H2A.Z is involved in premature aging and DSB repair initiation in muscle fibers.

Histone variants are key epigenetic players, but their functional and physiological roles remain poorly understood. Here, we show that depletion of the histone variant H2A.Z in mouse skeletal muscle causes oxidative stress, oxidation of proteins, accumulation of DNA damages, and both neuromuscular junction and mitochondria lesions that consequently lead to premature muscle aging and reduced life span. Investigation of the molecular mechanisms involved shows that H2A.Z is required to initiate DNA double strand break repair by recruiting Ku80 at DNA lesions. This is achieved via specific interactions of Ku80 vWA domain with H2A.Z. Taken as a whole, our data reveal that H2A.Z containing nucleosomes act as a molecular platform to bring together the proteins required to initiate and process DNA double strand break repair.

Decline of DNA damage response along with myogenic differentiation.

DNA integrity is incessantly confronted to agents inducing DNA lesions. All organisms are equipped with a network of DNA damage response mechanisms that will repair DNA lesions and restore proper cellular activities. Despite DNA repair mechanisms have been revealed in replicating cells, still little is known about how DNA lesions are repaired in postmitotic cells. Muscle fibers are highly specialized postmitotic cells organized in syncytia and they are vulnerable to age-related degeneration and atrophy after radiotherapy treatment. We have studied the DNA repair capacity of muscle fiber nuclei and compared it with the one measured in proliferative myoblasts here. We focused on the DNA repair mechanisms that correct ionizing radiation (IR)-induced lesions, namely the base excision repair, the nonhomologous end joining, and the homologous recombination (HR). We found that in the most differentiated myogenic cells, myotubes, these DNA repair mechanisms present weakened kinetics of recruitment of DNA repair proteins to IR-damaged DNA. For base excision repair and HR, this decline can be linked to reduced steady-state levels of key proteins involved in these processes.

Nucleolar reorganization after cellular stress is orchestrated by SMN shuttling between nuclear compartments.

Spinal muscular atrophy is an autosomal recessive neuromuscular disease caused by mutations in the multifunctional protein Survival of Motor Neuron, or SMN. Within the nucleus, SMN localizes to Cajal bodies, which are associated with nucleoli, nuclear organelles dedicated to the first steps of ribosome biogenesis. The highly organized structure of the nucleolus can be dynamically altered by genotoxic agents. RNAP1, Fibrillarin, and nucleolar DNA are exported to the periphery of the nucleolus after genotoxic stress and, once DNA repair is fully completed, the organization of the nucleolus is restored. We find that SMN is required for the restoration of the nucleolar structure after genotoxic stress. During DNA repair, SMN shuttles from the Cajal bodies to the nucleolus. This shuttling is important for nucleolar homeostasis and relies on the presence of Coilin and the activity of PRMT1.

Active mRNA degradation by EXD2 nuclease elicits recovery of transcription after genotoxic stress.

The transcriptional response to genotoxic stress involves gene expression arrest, followed by recovery of mRNA synthesis (RRS) after DNA repair. We find that the lack of the EXD2 nuclease impairs RRS and decreases cell survival after UV irradiation, without affecting DNA repair. Overexpression of wild-type, but not nuclease-dead EXD2, restores RRS and cell survival. We observe that UV irradiation triggers the relocation of EXD2 from mitochondria to the nucleus. There, EXD2 is recruited to chromatin where it transiently interacts with RNA Polymerase II (RNAPII) to promote the degradation of nascent mRNAs synthesized at the time of genotoxic attack. Reconstitution of the EXD2-RNAPII partnership on a transcribed DNA template in vitro shows that EXD2 primarily interacts with an elongation-blocked RNAPII and efficiently digests mRNA. Overall, our data highlight a crucial step in the transcriptional response to genotoxic attack in which EXD2 interacts with elongation-stalled RNAPII on chromatin to potentially degrade the associated nascent mRNA, allowing transcription restart after DNA repair.

XAB2 dynamics during DNA damage-dependent transcription inhibition.

Xeroderma Pigmentosum group A-binding protein 2 (XAB2) is a multifunctional protein playing a critical role in distinct cellular processes including transcription, splicing, DNA repair, and messenger RNA export. In this study, we demonstrate that XAB2 is involved specifically and exclusively in Transcription-Coupled Nucleotide Excision Repair (TC-NER) reactions and solely for RNA polymerase 2 (RNAP2)-transcribed genes. Surprisingly, contrary to all the other NER proteins studied so far, XAB2 does not accumulate on the local UV-C damage; on the contrary, it becomes more mobile after damage induction. XAB2 mobility is restored when DNA repair reactions are completed. By scrutinizing from which cellular complex/partner/structure XAB2 is released, we have identified that XAB2 is detached after DNA damage induction from DNA:RNA hybrids, commonly known as R-loops, and from the CSA and XPG proteins. This release contributes to the DNA damage recognition step during TC-NER, as in the absence of XAB2, RNAP2 is blocked longer on UV lesions. Moreover, we also demonstrate that XAB2 has a role in retaining RNAP2 on its substrate without any DNA damage.

