RESUMEN
The end joining of distant DNA double-strand ends (DSEs) can produce potentially deleterious rearrangements. We show that depletion of cohesion complex proteins specifically stimulates the end joining (both C-NHEJ and A-EJ) of distant, but not close, I-SceI-induced DSEs in S/G2 phases. At the genome level, whole-exome sequencing showed that ablation of RAD21 or Sororin produces large chromosomal rearrangements (translocation, duplication, deletion). Moreover, cytogenetic analysis showed that RAD21 silencing leads to the formation of chromosome fusions synergistically with replication stress, which generates distant single-ended DSEs. These data reveal a role for the cohesin complex in protecting against genome rearrangements arising from the ligation of distant DSEs in S/G2 phases (both long-range DSEs and those that are only a few kilobases apart), while keeping end joining fully active for close DSEs. Therefore, this role likely involves limitation of DSE motility specifically in S phase, rather than inhibition of the end-joining machinery itself.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN , Replicación del ADN , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/genética , Línea Celular , Proteínas Cromosómicas no Histona/genética , Aberraciones Cromosómicas , Proteínas de Unión al ADN , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Puntos de Control de la Fase G2 del Ciclo Celular , Reordenamiento Génico , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Interferencia de ARN , Puntos de Control de la Fase S del Ciclo Celular , Factores de Tiempo , Transfección , CohesinasRESUMEN
Hoyeraal-Hreidarsson syndrome (HHS) is a severe form of Dyskeratosis congenita characterized by developmental defects, bone marrow failure and immunodeficiency and has been associated with telomere dysfunction. Recently, mutations in Regulator of Telomere ELongation helicase 1 (RTEL1), a helicase first identified in Mus musculus as being responsible for the maintenance of long telomeres, have been identified in several HHS patients. Here we show that RTEL1 is required for the export and the correct cytoplasmic trafficking of the small nuclear (sn) RNA pre-U2, a component of the major spliceosome complex. RTEL1-HHS cells show abnormal subcellular partitioning of pre-U2, defects in the recycling of ribonucleotide proteins (RNP) in the cytoplasm and splicing defects. While most of these phenotypes can be suppressed by re-expressing the wild-type protein in RTEL1-HHS cells, expression of RTEL1 mutated variants in immortalized cells provokes cytoplasmic mislocalizations of pre-U2 and other RNP components, as well as splicing defects, thus phenocopying RTEL1-HHS cellular defects. Strikingly, expression of a cytoplasmic form of RTEL1 is sufficient to correct RNP mislocalizations both in RTEL1-HHS cells and in cells expressing nuclear mutated forms of RTEL1. This work unravels completely unanticipated roles for RTEL1 in RNP trafficking and strongly suggests that defects in RNP biogenesis pathways contribute to the pathology of HHS.
Asunto(s)
Núcleo Celular/metabolismo , Citoplasma/metabolismo , ADN Helicasas/fisiología , Precursores del ARN/metabolismo , ARN Nuclear Pequeño/metabolismo , Secuencia de Bases , Transporte Biológico , Northern Blotting , Cromatografía Liquida , ADN Helicasas/genética , Cartilla de ADN , Células HEK293 , Células HeLa , Humanos , Reacción en Cadena de la Polimerasa , ARN Interferente Pequeño , Espectrometría de Masas en TándemRESUMEN
BACKGROUND: Myb-Like, SWIRM, and MPN domains 1 (MYSM1) is a metalloprotease that deubiquitinates the K119-monoubiquitinated form of histone 2A (H2A), a chromatin marker associated with gene transcription silencing. Likewise, it has been reported that murine Mysm1 participates in transcription derepression of genes, among which are transcription factors involved in hematopoietic stem cell homeostasis, hematopoiesis, and lymphocyte differentiation. However, whether MYSM1 has a similar function in human subjects remains unclear. Here we describe a patient presenting with a complete lack of B lymphocytes, T-cell lymphopenia, defective hematopoiesis, and developmental abnormalities. OBJECTIVES: We sought to characterize the underlying genetic cause of this syndrome. METHODS: We performed genome-wide homozygosity mapping, followed by whole-exome sequencing. RESULTS: Genetic analysis revealed that this novel disorder is caused by a homozygous MYSM1 missense mutation affecting the catalytic site within the deubiquitinase JAB1/MPN/Mov34 (JAMM)/MPN domain. Remarkably, during the course of our study, the patient recovered a normal immunohematologic phenotype. Genetic analysis indicated that this improvement originated from a spontaneous genetic reversion of the MYSM1 mutation in a hematopoietic stem cell. CONCLUSIONS: We here define a novel human immunodeficiency and provide evidence that MYSM1 is essential for proper immunohematopoietic development in human subjects. In addition, we describe one of the few examples of spontaneous in vivo genetic cure of a human immunodeficiency.
