scholarly journals Structure of transcribed chromatin is a sensor of DNA damage

2015 ◽  
Vol 1 (6) ◽  
pp. e1500021 ◽  
Author(s):  
Nikolay A. Pestov ◽  
Nadezhda S. Gerasimova ◽  
Olga I. Kulaeva ◽  
Vasily M. Studitsky
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dna Supercoiling ◽  
Proximal Region ◽  
Cellular Systems ◽  
Dna Breaks ◽  
Specific Mechanism ◽  
Nucleosome Structure ◽  
Nucleosomal Dna ◽  
Promoter Proximal Region

Early detection and repair of damaged DNA is essential for cell functioning and survival. Although multiple cellular systems are involved in the repair of single-strand DNA breaks (SSBs), it remains unknown how SSBs present in the nontemplate strand (NT-SSBs) of DNA organized in chromatin are detected. The effect of NT-SSBs on transcription through chromatin by RNA polymerase II was studied. NT-SSBs localized in the promoter-proximal region of nucleosomal DNA and hidden in the nucleosome structure can induce a nearly quantitative arrest of RNA polymerase downstream of the break, whereas more promoter-distal SSBs moderately facilitate transcription. The location of the arrest sites on nucleosomal DNA suggests that formation of small intranucleosomal DNA loops causes the arrest. This mechanism likely involves relief of unconstrained DNA supercoiling accumulated during transcription through chromatin by NT-SSBs. These data suggest the existence of a novel chromatin-specific mechanism that allows the detection of NT-SSBs by the transcribing enzyme.

scholarly journals RNA Polymerase II Accumulation in the Promoter-Proximal Region of the Dihydrofolate Reductase and γ-Actin Genes

2003 ◽  
Vol 23 (6) ◽  
pp. 1961-1967 ◽  
Author(s):  
Chonghui Cheng ◽  
Phillip A. Sharp
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dihydrofolate Reductase ◽  
Mammalian Cells ◽  
Proximal Region ◽  
Heptad Repeat ◽  
Pol Ii ◽  
Actin Genes ◽  
Carboxyl Terminal ◽  
Promoter Proximal Region

ABSTRACT The carboxyl-terminal domain (CTD) of RNA polymerase II (Pol II) can be phosphorylated at serine 2 (Ser-2) and serine 5 (Ser-5) of the CTD heptad repeat YSPTSPS, and this phosphorylation is important in coupling transcription to RNA processing, including 5′ capping, splicing, and polyadenylation. The mammalian endogenous dihydrofolate reductase and γ-actin genes have been used to study the association of Pol II with different regions of transcribed genes (promoter-proximal compared to distal regions) and the phosphorylation status of its CTD. For both genes, Pol II is more concentrated in the promoter-proximal regions than in the interior regions. Moreover, different phosphorylation forms of Pol II are associated with distinct regions. Ser-5 phosphorylation of Pol II is concentrated near the promoter, while Ser-2 phosphorylation is observed throughout the gene. These results suggest that the accumulation of paused Pol II in promoter-proximal regions may be a common feature of gene regulation in mammalian cells.

scholarly journals CDK9-dependent RNA polymerase II pausing controls transcription initiation

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Saskia Gressel ◽  
Björn Schwalb ◽  
Tim Michael Decker ◽  
Weihua Qin ◽  
Heinrich Leonhardt ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Human Cell Line ◽  
Pause Duration ◽  
Proximal Region ◽  
Chromatin Interactions ◽  
Initiation Frequency ◽  
Polymerase Pausing ◽  
Promoter Proximal Region

Gene transcription can be activated by decreasing the duration of RNA polymerase II pausing in the promoter-proximal region, but how this is achieved remains unclear. Here we use a ‘multi-omics’ approach to demonstrate that the duration of polymerase pausing generally limits the productive frequency of transcription initiation in human cells (‘pause-initiation limit’). We further engineer a human cell line to allow for specific and rapid inhibition of the P-TEFb kinase CDK9, which is implicated in polymerase pause release. CDK9 activity decreases the pause duration but also increases the productive initiation frequency. This shows that CDK9 stimulates release of paused polymerase and activates transcription by increasing the number of transcribing polymerases and thus the amount of mRNA synthesized per time. CDK9 activity is also associated with long-range chromatin interactions, suggesting that enhancers can influence the pause-initiation limit to regulate transcription.

