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M6A RNA methylation regulates promoter proximal pausing of RNA Pol II (DRB-4sU-seq)

ABSTRACT: m6A RNA methylation regulates promoter proximal pausing of RNA Pol II (DRB-4sU-seq)

PROVIDER: PRJNA603117 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR10963282_1.fastq.gz	Fastqsanger.gz
	SRR10963282_2.fastq.gz	Fastqsanger.gz
	SRR10963283_1.fastq.gz	Fastqsanger.gz
	SRR10963283_2.fastq.gz	Fastqsanger.gz
	SRR10963284_1.fastq.gz	Fastqsanger.gz

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m6A RNA methylation regulates promoter proximal pausing of RNA Pol II (DRB-4sU-seq)

Project description:RNA modification has emerged as an important layer in the control of gene expression. The N6-methyladenosine (m6A) RNA modification is the most prevalent and influences several processes including pre-mRNA splicing, alternative polyadenylation, mRNA decay and translation. Here, we show that m6A regulates transcription by controlling promoter proximal pause release of RNA Pol II. Our results show that the m6A methyltransferase complex together with the nuclear reader Ythdc1 are recruited to gene promoters. Loss of m6A methyltransferase complex leads to increased levels of promoter proximal pausing of RNA Pol II, decrease in Ser2P occupancy on gene body, and affects nascent RNA transcription. Furthermore, we show that the binding of the complex is Spt6-dependent and can be predicted by collective features, with Pol II and pause factors (GAF and M1BP) binding being the most important. We also demonstrate that tethering Mettl3 to a heterologous gene promoter was sufficient to increase the release of RNAP II from the promoter, and this effect was dependent on its m6A catalytic domain. Collectively our data indicate that m6A RNA feeds back to the transcription machinery via its recruitment to the chromatin, and thus uncovering an important link between epigenetic modification of RNA and transcription.

2021-07-28 | GSE144245 | GEO

DRB-4SU seq of Ctrl and Mago condition in 2 replicates with fragmentation [Promoter-proximal pausing mediated by the exon junction complex regulates splicing]

Project description:DRB-4SU seq of Ctrl and Mago condition in 2 replicates with fragmentation [Promoter-proximal pausing mediated by the exon junction complex regulates splicing]

| PRJNA418417 | ENA

DRB-4sU seq in control and Mago depleted S2R+ cells [Promoter-proximal pausing mediated by the exon junction complex regulates splicing]

Project description:Promoter-proximal pausing of RNA polymerase II (Pol II) is a widespread transcriptional regulatory step across metazoans. Here we find that the nuclear exon junction complex (pre-EJC) is a critical and conserved regulator of this process. Depletion of pre-EJC subunits leads to a global decrease in Pol II pausing and to premature entry into elongation. This effect occurs, at least in part, via non-canonical recruitment of pre-EJC components at promoters. Failure to recruit the pre-EJC at promoters results in increased binding of the positive transcription elongation complex (P-TEFb) and in enhanced Pol II release. Notably, restoring pausing is sufficient to rescue exon skipping and the photoreceptor differentiation defect associated with depletion of pre-EJC components in vivo. We propose that the pre-EJC serves as an early transcriptional checkpoint to prevent premature entry into elongation, ensuring proper recruitment of RNA processing components that are necessary for exon definition.

2019-01-22 | GSE92379 | GEO

Genome-wide view of the impact of Spt5-Pol II inhibitors (SPIs) on transcription [4sU-seq]

Project description:We identified the first Spt5-Pol II inhibitors (SPIs). SPIs faithfully reproduced Spt5 knockdown effects on proximal-promoter-pausing, NF-κB activation and the expanded-repeat huntingtin gene in neuronal cells. Using SPIs we identified Spt5 target genes that responded with profoundly diverse kinetics and a novel regulatory element of proximal-promoter-pausing. To validate that the effects of SPIs are at the transcriptional level, cellular RNA was metabolically labeled with 4-thio-uridine (4sU) for 2 hours in the presence of DMSO or SPI-21 in the presence or absence of TNFα. Newly synthesized labeled RNA was then purified and subjected to RNA-seq.

