scholarly journals RNA polymerase II-independent recruitment of SPT6L at transcription start sites in Arabidopsis

2019 ◽  
Vol 47 (13) ◽  
pp. 6714-6725 ◽  
Author(s):  
Chen Chen ◽  
Jie Shu ◽  
Chenlong Li ◽  
Raj K Thapa ◽  
Vi Nguyen ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Elongation Factor ◽  
Proper Function ◽  
Gene Body ◽  
Transcription Start ◽  
Transcription Start Sites ◽  
Genome Wide ◽  
Yeast Mutants

Abstract SPT6 is a conserved elongation factor that is associated with phosphorylated RNA polymerase II (RNAPII) during transcription. Recent transcriptome analysis in yeast mutants revealed its potential role in the control of transcription initiation at genic promoters. However, the mechanism by which this is achieved and how this is linked to elongation remains to be elucidated. Here, we present the genome-wide occupancy of Arabidopsis SPT6-like (SPT6L) and demonstrate its conserved role in facilitating RNAPII occupancy across transcribed genes. We also further demonstrate that SPT6L enrichment is unexpectedly shifted, from gene body to transcription start site (TSS), when its association with RNAPII is disrupted. Protein domains, required for proper function and enrichment of SPT6L on chromatin, are subsequently identified. Finally, our results suggest that recruitment of SPT6L at TSS is indispensable for its spreading along the gene body during transcription. These findings provide new insights into the mechanisms underlying SPT6L recruitment in transcription and shed light on the coordination between transcription initiation and elongation.

scholarly journals RNA Polymerase II Independent Recruitment of SPT6 at Transcription Start Sites in Arabidopsis

2018 ◽  
Author(s):  
Chen Chen ◽  
Jie Shu ◽  
Chenlong Li ◽  
Raj K. Thapa ◽  
Vi Nguyen ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Potential Role ◽  
Elongation Factor ◽  
Gene Body ◽  
Transcription Start Sites ◽  
Genome Wide ◽  
Shed Light

SummarySPT6 is a conserved transcription regulator that is generally viewed as an elongation factor. However, emerging evidence show its potential role in the control of transcription initiation at genic and intragenic promoters. Here we first present the genome-wide occupancy of Arabidopsis SPT6-like (SPT6L) and demonstrate its conserved role in facilitating RNA Polymerase II (RNAPII) occupancy across transcribed genes. Further, we show that SPT6L enrichment is shifted, unexpectedly, from gene body to the transcription starting site (TSS) when its association with RNAPII is disrupted. Finally, we demonstrate that recruitment of SPT6L starts at TSS, and then spreads to the gene body during transcription. These findings refine the mechanisms underlying SPT6L recruitment in transcription and shed light on the role of SPT6L in transcription initiation.

scholarly journals Genome-wide R-loop analysis defines unique roles for DDX5, XRN2, and PRMT5 in DNA/RNA hybrid resolution

2020 ◽  
Vol 3 (10) ◽  
pp. e202000762
Author(s):  
Oscar D Villarreal ◽  
Sofiane Y Mersaoui ◽  
Zhenbao Yu ◽  
Jean-Yves Masson ◽  
Stéphane Richard
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Start Site ◽  
Transcription Initiation ◽  
Transcription Termination ◽  
Start Site ◽  
Loop Gain ◽  
Transcription Start ◽  
Genome Wide ◽  
Rna Polymerase Ii Transcription

DDX5, XRN2, and PRMT5 have been shown to resolve DNA/RNA hybrids (R-loops) at RNA polymerase II transcription termination sites at few genomic loci. Herein, we perform genome-wide R-loop mapping using classical DNA/RNA immunoprecipitation and high-throughput sequencing (DRIP-seq) of loci regulated by DDX5, XRN2, and PRMT5. We observed hundreds to thousands of R-loop gains and losses at transcribed loci in DDX5-, XRN2-, and PRMT5-deficient U2OS cells. R-loop gains were characteristic of highly transcribed genes located at gene-rich regions, whereas R-loop losses were observed in low-density gene areas. DDX5, XRN2, and PRMT5 shared many R-loop gain loci at transcription termination sites, consistent with their coordinated role in RNA polymerase II transcription termination. DDX5-depleted cells had unique R-loop gain peaks near the transcription start site that did not overlap with those of siXRN2 and siPRMT5 cells, suggesting a role for DDX5 in transcription initiation independent of XRN2 and PRMT5. Moreover, we observed that the accumulated R-loops at certain loci in siDDX5, siXRN2, and siPRMT5 cells near the transcription start site of genes led to antisense intergenic transcription. Our findings define unique and shared roles of DDX5, XRN2, and PRMT5 in DNA/RNA hybrid regulation.

