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

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE107257 | GEO | 2019/07/02

REPOSITORIES: GEO

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

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

| PRJNA419477 | ENA

Native Elongating Transcript Sequencing Reveals Human Transcriptional Activity at Nucleotide Resolution

Project description:Major features of transcription by human RNA polymerase II (Pol II) remain poorly defined due to a lack of quantitative approaches for visualizing Pol II progress at nucleotide resolution. We developed a simple and powerful approach for performing native elongating transcript sequencing (NET-seq) in human cells that globally maps strand-specific Pol II density at nucleotide resolution. NET-seq exposes a mode of antisense transcription that originates downstream and converges on transcription from the canonical promoter. Convergent transcription is associated with a distinctive chromatin configuration and is characteristic of lower-expressed genes. Integration of NET-seq with genomic footprinting data reveals stereotypic Pol II pausing coincident with transcription factor occupancy. Finally, exons retained in mature transcripts display Pol II pausing signatures that differ markedly from skipped exons, indicating an intrinsic capacity for Pol II to recognize exons with different processing fates.

2015-04-23 | GSE61332 | GEO

TEF-seq/NET-seq applied to factors Rpb3, Paf1, Spt6, Spt16, Cet1, Pcf11, Ssu72, Set2

Project description:Here we adapt native elongating transcript sequencing (NET-seq) to develop transcription elongation factor associated nascent elongating transcript sequencing (TEF-seq). In this the RNA polymerase II (Pol2) transcription elongation complex (TEC) is immunoprecipitated via associated transcription elongation factors (TEFs). Sequencing from the 3' end of the Pol2-associated nascent transcript shows the position of the final incorporated nucleotide giving strand-specific, single nucleotide resolution maps of the level of TEF association with Pol2 during transcription.

2021-01-18 | E-MTAB-4568 | biostudies-arrayexpress

Structural basis of human transcription-DNA repair coupling

Project description:Transcription-coupled DNA repair (TCR) efficiently removes bulky DNA lesions from transcribed parts of the genome and constitutes a major defence against DNA damage by UV irradiation. TCR begins when RNA polymerase II (Pol II) stalls at a DNA lesion and recruits the Cockayne syndrome protein CsB, the E3 ubiquitin ligase complex CRL4CsA, and the UV-stimulated scaffold protein A (UVSSA). Here we provide five high-resolution structures of Pol II transcription complexes with bound TCR factors and complementary biochemical data. Together with published work, our results converge on a model for the molecular mechanism of transcription-DNA repair coupling. In this model, Pol II stalling triggers the formation of an alternative transcription elongation complex that we call ECact. In ECact, CsB has replaced the elongation factor DSIF, binds the PAF1 complex, and repositions upstream DNA such that it contacts the elongation factor SPT6. The CsB translocase now pulls on the upstream DNA template, thereby pushing Pol II forward. If the lesion cannot be bypassed, Pol II gets stably stalled. CRL4CsA spans over the Pol II clamp to reach the Pol II jaw and direct ubiquitination of RPB1 residue lysine-1268. This triggers the recruitment of TFIIH to UVSSA, which is located at downstream DNA, and enables nucleotide excision repair. Finally, large-scale conformational changes in CRL4CsA enable ubiquitination of CsB and the release of TCR factors, thereby allowing Pol II to resume elongation and continue transcription over repaired DNA.

