RESUMEN
Transcription is a major contributor to genomic instability. The ribosomal RNA (rDNA) gene locus consists of a head-to-tail repeat of the most actively transcribed genes in the genome. RNA polymerase I (RNAPI) is responsible for massive rRNA production, and nascent rRNA is co-transcriptionally assembled with early assembly factors in the yeast nucleolus. In Saccharomyces cerevisiae, a mutant form of RNAPI bearing a fusion of the transcription factor Rrn3 with RNAPI subunit Rpa43 (CARA-RNAPI) has been described previously. Here, we show that the CARA-RNAPI allele results in a novel type of rRNA processing defect, associated with rDNA genomic instability. A fraction of the 35S rRNA produced in CARA-RNAPI mutant escapes processing steps and accumulates. This accumulation is increased in mutants affecting exonucleolytic activities of the exosome complex. CARA-RNAPI is synthetic lethal with monopolin mutants that are known to affect the rDNA condensation. CARA-RNAPI strongly impacts rDNA organization and increases rDNA copy number variation. Reduced rDNA copy number suppresses lethality, suggesting that the chromosome segregation defect is caused by genomic rDNA instability. We conclude that a constitutive association of Rrn3 with transcribing RNAPI results in the accumulation of rRNAs that escape normal processing, impacting rDNA organization and affecting rDNA stability.
Asunto(s)
ADN Ribosómico , Inestabilidad Genómica , Mutación , ARN Polimerasa I , Procesamiento Postranscripcional del ARN , ARN Ribosómico , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , ADN Ribosómico/genética , ADN Ribosómico/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , ARN Polimerasa I/metabolismo , ARN Polimerasa I/genética , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas del Complejo de Iniciación de Transcripción Pol1RESUMEN
Pervasive transcription is widespread in eukaryotes, generating large families of non-coding RNAs. Such pervasive transcription is a key player in the regulatory pathways controlling chromatin state and gene expression. Here, we describe long non-coding RNAs generated from the ribosomal RNA gene promoter called UPStream-initiating transcripts (UPS). In yeast, rDNA genes are organized in tandem repeats in at least two different chromatin states, either transcribed and largely depleted of nucleosomes (open) or assembled in regular arrays of nucleosomes (closed). The production of UPS transcripts by RNA Polymerase II from endogenous rDNA genes was initially documented in mutants defective for rRNA production by RNA polymerase I. We show here that UPS are produced in wild-type cells from closed rDNA genes but are hidden within the enormous production of rRNA. UPS levels are increased when rDNA chromatin states are modified at high temperatures or entering/leaving quiescence. We discuss their role in the regulation of rDNA chromatin states and rRNA production.
RESUMEN
Hematopoiesis is particularly sensitive to DNA damage. Myeloid tumor incidence increases in patients with DNA repair defects and after chemotherapy. It is not known why hematopoietic cells are highly vulnerable to DNA damage. Addressing this question is complicated by the paucity of mouse models of hematopoietic malignancies due to defective DNA repair. We show that DNA repair-deficient Mcm8- and Mcm9-knockout mice develop myeloid tumors, phenocopying prevalent myelodysplastic syndromes. We demonstrate that these tumors are preceded by a lifelong DNA damage burden in bone marrow and that they acquire proliferative capacity by suppressing signaling of the tumor suppressor and cell cycle controller RB, as often seen in patients. Finally, we found that absence of MCM9 and the tumor suppressor Tp53 switches tumorigenesis to lymphoid tumors without precedent myeloid malignancy. Our results demonstrate that MCM8/9 deficiency drives myeloid tumor development and establishes a DNA damage burdened mouse model for hematopoietic malignancies.
