5-Hydroxymethylcytosine
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5-Hydroxymethylcytosine (5-hmC) was recently identified as a relatively frequent base in eukaryotic genomes. Its physiological function is still unclear, but it is supposed to serve as an intermediate in DNA de novo demethylation. Using X-ray diffraction, we solved five structures of four variants of the d(CGCGAATTCGCG) dodecamer, containing either 5-hmC or 5-methylcytosine (5-mC) at position 3 or at position 9. The observed resolutions were between 1.42 and 1.99 Å. Cytosine modification in all cases influences neither the whole B-DNA double helix structure nor the modified base pair geometry. The additional hydroxyl group of 5-hmC with rotational freedom along the C5-C5A bond is preferentially oriented in the 3' direction. A comparison of thermodynamic properties of the dodecamers shows no effect of 5-mC modification and a sequence-dependent only slight destabilizing effect of 5-hmC modification. Also taking into account the results of a previous functional study [Münzel et al. (2011) (Improved synthesis and mutagenicity of oligonucleotides containing 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine. Chem. Eur. J., 17, 13782-13788)], we conclude that the 5 position of cytosine is an ideal place to encode epigenetic information. Like this, neither the helical structure nor the thermodynamics are changed, and polymerases cannot distinguish 5-hmC and 5-mC from unmodified cytosine, all these effects are making the former ones non-mutagenic.
Cytosine methylation patterns in higher eukaryotes are important in gene regulation. Along with 5-methylcytosine (5-mC), a newly discovered constituent of mammalian DNA, 5-hydroxymethylcytosine (5-hmC), is the other modified base in higher organisms. In this study we detected 5-hmC in plant protoplast DNA and demonstrated its increasing content during the first 72 hrs. of protoplast cultivation. In contrast to 5-hmC, the amount of 5-mC decreased during protoplast cultivation. It was also found that 5-hmC did not primarily arise as a product of oxidative DNA damage following protoplast culture.
The fundamental biochemical processes of 5-methylcytosine (5-mC) synthesis, maintenance, conversion and removal determine the time and spatial pattern of DNA methylation. This has a strong effect on a plethora of physiological aspects of cellular metabolism. While the presence of 5-mC within the promoter region can silence gene expression, its derivative - 5-hydroxymethylcytosine exerts an opposite effect. Dysregulations in the metabolism of 5-mC lead to an altered DNA methylation pattern which is linked with a disrupted epigenome, and are considered to play a significant part in the etiology of several human diseases. A summary of recent knowledge about the molecular processes participating in DNA methylation pattern shaping is provided here.
- MeSH
- 5-methylcytosin metabolismus MeSH
- DNA metabolismus MeSH
- lidé MeSH
- metylace DNA * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Five 2'-deoxyribonucleoside triphosphates (dNTPs) derived from epigenetic pyrimidines (5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, 5-hydroxymethyluracil, and 5-formyluracil) were prepared and systematically studied as substrates for nine DNA polymerases in competition with natural dNTPs by primer extension experiments. The incorporation of these substrates was evaluated by a restriction endonucleases cleavage-based assay and by a kinetic study of single nucleotide extension. All of the modified pyrimidine dNTPs were good substrates for the studied DNA polymerases that incorporated a significant percentage of the modified nucleotides into DNA even in the presence of natural nucleotides. 5-Methylcytosine dNTP was an even better substrate for most polymerases than natural dCTP. On the other hand, 5-hydroxymethyl-2'-deoxyuridine triphosphate was not the best substrate for SPO1 DNA polymerase, which naturally synthesizes 5hmU-rich genomes of the SPO1 bacteriophage. The results shed light onto the possibility of gene silencing through recycling and random incorporation of epigenetic nucleotides and into the replication of modified bacteriophage genomes.
