ELOF1 转录延伸因子

Somatic hypermutation (SHM) and class switch recombination (CSR) diversify immunoglobulin (Ig) genes and are initiated by the activation-induced deaminase (AID), a single-stranded DNA cytidine deaminase thought to engage its substrate during RNA polymerase II (RNAPII) transcription. Through a genetic screen, we identified numerous potential factors involved in SHM, including elongation factor 1 homolog (ELOF1), a component of the RNAPII elongation complex that functions in transcription-coupled nucleotide excision repair (TC-NER) and transcription elongation. Loss of ELOF1 compromises SHM, CSR, and AID action in mammalian B cells and alters RNAPII transcription by reducing RNAPII pausing downstream of transcription start sites and levels of serine 5 but not serine 2 phosphorylated RNAPII throughout transcribed genes. ELOF1 must bind to RNAPII to be a proximity partner for AID and to function in SHM and CSR, and TC-NER is not required for SHM. We propose that ELOF1 helps create the appropriate stalled RNAPII substrate on which AID acts.

Correct transcription is crucial for life. However, DNA damage severely impedes elongating RNA polymerase II, causing transcription inhibition and transcription-replication conflicts. Cells are equipped with intricate mechanisms to counteract the severe consequence of these transcription-blocking lesions. However, the exact mechanism and factors involved remain largely unknown. Here, using a genome-wide CRISPR–Cas9 screen, we identified the elongation factor ELOF1 as an important factor in the transcription stress response following DNA damage. We show that ELOF1 has an evolutionarily 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 transcription-blocking lesions and resume transcription. Additionally, ELOF1 modulates transcription to protect cells against transcription-mediated replication stress, thereby preserving genome stability. Thus, ELOF1 protects the transcription machinery from DNA damage via two distinct mechanisms. Two side-by-side papers report that the transcription elongation factor ELOF1 drives transcription-coupled repair and prevents replication stress.

In adaptive immunity, transcription-coupled damage (TCD) is introduced into antibody genes by activation-induced cytidine deaminase (AID) to diversify antibody repertoire. However, the coordination between transcription and DNA damage/repair remains elusive. Here, we find that transcription elongation factor 1 (ELOF1) stabilizes paused RNA polymerase II (RNAPII) at transcription barriers, providing a platform for transcription-coupled DNA damage/repair. Using a genetic screen, we discover that ELOF1 is required for AID targeting and that ELOF1 deficiency results in defective antibody class switch recombination and somatic hypermutation in mice. While downstream transcription-coupled repair factors are dispensable for AID damage, ELOF1 mechanistically facilitates both TCD and repair by stabilizing chromatin-bound RNAPII. In ELOF1-deficient cells, paused RNAPII tends to detach from chromatin and fails to recruit factors to induce or repair DNA damage. Our study places ELOF1 at the center of transcription-coupled DNA metabolism processes and suggests a transition of RNAPII from elongation to a DNA damage/repair scaffold.

Despite having been sequenced over a decade ago, the functional significance of much of the mammalian genome remains unknown. The mouse has become the preeminent mammalian model for identifying endogenous gene function in vivo. Here we characterize the phenotype of a loss-of function allele for the evolutionarily conserved transcription factor, Elongation Factor Homolog 1 (Elof1). Recent work utilizing the yeast homolog, Elf1, has demonstrated that Elf1 associates with the RNA polymerase II complex to promote elongation by relieving the association of the template DNA strand with bound histones. Loss of Elof1 results in developmental delay and morphological defects during early mouse development resulting in peri-gastrulation lethality. Although Elof1 is highly conserved we observe tissue specific expression during gastrulation and in adult murine tissues, suggesting there may be other genes with similar function in diverse tissues or that mElof1 has adopted lineage specific functions. To better understand its function in mammalian transcription, we examined splice variants and find that Elof1 regulates mutually exclusive exon use in vivo. Distinct from what has been demonstrated in yeast, we demonstrate that Elof1 is essential for viability during mammalian gastrulation which may be due to a role mediating tissue specific exclusive exon use, a regulatory function unique to higher eukaryotes.

The protein IWS1 (Interacts with SPT6 1) is implicated in transcription-associated processes, but a direct role in RNA polymerase (Pol) II function is unknown. Here, we use multi-omics kinetic analysis after rapid depletion of IWS1 in human cells to show that loss of IWS1 results in a global decrease of RNA synthesis and a global reduction in Pol II elongation velocity. We then resolve the cryo-EM structure of the activated Pol II elongation complex with bound IWS1 and elongation factor ELOF1 and show that IWS1 acts as a scaffold and positions downstream DNA within the cleft of Pol II. In vitro assays show that the disordered C-terminal region of IWS1 that contacts the cleft of Pol II is responsible for stimulation of Pol II activity and is aided by ELOF1. Finally, we find that the defect in transcription upon IWS1 depletion leads to a decrease of histone H3 tri-methylation at residue lysine-36 (H3K36me3), but that this secondary effect is an indirect function of IWS1. In summary, our structure-function analysis establishes IWS1 as a Pol II-associated elongation factor that acts globally to stimulate Pol II elongation velocity and ensure proper co-transcriptional histone methylation. Although IWS1 has been implicated in transcription-coupled processes, its direct role in RNA polymerase II function remained undefined. Here, the authors demonstrate that IWS1 enhances Pol II elongation velocity by acting as a structural scaffold and promoting co-transcriptional H3K36me3 deposition.

Transcription-coupled repair (TCR) is a vital nucleotide excision repair sub-pathway that removes DNA lesions from actively transcribed DNA strands. Binding of CSB to lesion-stalled RNA Polymerase II (Pol II) initiates TCR by triggering the recruitment of downstream repair factors. Yet it remains unknown how transcription factor IIH (TFIIH) is recruited to the intact TCR complex. Combining existing structural data with AlphaFold predictions, we build an integrative model of the initial TFIIH-bound TCR complex. We show how TFIIH can be first recruited in an open repair-inhibited conformation, which requires subsequent CAK module removal and conformational closure to process damaged DNA. In our model, CSB, CSA, UVSSA, elongation factor 1 (ELOF1), and specific Pol II and UVSSA-bound ubiquitin moieties come together to provide interaction interfaces needed for TFIIH recruitment. STK19 acts as a linchpin of the assembly, orienting the incoming TFIIH and bridging Pol II to core TCR factors and DNA. Molecular simulations of the TCR-associated CRL4CSA ubiquitin ligase complex unveil the interplay of segmental DDB1 flexibility, continuous Cullin4A flexibility, and the key role of ELOF1 for Pol II ubiquitination that enables TCR. Collectively, these findings elucidate the coordinated assembly of repair proteins in early TCR. Transcription-coupled repair (TCR) removes DNA lesions from actively transcribed strands. Here, the authors unveil the structural basis for coordinated TFIIH recruitment to the TCR machinery and the role of ubiquitin modifications in directing the response toward DNA repair or proteasomal degradation.

No abstract available

RNA polymerase II (RNAPII) is a central transcription enzyme that exists as multiple forms with or without accessory factors, and transcribes the genomic DNA packaged in chromatin. To understand how RNAPII functions in the human genome, we isolate transcribing RNAPII complexes from human nuclei by chromatin immunopurification, and determine the cryo-electron microscopy structures of RNAPII elongation complexes (ECs) associated with genomic DNA in distinct forms, without or with the elongation factors SPT4/5, ELOF1, and SPT6. This ChIP-cryoEM method also reveals the two EC-nucleosome complexes corresponding nucleosome disassembly/reassembly processes. In the structure of EC-downstream nucleosome, EC paused at superhelical location (SHL) −5 in the nucleosome, suggesting that SHL(−5) pausing occurs in a sequence-independent manner during nucleosome disassembly. In the structure of the EC-upstream nucleosome, EC directly contacts the nucleosome through the nucleosomal DNA-RPB4/7 stalk and the H2A-H2B dimer-RPB2 wall interactions, suggesting that EC may be paused during nucleosome reassembly. These representative EC structures transcribing the human genome provide mechanistic insights into understanding RNAPII transcription on chromatin. The authors establish the ChIP-CryoEM method, which combines chromatin immunopurification coupled with cryo-electron microscopy, to visualize native human RNAPII elongation complexes in action, with and without transcription elongation factors.

No abstract available

In eukaryotes, RNA polymerase (Pol) II transcribes chromatin and must move past nucleosomes, often resulting in nucleosome displacement. How Pol II unwraps the DNA from nucleosomes to allow transcription and how DNA rewraps to retain nucleosomes has been unclear. Here, we report the 3.0-angstrom cryo–electron microscopy structure of a mammalian Pol II-DSIF-SPT6-PAF1c-TFIIS-nucleosome complex stalled 54 base pairs within the nucleosome. The structure provides a mechanistic basis for nucleosome retention during transcription elongation where upstream DNA emerging from the Pol II cleft has rewrapped the proximal side of the nucleosome. The structure uncovers a direct role for Pol II and transcription elongation factors in nucleosome retention and explains how nucleosomes are retained to prevent the disruption of chromatin structure across actively transcribed genes. Description When Pol II meets nucleosome Eukaryotic cells organize their large genomes into a compacted structure called chromatin. The condensed structure of chromatin, with its fundamental unit the nucleosome, represents a challenge to nucleic acid–transacting machines including RNA polymerase II (Pol II), the enzyme responsible for the transcription of most protein-coding genes. How RNA Pol II overcomes nucleosomes without disrupting chromatin organization remains unknown. Using cryo–electron microscopy, Filipovski et al. provided structural snapshots of a complex between mammalian RNA Pol II and a nucleosome that show how previously transcribed DNA rewraps the nucleosome. The finding provides a structural basis of how nucleosomes, and consequently epigenetic marks, are retained during transcription. —DJ A high-resolution cryo–electron microscopy structure explains how nucleosomes are retained to prevent disruption of chromatin across actively transcribed genes.

Glioblastoma (GBM) is the most lethal primary brain cancer characterized by therapeutic resistance, which is promoted by GBM stem cells (GSCs). Here, we interrogated gene expression and whole genome CRISPR/Cas9 screening in a large panel of patient-derived GSCs, differentiated glioblastoma cells (DGCs), and neural stem cells (NSCs) to identify master regulators of GSC stemness, revealing an essential transcription state with increased RNA polymerase II-mediated transcription. The YY1 and transcriptional CDK9 complex was essential for GSC survival and maintenance in vitro and in vivo. YY1 interacted with CDK9 to regulate transcription elongation in GSCs. Genetic or pharmacological targeting of YY1-CDK9 complex elicited RNA m6A modification-dependent interferon responses, reduced regulatory T cell infiltration, and augmented efficacy of immune checkpoint therapy in glioblastoma. Collectively, these results suggest that YY1-CDK9 transcription elongation complex defines a targetable cell state with active transcription, suppressed interferon responses, and immunotherapy resistance in glioblastoma.

Understanding the complex network that regulates transcription elongation requires the quantitative analysis of RNA polymerase II (Pol II) activity in a wide variety of regulatory environments. We performed native elongating transcript sequencing (NET-seq) in 41 strains of Saccharomyces cerevisiae lacking known elongation regulators, including RNA processing factors, transcription elongation factors, chromatin modifiers, and remodelers. We found that the opposing effects of these factors balance transcription elongation and antisense transcription. Different sets of factors tightly regulate Pol II progression across gene bodies so that Pol II density peaks at key points of RNA processing. These regulators control where Pol II pauses with each obscuring large numbers of potential pause sites that are primarily determined by DNA sequence and shape. Antisense transcription varies highly across the regulatory landscapes analyzed, but antisense transcription in itself does not affect sense transcription at the same locus. Our findings collectively show that a diverse array of factors regulate transcription elongation by precisely balancing Pol II activity.

The MYC oncoprotein globally affects the function of RNA polymerase II (RNAPII). The ability of MYC to promote transcription elongation depends on its ubiquitylation. Here, we show that MYC and PAF1c (polymerase II-associated factor 1 complex) interact directly and mutually enhance each other's association with active promoters. PAF1c is rapidly transferred from MYC onto RNAPII. This transfer is driven by the HUWE1 ubiquitin ligase and is required for MYC-dependent transcription elongation. MYC and HUWE1 promote histone H2B ubiquitylation, which alters chromatin structure both for transcription elongation and double-strand break repair. Consistently, MYC suppresses double-strand break accumulation in active genes in a strictly PAF1c-dependent manner. Depletion of PAF1c causes transcription-dependent accumulation of double-strand breaks, despite widespread repair-associated DNA synthesis. Our data show that the transfer of PAF1c from MYC onto RNAPII efficiently couples transcription elongation with double-strand break repair to maintain the genomic integrity of MYC-driven tumor cells.

Significance Although plenty of studies have identified the RNA polymerase II (Pol II) pausing events in eukaryotes and demonstrated the regulatory roles of such events in gene expression and RNA processing, there was no study that systematically modeled the global landscape of Pol II pausing in the whole genome. Here, we have developed a deep learning framework that can accurately predict the Pol II pausing events from the contextual DNA sequences. Through applying our powerful computational approach to predict the pausing tendencies on interested regions in the human genome, we provided useful insights into understanding the relations between Pol II pausing events and alternative splicing, transcription factors, histone modifications, and DNA methylation. RNA polymerase II (Pol II) generally pauses at certain positions along gene bodies, thereby interrupting the transcription elongation process, which is often coupled with various important biological functions, such as precursor mRNA splicing and gene expression regulation. Characterizing the transcriptional elongation dynamics can thus help us understand many essential biological processes in eukaryotic cells. However, experimentally measuring Pol II elongation rates is generally time and resource consuming. We developed PEPMAN (polymerase II elongation pausing modeling through attention-based deep neural network), a deep learning-based model that accurately predicts Pol II pausing sites based on the native elongating transcript sequencing (NET-seq) data. Through fully taking advantage of the attention mechanism, PEPMAN is able to decipher important sequence features underlying Pol II pausing. More importantly, we demonstrated that the analyses of the PEPMAN-predicted results around various types of alternative splicing sites can provide useful clues into understanding the cotranscriptional splicing events. In addition, associating the PEPMAN prediction results with different epigenetic features can help reveal important factors related to the transcription elongation process. All these results demonstrated that PEPMAN can provide a useful and effective tool for modeling transcription elongation and understanding the related biological factors from available high-throughput sequencing data.

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.

Bulky 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. The transcription-coupled repair pathway removes transcription-blocking DNA lesions, but how transcription is restored following DNA repair is not clear. Here the authors reveal that the PAF1 complex, while dispensable for the repair process, restores transcription after DNA damage.

In addition to phosphodiester bond formation, RNA polymerase II has an RNA endonuclease activity, stimulated by TFIIS, which rescues complexes that have arrested and backtracked. How TFIIS affects transcription under normal conditions is poorly understood. We identified backtracking sites in human cells using a dominant-negative TFIIS (TFIISDN) that inhibits RNA cleavage and stabilizes backtracked complexes. Backtracking is most frequent within 2 kb of start sites, consistent with slow elongation early in transcription, and in 3' flanking regions where termination is enhanced by TFIISDN, suggesting that backtracked pol II is a favorable substrate for termination. Rescue from backtracking by RNA cleavage also promotes escape from 5' pause sites, prevents premature termination of long transcripts, and enhances activation of stress-inducible genes. TFIISDN slowed elongation rates genome-wide by half, suggesting that rescue of backtracked pol II by TFIIS is a major stimulus of elongation under normal conditions.

