Noncoding transcription influences the replication initiation program through chromatin regulation

Soudet, Julien; Gill, Jatinder Kaur; Stutz, Françoise

doi:10.1101/gr.239582.118

Cited by 22 publications

(27 citation statements)

References 75 publications

(141 reference statements)

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“…Replication origins tend to localize after TTS in yeast and upstream of promoters in humans, presumably to minimize transcription-replication conflicts [73,116,117]. The induction of transcription through origins, via defects in transcription termination at TTSs as well as at TSSs, leads to replication defects via dissociation or sliding of the pre-ORCs and MCMs [70][71][72][73][74] and changes in chromatin structure [118]. Furthermore, loss of origin function activates readthrough transcription in mammals and yeast.…”

Section: Discussionmentioning

confidence: 99%

Identification of a novel base J binding protein complex involved in RNA polymerase II transcription termination in trypanosomes

et al. 2020

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Base J, β-D-glucosyl-hydroxymethyluracil, is a modification of thymine DNA base involved in RNA Polymerase (Pol) II transcription termination in kinetoplastid protozoa. Little is understood regarding how specific thymine residues are targeted for J-modification or the mechanism of J regulated transcription termination. To identify proteins involved in J-synthesis, we expressed a tagged version of the J-glucosyltransferase (JGT) in Leishmania tarentolae, and identified four co-purified proteins by mass spectrometry: protein phosphatase (PP1), a homolog of Wdr82, a potential PP1 regulatory protein (PNUTS) and a protein containing a J-DNA binding domain (named JBP3). Gel shift studies indicate JBP3 is a J-DNA binding protein. Reciprocal tagging, co-IP and sucrose gradient analyses indicate PP1, JGT, JBP3, Wdr82 and PNUTS form a multimeric complex in kinetoplastids, similar to the mammalian PTW/PP1 complex involved in transcription termination via PP1 mediated dephosphorylation of Pol II. Using RNAi and analysis of Pol II termination by RNA-seq and RT-PCR, we demonstrate that ablation of PNUTS, JBP3 and Wdr82 lead to defects in Pol II termination at the 3'-end of polycistronic gene arrays in Trypanosoma brucei. Mutants also contain increased antisense RNA levels upstream of transcription start sites, suggesting an additional role of the complex in regulating termination of bi-directional transcription. In addition, PNUTS loss causes derepression of silent Variant Surface Glycoprotein genes involved in host immune evasion. Our results suggest a novel mechanistic link between base J and Pol II polycistronic transcription termination in kinetoplastids. Author summaryTrypanosoma brucei is a parasitic protozoan that causes African sleeping sickness in humans. The genome of T. brucei is organized into polycistronic gene clusters that contain multiple genes that are co-transcribed from a single promoter. We have recently described the presence of a modified DNA base J and variant of histone H3 (H3.V) at transcription termination sites within gene clusters where the loss of base J and H3.V leads to read-through transcription and the expression of downstream genes. We now PLOS Genetics | https://doi.identify a novel stable multimeric complex containing a J binding protein (JBP3), base J glucosyltransferase (JGT), PP1 phosphatase, PP1 interactive-regulatory protein (PNUTS) and Wdr82, which we refer to as PJW/PP1. A similar complex (PTW/PP1) has been shown to be involved in Pol II termination in humans and yeast. We demonstrate that PNUTS, JBP3 and Wdr82 mutants lead to read-through transcription in T. brucei. Our data suggest the PJW/PP1 complex regulates termination by recruitment to termination sites via JBP3-base J interactions and dephosphorylation of specific proteins (including Pol II and termination factors) by PP1. These findings significantly expand our understanding of mechanisms underlying transcription termination in eukaryotes, including organisms that utilize polycistronic transcription and novel epigenetic marks...

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Section: Discussionmentioning

confidence: 99%

Identification of a novel base J binding protein complex involved in RNA polymerase II transcription termination in trypanosomes

et al. 2020

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“…The nucleosomes profile data is from ref. []. NDR, nucleosome‐depleted region; tw, Termination‐window of the mRNA; NUT, Nrd1‐unterminated transcript; ARS, autonomously replicating sequence.…”

Section: Pervasive Transcription Distributes All Over Eukaryotic Genomesmentioning

confidence: 99%

“…The numbers in brackets refer to the number of ARS taken into account. The figure is adapted from our published work (Adapted under the terms and conditions of the Creative Commons Attribution license 4.0 . Copyright 2018, the Authors, published Cold Spring Harbor Laboratory Press), using additional unpublished data on H3K36me3 profiles at nucleosome resolution.…”

Section: Rnap II Transcription Locks the Chromatin Of Coding Genesmentioning

confidence: 99%

Regulation of Gene Expression and Replication Initiation by Non‐Coding Transcription: A Model Based on Reshaping Nucleosome‐Depleted Regions

