DNA (cytosine-5)-methyltransferase 1 Research Articles

Global DNA methylation, DNMT1, and MBD2 in patients with systemic lupus erythematosus

To investigate the associations of DNA methylation levels and mRNA expressions of DNA cytosine-5-methyltransferase 1 (DNMT1) and methyl CpG-binding domain 2 (MBD2) with systemic lupus erythematosus (SLE), 108 patients with SLE and 97 healthy controls were enrolled in this study. DNA and total RNA were extracted from the peripheral blood mononuclear cells of the SLE patients and the controls. The global methylation levels of DNA were measured in 63 patients with SLE and 68 healthy controls by the ELISA method. DNMT1 and MBD2 mRNA were also detected in 108 SLE patients and 97 controls using the quantitative real-time polymerase chain reaction method. The global methylation level of DNA was significantly decreased in the SLE patients in comparison with that in the controls (p < 0.001, 95% CI = 0.1573-0.5052). The patients with SLE have higher expressions of DNMT1 and MBD2 mRNA than the controls (p < 0.001, 95% CI = -0.0049 - -0.0019 and p = 0.001, 95% CI = -0.0119 - -0.0029, respectively). We also found that there were no significant differences in the methylation level and the expression of DNMT1 and MBD2 mRNA between the active and the inactive SLE patients. A positive correlation was also found between DNMT1 and MBD2 mRNA expressions in the SLE patients (p < 0.001). This study demonstrated that the patients with SLE had a significantly lower level of DNA methylation than the controls. The expression of both DNMT1 and MBD2 mRNA was significantly increased in the SLE patients compared with the controls. This study also showed a positive correlation between DNMT1 and MBD2 mRNA levels in the patients with SLE.

Global DNA methylation, DNMT1, and MBD2 in patients with rheumatoid arthritis

Objectives To investigate the associations of DNA methylation levels and mRNA expressions of DNA cytosine-5-methyltransferase 1 (DNMT1) and methyl CpG-binding domain 2 (MBD2) with rheumatoid arthritis (RA). Methods The global methylation status of DNA was measured in 65 patients with RA and 64 healthy controls by the ELISA method. DNMT1 and MBD2 mRNA were also detected in 177 RA patients and 95 controls using the quantitative real-time polymerase chain reaction method. Results The global methylation of DNA was significantly decreased in the RA patients compared to the controls ( p = 0.005, 95% CI = 0.0835–0.4503). The patients with RA had higher expressions of DNMT1 and MBD2 mRNA than the controls ( p < 0.001, 95% CI = −0.0024 to −0.0053 and p < 0.001, 95% CI = −0.0079 to −0.0167, respectively). We also found that the MBD2 mRNA level was not related to the disease activity of RA. However, the expression of DNMT1 mRNA tended to be associated with the disease activity of RA ( p = 0.08). The levels of DNA methylation and DNMT1 mRNA were significantly decreased in the patients with anti-CCP antibody compared with those without ( p = 0.005, 95% CI = −0.7333 to −0.1373 and p = 0.003, 95% CI = −0.0071 to −0.0022, respectively). The differences in the methylation level and expressions of DNMT1 and MBD2 were not significant between the patients treated with and without anti-TNFα biological agents (Enbrel or Humira). Conclusion This study demonstrated that the RA patients have a significantly lower level of DNA methylation than the controls. Moreover, RA patients have higher expressions of DNMT1 and MBD2 mRNA. The anti-TNFα biological agents do not seem to affect DNA methylation and mRNA expressions of DNMT1 and MBD2 in RA patients.

Fluorescence-based high-throughput assay for human DNA (cytosine-5)-methyltransferase 1

We have developed the first economical and rapid nonradioactive assay method that is suitable for high-throughput screening of the important pharmacological target human DNA (cytosine-5)-methyltransferase 1 (DNMT1). The method combines three key innovations: the use of a truncated form of the enzyme that is highly active on a 26-bp hemimethylated DNA duplex substrate, the introduction of the methylation site into the recognition sequence of a restriction endonuclease, and the use of a fluorogenic read-out method. The extent of DNMT1 methylation is reflected in the protection of the DNA substrate from endonuclease cleavage that would otherwise result in a large fluorescence increase. The assay has been validated in a high-throughput format, and trivial changes in the substrate sequence and endonuclease allow adaptation of the method to any bacterial or human DNA methyltransferase.