scholarly journals Transcriptional Pausing Caused by NELF Plays a Dual Role in Regulating Immediate-Early Expression of the junB Gene

2006 ◽  
Vol 26 (16) ◽  
pp. 6094-6104 ◽  
Author(s):  
Masatoshi Aida ◽  
Yexi Chen ◽  
Koichi Nakajima ◽  
Yuki Yamaguchi ◽  
Tadashi Wada ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Mrna Level ◽  
Proximal Region ◽  
Negative Regulation ◽  
Immediate Early ◽  
Dual Function ◽  
Early Expression ◽  
Transcriptional Pausing ◽  
Promoter Proximal Region

ABSTRACT Human 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole sensitivity-inducing factor (DSIF) and negative elongation factor (NELF) negatively regulate transcription elongation by RNA polymerase II (RNAPII) in vitro. However, the physiological roles of this negative regulation are not well understood. Here, by using a number of approaches to identify protein-DNA interactions in vivo, we show that DSIF- and NELF-mediated transcriptional pausing has a dual function in regulating immediate-early expression of the human junB gene. Before induction by interleukin-6, RNAPII, DSIF, and NELF accumulate in the promoter-proximal region of junB, mainly at around position +50 from the transcription initiation site. After induction, the association of these proteins with the promoter-proximal region continues whereas RNAPII and DSIF are also found in the downstream regions. Depletion of a subunit of NELF by RNA interference enhances the junB mRNA level both before and after induction, indicating that DSIF- and NELF-mediated pausing contributes to the negative regulation of junB expression, not only by inducing RNAPII pausing before induction but also by attenuating transcription after induction. These regulatory mechanisms appear to be conserved in other immediate-early genes as well.

Na+ and K+ Ions Differently Affect Nucleosome Structure, Stability, and Interactions with Proteins

2021 ◽  
pp. 1-11
Author(s):  
Tatyana V. Andreeva ◽  
Natalya V. Maluchenko ◽  
Anastasiia L. Sivkina ◽  
Oleg V. Chertkov ◽  
Maria E. Valieva ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Rna Polymerase Ii ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Inorganic Ions ◽  
Structure Stability ◽  
Ion Concentrations ◽  
Nucleosome Structure ◽  
Nucleosomal Dna ◽  
Stabilizing Effect

Inorganic ions are essential factors stabilizing nucleosome structure; however, many aspects of their effects on DNA transactions in chromatin remain unknown. Here, differential effects of K+ and Na+ on the nucleosome structure, stability, and interactions with protein complex FACT (FAcilitates Chromatin Transcription), poly(ADP-ribose) polymerase 1, and RNA polymerase II were studied using primarily single-particle Förster resonance energy transfer microscopy. The maximal stabilizing effect of K+ on a nucleosome structure was observed at ca. 80–150 mM, and it decreased slightly at 40 mM and considerably at >300 mM. The stabilizing effect of Na+ is noticeably lower than that of K+ and progressively decreases at ion concentrations higher than 40 mM. At 150 mM, Na+ ions support more efficient reorganization of nucleosome structure by poly(ADP-ribose) polymerase 1 and ATP-independent uncoiling of nucleosomal DNA by FACT as compared with K+ ions. In contrast, transcription through a nucleosome is nearly insensitive to K+ or Na+ environment. Taken together, the data indicate that K+ environment is more preserving for chromatin structure during various nucleosome transactions than Na+ environment.

scholarly journals WWP2 ubiquitylates RNA polymerase II for DNA-PK-dependent transcription arrest and repair at DNA breaks

