RESUMEN
DNA-protein crosslinks (DPCs) arise from enzymatic intermediates, metabolism or chemicals like chemotherapeutics. DPCs are highly cytotoxic as they impede DNA-based processes such as replication, which is counteracted through proteolysis-mediated DPC removal by spartan (SPRTN) or the proteasome. However, whether DPCs affect transcription and how transcription-blocking DPCs are repaired remains largely unknown. Here we show that DPCs severely impede RNA polymerase II-mediated transcription and are preferentially repaired in active genes by transcription-coupled DPC (TC-DPC) repair. TC-DPC repair is initiated by recruiting the transcription-coupled nucleotide excision repair (TC-NER) factors CSB and CSA to DPC-stalled RNA polymerase II. CSA and CSB are indispensable for TC-DPC repair; however, the downstream TC-NER factors UVSSA and XPA are not, a result indicative of a non-canonical TC-NER mechanism. TC-DPC repair functions independently of SPRTN but is mediated by the ubiquitin ligase CRL4CSA and the proteasome. Thus, DPCs in genes are preferentially repaired in a transcription-coupled manner to facilitate unperturbed transcription.
Asunto(s)
ADN Helicasas , Enzimas Reparadoras del ADN , Reparación del ADN , Proteínas de Unión a Poli-ADP-Ribosa , Proteolisis , ARN Polimerasa II , Transcripción Genética , Humanos , Proteínas Portadoras , ADN/metabolismo , ADN/genética , Daño del ADN , ADN Helicasas/metabolismo , ADN Helicasas/genética , Enzimas Reparadoras del ADN/metabolismo , Enzimas Reparadoras del ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Células HEK293 , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Receptores de Interleucina-17 , ARN Polimerasa II/metabolismo , ARN Polimerasa II/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
The brittle hair syndrome Trichothiodystrophy (TTD) is characterized by variable clinical features, including photosensitivity, ichthyosis, growth retardation, microcephaly, intellectual disability, hypogonadism, and anaemia. TTD-associated mutations typically cause unstable mutant proteins involved in various steps of gene expression, severely reducing steady-state mutant protein levels. However, to date, no such link to instability of gene-expression factors for TTD-associated mutations in MPLKIP/TTDN1 has been established. Here, we present seven additional TTD individuals with MPLKIP mutations from five consanguineous families, with a newly identified MPLKIP variant in one family. By mass spectrometry-based interaction proteomics, we demonstrate that MPLKIP interacts with core splicing factors and the lariat debranching protein DBR1. MPLKIP-deficient primary fibroblasts have reduced steady-state DBR1 protein levels. Using Human Skin Equivalents (HSEs), we observed impaired keratinocyte differentiation associated with compromised splicing and eventually, an imbalanced proteome affecting skin development and, interestingly, also the immune system. Our data show that MPLKIP, through its DBR1 stabilizing role, is implicated in mRNA splicing, which is of particular importance in highly differentiated tissue.
Asunto(s)
Síndromes de Tricotiodistrofia , Humanos , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Consanguinidad , Mutación , Fenotipo , Empalme del ARN , Síndromes de Tricotiodistrofia/genética , Síndromes de Tricotiodistrofia/metabolismoRESUMEN
The SWI/SNF family of ATP-dependent chromatin remodeling complexes is implicated in multiple DNA damage response mechanisms and frequently mutated in cancer. The BAF, PBAF and ncBAF complexes are three major types of SWI/SNF complexes that are functionally distinguished by their exclusive subunits. Accumulating evidence suggests that double-strand breaks (DSBs) in transcriptionally active DNA are preferentially repaired by a dedicated homologous recombination pathway. We show that different BAF, PBAF and ncBAF subunits promote homologous recombination and are rapidly recruited to DSBs in a transcription-dependent manner. The PBAF and ncBAF complexes promote RNA polymerase II eviction near DNA damage to rapidly initiate transcriptional silencing, while the BAF complex helps to maintain this transcriptional silencing. Furthermore, ARID1A-containing BAF complexes promote RNaseH1 and RAD52 recruitment to facilitate R-loop resolution and DNA repair. Our results highlight how multiple SWI/SNF complexes perform different functions to enable DNA repair in the context of actively transcribed genes.
