RESUMEN
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.
Asunto(s)
Envejecimiento Prematuro , Histonas , Fibras Musculares Esqueléticas , Animales , Ratones , Envejecimiento Prematuro/genética , ADN , Roturas del ADN de Doble Cadena , Histonas/genética , Histonas/metabolismo , Fibras Musculares Esqueléticas/metabolismo , NucleosomasRESUMEN
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.
Asunto(s)
Reparación del ADN , ADN Ribosómico/metabolismo , ARN Polimerasa I/metabolismo , Transcripción Genética , Línea Celular Transformada , ADN Ribosómico/genética , Humanos , ARN Polimerasa I/genética , Rayos UltravioletaRESUMEN
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.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/química , Proteínas de Neoplasias/química , Neoplasias/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas/química , Factor de Transcripción TFIIH/química , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Cristalografía por Rayos X , Reparación del ADN/efectos de los fármacos , Dimerización , Humanos , Ratones , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patología , Conformación Proteica/efectos de los fármacos , Multimerización de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Bibliotecas de Moléculas Pequeñas/farmacología , Factor de Transcripción TFIIH/genéticaRESUMEN
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.
RESUMEN
Transcription-coupled nucleotide excision repair (TC-NER) allows RNA polymerase II (RNAPII)-blocking lesions to be rapidly removed from the transcribed strand of active genes. Defective TCR in humans is associated with Cockayne syndrome (CS), typically caused by defects in either CSA or CSB. Here, we show that CSB contains a ubiquitin-binding domain (UBD). Cells expressing UBD-less CSB (CSB(del)) have phenotypes similar to those of cells lacking CSB, but these can be suppressed by appending a heterologous UBD, so ubiquitin binding is essential for CSB function. Surprisingly, CSB(del) remains capable of assembling nucleotide excision repair factors and repair synthesis proteins around damage-stalled RNAPII, but such repair complexes fail to excise the lesion. Together, our results indicate an essential role for protein ubiquitylation and CSB's UBD in triggering damage incision during TC-NER and allow us to integrate the function of CSA and CSB in a model for the process.
Asunto(s)
ADN Helicasas , Enzimas Reparadoras del ADN , Reparación del ADN , Ubiquitina/metabolismo , Secuencia de Aminoácidos , Línea Celular/efectos de la radiación , Núcleo Celular/metabolismo , Síndrome de Cockayne/genética , Síndrome de Cockayne/metabolismo , Daño del ADN , ADN Helicasas/genética , ADN Helicasas/metabolismo , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Humanos , Datos de Secuencia Molecular , Mutación , Proteínas de Unión a Poli-ADP-Ribosa , Regiones Promotoras Genéticas , Estructura Terciaria de Proteína , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Tetrahidrofolato Deshidrogenasa/genética , Ubiquitina/genética , Rayos UltravioletaRESUMEN
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.
Asunto(s)
Síndrome de Cockayne/genética , Síndrome de Cockayne/metabolismo , Daño del ADN , Reparación del ADN , Estrés Oxidativo , Transcripción Genética , Secuencia de Aminoácidos , Ciclo Celular , Línea Celular , Supervivencia Celular , Análisis por Conglomerados , Daño del ADN/efectos de la radiación , Expresión Génica , Perfilación de la Expresión Génica , Inestabilidad Genómica , Humanos , Mutación , Proteínas Recombinantes de Fusión , UbiquitinaciónRESUMEN
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.
Asunto(s)
Reparación del ADN , ADN de Cadena Simple/genética , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Proteína de la Xerodermia Pigmentosa del Grupo D/genética , Animales , Línea Celular , Daño del ADN , Humanos , Ratones , Ratones Transgénicos , Mutación , Rayos UltravioletaRESUMEN
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.
Asunto(s)
Cromatina/genética , Daño del ADN/genética , Metiltransferasas/genética , Animales , Cromatina/efectos de la radiación , Reparación del ADN/genética , Regulación de la Expresión Génica/efectos de los fármacos , N-Metiltransferasa de Histona-Lisina , Ácidos Hidroxámicos/farmacología , Hipersensibilidad , Ratones , Ratones Noqueados , ARN Polimerasa II/metabolismo , Activación Transcripcional , Rayos UltravioletaRESUMEN
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.
Asunto(s)
Reparación del ADN , Síndromes de Tricotiodistrofia , Animales , Síndrome de Cockayne , Humanos , Mutación , Factor de Transcripción TFIIH/genética , Factores de Transcripción/genética , Transcripción Genética , Síndromes de Tricotiodistrofia/genéticaRESUMEN
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.
Asunto(s)
Reparación del ADN/fisiología , ARN Polimerasa II/metabolismo , Factor de Transcripción TFIIH/metabolismo , Activación Transcripcional/fisiología , Factores de Elongación Transcripcional/metabolismo , Secuencia de Bases , Western Blotting , Línea Celular , Inmunoprecipitación de Cromatina , Clonación Molecular , Cartilla de ADN/genética , Recuperación de Fluorescencia tras Fotoblanqueo , Humanos , Espectrometría de Masas , Datos de Secuencia Molecular , Interferencia de ARN , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ADNRESUMEN
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.