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Drosophila FACT Regulates Promoter Proximal Pol II Pausing and Chromatin Architecture [RNA-seq]

Project description:The highly conserved histone chaperone FACT (Facilitates Chromatin Transcription) is thought to contribute to the disassembly and reassembly of nucleosomes in the wake of RNA Polymerase II (Pol II) passage through chromatin. However, FACT’s roles in chromatin biology and transcriptional regulation in vivo in higher eukaryotes are not well understood. Here, we report that depletion of FACT leads to a reduction in the duration of promoter-proximal pausing by Pol II in Drosophila S2 cells. In addition, FACT depletion leads to a modest decrease in the occupancy of histone H3, and to decrease in occupancy of histone H2A.v in promoter-proximal nucleosomes. Finally, we observed a dramatic 5’ to 3’ repositioning of the co-transcriptionally deposited histone modifications H3K4me3 and H3K36me3 in the FACT depleted cells. Taken together, our findings are consistent with the model that FACT contributes to the interplay between chromatin architecture and control of Pol II promoter-proximal pausing.

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DRB-4SU seq of Ctrl and Mago condition in 2 replicates with fragmentation [Promoter-proximal pausing mediated by the exon junction complex regulates splicing]

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m6A RNA methylation regulates promoter proximal pausing of RNA Pol II

Project description:m6A RNA methylation regulates promoter proximal pausing of RNA Pol II

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DRB-PRO-Seq for measuring Pol II transcriptional elongation rate alteration up on CDK12 and CDK13 inhibition by THZ531

Project description:We performed PRO-Seq to measure the Pol II elongation rate by measruing the distance of Pol II travels at viarous time points after relased from DRB induced pausing. We compared DMSO/DRB or THZ531/DRB co-treated sample to assess the effect of CDK12 and CDK13 inhibtion by THZ531 on transcriptional elongation rate.

2020-04-29 | GSE141370 | GEO

TFIID enables RNA polymerase II promoter-proximal pausing

Project description:RNA polymerase II (pol II) transcribes all protein-coding and many non-coding RNAs in the human genome. Pol II transcription initiation is governed by the Pre-Initiation Complex (PIC), which contains TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, pol II, and Mediator. After initiation, pol II enzymes typically pause after transcribing less than 100 bases, and paused polymerases represent a common regulatory intermediate. Accordingly, paused pol II has been implicated in enhancer function, development and homeostasis, and diseases ranging from cancer to viral pathogenesis. Precisely how pol II promoter-proximal pausing is enforced and regulated remains unclear; however, protein complexes such as NELF and DSIF increase pausing whereas the activity of CDK9 (P-TEFb complex) correlates with pause release. To address specific mechanistic questions about pol II pausing and its regulation, we reconstituted human pol II promoter-proximal pausing in vitro, entirely with purified factors (no extracts). As expected, NELF and DSIF increased pol II pausing in vitro, whereas P-TEFb promoted pause release. Unexpectedly, the PIC alone was sufficient to reconstitute pol II pausing, suggesting that pausing is an inherent property of the PIC. In agreement, pol II pausing was lost upon replacement of the TFIID complex with TATA-binding protein (TBP); moreover, pausing was dependent upon TFIID subunits TAF1 and TAF2. TAF1/2 bind genomic DNA downstream of the pol II initiation site, invoking a “complex interaction” model for pausing. Consistent with this model, PRO-Seq experiments revealed increased transcription upon acute depletion (t=60 min) of TAF1 and TAF2 in human cells, and pol II pausing was disrupted at thousands of genes. Similar results were obtained in TAF1-depleted Drosophila S2 cells. Collectively, these data establish the general transcription factor TFIID as a genome-wide regulator of pol II promoter-proximal pausing.

2020-05-28 | GSE132764 | GEO

Drosophila FACT Regulates Promoter Proximal Pol II Pausing and Chromatin Architecture [ChIP-seq]

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