Asunto(s)
Proteínas Cromosómicas no Histona , Estructuras R-Loop , Ensamble y Desensamble de Cromatina/genética , Proteínas Cromosómicas no Histona/metabolismo , ADN , Reparación del ADN/genética , Recombinación Homóloga/genética , HumanosRESUMEN
Cockayne syndrome is a rare inherited DNA repair multisystemic disorder. Here, we aim to raise awareness of the phenotypic resemblances between Cockayne syndrome and the neurodevelopmental disorder caused by pathogenic variants in MORC2, a gene also involved in DNA repair. Using exome sequencing, we identified a de novo pathogenic variant in MORC2 in our patient. Our patient's phenotype was characterized by multiple features evocative of Cockayne syndrome. Based on our patient's phenotype, in addition to the phenotypic description of patients with pathogenic variants in MORC2 reported in the literature, we suggest that pathogenic variants in this gene are associated with a Cockayne-like phenotype.
Asunto(s)
Síndrome de Cockayne , Trastornos del Neurodesarrollo , Humanos , Síndrome de Cockayne/genética , Fenotipo , Trastornos del Neurodesarrollo/genética , Secuenciación del Exoma , Factores de Transcripción/genéticaRESUMEN
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.
Asunto(s)
Reparación del ADN , Proteínas de Unión al ADN , ADN/genética , ADN/metabolismo , Daño del ADN , Replicación del ADN , Proteínas de Unión al ADN/genéticaRESUMEN
Correct transcription is crucial for life. However, DNA damage severely impedes elongating RNA polymerase II, causing transcription inhibition and transcription-replication conflicts. Cells are equipped with intricate mechanisms to counteract the severe consequence of these transcription-blocking lesions. However, the exact mechanism and factors involved remain largely unknown. Here, using a genome-wide CRISPR-Cas9 screen, we identified the elongation factor ELOF1 as an important factor in the transcription stress response following DNA damage. We show that ELOF1 has an evolutionarily conserved role in transcription-coupled nucleotide excision repair (TC-NER), where it promotes recruitment of the TC-NER factors UVSSA and TFIIH to efficiently repair transcription-blocking lesions and resume transcription. Additionally, ELOF1 modulates transcription to protect cells against transcription-mediated replication stress, thereby preserving genome stability. Thus, ELOF1 protects the transcription machinery from DNA damage via two distinct mechanisms.
Asunto(s)
Daño del ADN , Reparación del ADN , Inestabilidad Genómica , Factor 1 de Elongación Peptídica/metabolismo , Elongación de la Transcripción Genética , Sistemas CRISPR-Cas , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Evolución Molecular , Células HCT116 , Humanos , Factor 1 de Elongación Peptídica/genética , ARN Polimerasa II/metabolismo , Factor de Transcripción TFIIH/genética , Factor de Transcripción TFIIH/metabolismo , UbiquitinaciónRESUMEN
Trichothiodystrophy (TTD) is a rare hereditary neurodevelopmental disorder defined by sulfur-deficient brittle hair and nails and scaly skin, but with otherwise remarkably variable clinical features. The photosensitive TTD (PS-TTD) forms exhibits in addition to progressive neuropathy and other features of segmental accelerated aging and is associated with impaired genome maintenance and transcription. New factors involved in various steps of gene expression have been identified for the different non-photosensitive forms of TTD (NPS-TTD), which do not appear to show features of premature aging. Here, we identify alanyl-tRNA synthetase 1 and methionyl-tRNA synthetase 1 variants as new gene defects that cause NPS-TTD. These variants result in the instability of the respective gene products alanyl- and methionyl-tRNA synthetase. These findings extend our previous observations that TTD mutations affect the stability of the corresponding proteins and emphasize this phenomenon as a common feature of TTD. Functional studies in skin fibroblasts from affected individuals demonstrate that these new variants also impact on the rate of tRNA charging, which is the first step in protein translation. The extension of reduced abundance of TTD factors to translation as well as transcription redefines TTD as a syndrome in which proteins involved in gene expression are unstable.