Asunto(s)
Proteínas de Unión al ADN/genética , Síndromes de Inmunodeficiencia/genética , Factores de Transcripción/genética , Linfocitos B/citología , Diferenciación Celular , Hematopoyesis/genética , Humanos , Lactante , Linfopenia/genética , Masculino , Mutación , Linfocitos T/citología , Transactivadores , Proteasas Ubiquitina-EspecíficasRESUMEN
Hoyeraal-Hreidarsson syndrome (HHS), a severe variant of dyskeratosis congenita (DC), is characterized by early onset bone marrow failure, immunodeficiency and developmental defects. Several factors involved in telomere length maintenance and/or protection are defective in HHS/DC, underlining the relationship between telomere dysfunction and these diseases. By combining whole-genome linkage analysis and exome sequencing, we identified compound heterozygous RTEL1 (regulator of telomere elongation helicase 1) mutations in three patients with HHS from two unrelated families. RTEL1 is a DNA helicase that participates in DNA replication, DNA repair and telomere integrity. We show that, in addition to short telomeres, RTEL1-deficient cells from patients exhibit hallmarks of genome instability, including spontaneous DNA damage, anaphase bridges and telomeric aberrations. Collectively, these results identify RTEL1 as a novel HHS-causing gene and highlight its role as a genomic caretaker in humans.
Asunto(s)
ADN Helicasas/genética , Disqueratosis Congénita/genética , Retardo del Crecimiento Fetal/genética , Inestabilidad Genómica , Discapacidad Intelectual/genética , Microcefalia/genética , Homeostasis del Telómero/genética , Acortamiento del Telómero , Telómero/metabolismo , Células Cultivadas , Preescolar , Daño del ADN , ADN Helicasas/química , ADN Helicasas/deficiencia , ADN Helicasas/metabolismo , Replicación del ADN , Disqueratosis Congénita/metabolismo , Exoma , Femenino , Retardo del Crecimiento Fetal/metabolismo , Ligamiento Genético , Humanos , Lactante , Discapacidad Intelectual/metabolismo , Masculino , Microcefalia/metabolismo , Mutación , Alineación de Secuencia , Análisis de Secuencia de ARN , Telomerasa/genética , Telomerasa/metabolismo , Telómero/ultraestructuraRESUMEN
Ataxia telangiectasia-mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) kinases are two key regulators of DNA-damage responses (DDR) that are mainly activated in response to DNA double-strand breaks and single-stranded DNA damages, respectively. Seckel syndrome, a rare genetic disorder characterized by a microcephaly and a markedly reduced body size, has been associated with defective ATR-dependent DNA damage signaling. However, the only human genetic ATR defect reported so far is a hypomorphic splicing mutation identified in five related individuals with Seckel syndrome. Here, we report the first case of primary microcephaly with compound heterozygous mutations in ATR: a 540 kb genomic deletion on one allele and a missense mutation leading to splice dysregulation on the other, which ultimately lead to a sharp decrease in ATR expression. DNA combing technology revealed a profound spontaneous alteration of several DNA replication parameters in patient's cells and FISH analyses highlighted the genomic instability caused by ATR deficiency. Collectively, our results emphasize the crucial role for ATR in the control of DNA replication, and reinforce the complementary and nonredundant contributions of ATM and ATR in human cells to face DNA damages and warrant genome integrity.
