A CSB-PAF1C axis restores processive transcription elongation after DNA damage repair

AbstractBulky DNA lesions in transcribed strands block RNA polymerase II (RNAPII) elongation and induce a genome-wide transcriptional arrest. The transcription-coupled repair (TCR) pathway efficiently removes transcription-blocking DNA lesions, but how transcription is restored in the genome following DNA repair remains unresolved. Here, we find that the TCR-specific CSB protein loads the PAF1 complex (PAF1C) onto RNAPII in promoter-proximal regions in response to DNA damage. Although dispensable for TCR-mediated repair, PAF1C is essential for transcription recovery after UV irradiation. We find that PAF1C promotes RNAPII pause release in promoter-proximal regions and subsequently acts as a processivity factor that stimulates transcription elongation throughout genes. Our findings expose the molecular basis for a non-canonical PAF1C-dependent pathway that restores transcription throughout the human genome after genotoxic stress.

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A CSB-PAF1C axis restores processive transcription elongation after DNA damage repair

10.1101/2020.01.04.894808 ◽

2020 ◽

Cited By ~ 1

Author(s):

Diana van den Heuvel ◽

Cornelia G. Spruijt ◽

Román González-Prieto ◽

Angela Kragten ◽

Michelle T. Paulsen ◽

...

Keyword(s):

Dna Damage ◽

Rna Polymerase Ii ◽

Molecular Basis ◽

Rna Synthesis ◽

Transcription Elongation ◽

Genotoxic Stress ◽

Dna Lesions ◽

Damage Repair ◽

Processivity Factor ◽

Dependent Pathway

AbstractThe coordinated transcription of genes involves the regulated release of RNA polymerase II (RNAPII) from promoter-proximal sites into active elongation. DNA lesions in transcribed strands block elongation and induce a strong transcriptional arrest. The transcription-coupled repair (TCR) pathway efficiently removes transcription-blocking DNA lesions, but this is not sufficient to resume transcription. Through proteomics screens, we find that the TCR-specific CSB protein loads the evolutionary conserved PAF1 complex (PAF1C) onto RNAPII in promoter-proximal regions in response to DNA damage. PAF1C is dispensable for TCR-mediated repair, but is essential for recovery of RNA synthesis after UV irradiation, suggesting an uncoupling between DNA repair and transcription recovery. Moreover, we find that PAF1C promotes RNAPII pause release in promoter-proximal regions and subsequently acts as a processivity factor that stimulates transcription elongation throughout genes. Our findings expose the molecular basis for a non-canonical PAF1C-dependent pathway that restores transcription throughout the human genome after genotoxic stress.

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DNA damage stabilizes interaction of CSB with the transcription elongation machinery

The Journal of Cell Biology ◽

10.1083/jcb.200401056 ◽

2004 ◽

Vol 166 (1) ◽

pp. 27-36 ◽

Cited By ~ 84

Author(s):

Vincent van den Boom ◽

Elisabetta Citterio ◽

Deborah Hoogstraten ◽

Angelika Zotter ◽

Jean-Marc Egly ◽

...

Keyword(s):

Molecular Weight ◽

Dna Damage ◽

Dna Repair ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

High Molecular Weight ◽

Transcription Elongation ◽

Cockayne Syndrome ◽

Dna Lesions ◽

High Molecular Weight Complex

The Cockayne syndrome B (CSB) protein is essential for transcription-coupled DNA repair (TCR), which is dependent on RNA polymerase II elongation. TCR is required to quickly remove the cytotoxic transcription-blocking DNA lesions. Functional GFP-tagged CSB, expressed at physiological levels, was homogeneously dispersed throughout the nucleoplasm in addition to bright nuclear foci and nucleolar accumulation. Photobleaching studies showed that GFP-CSB, as part of a high molecular weight complex, transiently interacts with the transcription machinery. Upon (DNA damage-induced) transcription arrest CSB binding these interactions are prolonged, most likely reflecting actual engagement of CSB in TCR. These findings are consistent with a model in which CSB monitors progression of transcription by regularly probing elongation complexes and becomes more tightly associated to these complexes when TCR is active.