- MeSH
- 5-methylcytosin * MeSH
- deoxyribonukleosidy MeSH
- DNA-dependentní DNA-polymerasy metabolismus MeSH
- DNA metabolismus MeSH
- epigeneze genetická MeSH
- nukleotidy metabolismus MeSH
- pyrimidinové nukleotidy * MeSH
- pyrimidiny MeSH
- restrikční enzymy metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Germ cell tumors and particularly seminomas reflect the epigenomic features of their parental primordial germ cells (PGCs), including genomic DNA hypomethylation and expression of pluripotent cell markers. Because the DNA hypomethylation might be a result of TET dioxygenase activity, we examined expression of TET1-3 enzymes and the level of their product, 5-hydroxymethylcytosine (5hmC), in a panel of histologically characterized seminomas and non-seminomatous germ cell tumors. Expression of TET dioxygenase mRNAs was quantified by real-time PCR. TET1 expression and the level of 5hmC were examined immunohistochemically. Quantitative assessment of 5-methylcytosine (5mC) and 5hmC levels was done by the liquid chromatography-mass spectroscopy technique. We found highly increased expression of TET1 dioxygenase in most seminomas and strong TET1 staining in seminoma cells. Isocitrate dehydrogenase 1 and 2 mutations were not detected, suggesting the enzymatic activity of TET1. The levels of 5mC and 5hmC in seminomas were found decreased in comparison to non-seminomatous germ cell tumors and healthy testicular tissue. We propose that TET1 expression should be studied as a potential marker of seminomas and mixed germ cell tumors and we suggest that elevated expression of TET dioxygenase enzymes is associated with the maintenance of low DNA methylation levels in seminomas. This "anti-methylator" phenotype of seminomas is in contrast to the CpG island methylator phenotype (CIMP) observed in a fraction of tumors of various types.
- MeSH
- 5-methylcytosin analogy a deriváty analýza MeSH
- dioxygenasy analýza genetika MeSH
- DNA vazebné proteiny analýza genetika MeSH
- dospělí MeSH
- lidé MeSH
- metylace DNA * MeSH
- oxygenasy se smíšenou funkcí analýza genetika MeSH
- protoonkogenní proteiny analýza genetika MeSH
- regulace genové exprese u nádorů MeSH
- seminom genetika patologie MeSH
- testikulární nádory genetika patologie MeSH
- testis metabolismus patologie MeSH
- upregulace MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Prostate cancer is the second leading cause of male cancer-related deaths in Western countries, which is predominantly attributed to the metastatic castration-resistant stage of the disease (CRPC). There is an urgent need for better prognostic and predictive biomarkers, particularly for androgen receptor targeted agents and taxanes. METHODS: We have searched the PubMed database for original articles and meta-analyses providing information on blood-based markers for castration-resistant prostate cancer monitoring, risk group stratification and prediction of therapy response. RESULTS: The molecular markers are discussed along with the standard clinical parameters, such as prostate specific antigen, lactate dehydrogenase or C-reactive protein. Androgen receptor (AR) alterations are commonly associated with progression to CRPC. These include amplification of AR and its enhancer, point mutations and splice variants. Among DNA methylations, a novel 5-hydroxymethylcytosine activation marker of TOP2A and EZH2 has been identified for the aggressive disease. miR-375 is currently the most promising candidate among non-coding RNAs and sphingolipid analysis has recently emerged as a novel approach. CONCLUSIONS: The promising biomarkers have the potential to improve the care of metastatic prostate cancer patients, however, they need further validation for routine implementation.
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Recent studies have highlighted the significant role of 5-hydroxymethylcytosine (5hmC) in carcinogenesis. However, the specific role of 5hmC in osteosarcoma (OS) remains largely unexplored. The-re-fore, this study aimed to investigate the function of 5hmC and TET3 in OS. In this study, we found a decreased total level of 5hmC in OS tissues. The expression of the TET3 protein was also decreased in OS. Importantly, the decreased levels of TET3 were associated with a decreased disease-free survival (DFS) rate in patients. To investigate the role of TET3 and 5hmC in OS, we manipulated the levels of TET3 in MG-63 cells. Silencing TET3 in these cells resulted in a twofold increase in proliferation. Additio-nally, the level of 5hmC decreased in these cells. Con-versely, over-expression of TET3 in MG-63 cells led to the expected inhibition of proliferation and invasion, accompanied by an increase in 5hmC levels. In conclusion, both 5hmC and TET3 protein levels were decreased in OS. Additionally, the over-expression of TET3 inhibited the proliferation of MG-63 cells, while the suppression of TET3 had the opposite effect. These findings suggest that decreased levels of 5hmC and TET3 may serve as potential markers for OS.
