Processive methylation of hemimethylated CpG sites by mouse Dnmt1 DNA methyltransferase

DNA methyltransferase Dnmt1 ensures clonal transmission of lineage-specific DNA methylation patterns in a mammalian genome during replication. Dnmt1 is targeted to replication foci, interacts with PCNA, and favors methylating the hemimethylated form of CpG sites. To understand the underlying mechanism of its maintenance function, we purified recombinant forms of full-length Dnmt1, a truncated form of Dnmt1-(291-1620) lacking the binding sites for PCNA and DNA and examined their processivity using a series of long unmethylated and hemimethylated DNA substrates. Direct analysis of methylation patterns using bisulfite-sequencing and hairpin-PCR techniques demonstrated that full-length Dnmt1 methylates hemimethylated DNA with high processivity and a fidelity of over 95%, but unmethylated DNA with much less processivity. The truncated form of Dnmt1 showed identical properties to full-length Dnmt1 indicating that the N-terminal 290-amino acid residue region of Dnmt1 is not required for preferential activity toward hemimethylated sites or for processivity of the enzyme. Remarkably, our analyses also revealed that Dnmt1 methylates hemimethylated CpG sites on one strand of double-stranded DNA during a single processive run. Our findings suggest that these inherent enzymatic properties of Dnmt1 play an essential role in the faithful and efficient maintenance of methylation patterns in the mammalian genome.     

Accuracy of DNA methylation pattern preservation by the Dnmt1 methyltransferase

DNA methyltransferase 1 (Dnmt1) has a central role in copying the pattern of DNA methylation after replication which is one manifestation of epigenetic inheritance. With oligonculeotide substrates we show that mouse Dnmt1 has a 30- to 40-fold preference for hemimethylated DNA that is almost lost after addition of fully methylated oligonucleotides. Using long hemimethylated DNA substrates that carry defined methylation patterns and bisulfite analysis of the methylation reaction products, we show a 15-fold preference for hemimethylated CG sites. Dnmt1 moves along the DNA in a random walk methylating hemimethylated substrates with high processivity (>50 sites are visited on average which corresponds to linear diffusion over 6000 bp). The frequency of skipping sites is very low (<0.3%) and there is no detectable flanking sequence preference. CGCTC sites tend to terminate the processive methylation of DNA by Dnmt1. Unmethylated DNA is modified non-processively with a preference for methylation at CCGG sites. We simulate the propagation of methylation patterns using a stochastic model with the specificity of Dnmt1 observed here and conclude that either methylation of several sites is required to propagate the methylation information over several cellular generations or additional epigenetic information must be used.     

Bisulphite Differential Denaturation PCR for Analysis of DNA Methylation

Differential denaturation during PCR can be used to selectively amplify unmethylated DNA from a methylated DNA background. The use of differential denaturation in PCR is particularly suited to amplification of undermethylated sequences following treatment with bisulphite, since bisulphite selectively converts cytosines to uracil while methylated cytosines remain unreactive. Thus amplicons derived from unmethylated DNA retain less cytosines and their lower G + C content allows for their amplification at the lower melting temperatures, while limiting amplification of the corresponding methylated amplicons (Bisulphite Differential Denaturation PCR, BDD-PCR). Selective amplification of unmethylated DNA of four human genomic regions from three genes, GSTP1, BRCA1 and MAGE-A1, is demonstrated with selectivity observed at a ratio of down to one unmethylated molecule in 105 methylated molecules. BDD-PCR has the potential to be used to selectively amplify and detect aberrantly demethylated genes, such as oncogenes, in cancers. Additionally BDD-PCR can be effectively utilised in improving the specificity of methylation specific PCR (MSP) by limiting amplification of DNA that is not fully converted, thus preventing misinterpretation of the methylation versus non-conversion.      