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CDK12 globally stimulates RNA polymerase II transcription elongation and carboxyl-terminal domain phosphorylation

Nucleic Acids Research ◽

10.1093/nar/gkaa514 ◽

2020 ◽

Vol 48 (14) ◽

pp. 7712-7727

Author(s):

Michael Tellier ◽

Justyna Zaborowska ◽

Livia Caizzi ◽

Eusra Mohammad ◽

Taras Velychko ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcription Elongation ◽

Pol Ii ◽

Terminal Domain ◽

Carboxyl Terminal Domain ◽

Genome Wide ◽

A Genome ◽

Carboxyl Terminal ◽

Rna Polymerase Ii Transcription

Abstract Cyclin-dependent kinase 12 (CDK12) phosphorylates the carboxyl-terminal domain (CTD) of RNA polymerase II (pol II) but its roles in transcription beyond the expression of DNA damage response genes remain unclear. Here, we have used TT-seq and mNET-seq to monitor the direct effects of rapid CDK12 inhibition on transcription activity and CTD phosphorylation in human cells. CDK12 inhibition causes a genome-wide defect in transcription elongation and a global reduction of CTD Ser2 and Ser5 phosphorylation. The elongation defect is explained by the loss of the elongation factors LEO1 and CDC73, part of PAF1 complex, and SPT6 from the newly-elongating pol II. Our results indicate that CDK12 is a general activator of pol II transcription elongation and indicate that it targets both Ser2 and Ser5 residues of the pol II CTD.

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Elongation factor ELOF1 drives transcription-coupled repair and prevents genome instability

10.1101/2021.05.11.443558 ◽

2021 ◽

Author(s):

Marit Geijer ◽

Di Zhou ◽

Kathiresan Selvam ◽

Barbara Steurer ◽

Bastiaan Evers ◽

...

Keyword(s):

Dna Damage ◽

Rna Polymerase Ii ◽

Genome Stability ◽

Genome Instability ◽

Excision Repair ◽

Elongation Factor ◽

Transcription Inhibition ◽

Genome Wide ◽

A Genome ◽

Nucleotide Excision

Correct transcription is crucial for life. However, DNA damage severely impedes elongating RNA Polymerase II (Pol II), causing transcription inhibition and transcription-replication conflicts. Cells are equipped with intricate mechanisms to counteract the severe consequence of these transcription-blocking lesions (TBLs). However, the exact mechanism and factors involved remain largely unknown. Here, using a genome-wide CRISPR/cas9 screen, we identified elongation factor ELOF1 as an important new factor in the transcription stress response upon DNA damage. We show that ELOF1 has an evolutionary conserved role in Transcription-Coupled Nucleotide Excision Repair (TC-NER), where it promotes recruitment of the TC-NER factors UVSSA and TFIIH to efficiently repair TBLs and resume transcription. Additionally, ELOF1 modulates transcription to protect cells from transcription-mediated replication stress, thereby preserving genome stability. Thus, ELOF1 protects the transcription machinery from DNA damage by two distinct mechanisms.

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Genome wide mapping of DNA lesions by Repair Assisted Damage Detection sequencing – RADD-Seq

10.1101/2021.02.07.430183 ◽

2021 ◽

Author(s):

Noa Gilat ◽

Dena Fridman ◽

Hila Sharim ◽

Sapir Margalit ◽

Natalie R. Gassman ◽

...

Keyword(s):

Dna Damage ◽

Damage Detection ◽

Oxidative Dna Damage ◽

Dna Lesions ◽

Open Chromatin ◽

Dna Damage And Repair ◽

Genome Wide ◽

A Genome ◽

Wide Range ◽

Before And After

AbstractMapping DNA damage and its repair has immense potential in understanding environmental exposures, their genotoxicity, and their impact on human health. Monitoring changes in genomic stability also aids in the diagnosis of numerous DNA-related diseases, like cancer, and assists in monitoring their progression and prognosis. However, genome-wide maps of DNA damage distribution are challenging to produce. Here we describe the localization of DNA damage and repair loci by Repair Assisted Damage Detection sequencing – RADD-Seq. Based on the enrichment of damage lesions coupled with a pull-down assay and followed by next generation sequencing, this method is easy to perform and can produce compelling results with minimal coverage. RADD-seq enables the localization of both DNA damage and repair sites for a wide range of single-strand damage types. Using this technique, we created a genome-wide map of oxidative DNA damage loci before and after repair. Oxidative lesions were heterogeneously distributed along the human genome, with less damage occurring in tight chromatin regions. Furthermore, we showed repair is prioritized for highly expressed, essential genes and in open chromatin regions. RADD-seq sheds light on cellular repair mechanisms and capable of identifying genomic hotspots prone to mutation.