Asunto(s)
Diferenciación Celular/genética , Daño del ADN/genética , Regulación Leucémica de la Expresión Génica/genética , Neoplasias Hematológicas/metabolismo , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Envejecimiento/genética , Envejecimiento/metabolismo , Envejecimiento/fisiología , Animales , Apoptosis/genética , Médula Ósea/metabolismo , Médula Ósea/patología , Proliferación Celular/genética , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patología , Ratones , Ratones Noqueados , Proteínas de Mantenimiento de Minicromosoma/genética , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismo , Transducción de Señal/genética , Esplenomegalia/genética , Esplenomegalia/metabolismo , Proteína p53 Supresora de Tumor/genéticaRESUMEN
DNA replication stress (DRS) leads to the accumulation of stalled DNA replication forks leaving a fraction of genomic loci incompletely replicated, a source of chromosomal rearrangements during their partition in mitosis. MUS81 is known to limit the occurrence of chromosomal instability by processing these unresolved loci during mitosis. Here, we unveil that the endonucleases ARTEMIS and XPF-ERCC1 can also induce stalled DNA replication forks cleavage through non-epistatic pathways all along S and G2 phases of the cell cycle. We also showed that both nucleases are recruited to chromatin to promote replication fork restart. Finally, we found that rapid chromosomal breakage controlled by ARTEMIS and XPF is important to prevent mitotic segregation defects. Collectively, these results reveal that Rapid Replication Fork Breakage (RRFB) mediated by ARTEMIS and XPF in response to DRS contributes to DNA replication efficiency and limit chromosomal instability.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Fase G2/genética , Proteínas Nucleares/metabolismo , Fase S/genética , Línea Celular Tumoral , Segregación Cromosómica/fisiología , Roturas del ADN de Doble Cadena , Daño del ADN/fisiología , Reparación del ADN/fisiología , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Fibroblastos , Inestabilidad Genómica/fisiología , Holoenzimas/genética , Holoenzimas/metabolismo , Humanos , Proteínas Nucleares/genética , ARN Interferente Pequeño/metabolismoRESUMEN
Alteration in the DNA replication, repair or recombination processes is a highly relevant mechanism of genomic instability. Despite genomic aberrations manifested in hematologic malignancies, such a defect as a source of biomarkers has been underexplored. Here, we investigated the prognostic value of expression of 82 genes involved in DNA replication-repair-recombination in a series of 99 patients with chronic lymphocytic leukemia without detectable 17p deletion or TP53 mutation. We found that expression of the POLN gene, encoding the specialized DNA polymerase ν (Pol ν) correlates with time to relapse after first-line therapy with fludarabine. Moreover, we found that POLN was the only gene up-regulated in primary patients' lymphocytes when exposed in vitro to proliferative and pro-survival stimuli. By using two cell lines that were sequentially established from the same patient during the course of the disease and Pol ν knockout mouse embryonic fibroblasts, we reveal that high relative POLN expression is important for DNA synthesis and cell survival upon fludarabine treatment. These findings suggest that Pol ν could influence therapeutic resistance in chronic lymphocytic leukemia. (Patients' samples were obtained from the CLL 2007 FMP clinical trial registered at: clinicaltrials.gov identifer: 00564512).
Asunto(s)
ADN Polimerasa Dirigida por ADN/genética , Resistencia a Antineoplásicos/genética , Regulación Leucémica de la Expresión Génica , Leucemia Linfocítica Crónica de Células B/genética , Proteína p53 Supresora de Tumor/genética , Vidarabina/análogos & derivados , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Línea Celular Tumoral , ADN Polimerasa Dirigida por ADN/metabolismo , Progresión de la Enfermedad , Perfilación de la Expresión Génica , Humanos , Leucemia Linfocítica Crónica de Células B/diagnóstico , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/mortalidad , Ratones , Mutación , Pronóstico , Modelos de Riesgos Proporcionales , Vidarabina/farmacología , Vidarabina/uso terapéuticoRESUMEN
Chromosomal domains in Drosophila are marked by the insulator-binding proteins (IBPs) dCTCF/Beaf32 and cofactors that participate in regulating long-range interactions. Chromosomal borders are further enriched in specific histone modifications, yet the role of histone modifiers and nucleosome dynamics in this context remains largely unknown. Here, we show that IBP depletion impairs nucleosome dynamics specifically at the promoters and coding sequence of genes flanked by IBP binding sites. Biochemical purification identifies the H3K36 histone methyltransferase NSD/dMes-4 as a novel IBP cofactor, which specifically co-regulates the chromatin accessibility of hundreds of genes flanked by dCTCF/Beaf32. NSD/dMes-4 presets chromatin before the recruitment of transcriptional activators including DREF that triggers Set2/Hypb-dependent H3K36 trimethylation, nucleosome positioning, and RNA splicing. Our results unveil a model for how IBPs regulate nucleosome dynamics and gene expression through NSD/dMes-4, which may regulate H3K27me3 spreading. Our data uncover how IBPs dynamically regulate chromatin organization depending on distinct cofactors.
