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ARF alters PAF1 complex integrity to selectively repress oncogenic transcription programs upon p53 loss

ABSTRACT: 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. To investigate whether ARF regulates RNAPII and PAF1c occupancy at putative target genes identified through RNA-seq, we performed ChIP-seq (RNAPII, PAF1, and CTR9) in ARF (shARF) vs. control (shNT) knockdown MEFs. Our data show that loss of ARF increases RNAPII, PAF1, and CTR9 occupancy on upregulated genes, consistent with ARF-mediated gene-specific repression. Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for RNA polymerase subunit RPB3 and PAF1 complex subunits PAF1 and CTR9 in mouse embryonic fibroblasts.

ORGANISM(S): Mus musculus

PROVIDER: GSE217765 | GEO | 2024/10/10

REPOSITORIES: GEO

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ARF alters PAF1 complex integrity to selectively repress oncogenic transcription programs upon p53 loss

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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RNA Polymerase II-associated factor 1 regulates the release and phosphorylation of paused RNA Polymerase II

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2015-12-11 | E-GEOD-62171 | biostudies-arrayexpress

Systematic identification of Ctr9 regulome in ERα-positive breast cancer

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.

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Multiple direct and indirect roles of the Paf1 Complex in elongation, splicing, and histone post-translational modifications [4tU-seq]

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A CSB-PAF1 axis activates RNA polymerase II from promoter-proximal sites and restores efficient transcription elongation after DNA damage repair

Project description:The coordinated transcription of genes involves RNA polymerase II enzymes (RNAPII), which pull DNA through their active sites. DNA lesions in transcribed strands block RNAPII elongation and induce a strong transcriptional arrest. The transcription-coupled repair (TCR) pathway ensures the efficient removal of transcription-blocking DNA lesions, but this is not sufficient to overcome this arrest and resume transcription. Through proteomics screens, we find that the TCR-specific CSB protein loads the evolutionary conserved PAF1 complex (PAF1C) onto RNAPII in promoter-proximal regions specifically in response to DNA damage. PAF1C is dispensable for TCR-mediated repair,but is essential to resume RNA synthesis after UV irradiation, suggesting an unexpected uncoupling between DNA repair and transcription restart. Moreover, we find that PAF1C promotes RNAPII pause release in promoter-proximal regions and subsequently acts as a processivity factor that stimulates transcription elongation waves throughout genes. Our findings expose the molecular basis for a non-canonical PAF1C-dependent pathway that restores transcription throughout the human genome after genotoxic stress.

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