RESUMEN
Transcriptional heterogeneity due to plasticity of the epigenetic state of chromatin contributes to tumour evolution, metastasis and drug resistance1-3. However, the mechanisms that cause this epigenetic variation are incompletely understood. Here we identify micronuclei and chromosome bridges, aberrations in the nucleus common in cancer4,5, as sources of heritable transcriptional suppression. Using a combination of approaches, including long-term live-cell imaging and same-cell single-cell RNA sequencing (Look-Seq2), we identified reductions in gene expression in chromosomes from micronuclei. With heterogeneous penetrance, these changes in gene expression can be heritable even after the chromosome from the micronucleus has been re-incorporated into a normal daughter cell nucleus. Concomitantly, micronuclear chromosomes acquire aberrant epigenetic chromatin marks. These defects may persist as variably reduced chromatin accessibility and reduced gene expression after clonal expansion from single cells. Persistent transcriptional repression is strongly associated with, and may be explained by, markedly long-lived DNA damage. Epigenetic alterations in transcription may therefore be inherently coupled to chromosomal instability and aberrations in nuclear architecture.
Asunto(s)
Inestabilidad Cromosómica , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Micronúcleos con Defecto Cromosómico , Neoplasias , Transcripción Genética , Humanos , Cromatina/genética , Cromatina/metabolismo , Cromosomas/genética , Células Clonales/metabolismo , Daño del ADN/genética , Neoplasias/genética , Neoplasias/patología , Análisis de Expresión Génica de una Sola CélulaRESUMEN
DNA repair is critical for genome stability and is maintained through conserved pathways. Traditional genome-wide mammalian screens are both expensive and laborious. However, computational approaches circumvent these limitations and are a powerful tool to identify new DNA repair factors. By analyzing the evolutionary relationships between genes in the major DNA repair pathways, we uncovered functional relationships between individual genes and identified partners. Here we ranked 17,487 mammalian genes for coevolution with 6 distinct DNA repair pathways. Direct comparison to genetic screens for homologous recombination or Fanconi anemia factors indicates that our evolution-based screen is comparable, if not superior, to traditional screening approaches. Demonstrating the utility of our strategy, we identify a role for the DNA damage-induced apoptosis suppressor (DDIAS) gene in double-strand break repair based on its coevolution with homologous recombination. DDIAS knockdown results in DNA double-strand breaks, indicated by ATM kinase activation and 53BP1 foci induction. Additionally, DDIAS-depleted cells are deficient for homologous recombination. Our results reveal that evolutionary analysis is a powerful tool to uncover novel factors and functional relationships in DNA repair.
Asunto(s)
Reparación del ADN/genética , Estudio de Asociación del Genoma Completo/métodos , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Roturas del ADN de Doble Cadena , Evolución Molecular , Inestabilidad Genómica/genética , Recombinación Homóloga/genética , Humanos , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismoRESUMEN
Deficiency in several of the classical human RAD51 paralogs [RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3] is associated with cancer predisposition and Fanconi anemia. To investigate their functions, isogenic disruption mutants for each were generated in non-transformed MCF10A mammary epithelial cells and in transformed U2OS and HEK293 cells. In U2OS and HEK293 cells, viable ablated clones were readily isolated for each RAD51 paralog; in contrast, with the exception of RAD51B, RAD51 paralogs are cell-essential in MCF10A cells. Underlining their importance for genomic stability, mutant cell lines display variable growth defects, impaired sister chromatid recombination, reduced levels of stable RAD51 nuclear foci, and hyper-sensitivity to mitomycin C and olaparib, with the weakest phenotypes observed in RAD51B-deficient cells. Altogether these observations underscore the contributions of RAD51 paralogs in diverse DNA repair processes, and demonstrate essential differences in different cell types. Finally, this study will provide useful reagents to analyze patient-derived mutations and to investigate mechanisms of chemotherapeutic resistance deployed by cancers.