Asunto(s)
Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas del Ojo/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Elementos Aisladores/genética , Modelos Biológicos , Nucleosomas/fisiología , Animales , Western Blotting , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas del Ojo/genética , N-Metiltransferasa de Histona-Lisina/genética , Histonas/metabolismo , Análisis por Micromatrices , Datos de Secuencia Molecular , Análisis de Componente Principal , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ARN , Técnicas del Sistema de Dos HíbridosRESUMEN
Eukaryotic chromosomes are partitioned into topologically associating domains (TADs) that are demarcated by distinct insulator-binding proteins (IBPs) in Drosophila. Whether IBPs regulate specific long-range contacts and how this may impact gene expression remains unclear. Here we identify "indirect peaks" of multiple IBPs that represent their distant sites of interactions through long-range contacts. Indirect peaks depend on protein-protein interactions among multiple IBPs and their common cofactors, including CP190, as confirmed by high-resolution analyses of long-range contacts. Mutant IBPs unable to interact with CP190 impair long-range contacts as well as the expression of hundreds of distant genes that are specifically flanked by indirect peaks. Regulation of distant genes strongly correlates with RNAPII pausing, highlighting how this key transcriptional stage may trap insulator-based long-range interactions. Our data illustrate how indirect peaks may decipher gene regulatory networks through specific long-range interactions.
Asunto(s)
Inmunoprecipitación de Cromatina/métodos , Regulación de la Expresión Génica , Elementos Aisladores/fisiología , ARN Polimerasa II/metabolismo , Animales , Sitios de Unión , Factor de Unión a CCCTC , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Proteínas del Ojo/metabolismo , Redes Reguladoras de Genes , Mutación , Regiones Promotoras Genéticas , Unión Proteica , Mapeo de Interacción de Proteínas , Interferencia de ARN , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Rothmund-Thomson syndrome (RTS), a rare recessive autosomal disorder, presents genome instability and clinical heterogeneity with growth deficiency, skin and bone defects, premature aging symptoms and cancer susceptibility. A subset of RTS patients presents mutations of the RECQL4 gene, member of the RecQ family of DNA helicases, including the RECQL2 (BLM) and RECQL3 (WRN) genes, defective in the cancer prone Bloom and Werner syndromes, respectively. Analysis of the RECQL4 gene in six clinically diagnosed RTS patients shows five patients, including two siblings, with eight mutations mainly located in the helicase domain, three patients presenting two mutations. The alterations include four missense mutations, one nonsense mutation and the same frameshift deletion, g.2881delG in exon 9 found in three patients. Seven RECQL4 polymorphisms, two being new, have also been identified. Primary RTS fibroblasts from these RTS patients show no sensitivity to a wide variety of genotoxic agents including ionizing or ultraviolet irradiation, nitrogen mustard, 4NQO, 8-MOP, Cis-Pt, MMC, H2O2, HU, or UV plus caffeine which could be related to the RECQL4 alterations identified here. This is in contrast with the DNA damage sensitive Bloom and Werner cells and highlights the complexity of the numerous RecQ protein functions implicated in the different cellular pathways required for maintaining genomic integrity.