Asunto(s)
Alanina-ARNt Ligasa/genética , Metionina-ARNt Ligasa/genética , Síndromes de Tricotiodistrofia/genética , Alanina-ARNt Ligasa/metabolismo , Niño , Estabilidad de Enzimas/genética , Femenino , Humanos , Metionina-ARNt Ligasa/metabolismo , Síndromes de Tricotiodistrofia/enzimología , Síndromes de Tricotiodistrofia/patología , Secuenciación Completa del GenomaRESUMEN
Abnormal gut motility is a feature of several mitochondrial encephalomyopathies, and mutations in genes such as TYMP and POLG, have been linked to these rare diseases. The human genome encodes three DNA ligases, of which only one, ligase III (LIG3), has a mitochondrial splice variant and is crucial for mitochondrial health. We investigated the effect of reduced LIG3 activity and resulting mitochondrial dysfunction in seven patients from three independent families, who showed the common occurrence of gut dysmotility and neurological manifestations reminiscent of mitochondrial neurogastrointestinal encephalomyopathy. DNA from these patients was subjected to whole exome sequencing. In all patients, compound heterozygous variants in a new disease gene, LIG3, were identified. All variants were predicted to have a damaging effect on the protein. The LIG3 gene encodes the only mitochondrial DNA (mtDNA) ligase and therefore plays a pivotal role in mtDNA repair and replication. In vitro assays in patient-derived cells showed a decrease in LIG3 protein levels and ligase activity. We demonstrated that the LIG3 gene defects affect mtDNA maintenance, leading to mtDNA depletion without the accumulation of multiple deletions as observed in other mitochondrial disorders. This mitochondrial dysfunction is likely to cause the phenotypes observed in these patients. The most prominent and consistent clinical signs were severe gut dysmotility and neurological abnormalities, including leukoencephalopathy, epilepsy, migraine, stroke-like episodes, and neurogenic bladder. A decrease in the number of myenteric neurons, and increased fibrosis and elastin levels were the most prominent changes in the gut. Cytochrome c oxidase (COX) deficient fibres in skeletal muscle were also observed. Disruption of lig3 in zebrafish reproduced the brain alterations and impaired gut transit in vivo. In conclusion, we identified variants in the LIG3 gene that result in a mitochondrial disease characterized by predominant gut dysmotility, encephalopathy, and neuromuscular abnormalities.
Asunto(s)
ADN Ligasa (ATP)/genética , Enfermedades Gastrointestinales/genética , Motilidad Gastrointestinal/genética , Encefalomiopatías Mitocondriales/genética , Proteínas de Unión a Poli-ADP-Ribosa/genética , Animales , Femenino , Enfermedades Gastrointestinales/patología , Humanos , Masculino , Encefalomiopatías Mitocondriales/patología , Mutación , Linaje , Pez CebraRESUMEN
Brittle and "tiger-tail" hair is the diagnostic hallmark of trichothiodystrophy (TTD), a rare recessive disease associated with a wide spectrum of clinical features including ichthyosis, intellectual disability, decreased fertility, and short stature. As a result of premature abrogation of terminal differentiation, the hair is brittle and fragile and contains reduced cysteine content. Hypersensitivity to UV light is found in about half of individuals with TTD; all of these individuals harbor bi-allelic mutations in components of the basal transcription factor TFIIH, and these mutations lead to impaired nucleotide excision repair and basal transcription. Different genes have been found to be associated with non-photosensitive TTD (NPS-TTD); these include MPLKIP (also called TTDN1), GTF2E2 (also called TFIIEß), and RNF113A. However, a relatively large group of these individuals with NPS-TTD have remained genetically uncharacterized. Here we present the identification of an NPS-TTD-associated gene, threonyl-tRNA synthetase (TARS), found by next-generation sequencing of a group of uncharacterized individuals with NPS-TTD. One individual has compound heterozygous TARS variants, c.826A>G (p.Lys276Glu) and c.1912C>T (p.Arg638∗), whereas a second individual is homozygous for the TARS variant: c.680T>C (p.Leu227Pro). We showed that these variants have a profound effect on TARS protein stability and enzymatic function. Our results expand the spectrum of genes involved in TTD to include genes implicated in amino acid charging of tRNA, which is required for the last step in gene expression, namely protein translation. We previously proposed that some of the TTD-specific features derive from subtle transcription defects as a consequence of unstable transcription factors. We now extend the definition of TTD from a transcription syndrome to a "gene-expression" syndrome.