Asunto(s)
Proteínas de Ciclo Celular/genética , Replicación del ADN , ADN de Cadena Simple , Proteínas de Unión al ADN/genética , Inestabilidad Genómica , Microcefalia/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Supresoras de Tumor/genética , Alelos , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Secuencia de Bases , Western Blotting , Línea Celular , Niño , Hibridación Genómica Comparativa , Roturas del ADN de Doble Cadena , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente , Eliminación de Gen , Estudio de Asociación del Genoma Completo , Heterocigoto , Humanos , Hibridación Fluorescente in Situ , Masculino , Ratones , Datos de Secuencia Molecular , Mutación Missense , Empalme del ARN , Transducción de SeñalRESUMEN
BACKGROUND: Telomeres represent the tips of linear chromosomes. In human subjects telomere maintenance deficiency leads to dyskeratosis congenita (DC), a rare genetic disorder characterized by progressive bone marrow failure, accelerated aging, and cancer predisposition. Hoyeraal-Hreidarsson syndrome (HH) is a severe variant of DC in which an early onset of bone marrow failure leading to combined immunodeficiency is associated with microcephaly, cerebellar hypoplasia, and growth retardation. OBJECTIVES: Limited information is available on the cellular and molecular phenotypes of cells from patients with HH. We analyzed fibroblasts and whole blood cells from 5 patients with HH, 3 of them of unknown molecular origin. METHODS: Telomere length, cellular senescence rate, telomerase activity, telomeric aberration, and DNA repair pathways were investigated. RESULTS: Although patients' cells exhibit dysfunctional telomeres, sharp differences in the telomeric aberrations and telomere lengths were noted among these patients. In some patients the dysfunctional telomere phenotype was unprecedented and associated with either normal telomere length or with telomeric aberrations akin to fragile telomeres. This result is of particular importance because the molecular diagnosis of these patients is primarily based on telomere length, which therefore misses a subset of patients with telomere dysfunction. CONCLUSION: These observations provide the notions that (1) various telomere defects can lead to similar clinical features, (2) telomere dysfunction in cells from patients with DC/HH is not always associated with short telomeres, and (3) additional factors, likely involved in telomere protection rather than in length regulation, are responsible for a subset of DC/HH.
Asunto(s)
Disqueratosis Congénita/metabolismo , Retardo del Crecimiento Fetal/metabolismo , Fibroblastos/metabolismo , Discapacidad Intelectual/metabolismo , Microcefalia/metabolismo , Telómero/metabolismo , Preescolar , ADN/genética , Reparación del ADN , Disqueratosis Congénita/genética , Disqueratosis Congénita/patología , Femenino , Retardo del Crecimiento Fetal/genética , Retardo del Crecimiento Fetal/patología , Fibroblastos/enzimología , Fibroblastos/patología , Humanos , Lactante , Discapacidad Intelectual/genética , Discapacidad Intelectual/patología , Masculino , Microcefalia/genética , Microcefalia/patología , Fenotipo , Análisis de Secuencia de ADN , Telomerasa/metabolismo , Telómero/patología , Proteínas de Unión a Telómeros/genéticaRESUMEN
RTEL1 (regulator of telomere length helicase 1) is a DNA helicase that has been identified more than 10 years ago. Many works since, mainly in the nematode Caenorhabditis elegans and the mouse, have highlighted its role in chromosomal stability, maintenance of telomere length, and DNA repair. Recently, four laboratories have characterized RTEL1 mutations in patients with dyskeratosis congenita (DC) and Hoyeraal-Hreidarsson (HH) syndrome, a rare and severe variant of DC. We here summarize the current knowledge on RTEL1 and discuss the possible other functions that RTEL1 could play.
