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Hyperosmotic stress induces downstream-of-gene transcription and alters the RNA Polymerase II interactome despite widespread transcriptional repression

ABSTRACT: Stress-induced readthrough transcription results in the synthesis of thousands of downstream-of-gene (DoG) containing transcripts. The mechanisms underlying DoG formation during cellular stress remain unknown. Nascent transcription profiles during DoG induction in human cell lines using TT-TimeLapse-seq revealed that hyperosmotic stress induces widespread transcriptional repression. Yet, DoGs are produced regardless of the transcriptional level of their upstream genes. ChIP-seq confirmed that the stress-induced redistribution of RNA Polymerase (Pol) II correlates with the transcriptional output of genes. Stress-induced alterations in the Pol II interactome are observed by mass spectrometry. While subunits of the cleavage and polyadenylation machinery remained Pol II-associated, Integrator complex subunits dissociated from Pol II under stress conditions. Depleting the catalytic subunit of the Integrator complex, Int11, using siRNAs induces hundreds of readthrough transcripts, whose parental genes partially overlap those of stress-induced DoGs. Our results provide insights into the mechanisms underlying DoG production and how Integrator activity influences DoG transcription. This SuperSeries is composed of the SubSeries listed below.

ORGANISM(S): Homo sapiens

PROVIDER: GSE152063 | GEO | 2021/01/04

REPOSITORIES: GEO

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Comparative analysis reveals genomic features of stress-induced transcriptional readthrough

Project description:Transcription is a highly regulated process, and stress-induced changes in gene transcription have been shown to play a major role in responses and adaptation to stress. Numerous emerging genome-wide studies reveal prevalent transcription beyond known protein-coding gene loci, generating a variety of new classes of RNAs, most of unknown function. One such class, termed downstream of gene (DoG)-containing transcripts, was reported to result from transcriptional readthrough upon osmotic stress in human cell lines. However, how widespread the readthrough phenomenon is, and what its causes and consequences are, remain elusive. Here we present a systematic genome-wide mapping of transcriptional readthrough, using deep nuclear RNA-seq, comparing heat shock, osmotic and oxidative stress in NIH3T3 mouse fibroblast cells. We observe massive induction of transcriptional readthrough under all stress conditions, with significant, yet not complete overlap of readthrough-induced loci between different conditions. Importantly, our analyses suggest that stress-induced transcriptional readthrough is not a random failure process, but is rather differentially induced across different conditions. Additionally, analyzing public Pol-II occupancy data further supported our findings of stress-induced readthrough. We explore potential regulators and find a role for HSF1 in the induction of a subset of heat shock-induced readthrough transcripts. Furthermore, we examine genomic features of readthrough transcription, and observe a unique chromatin signature typical of DoG-producing regions, suggesting that readthrough transcription is associated with the maintenance of an open chromatin state.

2017-08-31 | GSE98906 | GEO

TT-TL-seq reveals transcriptional profiles that accompany DoG induction after hyperosmotic stress.

Project description:TT-TimeLapse-seq was used to investigate the nascent transcription profiles that accompany the induction of downstream-of-gene (DoG) containing transcripts in human cell lines. Data from HEK-293T cells exposed to hyperosmotic stress and cells transfected with siRNAs against Integrator subunit 11 are included.

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Integrator Recruits Protein Phosphatase 2A to Prevent Pause Release and Enable Transcription Termination

Project description:Efficient release of promoter-proximally paused Pol II into productive elongation is essential for gene expression. Recently, we reported that the Integrator complex can bind paused Pol II and drive premature transcription termination, potently attenuating the activity of target genes. Premature termination requires RNA cleavage by the endonuclease subunit of Integrator, but the roles of other Integrator subunits in gene regulation have yet to be elucidated. Here, we report that Integrator subunit 8 (IntS8) is critical for transcription repression through its association with Protein Phosphatase 2A (PP2A). We find that Integrator-bound PP2A dephosphorylates the Pol II C-terminal domain and Spt5, and prevents the transition to productive elongation. Blocking PP2A association with Integrator thus stimulates pause release and gene activation. These results reveal a second catalytic function associated with Integrator-mediated transcription termination, and suggest a new model for the control of productive elongation involving active competition between transcriptional kinases and phosphatases.

2020-10-06 | GSE150844 | GEO

Targeted protein degradation reveals Pol II heterogeneity and functional diversity

Project description:RNA polymerase II (Pol II) subunits are thought to be involved in various transcription-associated processes, but it is unclear whether they play different regulatory roles in modulating gene expression. Here, we performed nascent and mature transcript sequencing after the acute degradation of 12 mammalian Pol II subunits and profiled their genomic binding sites and protein interactomes to dissect their molecular functions. We found that Pol II subunits contribute differently to Pol II cellular localization and transcription process and preferentially regulate RNA processing (such as RNA splicing and 3’ end maturation). Genes sensitive to the depletion of different Pol II subunits tend to be involved in diverse biological functions and show different RNA half-lives. Sequences, associated protein factors, and RNA structures are correlated with Pol II subunit-mediated differential gene expression. These findings collectively suggest that the heterogeneity of Pol II and different genes appear to depend on some of the subunits.

2022-08-09 | PXD035866 | iProX

CAPTURE of the human U2 snRNA genes expands the repertoire of associated factors.

