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Saccharomyces cerevisiae

ABSTRACT: Novel transcriptional regulation by pruning RNA polymerase II during transcription elongation

PROVIDER: PRJDB20397 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	DRR656696_1.fastq.gz	Fastqsanger.gz
	DRR656696_2.fastq.gz	Fastqsanger.gz
	DRR656697_1.fastq.gz	Fastqsanger.gz
	DRR656697_2.fastq.gz	Fastqsanger.gz
	DRR656698_1.fastq.gz	Fastqsanger.gz

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Project description:The Paf1 complex (Paf1C) is a conserved transcription elongation factor that regulates transcription elongation efficiency, facilitates co-transcriptional histone modifications, and impacts molecular processes linked to RNA synthesis, such as polyA site selection. Coupling of the activities of Paf1C to transcription elongation requires its association with RNA polymerase II (Pol II). Mutational studies in yeast identified Paf1C subunits Cdc73 and Rtf1 as important mediators of Paf1C association with Pol II on active genes. While the interaction between Rtf1 and the general elongation factor Spt5 is relatively well-understood, the interactions involving Cdc73 have not been fully elucidated. Using a site-specific protein cross-linking strategy in yeast cells, we identified direct interactions between Cdc73 and two components of the Pol II elongation complex, the elongation factor Spt6 and the largest subunit of Pol II. Both of these interactions require the tandem SH2 domain of Spt6. We also show that Cdc73 and Spt6 can interact in vitro and that rapid depletion of Spt6 dissociates Paf1 from chromatin, altering patterns of Paf1C-dependent histone modifications genome-wide. These results reveal interactions between Cdc73 and the Pol II elongation complex and identify Spt6 as a key factor contributing to the occupancy of Paf1C at active genes in Saccharomyces cerevisiae.

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Locus-specific isolation of transcription elongation complexes from Saccharomyces cerevisiae

Project description:Transcription of mRNA products by RNA polymerase II (Pol II) is a multi-stage event subject to a multitude of regulatory processes. Transcription, RNA processing, and chromatin related factors all interact with Pol II to ensure proper timing and coordination of transcription and co-transcriptional processes. Many regulators must function simultaneously to coordinate these processes, yet few strategies exist to explore the full complement of factors regulating specific stages of transcription. To this end we developed a strategy to purify Pol II elongation complexes from specific loci of a single gene, namely the 5′ and 3′ regions, using sequences in the nascent RNA. Applying this strategy to Saccharomyces cerevisiae we determined the specific set of factors that interact with Pol II at precise stages during transcription. We identify many known region-specific factors as well as determine a role for the transcription termination factor Rai1 in regulating the early stages of transcription genome-wide. We also demonstrate a role for the ubiquitin ligase Bre1 in regulating Pol II dynamics during the latter stages of transcription. This strategy for gene and loci-specific isolation of transcription complexes will provide a useful tool to explore the host of factors that regulate the different stages of transcription and coordinate co-transcriptional processes.

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Project description:RNA polymerase II collision interrupts convergent transcription (ChIP-seq)

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Project description:RNA polymerase II collision interrupts convergent transcription (RNA-seq)

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The Paf1 complex broadly impacts the transcriptome of Saccharomyces cerevisiae

Project description:The polymerase associated factor 1 complex (Paf1C) is a multifunctional epigenetic regulator of RNA polymerase II (Pol II) transcription. Paf1C controls gene expression by stimulating the placement of co-transcriptional histone modifications, influencing nucleosome occupancy in coding regions, facilitating transcription termination, and regulating nuclear export of RNAs. In this study, we investigate the extent to which these functions of Paf1C combine to influence the Saccharomyces cerevisiae transcriptome. Using conditions that enrich for unstable transcripts, we show that deletion of PAF1 affects all classes of Pol II-transcribed RNAs including multiple classes of noncoding transcripts. Gene ontology analysis revealed that mRNAs encoding genes involved in iron and phosphate homeostasis were differentially affected by deletion of PAF1. We further investigated these two groups of mRNAs with the goal of identifying overarching mechanisms of up and down-regulation in cells lacking Paf1. Our results indicate that only a subset of the observed changes result from loss of Paf1C-promoted histone modifications. We also found that transcription of the FET4 gene is differentially regulated by Paf1 and an upstream CUT. Together these data highlight the complexity of the epigenetic regulation of Pol II transcription imposed by Paf1C and identify a role for Paf1C in promoting CUT transcription.

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