RESUMO
From biosynthesis to assembly into nucleosomes, histones are handed through a cascade of histone chaperones, which shield histones from non-specific interactions. Whether mechanisms exist to safeguard the histone fold during histone chaperone handover events or to release trapped intermediates is unclear. Using structure-guided and functional proteomics, we identify and characterize a histone chaperone function of DNAJC9, a heat shock co-chaperone that promotes HSP70-mediated catalysis. We elucidate the structure of DNAJC9, in a histone H3-H4 co-chaperone complex with MCM2, revealing how this dual histone and heat shock co-chaperone binds histone substrates. We show that DNAJC9 recruits HSP70-type enzymes via its J domain to fold histone H3-H4 substrates: upstream in the histone supply chain, during replication- and transcription-coupled nucleosome assembly, and to clean up spurious interactions. With its dual functionality, DNAJC9 integrates ATP-resourced protein folding into the histone supply pathway to resolve aberrant intermediates throughout the dynamic lives of histones.
Assuntos
Proteínas de Choque Térmico HSP40/metabolismo , Chaperonas de Histonas/metabolismo , Linhagem Celular Tumoral , Cromatina , Montagem e Desmontagem da Cromatina , Replicação do DNA , Proteínas de Choque Térmico HSP40/fisiologia , Proteínas de Choque Térmico HSP70/metabolismo , Células HeLa , Chaperonas de Histonas/fisiologia , Histonas/metabolismo , Humanos , Componente 2 do Complexo de Manutenção de Minicromossomo/metabolismo , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Nucleossomos , Ligação Proteica , Proteômica/métodosRESUMO
We investigated the role of TONSL, a mediator of homologous recombination repair (HRR), in stalled replication fork double-strand breaks (DSBs) in cancer. Publicly available clinical data (tumors from the ovary, breast, stomach and lung) were analyzed through KM Plotter, cBioPortal and Qomics. Cancer stem cell (CSC)-enriched cultures and bulk/general mixed cell cultures (BCCs) with RNAi were employed to determine the effect of TONSL loss in cancer cell lines from the ovary, breast, stomach, lung, colon and brain. Limited dilution assays and ALDH assays were used to quantify the loss of CSCs. Western blotting and cell-based homologous recombination assays were used to identify DNA damage derived from TONSL loss. TONSL was expressed at higher levels in cancer tissues than in normal tissues, and higher expression was an unfavorable prognostic marker for lung, stomach, breast and ovarian cancers. Higher expression of TONSL is partly associated with the coamplification of TONSL and MYC, suggesting its oncogenic role. The suppression of TONSL using RNAi revealed that it is required in the survival of CSCs in cancer cells, while BCCs could frequently survive without TONSL. TONSL dependency occurs through accumulated DNA damage-induced senescence and apoptosis in TONSL-suppressed CSCs. The expression of several other major mediators of HRR was also associated with worse prognosis, whereas the expression of error-prone nonhomologous end joining molecules was associated with better survival in lung adenocarcinoma. Collectively, these results suggest that TONSL-mediated HRR at the replication fork is critical for CSC survival; targeting TONSL may lead to the effective eradication of CSCs.