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Cockayne syndrome B protein acts as an ATP-dependent processivity factor that helps RNA polymerase II overcome nucleosome barriers

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2013379117 ◽

2020 ◽

Vol 117 (41) ◽

pp. 25486-25493 ◽

Cited By ~ 1

Author(s):

Jun Xu ◽

Wei Wang ◽

Liang Xu ◽

Jia-Yu Chen ◽

Jenny Chong ◽

...

Keyword(s):

Rna Polymerase Ii ◽

Neurological Diseases ◽

Transcription Elongation ◽

Cockayne Syndrome ◽

Stable Complex ◽

Loss Of Function ◽

Pol Ii ◽

Chromatin Remodelers ◽

Processivity Factor

While loss-of-function mutations in Cockayne syndrome group B protein (CSB) cause neurological diseases, this unique member of the SWI2/SNF2 family of chromatin remodelers has been broadly implicated in transcription elongation and transcription-coupled DNA damage repair, yet its mechanism remains largely elusive. Here, we use a reconstituted in vitro transcription system with purified polymerase II (Pol II) and Rad26, a yeast ortholog of CSB, to study the role of CSB in transcription elongation through nucleosome barriers. We show that CSB forms a stable complex with Pol II and acts as an ATP-dependent processivity factor that helps Pol II across a nucleosome barrier. This noncanonical mechanism is distinct from the canonical modes of chromatin remodelers that directly engage and remodel nucleosomes or transcription elongation factors that facilitate Pol II nucleosome bypass without hydrolyzing ATP. We propose a model where CSB facilitates gene expression by helping Pol II bypass chromatin obstacles while maintaining their structures.

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Targeting DNA Replication Stress and DNA Double-Strand Break Repair for Optimizing SCLC Treatment

Cancers ◽

10.3390/cancers11091289 ◽

2019 ◽

Vol 11 (9) ◽

pp. 1289 ◽

Cited By ~ 4

Author(s):

Xing Bian ◽

Wenchu Lin

Keyword(s):

Lung Cancer ◽

Dna Damage ◽

Dna Replication ◽

Dna Damage Repair ◽

Predictive Biomarkers ◽

Replication Stress ◽

Genotoxic Stress ◽

Repair System ◽

Damage Repair ◽

Repair Pathways

Small cell lung cancer (SCLC), accounting for about 15% of all cases of lung cancer worldwide, is the most lethal form of lung cancer. Despite an initially high response rate of SCLC to standard treatment, almost all patients are invariably relapsed within one year. Effective therapeutic strategies are urgently needed to improve clinical outcomes. Replication stress is a hallmark of SCLC due to several intrinsic factors. As a consequence, constitutive activation of the replication stress response (RSR) pathway and DNA damage repair system is involved in counteracting this genotoxic stress. Therefore, therapeutic targeting of such RSR and DNA damage repair pathways will be likely to kill SCLC cells preferentially and may be exploited in improving chemotherapeutic efficiency through interfering with DNA replication to exert their functions. Here, we summarize potentially valuable targets involved in the RSR and DNA damage repair pathways, rationales for targeting them in SCLC treatment and ongoing clinical trials, as well as possible predictive biomarkers for patient selection in the management of SCLC.

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Mutant huntingtin impairs PNKP and ATXN3, disrupting DNA repair and transcription

eLife ◽

10.7554/elife.42988 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 15

Author(s):

Rui Gao ◽

Anirban Chakraborty ◽

Charlene Geater ◽

Subrata Pradhan ◽

Kara L Gordon ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Rna Polymerase Ii ◽