- MeSH
- 5-methylcytosin * analogy a deriváty metabolismus MeSH
- demetylace DNA * MeSH
- dioxygenasy * metabolismus MeSH
- epigeneze genetická * MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- nádory kostí genetika metabolismus patologie MeSH
- osteosarkom genetika metabolismus patologie MeSH
- proliferace buněk * MeSH
- protoonkogenní proteiny metabolismus genetika MeSH
- regulace genové exprese u nádorů MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Histone to protamine exchange and the hyperacetylation of the remaining histones are hallmarks of spermiogenesis. Acetylation of histone H4 at lysine 12 (H4K12ac) was observed prior to full decondensation of sperm chromatin after fertilization suggesting an important role for the regulation of gene expression in early embryogenesis. Similarly, DNA methylation may contribute to gene silencing of several developmentally important genes. Following the identification of H4K12ac-binding promoters in sperm of fertile and subfertile patients, we aimed to investigate whether the depletion of histone-binding is associated with aberrant DNA methylation in sperm of subfertile men. Furthermore, we monitored the transmission of H4K12ac, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) from the paternal chromatin to the embryo applying mouse in vitro fertilization and immunofluorescence. RESULTS: Chromatin immunoprecipitation (ChIP) with anti-H4K12ac antibody was performed with chromatin isolated from spermatozoa of subfertile patients with impaired sperm chromatin condensation assessed by aniline blue staining. Fertile donors were used as control. DNA methylation analysis of selected H4K12ac-interacting promoters in spermatozoa was performed by pyrosequencing. Depletion of binding sites for H4K12ac was observed within the following developmentally important promoters: AFF4, EP300, LRP5, RUVBL1, USP9X, NCOA6, NSD1, and POU2F1. We found 5% to 10% hypomethylation within CpG islands of selected promoters in the sperm of fertile donors, and it was not significantly altered in the subfertile group. Our results demonstrate that the H4K12ac depletion in selected developmentally important promoters of subfertile patients was not accompanied by a change of DNA methylation. Using a murine model, immunofluorescence revealed that H4K12ac co-localize with 5mC in the sperm nucleus. During fertilization, when the pronuclei are formed, the paternal pronucleus exhibits a strong acetylation signal on H4K12, while in the maternal pronucleus, there is a permanent increase of H4K12ac until pronuclei fusion. Simultaneously, there is an increase of the 5hmC signal and a decrease of the 5mC signal. CONCLUSIONS: We suggest that aberrant histone acetylation within developmentally important gene promoters in subfertile men, but not DNA methylation, may reflect insufficient sperm chromatin compaction affecting the transfer of epigenetic marks to the oocyte.
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Syncytin-1 and 2, human fusogenic glycoproteins encoded by the env genes of the endogenous retroviral loci ERVWE1 and ERVFRDE1, respectively, contribute to the differentiation of multinucleated syncytiotrophoblast in chorionic villi. In non-trophoblastic cells, however, the expression of syncytins has to be suppressed to avoid potential pathogenic effects. Previously, we have shown that the transcriptional suppression of ERVWE1 promoter is controlled epigenetically by DNA methylation and chromatin modifications. In this study, we describe the aberrant expression of syncytin-1 in biopsies of testicular germ cell tumors. RESULTS: We found efficient expression and splicing of syncytin-1 in seminomas and mixed germ cell tumors with seminoma component. Although another fusogenic gene, syncytin-2 was also derepressed in seminomas, its expression was significantly lower than that of syncytin-1. Neither the transcription factor GCM1 nor the increased copy number of ERVWE1 were sufficient for this aberrant expression of syncytin-1 in seminomas. In accordance with our recent finding of the highly increased expression of TET1 dioxygenase in most seminomas, the ERVWE1 promoter was significantly hypomethylated in comparison with the matched controls. In contrast, 5-hydroxymethylcytosine levels were not detectable at the ERVWE1 promoter. We further describe that another endogenous retroviral element adjacent to ERVWE1 remains transcriptionally suppressed and two additional HERV-W family members are only slightly upregulated in seminomas. CONCLUSIONS: We conclude that DNA demethylation of the ERVWE1 promoter in seminomas is a prerequisite for syncytin-1 derepression. We propose the spliced syncytin-1 expression as a marker of seminoma and suggest that aberrant expression of endogenous retroviruses might be a correlate of the hypomethylated genome of seminomas.
- MeSH
- DNA virů metabolismus MeSH
- endogenní retroviry genetika MeSH
- epigeneze genetická MeSH
- genové produkty env biosyntéza MeSH
- lidé MeSH
- metylace DNA MeSH
- regulace genové exprese * MeSH
- seminom patologie virologie MeSH
- těhotenské proteiny biosyntéza MeSH
- testikulární nádory patologie virologie MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