Asunto(s)
Enfermedades del Cabello/patología , Mutación , Treonina-ARNt Ligasa/genética , Síndromes de Tricotiodistrofia/patología , Alelos , Secuencia de Aminoácidos , Estudios de Casos y Controles , Enfermedades del Cabello/genética , Humanos , Fenotipo , Homología de Secuencia , Factor de Transcripción TFIIH/genética , Síndromes de Tricotiodistrofia/genéticaRESUMEN
Aminoacyl-tRNA synthetases (ARSs) are essential enzymes responsible for charging tRNA molecules with cognate amino acids. Consistent with the essential function and ubiquitous expression of ARSs, mutations in 32 of the 37 ARS-encoding loci cause severe, early-onset recessive phenotypes. Previous genetic and functional data suggest a loss-of-function mechanism; however, our understanding of the allelic and locus heterogeneity of ARS-related disease is incomplete. Cysteinyl-tRNA synthetase (CARS) encodes the enzyme that charges tRNACys with cysteine in the cytoplasm. To date, CARS variants have not been implicated in any human disease phenotype. Here, we report on four subjects from three families with complex syndromes that include microcephaly, developmental delay, and brittle hair and nails. Each affected person carries bi-allelic CARS variants: one individual is compound heterozygous for c.1138C>T (p.Gln380∗) and c.1022G>A (p.Arg341His), two related individuals are compound heterozygous for c.1076C>T (p.Ser359Leu) and c.1199T>A (p.Leu400Gln), and one individual is homozygous for c.2061dup (p.Ser688Glnfs∗2). Measurement of protein abundance, yeast complementation assays, and assessments of tRNA charging indicate that each CARS variant causes a loss-of-function effect. Compared to subjects with previously reported ARS-related diseases, individuals with bi-allelic CARS variants are unique in presenting with a brittle-hair-and-nail phenotype, which most likely reflects the high cysteine content in human keratins. In sum, our efforts implicate CARS variants in human inherited disease, expand the locus and clinical heterogeneity of ARS-related clinical phenotypes, and further support impaired tRNA charging as the primary mechanism of recessive ARS-related disease.
Asunto(s)
Aminoacil-ARNt Sintetasas/genética , Enfermedad de Charcot-Marie-Tooth/etiología , Discapacidades del Desarrollo/etiología , Enfermedades del Cabello/etiología , Microcefalia/etiología , Mutación , Enfermedades de la Uña/etiología , Adulto , Secuencia de Aminoácidos , Enfermedad de Charcot-Marie-Tooth/enzimología , Enfermedad de Charcot-Marie-Tooth/patología , Discapacidades del Desarrollo/enzimología , Discapacidades del Desarrollo/patología , Femenino , Genes Recesivos , Predisposición Genética a la Enfermedad , Enfermedades del Cabello/enzimología , Enfermedades del Cabello/patología , Humanos , Masculino , Microcefalia/enzimología , Microcefalia/patología , Enfermedades de la Uña/enzimología , Enfermedades de la Uña/patología , Linaje , Fenotipo , Pronóstico , Homología de Secuencia , Adulto JovenRESUMEN
INTRODUCTION: Bladder cancer (urothelial carcinoma) is a common malignancy characterized by high recurrence rates and intense clinical follow-up, indicating the necessity for more effective therapies. Current treatment regimens include intra-vesical administration of mitomycin C (MMC) for non-muscle invasive disease and systemic cisplatin for muscle-invasive or metastatic disease. Hyperthermia, heating a tumor to 40-44°C, enhances the efficacy of these chemotherapeutics by various modes of action, one of which is inhibition of DNA repair via homologous recombination. Here, we explore whether ex vivo assays on freshly obtained bladder tumors can be applied to predict the response towards hyperthermia. MATERIAL AND METHODS: The cytochrome C release assay (apoptosis) and the RAD51 focus formation assay (DNA repair) were first established in the bladder cancer cell lines RT112 and T24 as measurements for hyperthermia efficiency, and subsequently tested in freshly obtained bladder tumors (n = 59). RESULTS: Hyperthermia significantly increased the fraction of apoptotic cells after cisplatin or MMC treatment in both RT112 and T24 cells and in most of the bladder tumors (8/10). The RAD51 focus formation assay detected both morphological and numerical changes of RAD51 foci upon hyperthermia in the RT112 and T24 cell lines. In 64% of 37 analyzed primary bladder tumor samples, hyperthermia induced similar morphological changes in RAD51 foci. CONCLUSION: The cytochrome C assay and the RAD51 focus formation assay are both feasible on freshly obtained bladder tumors, and could serve to predict the efficacy of hyperthermia together with cytotoxic agents, such as MMC or cisplatin.