Using LUMA: a Luminometric-Based Assay for Global DNA-Methylation

Changes in genomic DNA methylation are important events in normal and pathological cellular processes, contributing both to normal development and differentiation as well as to cancer and other diseases. We describe here a method to analyze global genomic DNA methylation, using a luminometric technology to quantitate methylation sensitive restriction digestions. The method is called LUminometric Methylation Assay (LUMA), and is based on a polymerase extension assay using the the Pyrosequencing? platform. The method is quantitative, highly reproducible and uses an internal control for DNA input. No modification of genomic DNA is needed and the total running time is only six hours. The method is suitable for analyzing clinical material, as well as determining dynamic changes in global methylation/demethylation events. This report describes the method in detail and gives an example of its application in epigenetic research.      

Long-Range Allosteric Interactions between the Folate and Methionine Cycles Stabilize DNA Methylation Reaction Rate

Several metabolites in the folate and methionine cycles influence the activities of distant enzymes involved in one-carbon metabolism. Many hypotheses have been advanced about the functional impact of these long-range interactions. Using both steady-state and fluctuation analyses of a mathematical model of methionine metabolism, we investigate the biochemical basis for several of these hypotheses. We show that the long-range interactions provide remarkable stabilization of the DNA methylation rate in the face of large fluctuations in methionine input. In particular, they enable the system to maintain methylation in the face of low and extremely low protein input. These interactions may therefore have evolved primarily to stabilize DNA methylation under conditions of methionine starvation. In silico experimentation allows us to evaluate the independent effects of various combinations of the long-range interactions, and thereby propose a plausible evolutionary scenario.      

Reactivation of Epigenetically Silenced Genes by DNA Methyltransferase Inhibitors: Basic Concepts and Clinical Applications

Hypermethylation of tumor suppressor genes is one of the most consistent hallmarks of human cancers. This epigenetic alteration has been associated with gene silencing and thus represents an important pathway for generating loss-of-function mutations. In this review, we survey the available literature on systematic, genome-wide approaches aimed at the identification of epigenetically silenced loci. These studies uncovered a variety of diverse genes, but a common signature for epigenetic reactivation has not been identified. Nevertheless, DNA methyltransferase inhibitors have shown significant clinical benefits, mostly in the therapy of leukemias. Recent analyses revealed substantial drug-induced methylation changes that can now be used as endpoints for the further refinement of clinical treatment schedules. Further optimization of epigenetic cancer therapies should be feasible through the use of novel DNA methyltransferase inhibitors with improved specificity. Rational design of epigenetic inhibitors might provide the foundation for a broader use of these drugs in the treatment of cancer.      

DNA Hypermethylation and Partial Gene Silencing of Human Thymine-DNA Glycosylase in Multiple Myeloma Cell Lines

Multiple myeloma (MM) has prominent features of karyotypic instability at the earliest stage, leading to extreme genetic abnormalities as the disease progresses. These successive genetic alterations can be attributed, in part, to defects in DNA repair pathways. A possible mechanism of dysregulation of the DNA repair pathway is epigenetic gene silencing. Therefore, we sought to determine the methylation status of enzymes involved in the base excision repair pathway in multiple myeloma cell lines. Here, we report the aberrant DNA methylation of TDG, one of the enzymes involved in base excision repair of damaged DNA, in several multiple myeloma cell lines but not in normal human plasma cells. DNA hypermethylation of TDG in the MM cell lines leads to lower gene expression levels that results in less efficient DNA repair activity in response to hydrogen peroxide-induced DNA damage. Expression of exogenous TDG can functionally compensate for lower repair activities of damaged DNA in the KAS-6/1 myeloma cell line, which has extensive DNA hypermethylation of the TDG promoter. Hypermethylation of DNA damage repair genes in MM cell lines may provide an explanation for the frequent genomic instability, as well as point mutations, that are encountered in MM.     

