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Genome-wide binding of Rpb1 (the largest subunit of the RNA polymerase II) to the chromatin state in bud27∆ and wild-type mutant strains (ChIP-seq)

ABSTRACT: Genome-wide binding of Rpb1 (the largest subunit of the RNA polymerase II) to the chromatin state in bud27∆ and wild-type mutant strains (ChIP-seq)

PROVIDER: PRJNA555509 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR9715590.fastq.gz	Fastqsanger.gz
	SRR9715591.fastq.gz	Fastqsanger.gz
	SRR9715592.fastq.gz	Fastqsanger.gz
	SRR9715593.fastq.gz	Fastqsanger.gz
	SRR9715594.fastq.gz	Fastqsanger.gz

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Genome-wide binding of Rpb1 (the largest subunit of the RNA polymerase II) to the chromatin state in bud27∆ and wild-type mutant strains (ChIP-seq)

Project description:Comparison of the average normalised ChIP-seq signal over protein-coding genes in bud27∆ mutants strain and wild-type.

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MudPIT analysis of proteins interacting with Saccharomyces cerevisiae RNA Polymerase II HA-Rpb1 C-Terminal Domain

Project description:The carboxy-terminal domain (CTD) of Rpb1, the largest component of the 12-subunit RNA polymerase II, consists of repeating Y1S2P3T4S5P6S7 heptapeptides (26 repeats in budding yeast). Each stage of transcription relies on the ordered recruitment and exchange of specific protein complexes that act on RNA polymerase II, its nascent transcripts, and the underlying chromatin. This dynamic process is orchestrated via patterned post-translational modifications of the CTD. To characterize the role of phosphorylation on Thr4, we examined the effect of Rpb1 alleles in which Thr4 was substituted with an alanine (T4A) or the phospho-mimic glutamate (T4E). Substitutions were made across all heptad repeats of the CTD. We affinity purified HA-tagged Rpb1 from Saccharomyces cerevisiae strains bearing WT, T4A, and T4E CTDs. A control strain (Z26) lacking the HA-tagged Rpb1 was subjected to an identical affinity enrichment procedure. Three biological replicates were acquired for each type of affinity purification and analyzed independently. After TCA-precipitation, proteins were urea-denatured, reduced, alkylated, then digested with endoproteinase LysC followed by trypsin. The resulting peptide mixtures were analyzed by Multidimensional Protein Identification Technology (MudPIT). Label-free quantitative proteomics was used to identify and quantify the relative abundance of affinity-enriched complexes.

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ChIP-seq analysis of Rpb1 from 12hr conjugating Oxytricha trifallax cells

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AOH1996, a small molecule ligand of PCNA, modulates PCNA interaction with RPB1

Project description:Targeting transcription replication conflicts, a major source of endogenous DNA double stranded breaks and genomic instability, could have important therapeutic implications. When transcription and DNA replication machineries encounter each other on chromosomes, one way to resolve the conflict is temporarily dislodging RNA polymerase II from the conflict sites to enable the replication fork to proceed. Proliferating cell nuclear antigen (PCNA), an essential component of replisomes, plays a critical role in this process. We discovered a small molecule ligand (AOH1996) of PCNA, which targets a binding pocket adjacent to the outer surface region of PCNA known to interact with PIP box and APIM motif proteins. Orally administrable, AOH1996 suppresses tumor growth but causes no discernable side effects. In addition, we found that AOH1996 treatment can stabilize interaction between PCNA and RPB1, the largest subunit of RNA polymerase II. To determine whether the effect of AOH1996 is mediated through affecting PCNA interaction with RPB1, we mutated Y418 within the APIM motif of RPB1 to an alanine in the SK-N-AS cell line by CRISPR and compared changes in protein expression profiles caused by AOH1996 in the parent and mutant SK-N-AS cells.

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KINETIC AND COMPOSITIONAL ANALYSIS OF RNA POLYMERASE II IN TRYPANOSOMA BRUCEI

Project description:Genome-wide analysis of nascent RNA synthesis gives kinetic information on the transcriptional status of RNA polymerase II (Pol II). In parallel, immunoprecipitation of the largest subunit of Pol II (RPB1) provides the protein composition of the enzyme complex in the presence or absence of the transcriptional inhibitor Actinomycin D.

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ChIP-seq analysis of Rpb1 from 12hr conjugating Oxytricha trifallax cells

Project description:To study the biogenesis of long non-coding RNAs transcribed during genome rearrangements in Oxytricha development, genome-wide localization pattern of Rpb1 (RNA Pol-II largest subunit) was studied from Oxytricha cells undergoing conjugation. Chromatin from 12hr conjugating O.trifallax cells was subjected to chromatin immunoprecipitation (ChIP) followed by sequencing of Input and ChIP samples via Illumina paired-end sequencing.

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Project description:The conserved transcription elongation factor Spt6 makes several contacts with the RNA Polymerase II (RNAPII) complex, including a high-affinity interaction between the Spt6 tandem SH2 domain (Spt6-tSH2) and phosphorylated residues of the Rpb1 subunit in a region linking the catalytic core and the heptad repeats at the C-terminal domain (CTD). This interaction contributes to generic localization of Spt6, but we show here that it also has gene-specific roles. Disrupting the interface affected transcription start site selection at a subset of genes whose expression is regulated by this choice, and this was accompanied by changes in the distinct pattern of Spt6 occupancy at these sites. Splicing efficiency was also impacted, as was apparent progression through introns that encode snoRNAs. Expression of genes that are particularly sensitive to efficient restoration of chromatin after transcription were affected, and a distinct role in maintaining +1 nucleosomes was identified, especially at ribosomal protein genes. The Spt6-tSH2:Rpb1 interface therefore has both genome-wide functions and local roles at subsets of genes where dynamic decisions regarding initiation, transcript processing, or termination are made. We propose that the Spt6-tSH2:Rpb1 interaction participates in coordinating appropriate responses to the varying local conditions encountered RNAPII at each gene, perhaps by modulating the elongation rate to allow reconfiguration of accessory factor activity or availability.

2021-12-20 | GSE184954 | GEO

Genome-wide maps of Bud27 binding to the chromatin [ChIP-seq]

Project description:The goal of this study is to analyse genome-wide the binding profile of Bud27 prefoldin-like to the genes or other genomic regions in order to know which genes could be transcriptionally regulated by this protein and to elucidate the contribution of Bud27 to the transcription. Average normalised ChIP-seq signal for Bud27 prefoldin-like binding in a S.cerevisiae wild-type strain.

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