RESUMO
Multiple proteins bind to telomeric DNA and are important for the role of telomeres in genome stability. A recent study established a broad-complex, tramtrack and bric-à-brac - zinc finger (BTB-ZF) protein, ZBTB10 (zinc finger and BTB domain-containing protein 10), as a telomeric variant repeat-binding protein at telomeres that use an alternative method for lengthening telomeres). ZBTB10 specifically interacts with the double-stranded telomeric variant repeat sequence TTGGGG by employing its tandem C2H2 zinc fingers (ZF1-2). Here, we solved the crystal structure of human ZBTB10 ZF1-2 in complex with a double-stranded DNA duplex containing the sequence TTGGGG to assess the molecular details of this interaction. Combined with calorimetric analysis, we identified the vital residues in TTGGGG recognition and determined the specific recognition mechanisms that are different from those of TZAP (telomere zinc finger-associated protein), a recently defined telomeric DNA-binding protein. Following these studies, we further identified a single amino-acid mutant (Arg767Gln) of ZBTB10 ZF1-2 that shows a preference for the telomeric DNA repeat TTAGGG sequence. We solved the cocrystal structure, providing a structural basis for telomeric DNA recognition by C2H2 ZF proteins.
Assuntos
Proteínas de Ligação a DNA , Proteínas Repressoras , Humanos , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ligação Proteica , Proteínas Repressoras/metabolismo , Telômero/metabolismo , Dedos de Zinco/genéticaRESUMO
Background and Objectives: ZBTB48 is a telomere-related protein that has been renamed telomeric zinc finger-associated protein (TZAP). It favorably binds to elongated telomeres to regulate their appropriate length. However, TZAP expression has not been investigated in hepatocellular carcinomas (HCC). Materials and Methods: The clinical significance of TZAP expression in 72 HCC was investigated. Additionally, its findings were supported by open big data and cancer cell lines. Results: TZAP expression level was not associated with the clinical parameters of HCC. TZAP expression induced a poorer survival result (overall survival, p = 0.020; disease-free survival, p = 0.012). TCGA data showed TZAP expression was more frequently found in HCCs with hepatitis C infection (p = 0.023). However, TCGA data revealed that TZAP expression did not predict HCC prognosis. In a cell line study, TZAP inhibition via siRNA suppressed PLC/PRF/5 cell growth; however, cell viability was increased in HepG2 cells. Conclusions: We presented the clinical and prognostic values of TZAP expression in HCC tissues and cancer cell lines. Additionally, the TCGA results also revealed a significant role for TZAP expression. TZAP expression may involve HCC progression and its prognosis.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Telômero/patologia , Dedos de Zinco , Prognóstico , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/genética , Proteínas de Ligação a DNA/genética , Fatores de Transcrição/genéticaRESUMO
Background and Objectives: Telomeric zinc finger-associated protein (TZAP) is a telomere regulation protein, previously known as ZBTB48. It binds preferentially to elongated telomeres, competing with telomeric repeat factors 1 and 2. TZAP expression may be associated with carcinogenesis, however; this study has not yet been performed in lung cancer. In this study, we examined the clinicopathological and prognostic values of TZAP expression in non-small cell lung cancer (NSCLC). Materials and Methods: Data were collected from The Cancer Genome Atlas. The clinical and prognostic values of TZAP for NSCLC were examined in adenocarcinoma (AD) and squamous cell carcinoma (SCC). Results: TZAP expression significantly increased in NSCLC tissues compared with normal tissues. In AD, TZAP expression was lower in patients with higher T stage (p = 0.005), and was associated with lymph node stage in SCC (p = 0.005). Survival analysis showed shorter disease-free survival in AD patients with lower TZAP expression (p = 0.047). TZAP expression did not have other clinical or prognostic value for AD and SCC. Conclusions: TZAP expression is a potential prognostic marker for NSCLC, especially in patients with AD.
Assuntos
Adenocarcinoma , Carcinoma Pulmonar de Células não Pequenas , Carcinoma de Células Escamosas , Neoplasias Pulmonares , Adenocarcinoma/genética , Adenocarcinoma/patologia , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma de Células Escamosas/genética , Proteínas de Ligação a DNA , Humanos , Pulmão/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Estadiamento de Neoplasias , Prognóstico , Telômero/patologia , Fatores de Transcrição , Dedos de Zinco/genéticaRESUMO
Background and Objectives: Telomeric zinc finger-associated protein (TZAP) is a telomere-associated factor that was previously called ZBTB48. This protein binds preferentially to long telomeres, competing with telomeric repeat factors 1 and 2. Genetic changes in TZAP may be associated with cancer pathogenesis; however, this relationship has not yet been elucidated for any type of cancer. In this study, we aimed to examine the clinicopathologic and prognostic value of TZAP expression in cervical cancer (CC). Materials and Methods: The data were extracted from The Cancer Genome Atlas cohorts by OncoLnc (21 cancer types, 7700 cancers). The prognostic value of TZAP for different stages of 264 CCs was examined using survival analysis. Results: The TZAP expression did not differ significantly between CC and normal matched tissues. Age, cancer stage, and viral infection were not associated with TZAP expression. Survival analysis revealed a shorter overall survival in CC patients with a lower TZAP expression (χ2 = 3.62, p = 0.057). The prognostic value of TZAP expression was greater in patients with N1 stage CC (χ2 = 5.64, p = 0.018). Conclusion: TZAP expression is a possible prognostic marker for CC, especially stage N1 CC.
