ARF alters PAF1 complex integrity to selectively repress oncogenic transcription programs upon p53 loss
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE217765 | GEO | 2024/10/10
REPOSITORIES: GEO
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Project description:In this study, we found that the ARF tumor suppressor directly binds the PAF1 subunit to block the assembly of PAF1 complex (PAF1c) and its interaction with RNAPII, thereby dampening PAF1c-dependent transcription in vitro and in cells. Using RNA-seq of control (shNT) and ARF knockdown (shARF) mouse embryonic fibroblasts (MEFs) we first define transcriptome changes upon ARF loss. Through downstream mechanistic analysis we then validate direct ARF-mediated inactivation of PAF1c-dependent RNAPII transcription and their cellular consequences.
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Project description:Release of promoter-proximal paused RNA polymerase II (Pol II) during early elongation is a critical step in transcriptional regulation in metazoan cells. Paused Pol II release is thought to require the kinase activity of cyclin-dependent kinase 9 (CDK9) for the phosphorylation of DRB sensitivity-inducing factor, negative elongation factor, and C-terminal domain (CTD) serine-2 of Pol II. We found that Pol II-associated factor 1 (PAF1) is a critical regulator of paused Pol II release, that positive transcription elongation factor b (P-TEFb) directly regulates the initial recruitment of PAF1 complex (PAF1C) to genes, and that the subsequent recruitment of CDK12 is dependent on PAF1C. These findings reveal cooperativity among P-TEFb, PAF1C, and CDK12 in pausing release and Pol II CTD phosphorylation. Comparison of the chromatin occupancy of [1] PAF1, CDC73, LEO1, CTR9, total Pol II, and CTD serine 2-phosphorylated Pol II by ChIP-seq in THP1 cells; [2] PAF1, Pol II, Pol II (ser-5p), CDK12, and CDK9 by ChIP-seq in control and PAF1 knockdown cells; [3] LEO1 and Pol II by ChIP-seq in control and flavopiridol treated THP1 cells.
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Project description:Ctr9, the key scaffold subunit of the human RNA polymerase II (RNAPII) associated factor complex (PAFc), has been demonstrated as a positive regulator of ERα-positive breast cancer progression and ERα-target gene expression. Previously, we found that knockdown of Ctr9 reduces ERα protein stability and decreases the occupancy of ERα and RNAPII at select ERα-target genes. However, the genome-wide regulation of the occupancy of ERα and RNAPII mediated by Ctr9 is still unclear. Here, we determined the genome-wide ERα and RNAPII occupancy in response to estrogen induction and/or Ctr9 knockdown by performing chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq). We found that loss of Ctr9 dramatically decreases the global occupancy of ERα and RNAPII, highlighting the significance of Ctr9 in regulating estrogen signaling in ERα-positive breast cancer cells. Combining this resource with previously published genomic data sets, we identified a unique subset of ERα and Ctr9 target genes, and further delineates the possible independent function of Ctr9 from other subunits in PAFc.
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Project description:Eukaryotes employ a set of conserved transcription elongation factors to modulate the behavior of RNA polymerase II (RNAPII). Disruptions of one such factor, the Paf1 complex (Paf1C), generate subunit-specific phenotypes, including distinct changes to co-transcriptional histone modifications. How individual Paf1C subunits impact transcription and coupled processes remains ambiguous. By comparing conditional depletion and steady-state deletion of Paf1C subunits, we determine direct and indirect contributions of Paf1C to gene expression in Saccharomyces cerevisiae. Through nascent transcript sequencing, RNAPII profiling, and mechanistic modeling of transcription elongation dynamics, we find evidence for unique roles of Paf1C subunits in regulating RNAPII processivity, elongation rate, mRNA stability, transcript splicing, and repression of antisense transcripts. Through genetic suppression, we attribute increased antisense transcription, but not other defects, of a PAF1 mutant to loss of H3 K36 methylation. This work comprehensively analyzes both the immediate and extended roles of Paf1C subunits in transcription elongation and transcript regulation.