Project description:In order to identify factors involved in transcription of human snRNA genes and 3’ end processing of the transcripts, we have carried out CRISPR affinity purification in situ of regulatory elements (CAPTURE), which is deadCas9-mediated pull-down, of the tandemly-repeated U2 snRNA genes in human cells. CAPTURE enriched many factors expected to be associated with human snRNA genes including pol II, NELF, CDK9, SPT5 and several subunits of the Integrator Complex. SPT6, Mediator subunits 16, 20 and 23, TAF15, CDK7, CDK9, Cyclin K, and the SPT16 subunit of FACT were also enriched. Several polyadenylation factors, including CPSF1, CPSF6 and CstF1-3 were also enriched by U2 gene CAPTURE. These add to the polyadenylation factors CstF2, Pcf11 and Ssu72 which we previously showed are associated with the human U1 and U2 genes. Analysis of genome-wide studies and ChIP-qPCR confirms the association of SPT6 with the U2 genes. In addition, SPT6 knockdown causes loss of subunit 3 of the Integrator complex from the U2 genes and transcriptional readthrough, indicating a functional role in snRNA gene expression. CAPTURE has therefore expanded the repertoire of transcription and RNA processing factors associated with these genes.

2022-05-18 | GSE197372 | GEO

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Project description:Using a modification of the yeast anchor away and PAR-CLIP protocols, we have mapped the position of Pol II genome-wide in living yeast cells after depletion of components of either the polyadenylation (pA) or non-pA termination complexes. Depletion of Ysh1 from the nucleus does not lead to readthrough transcription. In contrast, depletion of the termination factor Nrd1 leads to widespread runaway elongation of non-pA transcripts. Depletion of Sen1 also leads to readthrough at non-pA terminators but in contrast to Nrd1 this readthrough appears less processive. Examination of PAR-CLIP sequencing data of Pol II after removal of different transcriptional termination factors.

2014-07-24 | E-GEOD-56435 | biostudies-arrayexpress

The PAF1 complex promotes 3‘ processing of pervasive transcripts

Project description:The PAF1 complex (PAF1C) functions in multiple transcriptional processes involving RNA Polymerase II (Pol II). eRNAs and PROMPTs are pervasive transcripts transcribed by Pol II and rapidly degraded by the nuclear exosome complex after 3’ endonucleolytic cleavage by the Integrator complex (Integrator). Here we show that PAF1C has an unexpected role in the termination of eRNAs and PROMPTs that are cleaved 1-3 kb downstream of the transcription start site. Mechanistically, PAF1C facilitates recruitment of Integrator to sites of pervasive transcript cleavage, promoting timely cleavage and transcription termination. We also show that PAF1C recruits Integrator to coding genes, where PAF1C then dissociates from Integrator upon entry into processive elongation. Our results demonstrate an unexpected function for PAF1C in limiting the length and accumulation of pervasive transcripts that result from nonproductive transcription

2022-03-03 | PXD031531 | Pride

Integrator restraints paraspeckles assembly by promoting the 3’-end processing of the polyadenylated lncRNA NEAT1_1

Project description:Alternative 3’-end RNA processing provides an important source of transcriptome diversification, which impacts various biological processes and the etiology of diseases, including cancer. Here, we focused on a transcript isoform switch that leads to the readthrough expression of the long noncoding RNA NEAT1_2, at the expense of the shorter polyadenylated transcript NEAT1_1. Expression of NEAT1_2 is required for the formation of paraspeckles (PS), nuclear bodies that protect cancer cells from oncogene-induced replication stress and chemotherapy. Searching for proteins that interact with endogenous NEAT1 by RNA Affinity Purification coupled to mass spectrometry (RAP-MS), we identified factors involved in the 3’-end processing of polyadenylated (pA+) RNA as well as several components of the Integrator complex, known to process the 3’-ends of RNAs lacking polyadenylation sites. Unexpectedly, genetic perturbation experiments established that Integrator restraints NEAT1_2 expression, and thereby PS assembly, by promoting the 3’-end processing of pA+ NEAT1_1. Consistently, low expression levels of several Integrator subunits correlated with poorer prognosis of cancer patients exposed to chemotherapeutics. Our study identifies Integrator as a key regulator of PS biogenesis and highlights a previously unrecognized ability of the complex to process pA+ RNA. The data also establish a link between Integrator, cancer biology and chemosensitivity, which may be exploited therapeutically.

2020-10-14 | PXD015158 | Pride

The Integrator complex terminates promoter-proximal transcription at protein-coding genes

Project description:The transition of RNA polymerase II (Pol II) from initiation to productive elongation is a central, regulated step in metazoan gene expression. At many genes, Pol II pauses stably in early elongation, remaining engaged with the 25-60 nucleotide-long nascent RNA for many minutes while awaiting signals for release into the gene body. However, a number of genes display highly unstable promoter Pol II, suggesting that paused polymerase terminates transcription at these promoters and releases a short, non-functional RNA. Here, we investigate the mechanisms driving Pol II instability and discover that the Integrator complex targets paused Pol II and terminates transcription at selected genes. Specifically, Integrator utilizes its RNA endonuclease activity to cleave nascent RNA and destabilize elongating polymerase. Our findings uncover a previously unappreciated mechanism of metazoan gene repression, wherein promoter paused Pol II is prevented from entering productive elongation through regulated termination.

2019-11-27 | GSE114467 | GEO

Targeted protein degradation reveals Pol II heterogeneity and functional diversity [ChIP-Seq]

2022-08-09 | GSE179965 | GEO

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