Assuntos
Neoplasias , Reparo de DNA por Recombinação , Feminino , Humanos , Dano ao DNA , Reparo do DNA/genética , Replicação do DNA/genética , Recombinação Homóloga , Células-Tronco NeoplásicasRESUMO
SPONASTRIME dysplasia is an autosomal-recessive spondyloepimetaphyseal dysplasia characterized by spine (spondylar) abnormalities, midface hypoplasia with a depressed nasal bridge, metaphyseal striations, and disproportionate short stature. Scoliosis, coxa vara, childhood cataracts, short dental roots, and hypogammaglobulinemia have also been reported in this disorder. Although an autosomal-recessive inheritance pattern has been hypothesized, pathogenic variants in a specific gene have not been discovered in individuals with SPONASTRIME dysplasia. Here, we identified bi-allelic variants in TONSL, which encodes the Tonsoku-like DNA repair protein, in nine subjects (from eight families) with SPONASTRIME dysplasia, and four subjects (from three families) with short stature of varied severity and spondylometaphyseal dysplasia with or without immunologic and hematologic abnormalities, but no definitive metaphyseal striations at diagnosis. The finding of early embryonic lethality in a Tonsl-/- murine model and the discovery of reduced length, spinal abnormalities, reduced numbers of neutrophils, and early lethality in a tonsl-/- zebrafish model both support the hypomorphic nature of the identified TONSL variants. Moreover, functional studies revealed increased amounts of spontaneous replication fork stalling and chromosomal aberrations, as well as fewer camptothecin (CPT)-induced RAD51 foci in subject-derived cell lines. Importantly, these cellular defects were rescued upon re-expression of wild-type (WT) TONSL; this rescue is consistent with the hypothesis that hypomorphic TONSL variants are pathogenic. Overall, our studies in humans, mice, zebrafish, and subject-derived cell lines confirm that pathogenic variants in TONSL impair DNA replication and homologous recombination-dependent repair processes, and they lead to a spectrum of skeletal dysplasia phenotypes with numerous extra-skeletal manifestations.
Assuntos
Instabilidade Cromossômica , Dano ao DNA , Variação Genética , Anormalidades Musculoesqueléticas/patologia , NF-kappa B/genética , Osteocondrodisplasias/patologia , Adolescente , Adulto , Alelos , Animais , Células Cultivadas , Criança , Pré-Escolar , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Estudos de Associação Genética , Humanos , Camundongos , Camundongos Knockout , Anormalidades Musculoesqueléticas/genética , Osteocondrodisplasias/genética , Sequenciamento do Exoma , Adulto Jovem , Peixe-ZebraRESUMO
SPONASTRIME dysplasia is a rare, recessive skeletal dysplasia characterized by short stature, facial dysmorphism, and aberrant radiographic findings of the spine and long bone metaphysis. No causative genetic alterations for SPONASTRIME dysplasia have yet been determined. Using whole-exome sequencing (WES), we identified bi-allelic TONSL mutations in 10 of 13 individuals with SPONASTRIME dysplasia. TONSL is a multi-domain scaffold protein that interacts with DNA replication and repair factors and which plays critical roles in resistance to replication stress and the maintenance of genome integrity. We show here that cellular defects in dermal fibroblasts from affected individuals are complemented by the expression of wild-type TONSL. In addition, in vitro cell-based assays and in silico analyses of TONSL structure support the pathogenicity of those TONSL variants. Intriguingly, a knock-in (KI) Tonsl mouse model leads to embryonic lethality, implying the physiological importance of TONSL. Overall, these findings indicate that genetic variants resulting in reduced function of TONSL cause SPONASTRIME dysplasia and highlight the importance of TONSL in embryonic development and postnatal growth.
