Ubiquitination of DNA Damage-Stalled RNAPII Promotes Transcription-Coupled Repair.

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.

A disease-associated XPA allele interferes with TFIIH binding and primarily affects transcription-coupled nucleotide excision repair.

XPA is a central scaffold protein that coordinates the assembly of repair complexes in the global genome (GG-NER) and transcription-coupled nucleotide excision repair (TC-NER) subpathways. Inactivating mutations in XPA cause xeroderma pigmentosum (XP), which is characterized by extreme UV sensitivity and a highly elevated skin cancer risk. Here, we describe two Dutch siblings in their late forties carrying a homozygous H244R substitution in the C-terminus of XPA. They present with mild cutaneous manifestations of XP without skin cancer but suffer from marked neurological features, including cerebellar ataxia. We show that the mutant XPA protein has a severely weakened interaction with the transcription factor IIH (TFIIH) complex leading to an impaired association of the mutant XPA and the downstream endonuclease ERCC1-XPF with NER complexes. Despite these defects, the patient-derived fibroblasts and reconstituted knockout cells carrying the XPA-H244R substitution show intermediate UV sensitivity and considerable levels of residual GG-NER (~50%), in line with the intrinsic properties and activities of the purified protein. By contrast, XPA-H244R cells are exquisitely sensitive to transcription-blocking DNA damage, show no detectable recovery of transcription after UV irradiation, and display a severe deficiency in TC-NER-associated unscheduled DNA synthesis. Our characterization of a new case of XPA deficiency that interferes with TFIIH binding and primarily affects the transcription-coupled subpathway of nucleotide excision repair, provides an explanation of the dominant neurological features in these patients, and reveals a specific role for the C-terminus of XPA in TC-NER.

Effects of nongenetic factors on immune cell dynamics in early childhood: The Generation R Study.

BACKGROUND: Numbers of blood leukocyte subsets are highly dynamic in childhood and differ greatly between subjects. Interindividual variation is only partly accounted for by genetic factors. OBJECTIVE: We sought to determine which nongenetic factors affect the dynamics of innate leukocytes and naive and memory lymphocyte subsets. METHODS: We performed 6-color flow cytometry and linear mixed-effects modeling to define the dynamics of 62 leukocyte subsets from birth to 6 years of age in 1182 children, with 1 to 5 measurements per subject. Subsequently, we defined the effect of prenatal maternal lifestyle-related or immune-mediated determinants, birth characteristics, and bacterial/viral exposure-related determinants on leukocyte subset dynamics. RESULTS: Functionally similar leukocyte populations were grouped by using unbiased hierarchical clustering of patterns of age-related leukocyte dynamics. Innate leukocyte numbers were high at birth and predominantly affected by maternal low education level. Naive lymphocyte counts peaked around 1 year, whereas most memory lymphocyte subsets more gradually increased during the first 4 years of life. Dynamics of CD4+ T cells were predominantly associated with sex, birth characteristics, and persistent infections with cytomegalovirus (CMV) or EBV. CD8+ T cells were predominantly associated with CMV and EBV infections, and T-cell receptor γδ+ T cells were predominantly associated with premature rupture of membranes and CMV infection. B-cell subsets were predominantly associated with sex, breast-feeding, and Helicobacter pylori carriership. CONCLUSIONS: Our study identifies specific dynamic patterns of leukocyte subset numbers, as well as nongenetic determinants that affect these patterns, thereby providing new insights into the shaping of the childhood immune system.

No Interactive Effects of Sex and Persistent Cytomegalovirus on Immune Phenotypes in Young Children: The Generation R Study.

Persistent infections with cytomegalovirus (CMV) differentially affect the host immune phenotype in middle-aged males and females. Because CMV already impacts on T-cell memory at a young age, we studied whether these effects were modified by sex in 1,079 children with an average age of 6 years. Sex and CMV independently impacted on multiple B-cell and T-cell subsets. However, there was no significant effect of their interaction. Importantly, the effects of sex and CMV were in part explained by age and infection with other herpesviruses. Thus, immune aging is likely to be more complex, with involvement of hormonal changes with age, socioeconomic status, birth characteristics, and pathogen exposure.