Assuntos
Proteínas de Ligação a DNA/análise , Fatores de Transcrição/análise , Neoplasias do Colo do Útero/diagnóstico , Adulto , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/sangue , Distribuição de Qui-Quadrado , Proteínas de Ligação a DNA/sangue , Feminino , Humanos , Pessoa de Meia-Idade , Prognóstico , Estatísticas não Paramétricas , Fatores de Transcrição/sangue , Neoplasias do Colo do Útero/sangue , Neoplasias do Colo do Útero/fisiopatologiaRESUMO
BACKGROUND: Mesenchymal stromal cells (MSCs) were originally characterized by the ability to differentiate into different mesenchymal lineages in vitro, and their immunomodulatory and trophic functions have recently aroused significant interest in the application of MSCs in cell-based regenerative medicine. However, a major problem in clinical practice is the replicative senescence of MSCs, which limits the cell proliferation potential of MSCs after large-scale expansion. Telomeric zinc finger-associated protein (TZAP), a novel specific telomere-binding protein, was recently found to stimulate telomere trimming and prevent excessive telomere elongation. The aim of this study was to elucidate the role of TZAP in regulating MSCs senescence, differentiation and proliferation. METHOD: Primary porcine mesenchymal stromal cells (pMSCs) were isolated from the bone marrow of Tibet minipigs by a noninvasive method in combination with frequent medium changes (FMCs). The deterioration of the pMSCs' proliferation capacity and their resultant entry into senescence were analyzed by using CCK8 and EdU incorporation assays, SA-ß-gal staining and comparisons of the expression levels of cellular senescence markers (p16INK14 and p21) in pMSC cell lines with TZAP overexpression or knockout. The effects of TZAP overexpression or knockout on the differentiation potential of pMSCs were assessed by alizarin red S staining after osteogenic induction or by oil red O staining after adipogenic induction. The effect of TZAP overexpression and the involvement of the p53 signaling pathway were evaluated by detecting changes in ARF, MDM2, P53 and P21 protein levels in pMSCs. RESULTS: TZAP levels were significantly elevated in late-passage pMSCs compared to those in early-passage pMSCs. We also observed significantly increased levels of the senescence markers p16INK4A and p21. Overexpression of TZAP reduced the differentiation potential of the cells, leading to premature senescence in early-passage pMSCs, while knockout of TZAP led to the opposite phenotype in late-passage pMSCs. Furthermore, overexpression of TZAP activated the P53 pathway (ARF-MDM2-P53-P21WAF/CDKN1A) in vitro. TZAP also downregulated the expression levels of PPARγ and Cebpα, two key modulators of adipogenesis. CONCLUSIONS: This study demonstrates that the level of TZAP is closely related to differentiation potential in pMSCs and affects cellular senescence outcomes via the p53 pathway. Therefore, attenuation of intracellular TZAP levels could be a new strategy for improving the efficiency of pMSCs in cell therapy and tissue engineering applications.
Assuntos
Autorrenovação Celular , Senescência Celular , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Humanos , SuínosRESUMO
In multicellular organisms, regulation of telomere length in pluripotent stem cells is critical to ensure organism development and survival. Telomeres consist of repetitive DNA that are progressively lost with each cellular division. When telomeres become critically short, they activate a DNA damage response that results in cell cycle arrest. To counteract telomere attrition, pluripotent stem cells are equipped with telomere elongation mechanisms that ensure prolonged proliferation capacity and self-renewal capacity. Excessive telomere elongation can also be deleterious and is counteracted by a rapid telomere deletion mechanism termed telomere trimming. While the consequences of critically short telomeres are well established, we are only beginning to understand the mechanisms that counteract excessive telomere elongation. The balance between telomere elongation and shortening determine the telomere length set point in pluripotent stem cells and ensures sustained proliferative potential without causing chromosome instability.
Assuntos
Células-Tronco Pluripotentes/metabolismo , Homeostase do Telômero , Telômero/genética , Animais , Humanos , Células-Tronco Pluripotentes/citologia , Telômero/metabolismo , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismoRESUMO
Background and Objectives: ZBTB48 is a telomere-associated factor that has been renamed as telomeric zinc finger-associated protein (TZAP). It binds preferentially to long telomeres, competing with telomeric repeat factors 1 and 2. Materials and Methods: We analyzed the TZAP mutation in 128 breast carcinomas (BCs). In addition, its association with telomere length was investigated. Results: The TZAP mutation (c.1272 G > A, L424L) was found in 7.8% (10/128) of the BCs and was associated with the N0 stage. BCs with the TZAP mutation had longer telomeres than those without this mutation. Survival analysis showed that the TZAP mutation resulted in poorer overall survival. Conclusions: These results suggest that the TZAP mutation is a possible prognostic marker in BC.
