scholarly journals Continuous transcription initiation guarantees robust repair of transcribed genes and regulatory regions in response to DNA damage

2019 ◽  
Author(s):  
Anastasios Liakos ◽  
Dimitris Konstantopoulos ◽  
Matthieu D. Lavigne ◽  
Maria Fousteri
Keyword(s):  
Dna Damage ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
De Novo ◽  
Transcriptional Response ◽  
Genotoxic Stress ◽  
Chromatin Accessibility ◽  
Initiation Rate ◽  
Pol Ii ◽  
Active Genes

ABSTRACTInhibition of RNA synthesis caused by DNA damage-impaired RNA polymerase II (Pol II) elongation is found to conceal a local increase in de novo transcription, slowly progressing from Transcription Start Sites (TSSs) to gene ends. Although associated with accelerated repair of Pol II-encountered lesions and limited mutagenesis, it is still unclear how this mechanism is maintained during recovery from genotoxic stress. Here we uncover a surprising widespread gain in chromatin accessibility and preservation of the active histone mark H3K27ac after UV-irradiation. We show that the concomitant increase in Pol II release from promoter-proximal pause (PPP) sites of most active genes, PROMoter uPstream Transcripts (PROMPTs) and enhancer RNAs (eRNAs) favors unrestrained initiation, as demonstrated by the synthesis of short nascent RNAs, including TSS-associated RNAs (start-RNAs). In accordance, drug-inhibition of the transition into elongation replenished the post-UV reduced levels of pre-initiating pol II at TSSs. Continuous engagement of new Pol II thus ensures maximal transcription-driven DNA repair of active genes and non-coding regulatory loci. Together, our results reveal an unanticipated layer regulating the UV-triggered transcriptional-response and provide physiologically relevant traction to the emerging concept that transcription initiation rate is determined by pol II pause-release dynamics.

scholarly journals P-TEFb activation by RBM7 shapes a pro-survival transcriptional response to genotoxic stress

2018 ◽  
Author(s):  
Andrii Bugai ◽  
Alexandre J.C. Quaresma ◽  
Caroline C. Friedel ◽  
Tina Lenasi ◽  
Christopher R. Sibley ◽  
...  
Keyword(s):  
Dna Damage ◽  
Rna Polymerase Ii ◽  
Cell Fate ◽  
Rna Binding ◽  
Transcriptional Response ◽  
Genotoxic Stress ◽  
Binding Motif ◽  
Pol Ii ◽  
Small Nuclear Ribonucleoprotein ◽  
Stress Dependent

SUMMARYCellular DNA damage response (DDR) involves dramatic transcriptional alterations, the mechanisms of which remain ill-defined. Given the centrality of RNA polymerase II (Pol II) promoter-proximal pause release in transcriptional control, we evaluated its importance in DDR. Here we show that following genotoxic stress, the RNA-binding motif protein 7 (RBM7) stimulates Pol II elongation and promotes cell viability by activating the positive transcription elongation factor b (P-TEFb). This is mediated by genotoxic stress-enhanced binding of RBM7 to 7SK snRNA (7SK), the scaffold of the 7SK small nuclear ribonucleoprotein (7SK snRNP) which inhibits P-TEFb. In turn, P-TEFb relocates from 7SK snRNP to chromatin to induce transcription of short units including key DDR genes and multiple classes of non-coding RNAs. Critically, interfering with RBM7 or P-TEFb provokes cellular hypersensitivity to DNA damage-inducing agents through activation of apoptotic program. By alleviating the inhibition of P-TEFb, RBM7 thus facilitates Pol II elongation to enable a pro-survival transcriptional response that is crucial for cell fate upon genotoxic insult. Our work uncovers a new paradigm in stress-dependent control of Pol II pause release, and offers the promise for designing novel anti-cancer interventions using RBM7 and P-TEFb antagonists in combination with DNA-damaging chemotherapeutics.

scholarly journals Def1 interacts with TFIIH and modulates RNA polymerase II transcription