The role of vitamin D on circulating memory T cells in children: The Generation R study.

BACKGROUND: Previous studies have demonstrated that vitamin D affects T-cell function and maturation via the vitamin D receptor. However, no studies in children have been performed on this topic. Because most of the T-cell memory is formed in the first 5 years of life, we aimed to determine the association between serum 25-hydroxyvitamin D (25(OH)D) levels and numbers of circulatory naive, central memory (Tcm), and effector memory (Tem) T lymphocytes in a large population of healthy children. METHODS: Among 3189 children participating in a population-based prospective cohort, we measured 25(OH)D levels and performed detailed immunophenotyping of naive and memory T lymphocytes at a median age of 6.0 years (95% range 5.7-7.9). Detailed lymphocyte subsets were available in 986 children. Multivariable linear regression analyses were performed to determine the association between 25(OH)D and the maturation of T lymphocytes in children adjusted for cord blood 25(OH)D levels, herpes seropositivity, sociodemographic and lifestyle confounders. Furthermore, multivariable logistic regression analyses were performed to determine associations between 25(OH)D and childhood infections. RESULTS: Higher 25(OH)D levels were associated with higher numbers of Tem lymphocytes. Every 10 nmol/L higher 25(OH)D was associated with 2.20% (95% CI 0.54-3.89; P=.009) higher CD4TemRA, 1.50% (95% CI 0.38-2.62; P=.008) higher CD4TemRO, and 1.82% (95% CI 0.11-3.56; P=.037) higher CD8TemRA cell numbers. Generally, stronger associations were observed among boys. 25(OH)D levels were not significantly associated with naive, Tcm cell numbers, herpes seropositivity, or URTIs. CONCLUSIONS: Our results suggest that vitamin D enhances cellular immunity in young children.

Cytomegalovirus- and Epstein-Barr Virus-Induced T-Cell Expansions in Young Children Do Not Impair Naive T-cell Populations or Vaccination Responses: The Generation R Study.

BACKGROUND: Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) induce effector memory T-cell expansions, which are variable and potentially depend on the age at primary exposure and coinfections. We evaluated the T-cell compartment and herpesvirus infections in 6-year-old children. METHODS: T-cell subsets and immunoglobulin G seropositivity for CMV, EBV, herpes-simplex virus 1, and varicella-zoster virus were studied in 1079 6-year-old children. A random subgroup of 225 children was evaluated for CMV and EBV seropositivity before 2 years of age and for vaccination responses against measles and tetanus. RESULTS: CMV and EBV infections were associated with significant expansions of CD27(-) and CD27(+) effector memory T cells, respectively. These expansions were enhanced in CMV-EBV-coinfected children and were independent of varicella-zoster virus or herpes-simplex virus 1 coinfection. Naive and central memory T-cell numbers were not affected, nor were anti-tetanus and anti-measles immunoglobulin G levels. Children infected before 2 years of age showed smaller effector memory T-cell expansions than those infected between 2 and 6 years of age. CONCLUSIONS: CMV- and EBV-related T-cell expansions do not impair naive T-cell numbers or maintenance of protective responses against nonrelated pathogens. Duration of infection was not directly related to larger expansions of effector memory T cells in children, suggesting that other mechanisms affect these expansions at later age.

Determinants of Ethnic Differences in Cytomegalovirus, Epstein-Barr Virus, and Herpes Simplex Virus Type 1 Seroprevalence in Childhood.