Dna Damage Response ◽

Functional Decline ◽

Transcriptional Elongation ◽

Damage Repair ◽

Damage Response ◽

Cyclic Amp Response Element ◽

Atm Signaling

How huntingtin (HTT) triggers neurotoxicity in Huntington’s disease (HD) remains unclear. We report that HTT forms a transcription-coupled DNA repair (TCR) complex with RNA polymerase II subunit A (POLR2A), ataxin-3, the DNA repair enzyme polynucleotide-kinase-3'-phosphatase (PNKP), and cyclic AMP-response element-binding (CREB) protein (CBP). This complex senses and facilitates DNA damage repair during transcriptional elongation, but its functional integrity is impaired by mutant HTT. Abrogated PNKP activity results in persistent DNA break accumulation, preferentially in actively transcribed genes, and aberrant activation of DNA damage-response ataxia telangiectasia-mutated (ATM) signaling in HD transgenic mouse and cell models. A concomitant decrease in Ataxin-3 activity facilitates CBP ubiquitination and degradation, adversely impacting transcription and DNA repair. Increasing PNKP activity in mutant cells improves genome integrity and cell survival. These findings suggest a potential molecular mechanism of how mutant HTT activates DNA damage-response pro-degenerative pathways and impairs transcription, triggering neurotoxicity and functional decline in HD.

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High similarity among ChEC-seq datasets

10.1101/2021.02.04.429774 ◽

2021 ◽

Author(s):

Chitvan Mittal ◽

Matthew J. Rossi ◽

B. Franklin Pugh

Keyword(s):

Rna Polymerase Ii ◽

Transcriptional Regulators ◽

Micrococcal Nuclease ◽

High Similarity ◽

Dna Interactions ◽

Promoter Regions ◽

Genome Wide ◽

A Genome ◽

Protein Dna Interactions ◽

Negative Controls

AbstractChEC-seq is a method used to identify protein-DNA interactions across a genome. It involves fusing micrococcal nuclease (MNase) to a protein of interest. In principle, specific genome-wide interactions of the fusion protein with chromatin result in local DNA cleavages that can be mapped by DNA sequencing. ChEC-seq has been used to draw conclusions about broad gene-specificities of certain protein-DNA interactions. In particular, the transcriptional regulators SAGA, TFIID, and Mediator are reported to generally occupy the promoter/UAS of genes transcribed by RNA polymerase II in yeast. Here we compare published yeast ChEC-seq data performed with a variety of protein fusions across essentially all genes, and find high similarities with negative controls. We conclude that ChEC-seq patterning for SAGA, TFIID, and Mediator differ little from background at most promoter regions, and thus cannot be used to draw conclusions about broad gene specificity of these factors.

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The histone chaperone Spt6 is required for normal recruitment of the capping enzyme Abd1 to transcribed regions

10.1101/2021.05.13.444063 ◽

2021 ◽

Author(s):

Rajaraman Gopalakrishnan ◽

Fred Winston

Keyword(s):

Mass Spectrometry ◽

Rna Polymerase Ii ◽

Transcription Elongation ◽

Histone Chaperone ◽

Mrna Processing ◽

Wild Type ◽

Elongation Complex ◽

Protein Levels ◽

Genome Wide ◽

Capping Enzyme

The histone chaperone Spt6 is involved in promoting elongation of RNA polymerase II (RNAPII), maintaining chromatin structure, regulating co-transcriptional histone modifications, and controlling mRNA processing. These diverse functions of Spt6 are partly mediated through its interactions with RNAPII and other factors in the transcription elongation complex. In this study, we used mass spectrometry to characterize the differences in RNAPII interacting factors between wild-type cells and those depleted for Spt6, leading to the identification of proteins that depend on Spt6 for their interaction with RNAPII. The altered association of some of these factors could be attributed to changes in steady-state protein levels. However, Abd1, the mRNA cap methyltransferase, had decreased association with RNAPII after Spt6 depletion despite unchanged Abd1 protein levels, showing a requirement for Spt6 in mediating the Abd1-RNAPII interaction. Genome-wide studies showed that Spt6 is required for maintaining the level of Abd1 over transcribed regions, as well as the level of Spt5, another protein known to recruit Abd1 to chromatin. Abd1 levels were particularly decreased at the 5 ends of genes after Spt6 depletion, suggesting a greater need for Spt6 in Abd1 recruitment over these regions. Together, our results show that Spt6 is important in regulating the composition of the transcription elongation complex and reveal a previously unknown function for Spt6 in the recruitment of Abd1.

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