No abstract available

Abstract Transcription-coupled nucleotide excision repair (TC-NER or TCR) is initiated when the ATPase Cockayne syndrome protein B (CSB) recognizes a DNA lesion stalled RNA polymerase II (RNAPII) and forms a stable complex. Here, we report that poly(ADP-ribose) polymerase-1 (PARP1), that plays a key role in the lesion recognition step of global genomic NER, also facilitates the earliest step of TCR. PARP1, which is associated with RNAPII during normal transcription, interacts with and stabilizes CSB on the lesion-stalled RNAPII. CSB stimulates PARP1’s activity to form PAR, and in turn CSB is PARylated mainly at its N-terminal PAR-binding motif (PBM) to promote its stabilization with RNAPII, whereas its minor PARylation at the C-terminal domain suppresses its ATPase function, thus limiting the window of time for ATP-dependent lesion recognition by CSB. The loss of PARP1, treatment with inhibitors of PARP or poly(ADP-ribose) glycohydrolase (PARG) to prevent PAR synthesis or its catabolism to generate free PAR or engineering N-terminal PARylation-resistant CSB decrease the efficiency of cells for TCR. PARP1 mutant Caenorhabditis elegans larvae exhibit a pronounced TCR-deficient phenotype. Our findings uncover an evolutionarily conserved role of PARP1 and PAR metabolism in the initiation of TCR.

Transcription competes with other DNA-dependent processes, such as DNA repair, for access to its substrate, DNA. However, the principles governing the interplay between these processes remain poorly understood. Evidence suggests that the BRCA1-BARD1 complex, a key player in the DNA damage response, may act as a mediator of this crosstalk. In this study, we investigated the molecular mechanism underpinning the interaction between RNA polymerase II (RNAPII) and the BRCA1-BARD1 complex, as well as its functional implications. Our findings reveal that the BRCT repeat of BRCA1 binds the Ser5-phosphorylated CTD of RNAPII, utilising a mechanism previously established for other BRCT ligands. Furthermore, we demonstrate that this interaction is critical for the organisation of RNAPII into condensates with liquid-like properties. Analysis of disease-associated variants within the BRCT repeats further supports the biological relevance of this condensation. Collectively, our results suggest that the BRCA1-BARD1 complex may coordinate transcription and DNA repair by facilitating the organisation of RNAPII into transcription factories.

Significance The malfunction of CSB results in Cockayne syndrome (CS), a severe genetic degenerative disorder. CSB plays contrasting roles: It facilitates RNAPII to bypass minor obstacles or triggers TC-NER for RNAPII removal in the presence of bulky DNA damage. However, the mechanism by which CSB determines the fate of RNAPII remains unclear. Our identification of ARK2N and CK2 as crucial TC-NER facilitators, by enhancing the interaction between CSB and RNAPII, sheds light on this critical decision-making process. Notably, Ark2n−/− knockout mice exhibit increased sensitivity to UV damage and degenerative traits, suggesting its potential as a CS-associated gene. By highlighting the key role of ARK2N–CK2 in initiating TC-NER, our research bridges a longstanding gap in understanding this essential pathway.

The p53 tumour suppressor regulates the transcription initiation of selected genes by binding to specific DNA sequences at their promoters. Here we report a novel role of p53 in transcription elongation in human cells. Our data demonstrate that upon transcription elongation blockage, p53 is associated with genes that have not been reported as its direct targets. p53 could be co-immunoprecipitated with active forms of DNA-directed RNA polymerase II subunit 1 (RPB1), highlighting its association with the elongating RNA polymerase II. During a normal transcription cycle, p53 and RPB1 are localised at distinct regions of selected non-canonical p53 target genes and this pattern of localisation was changed upon blockage of transcription elongation. Additionally, transcription elongation blockage induced the proteasomal degradation of RPB1. Our results reveal a novel role of p53 in human cells during transcription elongation blockage that may facilitate the removal of RNA polymerase II from DNA.

During gene transcription, RNA polymerase II (RNAPII) traverses nucleosomes in chromatin, but the mechanism has remained elusive. Using cryo–electron microscopy, we obtained structures of the RNAPII elongation complex (EC) passing through a nucleosome in the presence of the transcription elongation factors Spt6, Spn1, Elf1, Spt4/5, and Paf1C and the histone chaperone FACT (facilitates chromatin transcription). The structures show snapshots of EC progression on DNA mediating downstream nucleosome disassembly, followed by its reassembly upstream of the EC, which is facilitated by FACT. FACT dynamically adapts to successively occurring subnucleosome intermediates, forming an interface with the EC. Spt6, Spt4/5, and Paf1C form a “cradle” at the EC DNA-exit site and support the upstream nucleosome reassembly. These structures explain the mechanism by which the EC traverses nucleosomes while maintaining the chromatin structure and epigenetic information. Description A passage through nucleosomes In the eukaryotic cell nucleus, genomic DNA is wrapped around histones to form nucleosomes, which are the basic units of the beads-on-a-string structure of chromatin. Although the nucleosomes are physical obstacles for the DNA-transcribing RNA polymerase II, it somehow passes through them while maintaining their structure. Ehara et al. obtained structural snapshots of the polymerase passaging through a nucleosome using cryo–electron microscopy. They found that the polymerase forms a huge elongation complex with multiple transcription elongation factors, which mediates the disassembly of the downstream nucleosome and its subsequent reassembly behind the polymerase with the aid of the histone chaperone FACT. —DJ Cryo-EM captures how the RNA polymerase II transcription elongation complex passes through a nucleosome with the aid of a histone chaperone. INTRODUCTION In the eukaryotic cell nucleus, genomic DNA is stored as chromatin, comprising multiple nucleosomes carrying various genetic and epigenetic information. Gene transcription by RNA polymerase II (RNAPII) intrinsically affects nucleosome structures because it requires temporary unfolding of the nucleosomes to read the DNA sequence. However, RNAPII transcribes genes while maintaining the nucleosome structures, suggesting the existence of a transcription-coupled mechanism to restore the nucleosomes. However, this mechanism has remained elusive. RATIONALE We designed nucleosomal DNA templates so that the RNAPII elongation complex (EC) would stall at the 42-, 49-, 58-, and 115-bp positions from the nucleosome entry. When EC stalls at these positions, its leading edge is near super helical locations (SHLs) –1, 0, +1, and +6, respectively, of the nucleosome. Transcription was conducted in the presence of the transcription elongation factors Spn1, Spt6, Spt4/5, Elf1, Paf1C, and TFIIS and the histone chaperone FACT. The ECs formed at these positions were analyzed by cryo–electron microscopy single-particle analysis. RESULTS We obtained six nucleosome-transcribing EC structures: EC42, EC49, EC49B, EC58hex, EC58oct, and EC115, where the numbers denote the DNA positions where EC stalled. The ECs contain the RNAPII-associated Spn1, Spt6, Spt4/5, Elf1, and Paf1C proteins, which constitute the EC downstream and/or upstream edge. The structures represent serial snapshots of the EC passage through the nucleosome. In EC42, the EC leading edge resides near SHL(–1) within the downstream nucleosome, in which an ~60-bp DNA segment is removed from the histone octamer surface. One of the H2A-H2B dimers is exposed and bound with the C-terminal tail of the Spt16 subunit of FACT. In EC49, the EC leading edge is just before the nucleosomal dyad [SHL(0)], and an ~70-bp DNA segment is removed from the histone octamer. As one of the H3-H4 dimers is exposed, the main body of FACT engages with the histones primarily through Spt16. An ~30-bp DNA segment is also removed from the distal end of the nucleosome, and thus only a third of the histone octamer surface is covered by DNA. This FACT-histone complex probably represents the state before its detachment from the DNA and its subsequent transfer. In EC49B, the nucleosome is shifted downstream by ~17 bp, which might have originated by the downstream transfer of the FACT-histone intermediate. By contrast, EC58hex and EC58oct, in which the EC leading edge has overrun the dyad of the original nucleosome, reveal a FACT-histone complex transferred upstream of the EC. In EC58hex, a FACT-histone hexamer [H2A-H2B-(H3-H4)2] complex is deposited onto the emerging DNA at the EC DNA exit site, and the resultant hexasome is wrapped by an ~40 bp DNA segment. In EC58oct, the remaining H2A-H2B dimer is deposited onto the histone hexamer to form the histone octamer (octasome), which is wrapped by an ~75-bp DNA segment. Finally, EC115 reveals a near-complete nucleosome on the EC upstream side, with the histone octamer covered by an ~120-bp DNA segment. At the rim of the EC DNA exit, the domains of Spt4, Spt5, Spt6, Leo1, and Rtf1 form a “cradle” that flexibly adapts to the subnucleosome intermediates in EC58hex, EC58oct, and EC115, supporting the sequential nucleosome reassembly process upstream of the EC. CONCLUSION The obtained structures visualize key steps of nucleosome traversal by the EC accompanied by the downstream nucleosome disassembly, followed by its reassembly upstream of the EC with the aid of FACT. When the EC passes through the nucleosomal dyad, the downstream-to-upstream transfer of the nucleosomal histones occurs. These views explain the mechanism by which the nucleosome structures are maintained during transcription. Transcription over a nucleosome mediated by the EC and FACT. Cryo–electron microscopy structures of the nucleosome-transcribing ECs: EC42, EC49, EC58hex, EC58oct, and EC115. EC49B is omitted in this figure. The EC contains the transcription elongation factors Spn1, Spt6, Spt4/5, Elf1, and Paf1C. These structures show snapshots of the EC progression on DNA mediating downstream nucleosome disassembly, followed by reassembly upstream of the EC with the aid of FACT.

Transcription-coupled DNA repair (TCR) removes bulky DNA lesions impeding RNA polymerase II (RNAPII) transcription. Recent studies have outlined the stepwise assembly of TCR factors CSB, CSA, UVSSA, and transcription factor IIH (TFIIH) around lesion-stalled RNAPII. However, the mechanism and factors required for the transition to downstream repair steps, including RNAPII removal to provide repair proteins access to the DNA lesion, remain unclear. Here, we identify STK19 as a TCR factor facilitating this transition. Loss of STK19 does not impact initial TCR complex assembly or RNAPII ubiquitylation but delays lesion-stalled RNAPII clearance, thereby interfering with the downstream repair reaction. Cryoelectron microscopy (cryo-EM) and mutational analysis reveal that STK19 associates with the TCR complex, positioning itself between RNAPII, UVSSA, and CSA. The structural insights and molecular modeling suggest that STK19 positions the ATPase subunits of TFIIH onto DNA in front of RNAPII. Together, these findings provide new insights into the factors and mechanisms required for TCR.

H2A.B is a distant histone H2A variant associated with actively transcribed regions of the genome, suggesting its positive role in promoting transcription. In the present study, we demonstrate that the RNA polymerase II elongation complex (EC) transcribes the nucleosome containing H2A.B more efficiently than that with canonical H2A in vitro. Our cryo-electron microscopy analysis of the H2A.B nucleosome during transcription revealed that the proximal H2A.B-H2B dimer is released from the nucleosome as the EC transcribes the proximal half of the nucleosomal DNA. This dissociation, which is not observed in the canonical H2A nucleosome, likely enhances the EC elongation efficiency in the H2A.B nucleosome. Mutational analyses suggested that the unique short C-terminal region of H2A.B alone enhances EC elongation efficiency when substituted for its counterpart in canonical H2A. Additionally, other regions of H2A.B contribute to this enhancement. These structural and biochemical findings provide new insights into the role of H2A.B in regulating gene expression. Histone variants are essential regulators of gene expression. This study demonstrates that the histone variant H2A.B enhances transcription efficiency in the nucleosome, with the proximal H2A.B-H2B dimer dissociating during transcription. RNA polymerase II elongation complex (EC) transcribes the H2A.B nucleosome more efficiently than the canonical H2A nucleosome in vitro. Cryo-electron microscopy analysis reveals that the proximal H2A.B-H2B dimer dissociates when EC transcribes the proximal half of the nucleosomal DNA. This dissociation is unique to H2A.B and is not observed in the canonical H2A nucleosome. Mutational analyses reveal H2A.B regions that enhance nucleosome transcription efficiency. RNA polymerase II elongation complex (EC) transcribes the H2A.B nucleosome more efficiently than the canonical H2A nucleosome in vitro. Cryo-electron microscopy analysis reveals that the proximal H2A.B-H2B dimer dissociates when EC transcribes the proximal half of the nucleosomal DNA. This dissociation is unique to H2A.B and is not observed in the canonical H2A nucleosome. Mutational analyses reveal H2A.B regions that enhance nucleosome transcription efficiency. H2A.B nucleosome are transcribed more efficiently by the RNA polymerase II elongation complex.

Fine-tuning DNA replication and transcription is crucial to prevent collisions between their machineries1. This is particularly important near promoters, where RNA polymerase II (RNAPII) initiates transcription and frequently arrests, forming R-loops2, 3–4. Arrested RNAPII can obstruct DNA replication, which often initiates near promoters5,6. The mechanisms that rescue arrested RNAPII during elongation to avoid conflicts with co-directional replisomes remain unclear. Here, using genome-wide approaches and genetic screens, we identify CFAP20 as part of a protective pathway that salvages arrested RNAPII in promoter-proximal regions, diverting it from the path of co-directional replisomes. CFAP20-deficient cells accumulate R-loops near promoters, which leads to defects in replication timing and dynamics. These defects stem from accelerated replication-fork speeds that cause a secondary reduction in origin activity. Co-depletion of the Mediator complex or removal of R-loop-engaged RNAPII restores normal replication. Our findings suggest that transcription-dependent fork stalling in cis induces accelerated fork progression in trans, generating single-stranded DNA gaps. We propose that CFAP20 facilitates RNAPII elongation under high levels of Mediator-driven transcription, thereby preventing replisome collisions. This study provides a transcription-centred view of transcription–replication encounters, revealing how locally arrested transcription complexes propagate genome-wide replication phenotypes and defining CFAP20 as a key factor that safeguards genome stability. CFAP20 has a key role in rescuing RNA polymerase II complexes that have arrested during DNA transcription, limiting the accumulation of R-loops and preventing collisions between the transcription and replication machinery.

DNA replication preferentially initiates close to active transcription start sites (TSSs) in the human genome. Transcription proceeds discontinuously with an accumulation of RNA polymerase II (RNAPII) in a paused state near the TSS. Consequently, replication forks inevitably encounter paused RNAPII soon after replication initiates. Hence, dedicated machinery may be needed to remove RNAPII and facilitate unperturbed fork progression. In this study, we discovered that Integrator, a transcription termination machinery involved in the processing of RNAPII transcripts, interacts with the replicative helicase at active forks and promotes the removal of RNAPII from the path of the replication fork. Integrator-deficient cells have impaired replication fork progression and accumulate hallmarks of genome instability including chromosome breaks and micronuclei. The Integrator complex resolves co-directional transcription-replication conflicts to facilitate faithful DNA replication.