Asunto(s)
Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Recombinación Homóloga/genética , Recombinasa Rad51/genética , Núcleo Celular/genética , Cromátides/genética , Daño del ADN/genética , Genoma Humano/genética , Células HEK293 , Humanos , MutaciónRESUMEN
RAD51 plays a central role in homologous recombination during double-strand break repair and in replication fork dynamics. Misregulation of RAD51 is associated with genetic instability and cancer. RAD51 is regulated by many accessory proteins including the highly conserved Shu complex. Here, we report the function of the human Shu complex during replication to regulate RAD51 recruitment to DNA repair foci and, secondly, during replication fork restart following replication fork stalling. Deletion of the Shu complex members, SWS1 and SWSAP1, using CRISPR/Cas9, renders cells specifically sensitive to the replication fork stalling and collapse caused by methyl methanesulfonate and mitomycin C exposure, a delayed and reduced RAD51 response, and fewer sister chromatid exchanges. Our additional analysis identified SPIDR and PDS5B as novel Shu complex interacting partners and genetically function in the same pathway upon DNA damage. Collectively, our study uncovers a protein complex, which consists of SWS1, SWSAP1, SPIDR and PDS5B, involved in DNA repair and provides insight into Shu complex function and composition.
Asunto(s)
Proteínas de Unión al Calcio/genética , Proteínas de Unión al ADN/genética , Recombinación Homóloga/genética , Proteínas Nucleares/genética , Rec A Recombinasas/genética , Factores de Transcripción/genética , Sistemas CRISPR-Cas/genética , Daño del ADN/genética , Reparación del ADN/genética , Replicación del ADN/genética , Inestabilidad Genómica/genética , Humanos , Complejos Multiproteicos/genética , Recombinasa Rad51/genética , Intercambio de Cromátides Hermanas/genéticaRESUMEN
Chromosomal aberrations are prevalent in cancer genomes, yet it remains challenging to resolve the long-range structure of rearranged chromosomes. A key problem is to determine the chromosomal origin of rearranged genomic segments, which requires chromosome-length haplotype information. Here we describe refLinker, a new computational method for whole-chromosome haplotype inference using external reference panels and Hi-C. We show that refLinker ensures consistent long-range phasing accuracy in both diploid human genomes and aneuploid cancers, including regions with loss-of-heterozygosity and high-level focal amplification. We further demonstrate the feasibility of complex genome reconstruction using haplotype-specific Hi-C contacts, revealing new karyotype features in two widely studied cancer cell lines. Together, these findings provide a new framework for the complete resolution of long-range chromosome structure in complex genomes and highlight the unique advantages of Hi-C data for reconstructing cancer genomes with chromosome-scale continuity.
RESUMEN
The progression of precancerous lesions to malignancy is often accompanied by increasing complexity of chromosomal alterations but how these alterations arise is poorly understood. Here we perform haplotype-specific analysis of chromosomal copy-number evolution in the progression of Barrett's esophagus (BE) to esophageal adenocarcinoma (EAC) on multiregional whole-genome sequencing data of BE with dysplasia and microscopic EAC foci. We identify distinct patterns of copy-number evolution indicating multigenerational chromosomal instability that is initiated by cell division errors but propagated only after p53 loss. While abnormal mitosis, including whole-genome duplication, underlies chromosomal copy-number changes, segmental alterations display signatures of successive breakage-fusion-bridge cycles and chromothripsis of unstable dicentric chromosomes. Our analysis elucidates how multigenerational chromosomal instability generates copy-number variation in BE cells, precipitates complex alterations including DNA amplifications, and promotes their independent clonal expansion and transformation. In particular, we suggest sloping copy-number variation as a signature of ongoing chromosomal instability that precedes copy-number complexity. These findings suggest copy-number heterogeneity in advanced cancers originates from chromosomal instability in precancerous cells and such instability may be identified from the presence of sloping copy-number variation in bulk sequencing data.
