Genetic Dissection of the RNA Polymerase II Transcription Cycle

Chou, Shao‐Pei; Ak, Alexander; Ej, Rice; La, Choate; Pe, Cohen; Danko, Charles G.

doi:10.1101/2021.05.23.445279

Cited by 6 publications

(4 citation statements)

References 82 publications

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“…Interestingly, it has recently been shown that the PIC alone is sufficient to establish RNAP pausing, and that rapid TAF1 depletion induces pause-release genome-wide [41]. We found other enriched motifs, but they mostly consisted of repetitive, highly G+C-rich sequences, similar to observations in other recent studies [20,42,43]. We examined the DNA sequences surrounding the nucleotide with the maximum PRO-seq read count in each pause peak, and observed a clear enrichment for cytosines at this position (see also [20,[42][43][44]; Supplementary Figs.…”

Section: Promoter-proximal Pause Sites Vary Across Cells and Pause-es...supporting

confidence: 90%

Model-based characterization of the equilibrium dynamics of transcription initiation and promoter-proximal pausing in human cells

Zhao

Liu

Siepel

2022

Preprint

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In metazoans, both transcription initiation and the escape of RNA polymerase (RNAP) from promoter-proximal pausing are key rate-limiting steps in gene expression. These processes play out at physically proximal sites on the DNA template and appear to influence one another through steric interactions, leading to a complex dynamic equilibrium in RNAP occupancy of the ~100 bp immediately downstream of the transcription start site. In this article, we examine the dynamics of these processes using a combination of statistical modeling, simulation, and analysis of real nascent RNA sequencing data. We develop a simple probabilistic model that jointly describes the kinetics of transcription initiation, pause-escape, and elongation, and the generation of nascent RNA sequencing read counts under steady-state conditions. We then extend this initial model to allow for variability across cells in promoter-proximal pause site locations and steric hindrance of transcription initiation from paused RNAPs. In an extensive series of simulations over a broad range of parameters, we show that this model enables accurate estimation of initiation and pause-escape rates even in the presence of collisions between RNAPs and variable elongation rates. Furthermore, we show by simulation and analysis of data for human cell lines that pause-escape is often more strongly rate-limiting than conventional “pausing indices” would suggest, that occupancy of the pause site is elevated at many genes, and that steric hindrance of initiation can lead to a pronounced reduction in apparent initiation rates. Our modeling framework is generally applicable for all types of nascent RNA sequencing data and can be applied to a variety of inference problems.

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Section: Promoter-proximal Pause Sites Vary Across Cells and Pause-es...supporting

confidence: 90%

Model-based characterization of the equilibrium dynamics of transcription initiation and promoter-proximal pausing in human cells

Zhao

Liu

Siepel

2022

Preprint

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“…18). After accounting for biological replication in this experiment 73 (7 replicates x 8 tissues x 9 histone marks), it would have taken 504 ChIP-seq assays to prepare this same dataset. Thus, using dHIT to interpret ChRO-seq data provides individual labs access to consortium scale annotation of functional elements in mammalian genomes, and this information has potential applications in precision diagnostic medicine and genome annotation.…”

Section: Genome Annotation Using a Single Functional Assaymentioning

confidence: 99%

Interdependence between histone marks and steps in Pol II transcription

Wang

Chivu

Choate

et al. 2020

Preprint

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We trained a sensitive machine learning tool to infer the distribution of histone marks using maps of nascent transcription. Transcription captured the variation in active histone marks and complex chromatin states, like bivalent promoters, down to single-nucleosome resolution and at an accuracy that rivaled the correspondence between independent ChIP-seq experiments. The relationship between active histone marks and transcription was conserved in all cell types examined, allowing individual labs to annotate active functional elements in mammals with similar richness as major consortia. Using imputation as an interpretative tool uncovered cell-type specific differences in how the PRC2-dependent repressive mark, H3K27me3, corresponds to transcription, and revealed that transcription initiation requires both chromatin accessibility and an active chromatin environment demonstrating that initiation is less promiscuous than previously thought.

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“…Very recent results of cryo-EM studies on human PICs, especially in structures visualizing TFIID, indicate that promoter classes may assemble PIC components in distinct fashion within a single organism 77 , yet these assembly pathways similarly position an upstream TSS proximal to the Pol II active site, consistent with proposals that human promoters could contain information for assembling PICs individually 105 . Recent results suggest that there may be plasticity in TSS selection from individual PICs upon mutation of Inr sites in mouse, potentially supporting a type of scanning in mammals 106 .…”

Section: Discussionmentioning

confidence: 93%

Ssl2/TFIIH function in Transcription Start Site Scanning by RNA Polymerase II in Saccharomyces cerevisiae

Zhao

Vvedenskaya

Lai

et al. 2021

Preprint

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Ssl2, as an essential subunit of the RNA Polymerase II (Pol II) general transcription factor TFIIH, promotes melting of promoter DNA through its ATPase-dependent DNA translocase activity within the Pol II pre-initiation complex. In Saccharomyces cerevisiae, after DNA melting, Ssl2 is proposed to drive Pol II scanning downstream for usable transcription start sites (TSSs) through this same ATP-dependent DNA translocase activity. However, how Pol II catalytic activity or activities of other general transcription factors integrate with Ssl2/TFIIH activity for promoter scanning and what ways Ssl2 modulates TSS selection by scanning have not been well studied. Here, we report the identification of ssl2 alleles conferring phenotypes in vivo consistent with altered Pol II initiation. We find that these ssl2 alleles alter TSS selection by scanning in ways that are distinct from how changes to Pol II or other GTF activities alter TSS selection by scanning. Specific predictions arise for interactions among initiation mutants depending on whether they function in promoter scanning through modulating initiation at an individual TSS or if they control the probability that a TSS is reached during the scanning process (scanning processivity). We test these predictions genetically and through analysis of transcription output at ADH1 and at promoters across the genome. Our data support a model whereby ssl2 alleles alter Ssl2 processivity and therefore the probability that TSSs are scanned. Examination of TSS usage genome-wide finds global effects of ssl2 alleles on TSS usage and the potential coupling of Pol II activity and Ssl2/TFIIH processivity during scanning. We propose that the initiation by promoter scanning is determined by the interaction of two functional networks, one controlling initiation efficiency and one controlling the processivity of scanning.

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Genetic Dissection of the RNA Polymerase II Transcription Cycle

Cited by 6 publications

References 82 publications

Model-based characterization of the equilibrium dynamics of transcription initiation and promoter-proximal pausing in human cells

Model-based characterization of the equilibrium dynamics of transcription initiation and promoter-proximal pausing in human cells

Interdependence between histone marks and steps in Pol II transcription

Ssl2/TFIIH function in Transcription Start Site Scanning by RNA Polymerase II in Saccharomyces cerevisiae

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