Cockayne Syndrome Group B Research Articles

Perspectives in the investigation of Cockayne syndrome group B neurological disease: the utility of patient-derived brain organoid models.

Cockayne syndrome (CS) group B (CSB), which results from mutations in the excision repair cross-complementation group 6 (ERCC6) genes, which produce CSB protein, is an autosomal recessive disease characterized by multiple progressive disorders including growth failure, microcephaly, skin photosensitivity, and premature aging. Clinical data show that brain atrophy, demyelination, and calcification are the main neurological manifestations of CS, which progress with time. Neuronal loss and calcification occur in various brain areas, particularly the cerebellum and basal ganglia, resulting in dyskinesia, ataxia, and limb tremors in CSB patients. However, the understanding of neurodevelopmental defects in CS has been constrained by the lack of significant neurodevelopmental and functional abnormalities observed in CSB-deficient mice. In this review, we focus on elucidating the protein structure and distribution of CSB and delve into the impact of CSB mutations on the development and function of the nervous system. In addition, we provide an overview of research models that have been instrumental in exploring CS disorders, with a forward-looking perspective on the substantial contributions that brain organoids are poised to further advance this field.

Cockayne Syndrome Linked to Elevated R-Loops Induced by Stalled RNA Polymerase II during Transcription Elongation

Mutations in the Cockayne Syndrome group B (CSB) gene cause cancer in mice, but premature aging and severe neurodevelopmental defects in humans. CSB, a member of the SWI/SNF family of chromatin remodelers, plays diverse roles in regulating gene expression and transcription-coupled nucleotide excision repair (TC-NER); however, these functions do not explain the distinct phenotypic differences observed between CSB-deficient mice and humans. During investigating Cockayne Syndrome-associated genome instability, we uncover an intrinsic mechanism that involves elongating RNA polymerase II (RNAPII) undergoing transient pauses at internal T-runs where CSB is required to propel RNAPII forward. Consequently, CSB deficiency retards RNAPII elongation in these regions, and when coupled with G-rich sequences upstream, exacerbates genome instability by promoting R-loop formation. These R-loop prone motifs are notably abundant in relatively long genes related to neuronal functions in the human genome, but less prevalent in the mouse genome. These findings provide mechanistic insights into differential impacts of CSB deficiency on mice versus humans and suggest that the manifestation of the Cockayne Syndrome phenotype in humans results from the progressive evolution of mammalian genomes.

CSB and SMARCAL1 compete for RPA32 at stalled forks and differentially control the fate of stalled forks in BRCA2-deficient cells.

CSB (Cockayne syndrome group B) and SMARCAL1 (SWI/SNF-related, matrix-associated, actin-dependent, regulator of chromatin, subfamily A-like 1) are DNA translocases that belong to the SNF2 helicase family. They both are enriched at stalled replication forks. While SMARCAL1 is recruited by RPA32 to stalled forks, little is known about whether RPA32 also regulates CSB's association with stalled forks. Here, we report that CSB directly interacts with RPA, at least in part via a RPA32C-interacting motif within the N-terminal region of CSB. Modeling of the CSB-RPA32C interaction suggests that CSB binds the RPA32C surface previously shown to be important for binding of UNG2 and SMARCAL1. We show that this interaction is necessary for promoting fork slowing and fork degradation in BRCA2-deficient cells but dispensable for mediating restart of stalled forks. CSB competes with SMARCAL1 for RPA32 at stalled forks and acts non-redundantly with SMARCAL1 to restrain fork progression in response to mild replication stress. In contrast to CSB stimulated restart of stalled forks, SMARCAL1 inhibits restart of stalled forks in BRCA2-deficient cells, likely by suppressing BIR-mediated repair of collapsed forks. Loss of CSB leads to re-sensitization of SMARCAL1-depleted BRCA2-deficient cells to chemodrugs, underscoring a role of CSB in targeted cancer therapy.

