RESUMO
Common fragile sites (CFSs) are conserved genomic regions prone to break under conditions of replication stress (RS). Thus, CFSs are hotspots for rearrangements in cancer and contribute to its chromosomal instability. Here, we have performed a global analysis of proteins that recruit to CFSs upon mild RS to identify novel players in CFS stability. To this end, we performed Chromatin Immunoprecipitation (ChIP) of FANCD2, a protein that localizes specifically to CFSs in G2/M, coupled to mass spectrometry to acquire a CFS interactome. Our strategy was validated by the enrichment of many known regulators of CFS maintenance, including Fanconi Anemia, DNA repair and replication proteins. Among the proteins identified with unknown functions at CFSs was the chromatin remodeler ATRX. Here we demonstrate that ATRX forms foci at a fraction of CFSs upon RS, and that ATRX depletion increases the occurrence of chromosomal breaks, a phenotype further exacerbated under mild RS conditions. Accordingly, ATRX depletion increases the number of 53BP1 bodies and micronuclei, overall indicating that ATRX is required for CFS stability. Overall, our study provides the first proteomic characterization of CFSs as a valuable resource for the identification of novel regulators of CFS stability.
Assuntos
Sítios Frágeis do Cromossomo , Instabilidade Genômica , Proteoma/metabolismo , Proteômica/métodos , Proteína Nuclear Ligada ao X/metabolismo , Quebra Cromossômica , Reparo do DNA , Replicação do DNA/genética , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Células HeLa , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Proteoma/genética , Interferência de RNA , Espectrometria de Massas em Tandem , Proteína Nuclear Ligada ao X/genéticaRESUMO
The generation of genetically engineered mouse models (GEMMs), including knock-out (KO) and knock-in (KI) models, often requires genomic screening of many mouse ES cell (mESC) clones by Southern blot. The use of large targeting constructs facilitates the recombination of exogenous DNA in a specific genomic locus, but limits the detection of its correct genomic integration by standard PCR methods. Genomic Long Range PCR (LR-PCR), using primers adjacent to the homology arms, has been used as an alternative to radioactive-based Southern blot screenings. However, LR-PCRs are often difficult and render many false positive and false negative results. Here, we propose an alternative screening method based on the detection of a genetic modification at the mRNA level, which we successfully optimized in two mouse models. This screening method consists of a reverse-transcription PCR (RT-PCR) using primers that match exons flanking the targeting construct. The detection of the expected modification in this PCR product confirms the integration at the correct genomic location and shows that the mutant mRNA is expressed. This is a simple and sensitive strategy to screen locus-specific recombination of targeting constructs which can also be useful to screen KO and KI mutant mice or cell lines including those generated by CRISPR/Cas9.
Assuntos
Células-Tronco Embrionárias/fisiologia , Recombinação Genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Southern Blotting , Genes BRCA1 , Camundongos TransgênicosRESUMO
Plk1-interacting checkpoint helicase (PICH) is a DNA translocase involved in resolving ultrafine anaphase DNA bridges and, therefore, is important to safeguard chromosome segregation and stability. PICH is overexpressed in various human cancers, particularly in lymphomas such as Burkitt lymphoma, which is caused by MYC translocations. To investigate the relevance of PICH in cancer development and progression, we have combined novel PICH-deficient mouse models with the Eµ-Myc transgenic mouse model, which recapitulates B-cell lymphoma development. We have observed that PICH deficiency delays the onset of MYC-induced lymphomas in Pich heterozygous females. Moreover, using a Pich conditional knockout mouse model, we have found that Pich deletion in adult mice improves the survival of Eµ-Myc transgenic mice. Notably, we show that Pich deletion in healthy adult mice is well tolerated, supporting PICH as a suitable target for anticancer therapies. Finally, we have corroborated these findings in two human Burkitt lymphoma cell lines and we have found that the death of cancer cells was accompanied by chromosomal instability. Based on these findings, we propose PICH as a potential therapeutic target for Burkitt lymphoma and for other cancers where PICH is overexpressed.
