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Global analysis of short RNAs reveals widespread promoter-proximal stalling and arrest of Pol II in Drosophila.

ABSTRACT: Emerging evidence indicates that gene expression in higher organisms is regulated by RNA polymerase II stalling during early transcription elongation. To probe the mechanisms responsible for this regulation, we developed methods to isolate and characterize short RNAs derived from stalled RNA polymerase II in Drosophila cells. Significant levels of these short RNAs were generated from more than one-third of all genes, indicating that promoter-proximal stalling is a general feature of early polymerase elongation. Nucleotide composition of the initially transcribed sequence played an important role in promoting transcriptional stalling by rendering polymerase elongation complexes highly susceptible to backtracking and arrest. These results indicate that the intrinsic efficiency of early elongation can greatly affect gene expression.

SUBMITTER: Nechaev S

PROVIDER: S-EPMC3435875 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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Global analysis of short RNAs reveals widespread promoter-proximal stalling and arrest of Pol II in Drosophila.

Nechaev Sergei S Fargo David C DC dos Santos Gilberto G Liu Liwen L Gao Yuan Y Adelman Karen K

Science (New York, N.Y.) 20091210 5963

Emerging evidence indicates that gene expression in higher organisms is regulated by RNA polymerase II stalling during early transcription elongation. To probe the mechanisms responsible for this regulation, we developed methods to isolate and characterize short RNAs derived from stalled RNA polymerase II in Drosophila cells. Significant levels of these short RNAs were generated from more than one-third of all genes, indicating that promoter-proximal stalling is a general feature of early polyme ...[more]

PMID: 20007866

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Global Analysis of Short RNAs Reveals Widespread Promoter-Proximal Stalling and Arrest of Pol II

Project description:We developed a novel approach to isolate and characterize, at nucleotide-level resolution, the RNAs derived from RNA polymerase II (Pol II) in early elongation complexes. We find that high numbers of these short RNAs are not correlated with gene expression but instead are indicative of promoter-proximally stalled polymerase. High amounts of these short RNAs are generated from more than one third of all genes, indicating that Pol II stalls in the promoter-proximal region on global scale. We also find that the nucleotide composition of the initially transcribed sequence plays an important role in promoting transcriptional stalling and that early elongation complexes are highly susceptible to backtracking and arrest. Global analysis of RNAs produced by promoter-proximal Pol II.

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Promoter elements associated with RNA Pol II stalling in the Drosophila embryo.

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NELF-mediated stalling of Pol II can enhance gene expression by blocking promoter-proximal nucleosome assembly.

Project description:The Negative Elongation Factor (NELF) is a transcription regulatory complex that induces stalling of RNA polymerase II (Pol II) during early transcription elongation and represses expression of several genes studied to date, including Drosophila Hsp70, mammalian proto-oncogene junB, and HIV RNA. To determine the full spectrum of NELF target genes in Drosophila, we performed a microarray analysis of S2 cells depleted of NELF and discovered that NELF RNAi affects many rapidly inducible genes involved in cellular responses to stimuli. Surprisingly, only one-third of NELF target genes were, like Hsp70, up-regulated by NELF-depletion, whereas the majority of target genes showed decreased expression levels upon NELF RNAi. Our data reveal that the presence of stalled Pol II at this latter group of genes enhances gene expression by maintaining a permissive chromatin architecture around the promoter-proximal region, and that loss of Pol II stalling at these promoters is accompanied by a significant increase in nucleosome occupancy and a decrease in histone H3 Lys 4 trimethylation. These findings identify a novel, positive role for stalled Pol II in regulating gene expression and suggest that there is a dynamic interplay between stalled Pol II and chromatin structure.

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Oxidative stress rapidly stabilizes promoter-proximal paused Pol II across the human genome.

Project description:Oxidative stress has pervasive effects on cells but how they respond transcriptionally upon the initial insult is incompletely understood. We developed a nuclear walk-on assay that semi-globally quantifies nascent transcripts in promoter-proximal paused RNA polymerase II (Pol II). Using this assay in conjunction with ChIP-Seq, in vitro transcription, and a chromatin retention assay, we show that within a minute, hydrogen peroxide causes accumulation of Pol II near promoters and enhancers that can best be explained by a rapid decrease in termination. Some of the accumulated polymerases slowly move or 'creep' downstream. This second effect is correlated with and probably results from loss of NELF association and function. Notably, both effects were independent of DNA damage and ADP-ribosylation. Our results demonstrate the unexpected speed at which a global transcriptional response can occur. The findings provide strong support for the residence time of paused Pol II elongation complexes being much shorter than estimated from previous studies.

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Project description:RNA polymerase II (RNA Pol II) is generally paused at promoter-proximal regions in most metazoans, and based on in vitro studies, this function has been attributed to the negative elongation factor (NELF). Here, we show that upon rapid depletion of NELF, RNA Pol II fails to be released into gene bodies, stopping instead around the +1 nucleosomal dyad-associated region. The transition to the 2nd pause region is independent of positive transcription elongation factor P-TEFb. During the heat shock response, RNA Pol II is rapidly released from pausing at heat shock-induced genes, while most genes are paused and transcriptionally downregulated. Both of these aspects of the heat shock response remain intact upon NELF loss. We find that NELF depletion results in global loss of cap-binding complex from chromatin without global reduction of nascent transcript 5' cap stability. Thus, our studies implicate NELF functioning in early elongation complexes distinct from RNA Pol II pause-release.

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Release of promoter-proximal paused Pol II in response to histone deacetylase inhibition.

Project description:A correlation between histone acetylation and transcription has been noted for a long time, but little is known about what step(s) in the transcription cycle is influenced by acetylation. We have examined the immediate transcriptional response to histone deacetylase (HDAC) inhibition, and find that release of promoter-proximal paused RNA polymerase II (Pol II) into elongation is stimulated, whereas initiation is not. Although histone acetylation is elevated globally by HDAC inhibition, less than 100 genes respond within 10 min. These genes are highly paused, are strongly associated with the chromatin regulators NURF and Trithorax, display a greater increase in acetylation of the first nucleosomes than other genes, and become transcriptionally activated by HDAC inhibition. Among these rapidly up-regulated genes are HDAC1 (Rpd3) and subunits of HDAC-containing co-repressor complexes, demonstrating feedback regulation upon HDAC inhibition. Our results suggest that histone acetylation stimulates transcription of paused genes by release of Pol II into elongation, and that increased acetylation is not a consequence of their enhanced expression. We propose that HDACs are major regulators of Pol II pausing and that this partly explains the presence of HDACs at active genes.

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