OBJECTIVE: To identify whether there are ethnic differences in cytomegalovirus (CMV), Epstein-Barr virus (EBV), and herpes simplex virus type 1 (HSV-1) seroprevalence rates in children at 6 years of age, and when present, to evaluate how these differences can be explained by sociodemographic and environmental factors. STUDY DESIGN: This study was embedded within a multi-ethnic population-based prospective cohort study. Serum IgG levels against CMV, EBV, and HSV-1 were measured by enzyme-linked immunosorbent assay in 4464 children (median age 6.0 years). Information on demographics and characteristics were assessed by questionnaires. Herpesvirus seroprevalences between Surinamese-Creole, Surinamese-Hindustani, Turkish, Moroccan, Cape Verdean Antillean, and Native Dutch children were compared. RESULTS: Non-Western ethnicity was an independent risk factor for CMV (aOR, 2.16; 95% CI 1.81-2.57), EBV (1.76; 1.48-2.09), and HSV-1 seropositivity (1.52; 1.39-1.66). Among the ethnic groups, CMV seroprevalences ranged between 29% and 65%, EBV between 43% and 69%, and HSV-1 between 13% and 39%. Low family net household income, low maternal educational level, crowding, and lifestyle factors explained up to 48% of the ethnic differences in HSV-1 seroprevalences, and up to 39% of the ethnic differences in EBV seroprevalences. These factors did not explain ethnic differences in CMV seroprevalences. CONCLUSIONS: Socioeconomic position and factors related to lifestyle explain only a part of the large ethnic differences in EBV and HSV-1 seroprevalences, whereas they do not explain ethnic differences in CMV seroprevalences in childhood.

Herpesvirus Infections and Transglutaminase Type 2 Antibody Positivity in Childhood: The Generation R Study.

OBJECTIVES: Persistent viral infections have been implicated in the etiology of autoimmune diseases in adulthood, but it is not known whether herpesviruses are associated with the development of celiac disease autoimmunity in childhood. We assessed whether herpesvirus infections are associated with transglutaminase type 2 antibody (TG2A) concentrations in children at 6 years of age. METHODS: The present study was embedded within a population-based prospective cohort study. Serum immunoglobulin G levels against Epstein-Barr virus, cytomegalovirus (CMV), and herpes simplex virus type 1 were measured by enzyme-linked immunosorbent assay , and TG2A concentrations with fluorescence enzyme immunoassay in 4420 children at 6 years of age. Children were categorized based on TG2A concentrations into negative (<7 U/mL), positive (≥7-70 U/mL), and strongly positive (≥70 U/mL), that is, 10 times upper limit normal. RESULTS: Fifty-nine children (1.3%) were TG2A positive, and of these 31 (53%) had concentrations 70 U/mL or more. Children with TG2A concentrations 70 U/mL or more were less often infected with CMV (adjusted odds ratio (aOR) 0.38, 95% CI 0.14-0.98, P = 0.04) and with any of the 3 viruses (aOR 0.38, 95% CI 0.18-0.78, P < 0.01) than children with TG2A negative concentrations. In addition, children with TG2A concentrations 70 U/mL or more were less often infected with 2 or more viruses than children with TG2A negative concentrations (aOR 0.15, 95% CI 0.03-0.65, P = 0.01). CONCLUSIONS: Both CMV single infection and combined CMV, Epstein-Barr virus and/or herpes simplex virus type 1 infections are inversely associated with strongly TG2A positivity. This may indicate a protective effect of herpesvirus infections in the pathogenesis of celiac disease autoimmunity.

Structural basis for RNA polymerase II ubiquitylation and inactivation in transcription-coupled repair.

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.

Transcription-coupled repair of DNA-protein cross-links depends on CSA and CSB.

Covalent DNA-protein cross-links (DPCs) are toxic DNA lesions that block replication and require repair by multiple pathways. Whether transcription blockage contributes to the toxicity of DPCs and how cells respond when RNA polymerases stall at DPCs is unknown. Here we find that DPC formation arrests transcription and induces ubiquitylation and degradation of RNA polymerase II. Using genetic screens and a method for the genome-wide mapping of DNA-protein adducts, DPC sequencing, we discover that Cockayne syndrome (CS) proteins CSB and CSA provide resistance to DPC-inducing agents by promoting DPC repair in actively transcribed genes. Consequently, CSB- or CSA-deficient cells fail to efficiently restart transcription after induction of DPCs. In contrast, nucleotide excision repair factors that act downstream of CSB and CSA at ultraviolet light-induced DNA lesions are dispensable. Our study describes a transcription-coupled DPC repair pathway and suggests that defects in this pathway may contribute to the unique neurological features of CS.