2019 ◽  
Vol 33 (11-12) ◽  
pp. 684-704 ◽  
Author(s):  
Pierre Caron ◽  
Tibor Pankotai ◽  
Wouter W. Wiegant ◽  
Maxim A.X. Tollenaere ◽  
Audrey Furst ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dna Breaks

scholarly journals DNAPKcs-dependent arrest of RNA polymerase II transcription in the presence of DNA breaks

2012 ◽  
Vol 19 (3) ◽  
pp. 276-282 ◽  
Author(s):  
Tibor Pankotai ◽  
Céline Bonhomme ◽  
David Chen ◽  
Evi Soutoglou
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dna Breaks ◽  
Rna Polymerase Ii Transcription

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 A Region within the RAP74 Subunit of Human Transcription Factor IIF Is Critical for Initiation but Dispensable for Complex Assembly

1999 ◽  
Vol 19 (11) ◽  
pp. 7377-7387 ◽  
Author(s):  
Delin Ren ◽  
Lei Lei ◽  
Zachary F. Burton
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Residual Activity ◽  
Dna Supercoiling ◽  
Single Amino Acid ◽  
Phosphodiester Bond ◽  
Promoter Escape ◽  
Transcription Factor Iif ◽  
Human Transcription Factor

ABSTRACT Human transcription factor IIF (TFIIF) is an α2β2 heterotetramer of RNA polymerase II-associating 74 (RAP74) and RAP30 subunits. Mutagenic analysis shows that the N-terminal region of RAP74 between L155 (leucine at codon 155) and M177 is important for initiation. Mutants in this region have reduced activity in transcription, but none are inactive. Single amino acid substitutions at hydrophobic residues L155, W164, I176, and M177 have similar activity to RAP74(1–158), from which all but three amino acids of this region are deleted. Residual activity can be explained because each of these mutants forms a complex with RAP30 and recruits RNA polymerase II into the preinitiation complex. Mutants are defective for formation of the first phosphodiester bond from the adenovirus major late promoter but do not appear to have an additional significant defect in promoter escape. Negative DNA supercoiling partially compensates for the defects of TFIIF mutants in initiation, indicating that TFIIF may help to untwist the DNA helix for initiation.

scholarly journals Nucleosomal Fluctuations Govern the Transcription Dynamics of RNA Polymerase II

Science ◽  
2009 ◽  
Vol 325 (5940) ◽  
pp. 626-628 ◽  
Author(s):  
Courtney Hodges ◽  
Lacramioara Bintu ◽  
Lucyna Lubkowska ◽  
Mikhail Kashlev ◽  
Carlos Bustamante
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Optical Tweezers ◽  
Direct Evidence ◽  
Eukaryotic Transcription ◽  
Pol Ii ◽  
The Core ◽  
Nucleosomal Dna ◽  
Dna Loop ◽  
Core Histones

RNA polymerase II (Pol II) must overcome the barriers imposed by nucleosomes during transcription elongation. We have developed an optical tweezers assay to follow individual Pol II complexes as they transcribe nucleosomal DNA. Our results indicate that the nucleosome behaves as a fluctuating barrier that locally increases pause density, slows pause recovery, and reduces the apparent pause-free velocity of Pol II. The polymerase, rather than actively separating DNA from histones, functions instead as a ratchet that rectifies nucleosomal fluctuations. We also obtained direct evidence that transcription through a nucleosome involves transfer of the core histones behind the transcribing polymerase via a transient DNA loop. The interplay between polymerase dynamics and nucleosome fluctuations provides a physical basis for the regulation of eukaryotic transcription.

scholarly journals Nucleosome surface containing nucleosomal DNA entry/exit site regulates H3-K36me3 via association with RNA polymerase II and Set2

2011 ◽  
Vol 17 (1) ◽  
pp. 65-81 ◽  
Author(s):  
Hirohito Endo ◽  
Yu Nakabayashi ◽  
Satoshi Kawashima ◽  
Takemi Enomoto ◽  
Masayuki Seki ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Exit Site ◽  
Nucleosomal Dna
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