Heat shock protein DNAJA2 regulates transcription-coupled repair by triggering CSB degradation via chaperone-mediated autophagy

Transcription-coupled nucleotide excision repair (TC-NER) is an important genome maintenance system that preferentially removes DNA lesions on the transcribed strand of actively transcribed genes, including non-coding genes. TC-NER involves lesion recognition by the initiation complex consisting of RNA polymerase II (Pol II) and Cockayne syndrome group B (CSB), followed by NER-catalyzed lesion removal. However, the efficient lesion removal requires the initiation complex to yield the right of way to the excision machinery, and how this occurs in a timely manner is unknown. Here we show that heat shock protein DNAJA2 facilitates the HSC70 chaperone-mediated autophagy (CMA) to degrade CSB during TC-NER. DNAJA2 interacts with and enables HSC70 to recognize sumoylated CSB. This triggers the removal of both CSB and Pol II from the lesion site in a manner dependent on lysosome receptor LAMP2A. Defects in DNAJA2, HSC70 or LAMP2A abolish CSB degradation and block TC-NER. Our findings discover DNAJA2-mediated CMA as a critical regulator of TC-NER, implicating the DNAJA2-HSC70-CMA axis factors in genome maintenance.

CSB Regulates Pathway Choice in Response to DNA Replication Stress Induced by Camptothecin.

Topoisomerase inhibitor camptothecin (CPT) induces fork stalling and is highly toxic to proliferating cells. However, how cells respond to CPT-induced fork stalling has not been fully characterized. Here, we report that Cockayne syndrome group B (CSB) protein inhibits PRIMPOL-dependent fork repriming in response to a low dose of CPT. At a high concentration of CPT, CSB is required to promote the restart of DNA replication through MUS81-RAD52-POLD3-dependent break-induced replication (BIR). In the absence of CSB, resumption of DNA synthesis at a high concentration of CPT can occur through POLQ-LIG3-, LIG4-, or PRIMPOL-dependent pathways, which are inhibited, respectively, by RAD51, BRCA1, and BRCA2 proteins. POLQ and LIG3 are core components of alternative end joining (Alt-EJ), whereas LIG4 is a core component of nonhomologous end joining (NHEJ). These results suggest that CSB regulates fork restart pathway choice following high-dosage CPT-induced fork stalling, promoting BIR but inhibiting Alt-EJ, NHEJ, and fork repriming. We find that loss of CSB and BRCA2 is a toxic combination to genomic stability and cell survival at a high concentration of CPT, which is likely due to accumulation of ssDNA gaps, underscoring an important role of CSB in regulating the therapy response in cancers lacking functional BRCA2.

Role of Cockayne Syndrome Group B Protein in Replication Stress: Implications for Cancer Therapy.

A variety of endogenous and exogenous insults are capable of impeding replication fork progression, leading to replication stress. Several SNF2 fork remodelers have been shown to play critical roles in resolving this replication stress, utilizing different pathways dependent upon the nature of the DNA lesion, location on the DNA, and the stage of the cell cycle, to complete DNA replication in a manner preserving genetic integrity. Under certain conditions, however, the attempted repair may lead to additional genetic instability. Cockayne syndrome group B (CSB) protein, a SNF2 chromatin remodeler best known for its role in transcription-coupled nucleotide excision repair, has recently been shown to catalyze fork reversal, a pathway that can provide stability of stalled forks and allow resumption of DNA synthesis without chromosome breakage. Prolonged stalling of replication forks may collapse to give rise to DNA double-strand breaks, which are preferentially repaired by homology-directed recombination. CSB plays a role in repairing collapsed forks by promoting break-induced replication in S phase and early mitosis. In this review, we discuss roles of CSB in regulating the sources of replication stress, replication stress response, as well as the implications of CSB for cancer therapy.

Cockayne syndrome group B protein uses its DNA translocase activity to promote mitotic DNA synthesis

Mitotic DNA synthesis, also known as MiDAS, has been suggested to be a form of RAD52-dependent break-induced replication (BIR) that repairs under-replicated DNA regions of the genome in mitosis prior to chromosome segregation. Cockayne syndrome group B (CSB) protein, a chromatin remodeler of the SNF2 family, has been implicated in RAD52-dependent BIR repair of stalled replication forks. However, whether CSB plays a role in MiDAS has not been characterized. Here, we report that CSB functions epistatically with RAD52 to promote MiDAS at common fragile sites in response to replication stress, and prevents genomic instability associated with defects in MiDAS. We show that CSB is dependent upon the conserved phenylalanine at position 796 (F796), which lies in the recently-reported pulling pin that is required for CSB’s translocase activity, to mediate MiDAS, suggesting that CSB uses its DNA translocase activity to promote MiDAS. Structural analysis reveals that CSB shares with a subset of SNF2 family proteins a translocase regulatory region (TRR), which is important for CSB’s function in MiDAS. We further demonstrate that phosphorylation of S1013 in the TRR regulates the function of CSB in MiDAS and restart of stalled forks but not in fork degradation in BRCA2-deficient cells and UV repair. Taken together, these results suggest that the DNA translocase activity of CSB in vivo is likely to be highly regulated by post-translational modification in a context-specific manner.