scholarly journals The telomeric Cdc13–Stn1–Ten1 complex regulates RNA polymerase II transcription

2019 ◽  
Vol 47 (12) ◽  
pp. 6250-6268 ◽  
Author(s):  
Olga Calvo ◽  
Nathalie Grandin ◽  
Antonio Jordán-Pla ◽  
Esperanza Miñambres ◽  
Noelia González-Polo ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Genome Stability ◽  
Elongation Factor ◽  
Yeast Saccharomyces Cerevisiae ◽  
Replication Fork Progression ◽  
Genome Wide ◽  
Length Regulation ◽  
Transcription Regulators ◽  
Rna Polymerase Ii Transcription

Abstract Specialized telomeric proteins have an essential role in maintaining genome stability through chromosome end protection and telomere length regulation. In the yeast Saccharomyces cerevisiae, the evolutionary conserved CST complex, composed of the Cdc13, Stn1 and Ten1 proteins, largely contributes to these functions. Here, we report genetic interactions between TEN1 and several genes coding for transcription regulators. Molecular assays confirmed this novel function of Ten1 and further established that it regulates the occupancies of RNA polymerase II and the Spt5 elongation factor within transcribed genes. Since Ten1, but also Cdc13 and Stn1, were found to physically associate with Spt5, we propose that Spt5 represents the target of CST in transcription regulation. Moreover, CST physically associates with Hmo1, previously shown to mediate the architecture of S-phase transcribed genes. The fact that, genome-wide, the promoters of genes down-regulated in the ten1-31 mutant are prefentially bound by Hmo1, leads us to propose a potential role for CST in synchronizing transcription with replication fork progression following head-on collisions.

scholarly journals JMJD5 couples with CDK9 to release the paused RNA polymerase II

2020 ◽  
Vol 117 (33) ◽  
pp. 19888-19895
Author(s):  
Haolin Liu ◽  
Srinivas Ramachandran ◽  
Nova Fong ◽  
Tzu Phang ◽  
Schuyler Lee ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Pol Ii ◽  
Transcription Start Sites ◽  
Terminal Domain ◽  
Carboxyl Terminal Domain ◽  
Pol Ii Pausing ◽  
Carboxyl Terminal ◽  
Heptad Repeats

More than 30% of genes in higher eukaryotes are regulated by RNA polymerase II (Pol II) promoter proximal pausing. Pausing is released by the positive transcription elongation factor complex (P-TEFb). However, the exact mechanism by which this occurs and whether phosphorylation of the carboxyl-terminal domain of Pol II is involved in the process remains unknown. We previously reported that JMJD5 could generate tailless nucleosomes at position +1 from transcription start sites (TSS), thus perhaps enable progression of Pol II. Here we find that knockout of JMJD5 leads to accumulation of nucleosomes at position +1. Absence of JMJD5 also results in loss of or lowered transcription of a large number of genes. Interestingly, we found that phosphorylation, by CDK9, of Ser2 within two neighboring heptad repeats in the carboxyl-terminal domain of Pol II, together with phosphorylation of Ser5 within the second repeat, HR-Ser2p (1, 2)-Ser5p (2) for short, allows Pol II to bind JMJD5 via engagement of the N-terminal domain of JMJD5. We suggest that these events bring JMJD5 near the nucleosome at position +1, thus allowing JMJD5 to clip histones on this nucleosome, a phenomenon that may contribute to release of Pol II pausing.

scholarly journals Pausing sites of RNA polymerase II on actively transcribed genes are enriched in DNA double-stranded breaks

2020 ◽  
Vol 295 (12) ◽  
pp. 3990-4000 ◽  
Author(s):  
Sandeep Singh ◽  
Karol Szlachta ◽  
Arkadi Manukyan ◽  
Heather M. Raimer ◽  
Manikarna Dinda ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Topoisomerase I ◽  
Cell Types ◽  
Dna Breaks ◽  
Transcription Start ◽  
Pol Ii ◽  
Transcription Start Sites