A stable XPG protein is required for proper ribosome biogenesis: Insights on the phenotype of combinate Xeroderma Pigmentosum/Cockayne Syndrome patients.

Nucleotide Excision Repair is one of the five DNA repair systems. More than 30 proteins are involved in this process, including the seven XP proteins. When mutated, the genes coding for these proteins are provoking the rare disease Xeroderma Pigmentosum, which causes cutaneous defects and a high prevalence of skin cancers in patients. The CSA and CSB proteins are also involved in Nucleotide Excision Repair, and their mutation leads to Cockayne Syndrome, another rare disease, causing dwarfism, neurodegeneration, and ultimately early death, but without high skin cancer incidence. Some mutations of ERCC5, the gene coding for XPG, may give rise to a combined Xeroderma Pigmentosum and Cockayne Syndrome. A defect in Nucleotide Excision Repair alone cannot explain all these phenotypes. XPG has been located in the nucleolus, where ribosome biogenesis happens. This energy-consuming process starts with the transcription of the ribosomal DNA in a long ribosomal RNA, the pre-rRNA 47S, by RNA Polymerase 1. 47S pre-rRNA undergoes several cleavages and modifications to form three mature products: the ribosomal RNAs 18S, 5.8S and 28S. In the cytoplasm, these three products will enter the ribosomes' composition, the producers of protein in our cells. Our work aimed to observe ribosome biogenesis in presence of an unstable XPG protein. By working on Xeroderma Pigmentosum/Cockayne Syndrome cell lines, meaning in the absence of XPG, we uncovered that the binding of UBF, as well as the number of unresolved R-loops, is increased along the ribosomal DNA gene body and flanking regions. Furthermore, ribosomal RNA maturation is impaired, with increased use of alternative pathways of maturation as well as an increase of immature precursors. These defective processes may explain the neurodegeneration observed when the XPG protein is heavily truncated, as ribosomal homeostasis and R-loops resolution are critical for proper neuronal development.

Active DNA damage eviction by HLTF stimulates nucleotide excision repair.

Nucleotide excision repair (NER) counteracts the onset of cancer and aging by removing helix-distorting DNA lesions via a "cut-and-patch"-type reaction. The regulatory mechanisms that drive NER through its successive damage recognition, verification, incision, and gap restoration reaction steps remain elusive. Here, we show that the RAD5-related translocase HLTF facilitates repair through active eviction of incised damaged DNA together with associated repair proteins. Our data show a dual-incision-dependent recruitment of HLTF to the NER incision complex, which is mediated by HLTF's HIRAN domain that binds 3'-OH single-stranded DNA ends. HLTF's translocase motor subsequently promotes the dissociation of the stably damage-bound incision complex together with the incised oligonucleotide, allowing for an efficient PCNA loading and initiation of repair synthesis. Our findings uncover HLTF as an important NER factor that actively evicts DNA damage, thereby providing additional quality control by coordinating the transition between the excision and DNA synthesis steps to safeguard genome integrity.

Cell-type specific concentration regulation of the basal transcription factor TFIIH in XPBy/y mice model.

BACKGROUND: The basal transcription/repair factor TFIIH is a ten sub-unit complex essential for RNA polymerase II (RNAP2) transcription initiation and DNA repair. In both these processes TFIIH acts as a DNA helix opener, required for promoter escape of RNAP2 in transcription initiation, and to set the stage for strand incision within the nucleotide excision repair (NER) pathway. METHODS: We used a knock-in mouse model that we generated and that endogenously expresses a fluorescent version of XPB (XPB-YFP). Using different microscopy, cellular biology and biochemistry approaches we quantified the steady state levels of this protein in different cells, and cells imbedded in tissues. RESULTS: Here we demonstrate, via confocal imaging of ex vivo tissues and cells derived from this mouse model, that TFIIH steady state levels are tightly regulated at the single cell level, thus keeping nuclear TFIIH concentrations remarkably constant in a cell type dependent manner. Moreover, we show that individual cellular TFIIH levels are proportional to the speed of mRNA production, hence to a cell's transcriptional activity, which we can correlate to proliferation status. Importantly, cancer tissue presents a higher TFIIH than normal healthy tissues. CONCLUSION: This study shows that TFIIH cellular concentration can be used as a bona-fide quantitative marker of transcriptional activity and cellular proliferation.