2017 ◽  
Vol 114 (50) ◽  
pp. 13230-13235 ◽  
Author(s):  
Nivedita Damodaren ◽  
Trevor Van Eeuwen ◽  
Joanna Zamel ◽  
Enrique Lin-Shiao ◽  
Nir Kalisman ◽  
...  
Keyword(s):  
Dna Damage ◽  
Heat Shock ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Excision Repair ◽  
Cell Extract ◽  
Pol Ii ◽  
Stress Responsive Genes

The DNA damage response is an essential process for the survival of living cells. In a subset of stress-responsive genes in humans, Elongin controls transcription in response to multiple stimuli, such as DNA damage, oxidative stress, and heat shock. Yeast Elongin (Ela1-Elc1), along with Def1, is known to facilitate ubiquitylation and degradation of RNA polymerase II (pol II) in response to multiple stimuli, yet transcription activity has not been examined. We have found that Def1 copurifies from yeast whole-cell extract with TFIIH, the largest general transcription factor required for transcription initiation and nucleotide excision repair. The addition of recombinant Def1 and Ela1-Elc1 enhanced transcription initiation in an in vitro reconstituted system including pol II, the general transcription factors, and TFIIS. Def1 also enhanced transcription restart from TFIIS-induced cleavage in a pol II transcribing complex. In the Δdef1 strain, heat shock genes were misregulated, indicating that Def1 is required for induction of some stress-responsive genes in yeast. Taken together, our results extend the understanding of the molecular mechanism of transcription regulation on cellular stress and reveal functional similarities to the mammalian system.

scholarly journals A hyperactive transcriptional state marks genome reactivation at the mitosis-G1 transition

2016 ◽  
Author(s):  
Chris C.-S. Hsiung ◽  
Caroline Bartman ◽  
Peng Huang ◽  
Paul Ginart ◽  
Aaron J. Stonestrom ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Single Molecule ◽  
Mammalian Cells ◽  
Transcriptional Activity ◽  
De Novo ◽  
Pol Ii ◽  
Mature Mrna ◽  
Genome Wide ◽  
Transcriptional State ◽  
Active Genes

AbstractDuring mitosis, RNA polymerase II (Pol II) and many transcription factors dissociate from chromatin, and transcription ceases globally. Transcription is known to restart in bulk by telophase, but whether de novo transcription at the mitosis-G1 transition is in any way distinct from later in interphase remains unknown. We tracked Pol II occupancy genome-wide in mammalian cells progressing from mitosis through late G1. Unexpectedly, during the earliest rounds of transcription at the mitosis-G1 transition, ~50% of active genes and distal enhancers exhibit a spike in transcription, exceeding levels observed later in G1 phase. Enhancer-promoter chromatin contacts are depleted during mitosis and restored rapidly upon G1 entry, but do not spike. Of the chromatin-associated features examined, histone H3 lysine 27 acetylation levels at individual loci in mitosis best predict the mitosis-G1 transcriptional spike. Single-molecule RNA imaging supports that the mitosis-G1 transcriptional spike can constitute the maximum transcriptional activity per DNA copy throughout the cell division cycle. The transcriptional spike occurs heterogeneously and propagates to cell-to-cell differences in mature mRNA expression. Our results raise the possibility that passage through the mitosis-G1 transition might predispose cells to diverge in gene expression states.

scholarly journals The histone chaperone FACT coordinates H2A.X-dependent signaling and repair of DNA damage

2018 ◽  
Author(s):  
Sandra Piquet ◽  
Florent Le Parc ◽  
Siau-Kun Bai ◽  
Odile Chevallier ◽  
Salomé Adam ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Function ◽  
De Novo ◽  
Genotoxic Stress ◽  
Chromatin Accessibility ◽  
Histone Chaperone ◽  
List Type ◽  
Repair Synthesis ◽  
Dna Damage Signaling ◽  
Damage Repair