The Dnmt1 DNA-(cytosine-C5)-methyltransferase methylates DNA processively with high preference for hemimethylated target sites

In the cell, Dnmt1 is the major enzyme in maintenance of the pattern of DNA methylation after DNA replication. Evidence suggests that the protein is located at the replication fork, where it could directly modify nascent DNA immediately after replication. To elucidate the potential mechanism of this process, we investigate the processivity of DNA methylation and accuracy of copying an existing pattern of methylation in this study using purified Dnmt1 and hemimethylated substrate DNA. We demonstrate that Dnmt1 methylates a hemimethylated 958-mer substrate in a highly processive reaction. Fully methylated and unmethylated CG sites do not inhibit processive methylation of the DNA. Extending previous work, we show that unmethylated sites embedded in a hemimethylated context are modified at an approximately 24-fold reduced rate, which demonstrates that the enzyme accurately copies existing patterns of methylation. Completely unmodified DNA is methylated even more slowly due to an allosteric activation of Dnmt1 by methylcytosine-containing DNA. Interestingly, Dnmt1 is not able to methylate hemimethylated CG sites on different strands of the DNA in a processive manner, indicating that Dnmt1 keeps its orientation with respect to the DNA while methylating the CG sites on one strand of the DNA.     

Evaluation of a Quantitative DNA Methylation Analysis Technique using Methylation-Sensitive/Dependent Restriction Enzymes and Real-Time PCR

DNA methylation in mammals has been shown to play many important roles in diverse biological phenomena. Several methods have been developed for the measurement of region-specific levels of DNA methylation. We sought a technique that could be used to quantitatively evaluate multiple independent loci in several tissues in a quick and cost-effective manner. Recently, a few quantitative techniques have been developed by employing the use of real-time PCR, though they require the additional step of sodium bisulfite conversion. Here we evaluate a technique that involves the digestion of non-sodium bisulfite-treated genomic DNA using methylation-sensitive and methylation-dependent restriction enzymes followed by real-time PCR. The utility of this method is tested by analyzing seventeen genomic regions of known tissue-specific levels of DNA methylation including three imprinted genes. We find that this approach generates rapid, reproducible and accurate results (range= ±5%) without the additional time required for bisulfite conversion. This approach is also adaptable for use with smaller amounts of starting material. We propose this method as a rapid, quantitative method for the analysis of DNA methylation at single sites or within small regions of DNA.      

Detection of RASSF1A and RAR? Hypermethylation in Serum DNA from Breast Cancer Patients

Breast cancer is fast emerging as the leading cancer amongst females, especially in young females in metropolitan cities in India. The epigenetic alterations involved in the onset and progression of breast cancer may serve as biomarkers for early detection and prognosis of the disease. Furthermore, using body fluids such as serum offers a non-invasive method to procure multiple samples for such analyses. In this study, we examined methylation status of two normally unmethylated but biologically significant cancer genes, RAS association domain family protein 1A (RASSF1A) and Retionic acid receptor ? (RAR?) by Methylation Specific PCR (MSP) in invasive ductal carcinomas of the breast and paired serum DNA. RASSF1A was found to be methylated in 17 of 20 (85%) breast tumors; while sera from 15 of 20 (75%) of the patients showed concordant methylated RASSF1A, with a sensitivity of 88%. RAR? was methylated in 2/20 (10%) breast tumors. A gene unmethylated in the tumor DNA was always found to be unmethylated in the matched serum DNA for both RASSF1A and RAR? genes; hence specificity was 100%. Immunohistochemical analysis of RAR? protein in 15 breast carcinoma patients harboring unmethylated RAR? in tumors and serum DNA showed the expression of RAR? protein in tumors and paired normal breast tissues, confirming the MSP findings, suggesting that RAR? promoter is functional in these cases. This study underscores the potential utility of DNA methylation based screening of serum, a readily accessible body fluid, as a surrogate marker for early detection of breast cancer.      