CSB-Dependent Cyclin-Dependent Kinase 9 Degradation and RNA Polymerase II Phosphorylation during Transcription-Coupled Repair.

DNA lesions block cellular processes such as transcription, inducing apoptosis, tissue failures, and premature aging. To counteract the deleterious effects of DNA damage, cells are equipped with various DNA repair pathways. Transcription-coupled repair specifically removes helix-distorting DNA adducts in a coordinated multistep process. This process has been extensively studied; however, once the repair reaction is accomplished, little is known about how transcription restarts. In this study, we show that, after UV irradiation, the cyclin-dependent kinase 9 (CDK9)/cyclin T1 kinase unit is specifically released from the HEXIM1 complex and that this released fraction is degraded in the absence of the Cockayne syndrome group B protein (CSB). We determine that UV irradiation induces a specific Ser2 phosphorylation of the RNA polymerase II and that this phosphorylation is CSB dependent. Surprisingly, CDK9 is not responsible for this phosphorylation but instead might play a nonenzymatic role in transcription restart after DNA repair.

Small molecule-based targeting of TTD-A dimerization to control TFIIH transcriptional activity represents a potential strategy for anticancer therapy.

The human transcription factor TFIIH is a large complex composed of 10 subunits that form an intricate network of protein-protein interactions critical for regulating its transcriptional and DNA repair activities. The trichothiodystrophy group A protein (TTD-A or p8) is the smallest TFIIH subunit, shuttling between a free and a TFIIH-bound state. Its dimerization properties allow it to shift from a homodimeric state, in the absence of a functional partner, to a heterodimeric structure, enabling dynamic binding to TFIIH. Recruitment of p8 at TFIIH stabilizes the overall architecture of the complex, whereas p8's absence reduces its cellular steady-state concentration and consequently decreases basal transcription, highlighting that p8 dimerization may be an attractive target for down-regulating transcription in cancer cells. Here, using a combination of molecular dynamics simulations to study p8 conformational stability and a >3000-member library of chemical fragments, we identified small-molecule compounds that bind to the dimerization interface of p8 and provoke its destabilization, as assessed by biophysical studies. Using quantitative imaging of TFIIH in living mouse cells, we found that these molecules reduce the intracellular concentration of TFIIH and its transcriptional activity to levels similar to that observed in individuals with trichothiodystrophy owing to mutated TTD-A Our results provide a proof of concept of fragment-based drug discovery, demonstrating the utility of small molecules for targeting p8 dimerization to modulate the transcriptional machinery, an approach that may help inform further development in anticancer therapies.

Mechanistic insights in transcription-coupled nucleotide excision repair of ribosomal DNA.

Nucleotide excision repair (NER) guarantees genome integrity against UV light-induced DNA damage. After UV irradiation, cells have to cope with a general transcriptional block. To ensure UV lesions repair specifically on transcribed genes, NER is coupled with transcription in an extremely organized pathway known as transcription-coupled repair. In highly metabolic cells, more than 60% of total cellular transcription results from RNA polymerase I activity. Repair of the mammalian transcribed ribosomal DNA has been scarcely studied. UV lesions severely block RNA polymerase I activity and the full transcription-coupled repair machinery corrects damage on actively transcribed ribosomal DNAs. After UV irradiation, RNA polymerase I is more bound to the ribosomal DNA and both are displaced to the nucleolar periphery. Importantly, the reentry of RNA polymerase I and the ribosomal DNA is dependent on the presence of UV lesions on DNA and independent of transcription restart.

A ubiquitylation site in Cockayne syndrome B required for repair of oxidative DNA damage, but not for transcription-coupled nucleotide excision repair.

Cockayne syndrome B (CSB), best known for its role in transcription-coupled nucleotide excision repair (TC-NER), contains a ubiquitin-binding domain (UBD), but the functional connection between protein ubiquitylation and this UBD remains unclear. Here, we show that CSB is regulated via site-specific ubiquitylation. Mass spectrometry analysis of CSB identified lysine (K) 991 as a ubiquitylation site. Intriguingly, mutation of this residue (K991R) does not affect CSB's catalytic activity or protein stability, but greatly affects genome stability, even in the absence of induced DNA damage. Moreover, cells expressing CSB K991R are sensitive to oxidative DNA damage, but proficient for TC-NER. K991 becomes ubiquitylated upon oxidative DNA damage, and while CSB K991R is recruited normally to such damage, it fails to dissociate in a timely manner, suggesting a requirement for K991 ubiquitylation in CSB activation. Interestingly, deletion of CSB's UBD gives rise to oxidative damage sensitivity as well, while CSB ΔUBD and CSB K991R affects expression of overlapping groups of genes, further indicating a functional connection. Together, these results shed new light on the regulation of CSB, with K991R representing an important separation-of-function-mutation in this multi-functional protein.