Assuntos
Linfoma de Burkitt , Adulto , Feminino , Animais , Humanos , Camundongos , Linfoma de Burkitt/genética , Linhagem Celular , Instabilidade Cromossômica , Modelos Animais de Doenças , Camundongos Knockout , Camundongos Transgênicos , DNARESUMO
Replication Stress (RS) is a type of DNA damage generated at the replication fork, characterized by single-stranded DNA (ssDNA) accumulation, and which can be caused by a variety of factors. Previous studies have reported elevated RS levels in aged cells. In addition, mouse models with a deficient RS response show accelerated aging. However, the relevance of endogenous or physiological RS, compared to other sources of genomic instability, for the normal onset of aging is unknown. We have performed long term survival studies of transgenic mice with extra copies of the Chk1 and/or Rrm2 genes, which we previously showed extend the lifespan of a progeroid ATR-hypomorphic model suffering from high levels of RS. In contrast to their effect in the context of progeria, the lifespan of Chk1, Rrm2 and Chk1/Rrm2 transgenic mice was similar to WT littermates in physiological settings. Most mice studied died due to tumors -mainly lymphomas- irrespective of their genetic background. Interestingly, a higher but not statistically significant percentage of transgenic mice developed tumors compared to WT mice. Our results indicate that supraphysiological protection from RS does not extend lifespan, indicating that RS may not be a relevant source of genomic instability on the onset of normal aging.
Assuntos
Quinase 1 do Ponto de Checagem/genética , Dano ao DNA , Longevidade/genética , Ribonucleosídeo Difosfato Redutase/genética , Animais , Quinase 1 do Ponto de Checagem/metabolismo , Replicação do DNA , Camundongos , Camundongos Transgênicos , Ribonucleosídeo Difosfato Redutase/metabolismoRESUMO
Unrepaired DNA damage during embryonic development can be potentially inherited by a large population of cells. However, the quality control mechanisms that minimize the contribution of damaged cells to developing embryos remain poorly understood. Here, we uncovered an ATR- and CHK1-mediated transcriptional response to replication stress (RS) in mouse embryonic stem cells (ESCs) that induces genes expressed in totipotent two-cell (2C) stage embryos and 2C-like cells. This response is mediated by Dux, a multicopy retrogene defining the cleavage-specific transcriptional program in placental mammals. In response to RS, DUX triggers the transcription of 2C-like markers such as murine endogenous retrovirus-like elements (MERVL) and Zscan4. This response can also be elicited by ETAA1-mediated ATR activation in the absence of RS. ATR-mediated activation of DUX requires GRSF1-dependent post-transcriptional regulation of Dux mRNA. Strikingly, activation of ATR expands ESCs fate potential by extending their contribution to both embryonic and extra-embryonic tissues. These findings define a novel ATR dependent pathway involved in maintaining genome stability in developing embryos by controlling ESCs fate in response to RS.
Assuntos
Quinase 1 do Ponto de Checagem/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Diferenciação Celular , Proliferação de Células/fisiologia , Células Cultivadas , Quinase 1 do Ponto de Checagem/genética , Quimera , Cromatografia Líquida , Clonagem Molecular , Dano ao DNA , Células-Tronco Embrionárias , Regulação da Expressão Gênica , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de RNA , Espectrometria de Massas em TandemRESUMO
PICH is a DNA translocase necessary for the resolution of ultrafine anaphase DNA bridges and to ensure the fidelity of chromosomal segregation. Here, we report the generation of an animal model deficient for PICH that allowed us to investigate its physiological relevance. Pich KO mice lose viability during embryonic development due to a global accumulation of DNA damage. However, despite the presence of chromosomal instability, extensive p53 activation, and increased apoptosis throughout the embryo, Pich KO embryos survive until day 12.5 of embryonic development. The absence of p53 failed to improve the viability of the Pich KO embryos, suggesting that the observed developmental defects are not solely due to p53-induced apoptosis. Moreover, Pich-deficient mouse embryonic fibroblasts exhibit chromosomal instability and are resistant to RASV12/E1A-induced transformation. Overall, our data indicate that PICH is essential to preserve chromosomal integrity in rapidly proliferating cells and is therefore critical during embryonic development and tumorigenesis.