The MethDB DAS Server: Adding an Epigenetic Information Layer to the Human Genome

The DNA methylation database MethDB (http://www.methdb.net) was developed in order to standardize and collect the dispersed data about this epigenetic phenomenon in a common resource. In the first version of MethDB, data was gathered by annotators and the database could only be queried. In a second step, we added an on-line data submission system that is open to the public. Here we present the DAS annotation server of MethDB that allows integration of MethDB into the network of biological databases via the Distributed Annotation System (DAS) and the representation of DNA methylation data as an epigenetic information layer to the human genome. In order to validate our system and to incorporate the data of the first large scale methylation analysis of the human genome, we assembled the 31312 sequences of the human CpG island tagging project into 13786 CpG islands and imported them into MethDB. The database contains now 19905 methylation content data and 5382 methylation patterns or profiles for 48 species, 1511 individuals, 198 tissues and cell lines, and 79 phenotypes.      

The activity of the murine DNA methyltransferase Dnmt1 is controlled by interaction of the catalytic domain with the N-terminal part of the enzyme leading to an allosteric activation of the enzyme after binding to methylated DNA

The mammalian DNA methyltransferase Dnmt1 is responsible for the maintenance of the pattern of DNA methylation in vivo. It is a large multidomain enzyme comprising 1620 amino acid residues. We have purified and characterized individual domains of Dnmt1 (NLS-containing domain, NlsD, amino acid residues: 1-343; replication foci-directing domain, 350-609; Zn-binding domain (ZnD), 613-748; polybromo domain, 746-1110; and the catalytic domain (CatD), 1124-1620). CatD, ZnD and NlsD bind to DNA, demonstrating the existence of three independent DNA-binding sites in Dnmt1. CatD shows a preference for binding to hemimethylated CpG-sites; ZnD prefers methylated CpGs; and NlsD specifically binds to CpG-sites, but does not discriminate between unmethylated and methylated DNA. These results are not compatible with the suggestion that the target recognition domain of Dnmt1 resides in the N terminus of the enzyme. We show by protein-protein interaction assays that ZnD and CatD interact with each other. The isolated catalytic domain does not methylate DNA, neither alone nor in combination with other domains. Full-length Dnmt1 was purified from baculovirus-infected insect cells. Under the experimental conditions, Dnmt1 has a strong (50-fold) preference for hemimethylated DNA. Dnmt1 is stimulated to methylate unmodified CpG sites by the addition of fully methylated DNA. This effect is dependent on Zn, suggesting that binding of methylated DNA to ZnD triggers the allosteric activation of the catalytic center of Dnmt1. The allosteric activation model can explain kinetic data obtained by others. It suggests that Dnmt1 might be responsible for spreading of methylation, a process that is observed during aging and carcenogenesis but may be important for de novo methylation of DNA.     

Enzymatic properties of recombinant Dnmt3a DNA methyltransferase from mouse: the enzyme modifies DNA in a non-processive manner and also methylates non-CpG [correction of non-CpA] sites

We present the first in vitro study investigating the catalytic properties of a mammalian de novo DNA methyltransferase. Dnmt3a from mouse was cloned and expressed in Escherichia coli. It was shown to be catalytically active in E. coli cells in vivo. The methylation activity of the purified protein was highest at pH 7.0 and 30 mM KCl. Our data show that recombinant Dnmt3a protein is indeed a de novo methyltransferase, as it catalyzes the transfer of methyl groups to unmethylated substrates with similar efficiency as to hemimethylated substrates. With oligonucleotide substrates, the catalytic activity of Dnmt3a is similar to that of Dnmt1: the K(m) values for the unmethylated and hemimethylated oligonucleotide substrates are 2.5 microM, and the k(cat) values are 0.05 h(-1) and 0.07 h(-1), respectively. The enzyme catalyzes the methylation of DNA in a distributive manner, suggesting that Dnmt3a and Dnmt1 may cooperate during de novo methylation of DNA. Further, we investigated the methylation activity of Dnmt3a at non-canonical sites. Even though the enzyme shows maximum activity at CpG sites, with oligonucleotide substrates, a high methylation activity was also found at CpA sites, which are modified only twofold slower than CpG sites. Therefore, the specificity of Dnmt3a is completely different from that of the maintenance methyltransferase Dnmt1, which shows a 40 to 50-fold preference for hemimethylated over unmethylated CpG sites and has almost no methylation activity at non-CpG sites.     