Camptothecin compromises transcription recovery and cell survival against cisplatin and ultraviolet irradiation regardless of transcription-coupled nucleotide excision repair

DNA-damaging anti-cancer drugs are used clinically to induce cell death by causing DNA strand breaks or DNA replication stress. Camptothecin (CPT) and cisplatin are commonly used anti-cancer drugs, and their combined use enhances the anti-tumour effects. However, the mechanism underlying this enhanced effect has not been well studied. In this study, we analysed the combined effect of CPT and cisplatin or ultraviolet (UV) and found that CPT suppresses transcription recovery after UV damage and induces the disappearance of the Cockayne syndrome group B (CSB) protein, a transcription-coupled nucleotide excision repair (TC-NER) factor. This CPT-induced disappearance of CSB expression was suppressed by proteasome and transcription inhibitors. Moreover, CSB ubiquitination was detected after CPT treatment in a transcription-dependent manner, suggesting that the transcription stress caused by CPT induces CSB ubiquitination, resulting in CSB undetectability. However, Cockayne syndrome group A (CSA) and CUL4A were not involved in the CPT-induced CSB undetectability, suggesting that CSB ubiquitination caused by CPT is regulated differently from the UV response. However, cisplatin or UV sensitivity was enhanced by CPT even in CSB- or CSA-knockout cells. Furthermore, the excessive CSB expression, which suppressed CSB ubiquitination, did not cancel the combined effect of CPT. These results suggest that CPT blocks the repair of cisplatin or UV-induced DNA damage regardless of TC-NER status. CPT possibly compromised the alternative repair pathways other than TC-NER, leading to the suppression of transcription recovery and enhancement of cell killing.

Cockayne syndrome group B protein regulates fork restart, fork progressionand MRE11-dependent fork degradation in BRCA1/2-deficient cells.

Cockayne syndrome group B (CSB) protein has been implicated in the repair of a variety of DNA lesions that induce replication stress. However, little is known about its role at stalled replication forks. Here, we report that CSB is recruited to stalled forks in a manner dependent upon its T1031 phosphorylation by CDK. While dispensable for MRE11 association with stalled forks in wild-type cells, CSB is required for further accumulation of MRE11 at stalled forks in BRCA1/2-deficient cells. CSB promotes MRE11-mediated fork degradation in BRCA1/2-deficient cells. CSB possesses an intrinsic ATP-dependent fork reversal activity in vitro, which is activated upon removal of its N-terminal region that is known to autoinhibit CSB’s ATPase domain. CSB functions similarly to fork reversal factors SMARCAL1, ZRANB3 and HLTF to regulate slowdown in fork progression upon exposure to replication stress, indicative of a role of CSB in fork reversal in vivo. Furthermore, CSB not only acts epistatically with MRE11 to facilitate fork restart but also promotes RAD52-mediated break-induced replication repair of double-strand breaks arising from cleavage of stalled forks by MUS81 in BRCA1/2-deficient cells. Loss of CSB exacerbates chemosensitivity in BRCA1/2-deficient cells, underscoring an important role of CSB in the treatment of cancer lacking functional BRCA1/2.

Whole Exome Sequencing Identifies a Novel Homozygous Missense Mutation in the CSB Protein-Encoding ERCC6 Gene in a Taiwanese Boy with Cockayne Syndrome.