ELL, a novel TFIIH partner, is involved in transcription restart after DNA repair.

DNA lesions that block transcription may cause cell death even when repaired, if transcription does not restart to reestablish cellular metabolism. However, transcription resumption after individual DNA-lesion repair remains poorly described in mechanistic terms and its players are largely unknown. The general transcription factor II H (TFIIH) is a major actor of both nucleotide excision repair subpathways of which transcription-coupled repair highlights the interplay between DNA repair and transcription. Using an unbiased proteomic approach, we have identified the protein eleven-nineteen lysine-rich leukemia (ELL) as a TFIIH partner. Here we show that ELL is recruited to UV-damaged chromatin in a Cdk7- dependent manner (a component of the cyclin-dependent activating kinase subcomplex of TFIIH). We demonstrate that depletion of ELL strongly hinders RNA polymerase II (RNA Pol II) transcription resumption after lesion removal and DNA gap filling. Lack of ELL was also observed to increase RNA Pol II retention to the chromatin during this process. Identifying ELL as an essential player for RNA Pol II restart during cellular DNA damage response opens the way to obtaining a mechanistic description of transcription resumption after DNA repair.

Histone methyltransferase DOT1L drives recovery of gene expression after a genotoxic attack.

UV-induced DNA damage causes repression of RNA synthesis. Following the removal of DNA lesions, transcription recovery operates through a process that is not understood yet. Here we show that knocking-out of the histone methyltransferase DOT1L in mouse embryonic fibroblasts (MEF(DOT1L)) leads to a UV hypersensitivity coupled to a deficient recovery of transcription initiation after UV irradiation. However, DOT1L is not implicated in the removal of the UV-induced DNA damage by the nucleotide excision repair pathway. Using FRAP and ChIP experiments we established that DOT1L promotes the formation of the pre-initiation complex on the promoters of UV-repressed genes and the appearance of transcriptionally active chromatin marks. Treatment with Trichostatin A, relaxing chromatin, recovers both transcription initiation and UV-survival. Our data suggest that DOT1L secures an open chromatin structure in order to reactivate RNA Pol II transcription initiation after a genotoxic attack.

In vivo interactions of TTDA mutant proteins within TFIIH.

Trichothiodystrophy group A (TTD-A) patients carry a mutation in the transcription factor II H (TFIIH) subunit TTDA. Using a novel in vivo tripartite split-GFP system, we show that TTDA interacts with the TFIIH subunit p52 and the p52-TTDA-GFP product is incorporated into TFIIH. p52-TTDA-GFP is able to bind DNA and is recruited to UV-damaged DNA. Furthermore, we show that two patient-mutated TTDA proteins can interact with p52, are able to bind to the DNA and can localize to damaged DNA. Our findings give new insights into the behavior of TTDA within the context of a living cell and thereby shed light on the complex phenotype of TTD-A patients.

Disruption of TTDA results in complete nucleotide excision repair deficiency and embryonic lethality.

The ten-subunit transcription factor IIH (TFIIH) plays a crucial role in transcription and nucleotide excision repair (NER). Inactivating mutations in the smallest 8-kDa TFB5/TTDA subunit cause the neurodevelopmental progeroid repair syndrome trichothiodystrophy A (TTD-A). Previous studies have shown that TTDA is the only TFIIH subunit that appears not to be essential for NER, transcription, or viability. We studied the consequences of TTDA inactivation by generating a Ttda knock-out (Ttda(-/-) ) mouse-model resembling TTD-A patients. Unexpectedly, Ttda(-/-) mice were embryonic lethal. However, in contrast to full disruption of all other TFIIH subunits, viability of Ttda(-/-) cells was not affected. Surprisingly, Ttda(-/-) cells were completely NER deficient, contrary to the incomplete NER deficiency of TTD-A patient-derived cells. We further showed that TTD-A patient mutations only partially inactivate TTDA function, explaining the relatively mild repair phenotype of TTD-A cells. Moreover, Ttda(-/-) cells were also highly sensitive to oxidizing agents. These findings reveal an essential role of TTDA for life, nucleotide excision repair, and oxidative DNA damage repair and identify Ttda(-/-) cells as a unique class of TFIIH mutants.

DNA repair: a changing geography? (1964-2008).