Assuntos
Fibroblastos/patologia , Genes Letais , Mutação , NF-kappa B/genética , Osteocondrodisplasias/patologia , Adolescente , Adulto , Animais , Células Cultivadas , Criança , Pré-Escolar , Dano ao DNA , Derme/metabolismo , Derme/patologia , Feminino , Fibroblastos/metabolismo , Humanos , Lactente , Recém-Nascido , Camundongos , Camundongos Endogâmicos C57BL , Osteocondrodisplasias/genética , Sequenciamento do Exoma/métodos , Adulto JovemRESUMO
BACKGROUND: It has been reported that high expression levels of miR-197 can predict coronary artery disease (CAD). Our bioinformatics analysis showed that miR-197 may bind to long non-coding RNA (lncRNA) TONSL-AS1. This study aimed to investigate the role of TONSL-AS1 in CAD. METHODS: This study included 60 CAD patients and 60 healthy controls. Coronary angiography was performed to diagnose CAD. The interaction between TONSL-AS1 and miR-197 was predicted by IntaRNA2.0. Western-blot analysis was performed to illustrate the effect of MTONSL-AS1, miR-197 and BCL2 on human primary coronary artery endothelial cells (HCAECs). Cell migration assay was performed to explore the roles of MTONSL-AS1, miR-197 and BCL2 in regulating cell migration. Cell apoptosis assay was performed to investigate the role of MTONSL-AS1, miR-197 and BCL2 in regulating the apoptosis of HCAECs. RESULT: Significant differences in high-density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), and gensini score were observed in patients with CAD. In addition, TONSL-AS1 was downregulated in CAD. Follow-up study revealed that low expression levels of TONSL-AS1 and high expression levels of miR-197 predicted poor survival of CAD patients. Overexpression experiments showed that TONSL-AS1 and miR-197 had no significant effect on the expression of each other. We speculated that MAFG-AS1 may sponge miR-145. Moreover, overexpression of TONSL-AS1 increased, while overexpression of miR-197 decreased the expression levels of BCL2. Furthermore, overexpression of TONSL-AS1 attenuated the effects of overexpression of miR-197 on migration and apoptosis of HCAECs. CONCLUSIONS: Therefore, the expression of TONSL-AS1 predicted the survival of CAD patients and it sponged miR-197 to inhibit the apoptosis of HCAECs.
Assuntos
Doença da Artéria Coronariana/metabolismo , Vasos Coronários/metabolismo , Células Endoteliais/metabolismo , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Adulto , Idoso , Apoptose , Estudos de Casos e Controles , Movimento Celular , Células Cultivadas , HDL-Colesterol/sangue , LDL-Colesterol/sangue , Angiografia Coronária , Doença da Artéria Coronariana/diagnóstico por imagem , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/patologia , Vasos Coronários/patologia , Células Endoteliais/patologia , Feminino , Humanos , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Longo não Codificante/genéticaRESUMO
Long non-coding RNAs (lncRNAs) are reported to play a significant role in various malignant tumors, yet their potential functions in gastric cancer are not clear. In this study, we found a novel lncRNA, named TONSL-AS1, was downregulated in gastric cancer tissues and cell lines compared with the normal. TONSL-AS1 inhibited cell migration, invasion and proliferation in SGC-7901, MGC-803â¯cells. Furthermore, TONSL-AS1 could suppress cell tumorigenesis in vivo. Mechanistically, TONSL-AS1's genomic neighboring gene TONSL, which was reported as a tumor suppress gene, was upregulated by TONSL. Additionally, the TONSL-AS1 was positively associated with TONSL in cancer tissues. Our study revealed that the tumor-inhibiting effect of TONSL-AS1 in gastric cancer cells was associated with TONSL. In general, our results indicated that TONSL-AS1 works as a tumor suppressor lncRNA, which may be a new therapeutic target for gastric cancer.
Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Genes Supressores de Tumor , NF-kappa B/fisiologia , Proteínas de Neoplasias/fisiologia , RNA Neoplásico/fisiologia , Neoplasias Gástricas/genética , Adulto , Idoso , Animais , Carcinogênese , Adesão Celular , Divisão Celular , Linhagem Celular Tumoral , Movimento Celular , Progressão da Doença , Regulação para Baixo , Feminino , Genes Reporter , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , NF-kappa B/genética , Proteínas de Neoplasias/genética , Interferência de RNA , RNA Neoplásico/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Organismos Livres de Patógenos EspecíficosRESUMO
The complete and faithful duplication of the genome is an essential prerequisite for proliferating cells to maintain genome integrity. This objective is greatly challenged by DNA damage encountered during replication, which causes fork stalling and in certain cases, fork breakage. DNA damage tolerance (DDT) pathways mitigate the effects on fork stability induced by replication fork stalling by mediating damage-bypass and replication fork restart. These DDT mechanisms, largely relying on homologous recombination (HR) and specialized polymerases, can however contribute to genome rearrangements and mutagenesis. There is a profound connection between replication and recombination: recombination proteins protect replication forks from nuclease-mediated degradation of the nascent DNA strands and facilitate replication completion in cells challenged by DNA damage. Moreover, in case of fork collapse and formation of double strand breaks (DSBs), the recombination factors present or recruited to the fork facilitate HR-mediated DSB repair, which is primarily error-free. Disruption of HR is inexorably linked to genome instability, but the premature activation of HR during replication often leads to genome rearrangements. Faithful replication necessitates the downregulation of HR and disruption of active RAD51 filaments at replication forks, but upon persistent fork stalling, building up of HR is critical for the reorganization of the replication fork and for filling-in of the gaps associated with discontinuous replication induced by DNA lesions. Here we summarize and reflect on our understanding of the mechanisms that either suppress recombination or locally enhance it during replication, and the principles that underlie this regulation.