Background: Cockayne syndrome (CS) is a rare form of dwarfism that is characterized by progressive premature aging. CS is typically caused by mutations in the excision repair cross-complementing protein group 6 (ERCC6) gene that encodes the CS group B (CSB) protein. Using whole exome sequencing, we recently identified a novel homozygous missense mutation (Leu536Trp) in CSB in a Taiwanese boy with CS. Since the current database (Varsome) interprets this variant as likely pathogenic, we utilized a bioinformatic tool to investigate the impact of Leu536Trp as well as two other variants (Arg453Ter, Asp532Gly) in similar articles on the CSB protein structure stability. Methods: We used iterative threading assembly refinement (I-TASSER) to generate a predictive 3D structure of CSB. We calculated the change of mutation energy after residues substitution on the protein stability using I-TASSER as well as the artificial intelligence program Alphafold. Results: The Asp532Gly variant destabilized both modeled structures, while the Leu536Trp variant showed no effect on I-TASSER’s model but destabilized the Alphafold’s modeled structure. Conclusions: We propose here the first case of CS associated with a novel homozygous missense mutation (Leu536Trp) in CSB. Furthermore, we suggest that the Asp532Gly and Leu536Trp variants are both pathogenic after bioinformatic analysis of protein stability.

Neuroblastoma Cells Depend on CSB for Faithful Execution of Cytokinesis and Survival.

Neuroblastoma, the most common extra-cranial solid tumor of early childhood, is one of the major therapeutic challenges in child oncology: it is highly heterogenic at a genetic, biological, and clinical level. The high-risk cases have one of the least favorable outcomes amongst pediatric tumors, and the mortality rate is still high, regardless of the use of intensive multimodality therapies. Here, we observed that neuroblastoma cells display an increased expression of Cockayne Syndrome group B (CSB), a pleiotropic protein involved in multiple functions such as DNA repair, transcription, mitochondrial homeostasis, and cell division, and were recently found to confer cell robustness when they are up-regulated. In this study, we demonstrated that RNAi-mediated suppression of CSB drastically impairs tumorigenicity of neuroblastoma cells by hampering their proliferative, clonogenic, and invasive capabilities. In particular, we observed that CSB ablation induces cytokinesis failure, leading to caspases 9 and 3 activation and, subsequently, to massive apoptotic cell death. Worthy of note, a new frontier in cancer treatment, already proved to be successful, is cytokinesis-failure-induced cell death. In this context, CSB ablation seems to be a new and promising anticancer strategy for neuroblastoma therapy.

Transcription-coupled nucleotide excision repair: New insights revealed by genomic approaches

Elongation of RNA polymerase II (Pol II) is affected by many factors including DNA damage. Bulky damage, such as lesions caused by ultraviolet (UV) radiation, arrests Pol II and inhibits gene transcription, and may lead to genome instability and cell death. Cells activate transcription-coupled nucleotide excision repair (TC-NER) to remove Pol II-impeding damage and allow transcription resumption. TC-NER initiation in humans is mediated by Cockayne syndrome group B (CSB) protein, which binds to the stalled Pol II and promotes assembly of the repair machinery. Given the complex nature of the TC-NER pathway and its unique function at the interface between transcription and repair, new approaches are required to gain in-depth understanding of the mechanism. Advances in genomic approaches provide an important opportunity to investigate how TC-NER is initiated upon damage-induced Pol II stalling and what factors are involved in this process. In this Review, we discuss new mechanisms of TC-NER revealed by genome-wide DNA damage mapping and new TC-NER factors identified by high-throughput screening. As TC-NER conducts strand-specific repair of mutagenic damage, we also discuss how this repair pathway causes mutational strand asymmetry in the cancer genome.

Cockayne Syndrome Group B (CSB): The Regulatory Framework Governing the Multifunctional Protein and Its Plausible Role in Cancer.

Cockayne syndrome (CS) is a DNA repair syndrome characterized by a broad spectrum of clinical manifestations such as neurodegeneration, premature aging, developmental impairment, photosensitivity and other symptoms. Mutations in Cockayne syndrome protein B (CSB) are present in the vast majority of CS patients and in other DNA repair-related pathologies. In the literature, the role of CSB in different DNA repair pathways has been highlighted, however, new CSB functions have been identified in DNA transcription, mitochondrial biology, telomere maintenance and p53 regulation. Herein, we present an overview of identified structural elements and processes that impact on CSB activity and its post-translational modifications, known to balance the different roles of the protein not only during normal conditions but most importantly in stress situations. Moreover, since CSB has been found to be overexpressed in a number of different tumors, its role in cancer is presented and possible therapeutic targeting is discussed.