Poli et al. present genetic and proteomic analyses from budding yeast that uncover links between the DNA replication checkpoint sensor Mec1–Ddc2 (ATR–ATRIP), the chromatin remodeling complex INO80C, and the transcription complex PAF1C. A subset of chromatin-bound RNAPII is degraded in a manner dependent on Mec1, INO80, and PAF1 complexes in cells exposed to hydroxyurea.

Abstract Transcripts produced by RNA polymerase II (RNAPII) are fundamental for cellular responses to environmental changes. It is therefore no surprise that there exist multiple avenues for the regulation of this process. To explore the regulation mediated by RNAPII-interacting proteins, we used a small interfering RNA (siRNA)-based screen to systematically evaluate their influence on RNA synthesis. We identified several proteins that strongly affected RNAPII activity. We evaluated one of the top hits, SCAF1 (SR-related C-terminal domain-associated factor 1), using an auxin-inducible degradation system and sequencing approaches. In agreement with our screen results, acute depletion of SCAF1 decreased RNA synthesis, and showed an increase of Serine-2 phosphorylated-RNAPII (pS2-RNAPII). We found that the accumulation of pS2-RNAPII within the gene body occurred at GC-rich regions and was indicative of stalled RNAPII complexes. The accumulation of stalled RNAPII complexes was accompanied by reduced recruitment of initiating RNAPII, explaining the observed global decrease in transcriptional output. Furthermore, upon SCAF1 depletion, RNAPII complexes showed increased association with components of the proteasomal-degradation machinery. We concluded that in cells lacking SCAF1, RNAPII undergoes a rather interrupted passage, resulting in intervention by the proteasomal-degradation machinery to clear stalled RNAPII. While cells survive the compromised transcription caused by absence of SCAF1, further inhibition of proteasomal-degradation machinery is synthetically lethal.

Ewing sarcoma (EwS) is an aggressive pediatric bone cancer with limited treatment options, highlighting a drastic need for novel therapeutics. EwS is driven by translocation of the EWSR1 low complexity domain and most often the DNA binding domain of FLI1, making up the oncogenic fusion protein EWS::FLI1. Accumulating evidence suggests that EWS::FLI1 alters the biophysical properties of EWS and EWS::FLI1 phase separated condensates. We have previously shown that EWS::FLI1 prevents the release of the double strand break (DSB) repair protein BRCA1 from RNA Polymerase II (RNAPII) complexes, inhibiting BRCA1 mediated DSB repair. However, how EWS::FLI1 alters protein dynamics at the RNAPII complexes is not understood. In this work we show that EWS::FLI1 co-localization to RNAPII C-Terminal Domain (CTD) protein hubs causes enhanced phase separation, imitating a hardening of RNAPII CTD condensates. Additionally, we find that treatment with DNA damaging agents decreases BRCA1 occupancy at RNAPII CTD protein hubs. Expression of EWS::FLI1 in this in cellulo system suggests inhibition of BRCA1 release from RNAPII phase separated entities upon DNA damage. Overall, this data indicates that EWS::FLI1 mediated changes in phase state alters heterotypic protein-protein interactions at the RNAPII complexes. Furthermore, we find that RNAPII CTD phase separated entities are refractory to THZ1 drug treatment. This underscores the physiological significance condensate biology plays in drug efficacy and drug resistance. In conclusion, our results prompt further investigation into EWS::FLI1 affects on protein-protein interactions at RNAPII condensates. Our findings are novel as we look at heterotypic phase condensate biology in cellulo, whereas most phase studies are conducted in vitro and focus on homotypic interactions. Moreover, though much work was conducted on BRCA1 and RNAPII interaction and damage-induced release in the 1990s and early 2000s, little is understood about the relationship of BRCA1 and RNAPII and certainly not in the context of EWS::FLI1. Importantly, this work provides a basis for the physiological role of condensate biology in EWS::FLI1 mediated BRCA1 and RNAPII protein-protein dynamics. Citation Format: Aiola Stoja, Xiaoping Xu, David S Libich, Alexander J. R. Bishop. EWS::FLI1 alters DNA damage induced protein dynamics at the RNAPII CTD [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pediatric Cancer Research; 2024 Sep 5-8; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl):Abstract nr B081.

Transcription-coupled nucleotide excision repair (TC-NER) is initiated by the stalling of elongating RNA polymerase II (RNAPIIo) at DNA lesions. The ubiquitination of RNAPIIo in response to DNA damage is an evolutionarily conserved event, but its function in mammals is unknown. Here, we identified a single DNA damage-induced ubiquitination site in RNAPII at RPB1-K1268, which regulates transcription recovery and DNA damage resistance. Mechanistically, RPB1-K1268 ubiquitination stimulates the association of the core-TFIIH complex with stalled RNAPIIo through a transfer mechanism that also involves UVSSA-K414 ubiquitination. We developed a strand-specific ChIP-seq method, which revealed RPB1-K1268 ubiquitination is important for repair and the resolution of transcriptional bottlenecks at DNA lesions. Finally, RPB1-K1268R knockin mice displayed a short life-span, premature aging, and neurodegeneration. Our results reveal RNAPII ubiquitination provides a two-tier protection mechanism by activating TC-NER and, in parallel, the processing of DNA damage-stalled RNAPIIo, which together prevent prolonged transcription arrest and protect against neurodegeneration.

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Cockayne syndrome group B protein (CSB), a member of the SWI/SNF superfamily, resides in an elongating RNA polymerase II (RNAPII) complex and regulates transcription elongation. CSB contains a C-terminal winged helix domain (WHD) that binds to ubiquitin and plays an important role in DNA repair. However, little is known about the role of the CSB-WHD in transcription regulation. Here, we report that CSB is dependent upon its WHD to regulate RNAPII abundance at promoter proximal pause (PPP) sites of several actively transcribed genes, a key step in the regulation of transcription elongation. We show that two ubiquitin binding-defective mutations in the CSB-WHD, which impair CSB’s ability to promote cell survival in response to treatment with cisplatin, have little impact on its ability to stimulate RNAPII occupancy at PPP sites. In addition, we demonstrate that two cancer-associated CSB mutations, which are located on the opposite side of the CSB-WHD away from its ubiquitin-binding pocket, impair CSB’s ability to promote RNAPII occupancy at PPP sites. Taken together, these results suggest that CSB promotes RNAPII association with PPP sites in a manner requiring the CSB-WHD but independent of its ubiquitin-binding activity. These results further imply that CSB-mediated RNAPII occupancy at PPP sites is mechanistically separable from CSB-mediated repair of cisplatin-induced DNA damage.

In this study, Dehmer et al. show that USP11 complexes with TCEAL1 to competitively antagonize the interaction of TFIIS with RNAPII and chromatin, thereby buffering TFIIS's function in RNAPII backtracking and maintaining expression of the nuclear RNA polymerase subunit RPB8. In this manner, USP11–TCEAL not only promotes transcription elongation but also supports an oncogenic transcriptional program in neuroblastoma.

Integrator is a multi-subunit protein complex responsible for premature transcription termination of coding and non-coding RNAs in Metazoans. This is achieved via Integrator’s two enzymatic activities, RNA endonuclease and protein phosphatase, acting on the promoter-proximally paused RNA Polymerase II (RNAPII). Yet, it remains unclear how Integrator assembly and recruitment are regulated and what are the functions of many of its core subunits. Here we report two cryo-EM reconstructions of large Integrator sub-complexes: INTS10/13/14/15 (Arm module) and INTS5/8/10/15, which allowed integrative modelling of the fully-assembled Integrator bound to the RNAPII paused elongating complex (PEC). INTS13/14 are positioned near the DNA upstream from the transcription pause site, suggesting a potential role in the chromatin context. An in silico protein interaction screen of over 1500 transcription factors (TFs), identified Zinc Finger Protein 655 (ZNF655) as a direct interacting partner of INTS13 that associates with a fully assembled, 17-subunit Integrator complex. We propose a model wherein the Arm module acts as a platform for the recruitment of TFs that could modulate the stability of the Integrator’s association at specific loci and modulate transcription attenuation of the target genes.

The Integrator complex plays essential roles in RNA polymerase II (RNAPII) transcription termination and RNA processing. Here, we identify INTS6, a subunit of the Integrator complex, as a novel gene associated with neurodevelopmental disorders (NDDs). Through analysis of large NDD cohorts and international collaborations, we identified 23 families harboring monoallelic likely gene-disruptive or de novo missense variants in INTS6. Phenotypic characterization revealed shared features, including language and motor delays, autism, intellectual disability, and sleep disturbances. Using a nervous-system conditional KO (cKO) mouse model, we show that Ints6 deficiency disrupts early neurogenesis, cortical lamination, and synaptic development. Ints6 cKO mice had a thickened ventricular zone/subventricular zone, thinning of the cortical plate, reduced neuronal differentiation, and increased apoptosis in cortical layer 6. Behavioral assessments of heterozygous mice revealed deficits in social novelty preference, spatial memory, and hyperactivity, mirroring phenotypes observed in individuals with INTS6 variants. Molecular analyses further revealed that INTS6 deficiency alters RNAPII dynamics, disrupts transcriptional regulation, and impairs synaptic gene expression. Treatment with a CDK9 inhibitor (CDK9i) reduced RNAPII phosphorylation, thereby limiting its binding to target genes. Notably, CDK9i reversed neurosphere overproliferation and rescued the abnormal dendritic spine phenotype caused by Ints6 deficiency. This work advances understanding of INTS-related NDD pathogenesis and highlights potential therapeutic targets for intervention.

Abstract Eradicating HIV-1 is complicated by latently infected CD4+T cells harboring dormant proviruses capable of reactivation. Through a pooled shRNAmir screen targeting human chromatin regulators, we identified EP400, a member of the p400 chromatin remodeling complex, as a potent inhibitor of HIV-1 transcription in Jurkat and primary CD4+T cells. EP400 and its complex partner DMAP1 co-localize with paused RNA Polymerase II (RNAPII) at transcriptional start sites of protein-coding genes and their depletion modestly reduced RNAPII pausing. At the HIV-1 locus, EP400 and DMAP1 were co-recruited with RNAPII across the entire HIV-1 genome, and their depletion markedly increases RNAPII pause release. Together this suggests that EP400 may play a role in limiting HIV-1 transcriptional elongation. Additionally, EP400 depletion increased expression of key T-cell factors known to activate HIV-1 transcription. Therefore, the p400 complex reduces efficient HIV-1 transcriptional elongation and contributes to a CD4+T cell state unfavorable for HIV-1 transcription.

The mechanisms that control the dynamic composition of RNAPII elongation complexes govern major transitions in the transcription cycle yet are poorly understood. Here, we show that the transcription elongation factor Spt5 determines elongation complex composition to promote productive elongation and the transition to termination. Using an unbiased genetic screen and genomic approaches in Saccharomyces cerevisiae, we provide evidence that dephosphorylation of the Spt5 C-terminal repeat domain (CTR) by Glc7/PP1 is required to dislodge the Paf1 complex (Paf1C) from RNAPII near the cleavage and polyadenylation site (CPS). Mutations in Paf1C or the Spt5 CTR that dissociate Paf1C from RNAPII bypass the requirement for two critical regulators of Glc7 in the cleavage and polyadenylation factor that promote Glc7 enrichment at the 3’ ends of genes. Depletion of Glc7 causes aberrant retention of Paf1C past the CPS and a dramatic increase in readthrough transcription, which is fully suppressed by Paf1C mutations. Our results demonstrate that Paf1C retention antagonizes transcription termination and that Glc7-mediated restructuring of the RNAPII elongation complex to evict Paf1C at the CPS is a critical step in the transition from elongation to termination.

Abstract The chromatin landscape surrounding integrated HIV proviruses critically shapes viral transcription. We systematically examined ATP-dependent chromatin remodeling complexes (SWI/SNF, ISWI, CHD, and INO80) as regulators of HIV expression and identified the p400 complex, a member of the INO80 family, as a potent repressor. Depleting p400 subunits, including the EP400 ATPase and DMAP1, markedly increased HIV transcription and RNAPII elongation at the proviral locus. Mechanistically, EP400 associates with the RNAPII C-terminal domain, while DMAP1 directly engages the viral transactivator Tat, with repression requiring simultaneous interactions among EP400, DMAP1, and Tat. Loss of either EP400 or DMAP1 selectively increased infection and transcription of Tat-competent, but not Tat-deficient, viruses. Although p400 is recruited to active HIV chromatin via RNAPII in a Tat-independent manner, it restrains elongation once Tat accumulates during reactivation. DMAP1 binding to Tat’s basic domain blocks Tat-TAR RNA interaction, thereby limiting p-TEFb-mediated RNAPII Ser2 phosphorylation and elongation. Thus, the p400 complex functions as a host restriction factor that limits Tat-dependent HIV transcription via a Tat-dependent proximal mechanism, highlighting the p400-Tat interface as a potential target for HIV cure strategies.

NRDE2 is a highly conserved protein that is implicated in post-transcriptional gene silencing in S. pombe and C. elegans while in mammals, it has been shown to modulate splicing. To determine whether NRDE2 may be implicated in other processes in humans, we performed tandem affinity purification followed by proteomic analysis of NRDE2 from nuclear extracts in HEK293T and HeLa cells. Our data show that, in addition to its well-characterized partner, MTR4 helicase (MTREX), as well as several splicing factors, NRDE2 also interacts with chromatin-associated factors involved in transcription, including the Polymerase-Associated Factor 1 (PAF1) complex and elongating forms of RNA polymerase II (RNAPII). To further investigate the function of NRDE2 in gene expression, we performed RNA-seq following its transient depletion. Differential expression analysis showed that loss of NRDE2 modulated the expression of thousands of genes. While effects on splicing, including intron retention, were detected, as described previously, our analysis revealed that the impact of NRDE2 on intron retention is more widespread than previously thought. Moreover, intron retention was also highly associated with down-regulation of mRNA expression. Taken together, these results suggest that NRDE2 associates with the transcription and splicing machineries and affects RNA processing.

Zinc ribbons, one of the largest fold groups among zinc fingers, often include proteins involved in the transcription machinery. Here, we identify and characterize one such zinc ribbon‐bearing protein in the apicomplexan parasite Toxoplasma gondii, annotated as putative transcription elongation factor 1 (ELF1), with predicted functions in transcription and chromatin maintenance. We show that this ELF1 homolog, referred to as T. gondii ELF1‐like divergent (TgELD), is expressed in both tachyzoite and bradyzoite developmental stages. TgELD associates with the cytoskeleton in the tachyzoites, while it transiently becomes a part of the cyst wall in the early bradyzoites, followed by a cytosolic and peripheral localization in late bradyzoites. TgELD is phosphorylated by a casein kinase 2‐like protein, which has potential implications for its localization and function in the parasite.

