tumor Suppressor ARID1A Controls Global Transcription via Pausing of RNA Polymerase II

“…4A–D). There is broad consensus that most of the promoter-proximal RNAPII signal represents the paused form of polymerase (Adelman and Lis, 2012; Henriques et al, 2013; Levine, 2011; Williams et al, 2015). After transcription initiation, an early RNAPII elongation complex begins mRNA synthesis, only to arrest within the first 30–50bp.…”

“…The mechanism of RNAPII pausing has emerged, over the last decade, as a fundamental regulatory step of transcription in higher eukaryotes (Adelman and Lis, 2012). It must be noted that pausing of RNAPII is not simply a brake for transcription, as paused RNAPII in ES cells is required to maintain cell homeostasis and properly respond to environmental stimuli and cues (Henriques et al, 2013; Shao and Zeitlinger, 2017; Williams et al, 2015). In the absence of properly functioning pausing machinery, such as upon depletion of NELF, thousands of genes were shown to respond with transcriptional attenuation (Williams et al, 2015).…”

“…Paused RNA polymerase is centrally regulated by the negative elongation factor, NELF, which prevents productive elongation and determines arrest of the RNAPII holoenzyme within the first 100bp from the Transcription Start Site (TSS). Depletion of NELF and loss of paused RNAPII result in overall loss of polymerase from the gene locus and transcriptional downregulation (Gilchrist et al, 2010; Williams et al, 2015). While several complexes that allow for coordinated release of paused polymerase have been identified (Gardini et al, 2014; Patel et al, 2013; Rahl et al, 2010), little is known about additional mechanisms and proteins that actively enforce pausing genome-wide (Chen et al, 2015).…”

The point of no return: The poly(A)-associated elongation checkpoint

“…[6][7][8] In addition, pausing at this checkpoint can ensure synchronous activation during development 9 and control of signaling networks in embryonic stem cells. 10 Global Run-On Sequencing (GRO-seq) analysis 11 of actively-engaged pol II after treatment of HeLa cells with the CDK9 inhibitors DRB, KM05283, and Flavopiridol indicates that the vast majority of pol II-transcribed genes have a kinasedependent early-elongation checkpoint. 12,13 The CDK9 inhibitors also induce an increase in pol II levels just downstream of the transcription start site (TSS), which is thought to result from pol II piling up because it is unable to negotiate its way through the early elongation checkpoint.…”