SUMMARYSafeguarding cell function and identity following a genotoxic stress challenge entails a tight coordination of DNA damage signaling and repair with chromatin maintenance. How this coordination is achieved and with what impact on chromatin integrity remains elusive. Here, by investigating the mechanisms governing the distribution of H2A.X in mammalian chromatin, we demonstrate that this histone variant is deposited de novo at sites of DNA damage in a repair synthesis-coupled manner. Our mechanistic studies further identify the histone chaperone FACT (Facilitates Chromatin Transcription) as responsible for the deposition of newly synthesized H2A.X. Functionally, FACT potentiates H2A.X-dependent signaling of DNA damage and, together with ANP32E (Acidic Nuclear Phosphoprotein 32 Family Member E), orchestrates a H2A.Z/H2A.X exchange reaction that reshapes the chromatin landscape at repair sites. We propose that this mechanism promotes chromatin accessibility and helps tailoring DNA damage signaling to repair progression.HIGHLIGHTSH2A.X, but not H2A.Z, is deposited de novo at sites of DNA damage repairFACT promotes new H2A.X deposition coupled to repair synthesisFACT and ANP32E chaperones orchestrate H2A.Z/H2A.X exchange in damaged chromatinFACT stimulates H2A.X-dependent signaling of DNA damage

scholarly journals DNA damage shifts circadian clock time via Hausp-dependent Cry1 stabilization

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Stephanie J Papp ◽  
Anne-Laure Huber ◽  
Sabine D Jordan ◽  
Anna Kriebs ◽  
Madelena Nguyen ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Circadian Clock ◽  
Transcriptional Response ◽  
Genotoxic Stress ◽  
Genomic Integrity ◽  
Clock Time ◽  
Dna Repair Enzymes ◽  
Repair Enzymes ◽  
Specific Protease

The circadian transcriptional repressors cryptochrome 1 (Cry1) and 2 (Cry2) evolved from photolyases, bacterial light-activated DNA repair enzymes. In this study, we report that while they have lost DNA repair activity, Cry1/2 adapted to protect genomic integrity by responding to DNA damage through posttranslational modification and coordinating the downstream transcriptional response. We demonstrate that genotoxic stress stimulates Cry1 phosphorylation and its deubiquitination by Herpes virus associated ubiquitin-specific protease (Hausp, a.k.a Usp7), stabilizing Cry1 and shifting circadian clock time. DNA damage also increases Cry2 interaction with Fbxl3, destabilizing Cry2. Thus, genotoxic stress increases the Cry1/Cry2 ratio, suggesting distinct functions for Cry1 and Cry2 following DNA damage. Indeed, the transcriptional response to genotoxic stress is enhanced in Cry1−/− and blunted in Cry2−/− cells. Furthermore, Cry2−/− cells accumulate damaged DNA. These results suggest that Cry1 and Cry2, which evolved from DNA repair enzymes, protect genomic integrity via coordinated transcriptional regulation.

scholarly journals Regulation of Pkc1 Hyper-Phosphorylation by Genotoxic Stress

2021 ◽  
Vol 7 (10) ◽  
pp. 874
Author(s):  
Li Liu ◽  
Jiri Veis ◽  
Wolfgang Reiter ◽  
Edwin Motari ◽  
Catherine E. Costello ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Wall ◽  
Protein Kinase ◽  
Transcriptional Response ◽  
Genotoxic Stress ◽  
Telomere Maintenance ◽  
Phosphorylation Sites ◽  
Casein Kinase 1 ◽  
Checkpoint Kinases ◽  
Radiation Repair