Asunto(s)
Adenocarcinoma , Esófago de Barrett , Neoplasias Esofágicas , Lesiones Precancerosas , Humanos , Esófago de Barrett/genética , Esófago de Barrett/patología , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Adenocarcinoma/genética , Adenocarcinoma/patología , Inestabilidad Cromosómica/genética , Lesiones Precancerosas/genética , Lesiones Precancerosas/patología , Genómica , Progresión de la EnfermedadRESUMEN
Haplotype phase represents the collective genetic variation between homologous chromosomes and is an essential feature of non-haploid genomes. Here we describe a computational strategy to reliably determine complete whole-chromosome haplotypes using a combination of bulk long-range sequencing and Hi-C sequencing. We demonstrate that this strategy can resolve the haplotypes of parental chromosomes in diploid human genomes with high precision (>99%) and completeness (>98%) and assemble the syntenic structure of rearranged chromosomes in aneuploid cancer genomes at base pair level resolution. Our work enables direct interrogation of chromosome-specific alterations and chromatin reorganization using bulk DNA sequencing.
Asunto(s)
Haplotipos/genética , Análisis de Secuencia de ADN , Aneuploidia , Cromosomas Humanos/genética , Diploidia , Dosificación de Gen , Ligamiento Genético , Genoma Humano , Humanos , Estadística como AsuntoRESUMEN
Homologous recombination (HR) mediates the error-free repair of DNA double-strand breaks to maintain genomic stability. Here we characterize C17orf53/MCM8IP, an OB-fold containing protein that binds ssDNA, as a DNA repair factor involved in HR. MCM8IP-deficient cells exhibit HR defects, especially in long-tract gene conversion, occurring downstream of RAD51 loading, consistent with a role for MCM8IP in HR-dependent DNA synthesis. Moreover, loss of MCM8IP confers cellular sensitivity to crosslinking agents and PARP inhibition. Importantly, we report that MCM8IP directly associates with MCM8-9, a helicase complex mutated in primary ovarian insufficiency, and RPA1. We additionally show that the interactions of MCM8IP with MCM8-9 and RPA facilitate HR and promote replication fork progression and cellular viability in response to treatment with crosslinking agents. Mechanistically, MCM8IP stimulates the helicase activity of MCM8-9. Collectively, our work identifies MCM8IP as a key regulator of MCM8-9-dependent DNA synthesis during DNA recombination and replication.
Asunto(s)
Daño del ADN , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Reparación del ADN por Recombinación , Línea Celular Tumoral , Supervivencia Celular/genética , Cromatina/genética , Cromatina/metabolismo , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/genética , Células HCT116 , Células HEK293 , Humanos , Proteínas de Mantenimiento de Minicromosoma/genética , Mutación , Unión Proteica , Recombinasa Rad51/metabolismo , Proteína de Replicación A/genética , Proteína de Replicación A/metabolismoRESUMEN
High-grade epithelial ovarian carcinomas containing mutated BRCA1 or BRCA2 (BRCA1/2) homologous recombination (HR) genes are sensitive to platinum-based chemotherapy and PARP inhibitors (PARPi), while restoration of HR function due to secondary mutations in BRCA1/2 has been recognized as an important resistance mechanism. We sequenced core HR pathway genes in 12 pairs of pretreatment and postprogression tumor biopsy samples collected from patients in ARIEL2 Part 1, a phase II study of the PARPi rucaparib as treatment for platinum-sensitive, relapsed ovarian carcinoma. In 6 of 12 pretreatment biopsies, a truncation mutation in BRCA1, RAD51C, or RAD51D was identified. In five of six paired postprogression biopsies, one or more secondary mutations restored the open reading frame. Four distinct secondary mutations and spatial heterogeneity were observed for RAD51CIn vitro complementation assays and a patient-derived xenograft, as well as predictive molecular modeling, confirmed that resistance to rucaparib was associated with secondary mutations.Significance: Analyses of primary and secondary mutations in RAD51C and RAD51D provide evidence for these primary mutations in conferring PARPi sensitivity and secondary mutations as a mechanism of acquired PARPi resistance. PARPi resistance due to secondary mutations underpins the need for early delivery of PARPi therapy and for combination strategies. Cancer Discov; 7(9); 984-98. ©2017 AACR.See related commentary by Domchek, p. 937See related article by Quigley et al., p. 999See related article by Goodall et al., p. 1006This article is highlighted in the In This Issue feature, p. 920.