This article aims to explain the current state of DNA Repair studies' global geography by focusing on the genesis of the community. Bibliometric data is used to localize scientific activities related to DNA Repair at the city level. The keyword "DNA Repair" was introduced first by American scientists. It started to spread after 1964 that is to say, after P. Howard-Flanders (Yale University), P. Hanawalt (Stanford University) and R. Setlow (Oak Ridge Laboratories) found evidence for Excision Repair mechanisms. It was the first stage in the emergence of an autonomous scientific community. In this article, we will try to assess to what extent the geo-history of this scientific field is determinant in understanding its current geography. In order to do so, we will localize the places where the first "DNA Repair" publications were signed fifty years ago and the following spatial diffusion process, which led to the current geography of the field. Then, we will focus on the evolution of the research activity of "early entrants" in relation to the activity of "latecomers". This article is an opportunity to share with DNA Repair scientists some research results of a dynamic field in Science studies: spatial scientometrics.

Mutations in TFIIH causing trichothiodystrophy are responsible for defects in ribosomal RNA production and processing.

The basal transcription/repair factor II H (TFIIH), found mutated in cancer-prone or premature aging diseases, plays a still unclear role in RNA polymerase I transcription. Furthermore, the impact of this function on TFIIH-related diseases, such as trichothiodystrophy (TTD), remains to be explored. Here, we studied the involvement of TFIIH during the whole process of ribosome biogenesis, from RNAP1 transcription to maturation steps of the ribosomal RNAs. Our results show that TFIIH is recruited to the ribosomal DNA in an active transcription-dependent manner and functions in RNAP1 transcription elongation through ATP hydrolysis of the XPB subunit. Remarkably, we found a TFIIH allele-specific effect, affecting RNAP1 transcription and/or the pre-rRNA maturation process. Interestingly, this effect was observed in mutant TFIIH-TTD cells and also in the brains of TFIIH-TTD mice. Our findings provide evidence that defective ribosome synthesis represents a new faulty mechanism involved in the pathophysiology of TFIIH-related diseases.

Isoform-specific anti-MeCP2 antibodies confirm that expression of the e1 isoform strongly predominates in the brain.

Rett syndrome is a neurological disorder caused by mutations in the MECP2 gene.  MeCP2 transcripts are alternatively spliced to generate two protein isoforms (MeCP2_e1 and MeCP2_e2) that differ at their N-termini. Whilst mRNAs for both forms are expressed ubiquitously, the one for MeCP2_e1 is more abundant than for MeCP2_e2 in the central nervous system. In transfected cells, both protein isoforms are nuclear and colocalize with densely methylated heterochromatic foci. With a view to understanding the physiological contribution of each isoform, and their respective roles in the pathogenesis of Rett syndrome, we set out to generate isoform-specific anti-MeCP2 antibodies. To this end, we immunized rabbits against the peptides corresponding to the short amino-terminal portions that are different between the two isoforms. The polyclonal antibodies thus obtained specifically detected their respective isoforms of MeCP2 in Neuro2a (N2A) cells transfected to express either form. Both antisera showed comparable sensitivities when used for Western blot or immunofluorescence, and were highly specific for their respective isoform. When those antibodies were used on mouse tissues, specific signals were easily detected for Mecp2_e1, whilst Mecp2_e2 was very difficult to detect by Western blot, and even more so by immunofluorescence. Our results thus suggest that brain cells express low amounts of the Mecp2-e2 isoform. Our findings are compatible with recent reports showing that MeCP2_e2 is dispensable for healthy brain function, and that it may be involved in the regulation of neuronal apoptosis and embryonic development.

Generation of DNA single-strand displacement by compromised nucleotide excision repair.

Nucleotide excision repair (NER) is a precisely coordinated process essential to avoid DNA damage-induced cellular malfunction and mutagenesis. Here, we investigate the mechanistic details and effects of the NER machinery when it is compromised by a pathologically significant mutation in a subunit of the repair/transcription factor TFIIH, namely XPD. In contrast to previous studies, we find that no single- or double-strand DNA breaks are produced at early time points after UV irradiation of cells bearing a specific XPD mutation, despite the presence of a clear histone H2AX phosphorylation (γH2AX) signal in the UV-exposed areas. We show that the observed γH2AX signal can be explained by the presence of longer single-strand gaps possibly generated by strand displacement. Our in vivo measurements also indicate a strongly reduced TFIIH-XPG binding that could promote single-strand displacement at the site of UV lesions. This finding not only highlights the crucial role of XPG's interactions with TFIIH for proper NER, but also sheds new light on how a faulty DNA repair process can induce extreme genomic instability in human patients.