2024-05-15 | GSE255362 | GEO
Project description:Eukaryotes employ a set of conserved transcription elongation factors to modulate the behavior of RNA polymerase II (RNAPII). Disruptions of one such factor, the Paf1 complex (Paf1C), generate subunit-specific phenotypes, including distinct changes to co-transcriptional histone modifications. How individual Paf1C subunits impact transcription and coupled processes remains ambiguous. By comparing conditional depletion and steady-state deletion of Paf1C subunits, we determine direct and indirect contributions of Paf1C to gene expression in Saccharomyces cerevisiae. Through nascent transcript sequencing, RNAPII profiling, and mechanistic modeling of transcription elongation dynamics, we find evidence for unique roles of Paf1C subunits in regulating RNAPII processivity, elongation rate, mRNA stability, transcript splicing, and repression of antisense transcripts. Through genetic suppression, we attribute increased antisense transcription, but not other defects, of a PAF1 mutant to loss of H3 K36 methylation. This work comprehensively analyzes both the immediate and extended roles of Paf1C subunits in transcription elongation and transcript regulation.
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Project description:Eukaryotes employ a set of conserved transcription elongation factors to modulate the behavior of RNA polymerase II (RNAPII). Disruptions of one such factor, the Paf1 complex (Paf1C), generate subunit-specific phenotypes, including distinct changes to co-transcriptional histone modifications. How individual Paf1C subunits impact transcription and coupled processes remains ambiguous. By comparing conditional depletion and steady-state deletion of Paf1C subunits, we determine direct and indirect contributions of Paf1C to gene expression in Saccharomyces cerevisiae. Through nascent transcript sequencing, RNAPII profiling, and mechanistic modeling of transcription elongation dynamics, we find evidence for unique roles of Paf1C subunits in regulating RNAPII processivity, elongation rate, mRNA stability, transcript splicing, and repression of antisense transcripts. Through genetic suppression, we attribute increased antisense transcription, but not other defects, of a PAF1 mutant to loss of H3 K36 methylation. This work comprehensively analyzes both the immediate and extended roles of Paf1C subunits in transcription elongation and transcript regulation.
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Project description:Our structural and biochemical studies show that S. cerevisiae RNA Polymerase II (RNAPII) homodimerizes through the stalk domain (formed by the Rpb4-Rpb7 subunits). To explore the biological impact of disrupting this interaction we introduced a triple point mutation at the RNAPII dimerization interface in Rpb7 (Q96A, H97A, F109K). To test the impact of the dimerziation mutant on RNAPII occupancy, we performed ChIP-seq using a monoclonal antibody against the Rpb3 subunit (1Y26, abcam) of RNAPII, in WT and Rpb7-QHF cells.
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Project description:Chromatin places fundamental physical constraints on transcription (Gamarra and Narlikar, 2021). The PAF1 complex (PAF1C), a hexamer of PAF1, LEO1, CTR9, SKI8, CDC73 and RTF1, plays a critical role in transcription with incompletely understood mechanisms. During transcriptional elongation, PAF1C is one of the positive elongation factors in complex with RNA polymerase II (Pol II) to facilitate elongation through chromatin (Vos et al., 2018), but it is unclear yet if negative elongation factors are needed concurrently to restrain elongation. Here we show that besides decreasing elongation rate, LEO1 knockout in human K562 cells increases transcriptional readthrough and cellular level of C-terminal domain (CTD) phosphorylated Pol II while increases and decreases transcriptional output of several hundred genes, respectively. Mechanistic analyses taking proteomic, functional genomic and biochemical approaches discovered that PAF1C regulates transcriptional termination in part through recruiting PNUTS-PP1γ complex and facilitates Pol II transcriptional re-initiation through recruiting TOX4-PP1α complex. Moreover, Paf1 conditional knockout in mice severely blocks T cell development, increases cellular level of CTD phosphorylated Pol II, mainly decreases Pol II occupancy and transcriptional output in double positive T cells, and importantly, the regulation of re-initiation by PAF1C through TOX4-PP1α complex is conserved between mouse and human. Our results also suggest that PNUTS-PP1γ and TOX4-PP1α bind PAF1C to restrain Pol II elongation through chromatin. These findings not only establish PAF1C as a critical regulator of transcriptional termination and re-initiation besides elongation but also advanced current understanding of elongation.