Asunto(s)
Hipertermia Inducida , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Línea Celular Tumoral , Cisplatino/uso terapéutico , Citocromos c/metabolismo , Humanos , Mitomicina/uso terapéutico , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
Histone acetylation influences protein interactions and chromatin accessibility and plays an important role in the regulation of transcription, replication, and DNA repair. Conversely, DNA damage affects these crucial cellular processes and induces changes in histone acetylation. However, a comprehensive overview of the effects of DNA damage on the histone acetylation landscape is currently lacking. To quantify changes in histone acetylation, we developed an unbiased quantitative mass spectrometry analysis on affinity-purified acetylated histone peptides, generated by differential parallel proteolysis. We identify a large number of histone acetylation sites and observe an overall reduction of acetylated histone residues in response to DNA damage, indicative of a histone-wide loss of acetyl modifications. This decrease is mainly caused by DNA damage-induced replication stress coupled to specific proteasome-dependent loss of acetylated histones. Strikingly, this degradation of acetylated histones is independent of ubiquitylation but requires the PA200-proteasome activator, a complex that specifically targets acetylated histones for degradation. The uncovered replication stress-induced degradation of acetylated histones represents an important chromatin-modifying response to cope with replication stress.
Asunto(s)
Cromatina/genética , Daño del ADN/genética , Proteínas Nucleares/genética , Complejo de la Endopetidasa Proteasomal/genética , Acetilación , Secuencia de Aminoácidos/genética , Reparación del ADN/genética , Replicación del ADN/genética , Histonas/genética , Humanos , Proteolisis , Ubiquitinación/genéticaRESUMEN
BACKGROUND: Fanconi anemia (FA) is an inherited genomic instability disorder with congenital and developmental abnormalities, bone marrow failure and predisposition to cancer early in life, and cellular sensitivity to DNA interstrand crosslinks. CASE PRESENTATION: A fifty-one-year old female patient, initially diagnosed with FA in childhood on the basis of classic features and increased chromosomal breakage, and remarkable sun-sensitivity is described. She only ever had mild haematological abnormalities and no history of malignancy. To identify and characterise the genetic defect in this lady, who is one of the oldest reported FA patients, we used whole-exome sequencing for identification of causative mutations, and functionally characterized the cellular phenotype. Detection of the novel splice site mutation c.793-2A > G and the previously described missense mutation c.1765C > T (p.Arg589Trp) in XPF/ERCC4/FANCQ assign her as the third individual of complementation group FA-Q. Ectopic expression of wildtype, but not mutant, XPF/ERCC4/FANCQ, in patient-derived fibroblasts rescued cellular resistance to DNA interstrand-crosslinking agents. Patient derived FA-Q cells showed impaired nuclear excision repair capacity. However, mutated XPF/ERCC4/FANCQ protein in our patient's cells, as in the two other patients with FA-Q, was detectable on chromatin, in contrast to XP-F cells, where missense-mutant protein failed to properly translocate to the nucleus. CONCLUSIONS: Patients with FA characteristics and UV sensitivity should be tested for mutations in XPF/ERCC4/FANCQ. The missense mutation p.Arg589Trp was previously detected in patients diagnosed with Xeroderma pigmentosum or Cockayne syndrome. Hence, phenotypic manifestations associated with this XPF/ERCC4/ FANCQ mutation are highly variable.