Asunto(s)
ADN Helicasas/fisiología , Inestabilidad Genómica/fisiología , Animales , ADN Helicasas/genética , Reparación del ADN , Disqueratosis Congénita/genética , Retardo del Crecimiento Fetal/genética , Fenómenos Genéticos/genética , Fenómenos Genéticos/fisiología , Inestabilidad Genómica/genética , Humanos , Discapacidad Intelectual/genética , Ratones , Microcefalia/genética , Mutación , Telómero/fisiologíaRESUMEN
Telomeres are nucleoprotein structures at the end of linear chromosomes. Their length, structure, and integrity are regulated by the telomerase complex, the shelterins and components of the DNA damage response. In human subjects, defects in telomere maintenance are responsible for Dyskeratosis Congenita (DC), a rare genetic disorder characterized by aplastic anaemia, premature aging and predisposition to cancer. Recent data from the study of patients with Hoyeraal-Hreidarsson syndrome, a severe variant of DC, demonstrate the great molecular heterogeneity of this disease. While most cases of DC are associated with defects in factors involved in telomere length regulation, some severe forms of the disease seem to be rather associated with defects in telomere replication and protection.
Asunto(s)
Disqueratosis Congénita/genética , Telómero/fisiología , Replicación del ADN/genética , Replicación del ADN/fisiología , Disqueratosis Congénita/diagnóstico , Disqueratosis Congénita/etiología , Retardo del Crecimiento Fetal/diagnóstico , Retardo del Crecimiento Fetal/genética , Humanos , Discapacidad Intelectual/diagnóstico , Discapacidad Intelectual/genética , Microcefalia/diagnóstico , Microcefalia/genética , Modelos Biológicos , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/fisiología , Telomerasa/genética , Telomerasa/metabolismo , Telómero/genética , Telómero/metabolismoRESUMEN
The forkhead box G1 (FOXG1)gene has recently been associated with the congenital variant of Rett syndrome, and so far 17 mutations have been reported. We screened the coding region in 150 patients affected by postnatal microcephaly, and identified two mutations: the c.326C>T (p.P109L) substitution inherited from the healthy father; and the de novo c.730C>T transition, which induces the p.R244C mutation within the DNA-binding forkhead domain. This latter mutation is carried by an 8-year-old girl, who presented a phenotype reminiscent of the congenital variant of Rett syndrome. Immunofluorescence analysis of the wild-type protein revealed a homogeneous nuclear staining excluding the nucleoli, while the p.R244C mutant showed abnormal nuclear foci in a large proportion of cells, suggesting that its mislocalization may reduce and/or impair target recognition. Interestingly, this missense mutation results in a mislocalization of FoxG1 to specific nuclear foci referred to as nuclear speckles, and affects the cyclin-dependent kinase inhibitor p21 CDKN1A expression. Because CDKL5, which is involved in the early-onset variant of Rett syndrome, is also located in these speckles, we suggest that disregulation of the dynamic behaviour of nuclear speckles may functionally link these two proteins, which are both involved in atypical forms of Rett syndrome.