DNA double-stranded breaks (DSBs) are strongly associated with active transcription, and promoter-proximal pausing of RNA polymerase II (Pol II) is a critical step in transcriptional regulation. Mapping the distribution of DSBs along actively expressed genes and identifying the location of DSBs relative to pausing sites can provide mechanistic insights into transcriptional regulation. Using genome-wide DNA break mapping/sequencing techniques at single-nucleotide resolution in human cells, we found that DSBs are preferentially located around transcription start sites of highly transcribed and paused genes and that Pol II promoter-proximal pausing sites are enriched in DSBs. We observed that DSB frequency at pausing sites increases as the strength of pausing increases, regardless of whether the pausing sites are near or far from annotated transcription start sites. Inhibition of topoisomerase I and II by camptothecin and etoposide treatment, respectively, increased DSBs at the pausing sites as the concentrations of drugs increased, demonstrating the involvement of topoisomerases in DSB generation at the pausing sites. DNA breaks generated by topoisomerases are short-lived because of the religation activity of these enzymes, which these drugs inhibit; therefore, the observation of increased DSBs with increasing drug doses at pausing sites indicated active recruitment of topoisomerases to these sites. Furthermore, the enrichment and locations of DSBs at pausing sites were shared among different cell types, suggesting that Pol II promoter-proximal pausing is a common regulatory mechanism. Our findings support a model in which topoisomerases participate in Pol II promoter-proximal pausing and indicated that DSBs at pausing sites contribute to transcriptional activation.

scholarly journals Live-cell single particle imaging reveals the role of RNA polymerase II in histone H2A.Z eviction

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Anand Ranjan ◽  
Vu Q Nguyen ◽  
Sheng Liu ◽  
Jan Wisniewski ◽  
Jee Min Kim ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Single Molecule ◽  
Transcription Initiation ◽  
General Mechanism ◽  
Pol Ii ◽  
Promoter Escape ◽  
Genome Wide ◽  
A Genome ◽  
Particle Imaging

The H2A.Z histone variant, a genome-wide hallmark of permissive chromatin, is enriched near transcription start sites in all eukaryotes. H2A.Z is deposited by the SWR1 chromatin remodeler and evicted by unclear mechanisms. We tracked H2A.Z in living yeast at single-molecule resolution, and found that H2A.Z eviction is dependent on RNA Polymerase II (Pol II) and the Kin28/Cdk7 kinase, which phosphorylates Serine 5 of heptapeptide repeats on the carboxy-terminal domain of the largest Pol II subunit Rpb1. These findings link H2A.Z eviction to transcription initiation, promoter escape and early elongation activities of Pol II. Because passage of Pol II through +1 nucleosomes genome-wide would obligate H2A.Z turnover, we propose that global transcription at yeast promoters is responsible for eviction of H2A.Z. Such usage of yeast Pol II suggests a general mechanism coupling eukaryotic transcription to erasure of the H2A.Z epigenetic signal.

scholarly journals CUT&RUN detects distinct DNA footprints of RNA polymerase II near the transcription start sites

2020 ◽  
Vol 28 (3-4) ◽  
pp. 381-393
Author(s):  
Michi Miura ◽  
Honglin Chen
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Chromatin Immunoprecipitation ◽  
Biological Significance ◽  
Micrococcal Nuclease ◽  
Dna Fragments ◽  
Transcription Start ◽  
Transcription Start Sites ◽  
Human Lung Carcinoma Cell

AbstractCUT&RUN is a powerful tool to study protein-DNA interactions in vivo. DNA fragments cleaved by the targeted micrococcal nuclease identify the footprints of DNA-binding proteins on the chromatin. We performed CUT&RUN on human lung carcinoma cell line A549 maintained in a multi-well cell culture plate to profile RNA polymerase II. Long (> 270 bp) DNA fragments released by CUT&RUN corresponded to the bimodal peak around the transcription start sites, as previously seen with chromatin immunoprecipitation. However, we found that short (< 120 bp) fragments identify a well-defined peak localised at the transcription start sites. This distinct DNA footprint of short fragments, which constituted only about 5% of the total reads, suggests the transient positioning of RNA polymerase II before promoter-proximal pausing, which has not been detected in the physiological settings by standard chromatin immunoprecipitation. We showed that the positioning of the large-size-class DNA footprints around the short-fragment peak was associated with the directionality of transcription, demonstrating the biological significance of distinct CUT&RUN footprints of RNA polymerase II.