2022-02-17 | PXD025328 | Pride

Dynamics of transcription elongation are finely-tuned by dozens of regulatory factors

Project description:Understanding the complex network and dynamics that regulate transcription elongation requires quantitative analysis of RNA polymerase II (Pol II) behavior in a wide variety of regulatory environments. We performed native elongating transcript sequencing (NET-seq) in 42 strains of S. cerevisiae, each missing a known elongation regulator including RNA processing factors, transcription elongation factors, histone variants, chromatin modifiers, and chromatin remodelers and chaperones. For each strain, we determined differentially expressed genes, measured levels of antisense transcription, examined sites of high polymerase density, and identified single-nucleotide loci of Pol II pausing. For each elongation phenotype, we found that wild-type Pol II activity sits in the middle of the dynamic range, indicating that elongation is balanced by the opposing effects of many factors. We find that Pol II elongation at sites of RNA processing is co-transcriptionally controlled by key regulators. Furthermore, Pol II pauses frequently across gene bodies at locations that vary across deletion strains. Meta-analysis of all datasets finds that commonly differentially-expressed genes across the deletion strains exhibit more extreme antisense transcription and polymerase pausing, revealing a link between elongation activity and gene regulation. Thus, regulation of transcription elongation impacts co-transcriptional processing and overall gene expression through precisely balancing Pol II activity by a diverse array of factors.

2021-08-11 | GSE159603 | GEO

Coordination of widespread transcription at enhancers and target genes through BRD4 (His-NET-Seq)

Project description:Gene transcription by RNA polymerase II (Pol II) is under the control of promoters and distal regulatory elements known as enhancers. Enhancers are themselves transcribed by Pol II which correlates with their activity. How enhancer transcription is regulated and coordinated with transcription at target genes has remained unclear. Here, we developed a high-sensitive native elongating transcript sequencing approach, called HiS-NET-seq, to provide an extended high-resolution view on transcription, especially at lowly transcribed regions such as enhancers. HiS-NET-seq uncovers new transcriptional enhancers in human cells. An integrated multi-omics analysis shows that transcription at most enhancers depends on the BET protein BRD4. We provide evidence for a direct BRD4-mediated coupling of Pol II transcription at enhancers and target genes. Specifically, BRD4 links elongation activation at enhancers and genes by maintaining their proximity. These studies reveal that transcription at enhancers and target genes is coordinated at the level of elongation.

2023-07-06 | GSE214593 | GEO

Coordination of widespread transcription at enhancers and target genes through BRD4 (HiChIP)

2023-07-06 | GSE214591 | GEO

Coordination of widespread transcription at enhancers and target genes through BRD4 (ChIP-Rx)

2023-07-06 | GSE214590 | GEO

Allosteric transcription stimulation by super elongation complex

Project description:The super elongation complex (SEC) contains the positive transcription elongation factor b (P-TEFb) and a subcomplex, ELL2-EAF1, which stimulates transcription elongation by RNA polymerase II (Pol II). Here we report the cryo-EM structure of ELL2-EAF1 bound to a Pol II elongation complex at 2.8 Å resolution. The ELL2-EAF1 dimerization module directly binds the Pol II lobe, explaining how SEC delivers P-TEFb to Pol II. The same site on the lobe also binds the initiation factor TFIIF, consistent with SEC binding only after the transition from transcription initiation to elongation. Structure-guided functional analysis shows that elongation stimulation requires the dimerization module and an ELL2 protein linker that tethers this module to the Pol II protrusion. Our results show that SEC stimulates elongation allosterically and indicate that this stimulation involves stabilization of a further closed conformation of the Pol II active center cleft.

2021-07-15 | PXD024457 | Pride

Global Analysis of Short RNAs Reveals Widespread Promoter-Proximal Stalling and Arrest of Pol II

Project description:We developed a novel approach to isolate and characterize, at nucleotide-level resolution, the RNAs derived from RNA polymerase II (Pol II) in early elongation complexes. We find that high numbers of these short RNAs are not correlated with gene expression but instead are indicative of promoter-proximally stalled polymerase. High amounts of these short RNAs are generated from more than one third of all genes, indicating that Pol II stalls in the promoter-proximal region on global scale. We also find that the nucleotide composition of the initially transcribed sequence plays an important role in promoting transcriptional stalling and that early elongation complexes are highly susceptible to backtracking and arrest. Global analysis of RNAs produced by promoter-proximal Pol II.

2009-12-11 | E-GEOD-18643 | biostudies-arrayexpress

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