Asunto(s)
Núcleo Celular/metabolismo , Factores de Transcripción Forkhead/genética , Mutación Missense , Proteínas del Tejido Nervioso/genética , Secuencia de Aminoácidos , Línea Celular Tumoral , Núcleo Celular/química , Niño , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Femenino , Factores de Transcripción Forkhead/análisis , Humanos , Lactante , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/análisis , Proteínas Serina-Treonina Quinasas/genética , Síndrome de Rett/genética , Alineación de SecuenciaRESUMEN
Mutations in the FOXG1 gene have been shown to cause congenital variant of Rett syndrome. To date, point mutations have been reported only in female patients. We screened the entire coding region of the gene for mutations in 50 boys with congenital encephalopathy, postnatal microcephaly, and complex movement disorders, a clinical picture very similar to that described in girls with FOXG1 mutations. We found one boy carrying the de novo c.256_257dupC frameshift mutation. He presented the association of postnatal microcephaly, severe axial dystonia with severe feeding difficulties with protruding tongue movements during the first year of life that subsequently evolved into dyskinetic movement disorders with hand stereotypies. In contrast to his severe motor impairment, he developed nonverbal communication skills and relative good eye contact. Brain MRI showed frontal gyral simplification with dramatic myelination delay most prominent in both frontal lobes. Altogether the presentation in this male patient is highly reminiscent of that observed in FOXG1-mutated females with the congenital variant of Rett syndrome. This new case confirms the prediction that congenital variant of Rett syndrome should be found also in males, with the characteristic hallmarks consisting of postnatal microcephaly, dyskinetic movement disorder with Rett-like features, i.e., hand stereotypies, and frontal gyral simplification with myelination delay. FOXG1 screening should be considered in individuals with these clinical features.
Asunto(s)
Factores de Transcripción Forkhead/genética , Mutación del Sistema de Lectura , Proteínas del Tejido Nervioso/genética , Síndrome de Rett/genética , Secuencia de Bases , Preescolar , Deleción Cromosómica , Cromosomas Humanos Par 14/genética , Análisis Mutacional de ADN , Cartilla de ADN/genética , Femenino , Humanos , Imagen por Resonancia Magnética , Masculino , Microcefalia/genética , Microcefalia/patología , Trastornos del Movimiento/genética , Síndrome de Rett/diagnóstico , Síndrome de Rett/patologíaRESUMEN
DNA double-strand breaks (DSBs) are highly toxic lesions that can lead to profound genome rearrangements and/or cell death. They routinely occur in genomes due to endogenous or exogenous stresses. Efficient repair systems, canonical non-homologous end-joining and homologous recombination exist in the cell and not only ensure the maintenance of genome integrity but also, via specific programmed DNA double-strand breaks, permit its diversity and plasticity. However, these repair systems need to be tightly controlled because they can also generate genomic rearrangements. Thus, when DSB repair is not properly regulated, genome integrity is no longer guaranteed. In this review, we will focus on non-programmed genome rearrangements generated by DSB repair, in somatic cells. We first discuss genome rearrangements induced by homologous recombination and end-joining. We then discuss recently described rearrangement mechanisms, driven by microhomologies, that do not involve the joining of DNA ends but rather initiate DNA synthesis (microhomology-mediated break-induced replication, fork stalling and template switching and microhomology-mediated template switching). Finally, we discuss chromothripsis, which is the shattering of a localized region of the genome followed by erratic rejoining.
Asunto(s)
Roturas del ADN de Doble Cadena , Reordenamiento Génico , Modelos Genéticos , Estrés Fisiológico , Regulación hacia Arriba , Animales , Cromotripsis , Reparación del ADN por Unión de Extremidades , Reparación del ADN , Inestabilidad Genómica , Humanos , Estrés Oxidativo , Reparación del ADN por RecombinaciónRESUMEN
DNA double-strand breaks (DSBs) are highly lethal lesions that jeopardize genome integrity. However, DSBs are also used to generate diversity during the physiological processes of meiosis or establishment of the immune repertoire. Therefore, DSB repair must be tightly controlled. Two main strategies are used to repair DSBs: homologous recombination (HR) and non-homologous end joining (NHEJ). HR is generally considered to be error-free, whereas NHEJ is considered to be error-prone. However, recent data challenge these assertions. Here, we present the molecular mechanisms involved in HR and NHEJ and the recently described alternative end-joining mechanism, which is exclusively mutagenic. Whereas NHEJ is not intrinsically error-prone but adaptable, HR has the intrinsic ability to modify the DNA sequence. Importantly, in both cases the initial structure of the DNA impacts the outcome. Finally, the consequences and applications of these repair mechanisms are discussed. Both HR and NHEJ are double-edged swords, essential for maintenance of genome stability and diversity but also able to generate genome instability.