SUMMARY The polymerase-associated factor 1 (Paf1) complex (Paf1C) is a conserved protein complex with critical functions during eukaryotic transcription. Previous studies showed that Paf1C is multi-functional, controlling specific aspects of transcription ranging from RNA polymerase II (RNAPII) processivity to histone modifications. However, it is unclear how specific Paf1C subunits directly impact transcription and coupled processes. We have compared conditional depletion to steady-state deletion for each Paf1C subunit to determine the direct and indirect contributions to gene expression in Saccharomyces cerevisiae. Using nascent transcript sequencing, RNAPII profiling, and modeling of transcription elongation dynamics, we have demonstrated direct effects of Paf1C subunits on RNAPII processivity and elongation rate and indirect effects on transcript splicing and repression of antisense transcripts. Further, our results suggest that the direct transcriptional effects of Paf1C cannot be readily assigned to any particular histone modification. This work comprehensively analyzes both the immediate and the extended roles of each Paf1C subunit in transcription elongation and transcript regulation.

In transcription-coupled repair, stalled RNA polymerase II (Pol II) is recognized by CSB and CRL4CSA, which co-operate with UVSSSA and ELOF1 to recruit TFIIH for nucleotide excision repair (TC-NER). To explore the mechanism of TC-NER, we recapitulated this reaction in vitro. When a plasmid containing a site-specific lesion is transcribed in frog egg extract, error-free repair is observed that depends on CSB, CRL4CSA, UVSSA, and ELOF1. Repair also depends on STK19, a factor previously implicated in transcription recovery after UV exposure. A 1.9 Å cryo-electron microscopy structure shows that STK19 joins the TC-NER complex by binding CSA and the RPB1 subunit of Pol II. Furthermore, AlphaFold predicts that STK19 interacts with the XPD subunit of TFIIH, and disrupting this interface impairs cell-free repair. Molecular modeling suggests that STK19 positions TFIIH ahead of Pol II for lesion verification. In summary, our analysis of cell-free TC-NER suggests that STK19 couples RNA polymerase II stalling to downstream repair events.

During transcription-coupled DNA repair (TCR), RNA polymerase II (Pol II) transitions from a transcriptionally active state to an arrested state that allows for removal of DNA lesions. This transition requires site-specific ubiquitylation of Pol II by the CRL4CSA ubiquitin ligase, a process that is facilitated by ELOF1 in an unknown way. Using cryogenic electron microscopy, biochemical assays and cell biology approaches, we found that ELOF1 serves as an adaptor to stably position UVSSA and CRL4CSA on arrested Pol II, leading to ligase neddylation and activation of Pol II ubiquitylation. In the presence of ELOF1, a transcription factor IIS (TFIIS)-like element in UVSSA gets ordered and extends through the Pol II pore, thus preventing reactivation of Pol II by TFIIS. Our results provide the structural basis for Pol II ubiquitylation and inactivation in TCR. Here the authors visualize the workings of ELOF1 in transcription-coupled DNA repair, showing that ELOF1 repositions repair factors on the surface of DNA damage-stalled RNA polymerase II to facilitate its ubiquitylation by the CRL4CSA E3 ligase and inactivation by UVSSA.

Transcription-coupled nucleotide excision repair (TC-NER) is a highly conserved DNA repair pathway that removes bulky lesions in the transcribed genome. Cockayne syndrome B protein (CSB), or its yeast ortholog Rad26, has been known for decades to play important roles in the lesion-recognition steps of TC-NER. Another conserved protein ELOF1, or its yeast ortholog Elf1, was recently identified as a core transcription-coupled repair factor. How Rad26 distinguishes between RNA polymerase II (Pol II) stalled at a DNA lesion or other obstacles and what role Elf1 plays in this process remains unknown. Here, we present cryo-EM structures of Pol II-Rad26 complexes stalled at different obstacles that show that Rad26 uses a universal mechanism to recognize a stalled Pol II but interacts more strongly with a lesion-arrested Pol II. A cryo-EM structure of lesion-arrested Pol II-Rad26 bound to Elf1 revealed that Elf1 induces new interactions between Rad26 and Pol II when the complex is stalled at a lesion. Biochemical and genetic data support the importance of the interplay between Elf1 and Rad26 in TC-NER initiation.

Stalling of elongating RNA polymerase II (RNAPII) at DNA lesions blocks transcription and triggers transcription-coupled repair (TCR). However, the mechanisms determining the fate of stalled RNAPII remain incompletely understood. Here, we develop a time-resolved assay to track RNAPII clearance and degradation at UV-induced lesions. We show that RNAPII ubiquitylation by CSB and the CRL4CSA ubiquitin ligase is essential, as loss of these proteins causes persistent RNAPII accumulation at damage sites. Downstream of CSB/CRL4CSA-mediated ubiquitylation, two distinct pathways mediate RNAPII removal. The primary rapid route relies on TFIIH, with its XPD helicase activity driving RNAPII dissociation after proper recruitment and positioning by ELOF1, UVSSA, and STK19. A secondary slow pathway is mediated by the ubiquitin-dependent segregase VCP, which compensates for impaired TFIIH function. While VCP contributes only minimally in TCR-proficient cells, inhibition of VCP in TFIIH-deficient contexts completely abrogates RNAPII clearance. Together, these findings establish a hierarchical program in which CSB/CRL4CSA-mediated ubiquitylation initiates RNAPII processing, TFIIH/XPD helicase activity provides the main clearance mechanism, and VCP-dependent extraction acts as a backup when TFIIH fails. This mechanistic framework explains how cells resolve DNA lesion-stalled RNAPII during normal and compromised TCR. DNA damage stalls RNA polymerase II and halts gene expression. Here, the authors reveal how cells clear stalled polymerase by two hierarchical pathways, in which CSB/CRL4CSA -triggered ubiquitylation enables rapid TFIIH-driven removal, and with VCP-mediated extraction acting as a backup when this fails.

A study in Molecular Cell by Ramadhin et al.1 and two studies in Cell by van den Heuvel et al.2 and by Mevissen et al.3 show that STK19 is a key protein in transcription-coupled nucleotide excision repair (TC-NER) in mammalian cells by mediating the initial repair protein complex formation and subsequent TFIIH recruitment, thereby enabling the vital damage verification step.

Global Genomic Repair (GGR) and Transcription-Coupled Repair (TCR) have been viewed, respectively, as major and minor sub-pathways of the nucleotide excision repair (NER) process that removes bulky lesions from the genome. Here we applied a next generation sequencing assay, CPD-seq, in E. coli to measure the levels of cyclobutane pyrimidine dimer (CPD) lesions before, during, and after UV-induced genotoxic stress, and, therefore, to determine the rate of genomic recovery by NER at a single nucleotide resolution. We find that active transcription is necessary for the repair of not only the template strand (TS), but also the non-template strand (NTS), and that the bulk of TCR is independent of Mfd – a DNA translocase that is thought to be necessary and sufficient for TCR in bacteria. We further show that repair of both TS and NTS is enhanced by increased readthrough past Rho-dependent terminators. We demonstrate that UV-induced genotoxic stress promotes global antitermination so that TCR is more accessible to the antisense, intergenic, and other low transcribed regions. Overall, our data suggest that GGR and TCR are essentially the same process required for complete repair of the bacterial genome. Transcription-Coupled DNA repair has been classically defined as the preferential repair of the template strand (TS) over the non-template strand (NTS). Here the authors challenge this classic model of TCR by using a genome-wide repair assay, CPD-seq, as well as RNA-seq, to show that TCR occurs across the entire E. coli genome – including NTS and intergenic regions.

Transcription-coupled repair (TCR) is a dedicated pathway for the preferential repair of bulky transcription-blocking DNA lesions. These lesions stall the elongating RNA-polymerase II (RNAPII) triggering the recruitment of TCR proteins at the damaged site. UV-stimulated scaffold protein A (UVSSA) is a recently identified cofactor which is involved in stabilization of the TCR complex, recruitment of DNA-repair machinery and removal/restoration of RNAPII from the lesion site. Mutations in UVSSA render the cells TCR-deficient and have been linked to UV-sensitive syndrome. Human UVSSA is a 709-residue long protein with two short conserved domains; an N-terminal (residues 1-150) and a C-terminal (residues 495-605) domain, while the rest of the protein is predicted to be intrinsically disordered. The protein is well conserved in eukaryotes, however; none of its homologs have been characterized yet. Here, we have purified the recombinant human UVSSA and have characterized it using bioinformatics, biophysical and biochemical techniques. Using EMSA, SPR and fluorescence-based methods, we have shown that human UVSSA interacts with DNA and RNA. Furthermore, we have mapped the nucleic acid binding regions using several recombinant protein fragments containing either the N-terminal or the C-terminal domains. Our data indicate that UVSSA possesses at least two nucleic acid binding regions; the N-terminal domain and a C-terminal tail region (residues 606-662). These regions, far apart in sequence space, are predicted to be in close proximity in structure-space suggesting a coherent interaction with target DNA/RNA. The study may provide functional clues about the novel family of UVSSA proteins.

Eukaryotic transcription-coupled repair (TCR) is an important and well-conserved sub-pathway of nucleotide excision repair that preferentially removes DNA lesions from the template strand that block translocation of RNA polymerase II (Pol II). Cockayne syndrome group B (CSB, also known as ERCC6) protein in humans (or its yeast orthologues, Rad26 in Saccharomyces cerevisiae and Rhp26 in Schizosaccharomyces pombe) is among the first proteins to be recruited to the lesion-arrested Pol II during the initiation of eukaryotic TCR. Mutations in CSB are associated with the autosomal-recessive neurological disorder Cockayne syndrome, which is characterized by progeriod features, growth failure and photosensitivity. The molecular mechanism of eukaryotic TCR initiation remains unclear, with several long-standing unanswered questions. How cells distinguish DNA lesion-arrested Pol II from other forms of arrested Pol II, the role of CSB in TCR initiation, and how CSB interacts with the arrested Pol II complex are all unknown. The lack of structures of CSB or the Pol II–CSB complex has hindered our ability to address these questions. Here we report the structure of the S. cerevisiae Pol II–Rad26 complex solved by cryo-electron microscopy. The structure reveals that Rad26 binds to the DNA upstream of Pol II, where it markedly alters its path. Our structural and functional data suggest that the conserved Swi2/Snf2-family core ATPase domain promotes the forward movement of Pol II, and elucidate key roles for Rad26 in both TCR and transcription elongation.

Persisters constitute a population of temporarily antibiotic-tolerant variants in an isogenic bacterial population and are considered an important cause of relapsing infections. It is currently unclear how cellular damage inflicted by antibiotic action is reversed upon persister state exit and how this relates to antibiotic resistance development. We demonstrate that persisters, upon fluoroquinolone treatment, accumulate oxidative damage which is repaired through nucleotide excision repair. Detection of the damage occurs via transcription-coupled repair using UvrD-mediated backtracking or Mfd-mediated displacement of the RNA polymerase. This competition results in heterogeneity in persister awakening lags. Most persisters repair the oxidative DNA damage, displaying a mutation rate equal to the untreated population. However, the promutagenic factor Mfd increases the mutation rate in a persister subpopulation. Our data provide in-depth insight in the molecular mechanisms underlying persister survival and pinpoints Mfd as an important molecular factor linking persistence to resistance development.

Significance Transcription-coupled repair (TCR) involves four core proteins: CSA, CSB, USP7, and UVSSA. CSA and CSB are mutated in the severe human neurocutaneous disease Cockayne syndrome. In contrast UVSSA is a mild photosensitive disease in which a mutated protein sequence prevents recruitment of USP7 protease to deubiquitinate and stabilize CSB. We deleted the UVSSA protein using CRISPR-Cas9 in an aneuploid cell line, HEK293, and determined the functional consequences. The knockout cell line was sensitive to transcription-blocking lesions but not sensitive to oxidative agents or PARP inhibitors, unlike CSB. Knockout of UVSSA also activated ATM, like CSB, in transcription-arrested cells. The phenotype of UVSSA, especially its rarity, suggests that many TCR-deficient patients and tumors fail to be recognized clinically.

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Abstract Transcription-coupled nucleotide excision repair factor Cockayne syndrome protein B (CSB) was suggested to function in the repair of oxidative DNA damage. However thus far, no clear role for CSB in base excision repair (BER), the dedicated pathway to remove abundant oxidative DNA damage, could be established. Using live cell imaging with a laser-assisted procedure to locally induce 8-oxo-7,8-dihydroguanine (8-oxoG) lesions, we previously showed that CSB is recruited to these lesions in a transcription-dependent but NER-independent fashion. Here we showed that recruitment of the preferred 8-oxoG-glycosylase 1 (OGG1) is independent of CSB or active transcription. In contrast, recruitment of the BER-scaffolding protein, X-ray repair cross-complementing protein 1 (XRCC1), to 8-oxoG lesions is stimulated by CSB and transcription. Remarkably, recruitment of XRCC1 to BER-unrelated single strand breaks (SSBs) does not require CSB or transcription. Together, our results suggest a specific transcription-dependent role for CSB in recruiting XRCC1 to BER-generated SSBs, whereas XRCC1 recruitment to SSBs generated independently of BER relies predominantly on PARP activation. Based on our results, we propose a model in which CSB plays a role in facilitating BER progression at transcribed genes, probably to allow XRCC1 recruitment to BER-intermediates masked by RNA polymerase II complexes stalled at these intermediates.

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DNA–protein crosslinks (DPCs) arise from enzymatic intermediates, metabolism or chemicals like chemotherapeutics. DPCs are highly cytotoxic as they impede DNA-based processes such as replication, which is counteracted through proteolysis-mediated DPC removal by spartan (SPRTN) or the proteasome. However, whether DPCs affect transcription and how transcription-blocking DPCs are repaired remains largely unknown. Here we show that DPCs severely impede RNA polymerase II-mediated transcription and are preferentially repaired in active genes by transcription-coupled DPC (TC-DPC) repair. TC-DPC repair is initiated by recruiting the transcription-coupled nucleotide excision repair (TC-NER) factors CSB and CSA to DPC-stalled RNA polymerase II. CSA and CSB are indispensable for TC-DPC repair; however, the downstream TC-NER factors UVSSA and XPA are not, a result indicative of a non-canonical TC-NER mechanism. TC-DPC repair functions independently of SPRTN but is mediated by the ubiquitin ligase CRL4CSA and the proteasome. Thus, DPCs in genes are preferentially repaired in a transcription-coupled manner to facilitate unperturbed transcription. Three studies identify a transcription-coupled DNA–protein crosslink repair pathway that depends on the Cockayne syndrome proteins and the proteasome.

Nucleotide excision repair is a highly versatile DNA repair system responsible for elimination of a wide variety of lesions from the genome. It is comprised of two subpathways: transcription-coupled repair that accomplishes efficient removal of damage blocking transcription and global genome repair. Recently, the basic mechanism of global genome repair has emerged from biochemical studies. However, little is known about transcription-coupled repair in eukaryotes. Here we report the identification of a novel protein designated XAB2 (XPA-binding protein 2) that was identified by virtue of its ability to interact with XPA, a factor central to both nucleotide excision repair subpathways. The XAB2 protein of 855 amino acids consists mainly of 15 tetratricopeptide repeats. In addition to interacting with XPA, immunoprecipitation experiments demonstrated that a fraction of XAB2 is able to interact with the transcription-coupled repair-specific proteins CSA and CSB as well as RNA polymerase II. Furthermore, antibodies against XAB2 inhibited both transcription-coupled repair and transcription in vivo but not global genome repair when microinjected into living fibroblasts. These results indicate that XAB2 is a novel component involved in transcription-coupled repair and transcription.