2024-11-06 | GSE188412 | GEO
Project description:Chromatin places fundamental physical constraints on transcription (Gamarra and Narlikar, 2021). The PAF1 complex (PAF1C), a hexamer of PAF1, LEO1, CTR9, SKI8, CDC73 and RTF1, plays a critical role in transcription with incompletely understood mechanisms. During transcriptional elongation, PAF1C is one of the positive elongation factors in complex with RNA polymerase II (Pol II) to facilitate elongation through chromatin (Vos et al., 2018), but it is unclear yet if negative elongation factors are needed concurrently to restrain elongation. Here we show that besides decreasing elongation rate, LEO1 knockout in human K562 cells increases transcriptional readthrough and cellular level of C-terminal domain (CTD) phosphorylated Pol II while increases and decreases transcriptional output of several hundred genes, respectively. Mechanistic analyses taking proteomic, functional genomic and biochemical approaches discovered that PAF1C regulates transcriptional termination in part through recruiting PNUTS-PP1γ complex and facilitates Pol II transcriptional re-initiation through recruiting TOX4-PP1α complex. Moreover, Paf1 conditional knockout in mice severely blocks T cell development, increases cellular level of CTD phosphorylated Pol II, mainly decreases Pol II occupancy and transcriptional output in double positive T cells, and importantly, the regulation of re-initiation by PAF1C through TOX4-PP1α complex is conserved between mouse and human. Our results also suggest that PNUTS-PP1γ and TOX4-PP1α bind PAF1C to restrain Pol II elongation through chromatin. These findings not only establish PAF1C as a critical regulator of transcriptional termination and re-initiation besides elongation but also advanced current understanding of elongation.
2024-11-06 | GSE188415 | GEO
Project description:Chromatin places fundamental physical constraints on transcription (Gamarra and Narlikar, 2021). The PAF1 complex (PAF1C), a hexamer of PAF1, LEO1, CTR9, SKI8, CDC73 and RTF1, plays a critical role in transcription with incompletely understood mechanisms. During transcriptional elongation, PAF1C is one of the positive elongation factors in complex with RNA polymerase II (Pol II) to facilitate elongation through chromatin (Vos et al., 2018), but it is unclear yet if negative elongation factors are needed concurrently to restrain elongation. Here we show that besides decreasing elongation rate, LEO1 knockout in human K562 cells increases transcriptional readthrough and cellular level of C-terminal domain (CTD) phosphorylated Pol II while increases and decreases transcriptional output of several hundred genes, respectively. Mechanistic analyses taking proteomic, functional genomic and biochemical approaches discovered that PAF1C regulates transcriptional termination in part through recruiting PNUTS-PP1γ complex and facilitates Pol II transcriptional re-initiation through recruiting TOX4-PP1α complex. Moreover, Paf1 conditional knockout in mice severely blocks T cell development, increases cellular level of CTD phosphorylated Pol II, mainly decreases Pol II occupancy and transcriptional output in double positive T cells, and importantly, the regulation of re-initiation by PAF1C through TOX4-PP1α complex is conserved between mouse and human. Our results also suggest that PNUTS-PP1γ and TOX4-PP1α bind PAF1C to restrain Pol II elongation through chromatin. These findings not only establish PAF1C as a critical regulator of transcriptional termination and re-initiation besides elongation but also advanced current understanding of elongation.
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