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RNA Polymerase II transcription independent of TBP [NET-seq]

ABSTRACT: RNA Polymerase II transcription independent of TBP [NET-seq]

PROVIDER: PRJNA851782 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR19785030.fastq.gz	Fastqsanger.gz
	SRR19785031.fastq.gz	Fastqsanger.gz
	SRR19785032.fastq.gz	Fastqsanger.gz
	SRR19785033.fastq.gz	Fastqsanger.gz
	SRR19785034.fastq.gz	Fastqsanger.gz

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RNA Polymerase II transcription independent of TBP [NET-seq]

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RNA Polymerase II transcription independent of TBP (CUT&Tag)

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RNA Polymerase II transcription independent of TBP (CUT&Tag)

Project description:Using CUT&Tag, a chromatin profiling technique, we show that TBP depletion via IAA surprisingly does not affect RNA Pol II transcription but affects RNA Pol III transcription. Additionally, induction of genes via heat shock and retinoic acid treatment does not require TBP. We also show that a metazoan specific paralog TRF2 does not compensate for TBP for RNA Pol II transcription and that the TFIID subunit of the Pre-initiation Complex can still form with specific subunits still binding onto DNA when TBP is depleted.

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Differential dependencies of human RNA polymerase II promoters on TBP, TAF1, TFIIB, and XPB.

Project description:General transcription factors (GTFs) are required for RNA polymerase II (Pol II) to initiate transcription at promoters. In this study, we determined the effects of acute depletion of TBP, TAF1, TAF4, TFIIB, and XPB in HAP1 cells. We performed precision nuclear run-on sequencing (PRO-Seq) and quantified nascent transcripts arising from more than 70,000 promoters. The average dependencies for each factor across all promoters varied widely even though levels of depletions were similar. Many of the effects could be attributed to the presence or absence of core promoter elements. Depletion of TBP had a large effect on only a small fraction of Pol II and Pol III promoters. TFIIB depletion also led to readthrough transcription downstream of the 3′ ends of genes. We conclude that promoter activity is influenced by recruitment of TFIID, sequence-specific transcription factors, and interaction of the preinitiation complex (PIC) with the +1 nucleosome.

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NET-prism enables RNA polymerase-dedicated transcriptional interrogation at nucleotide resolution

Project description:The advent of quantitative approaches that enable interrogation of transcription at single nucleotide resolution has allowed a novel understanding of transcriptional regulation previously undefined. To better map transcription genome-wide at base pair resolution and with transcription/elongation factor dependency we developed an adapted NET-seq protocol called NET-prism (Native Elongating Transcription by Polymerase-Regulated Immunoprecipitants in the Mammalian genome). NET-prism introduces an immunoprecipitation to capture RNA Pol II – associated proteins, which reveals the interaction of these proteins with active RNA Pol II. Application of NET-prism on different Pol II variants (Pol II S2ph, Pol II S5ph), elongation factors (Spt6, Ssrp1), splicing factors (Sf1), and components of the pre-initiation complex (PIC) (TFIID, and Mediator) reveals diverse Pol II signals, at a single nucleotide resolution, with regards to directionality and intensity over promoters, splice sites, and enhancers/super-enhancers. NET-prism will be broadly applicable as it exposes transcription factor/Pol II dependent topographic specificity and thus, a new degree of regulatory complexity.

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ChIP-chip from H9 hESCs for RNA Polymerase II, TBP, TAF3, TAF5, TAF7, and TAF11

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