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Summary Transcription-coupled DNA repair (TCR) is a subpathway of nucleotide excision repair (NER) that is triggered when RNA polymerase is stalled by DNA damage. Lesions targeted by TCR are repaired more quickly than lesions repaired by the transcription-independent “global” NER pathway, but the mechanism underlying this rate enhancement is not understood. Damage recognition during bacterial NER depends upon UvrA, which binds to the damage and loads UvrB onto the DNA. Bacterial TCR additionally requires the Mfd protein, a DNA translocase that removes the stalled transcription complexes. We have determined the properties of Mfd, UvrA, and UvrB that are required for the elevated rate of repair observed during TCR. We show that TCR and global NER differ in their requirements for damage recognition by UvrA, indicating that Mfd acts at the very earliest stage of the repair process and extending the functional similarities between TCR in bacteria and eukaryotes.

Transcription-coupled repair (TCR) removes DNA lesions from the transcribed strand of active genes. Stalling of RNA polymerase II (RNAPII) at DNA lesions initiates TCR through the recruitment of the CSB and CSA proteins. The full repertoire of proteins required for human TCR – particularly in a chromatin context - remains to be determined. Studies in mice have revealed that the nucleosome-binding protein HMGN1 is required to enhance the repair of UV-induced lesions in transcribed genes. However, whether HMGN1 is required for human TCR remains unaddressed. Here, we show that knockout or knockdown of HMGN1, either alone or in combination with HMGN2, does not render human cells sensitive to UV light or Illudin S-induced transcription-blocking DNA lesions. Moreover, transcription restart after UV irradiation was not impaired in HMGN-deficient cells. In contrast, TCR-deficient cells were highly sensitive to DNA damage and failed to restart transcription. Furthermore, GFP-tagged HMGN1 was not recruited to sites of UV-induced DNA damage under conditions where GFP-CSB readily accumulated. In line with this, HMGN1 did not associate with the TCR complex, nor did TCR proteins require HMGN1 to associate with DNA damage-stalled RNAPII. Together, our findings suggest that HMGN1 and HMGN2 are not required for human TCR.

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Summary Elongating RNA polymerase II (RNAPII) stalls at transcription-blocking lesions in the DNA template strand and is removed by transcription-coupled DNA repair (TCR) factors. Here, we present a protocol to measure RNAPII clearance during TCR at sites of localized UV-induced DNA damage in adherent cells. We describe how to induce local UV damage and visualize the damaged area and chromatin-bound RNAPII levels using immunofluorescence staining. This approach can quantify the clearance of DNA damage-stalled RNAPII and its dependence on TCR factors. For complete details on the use and execution of this protocol, please refer to van den Heuvel et al.1

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Stalled RNA polymerase II (RNAPII) threatens genome integrity, yet how cells resolve transcription blocks at difficult-to-terminate sites is unclear. Leveraging the compact genome of fission yeast and termination defects associated with the non-canonical function of Dcr1, we unravel the recognition and release mechanisms of stalled RNAPII. Through dual recognition, Dcr1 senses the difficult-to-terminate context-stalled RNAPII and accumulated R-loops-and recruits the termination factor Dhp1 to ensure efficient RNAPII release. Failure of this mechanism causes termination defects that impede replication forks, necessitating DNA polymerase delta (DNAPδ)-mediated replication fork restart at stalled sites. Moreover, Dcr1 promotes genome stability by repurposing its hybrid-recognition ability to engage Rad51, thereby biasing DNA repair toward high-fidelity homologous recombination. Our work defines a key chromatin context and mechanisms governing RNAPII termination, establishing Dcr1 as a molecular hub that directly couples the fidelity of transcription termination to the stability of the genome during replication and repair.

Deregulation of RNA Polymerase II (RNAPII) by oncogenic signaling leads to collisions of RNAPII with DNA synthesis machinery (transcription-replication conflicts, TRCs). TRCs can result in DNA damage and are thought to underlie genomic instability in tumor cells. Here we provide evidence that elongating RNAPII nucleates activation of the ATM kinase at TRCs to stimulate DNA repair. We show the ATPase WRNIP1 associates with RNAPII and limits ATM activation during unperturbed cell cycle. WRNIP1 binding to elongating RNAPII requires catalytic activity of the ubiquitin ligase HUWE1. Mutation of HUWE1 induces TRCs, promotes WRNIP1 dissociation from RNAPII and binding to the replisome, stimulating ATM recruitment and activation at RNAPII. TRCs and translocation of WRNIP1 are rapidly induced in response to hydroxyurea treatment to activate ATM and facilitate subsequent DNA repair. We propose that TRCs can provide a controlled mechanism for stalling of replication forks and ATM activation, instrumental in cellular response to replicative stress.

Stalled replication forks, susceptible to nucleolytic threats, necessitate protective mechanisms involving pivotal factors such as the tumor suppressors BRCA1 and BRCA2. Here, we demonstrate that, upon replication stress, RNA polymerase II (RNAPII) is recruited to stalled forks, actively promoting the transient formation of RNA-DNA hybrids. These hybrids act as safeguards, preventing premature engagement by the DNA2 nuclease and uncontrolled DNA2-mediated degradation of nascent DNA. Furthermore, we provide evidence that DExD box polypeptide 39A (DDX39A), serving as an RNA-DNA resolver, unwinds these structures and facilitates regulated DNA2 access to stalled forks. This orchestrated process enables controlled DNA2-dependent stalled fork processing and restart. Finally, we reveal that loss of DDX39A enhances stalled fork protection in BRCA1/2-deficient cells, consequently conferring chemoresistance. Our results suggest that the dynamic regulation of RNA-DNA hybrid formation at stalled forks by RNAPII and DDX39A precisely governs the timing of DNA2 activation, contributing to stalled fork protection, processing, and restart, ultimately promoting genome stability.

Platinum-based compounds and ultraviolet (UV) irradiation produce bulky DNA lesions that stall RNA polymerase II (RNAPII), activating transcription-coupled nucleotide excision repair (TC-NER), RNAPII degradation, and global transcriptional shutdown. However, the consequences of RNAPII bypassing such lesions remain unclear. We identified the acetyltransferase p300 as a key regulator of TC-NER-dependent RNAPII removal from damaged chromatin via a USP7-dependent mechanism. Loss of p300 permits RNAPII to bypass transcription-blocking lesions, sustaining transcription and full-length mRNA production despite DNA damage. This leads to continued translation, endoplasmic reticulum (ER) stress, and activation of the unfolded protein response (UPR), compromising cell viability. Notably, this stress response resensitizes tumors resistant to platinum-based chemotherapy. Our findings reveal a vulnerability in tumor cells that evade transcriptional shutdown and define a synthetic lethal interaction between p300 inhibition and platinum-induced DNA damage, offering a targeted strategy to overcome chemoresistance.

Temperature profoundly affects the kinetics of biochemical reactions, yet how large molecular complexes such as the transcription machinery accommodate changing temperatures to maintain cellular function is poorly understood. Here, we developed plant native elongating transcripts sequencing (plaNET-seq) to profile genome-wide nascent RNA polymerase II (RNAPII) transcription during the cold-response of Arabidopsis thaliana with single-nucleotide resolution. Combined with temporal resolution, these data revealed transient genome-wide reprogramming of nascent RNAPII transcription during cold, including characteristics of RNAPII elongation and thousands of non-coding transcripts connected to gene expression. Our results suggest a role for promoter-proximal RNAPII stalling in predisposing genes for transcriptional activation during plant-environment interactions. At gene 3’-ends, cold initially facilitated transcriptional termination by limiting the distance of read-through transcription. Within gene bodies, cold reduced the kinetics of co-transcriptional splicing leading to increased intragenic stalling. Our data resolved multiple distinct mechanisms by which temperature transiently altered the dynamics of nascent RNAPII transcription and associated RNA processing, illustrating potential biotechnological solutions and future focus areas to promote food security in the context of a changing climate.

Adult stem cells persist in mammalian tissues by entering a state of reversible quiescence/ G0, associated with low transcription. Using cultured myoblasts and muscle stem cells, we report that in G0, global RNA content and synthesis are substantially repressed, correlating with decreased RNA Polymerase II (RNAPII) expression and activation. Integrating RNAPII occupancy and transcriptome profiling, we identify repressed networks and a role for promoter-proximal RNAPII pausing in G0. Strikingly, RNAPII shows enhanced pausing in G0 on repressed genes encoding regulators of RNA biogenesis (Nucleolin, Rps24, Ctdp1); release of pausing is associated with their increased expression in G1. Knockdown of these transcripts in proliferating cells leads to induction of G0 markers, confirming the importance of their repression in establishment of G0. A targeted screen of RNAPII regulators revealed that knockdown of Aff4 (positive regulator of elongation) unexpectedly enhances expression of G0-stalled genes and hastens S phase; NELF, a regulator of pausing appears to be dispensable. We propose that RNAPII pausing contributes to transcriptional control of a subset of G0-repressed genes to maintain quiescence and impacts the timing of the G0-G1 transition.

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The CGG triplet repeat binding protein 1 (CGGBP1) binds to CGG repeats and has several important cellular functions, but how this DNA sequence-specific binding factor affects transcription and replication processes is an open question. Here, we show that CGGBP1 binds human gene promoters containing short (< 5) CGG-repeat tracts prone to R-loop formation. Loss of CGGBP1 leads to deregulated transcription, transcription–replication–conflicts (TRCs) and accumulation of Serine-5 phosphorylated RNA polymerase II (RNAPII), indicative of promoter-proximal stalling and a defect in transcription elongation. Consistently, an episomal CGG-repeat-containing model locus as well as endogenous genes show deregulated transcription, R-loop accumulation and increased RNAPII chromatin occupancy in CGGBP1-depleted cells. We identify the DEAD-box RNA:DNA helicases DDX41 and DHX15 as interaction partners specifically recruited by CGGBP1. Co-depletion experiments show that DDX41 and CGGBP1 work in the same pathway to unwind R-loops and avoid TRCs. Together, our work shows that short trinucleotide repeats are a source of genome-destabilizing secondary structures, and cells rely on specific DNA-binding factors to maintain proper transcription and replication coordination at short CGG repeats. CGGBP1 binds to short CGG repeats in gene promoters, recruiting RNA:DNA helicases like DDX41 to ensure proper transcription and replication activities. CGGBP1-depleted cells fail to recruit such helicases and accumulate R-loops and transcription-replication conflicts, threatening genome integrity. CGGBP1 binds to short tracts of CGG repeats in human gene promoters. CGGBP1 interacts with and recruits a subset of DEAD-box RNA:DNA helicases including DDX41 and DHX15. DDX41 and CGGBP1 work in the same pathway to unwind R-loops and prevent TRCs. CGGBP1 binds to short tracts of CGG repeats in human gene promoters. CGGBP1 interacts with and recruits a subset of DEAD-box RNA:DNA helicases including DDX41 and DHX15. DDX41 and CGGBP1 work in the same pathway to unwind R-loops and prevent TRCs. CGGBP1 binds to short CGG repeats in gene promoters, recruiting RNA:DNA helicases like DDX41 to ensure proper transcription and replication activities. CGGBP1-depleted cells fail to recruit such helicases and accumulate R-loops and transcription-replication conflicts, threatening genome integrity.

The DNA Damage Response (DDR) is a highly regulated process that safeguards genomic integrity against DNA lesions. Increasing evidence supports a reciprocal relationship between damaged chromatin architecture and the signalling pathways that coordinate the DDR. However, the mechanisms underlying this interplay in response to transcription-blocking DNA lesions remain largely unexplored. Here, we show that stalling of RNA polymerase II (RNAPII) at such lesions induces local chromatin acetylation, mediated primarily by the histone acetyltransferase p300. The resulting chromatin relaxation stimulates the dissociation of mature co-transcriptional spliceosomes from nascent RNA and promotes RNA:DNA hybrid (R-loop) formation, leading to ATM activation. In turn, activated ATM modulates chromatin conformation by phosphorylating histone H2A.X and triggering p38MAPK/MSK1-dependent histone H3S10 phosphorylation. Our findings highlight the cross-regulation between chromatin state and ATM signalling as a key component of the cellular response to transcription stress. How chromatin responds to transcription-blocking DNA lesions is unclear. This study reveals that RNA polymerase II (RNAPII) stalling triggers histone acetylation, promoting ATM activation that in turn modifies chromatin in a regulatory feedback loop. DNA lesion-induced stalling of elongating RNAPII stimulates upstream nucleosome acetylation by the p300 histone acetyltransferase. Chromatin acetylation leads to destabilization and release of mature co-transcriptional spliceosomes from the nascent transcript. Unspliced, spliceosome-free RNA forms R-loops that activate ATM signalling. ATM modulates chromatin structure, both directly (e.g., via H2A.X phosphorylation) and indirectly through the p38/MSK1–H3S10 phosphorylation pathway. DNA lesion-induced stalling of elongating RNAPII stimulates upstream nucleosome acetylation by the p300 histone acetyltransferase. Chromatin acetylation leads to destabilization and release of mature co-transcriptional spliceosomes from the nascent transcript. Unspliced, spliceosome-free RNA forms R-loops that activate ATM signalling. ATM modulates chromatin structure, both directly (e.g., via H2A.X phosphorylation) and indirectly through the p38/MSK1–H3S10 phosphorylation pathway. Lesion-mediated stalling of RNA Pol II triggers p300-dependent histone acetylation, R-loop formation, and chromatin modulation via a regulatory feedback loop.