Asunto(s)
Factor de Transcripción TFIIH/metabolismo , Factores de Transcripción/metabolismo , Síndromes de Tricotiodistrofia/genética , Síndromes de Tricotiodistrofia/metabolismo , Línea Celular , ADN/metabolismo , Daño del ADN , Fibroblastos , Humanos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Dominios y Motivos de Interacción de Proteínas , Subunidades de Proteína , Proteínas Adaptadoras de la Señalización Shc/genética , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Factor de Transcripción TFIIH/genética , Factores de Transcripción/genética , TransfecciónRESUMEN
The structure specific flap endonuclease 1 (FEN1) plays an essential role in long-patch base excision repair (BER) and in DNA replication. We have generated a fluorescently tagged FEN1 expressing mouse which allows monitoring the localization and kinetics of FEN1 in response to DNA damage in living cells and tissues. The expression of FEN1, which is tagged at its C-terminal end with enhanced yellow fluorescent protein (FEN1-YFP), is under control of the endogenous Fen1 transcriptional regulatory elements. In line with its role in processing of Okazaki fragments during DNA replication, we found that FEN1-YFP expression is mainly observed in highly proliferating tissue. Moreover, the FEN1-YFP fusion protein allowed us to investigate repair kinetics in cells challenged with local and global DNA damage. In vivo multi-photon fluorescence microscopy demonstrates rapid localization of FEN1 to local laser-induced DNA damage sites in nuclei, providing evidence of a highly mobile protein that accumulates fast at DNA lesion sites with high turnover rate. Inhibition of poly (ADP-ribose) polymerase 1 (PARP1) disrupts FEN1 accumulation at sites of DNA damage, indicating that PARP1 is required for FEN1 recruitment to DNA repair intermediates in BER.
Asunto(s)
Reparación del ADN , Endonucleasas de ADN Solapado/metabolismo , Animales , Proteínas Bacterianas/genética , Encéfalo/metabolismo , Células Cultivadas , Daño del ADN , Endonucleasas de ADN Solapado/análisis , Endonucleasas de ADN Solapado/genética , Técnicas de Sustitución del Gen , Cinética , Proteínas Luminiscentes/genética , Ratones , Poli(ADP-Ribosa) Polimerasa-1 , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Antígeno Nuclear de Célula en Proliferación/análisis , Fase SRESUMEN
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.
Asunto(s)
Daño del ADN , Reparación del ADN , Daño del ADN/genética , Reparación del ADN por Unión de Extremidades , Diferenciación Celular/genética , ADN/metabolismoRESUMEN
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.
Asunto(s)
Atrofia Muscular Espinal , Proteínas de Unión al ARN , Humanos , Proteínas de Unión al ARN/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Nucléolo Celular/metabolismo , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Neuronas Motoras/metabolismo , Proteínas del Complejo SMN/metabolismo , Cuerpos Enrollados/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas Represoras/metabolismoRESUMEN
Radiosensitive T-B- severe combined immunodeficiency (RS-SCID) is caused by defects in the nonhomologous end-joining (NHEJ) DNA repair pathway, which results in failure of functional V(D)J recombination. Here we have identified the first human RS-SCID patient to our knowledge with a DNA-PKcs missense mutation (L3062R). The causative mutation did not affect the kinase activity or DNA end-binding capacity of DNA-PKcs itself; rather, the presence of long P-nucleotide stretches in the immunoglobulin coding joints indicated that it caused insufficient Artemis activation, something that is dependent on Artemis interaction with autophosphorylated DNA-PKcs. Moreover, overall end-joining activity was hampered, suggesting that Artemis-independent DNA-PKcs functions were also inhibited. This study demonstrates that the presence of DNA-PKcs kinase activity is not sufficient to rule out a defect in this gene during diagnosis and treatment of RS-SCID patients. Further, the data suggest that residual DNA-PKcs activity is indispensable in humans.