Soudet

Stutz

2019

BioEssays

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RNA polymerase II (RNAP II) non-coding transcription is now known to cover almost the entire eukaryotic genome, a phenomenon referred to as pervasive transcription. As a consequence, regions previously thought to be non-transcribed are subject to the passage of RNAP II and its associated proteins for histone modification. This is the case for the nucleosome-depleted regions (NDRs), which provide key sites of entry into the chromatin for proteins required for the initiation of coding gene transcription and DNA replication. In this review, recent data on the effects of pervasive transcription through NDRs are summarized and a model is proposed to explain how RNAP II-driven transcription is able to modify the nucleosomes flanking the NDRs, leading to nucleosome repositioning and NDR closure. Even though much of the mechanistic detail underlying these events remains to be elucidated, such a model provides a basis to explain how non-coding transcription through NDRs can regulate the initiation of coding gene expression and DNA replication.

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“…Replication origins tend to localize after TTS in yeast and upstream of promoters in humans, presumably to minimize transcription-replication conflicts (71, 110, 111). The induction of transcription through origins, via defects in transcription termination at TTSs as well as at TSSs, leads to replication defects via dissociation or sliding of the pre-ORCs and MCMs (68–72) and changes in chromatin structure (112). Furthermore, loss of origin function activates readthrough transcription in mammals and yeast.…”

Section: Discussionmentioning

confidence: 99%

Identification of a Novel Base J Binding Protein Complex Involved in RNA Polymerase II Transcription Termination in Trypanosomes

Sabatini

Kieft

Zhang

et al. 2019

Preprint

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19 Base J, β-D-glucosyl-hydroxymethyluracil, is a modification of thymine DNA base involved in RNA 20 Polymerase (Pol) II transcription termination in kinetoplastid protozoa. Little is understood regarding how 21 specific thymine residues are targeted for J-modification or the mechanism of J regulated transcription 22 termination. To identify proteins involved in J-synthesis, we expressed a tagged version of the J-23 glucosyltransferase (JGT) in Leishmania tarentolae, and identified four co-purified proteins by mass 24 spectrometry: protein phosphatase (PP1), a homolog of Wdr82, a potential PP1 regulatory protein 25 (PNUTS) and a protein containing a J-DNA binding domain (named JBP3). Gel shift studies indicate 26 JBP3 is a J-DNA binding protein. Reciprocal tagging, co-IP and sucrose gradient analyses indicate PP1, 27 JGT, JBP3, Wdr82 and PNUTS form a multimeric complex in kinetoplastids, similar to the mammalian 28 PTW/PP1 complex involved in transcription termination via PP1 mediated dephosphorylation of Pol II. 29 Using RNAi and analysis of Pol II termination by RNA-seq and RT-PCR, we demonstrate that ablation of 30 PNUTS, JBP3 and Wdr82 lead to defects in Pol II termination at the 3'-end of polycistronic gene arrays in 31 Trypanosoma brucei. Mutants also contain increased antisense RNA levels upstream of promoters, 32 suggesting an additional role of the complex in regulating termination of bi-directional transcription. In 33 addition, PNUTS loss causes derepression of silent Variant Surface Glycoprotein genes important for 34 host immune evasion. Our results provide the first direct mechanistic link between base J and regulation 35 of Pol II termination and suggest a novel molecular model for the role of the CTD of Pol II in terminating 36 polycistronic transcription in trypanosomatids. 37 38 Author Summary 39 Trypanosoma brucei is an early-diverged parasitic protozoan that causes African sleeping 40 sickness in humans. The genome of T. brucei is organized into polycistronic gene clusters that contain 41 multiple genes that are co-transcribed from a single promoter. We have recently described the presence 42 of a modified DNA base J and variant of histone H3 (H3.V) at transcription termination sites within gene 43 clusters where the loss of base J and H3.V leads to read-through transcription and the expression of 44 downstream genes. We now identify a novel stable multimeric complex containing a J binding protein . CC-BY 4.0 International license is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/753004 doi: bioRxiv preprint 45 (JBP3), base J glucosyltransferase (JGT), PP1 phosphatase, PP1 interactive-regulatory protein (PNUTS) 46 and Wdr82, which we refer to as PJW/PP1. A similar complex (PTW/PP1) has been shown to be involved 47 in Pol II termination in humans and yeast. We demonstrate that PNUTS, JBP3 and Wdr82 mutants lead 48 to read-through transcription in T. brucei. Our data suggest the PJW/PP1 complex reg...

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Noncoding transcription influences the replication initiation program through chromatin regulation

Cited by 22 publications

References 75 publications

Identification of a novel base J binding protein complex involved in RNA polymerase II transcription termination in trypanosomes

Identification of a novel base J binding protein complex involved in RNA polymerase II transcription termination in trypanosomes

Regulation of Gene Expression and Replication Initiation by Non‐Coding Transcription: A Model Based on Reshaping Nucleosome‐Depleted Regions

Identification of a Novel Base J Binding Protein Complex Involved in RNA Polymerase II Transcription Termination in Trypanosomes

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