Transcription and replication both utilize genomic DNA as a template, creating the potential for frequent interference between the two machineries. Such transcription–replication conflicts (TRCs) can compromise genome stability due to the abnormal accumulation and persistence of R-loops—three-stranded nucleic acid structures comprising a DNA:RNA hybrid and a displaced single-stranded DNA strand (Bhowmick R., et al., Mol Cell. 2023). To safeguard genome integrity, cells deploy multiple R-loop resolution factors that prevent R-loop-associated replication stress (Brickner J. R., et al., Mol Cell. 2022). Persistent R-loops are also associated with the stalling of RNA polymerase II (RNAPII) on chromatin, which further impedes replication fork progression (Zardoni L., et al., NAR 2021). Our observation of cells depleted of the chromatin remodelerHELLS, which displayed an accumulation of R-loops and stalled RNAPII on highly transcribed genes (Tameni A., Mallia S., et al., NAR 2024) in aggressive anaplastic large cell lymphoma (ALCL), led us to hypothesize that HELLS-dependent R-loop accumulation can induce replication stress and TRCs. To test replication forks progression, we performed DNA fiber analysis by sequentially labeling the cells with two thymidine analogs, 5-chloro-2′-deoxyuridine (CldU, red) and 5-iodo-2′-deoxyuridine (IdU, green). While control cells showed a CIdU/ldU ratio close to 1, indicating the absence of stalled forks, TLBR-2 and MAC2A HELLS-KD cells displayed an CIdU/ldU ratio close to 2, strongly indicating the presence of stalled forks in absence of HELLS. Then, we performed proximity-ligation assays (PLA) for measuring the presence of TRCs. Specifically, we used antibodies against phospho-forms of RNAPII, pSer5-RNAPII and pSer2-RNAPII, corresponding to initiation and elongation transcriptional complexes respectively and PCNA, as key component of DNA replicative machinery. We found that PLA signal between PCNA and pSer5-RNAPII, but not pSer2 RNAPII, increased in HELLS-KD cells compared to control, suggesting that the depletion of HELLS increases the collision between RNAPII and replicative complex at promoter-proximal sites before pause release. Importantly, ectopic expression of RNaseH1 in HELLS-KD cells reduced R-loop-dependent TRCs, confirming the functional involvement of R-loops in this process. Despite the large number of genes demonstrated or proposed to regulate R-loop homeostasis and, therefore, to impact TRCs and in turn on genome integrity, our knowledge of specific HELLS-regulated factors is still limited. To explore the mechanism contributing to TRCs and thus to genome instability, we performed gene set enrichment analysis focusing on genes belonging to functional categories related to chromatin regulation previously identified as HELLS transcriptional direct genes. Among them, mini chromosome maintenance 5 (MCM5) emerged as a potential candidate. Inducible knockdown of MCM5 significantly reduced cell proliferation, increased TRCs levels in lymphoma cells, as measured by pSer5-RNAPII–PCNA PLA foci, led to forks stall and to enhanced nuclear R-loop accumulation, phenocopying HELLS depletion. Notably, the ectopic overexpression of MCM5 in TLBR-2 HELLS-KDcells fully rescues cell proliferation reducing R-loop formation and the presence of TRCs. Additionally, perturbations observed upon MCM5-KD were dramatically reduced when R-loops were suppressed by overexpression of RNaseH1 indicating that, in absence of MCM5, the persistence of R-loop disrupts the replication fork process through TRCs, mimicking HELLS. These data highligh MCM5 as the downstream target of HELLS in replicative processes. Integrative analysis of MCM5, and RNAPII chromatin occupancy (ChIP-seq), alongside chromatin accessibility profiles (ATAC-seq) in TLBR-2 cells, further revealed that MCM5 was excluded from accessible, actively transcribed loci, confirming the not-transcriptional role of MCM5. Collectively, these findings position the axis HELLS/MCM5 as a critical regulator of replicative processes in aggressive ALCL. Disruption of HELLS/MCM5 activity induces aberrant TRCs and R-loop accumulation, ultimately compromising genome integrity and highlighting a potential therapeutic vulnerability in lymphoma cells.

Transcription stress has been linked to DNA damage -driven aging, yet the underlying mechanism remains unclear. Here, we demonstrate that Tcea1−/− cells, which harbor a TFIIS defect in transcription elongation, exhibit RNAPII stalling at oxidative DNA damage sites, impaired transcription, accumulation of R-loops, telomere uncapping, chromatin bridges, and genome instability, ultimately resulting in cellular senescence. We found that R-loops at telomeres causally contribute to the release of telomeric DNA fragments in the cytoplasm of Tcea1−/− cells and primary cells derived from naturally aged animals triggering a viral-like immune response. TFIIS-defective cells release extracellular vesicles laden with telomeric DNA fragments that target neighboring cells, which consequently undergo cellular senescence. Thus, transcription stress elicits paracrine signals leading to cellular senescence, promoting aging. Cellular senescence and the process of transcription are intimately linked, yet the mechanisms involved remain unclear. Here the authors show that a defect in TFIIS leads to telomere dysfunction, genome instability and the release of vesicles that induce senescence to neighboring cells.

Centromere sequences are not conserved between species, and there is compelling evidence for epigenetic regulation of centromere identity, with location being dictated by the presence of chromatin containing the histone H3 variant CENP-A. Paradoxically, in most organisms CENP-A chromatin generally occurs on particular sequences. To investigate the contribution of primary DNA sequence to establishment of CENP-A chromatin in vivo, we utilised the fission yeast Schizosaccharomyces pombe. CENP-ACnp1 chromatin is normally assembled on ∼10 kb of central domain DNA within these regional centromeres. We demonstrate that overproduction of S. pombe CENP-ACnp1 bypasses the usual requirement for adjacent heterochromatin in establishing CENP-ACnp1 chromatin, and show that central domain DNA is a preferred substrate for de novo establishment of CENP-ACnp1 chromatin. When multimerised, a 2 kb sub-region can establish CENP-ACnp1 chromatin and form functional centromeres. Randomization of the 2 kb sequence to generate a sequence that maintains AT content and predicted nucleosome positioning is unable to establish CENP-ACnp1 chromatin. These analyses indicate that central domain DNA from fission yeast centromeres contains specific information that promotes CENP-ACnp1 incorporation into chromatin. Numerous transcriptional start sites were detected on the forward and reverse strands within the functional 2 kb sub-region and active promoters were identified. RNAPII is enriched on central domain DNA in wild-type cells, but only low levels of transcripts are detected, consistent with RNAPII stalling during transcription of centromeric DNA. Cells lacking factors involved in restarting transcription—TFIIS and Ubp3—assemble CENP-ACnp1 on central domain DNA when CENP-ACnp1 is at wild-type levels, suggesting that persistent stalling of RNAPII on centromere DNA triggers chromatin remodelling events that deposit CENP-ACnp1. Thus, sequence-encoded features of centromeric DNA create an environment of pervasive low quality RNAPII transcription that is an important determinant of CENP-ACnp1 assembly. These observations emphasise roles for both genetic and epigenetic processes in centromere establishment.

DNA-protein crosslinks (DPCs) are highly toxic DNA lesions that are relevant to multiple human diseases. They are caused by various endogenous and environmental agents, and from the actions of enzymes such as topoisomerases. DPCs impede DNA polymerases, triggering replication-coupled DPC repair. Until recently the consequences of DPC blockade of RNA polymerases remained unclear. New methodologies for studying DPC repair have enabled the discovery of a transcription-coupled (TC) DPC repair pathway. Briefly, RNA polymerase II (RNAPII) stalling initiates TC-DPC repair, leading to sequential engagement of Cockayne syndrome (CS) proteins CSB and CSA, and to proteasomal degradation of the DPC. Deficient TC-DPC repair caused by loss of CSA or CSB function may help to explain the complex clinical presentation of CS patients.

The response to DNA damage is critical for cellular homeostasis, tumor suppression, immunity and gametogenesis. In order to provide an unbiased and global view of the DNA damage response in human cells, we undertook 28 CRISPR/Cas9 screens against 25 genotoxic agents in the retinal pigment epithelium-1 (RPE1) cell line. These screens identified 840 genes whose loss causes either sensitivity or resistance to DNA damaging agents. Mining this dataset, we uncovered that ERCC6L2, which is mutated in a bone-marrow failure syndrome, codes for a canonical non-homologous end-joining pathway factor; that the RNA polymerase II component ELOF1 modulates the response to transcription-blocking agents and that the cytotoxicity of the G-quadruplex ligand pyridostatin involves trapping topoisomerase II on DNA. This map of the DNA damage response provides a rich resource to study this fundamental cellular system and has implications for the development and use of genotoxic agents in cancer therapy.

Mutations in DNA damage response (DDR) genes endanger genome integrity and predispose to cancer and genetic disorders. Here, using CRISPR-dependent cytosine base editing screens, we identify > 2,000 sgRNAs that generate nucleotide variants in 86 DDR genes, resulting in altered cellular fitness upon DNA damage. Among those variants, we discover loss- and gain-of-function mutants in the Tudor domain of the DDR regulator 53BP1 that define a non-canonical surface required for binding the deubiquitinase USP28. Moreover, we characterize variants of the TRAIP ubiquitin ligase that define a domain, whose loss renders cells resistant to topoisomerase I inhibition. Finally, we identify mutations in the ATM kinase with opposing genome stability phenotypes and loss-of-function mutations in the CHK2 kinase previously categorized as variants of uncertain significance for breast cancer. We anticipate that this resource will enable the discovery of additional DDR gene functions and expedite studies of DDR variants in human disease.

Despite recent advances in the use of immunotherapy, only a minority of patients with small cell lung cancer (SCLC) respond to immune checkpoint blockade (ICB). Here, we show that targeting the DNA damage response (DDR) proteins PARP and checkpoint kinase 1 (CHK1) significantly increased protein and surface expression of PD-L1. PARP or CHK1 inhibition remarkably potentiated the antitumor effect of PD-L1 blockade and augmented cytotoxic T-cell infiltration in multiple immunocompetent SCLC in vivo models. CD8+ T-cell depletion reversed the antitumor effect, demonstrating the role of CD8+ T cells in combined DDR-PD-L1 blockade in SCLC. We further demonstrate that DDR inhibition activated the STING/TBK1/IRF3 innate immune pathway, leading to increased levels of chemokines such as CXCL10 and CCL5 that induced activation and function of cytotoxic T lymphocytes. Knockdown of cGAS and STING successfully reversed the antitumor effect of combined inhibition of DDR and PD-L1. Our results define previously unrecognized innate immune pathway-mediated immunomodulatory functions of DDR proteins and provide a rationale for combining PARP/CHK1 inhibitors and immunotherapies in SCLC. SIGNIFICANCE: Our results define previously unrecognized immunomodulatory functions of DDR inhibitors and suggest that adding PARP or CHK1 inhibitors to ICB may enhance treatment efficacy in patients with SCLC. Furthermore, our study supports a role of innate immune STING pathway in DDR-mediated antitumor immunity in SCLC.See related commentary by Hiatt and MacPherson, p. 584.This article is highlighted in the In This Issue feature, p. 565.

Here, we report that genome editing by CRISPR–Cas9 induces a p53-mediated DNA damage response and cell cycle arrest in immortalized human retinal pigment epithelial cells, leading to a selection against cells with a functional p53 pathway. Inhibition of p53 prevents the damage response and increases the rate of homologous recombination from a donor template. These results suggest that p53 inhibition may improve the efficiency of genome editing of untransformed cells and that p53 function should be monitored when developing cell-based therapies utilizing CRISPR–Cas9. CRISPR–Cas9-induced DNA damage triggers p53 to limit the efficiency of gene editing in immortalized human retinal pigment epithelial cells.

Laser micro-irradiation across the nucleus rapidly generates localized chromatin-associated DNA lesions permitting analysis of repair protein recruitment in living cells. Recruitment of three fluorescently-tagged base excision repair factors [DNA polymerase β (pol β), XRCC1 and PARP1], known to interact with one another, was compared in gene-deleted mouse embryonic fibroblasts and in those expressing the endogenous factor. A low energy micro-irradiation (LEMI) forming direct single-strand breaks and a moderate energy (MEMI) protocol that additionally creates oxidized bases were compared. Quantitative characterization of repair factor recruitment and sensitivity to clinical PARP inhibitors (PARPi) was dependent on the micro-irradiation protocol. PARP1 recruitment was biphasic and generally occurred prior to pol β and XRCC1. After LEMI, but not after MEMI, pol β and XRCC1 recruitment was abolished by the PARPi veliparib. Consistent with this, pol β and XRCC1 recruitment following LEMI was considerably slower in PARP1-deficient cells. Surprisingly, the recruitment half-times and amplitudes for pol β were less affected by PARPi than were XRCC1 after MEMI suggesting there is a XRCC1-independent component for pol β recruitment. After LEMI, but not MEMI, pol β dissociation was more rapid than that of XRCC1. Unexpectedly, PARP1 dissociation was slowed in the absence of XRCC1 as well with a PARPi after LEMI but not MEMI, suggesting that XRCC1 facilitates PARP1 dissociation from specific DNA lesions. XRCC1-deficient cells showed pronounced hypersensitivity to the PARPi talazoparib correlating with its known cytotoxic PARP1 trapping activity. In contrast to DNA methylating agents, PARPi only minimally sensitized pol β and XRCC1-deficient cells to oxidative DNA damage suggesting differential binding of PARP1 to alternate repair intermediates. In summary, pol β, XRCC1, and PARP1 display recruitment kinetics that exhibit correlated and unique properties that depend on the DNA lesion and PARP activity revealing that there are multiple avenues utilized in the repair of chromatin-associated DNA.

Summary In response to transcription-blocking DNA damage, cells orchestrate a multi-pronged reaction, involving transcription-coupled DNA repair, degradation of RNA polymerase II (RNAPII), and genome-wide transcription shutdown. Here, we provide insight into how these responses are connected by the finding that ubiquitylation of RNAPII itself, at a single lysine (RPB1 K1268), is the focal point for DNA-damage-response coordination. K1268 ubiquitylation affects DNA repair and signals RNAPII degradation, essential for surviving genotoxic insult. RNAPII degradation results in a shutdown of transcriptional initiation, in the absence of which cells display dramatic transcriptome alterations. Additionally, regulation of RNAPII stability is central to transcription recovery—persistent RNAPII depletion underlies the failure of this process in Cockayne syndrome B cells. These data expose regulation of global RNAPII levels as integral to the cellular DNA-damage response and open the intriguing possibility that RNAPII pool size generally affects cell-specific transcription programs in genome instability disorders and even normal cells.

The ability of the humoral immune system to generate Abs capable of specifically binding a myriad of Ags is critically dependent on the somatic hypermutation program. This program induces both templated mutations (i.e., gene conversion) and untemplated mutations. In humans, somatic hypermutation is widely believed to result in untemplated point mutations. In this study, we demonstrate detection of large-scale templated events that occur in human memory B cells and circulating plasmablasts. We find that such mutations are templated intrachromosomally from IGHV genes and interchromosomally from IGHV pseudogenes as well as other homologous regions unrelated to IGHV genes. These same donor regions are used in multiple individuals, and they predominantly originate from chromosomes 14, 15, and 16. In addition, we find that exogenous sequences placed at the IgH locus, such as LAIR1, undergo templated mutagenesis and that homology appears to be the major determinant for donor choice. Furthermore, we find that donor tracts originate from areas in proximity with open chromatin, which are transcriptionally active, and are found in spatial proximity with the IgH locus during the germinal center reaction. These donor sequences are inserted into the Ig gene segment in association with overlapping activation-induced cytidine deaminase hotspots. Taken together, these studies suggest that diversity generated during the germinal center response is driven by untemplated point mutations as well as templated mutagenesis using local and distant regions of the genome. Visual Abstract Key Points Novel computational script TRACE can detect gene conversion from across the genome. TRACE detects gene conversion donors from both intra- and interchromosomal sources. TRACE outputs correlate with germinal center B cell physiology.