A Potential Role for Epigenetic Modulatory Drugs in the Enhancement of Cancer/Germ-Line Antigen Vaccine Efficacy

The discovery of epigenetic silencing as a key mechanism of tumor suppressor gene inactivation in human cancer has led to great interest in utilizing epigenetic modulatory drugs as cancer therapeutics. It is less appreciated that medically important tumor-associated antigens, particularly the Cancer Testis or Cancer/Germ-line family of antigens (CG antigens), which are being actively tested as cancer vaccine targets, are epigenetically activated in many human cancers. However, a major limitation to the therapeutic value of CG antigen-directed vaccines is the limited and heterogeneous expression of CG antigens in tumors. Recent work has begun to dissect the specific epigenetic mechanisms controlling differential expression of CG antigen genes in human cancers. From a clinical perspective, convincing data indicate that epigenetic modulatory agents, including DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors, robustly promote the expression of CG antigens, as well as class I major histocompatibility complex (MHC I) and other immune co-stimulatory molecules, in tumors. Importantly, the effects of these agents on CG antigen gene expression often show marked specificity for tumor cells as compared to normal cells. Taken together, these data encourage clinical evaluation of combination therapies involving epigenetic modulatory drugs and CG antigen-directed tumor vaccines for the treatment of human malignancies.     

Cooperativity between DNA methyltransferases in the maintenance methylation of repetitive elements.

We used mouse embryonic stem (ES) cells with systematic gene knockouts for DNA methyltransferases to delineate the roles of DNA methyltransferase 1 (Dnmt1) and Dnmt3a and -3b in maintaining methylation patterns in the mouse genome. Dnmt1 alone was able to maintain methylation of most CpG-poor regions analyzed. In contrast, both Dnmt1 and Dnmt3a and/or Dnmt3b were required for methylation of a select class of sequences which included abundant murine LINE-1 promoters. We used a novel hemimethylation assay to show that even in wild-type cells these sequences contain high levels of hemimethylated DNA, suggestive of poor maintenance methylation. We showed that Dnmt3a and/or -3b could restore methylation of these sequences to pretreatment levels following transient exposure of cells to 5-aza-CdR, whereas Dnmt1 by itself could not. We conclude that ongoing de novo methylation by Dnmt3a and/or Dnmt3b compensates for inefficient maintenance methylation by Dnmt1 of these endogenous repetitive sequences. Our results reveal a previously unrecognized degree of cooperativity among mammalian DNA methyltransferases in ES cells.     

CD44 and PTGS2 Methylation are Independent Prognostic Markers for Biochemical Recurrence Among Prostate Cancer Patients with Clinically Localized Disease

Up to 30% of men with clinically localized disease who receive radical prostatectomy develop a biochemical recurrence. Gene methylation in tumor tissue may distinguish men with aggressive cancer. This study evaluated methylation of GSTP1, RARβ2, CD44 and PTGS2 with biochemical recurrence among 60 patients who underwent radical prostatectomy using logistic regression and Kaplan Meier time to event analysis. Methylation of GSTP1 and RARβ2 was not associated with recurrence, however, CD44 and PTGS2 methylation were significant predictors. In multivariate models adjusting for Gleason grade, methylation profile of CD44 and PTGS2 combined was an independent predictor of biochemical recurrence (associated with 9-fold increased risk). In addition, Kaplan Meier analysis showed CD44 and PTGS2 methylation was associated with shorter time to recurrence. CD44 and PTGS2 methylation may predict biochemical recurrence in prostate cancer patients undergoing radical prostatectomy and if validated in larger studies, may identify patients with aggressive cancer.