Assuntos
Replicação do DNA , Recombinação Genética , Animais , Dano ao DNA , Reparo do DNA , HumanosRESUMO
PURPOSE: The tonsoku-like DNA repair protein (TONSL) encoded by the TONSL gene, located on chromosome 8q24.3, is crucial for repairing DNA double-strand breaks through homologous recombination. However, TONSL overexpression in lung adenocarcinoma (LUAD) promotes tumor development, leading to a poor prognosis. METHODS: TONSL was verified as a reliable prognostic marker for LUAD using bioinformatics, and clinical features related to LUAD prognosis were screened from the TCGA database to establish the relationship between risk factors and TONSL expression. In addition, TONSL expression in normal and LUAD tissues was verified using real-time quantitative polymerase chain reaction and immunohistochemistry. To elucidate the possible functions of TONSL, TONSL-related differentially expressed genes were screened, and functional enrichment analysis was performed. Subsequently, siRNA was used to knock down TONSL expression in lung cancer cells for cytobehavioral experiments. The effects of TONSL expression on tumor immune escape were analyzed using the ESTIMATE algorithm and tumor immune-infiltration analysis. In addition, the half-maximal inhibitory concentration of LUAD with varying TONSL expression levels in response to first-line chemotherapeutic drugs and epidermal growth factor receptor-tyrosine kinase inhibitors was analyzed for drug sensitivity. RESULTS: Up-regulation of TONSL in LUAD promotes the proliferation, migration, and invasion of lung cancer cells, thereby contributing to a poor prognosis. Furthermore, TONSL overexpression promotes immune escape and drug sensitivity in LUAD. CONCLUSION: TONSL serves as a reliable prognostic marker for LUAD, and its up-regulation is associated with increased immune escape and drug sensitivity. These findings suggest that TONSL holds potential as a novel therapeutic target for LUAD.
RESUMO
Dysregulation of long non-coding RNAs (lncRNAs) results in development of human diseases, including hepatocellular carcinoma (HCC). lncRNA TONSL-AS1 has been reported to act as a tumor suppressor in gastric cancer. The present study aimed to investigate the role of TONSL-AS1 in hepatocellular carcinoma (HCC). Reverse transcription-quantitative PCR analysis was performed to detect the expression levels of TONSL-AS1 and microRNA (miRNA/miR)-135a in HCC tissues and paired adjacent normal tissues. A 5-year follow-up study was performed to determine the prognostic value of TONSL-AS1 in HCC. The association between miR-135a and TONSL-AS1 was assessed via overexpression experiments. The Cell Counting Kit-8 assay was performed to assess cell proliferation. The results demonstrated that TONSL-AS1 expression was downregulated in HCC tissues, which was associated with a lower survival rate in patients with HCC. TONSL-AS1 and miR-135a were predicted to interact with each other, whereby overexpression of miR-135a downregulated TONSL-AS1 expression. The results demonstrated that TONSL-AS1 and miR-135a were inversely correlated with each other. Notably, overexpression of TONSL-AS1 inhibited HCC cell proliferation, while overexpression of miR-135a promoted HCC cell proliferation and decreased the effect of overexpression of TONSL-AS1 on cell proliferation. Taken together, the results of the present study suggest that miR-135a expression is upregulated in HCC and targets lncRNA TONSL-AS1 to suppress cell proliferation.
