Regulating RNA polymerase pausing and transcription elongation in embryonic stem cells

Abstract

Transitions between pluripotent stem cells and differentiated cells are executed by key transcription regulators. Comparative measurements of RNA polymerase distribution over the genome's primary transcription units in different cell states can identify the genes and steps in the transcription cycle that are regulated during such transitions. To identify the complete transcriptional profiles of RNA polymerases with high sensitivity and resolution, as well as the critical regulated steps upon which regulatory factors act, we used genome-wide nuclear run-on (GRO-seq) to map the density and orientation of transcriptionally engaged RNA polymerases in mouse embryonic stem cells (ESCs) and mouse embryonic fibroblasts (MEFs). In both cell types, progression of a promoter-proximal, paused RNA polymerase II (Pol II) into productive elongation is a rate-limiting step in transcription of~40% of mRNA-encoding genes. Importantly, quantitative comparisons between cell types reveal that transcription is controlled frequently at paused Pol II's entry into elongation. Furthermore, ''bivalent'' ESC genes (exhibiting both active and repressive histone modifications) bound by Polycomb group complexes PRC1 (Polycomb-repressive complex 1) and PRC2 show dramatically reduced levels of paused Pol II at promoters relative to an average gene. In contrast, bivalent promoters bound by only PRC2 allow Pol II pausing, but it is confined to extremely 59 proximal regions. Altogether, these findings identify rate-limiting targets for transcription regulation during cell differentiation.

Transcription-associated processes cause DNA double-strand breaks and translocations in neural stem/progenitor cells

“…Aggregate patterns of transcription profiles showed enrichment of reads within 1.5 kb of TSSs genome-wide (Fig. S1B, Right), consistent with previously published GRO-seq results obtained in other cell types (30,31). To perform HTGTS for DSB identification, we used a Cas9:sgRNAbased approach to introduce HTGTS bait DSBs into primary NSPCs isolated from Xrcc4 −/− p53 −/− mice, as previously described (4).…”

“…To assess whether transcription-associated DSBs occur and form translocations in NSPCs, we first identified actively transcribed genes by performing GRO-seq (8,30,31) in Xrcc4 −/− p53 −/− NSPCs. In these analyses, a Pearson correlation coefficient analysis showed high reproducibility among three independent experiments (Fig.…”

Embryonic stem cell and induced pluripotent stem cell: an epigenetic perspective

“…Instead, the majority of promoters are marked by H3K4me3, regardless of the gene expression status [63,64]. Although the presence of both H3K4me3 and Pol II at promoter is an indication of transcription allowance [63,64], productive transcription relies on the rate-limiting step of RNA Pol II releasing from the promoter [63,65,66]. The inactive state of some H3K4me3-marked genes can also be attributed to the coexistence of repressive modifications, such as H3K27me3 (see below).…”

Enforcing the pause: Transcription factor Sp3 limits productive elongation by RNA polymerase II

“…The kinase activity of P-TEFb Drosophila cells revealed that loss of NELF-mediated pausing reduced RNA Pol II promoter occupancy at most, but not all, promoters, 2 globally implicating NELF in inhibition of early elongation. The high sensitivity of the global runon-sequencing (GRO-seq) technique, 51 allowed the determination that even active genes undergo transient pausing of RNA Pol II, 47,52 suggesting that NELF-mediated pausing may be a general feature of the transcription mechanism. Furthermore, these studies suggest that the efficiency of pause release, rather than the establishment of pausing, plays a major role in determining gene expression levels.…”

“…47 Following initiation, RNA Pol II falls under the control of negative elongation factors, including DRB (5,6-ichlorobenzimidazole riboside) sensitivity-inducing factor (DSIF) and the negative elongation factor complex (NELF). NELF is the major factor associated with the early pausing of RNA Pol II.…”