DNA hypomethylation induced by tributyltin, triphenyltin, and a mixture of these in Sebastiscus marmoratus liver

Tributyltin (TBT) and triphenyltin (TPT) coexist in freshwater and marine environments. However, the effects of TBT, TPT, and a mixture of the two on DNA methylation in marine fish livers and the mechanism involved remain to be elucidated. Previous study have proved that abnormal methylation patterns are induced by the balance of transmethylation reaction including the tissue level of S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH) or the activity of DNA (cytosine-5) methyltransferase 1 (DNMT1). Therefore, in the present study, we assessed their ability to cause hepatic DNA hypomethylation in Sebastiscus marmoratus liver and the related mechanism. The results showed that TBT, TPT, and a mixture of the two significantly induced DNA hypomethylation in the fish livers in a dose-dependent manner. Using Pearson correlation coefficient analysis, we identified strong linear correlations between S-adenosylhomocysteine, S-adenosylmethionine, or the SAM to SAH ratio and the hepatic genome-wide 5-methylcytosine content of the DNA, but no correlation between the latter and the DNMT1 expression level. It is therefore proposed that the organotins hypomethylation induced in the marine fish livers was due to altering the balance of the substrate and the product in transmethylation reactions.

CXXC Domain of Human DNMT1 Is Essential for Enzymatic Activity

DNA cytosine methylation is one of the major epigenetic gene silencing marks in the human genome facilitated by DNA methyltransferases. DNA cytosine-5 methyltransferase 1 (DNMT1) performs maintenance methylation in somatic cells. In cancer cells, DNMT1 is responsible for the aberrant hypermethylation of CpG islands and the silencing of tumor suppressor genes. Here we show that the catalytically active recombinant DNMT1, lacking 580 amino acids from the amino terminus, binds to unmethylated DNA with higher affinity than hemimethylated or methylated DNA. To further understand the binding domain of enzyme, we have used gel shift assay. We have demonstrated that the CXXC region (C is cysteine; X is any amino acid) of DNMT1 bound specifically to unmethylated CpG dinucleotides. Furthermore, mutation of the conserved cysteines abolished CXXC mediated DNA binding. In transfected COS-7 cells, CXXC deleted DNMT1 (DNMT1 (DeltaCXXC)) localized on replication foci. Both point mutant and DNMT1 (DeltaCXXC) enzyme displayed significant reduction in catalytic activity, confirming that this domain is crucial for enzymatic activity. A permanent cell line with DNMT1 (DeltaCXXC) displayed partial loss of genomic methylation on rDNA loci, despite the presence of endogenous wild-type enzyme. Thus, the CXXC domain encompassing the amino terminus region of DNMT1 cooperates with the catalytic domain for DNA methyltransferase activity.

Transcript levels of DNA methyltransferases DNMT1, DNMT3A and DNMT3B in CD4+T cells from patients with systemic lupus erythematosus

Global DNA hypomethylation in CD4(+) T cells has been detected in systemic lupus erythematosus (SLE) and it seems to be linked to its pathogenesis. We investigated the relationship between overall DNA methylation and the expression of three DNA (cytosine-5) methyltransferases involved in the DNA methylation process. The DNA deoxymethylcytosine (dmC) content of purified CD4(+) T cells from 29 SLE patients and 30 healthy controls was measured by means of an enzyme-linked immunosorbent assay (ELISA). The transcript levels of DNA cytosine-5-methyltransferase 1 (DNMT1), DNA cytosine-5-methyltransferase 3A (DNMT3A) and DNA cytosine-5-methyltransferase 3B (DNMT3B) were quantified by real-time reverse transcription-polymerase chain reaction (RT-PCR). Association studies were also carried out with several laboratory parameters, as well as with the patients' clinical manifestations. SLE patients had a significantly lower CD4(+) T-cell DNA dmC content than controls (0.802 +/- 0.134 versus 0.901 +/- 0.133) (P = 0.007). No differences in transcript levels were observed for DNMT1, DNMT3A and DNMT3B between patients and controls. The simultaneous association of low complement counts with lymphopenia, high titres of anti-double-stranded DNA (anti-dsDNA), or an SLE disease activity index (SLEDAI) of > 5, resulted in the increase of at least one of the three DNA methyltransferases. It is possible that patients were reacting indirectly to an underlying DNA hypomethylation status by increasing the mRNA levels of DNA methyltransferases when the disease was being definitely active.

DNA (cytosine-5)-methyltransferase 1 as a mediator of mutant p53-determined p16ink4A down-regulation

In cancer, gene silencing via hypermethylation is as common as genetic mutations in p53. Understanding the relationship between mutant p53 and hypermethylation of other tumor suppressor genes is essential when elucidate mechanisms of tumor development. In this study, two isogenic human B lymphoblast cell lines with different p53 status include TK6 containing wild-type p53 and WTK1 with mutant p53 were used and contrasted. Lower levels of p16(ink4A) protein were detected in WTK1 cells than in TK6 cells, which were accompanied by increased DNA (cytosine-5)-methyltransferase 1 (DNMT1) gene expression as well as hypermethylation of the p16 ( ink4A ) promoter. siRNA experiments to transiently knock down wild-type p53 in TK6 cells resulted in increase of DNMT1 expression as well as decrease of p16(ink4A) protein. Conversely, siRNA knockdown of mutant p53 in WTK1 cells did not alter either DNMT1 or p16(ink4A) protein levels. Furthermore, loss of suppression function of mutant p53 to DNMT1 in WTK1 was caused by the attenuation of its binding ability to the DNMT1 promoter. In summary, we provide evidences to elucidate the relationship between mutant p53 and DNMT1. Our results indicate that mutant p53 loses its ability to suppress DNMT1 expression, and thus enhances methylation levels of the p16 ( ink4A ) promoter and subsequently down-regulates p16(ink4A )protein.