Current and emerging roles of Cockayne syndrome group B (CSB) protein.

Cockayne syndrome (CS) is a segmental premature aging syndrome caused primarily by defects in the CSA or CSB genes. In addition to premature aging, CS patients typically exhibit microcephaly, progressive mental and sensorial retardation and cutaneous photosensitivity. Defects in the CSB gene were initially thought to primarily impair transcription-coupled nucleotide excision repair (TC-NER), predicting a relatively consistent phenotype among CS patients. In contrast, the phenotypes of CS patients are pleiotropic and variable. The latter is consistent with recent work that implicates CSB in multiple cellular systems and pathways, including DNA base excision repair, interstrand cross-link repair, transcription, chromatin remodeling, RNAPII processing, nucleolin regulation, rDNA transcription, redox homeostasis, and mitochondrial function. The discovery of additional functions for CSB could potentially explain the many clinical phenotypes of CSB patients. This review focuses on the diverse roles played by CSB in cellular pathways that enhance genome stability, providing insight into the molecular features of this complex premature aging disease.

Genome-wide role of Rad26 in promoting transcription-coupled nucleotide excision repair in yeast chromatin

Transcription-coupled nucleotide excision repair (TC-NER) is an important DNA repair mechanism that removes RNA polymerase (RNAP)-stalling DNA damage from the transcribed strand (TS) of active genes. TC-NER deficiency in humans is associated with the severe neurological disorder Cockayne syndrome. Initiation of TC-NER is mediated by specific factors such as the human Cockayne syndrome group B (CSB) protein or its yeast homolog Rad26. However, the genome-wide role of CSB/Rad26 in TC-NER, particularly in the context of the chromatin organization, is unclear. Here, we used single-nucleotide resolution UV damage mapping data to show that Rad26 and its ATPase activity is critical for TC-NER downstream of the first (+1) nucleosome in gene coding regions. However, TC-NER on the transcription start site (TSS)-proximal half of the +1 nucleosome is largely independent of Rad26, likely due to high occupancy of the transcription initiation/repair factor TFIIH in this nucleosome. Downstream of the +1 nucleosome, the combination of low TFIIH occupancy and high occupancy of the transcription elongation factor Spt4/Spt5 suppresses TC-NER in Rad26-deficient cells. We show that deletion of SPT4 significantly restores TC-NER across the genome in a rad26∆ mutant, particularly in the downstream nucleosomes. These data demonstrate that the requirement for Rad26 in TC-NER is modulated by the distribution of TFIIH and Spt4/Spt5 in transcribed chromatin and Rad26 mainly functions downstream of the +1 nucleosome to remove TC-NER suppression by Spt4/Spt5.

USP7-mediated deubiquitination differentially regulates CSB but not UVSSA upon UV radiation-induced DNA damage

ABSTRACT Cockayne syndrome group B (CSB) protein participates in transcription-coupled nucleotide excision repair. The stability of CSB is known to be regulated by ubiquitin-specific protease 7 (USP7). Yet, whether USP7 acts as a deubiquitinating enzyme for CSB is not clear. Here, we demonstrate that USP7 deubiquitinates CSB to maintain its levels after ultraviolet (UV)-induced DNA damage. While both CSB and UV-stimulated scaffold protein A (UVSSA) exhibit a biphasic decrease and recovery upon UV irradiation, only CSB recovery depends on USP7, which physically interacts with and deubiquitinates CSB. Meanwhile, CSB overexpression stabilizes UVSSA, but decrease UVSSA’s presence in nuclease-releasable/soluble chromatin, and increase the presence of ubiquitinated UVSSA in insoluble chromatin alongside CSB-ubiquitin conjugates. Remarkably, CSB overexpression also decreases CSB association with USP7 and UVSSA in soluble chromatin. UVSSA exists in several ubiquitinated forms, of which mono-ubiquitinated form and other ubiquitinated UVSSA forms are detectable upon 6xHistidine tag-based purification. The ubiquitinated UVSSA forms, however, are not cleavable by USP7 in vitro. Furthermore, USP7 disruption does not affect RNA synthesis but decreases the recovery of RNA synthesis following UV exposure. These results reveal a role of USP7 as a CSB deubiquitinating enzyme for fine-tuning the process of TC-NER in human cells.