The cell wall integrity (CWI) signaling pathway is best known for its roles in cell wall biogenesis. However, it is also thought to participate in the response to genotoxic stress. The stress-activated protein kinase Mpk1 (Slt2, is activated by DNA damaging agents through an intracellular mechanism that does not involve the activation of upstream components of the CWI pathway. Additional observations suggest that protein kinase C (Pkc1), the top kinase in the CWI signaling cascade, also has a role in the response to genotoxic stress that is independent of its recognized function in the activation of Mpk1. Pkc1 undergoes hyper-phosphorylation specifically in response to genotoxic stress; we have found that this requires the DNA damage checkpoint kinases Mec1 (Mitosis Entry Checkpoint) and Tel1 (TELomere maintenance), but not their effector kinases. We demonstrate that the casein kinase 1 (CK1) ortholog, Hrr25 (HO and Radiation Repair), previously implicated in the DNA damage transcriptional response, associates with Pkc1 under conditions of genotoxic stress. We also found that the induced association of Hrr25 with Pkc1 requires Mec1 and Tel1, and that Hrr25 catalytic activity is required for Pkc1-hyperphosphorylation, thereby delineating a pathway from the checkpoint kinases to Pkc1. We used SILAC mass spectrometry to identify three residues within Pkc1 the phosphorylation of which was stimulated by genotoxic stress. We mutated these residues as well as a collection of 13 phosphorylation sites within the regulatory domain of Pkc1 that fit the consensus for CK1 sites. Mutation of the 13 Pkc1 phosphorylation sites blocked hyper-phosphorylation and diminished RNR3 (RiboNucleotide Reductase) basal expression and induction by genotoxic stress, suggesting that Pkc1 plays a role in the DNA damage transcriptional response.

Epigenetic reprogramming of host and viral genes by Human Cytomegalovirus infection in Kasumi-3 myeloid progenitor cells at early times post-infection

2021 ◽  
Author(s):  
Eleonora Forte ◽  
Fatma Ayaloglu Butun ◽  
Christian Marinaccio ◽  
Matthew J. Schipma ◽  
Andrea Piunti ◽  
...  
Keyword(s):  
Gene Expression ◽  
Post Infection ◽  
De Novo ◽  
Critical Role ◽  
Myeloid Cells ◽  
Viral Gene Expression ◽  
Chromatin Accessibility ◽  
Viral Gene ◽  
Dynamic Changes ◽  
Pol Ii

HCMV establishes latency in myeloid cells. Using the Kasumi-3 latency model, we previously showed that lytic gene expression is activated prior to establishment of latency in these cells. The early events in infection may have a critical role in shaping establishment of latency. Here, we have used an integrative multi-omics approach to investigate dynamic changes in host and HCMV gene expression and epigenomes at early times post infection. Our results show dynamic changes in viral gene expression and viral chromatin. Analyses of Pol II, H3K27Ac and H3K27me3 occupancy of the viral genome showed that 1) Pol II occupancy was highest at the MIEP at 4 hours post infection. However, it was observed throughout the genome; 2) At 24 hours, H3K27Ac was localized to the major immediate early promoter/enhancer and to a possible second enhancer in the origin of replication OriLyt; 3) viral chromatin was broadly accessible at 24 hpi. In addition, although HCMV infection activated expression of some host genes, we observed an overall loss of de novo transcription. This was associated with loss of promoter-proximal Pol II and H3K27Ac, but not with changes in chromatin accessibility or a switch in modification of H3K27. Importance. HCMV is an important human pathogen in immunocompromised hosts and developing fetuses. Current anti-viral therapies are limited by toxicity and emergence of resistant strains. Our studies highlight emerging concepts that challenge current paradigms of regulation of HCMV gene expression in myeloid cells. In addition, our studies show that HCMV has a profound effect on de novo transcription and the cellular epigenome. These results may have implications for mechanisms of viral pathogenesis.

scholarly journals The cryoelectron microscopy structure of the human CDK-activating kinase

2020 ◽  
Vol 117 (37) ◽  
pp. 22849-22857 ◽  
Author(s):  
Basil J. Greber ◽  
Juan M. Perez-Bertoldi ◽  
Kif Lim ◽  
Anthony T. Iavarone ◽  
Daniel B. Toso ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Rational Design ◽  
Control Cell ◽  
3D Structure ◽  
Structural Basis ◽  
Pol Ii ◽  
Insight Into ◽  
Cdk Activating Kinase