Assuntos
Neoplasias da Mama/complicações , Proteínas de Ligação a DNA/genética , Mutação/genética , Fatores de Transcrição/genética , Adulto , Neoplasias da Mama/genética , Distribuição de Qui-Quadrado , Feminino , Humanos , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase/métodos , Prognóstico , Estatísticas não Paramétricas , Telômero/genética , Telômero/patologiaRESUMO
BACKGROUND: N6-methyladenosine (m6A), the most abundant internal modification on eukaryotic mRNA, and N6, 2'-O-dimethyladenosine (m6Am), are epitranscriptomic marks that function in multiple aspects of posttranscriptional regulation. Fat mass and obesity-associated protein (FTO) can remove both m6A and m6Am; however, little is known about how FTO achieves its substrate selectivity. RESULTS: Here, we demonstrate that ZBTB48, a C2H2-zinc finger protein that functions in telomere maintenance, associates with FTO and binds both mRNA and the telomere-associated regulatory RNA TERRA to regulate the functional interactions of FTO with target transcripts. Specifically, depletion of ZBTB48 affects targeting of FTO to sites of m6A/m6Am modification, changes cellular m6A/m6Am levels and, consequently, alters decay rates of target RNAs. ZBTB48 ablation also accelerates growth of HCT-116 colorectal cancer cells and modulates FTO-dependent regulation of Metastasis-associated protein 1 (MTA1) transcripts by controlling the binding to MTA1 mRNA of the m6A reader IGF2BP2. CONCLUSIONS: Our findings thus uncover a previously unknown mechanism of posttranscriptional regulation in which ZBTB48 co-ordinates RNA-binding of the m6A/m6Am demethylase FTO to control expression of its target RNAs.
Assuntos
Adenosina , Dioxigenase FTO Dependente de alfa-Cetoglutarato , Humanos , Adenosina/análogos & derivados , Adenosina/metabolismo , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Células HCT116 , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Telômero/metabolismo , Telômero/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Dedos de ZincoRESUMO
Human herpesvirus 6A and 6B are two closely related viruses that infect almost all humans. In contrast to most herpesviruses, HHV-6A/B can integrate their genomes into the telomeres during the infection process. Both viruses can also integrate in germ cells and subsequently be inherited in children. How HHV-6A/B integrate into host telomeres and the consequences of this remain a subject of active research. Here, we developed a method to measure telomere length by quantitative fluorescence in situ hybridization, confocal microscopy, and computational processing. This method was validated using a panel of HeLa cells having short or long telomeres. These cell lines were infected with HHV-6A, revealing that the virus could efficiently integrate into telomeres independent of their length. Furthermore, we assessed the telomere lengths after HHV-6A integration and found that the virus-containing telomeres display a variety of lengths, suggesting that either telomere length is restored after integration or telomeres are not shortened by integration. Our results highlight new aspects of HHV-6A/B biology and the role of telomere length on virus integration.
Assuntos
Herpesvirus Humano 6 , Infecções por Roseolovirus , Criança , Células HeLa , Herpesvirus Humano 6/genética , Humanos , Hibridização in Situ Fluorescente , Infecções por Roseolovirus/genética , Telômero , Integração ViralRESUMO
PURPOSE: The zinc finger protein, ZBTB48, is a telomere-associated protein. It was renamed as telomeric zinc finger-associated protein (TZAP) binding to elongated telomeres. However, its expression level was not measured in cancers. PATIENTS AND METHODS: We analyzed TZAP mRNA levels in 60 colorectal cancers (CRC) and its correlation with telomere length and TERT was studied. RESULTS: TZAP mRNA in CRC was higher statistically than that in paired non-cancerous tissues (p = 0.033). Higher TZAP was found in carcinoembryonic antigen (CEA)-positive CRCs (>5 ng/mL) (p = 0.012). Shorter telomere was found in CRCs with high TZAP expression than that with low TZAP expression (p = 0.010). According to quantitative correlation analysis, TZAP has a correlation with age (r = -0.349, p = 0.007), TERT (r = 0.279, p = 0.041) and telomere length (r = -0.305, p = 0.021). TZAP expression did not harbor prognostic value in CRC. Inhibition of TZAP expression by siRNA suppresses cell growth in HT29 cells; however, it resulted in increased cell viability in HCT116 cells. TZAP inhibition induces a decrease in mRNA levels of TERT in both HT29 and HCT116 cells. TCGA data analysis showed higher expression of TZAP showed poorer overall survival in colon cancer (p = 0.001); however, it did not have a significance in rectal cancer (p = 0.951). CONCLUSION: We suggested that TZAP may be a possible biomarker for CRC.