Asunto(s)
Proteínas de Unión al ADN/genética , Anemia de Fanconi/genética , Mutación Missense , Trastornos por Fotosensibilidad/genética , Secuencia de Aminoácidos , Línea Celular , Línea Celular Tumoral , Daño del ADN , Reparación del ADN , Anemia de Fanconi/diagnóstico , Femenino , Fibroblastos , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Persona de Mediana Edad , Trastornos por Fotosensibilidad/diagnóstico , Sistema SolarRESUMEN
PURPOSE: Hyperthermia (40-44 °C) effectively sensitises tumours to radiotherapy by locally altering tumour biology. One of the effects of heat at the cellular level is inhibition of DNA repair by homologous recombination via degradation of the BRCA2-protein. This suggests that hyperthermia can expand the group of patients that benefit from PARP-inhibitors, a drug exploiting homologous recombination deficiency. Here, we explore whether the molecular mechanisms that cause heat-mediated degradation of BRCA2 are conserved in cell lines from various origins and, most importantly, whether, BRCA2 protein levels can be attenuated by heat in freshly biopted human tumours. EXPERIMENTAL DESIGN: Cells from four established cell lines and from freshly biopsied material of cervical (15), head- and neck (9) or bladder tumours (27) were heated to 42 °C for 60 min ex vivo. In vivo hyperthermia was studied by taking two biopsies of the same breast or cervical tumour: one before and one after treatment. BRCA2 protein levels were measured by immunoblotting. RESULTS: We found decreased BRCA2-levels after hyperthermia in all established cell lines and in 91% of all tumours treated ex vivo. For tumours treated with hyperthermia in vivo, technical issues and intra-tumour heterogeneity prevented obtaining interpretable results. CONCLUSIONS: This study demonstrates that heat-mediated degradation of BRCA2 occurs in tumour material directly derived from patients. Although BRCA2-degradation may not be a practical biomarker for heat deposition in situ, it does suggest that application of hyperthermia could be an effective method to expand the patient group that could benefit from PARP-inhibitors.
Asunto(s)
Proteína BRCA2/metabolismo , Hipertermia Inducida , Neoplasias/metabolismo , Neoplasias/terapia , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Línea Celular Tumoral , Terapia Combinada , Femenino , Calor , Humanos , ProteolisisRESUMEN
The rare recessive developmental disorder Trichothiodystrophy (TTD) is characterized by brittle hair and nails. Patients also present a variable set of poorly explained additional clinical features, including ichthyosis, impaired intelligence, developmental delay and anemia. About half of TTD patients are photosensitive due to inherited defects in the DNA repair and transcription factor II H (TFIIH). The pathophysiological contributions of unrepaired DNA lesions and impaired transcription have not been dissected yet. Here, we functionally characterize the consequence of a homozygous missense mutation in the general transcription factor II E, subunit 2 (GTF2E2/TFIIEß) of two unrelated non-photosensitive TTD (NPS-TTD) families. We demonstrate that mutant TFIIEß strongly reduces the total amount of the entire TFIIE complex, with a remarkable temperature-sensitive transcription defect, which strikingly correlates with the phenotypic aggravation of key clinical symptoms after episodes of high fever. We performed induced pluripotent stem (iPS) cell reprogramming of patient fibroblasts followed by in vitro erythroid differentiation to translate the intriguing molecular defect to phenotypic expression in relevant tissue, to disclose the molecular basis for some specific TTD features. We observed a clear hematopoietic defect during late-stage differentiation associated with hemoglobin subunit imbalance. These new findings of a DNA repair-independent transcription defect and tissue-specific malfunctioning provide novel mechanistic insight into the etiology of TTD.