Activation-induced cytidine deaminase (AID)-dependent DNA cleavage are the initial event of antibody gene-diversification processes such as class switch recombination (CSR) and somatic hypermutation (SHM). We previously reported the requirement of an AID-dependent decrease of topoisomerase 1 (Top1) for efficient DNA cleavage, but the underlying molecular mechanism has remained elusive. This study focuses on HuR/ELAVL1, a protein that binds to AU-rich elements in RNA. HuR-knockout (KO) CH12 cells derived from murine B lymphoma cells were found to have lower CSR and hypermutation efficiencies due to decreased AID-dependent DNA cleavage levels. The HuR-KO CH12 cells do not show impairment in cell cycles and Myc expression, which have been reported in HuR-reduced spleen B cells. Furthermore, drugs that scavenge reactive oxygen species (ROS) do not rescue the lower CSR in HuR-KO CH12 cells, meaning that ROS or decreased c-Myc protein amount is not the reason for the deficiencies of CSR and hypermutation in HuR-KO CH12 cells. We show that HuR binds to Top1 mRNA and that complete deletion of HuR abolishes AID-dependent repression of Top1 protein synthesis in CH12 cells. Additionally, reduction of CSR to IgG3 in HuR-KO cells is rescued by knockdown of Top1, indicating that elimination of the AID-dependent Top1 decrease is the cause of the inefficiency of DNA cleavage, CSR, and hypermutation in HuR-KO cells. These results show that HuR is required for initiation of antibody diversification and acquired immunity through the regulation of AID-dependent DNA cleavage by repressing Top1 protein synthesis.

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Insertional diversification of the immunoglobulin heavy chain (IgH) switch and variable regions occurs when nucleic acid sequences from non-IgH sites are integrated into these regions. This unconventional antibody diversification mechanism could contribute to lymphomagenesis. We present an analysis of IgH/BCR VDJ (IGHV) sequencing data to reveal insertion events in chronic lymphoproliferative disorder patients and the development of a targeted switch region sequencing approach. The targeted switch region sequencing successfully demonstrated inserts in polyclonal cells, and we describe insertional diversification events occurring in the variable region in chronic lymphoproliferative disorders.

Significance Heterogeneous nuclear ribonucleoprotein K (hnRNP K), an RNA-binding protein, is the cofactor of activation-induced cytidine deaminase (AID) that induces DNA breaks in immunoglobulin (Ig) genes. Here, we elucidated that the GXXG and RGG RNA-binding motifs were critically necessary for class switch recombination and somatic hypermutation. Nuclear localization of hnRNP K and interaction with AID were also dependent on all of the RNA-binding motifs. This study clearly demonstrated that hnRNP K contributed to AID-dependent DNA breaks through its RNA-binding capacity, suggesting the possibility that hnRNP K holds and presents some editing target RNAs to AID, which eventually induces DNA breaks in Ig genes. Activation-induced cytidine deaminase (AID) is the key enzyme for class switch recombination (CSR) and somatic hypermutation (SHM) to generate antibody memory. Previously, heterogeneous nuclear ribonucleoprotein K (hnRNP K) was shown to be required for AID-dependent DNA breaks. Here, we defined the function of major RNA-binding motifs of hnRNP K, GXXGs and RGGs in the K-homology (KH) and the K-protein-interaction (KI) domains, respectively. Mutation of GXXG, RGG, or both impaired CSR, SHM, and cMyc/IgH translocation equally, showing that these motifs were necessary for AID-dependent DNA breaks. AID–hnRNP K interaction is dependent on RNA; hence, mutation of these RNA-binding motifs abolished the interaction with AID, as expected. Some of the polypyrimidine sequence-carrying prototypical hnRNP K-binding RNAs, which participate in DNA breaks or repair bound to hnRNP K in a GXXG and RGG motif-dependent manner. Mutation of the GXXG and RGG motifs decreased nuclear retention of hnRNP K. Together with the previous finding that nuclear localization of AID is necessary for its function, lower nuclear retention of these mutants may worsen their functional deficiency, which is also caused by their decreased RNA-binding capacity. In summary, hnRNP K contributed to AID-dependent DNA breaks with all of its major RNA-binding motifs.

Recent reports have underlined the potential of gamma (γ)-rays as tools for seed priming, a process used in seed industry to increase seed vigor and to enhance plant tolerance to biotic/abiotic stresses. However, the impact of γ-rays on key aspects of plant metabolism still needs to be carefully evaluated. In the present study, rice seeds were challenged with different doses of γ-rays and grown in absence/presence of NaCl to assess the impact of these treatments on the early stages of plant life. Enhanced germination efficiency associated with increase in radicle and hypocotyl length was observed, while at later stages no increase in plant tolerance to salinity stress was evident. APX, CAT, and GR were enhanced at transcriptional level and in terms of enzyme activity, indicating the activation of antioxidant defence. The profiles of DNA damage accumulation were obtained using SCGE and the implication of TC-NER pathway in DNA damage sensing and repair mechanisms is discussed. OsXPB2, OsXPD, OsTFIIS, and OsTFIIS-like genes showed differential modulation in seedlings and plantlets in response to γ-irradiation and salinity stress. Altogether, the synergistic exposure to γ-rays and NaCl resulted in enhanced oxidative stress and proper activation of antioxidant mechanisms, thus being compatible with plant survival.

Cells possess a complex DNA damage response (DDR) system, to prevent detrimental mutations from accumulating and being passed on. The genes encoding various DDR components are frequently mutated in cancer cells and provide a potential site for therapeutic intervention. DNA damaging agents, with one exception, are repaired by the global genome repair (GG-NER). The one exception are Illudofulvenes, which hide from the GG-NER and can only be repaired by the transcriptional-coupled repair (TC-NER) pathway. Many cancers are deficient in the TC-NER pathway and cannot properly repair DNA damage. In TC-NER deficient cancer cells the TC-NER polymerase complex becomes blocked by the illudofulvene ITX-0121, disengages from DNA, and the irreversible process of apoptosis. These cancers are 10-fold more sensitive to ITX-0121 than are cancer cells with a functional TC-NER pathway. In contrast, non-replicating or normal cells are minimally affected by and this is reflected in the lack of systemic toxicity in humans administered an illudofulvene. Analysis of the GENIE Database indicates the incidence of TC-NER deficient cancers in solid tumors is ∼13%, and includes all major histological classifications (or ∼175, 000 new patients annually). The highest incidence of TC-NER gene mutations/deletions was for the ERCC2 and ERCC5 genes with an incidence of 2.1% for each. Surprisingly, many patients with an ERCC2 or ERCC5 mutation had a second mutation in another ERCC gene (often ERCC6). The ERCC8 gene has the lowest incidence (0.4%) and the ERCC1 gene had the second lowest incidence of mutations (0.5%), but 65% of ERCC1 mutations were associated with a mutation in another ERCC gene. Based on studies involving double knockouts of TC-NER genes, these cancers have a 20-fold increase in sensitivity to ITX-0121. Histological classifications of cancers with the highest incidence of TC-NER deficiency (>25%) in the GENIE database included bladder, colorectal, endometrial, ovarian, and melanoma. In contrast, bone, renal, and thyroid cancers had a relatively low incidence of <8%. We developed a companion diagnostic to identify TC-NER deficient patients based on this data. Based on prior studies, we believe the MTD of ITX-0121 in humans to be >1.2 mg/kg, but only 0.4 mg/kg will be sufficient for clinical efficacy in TC-NER deficient cancers. Hence, despite administering a cytotoxic agent, one will treat TC-NER deficient cancer patients at a non-toxic dose. Toxicity (if it occurs) should be limited to transient thrombocytopenia based on prior studies. In summary, we are utilizing a Precision Medicine approach to develop a drug for an indication defined by a specific molecular alteration across cancer types, which in this case is the presence of TC-NER deficiency as identified by monoallelic mutations in specific genes coding for proteins in the TC-NER pathway. Michael J. Kelner. Analysis of the Genie database for TC-NER deficient cancers sensitive to ITX-0121 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1125.

ERCC6, also known as CSB (Cockayne Syndrome B), is a key protein involved in transcription-coupled nucleotide excision repair (TC-NER), a DNA repair process that removes lesions blocking RNA polymerase. ERCC6’s multifaceted roles include chromatin remodeling, transcription regulation, oxidative stress response, and coordination with other DNA repair proteins. Mutations in ERCC6 lead to Cockayne Syndrome and other neurodegenerative disorders, but some variants, such as M1097V, have been associated with cancer risk, particularly prostate cancer (PCa) in African Americans (AAs) in Louisiana. Recent studies have explored the functional impact of ERCC6 variants in PCa, especially among AAs, who face higher incidence and more aggressive forms of the disease. A notable finding is that the M1097V variant increases cellular tolerance to UV damage, suggesting not only a possible evolutionary benefit but also a potential risk for mutagenesis when exposed to complex environmental carcinogens. Other ERCC6 mutations, such as S636N (also only found in Louisiana PCa), located near regulatory regions, may alter repair activity, though their effects remain unclear. Given the high mutation burden in mismatch repair (MMR) and NER genes observed in AA patients with PCa, a synthetic lethality strategy targeting both TC-NER and homologous recombination repair (HRR) pathways could be effective. This includes combining agents like CDDP (cisplatin) with inhibitors of RAD54, such as J54. These approaches may offer alternatives to androgen deprivation therapy (ADT), which is often ineffective in advanced or treatment-resistant PCa common among AA men. This work underscores the importance of integrating genetic, environmental, and therapeutic insights to address PCa disparities.

Abstract Transcription-coupled nucleotide excision repair (TC-NER) is a dedicated DNA repair pathway that removes transcription-blocking DNA lesions (TBLs). TC-NER is initiated by the recognition of lesion-stalled RNA Polymerase II by the joint action of the TC-NER factors Cockayne Syndrome protein A (CSA), Cockayne Syndrome protein B (CSB) and UV-Stimulated Scaffold Protein A (UVSSA). However, the exact recruitment mechanism of these factors toward TBLs remains elusive. Here, we study the recruitment mechanism of UVSSA using live-cell imaging and show that UVSSA accumulates at TBLs independent of CSA and CSB. Furthermore, using UVSSA deletion mutants, we could separate the CSA interaction function of UVSSA from its DNA damage recruitment activity, which is mediated by the UVSSA VHS and DUF2043 domains, respectively. Quantitative interaction proteomics showed that the Spt16 subunit of the histone chaperone FACT interacts with UVSSA, which is mediated by the DUF2043 domain. Spt16 is recruited to TBLs, independently of UVSSA, to stimulate UVSSA recruitment and TC-NER-mediated repair. Spt16 specifically affects UVSSA, as Spt16 depletion did not affect CSB recruitment, highlighting that different chromatin-modulating factors regulate different reaction steps of the highly orchestrated TC-NER pathway.

The genome of living cells is constantly challenged by DNA lesions that interfere with cellular processes such as transcription and replication. A manifold of mechanisms act in concert to ensure adequate DNA repair, gene expression, and genome stability. Bulky DNA lesions, such as those induced by UV light or the DNA-damaging agent 4-nitroquinoline oxide, act as transcriptional and replicational roadblocks and thus represent a major threat to cell metabolism. When located on the transcribed strand of active genes, these lesions are handled by transcription-coupled nucleotide excision repair (TC-NER), a yet incompletely understood NER sub-pathway. Here, using a genetic screen in the yeast Saccharomyces cerevisiae, we identified histone variant H2A.Z as an important component to safeguard transcription and DNA integrity following UV irradiation. In the absence of H2A.Z, repair by TC-NER is severely impaired and RNA polymerase II clearance reduced, leading to an increase in double-strand breaks. Thus, H2A.Z is needed for proficient TC-NER and plays a major role in the maintenance of genome stability upon UV irradiation.

Abstract Ultraviolet (UV) induces distorting lesions to the DNA that can lead to stalling of the RNA polymerase II (RNAP II) and that are removed by transcription-coupled nucleotide excision repair (TC-NER). In humans, mutations in the TC-NER genes CSA and CSB lead to severe postnatal developmental defects in Cockayne syndrome patients. In Caenorhabditis elegans, mutations in the TC-NER genes csa-1 and csb-1, lead to developmental growth arrest upon UV treatment. We conducted a genetic suppressor screen in the nematode to identify mutations that could suppress the developmental defects in csb-1 mutants. We found that mutations in the ERK1/2 MAP kinase mpk-1 alleviate the developmental retardation in TC-NER mutants, while constitutive activation of the RAS-MAPK pathway exacerbates the DNA damage-induced growth arrest. We show that MPK-1 act via insulin/insulin-like signaling pathway and regulates the FOXO transcription factor DAF-16 to mediate the developmental DNA damage response.

e15149 Background: Cells possess a complex DNA damage response (DDR) system to prevent detrimental mutations from accumulating. The genes encoding various DDR components are frequently mutated in cancer cells and provide potential sites for therapeutic intervention. DNA damaging agents, with one exception, are repaired by the global genome nucleotide excision repair (GG-NER) pathway. Our novel class of drugs, Illudins, are the exception as Illudin-induced DNA damage is not recognized by GG-NER and can only be repaired by the transcription-coupled nucleotide excision repair (TC-NER) pathway. Studies utilizing isogeneic cell lines completely deficient in specific TC-NER proteins (e.g. homozygous ERCC6 knockout) indicate these cells are extremely sensitive to Illudins (up to 30-fold). In contrast, cells deficient in GG-NER activity are not sensitive to our drugs. Moreover, non-replicating or normal cells are minimally affected by Illudins, as evidenced by the lack of systemic toxicity in humans treated with one of our drugs. In TC-NER deficient cancer cells, however, our drug ITX-0121 blocks the TC-NER polymerase complex, which disengages from DNA, and initiates the irreversible process of apoptosis or cellular suicide. Many cancers exhibit deficiencies in DDR mechanisms and cannot properly repair DNA damage. Methods: Analysis of the GENIE Database indicates that the overall incidence of TC-NER deficient cancers in solid tumors is ~10%, and includes all major histological classifications (breast, ovarian, endometrial, prostate, renal, bladder, bone, colorectal, liver, lung, skin, pancreatic, thyroid, etc.) or ~175,000 new patients annually. These cancers, however, possess monoallelic mutations, as opposed to biallelic, and thus have some residual TC-NER functionality. To determine the relative sensitivity of cancers with monoallelic alterations in TC-NER genes, we generated colorectal HCT-116 cells with a monoallelic deletion in a single TC-NER gene. For example, we are generating daughter cell lines with small monoallelic deletions in ERCC2, ERCC3, ERCC4, ERCC6, and ELOF1 genes. Results: We are comparing sensitivity of these daughter lines to our lead drug ITX-0121, as well as to other DNA damaging agents such as cisplatin. Preliminary results indicate a monoallelic alteration in a TC-NER gene can confer > 6-fold sensitivity to ITX-0121, which is clinically relevant based on PK results. Based on these PK results, we believe the maximum tolerated dose (MTD) of ITX-0121 in patients to be > 1.2 mg/kg; yet only 0.4 mg/kg will be sufficient for clinical efficacy against cancers that are phenotypically TC-NER deficient. Conclusions: Thus, despite the administration of a cytotoxic agent, TC-NER deficient cancer patients can be treated at a non-toxic dose; if toxicity occurs, it should be limited to transient thrombocytopenia, as indicated by prior studies.