Asunto(s)
Daño del ADN/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Mutágenos/toxicidad , Mutación/genética , RecQ Helicasas/genética , Síndrome Rothmund-Thomson/genética , Adolescente , Adulto , Células Cultivadas , Niño , Daño del ADN/genética , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Masculino , Radiación Ionizante , Síndrome Rothmund-Thomson/metabolismo , HermanosRESUMEN
Immunosuppressed renal transplant recipients (RTRs) are predisposed to non-melanoma skin cancers (NMSCs), predominantly squamous cell carcinomas (SCCs). We have analyzed skin lesions from RTRs with aggressive tumors for p53 gene modifications, the presence of Human Papillomas Virus (HPV) DNA in relation to the p53 codon 72 genotype and polymorphisms of the XPD repair gene. We detected 24 p53 mutations in 15/25 (60%) NMSCs, 1 deletion and 23 base substitutions, the majority (78%) being UV-specific C to T transitions at bipyrimidine sites. Importantly, 35% (6/17) are tandem mutations, including 4 UV signature CC to TT transitions possibly linked to modulated DNA repair caused by the immunosuppressive drug cyclosporin A (CsA). We found 8 p53 mutations in 7/17 (41%) precancerous actinic keratosis (AK), suggesting that p53 mutations are early events in RTR skin carcinogenesis. Immunohistochemical analysis shows a good correlation between p53 accumulation and mutations. HPV DNA was detected in 78% of skin lesions (60% Basal Cell Carcinomas, 82%AK and 79% SCCs). Thus, immunosuppression has increased the risk of infections by HPVs, predominantly epidermodysplasia verruciformis, speculated to play a role in skin cancer development. No association is found between HPV status and p53 mutation. Moreover, p53 codon 72 or frequencies of three XPD genotypes of RTRs are comparable with control populations. The p53 mutation spectrum, presenting a high level of CC to TT mutations, shows that the UV component of sunlight is the major risk factor and modulated DNA repair by immunosuppressive drug treatment may be significant in the skin carcinogenesis of RTRs.
Asunto(s)
Carcinoma de Células Escamosas/epidemiología , Genes p53 , Terapia de Inmunosupresión/efectos adversos , Trasplante de Riñón/inmunología , Polimorfismo Genético , Neoplasias Cutáneas/epidemiología , Rayos Ultravioleta , Carcinoma de Células Escamosas/genética , Codón , ADN Viral/genética , Genotipo , Humanos , Factores de Riesgo , Neoplasias Cutáneas/genéticaRESUMEN
Altered sonic hedgehog (SHH) signaling is crucial in the development of basal cell carcinomas (BCC), the most common human cancer. Mutations in SHH signal transducers, PATCHED and SMOOTHENED, have already been identified, but SHH mutations are extremely rare; only 1 was detected in 74 sporadic BCCs. We present data showing unique SHH mutations in BCCs from repair-deficient, skin cancer-prone xeroderma pigmentosum (XP) patients, which are characterized by high levels of UV-specific mutations in key genes involved in skin carcinogenesis, including PATCHED and SMOOTHENED. Thus, 6 UV-specific SHH mutations were detected in 5 of 33 XP BCCs. These missense SHH alterations are not activating mutations for its postulated proto-oncogene function, as the mutant SHH proteins do not show transforming activity and induce differentiation or stimulate proliferation to the same level as the wild-type protein. Structural modeling studies of the 4 proteins altered at the surface residues, G57S, G64K, D147N, and R155C, show that they do not effect the protein conformation. Interestingly, they are all located on one face of the compact SHH protein suggesting that they may have altered affinity for different partners, which may be important in altering other functions. Additional functional analysis of the SHH mutations found in vivo in XP BCCs will help shed light on the role of SHH in skin carcinogenesis. In conclusion, we report for the first time, significant levels of SHH mutations found only in XP BCCs and none in squamous cell carcinomas, indicating their importance in the specific development of BCCs.