Unscheduled DNA Synthesis at Sites of Local UV-induced DNA Damage to Quantify Global Genome Nucleotide Excision Repair Activity in Human Cells.

Nucleotide excision repair (NER) removes a wide variety of structurally unrelated lesions from the genome, including UV-induced photolesions such as 6-4 pyrimidine-pyrimidone photoproducts (6-4PPs) and cyclobutane pyrimidine dimers (CPDs). NER removes lesions by excising a short stretch of single-stranded DNA containing the damaged DNA, leaving a single-stranded gap that is resynthesized in a process called unscheduled DNA synthesis (UDS). Measuring UDS after UV irradiation in non-dividing cells provides a measure of the overall NER activity, of which approximately 90% is carried out by the global genome repair (GGR) sub pathway. Here, we present a protocol for the microscopy-based analysis and quantification of UDS as a measurement for GGR activity. Following local UV-C irradiation, serum-starved human cells are supplemented with the thymidine analogue 5-ethynyl-2'-deoxyuridine (EdU), which is incorporated into repair patches following NER-dependent dual incision. The incorporated nucleotide analogue is coupled to a fluorophore using Click-iT chemistry, followed by immunodetection of CPD photolesions to simultaneously visualize both signals by fluorescence microscopy. Accompanying this protocol is a custom-built ImageJ plug-in to analyze and quantify UDS signals at sites of CPD-marked local damage. The local UDS assay enables an effective and sensitive fluorescence-based quantification of GGR activity in single cells with application in basic research to better understand the regulatory mechanism in NER, as well as in diagnostics to characterize fibroblasts from individuals with NER-deficiency disorder. Graphical abstract.

XPC-PARP complexes engage the chromatin remodeler ALC1 to catalyze global genome DNA damage repair.

Cells employ global genome nucleotide excision repair (GGR) to eliminate a broad spectrum of DNA lesions, including those induced by UV light. The lesion-recognition factor XPC initiates repair of helix-destabilizing DNA lesions, but binds poorly to lesions such as CPDs that do not destabilize DNA. How difficult-to-repair lesions are detected in chromatin is unknown. Here, we identify the poly-(ADP-ribose) polymerases PARP1 and PARP2 as constitutive interactors of XPC. Their interaction results in the XPC-stimulated synthesis of poly-(ADP-ribose) (PAR) by PARP1 at UV lesions, which in turn enables the recruitment and activation of the PAR-regulated chromatin remodeler ALC1. PARP2, on the other hand, modulates the retention of ALC1 at DNA damage sites. Notably, ALC1 mediates chromatin expansion at UV-induced DNA lesions, leading to the timely clearing of CPD lesions. Thus, we reveal how chromatin containing difficult-to-repair DNA lesions is primed for repair, providing insight into mechanisms of chromatin plasticity during GGR.

Functional assessment of variants in the TSC1 and TSC2 genes identified in individuals with Tuberous Sclerosis Complex.

The effects of missense changes and small in-frame deletions and insertions on protein function are not easy to predict, and the identification of such variants in individuals at risk of a genetic disease can complicate genetic counselling. One option is to perform functional tests to assess whether the variants affect protein function. We have used this strategy to characterize variants identified in the TSC1 and TSC2 genes in individuals with, or suspected of having, Tuberous Sclerosis Complex (TSC). Here we present an overview of our functional studies on 45 TSC1 and 107 TSC2 variants. Using a standardized protocol we classified 16 TSC1 variants and 70 TSC2 variants as pathogenic. In addition we identified eight putative splice site mutations (five TSC1 and three TSC2). The remaining 24 TSC1 and 34 TSC2 variants were classified as probably neutral.

Dealing with transcription-blocking DNA damage: Repair mechanisms, RNA polymerase II processing and human disorders.