Generation of splice switching oligonucleotides targeting the Cockayne syndrome group B gene product in order to change the diseased cell state

Cockayne syndrome (CS) is a severe disorder with no effective treatment. The Cockayne syndrome group B (CSB) gene is one gene responsible for CS and also causes UV sensitive syndrome (UVSS), a disorder that causes mild symptoms. How the CSB gene determines a patient's fate is unknown, but one intriguing point is that in UVSS patient cell, there are nonsense mutations in both alleles at the same position in each upstream region of the PiggyBac transposable element derived 3 (PGBD3) inserted region. In contrast, in CS patient cells, there is at least one allele with several mutations downstream of the PGBD3 inserted region, or there are homozygous mutations in exon 1. Here, we designed and synthesized 24 splice switching oligonucleotides (SSOs) to skip exon 3 in CSB mRNA. Use of these SSOs induced a frame shift in order to generate an alternative stop codon at the upstream region of the PGBD3 invasion site. As a result, a reduction of mitochondrial membrane potential following H2O2 treatment in CS cell was recovered. It was demonstrated that up-regulation of several gene expression brought about by SSOs are related to mitochondrial dysfunction in CS cells.

CSB: An Emerging Actionable Target for Cancer Therapy

The DNA repair protein Cockayne syndrome group B (CSB) is frequently found overexpressed in cancer cells. High CSB levels favor tumor cell proliferation whilst inhibiting apoptosis. Conversely, the suppression of CSB has significant anticancer effects. In this manuscript we describe CSB downregulation as a potential new therapeutic approach in cancer.

CSB ablation induced apoptosis is mediated by increased endoplasmic reticulum stress response.

The DNA repair protein Cockayne syndrome group B (CSB) has been recently identified as a promising anticancer target. Suppression, by antisense technology, of this protein causes devastating effects on tumor cells viability, through a massive induction of apoptosis, while being non-toxic to non-transformed cells. To gain insights into the mechanisms underlying the pro-apoptotic effects observed after CSB ablation, global gene expression patterns were determined, to identify genes that were significantly differentially regulated as a function of CSB expression. Our findings revealed that response to endoplasmic reticulum stress and response to unfolded proteins were ranked top amongst the cellular processes affected by CSB suppression. The major components of the endoplasmic reticulum stress-mediated apoptosis pathway, including pro-apoptotic factors downstream of the ATF3-CHOP cascade, were dramatically up-regulated. Altogether our findings add new pieces to the understanding of CSB mechanisms of action and to the molecular basis of CS syndrome.

Stabilization of Ultraviolet (UV)-stimulated Scaffold Protein A by Interaction with Ubiquitin-specific Peptidase 7 Is Essential for Transcription-coupled Nucleotide Excision Repair

UV-sensitive syndrome is an autosomal recessive disorder characterized by hypersensitivity to UV light and deficiency in transcription-coupled nucleotide excision repair (TC-NER), a subpathway of nucleotide excision repair that rapidly removes transcription-blocking DNA damage. UV-sensitive syndrome consists of three genetic complementation groups caused by mutations in the CSA, CSB, and UVSSA genes. UV-stimulated scaffold protein A (UVSSA), the product of UVSSA, which is required for stabilization of Cockayne syndrome group B (CSB) protein and reappearance of the hypophosphorylated form of RNA polymerase II after UV irradiation, forms a complex with ubiquitin-specific peptidase 7 (USP7). In this study, we demonstrated that the deubiquitination activity of USP7 is suppressed by its interaction with UVSSA. The interaction required the tumor necrosis factor receptor-associated factor domain of USP7 and the central region of UVSSA and was disrupted by an amino acid substitution in the tumor necrosis factor receptor-associated factor-binding motif of UVSSA. Cells expressing mutant UVSSA were highly sensitive to UV irradiation and defective in recovery of RNA synthesis after UV irradiation. These results indicate that the interaction between UVSSA and USP7 is important for TC-NER. Furthermore, the mutant UVSSA was rapidly degraded by the proteasome, and CSB was also degraded after UV irradiation as observed in UVSSA-deficient cells. Thus, stabilization of UVSSA by interaction with USP7 is essential for TC-NER.