Asunto(s)
Factores de Transcripción TFII/genética , Síndromes de Tricotiodistrofia/genética , Diferenciación Celular/genética , Reprogramación Celular/genética , ADN Helicasas/genética , Reparación del ADN , Femenino , Humanos , Células Madre Pluripotentes Inducidas/patología , Masculino , Mutación , Mutación Missense , Especificidad de Órganos , Linaje , Factores de Transcripción TFII/metabolismo , Transcripción Genética , Síndromes de Tricotiodistrofia/metabolismo , Síndromes de Tricotiodistrofia/patologíaRESUMEN
Bladder cancer has a high incidence with significant morbidity and mortality. Attenuated expression of the DNA damage response protein Xeroderma Pigmentosum complementation group C (XPC) has been described in bladder cancer. XPC plays an essential role as the main initiator and damage-detector in global genome nucleotide excision repair (NER) of UV-induced lesions, bulky DNA adducts and intrastrand crosslinks, such as those made by the chemotherapeutic agent Cisplatin. Hence, XPC protein might be an informative biomarker to guide personalized therapy strategies in a subset of bladder cancer cases. Therefore, we measured the XPC protein expression level and functional NER activity of 36 bladder tumors in a standardized manner. We optimized conditions for dissociation and in vitro culture of primary bladder cancer cells and confirmed attenuated XPC expression in approximately 40% of the tumors. However, NER activity was similar to co-cultured wild type cells in all but one of 36 bladder tumors. We conclude, that (i) functional NER deficiency is a relatively rare phenomenon in bladder cancer and (ii) XPC protein levels are not useful as biomarker for NER activity in these tumors.
Asunto(s)
Reparación del ADN , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Vejiga Urinaria/genética , Vejiga Urinaria/patología , Xerodermia Pigmentosa/genética , ADN/genética , Proteínas de Unión al ADN/análisis , Humanos , Células Tumorales Cultivadas , Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/patología , Xerodermia Pigmentosa/patologíaRESUMEN
Mutations in ERCC6 are associated with growth failure, intellectual disability, neurological dysfunction and deterioration, premature aging, and photosensitivity. We describe siblings with biallelic ERCC6 mutations (NM_000124.2:c. [543+4delA];[2008C>T]) and brain hypomyelination, microcephaly, cognitive decline, and skill regression but without photosensitivity or progeria. DNA repair assays on cultured skin fibroblasts confirmed a defect of transcription-coupled nucleotide excision repair and increased ultraviolet light sensitivity. This report expands the disease spectrum associated with ERCC6 mutations.
Asunto(s)
Encéfalo/patología , Encéfalo/fisiopatología , ADN Helicasas/genética , Enzimas Reparadoras del ADN/genética , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Enfermedades del Sistema Nervioso/genética , Empalme Alternativo , Biomarcadores/metabolismo , Niño , Preescolar , ADN Helicasas/metabolismo , Análisis Mutacional de ADN , Enzimas Reparadoras del ADN/metabolismo , Facies , Femenino , Expresión Génica , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/diagnóstico , Humanos , Intrones , Imagen por Resonancia Magnética , Masculino , Mutación , Enfermedades del Sistema Nervioso/diagnóstico , Linaje , Fenotipo , Proteínas de Unión a Poli-ADP-Ribosa , HermanosRESUMEN
Complete human genome sequencing was used to identify the causative mutation in a family with Pollitt syndrome (MIM #275550), comprising two non-consanguineous parents and their two affected children. The patient's symptoms were reminiscent of the non-photosensitive form of recessively inherited trichothiodystrophy (TTD). A mutation in the TTDN1/C7orf11 gene, a gene that is known to be involved in non-photosensitive TTD, had been excluded by others by Sanger sequencing. Unexpectedly, we did find a homozygous single-base pair deletion in the coding region of this gene, a mutation that is known to cause non-photosensitive TTD. The deleterious variant causing a frame shift at amino acid 93 (C326delA) followed the right mode of inheritance in the family and was independently validated using conventional DNA sequencing. We expect this novel DNA sequencing technology to help redefine phenotypic and genomic variation in patients with (mono) genetic disorders in an unprecedented manner.
RESUMEN
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.