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ABSTRACT: RNA expression profiling for Paf1 complex mutant strains

PROVIDER: PRJNA254522 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR1508480.fastq.gz	Fastqsanger.gz
	SRR1508481.fastq.gz	Fastqsanger.gz
	SRR1508482.fastq.gz	Fastqsanger.gz
	SRR1508483.fastq.gz	Fastqsanger.gz
	SRR1508484.fastq.gz	Fastqsanger.gz

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Schizosaccharomyces pombe

Project description:RNA expression profiling for Paf1 complex mutant strains [miRNA]

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Post-Transcriptional Role for the Yeast Paf1-RNA Polymerase II Complex is Revealed by Identification of Primary Targets

Project description:To identify the direct targets of the Paf1/RNA polymerase II complex we compared expression profiles of isogenic wild type and paf1 and ctr9 mutant strains. We also created a Tet-regulated form of Paf1 and monitored expression patterns after shut off of Paf1. Samples were isolated at one hour intervals from 1 to 8 hours after shut off. Keywords: single channel nucleotide

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Gene expression profiling using RNA-sequencing in mouse embryonic stem cells under control conditions or Phf5a knockdown

Project description:Phf5a regulates transcription elongation in mouse embryonic stem cells (ESCs), through regulation of the Paf1 complex. In this study we assayed gene expression profiling using RNA-sequncing in mouse ESCs under conditions of self-renewal, Phf5a knockdown and Paf1 knockdown. These results revealed that Phf5a and Paf1 regulate similar gene expression signatures in mouse ESCs.

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Nascent transript profiling upon in mouse embryonic stem cells under control conditions or Phf5a knockdown

Project description:Phf5a regulates transcription elongation in mouse embryonic stem cells (ESCs), through regulation of the Paf1 complex. In this study we assayed nascent RNA profiling using global run-on sequncing in mouse ESCs under conditions of self-renewal, differentiation and Phf5a knockdown. These results revealed that genes positively regulated by Paf1 in ESCs exibit RNA elongation pausing on their promoters. We used conditions of self-renewal, differentiation, Phf5a knockdown and Paf1 knockdown to monitor nascent transcript expression changes during Phf5a depletion using global run-on RNA sequencing (GRO-seq). These results revealed that Phf5a through the control of Paf1 complex regulates pause release and elongation of pluripoency-associated genes in embryonic stem cells.

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PAF1, a molecular regulator of promoter-proximal pausing by RNA Polymerase II

Project description:The control of promoter-proximal pausing and the release of RNA polymerase II (RNA Pol II) is a widely used mechanism for regulating gene expression in metazoans, especially for genes that respond to environmental and developmental cues. Here, we identify Pol II associated Factor 1 (PAF1) as a major regulator of promoter-proximal pausing. Knockdown of PAF1 leads to increased release of paused Pol II into gene bodies at thousands of genes. Genes with the highest levels of paused Pol II exhibit the largest redistribution of Pol II from the promoter-proximal region into the gene body in the absence of PAF1. PAF1 depletion results in increased nascent transcription and increased levels of phosphorylation of Pol II’s c-terminal domain on serine 2 (Ser2P). These changes can be explained by the recruitment of the Ser2P kinase Super Elongation Complex (SEC) effecting increased release of paused Pol II into productive elongation, thus establishing a novel function for PAF1 as a major regulator of pausing in metazoans. ChIP-seq of Pol II of different forms, SEC subunits, PAFc subunits and H2Bub in human cell lines targeted by PAF1 or scramble shRNA. ChIP-seq of total Pol II in HCT116 cells targeted by BRE1A or scramble shRNA. ChIP-seq of total Pol II in S2 cells targeted by Paf1 or LacZ RNAi. Total RNA-seq, nascent RNA-seq and GRO-seq in HCT116 cells targeted by PAF1 or scramble shRNA.

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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. 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.

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In Vitro Transcription Recycling Assay Identifies PAF1 as a Driver of RNA Pol II Recycling

Project description:RNA Polymerase II transcriptional recycling is an underappreciated mechanism for which the required factors and contributions to overall gene expression levels are poorly understood. We describe an in vitro methodology facilitating unbiased identification of novel RNA Pol II transcriptional recycling factors and quantitative measurement of transcriptional output from recycled transcriptional components. Proof-of-principle experiments identified PAF1 complex components among recycling factors and detected defective transcriptional output from RNA Pol II recycling following PAF1 depletion. Dynamic ChIP-seq confirmed in vivo PAF1 complex cycling through gene bodies with RNA Pol II and recycling back to the transcription start sites, while PAF1 silencing triggered defective RNA Pol II recycling. Prostate tumors exhibited enhanced transcriptional recycling, which was attenuated by antibody-based PAF1 depletion. These findings identify RNA Pol II recycling as a potential target in cancer and demonstrate the assay’s applicability to characterize RNA Pol II recycling in cells and tissues from other disease states.

2021-10-07 | GSE133655 | GEO

Spt6 regulates intragenic and antisense transcription, nucleosome positioning, and histone modifications genome-wide in fission yeast [ChIP-seq]

Project description:Spt6 is a highly conserved histone chaperone that interacts directly with both RNA polymerase II and histones to regulate gene expression. To gain a comprehensive understanding of the requirements for this critical factor, we have performed genome-wide analyses of transcription, chromatin structure, and histone modifications in an S. pombe spt6 mutant. Our results demonstrate several dramatic changes to transcription and chromatin structure in the spt6 mutant, including an elevation of antisense transcripts at over 70 percent of all genes and general loss of the +1 nucleosome. Furthermore, Spt6 is required for the trimethylation of histone H3 on lysines 4 and 36, marks associated with active transcription. Taken together, our results indicate that Spt6 is critical for the accuracy of transcription and the integrity of chromatin, likely via its direct interactions with RNA polymerase II and histones. ChIP-seq experiments were performed on wild type and spt6-1 strains on the following proteins: RNA polymerase II (Rpb1), Paf1 Complex (Ctr9), COMPASS (Swd1), Set2, Spt6, histones H2B and H3, histone modifications H3K4me3 and H3K36me3. Experiments were performed in replicates and matching inputs were also sequenced.

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