Transcription-blocking DNA lesions (TBLs) in genomic DNA are triggered by a wide variety of DNA-damaging agents. Such lesions cause stalling of elongating RNA polymerase II (RNA Pol II) enzymes and fully block transcription when unresolved. The toxic impact of DNA damage on transcription progression is commonly referred to as transcription stress. In response to RNA Pol II stalling, cells activate and employ transcription-coupled repair (TCR) machineries to repair cytotoxic TBLs and resume transcription. Increasing evidence indicates that the modification and processing of stalled RNA Pol II is an integral component of the cellular response to and the repair of TBLs. If TCR pathways fail, the prolonged stalling of RNA Pol II will impede global replication and transcription as well as block the access of other DNA repair pathways that may act upon the TBL. Consequently, such prolonged stalling will trigger profound genome instability and devastating clinical features. In this review, we will discuss the mechanisms by which various types of TBLs are repaired by distinct TCR pathways and how RNA Pol II processing is regulated during these processes. We will also discuss the clinical consequences of transcription stress and genotype-phenotype correlations of related TCR-deficiency disorders.

Transcription-Coupled DNA Repair: From Mechanism to Human Disorder.

DNA lesions pose a major obstacle during gene transcription by RNA polymerase II (RNAPII) enzymes. The transcription-coupled DNA repair (TCR) pathway eliminates such DNA lesions. Inherited defects in TCR cause severe clinical syndromes, including Cockayne syndrome (CS). The molecular mechanism of TCR and the molecular origin of CS have long remained enigmatic. Here we explore new advances in our understanding of how TCR complexes assemble through cooperative interactions between repair factors stimulated by RNAPII ubiquitylation. Mounting evidence suggests that RNAPII ubiquitylation activates TCR complex assembly during repair and, in parallel, promotes processing and degradation of RNAPII to prevent prolonged stalling. The fate of stalled RNAPII is therefore emerging as a crucial link between TCR and associated human diseases.

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

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.

ELOF1 is a transcription-coupled DNA repair factor that directs RNA polymerase II ubiquitylation.

Cells employ transcription-coupled repair (TCR) to eliminate transcription-blocking DNA lesions. DNA damage-induced binding of the TCR-specific repair factor CSB to RNA polymerase II (RNAPII) triggers RNAPII ubiquitylation of a single lysine (K1268) by the CRL4CSA ubiquitin ligase. How CRL4CSA is specifically directed towards K1268 is unknown. Here, we identify ELOF1 as the missing link that facilitates RNAPII ubiquitylation, a key signal for the assembly of downstream repair factors. This function requires its constitutive interaction with RNAPII close to K1268, revealing ELOF1 as a specificity factor that binds and positions CRL4CSA for optimal RNAPII ubiquitylation. Drug-genetic interaction screening also revealed a CSB-independent pathway in which ELOF1 prevents R-loops in active genes and protects cells against DNA replication stress. Our study offers key insights into the molecular mechanisms of TCR and provides a genetic framework of the interplay between transcriptional stress responses and DNA replication.

Human HMGN1 and HMGN2 are not required for transcription-coupled DNA repair.

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.

Publisher Correction: The cooperative action of CSB, CSA, and UVSSA target TFIIH to DNA damage-stalled RNA polymerase II.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The cooperative action of CSB, CSA, and UVSSA target TFIIH to DNA damage-stalled RNA polymerase II.

The response to DNA damage-stalled RNA polymerase II (RNAPIIo) involves the assembly of the transcription-coupled repair (TCR) complex on actively transcribed strands. The function of the TCR proteins CSB, CSA and UVSSA and the manner in which the core DNA repair complex, including transcription factor IIH (TFIIH), is recruited are largely unknown. Here, we define the assembly mechanism of the TCR complex in human isogenic knockout cells. We show that TCR is initiated by RNAPIIo-bound CSB, which recruits CSA through a newly identified CSA-interaction motif (CIM). Once recruited, CSA facilitates the association of UVSSA with stalled RNAPIIo. Importantly, we find that UVSSA is the key factor that recruits the TFIIH complex in a manner that is stimulated by CSB and CSA. Together these findings identify a sequential and highly cooperative assembly mechanism of TCR proteins and reveal the mechanism for TFIIH recruitment to DNA damage-stalled RNAPIIo to initiate repair.