Cockayne syndrome (CS) is a disorder characterized by neurodegeneration and a segmental progeroid phenotype, resulting from mutations in ERCC8/CSA or ERCC6/CSB genes. These genes encode proteins essential for the DNA repair pathway known as transcription-coupled nucleotide excision repair (TC-NER). To further investigate the biological pathways associated with this phenotype, we analyzed transcriptome datasets specific to CS. We conducted RNA-seq on the Csa-/- mouse model at three different age timepoints, and re-analyzed 8 microarray- or RNA-seq based CS transcriptomes present in Gene Expression Omnibus that contained appropriate isogenic controls. We identified differentially expressed genes in each dataset, which were subsequently used for pathway enrichment analysis. Our findings revealed that gene expression of CCL2 and VCAN was altered in the majority of the CS transcriptomes analyzed. Over-representation enrichment analyses of human CS transcriptomes revealed significant changes in genes related to the MAPK, ERK1/2, PI3K-Akt pathways, alongside pathways related to neuronal processes and extracellular matrix metabolism. Additionally, gene-set enrichment analysis of nervous tissue CS datasets highlighted terms related to inflammation and synapse biology. These pathways and processes may contribute to the neurological dysfunction and overall phenotype of CS, presenting promising avenues for future research into the etiology and potential treatments for this aging-related disorder.

Ultraviolet (UV) radiation is a major environmental factor that induces DNA lesions. Cells have evolved repair pathways, in which the transcription-coupled nucleotide excision repair (TC-NER) has a central role in removing the lesions. Here we demonstrate that DGCR8, known as a crucial component in microRNA biogenesis, coordinates the UV-induced formation of the TC-NER complex by interacting with TC-NER factors. These interactions could depend on the phosphorylation of Serine 153 of DGCR8, potentially serving as a functional switch from miRNA biogenesis to the TC-NER process. Interestingly, DGCR8 is also involved in recruiting chromatin remodelers, SPT16 and SMARCA5, for the TC-NER initiation, regulating UV-induced DNA/RNA hybrids (R-loops), and modulating DNA replication through the ATR-CHK1 checkpoint pathway. These findings reveal a novel essential regulator of TC-NER independently of miRNA processing and provide new insights into the relevant biological processes and pathological mechanisms.

Abstract The mycotoxin, aflatoxin B1 (AFB1), is a potent mutagen that contaminates agricultural food supplies. After ingestion, AFB1 is oxidized into a reactive electrophile that alkylates DNA, forming bulky lesions such as the genotoxic formamidopyrimidine lesion, AFB1-Fapy dG. This lesion is mainly repaired by nucleotide excision repair (NER) in bacteria; however, in humans the picture is less clear. We report a plasmid-based host cell reactivation assay containing a site-specific AFB1-Fapy dG lesion and present evidence that this lesion is mainly repaired by transcription-coupled NER (TC-NER) in human cells. Using a combination of isogenic knockout cell lines and immortalized fibroblasts from xeroderma pigmentosum and Cockayne syndrome patients, we show that the TC-NER factors CSA, CSB, and UVSSA are required for efficient AFB1-Fapy dG repair, while the global-genome NER protein, XPC, is dispensable. Furthermore, knockout of CSB or UVSSA impairs AFB1-Fapy dG repair to a similar degree as knockout of the core NER nuclease, XPF. Our data indicate that TC-NER is the major repair pathway for AFB1-Fapy dG adducts in human cells.

Serine/threonine protein kinase 19 (STK19) has been reported to phosphorylate and activate oncogenic NRAS to promote melanomagenesis. However, concerns have been raised about whether STK19 is a kinase. STK19 has also been identified as a putative factor involved in the transcription-coupled nucleotide excision repair (TC-NER) pathway. In this study, we determined the 1.32 Å crystal structure of human STK19. The structure reveals that STK19 is a winged helix (WH) protein consisting of three tandem WH domains. STK19 binds more strongly to double-stranded DNA and RNA (dsDNA/dsRNA) than to ssDNA. A positively charged patch centered on helix WH3-H1 contributes to dsDNA binding, which is unusual because the WH domain typically uses helix H3 as the recognition helix. Importantly, mutations of the conserved residues in the basic patch, K186N, R200W, and R215W, are found in cancer patients, and these mutations compromise STK19 DNA binding. Other mutations have been predicted to produce a similar effect, including two mutations that disrupt the nuclear localization signal (NLS) motif. These mutations may indirectly impact the DNA binding capacity of STK19 by interfering with its nuclear localization.

In bacteria, mutations lead to the evolution of antibiotic resistance, which is one the main public health problems of the 21st century. Therefore, determining which cellular processes most frequently contribute to mutagenesis, especially in cells that have not been exposed to exogenous DNA damage, is critical. Here, we show that endogenous oxidative stress is a key driver of mutagenesis and the subsequent development of antibiotic resistance. This is the case for all classes of antibiotics tested and across highly divergent species, including patient-derived strains. We show that the transcription-coupled repair pathway, which uses the nucleotide excision repair proteins (TC-NER), is responsible for endogenous oxidative stress-dependent mutagenesis and subsequent evolution. This strongly suggests that a majority of mutations arise through transcription-associated processes rather than the replication fork. In addition to determining that the NER proteins play a critical role in mutagenesis and evolution, we also identify the DNA polymerases responsible for this process. Our data strongly suggest that cooperation between three different mutagenic DNA polymerases, likely at the last step of TC-NER, is responsible for mutagenesis and evolution. Overall, our work identifies that a highly conserved pathway drives mutagenesis due to endogenous oxidative stress, which has broad implications for all diseases of evolution, including antibiotic resistance development.

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Significance Nucleotide excision DNA repair (NER) removes a large variety of genomic lesions. NER can be initiated through two distinct pathways: global genome repair (GG-NER) and transcription-coupled repair (TC-NER). Both pathways subsequently funnel into a common pathway that involves recruitment of the transcription factor IIH (TFIIH) complex and the central scaffold protein XPA that enables full NER complex assembly. Although downstream steps after damage recognition are thought to be identical, we identify a disease-associated mutation in XPA that severely weakens the interaction with the TFIIH complex causing TC-NER to be affected to a greater extent than GG-NER. This differential impact on GG-NER and TC-NER suggests unanticipated mechanistic differences in the transition from lesion recognition to dual incision for the two pathways of NER.

Trypanosoma cruzi is the etiological agent of Chagas disease and a peculiar eukaryote with unique biological characteristics. DNA damage can block RNA polymerase, activating transcription-coupled nucleotide excision repair (TC-NER), a DNA repair pathway specialized in lesions that compromise transcription. If transcriptional stress is unresolved, arrested RNA polymerase can activate programmed cell death. Nonetheless, how this parasite modulates these processes is unknown. Here, we demonstrate that T. cruzi cell death after UV irradiation, a genotoxic agent that generates lesions resolved by TC-NER, depends on active transcription and is signaled mainly by an apoptotic-like pathway. Pre-treated parasites with α-amanitin, a selective RNA polymerase II inhibitor, become resistant to such cell death. Similarly, the gamma pre-irradiated cells are more resistant to UV when the transcription processes are absent. The Cockayne Syndrome B protein (CSB) recognizes blocked RNA polymerase and can initiate TC-NER. Curiously, CSB overexpression increases parasites' cell death shortly after UV exposure. On the other hand, at the same time after irradiation, the single-knockout CSB cells show resistance to the same treatment. UV-induced fast death is signalized by the exposition of phosphatidylserine to the outer layer of the membrane, indicating a cell death mainly by an apoptotic-like pathway. Furthermore, such death is suppressed in WT parasites pre-treated with inhibitors of ataxia telangiectasia and Rad3-related (ATR), a key DDR kinase. Signaling for UV radiation death may be related to R-loops since the overexpression of genes associated with the resolution of these structures suppress it. Together, results suggest that transcription blockage triggered by UV radiation activates an ATR-dependent apoptosis-like mechanism in T. cruzi, with the participation of CSB protein in this process.

Abstract DNA damage severely impedes gene transcription by RNA polymerase II (Pol II), causing cellular dysfunction. Transcription-Coupled Nucleotide Excision Repair (TC-NER) specifically removes such transcription-blocking damage. TC-NER initiation relies on the CSB, CSA and UVSSA proteins; loss of any results in complete TC-NER deficiency. Strikingly, UVSSA deficiency results in UV-Sensitive Syndrome (UVSS), with mild cutaneous symptoms, while loss of CSA or CSB activity results in the severe Cockayne Syndrome (CS), characterized by neurodegeneration and premature aging. Thus far the underlying mechanism for these contrasting phenotypes remains unclear. Live-cell imaging approaches reveal that in TC-NER proficient cells, lesion-stalled Pol II is swiftly resolved, while in CSA and CSB knockout (KO) cells, elongating Pol II remains damage-bound, likely obstructing other DNA transacting processes and shielding the damage from alternative repair pathways. In contrast, in UVSSA KO cells, Pol II is cleared from the damage via VCP-mediated proteasomal degradation which is fully dependent on the CRL4CSA ubiquitin ligase activity. This Pol II degradation might provide access for alternative repair mechanisms, such as GG-NER, to remove the damage. Collectively, our data indicate that the inability to clear lesion-stalled Pol II from the chromatin, rather than TC-NER deficiency, causes the severe phenotypes observed in CS.

Abstract Accumulation of DNA damage resulting from reactive oxygen species was proposed to cause neurological and degenerative disease in patients, deficient in nucleotide excision repair (NER) or its transcription-coupled subpathway (TC-NER). Here, we assessed the requirement of TC-NER for the repair of specific types of oxidatively generated DNA modifications. We incorporated synthetic 5′,8-cyclo-2′-deoxypurine nucleotides (cyclo-dA, cyclo-dG) and thymine glycol (Tg) into an EGFP reporter gene to measure transcription-blocking potentials of these modifications in human cells. Using null mutants, we further identified the relevant DNA repair components by a host cell reactivation approach. The results indicated that NTHL1-initiated base excision repair is by far the most efficient pathway for Tg. Moreover, Tg was efficiently bypassed during transcription, which effectively rules out TC-NER as an alternative repair mechanism. In a sharp contrast, both cyclopurine lesions robustly blocked transcription and were repaired by NER, wherein the specific TC-NER components CSB/ERCC6 and CSA/ERCC8 were as essential as XPA. Instead, repair of classical NER substrates, cyclobutane pyrimidine dimer and N-(deoxyguanosin-8-yl)-2-acetylaminofluorene, occurred even when TC-NER was disrupted. The strict requirement of TC-NER highlights cyclo-dA and cyclo-dG as candidate damage types, accountable for cytotoxic and degenerative responses in individuals affected by genetic defects in this pathway.

NER operates through two distinct pathways: global genome repair (GGR) and transcription-coupled repair (TC-NER). TC-NER, as its name suggests, is specifically dedicated to rectifying lesions that obstruct the progression of RNA polymerase during active transcription. This process is imperative for the maintenance of cellular homeostasis, as lesions encountered by the transcription machinery can lead to stalling, erroneous transcription, and ultimately, deleterious cellular outcomes. At the heart of the TC-NER pathway lies ERCC6, a helicase orchestrating the repair of DNA lesions within transcribed regions of the genome. ERCC6 (AKA CS-B) plays a pivotal role in detecting and coordinating the repair of transcription-blocking lesions and has been implicated in a plethora of cellular processes underscoring its multifaceted nature and its broader impact on genome stability and cellular health, as exemplified by the severity of syndromes in individuals with loss-of-function mutations. In the US, compared to Caucasians (CC), AA are at higher risk for developing PCa and have more aggressive disease that is refractory to treatment, to some extent explained by genetic differences in some cases attributed to alterations of their “Repairome”. GWAS revealed that, unlike CC, 89% of AA-PCa have at least one mutation in NER pathway genes. A defect in ERCC6 activity, either through reduced expression or mutation (e.g., M1097V found frequently in AA) may result in impaired TC-NER. This may be compounded by aging or obesity-related oxidative stress, resulting in progressive accumulation of damaged bases (e.g., 8-OG) and consequent (cancer-propensity) mutations, the correction of which involves BER or NER. This was investigated in part with PCa cell lines engineered via CRISPR-SDM to carry that particular genomic mutation, by studying their sensitivity to UV and the efficiency of removal of CPDs in vivo. In addition, we have begun investigation of its function in the variants (engineered and naturally found in PCa. Lines) in vitro, after IP from cells, by studying its intrinsic ATPase activity. We have also identified the kinase NEK1 as an important novel interactor and regulator of ERCC6. We previously identified NEK1 as being an early regulator of the adaptive response of PCa cells to ADT. Citation Format: Oluwatobi M. Ogundepo, Arrigo De Benedetti. NEK1 phosphorylation modulates ERCC6 in transcription coupled nucleotide excision repair with implications for prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 408.

Abstract DNA–protein crosslinks (DPCs) form following exposure to various alkylating agents, including environmental carcinogens, cancer chemotherapeutics, and reactive aldehydes. If not repaired, DPCs can interfere with key biological processes such as transcription and replication and activate programmed cell death. A growing body of evidence implicates nucleotide excision repair (NER), homologous recombination, and other mechanisms in the removal of DPCs. However, the effects of genomic context on DPC formation and removal have not been comprehensively addressed. Using a combination of next-generation sequencing and DPC enrichment via protein precipitation, we show that DPCs induced following exposure to formaldehyde are non-randomly distributed across the human genome, based on chromatin state. The data further show that the efficiency of DPC removal correlates with transcription at loci transcribed by RNA polymerase II. Data presented herein indicate that efficient removal of chromosomal DPCs requires both the Cockayne syndrome group B gene as well as “downstream” TC-NER factor xeroderma pigmentosum group A gene. In contrast, loci transcribed by RNA polymerase I showed no evidence of transcription-coupled DPC removal. Taken together, our results indicate that complex interactions between chromatin organization, transcriptional activity, and numerous DNA repair pathways dictate genomic patterns of DPC formation and removal.

Due to a demonstrated lack of DNA repair deficiencies, clear cell renal cell carcinoma (ccRCC) has not benefitted from targeted synthetic lethality-based therapies. We investigated whether nucleotide excision repair (NER) deficiency is present in an identifiable subset of ccRCC cases that would render those tumors sensitive to therapy targeting this specific DNA repair pathway aberration. We used functional assays that detect UV-induced 6–4 pyrimidine-pyrimidone photoproducts to quantify NER deficiency in ccRCC cell lines. We also measured sensitivity to irofulven, an experimental cancer therapeutic agent that specifically targets cells with inactivated transcription-coupled nucleotide excision repair (TC-NER). In order to detect NER deficiency in clinical biopsies, we assessed whole exome sequencing data for the presence of an NER deficiency associated mutational signature previously identified in ERCC2 mutant bladder cancer. Functional assays showed NER deficiency in ccRCC cells. Some cell lines showed irofulven sensitivity at a concentration that is well tolerated by patients. Prostaglandin reductase 1 (PTGR1), which activates irofulven, was also associated with this sensitivity. Next generation sequencing data of the cell lines showed NER deficiency-associated mutational signatures. A significant subset of ccRCC patients had the same signature and high PTGR1 expression. ccRCC cell line-based analysis showed that NER deficiency is likely present in this cancer type. Approximately 10% of ccRCC patients in the TCGA cohort showed mutational signatures consistent with ERCC2 inactivation associated NER deficiency and also substantial levels of PTGR1 expression. These patients may be responsive to irofulven, a previously abandoned anticancer agent that has minimal activity in NER-proficient cells.