RESUMO
BACKGROUND: LncRNA TONSL-AS1 has been characterized as a critical player in gastric cancer. By analyze the TCGA dataset, we observed the upregulation of TONSL-AS1 in ovarian epithelial carcinoma (EOC). We therefore investigated the involvement of TONSL-AS1 in EOC. METHODS: The differential expression of TONSL-AS1 in EOC was first explored by analyzing the TCGA dataset. The effects of overexpression of TONSL-AS1 and miR-490-3p on the expression of CDK1 mRNA and protein in OVCAR3 cells were evaluated by qPCR and western blot, respectively. CCK-8 assay was performed to investigate the effects of overexpression of TONSL-AS1, miR-490-3p and CDK1 on proliferation of OVCAR3 cells. RESULTS: We observed that TONSL-AS1 was upregulated in EOC tumor tissues from EOC patients, and its high expression level was correlated with poor survival. Dual luciferase assay and RNA interaction prediction showed the direct interaction between TONSL-AS1 and miR-490-3p. However, overexpression of miR-490-3p did not affect the expression of TONSL-AS1. Instead, overexpression of TONSL-AS1 resulted in the upregulation of CDK1, a target of miR-490-3p, in EOC cells. Overexpression of TONSL-AS1 and CDK1 resulted in increased proliferation rate of EOC cells. Overexpression of miR-490-3p played an opposite role and reduced the effects of overexpression of TONSL -AS1 and CDK1. CONCLUSIONS: Therefore, TONSL-AS1 may regulate miR-490-3p/CDK1 to affect EOC cell proliferation.
Assuntos
Carcinoma Epitelial do Ovário/genética , MicroRNAs/metabolismo , NF-kappa B/genética , RNA Longo não Codificante/genética , Idoso , Carcinoma Epitelial do Ovário/metabolismo , Carcinoma Epitelial do Ovário/patologia , Proliferação de Células , Feminino , Humanos , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , RNA Antissenso/genética , RNA Antissenso/metabolismo , RNA Longo não Codificante/metabolismo , Análise de Sobrevida , TransfecçãoRESUMO
BACKGROUND: TONSL has been suggested to function as an oncogene in lung, esophageal and cervical cancer. This study was aimed to identify the expression of TONSL and its role in hepatocellular carcinoma (HCC). METHODS: By data mining in the Cancer Genome Atlas (TCGA) and Human Protein Atlas (HPA) databases, the expression profile of TONSL, its clinical significance, the potential mechanisms of its dysregulation and its underlying biological function in HCC were investigated. RESULTS: TONSL was significantly upregulated in HCC tissues relative to normal liver tissues (P < 0.05). High TONSL expression was significantly correlated with advanced TNM stage, poorly differentiated tumors, vascular invasion, elevated serum alpha-fetoprotein expression and a worse prognosis (all P < 0.05). Multivariate analysis further confirmed that TONSL overexpression was an independent risk factor for poor overall survival (OS) and recurrence-free survival (RFS) in HCC (all P < 0.05). Additionally, 16% of HCC cases (n = 370) had TONSL DNA amplification. The total methylation level of TONSL was moderately and negatively correlated with its mRNA expression (P < 0.05). TONSL was predictively targeted by miR-133b, which was downregulated in HCC and negatively related to TONSL mRNA expression (all P < 0.05). Kaplan-Meier analyses demonstrated that low miR-133b expression was significantly associated with poor OS and RFS (all P < 0.05). Moreover, gene set enrichment analysis revealed that cases with TONSL overexpression were enriched in cell cycle regulation pathways (all P < 0.05). CONCLUSIONS: TONSL holds promise for serving as a prognostic biomarker for HCC. DNA amplification, hypomethylation and miR-133b downregulation could be the mechanisms associated with TONSL upregulation in HCC. TONSL might function as an oncogene via cell cycle regulation pathways in HCC.