Predicting aberrant CpG island methylation

Epigenetic silencing associated with aberrant methylation of promoter region CpG islands is one mechanism leading to loss of tumor suppressor function in human cancer. Profiling of CpG island methylation indicates that some genes are more frequently methylated than others, and that each tumor type is associated with a unique set of methylated genes. However, little is known about why certain genes succumb to this aberrant event. To address this question, we used Restriction Landmark Genome Scanning to analyze the susceptibility of 1,749 unselected CpG islands to de novo methylation driven by overexpression of DNA cytosine-5-methyltransferase 1 (DNMT1). We found that although the overall incidence of CpG island methylation was increased in cells overexpressing DNMT1, not all loci were equally affected. The majority of CpG islands (69.9%) were resistant to de novo methylation, regardless of DNMT1 overexpression. In contrast, we identified a subset of methylation-prone CpG islands (3.8%) that were consistently hypermethylated in multiple DNMT1 overexpressing clones. Methylation-prone and methylation-resistant CpG islands were not significantly different with respect to size, C+G content, CpG frequency, chromosomal location, or promoter association. We used DNA pattern recognition and supervised learning techniques to derive a classification function based on the frequency of seven novel sequence patterns that was capable of discriminating methylation-prone from methylation-resistant CpG islands with 82% accuracy. The data indicate that CpG islands differ in their intrinsic susceptibility to de novo methylation, and suggest that the propensity for a CpG island to become aberrantly methylated can be predicted based on its sequence context.

Recombinant Human DNA (Cytosine-5) Methyltransferase: III. ALLOSTERIC CONTROL, REACTION ORDER, AND INFLUENCE OF PLASMID TOPOLOGY AND TRIPLET REPEAT LENGTH ON METHYLATION OF THE FRAGILE X CGG·CCG SEQUENCE

Steady-state kinetic analyses revealed that the methylation reaction of the human DNA (cytosine-5) methyltransferase 1 (DNMT1) is repressed by the N-terminal domain comprising the first 501 amino acids, and that repression is relieved when methylated DNA binds to this region. DNMT1 lacking the first 501 amino acids retains its preference for hemimethylated DNA. The methylation reaction proceeds by a sequential mechanism, and either substrate (S-adenosyl-l-methionine and unmethylated DNA) may be the first to bind to the active site. However, initial binding of S-adenosyl-l-methionine is preferred. The binding affinities of DNA for both the regulatory and the catalytic sites increase in the presence of methylated CpG dinucleotides and vary considerably (more than one hundred times) according to DNA sequence. DNA topology strongly influences the reaction rates, which increased with increasing negative superhelical tension. These kinetic data are consistent with the role of DNMT1 in maintaining the methylation patterns throughout development and suggest that the enzyme may be involved in the etiology of fragile X, a syndrome characterized by de novo methylation of a greatly expanded CGG.CCG triplet repeat sequence.

The DNMT1 Target Recognition Domain Resides in the N Terminus

DNA-cytosine-5-methyltransferase 1 (DNMT1) is the enzyme believed to be responsible for maintaining the epigenetic information encoded by DNA methylation patterns. The target recognition domain of DNMT1, the domain responsible for recognizing hemimethylated CGs, is unknown. However, based on homology with bacterial cytosine DNA methyltransferases it has been postulated that the entire catalytic domain, including the target recognition domain, is localized to 500 amino acids at the C terminus of the protein. The N-terminal domain has been postulated to have a regulatory role, and it has been suggested that the mammalian DNMT1 is a fusion of a prokaryotic methyltransferase and a mammalian DNA-binding protein. Using a combination of in vitro translation of different DNMT1 deletion mutant peptides and a solid-state hemimethylated substrate, we show that the target recognition domain of DNMT1 resides in the N terminus (amino acids 122-417) in proximity to the proliferating cell nuclear antigen binding site. Hemimethylated CGs were not recognized specifically by the postulated catalytic domain. We have previously shown that the hemimethylated substrates utilized here act as DNMT1 antagonists and inhibit DNA replication. Our results now indicate that the DNMT1-PCNA interaction can be disrupted by substrate binding to the DNMT1 N terminus. These results point toward new directions in our understanding of the structure-function of DNMT1.