Asunto(s)
Proteína Quinasa Activada por ADN/genética , Mutación Missense , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Tolerancia a Radiación , Recombinación Genética , Inmunodeficiencia Combinada Grave/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Preescolar , ADN Ligasa (ATP) , ADN Ligasas/genética , ADN Ligasas/metabolismo , Análisis Mutacional de ADN , Reparación del ADN , Proteína Quinasa Activada por ADN/metabolismo , Proteínas de Unión al ADN , Endonucleasas , Femenino , Fibroblastos/citología , Fibroblastos/fisiología , Fibroblastos/efectos de la radiación , Genotipo , Humanos , Lactante , Masculino , Datos de Secuencia Molecular , Linaje , Alineación de Secuencia , Inmunodeficiencia Combinada Grave/diagnósticoRESUMEN
The DNA-dependent protein kinase catalytic subunit (DNA-PK(CS)) plays an important role during the repair of DNA double-strand breaks (DSBs). It is recruited to DNA ends in the early stages of the nonhomologous end-joining (NHEJ) process, which mediates DSB repair. To study DNA-PK(CS) recruitment in vivo, we used a laser system to introduce DSBs in a specified region of the cell nucleus. We show that DNA-PK(CS) accumulates at DSB sites in a Ku80-dependent manner, and that neither the kinase activity nor the phosphorylation status of DNA-PK(CS) influences its initial accumulation. However, impairment of both of these functions results in deficient DSB repair and the maintained presence of DNA-PK(CS) at unrepaired DSBs. The use of photobleaching techniques allowed us to determine that the kinase activity and phosphorylation status of DNA-PK(CS) influence the stability of its binding to DNA ends. We suggest a model in which DNA-PK(CS) phosphorylation/autophosphorylation facilitates NHEJ by destabilizing the interaction of DNA-PK(CS) with the DNA ends.
Asunto(s)
Dominio Catalítico , Roturas del ADN de Doble Cadena , Reparación del ADN , Proteína Quinasa Activada por ADN/metabolismo , Animales , Antígenos Nucleares/metabolismo , Células CHO , Cricetinae , Cricetulus , ADN/metabolismo , Proteína Quinasa Activada por ADN/química , Proteínas de Unión al ADN/metabolismo , Humanos , Autoantígeno Ku , Rayos Láser , Fosforilación , FotoblanqueoRESUMEN
Schimke immuno-osseous dysplasia (SIOD) is a multisystemic disorder with prominent skeletal, renal, immunological, and ectodermal abnormalities. It is caused by mutations of SMARCAL1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a-like 1), which encodes a DNA stress response protein. To determine the relationship of this function to the SIOD phenotype, we profiled the cancer prevalence in SIOD and assessed if defects of nucleotide excision repair (NER) and nonhomologous end joining (NHEJ), respectively, explained the ectodermal and immunological features of SIOD. Finally, we determined if Smarcal1(del/del) mice had hypersensitivity to irinotecan (CPT-11), etoposide, and hydroxyurea (HU) and whether exposure to these agents induced features of SIOD. Among 71 SIOD patients, three had non-Hodgkin lymphoma (NHL) and one had osteosarcoma. We did not find evidence of defective NER or NHEJ; however, Smarcal1-deficient mice were hypersensitive to several genotoxic agents. Also, CPT-11, etoposide, and HU caused decreased growth and loss of growth plate chondrocytes. These data, which identify an increased prevalence of NHL in SIOD and confirm hypersensitivity to DNA damaging agents in vivo, provide guidance for the management of SIOD patients.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , ADN Helicasas/genética , Linfoma no Hodgkin/tratamiento farmacológico , Linfoma no Hodgkin/genética , Animales , Línea Celular , Reparación del ADN por Unión de Extremidades , Reparación del ADN , Humanos , Etiquetado Corte-Fin in Situ , RatonesRESUMEN
Studies based on cell-free systems and on in vitro-cultured living cells support the concept that many cellular processes, such as transcription initiation, are highly dynamic: individual proteins stochastically bind to their substrates and disassemble after reaction completion. This dynamic nature allows quick adaptation of transcription to changing conditions. However, it is unknown to what extent this dynamic transcription organization holds for postmitotic cells embedded in mammalian tissue. To allow analysis of transcription initiation dynamics directly into living mammalian tissues, we created a knock-in mouse model expressing fluorescently tagged TFIIH. Surprisingly and in contrast to what has been observed in cultured and proliferating cells, postmitotic murine cells embedded in their tissue exhibit a strong and long-lasting transcription-dependent immobilization of TFIIH. This immobilization is both differentiation driven and development dependent. Furthermore, although very statically bound, TFIIH can be remobilized to respond to new transcriptional needs. This divergent spatiotemporal transcriptional organization in different cells of the soma revisits the generally accepted highly dynamic concept of the kinetic framework of transcription and shows how basic processes, such as transcription, can be organized in a fundamentally different fashion in intact organisms as previously deduced from in vitro studies.
Asunto(s)
Diferenciación Celular , Regulación del Desarrollo de la Expresión Génica , Factor de Transcripción TFIIH/metabolismo , Transcripción Genética , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Células Cultivadas , Cerebelo/citología , Cerebelo/metabolismo , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Inmunoprecipitación de Cromatina , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Fibroblastos/citología , Recuperación de Fluorescencia tras Fotoblanqueo , Cinética , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Endogámicos C57BL , Técnicas de Cultivo de Órganos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factor de Transcripción TFIIH/genéticaRESUMEN
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.
Asunto(s)
Factores de Transcripción , Transcripción Genética , Daño del ADN , Reparación del ADN , ARN Polimerasa II/metabolismo , Factores de Transcripción/metabolismoRESUMEN
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.