scholarly journals Properties of an Intergenic Terminator and Start Site Switch That Regulate IMD2 Transcription in Yeast

2008 ◽  
Vol 28 (12) ◽  
pp. 3883-3893 ◽  
Author(s):  
M. Harley Jenks ◽  
Thomas W. O'Rourke ◽  
Daniel Reines
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Initiation Site ◽  
Start Codon ◽  
Start Site ◽  
Transcription Start ◽  
Transcription Start Sites ◽  
Short Transcript ◽  
Alternative Transcription ◽  
Polyadenylation Sites

ABSTRACT The IMD2 gene in Saccharomyces cerevisiae is regulated by intracellular guanine nucleotides. Regulation is exerted through the choice of alternative transcription start sites that results in synthesis of either an unstable short transcript terminating upstream of the start codon or a full-length productive IMD2 mRNA. Start site selection is dictated by the intracellular guanine nucleotide levels. Here we have mapped the polyadenylation sites of the upstream, unstable short transcripts that form a heterogeneous family of RNAs of ≈200 nucleotides. The switch from the upstream to downstream start sites required the Rpb9 subunit of RNA polymerase II. The enzyme's ability to locate the downstream initiation site decreased exponentially as the start was moved downstream from the TATA box. This suggests that RNA polymerase II's pincer grip is important as it slides on DNA in search of a start site. Exosome degradation of the upstream transcripts was highly dependent upon the distance between the terminator and promoter. Similarly, termination was dependent upon the Sen1 helicase when close to the promoter. These findings extend the emerging concept that distinct modes of termination by RNA polymerase II exist and that the distance of the terminator from the promoter, as well as its sequence, is important for the pathway chosen.

scholarly journals CUT&RUN detects distinct DNA footprints of RNA polymerase II near the transcription start sites

2020 ◽  
Author(s):  
Michi Miura ◽  
Honglin Chen
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Chromatin Immunoprecipitation ◽  
Biological Significance ◽  
Micrococcal Nuclease ◽  
Dna Fragments ◽  
Transcription Start ◽  
Transcription Start Sites ◽  
Human Lung Carcinoma Cell

AbstractCUT&RUN is a powerful tool to study protein-DNA interactions in vivo. DNA fragments cleaved by the targeted micrococcal nuclease identify the footprints of DNA-binding proteins on the chromatin. We performed CUT&RUN on human lung carcinoma cell line A549 maintained in a multi-well cell culture plate to profile RNA polymerase II. Long (>270 bp) DNA fragments released by CUT&RUN corresponded to the bimodal peak around the transcription start sites, as previously seen with chromatin immunoprecipitation. However, we found that short (<120 bp) fragments identify a well-defined peak localised at the transcription start sites. This distinct DNA footprint of short fragments, which constituted only about 5% of the total reads, suggests the transient positioning of RNA polymerase II before promoter-proximal pausing, which has not been detected in the physiological settings by standard chromatin immunoprecipitation. We showed that the positioning of the large-size-class DNA footprints around the short-fragment peak was associated with the directionality of transcription, demonstrating the biological significance of distinct CUT&RUN footprints of RNA polymerase II.

scholarly journals Spt4 Facilitates the Movement of RNA Polymerase II through the +2 Nucleosomal Barrier

2021 ◽  
Author(s):  
Ülkü Uzun ◽  
Thomas Brown ◽  
Harry Fischl ◽  
Andrew Angel ◽  
Jane Mellor
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Nucleosome Positioning ◽  
Gene Body ◽  
Precise Position ◽  
Transcription Elongation Factor

AbstractSpt4 is a transcription elongation factor, with homologues in organisms with nucleosomes. Structural and in vitro studies implicate Spt4 in transcription through nucleosomes, yet the in vivo function of Spt4 is unclear. Here we assessed the precise position of Spt4 during transcription and the consequences of loss of Spt4 on RNA polymerase II (RNAPII) dynamics and nucleosome positioning in Saccharomyces cerevisiae. In the absence of Spt4, the spacing between gene-body nucleosomes increases and RNAPII accumulates upstream of the nucleosomal dyad, most dramatically at nucleosome +2. Spt4 associates with elongating RNAPII early in transcription and its association dynamically changes depending on nucleosome positions. Together, our data show that Spt4 regulates early elongation dynamics, participates in co-transcriptional nucleosome positioning, and promotes RNAPII movement through the gene-body nucleosomes, especially the +2 nucleosome.

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