The human CDK-activating kinase (CAK), a complex composed of cyclin-dependent kinase (CDK) 7, cyclin H, and MAT1, is a critical regulator of transcription initiation and the cell cycle. It acts by phosphorylating the C-terminal heptapeptide repeat domain of the RNA polymerase II (Pol II) subunit RPB1, which is an important regulatory event in transcription initiation by Pol II, and it phosphorylates the regulatory T-loop of CDKs that control cell cycle progression. Here, we have determined the three-dimensional (3D) structure of the catalytic module of human CAK, revealing the structural basis of its assembly and providing insight into CDK7 activation in this context. The unique third component of the complex, MAT1, substantially extends the interaction interface between CDK7 and cyclin H, explaining its role as a CAK assembly factor, and it forms interactions with the CDK7 T-loop, which may contribute to enhancing CAK activity. We have also determined the structure of the CAK in complex with the covalently bound inhibitor THZ1 in order to provide insight into the binding of inhibitors at the CDK7 active site and to aid in the rational design of therapeutic compounds.

Structural Biology of RNA Polymerase II Transcription: 20 Years On

2020 ◽  
Vol 36 (1) ◽  
pp. 1-34 ◽  
Author(s):  
Sara Osman ◽  
Patrick Cramer
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Focal Point ◽  
Dna Lesions ◽  
Cellular Regulation ◽  
Pol Ii ◽  
Associated Proteins ◽  
Rna Polymerase Ii Transcription

Gene transcription by RNA polymerase II (Pol II) is the first step in the expression of the eukaryotic genome and a focal point for cellular regulation during development, differentiation, and responses to the environment. Two decades after the determination of the structure of Pol II, the mechanisms of transcription have been elucidated with studies of Pol II complexes with nucleic acids and associated proteins. Here we provide an overview of the nearly 200 available Pol II complex structures and summarize how these structures have elucidated promoter-dependent transcription initiation, promoter-proximal pausing and release of Pol II into active elongation, and the mechanisms that Pol II uses to navigate obstacles such as nucleosomes and DNA lesions. We predict that future studies will focus on how Pol II transcription is interconnected with chromatin transitions, RNA processing, and DNA repair.

scholarly journals Sir2 Silences Gene Transcription by Targeting the Transition between RNA Polymerase II Initiation and Elongation

2008 ◽  
Vol 28 (12) ◽  
pp. 3979-3994 ◽  
Author(s):  
Lu Gao ◽  
David S. Gross
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Transcriptional Silencing ◽  
Pol Ii ◽  
Initiation Factors ◽  
Lysine Deacetylase ◽  
Evolutionarily Conserved ◽  
Mrna Capping ◽  
Capping Enzyme

ABSTRACT It is well accepted that for transcriptional silencing in budding yeast, the evolutionarily conserved lysine deacetylase Sir2, in concert with its partner proteins Sir3 and Sir4, establishes a chromatin structure that prevents RNA polymerase II (Pol II) transcription. However, the mechanism of repression remains controversial. Here, we show that the recruitment of Pol II, as well as that of the general initiation factors TBP and TFIIH, occurs unimpeded to the silent HMR a 1 and HMLα1/HMLα2 mating promoters. This, together with the fact that Pol II is Ser5 phosphorylated, implies that SIR-mediated silencing is permissive to both preinitiation complex (PIC) assembly and transcription initiation. In contrast, the occupancy of factors critical to both mRNA capping and Pol II elongation, including Cet1, Abd1, Spt5, Paf1C, and TFIIS, is virtually abolished. In agreement with this, efficiency of silencing correlates not with a restriction in Pol II promoter occupancy but with a restriction in capping enzyme recruitment. These observations pinpoint the transition between polymerase initiation and elongation as the step targeted by Sir2 and indicate that transcriptional silencing is achieved through the differential accessibility of initiation and capping/elongation factors to chromatin. We compare Sir2-mediated transcriptional silencing to a second repression mechanism, mediated by Tup1. In contrast to Sir2, Tup1 prevents TBP, Pol II, and TFIIH recruitment to the HMLα1 promoter, thereby abrogating PIC formation.

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