DNA Methylation-Dependent Epigenetic Regulation of Gene Expression in MCF-7 Breast Cancer Cells

To identify epigenetically-regulated genes in breast cancer, MCF-7 cells were exposed to 250nM 5-aza or 5-aza + 50nM TSA for 3 weeks followed by a 5 week recovery period after treatment withdrawal and gene expression patterns were examined by microarray analysis. We identified 20 genes that are associated with a >2-fold increase in expression in response to the demethylating treatment but returned to control levels after treatment withdrawal. RT-PCR verified that the genes identified were expressed at low or undetectable levels in control MCF-7 cells, but increased expression in treated cells. Most of these putative epigentically-regulated genes in MCF-7 cells do not contain CpG islands. In fact, these genes could be classified based upon their promoter CpG features, including genes with: (i) typical CpG features (CpG islands), (ii) intermediate CpG features (weak CpG islands), and (iii) atypical CpG features (no CpG islands). Prototype genes from each class (including CpG-deficient genes) were shown to be methylation-sensitive (subject to CpG methylation and responsive to demethylating agents), suggesting that not all gene targets of DNA methylation in breast cancer will contain a CpG island. Based upon the results of the current study and observations from the literature, we propose expansion of the current model for methylation-dependent regulation of gene expression to include genes lacking typical CpG islands. The expanded model we propose recognizes that all promoter CpG dinucleotides represent legitimate targets for DNA methylation and that the methylation of specific CpG dinucleotides in critical domains of regulatory regions can result in gene silencing.      

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.      

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.      

An improved version of the DNA Methylation database (MethDB)

Cytosine methylation is a characteristic property of prokaryotic and eukaryotic genomes. In the latter, it is indispensable for a healthy development of the organism and uncontrolled changes in the distribution of 5-methylcytosine (5mC) have been linked to severe disorders, in particular cancer. The growing scientific interest in DNA methylation has led to a considerable amount of data about this epigenetic phenomenon. In order to make these data readily available, we have established a dedicated database. The DNA Methylation database (MethDB) is currently the only public database for DNA methylation (http://www.methdb.net). This constantly growing database has become a key resource in the field of DNA methylation research. The database contains currently methylation patterns, profiles and total methylation content data for 46 species, 160 tissues and 72 phenotypes coming from a total of 6667 experiments (as of September 4, 2002). About 14% of the data have not been published elsewhere. These data can be conveniently searched and represented in different ways. Recently, we have included an on-line submission tool that permits the scientific public to directly enter new data into MethDB.     

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.     

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.      

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.      

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.     

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.      

Reduced MeCP2 Expression is Frequent in Autism Frontal Cortex and Correlates with Aberrant MECP2 Promoter Methylation

Mutations in MECP2, encoding methyl CpG binding protein 2 (MeCP2), cause most cases of Rett syndrome (RTT), an X-linked neurodevelopmental disorder. Both RTT and autism are “pervasive developmental disorders” and share a loss of social, cognitive and language skills and a gain in repetitive stereotyped behavior, following apparently normal perinatal development. Although MECP2 coding mutations are a rare cause of autism, MeCP2 expression defects were previously found in autism brain. To further study the role of MeCP2 in autism spectrum disorders (ASDs), we determined the frequency of MeCP2 expression defects in brain samples from autism and other ASDs. We also tested the hypotheses that MECP2 promoter mutations or aberrant promoter methylation correlate with reduced expression in cases of idiopathic autism. MeCP2 immunofluorescence in autism and other neurodevelopmental disorders was quantified by laser scanning cytometry and compared with control postmortem cerebral cortex samples on a large tissue microarray. A significant reduction in MeCP2 expression compared to age-matched controls was found in 11/14 autism (79%), 9/9 RTT (100%), 4/4 Angelman syndrome (100%), 3/4 Prader-Willi syndrome (75%), 3/5 Down syndrome (60%), and 2/2 attention deficit hyperactivity disorder (100%) frontal cortex samples. One autism female was heterozygous for a rare MECP2 promoter variant that correlated with reduced MeCP2 expression. A more frequent occurrence was significantly increased MECP2 promoter methylation in autism male frontal cortex compared to controls. Furthermore, percent promoter methylation of MECP2 significantly correlated with reduced MeCP2 protein expression. These results suggest that both genetic and epigenetic defects lead to reduced MeCP2 expression and may be important in the complex etiology of autism.     

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.