DNA Methylation Of Repetitive Elements Research Articles

DNA methylation in ES cells requires the lysine methyltransferase G9a but not its catalytic activity

Histone H3K9 methylation is required for DNA methylation and silencing of repetitive elements in plants and filamentous fungi. In mammalian cells however, deletion of the H3K9 histone methyltransferases (HMTases) Suv39h1 and Suv39h2 does not affect DNA methylation of the endogenous retrovirus murine leukaemia virus, indicating that H3K9 methylation is dispensable for DNA methylation of retrotransposons, or that a different HMTase is involved. We demonstrate that embryonic stem (ES) cells lacking the H3K9 HMTase G9a show a significant reduction in DNA methylation of retrotransposons, major satellite repeats and densely methylated CpG-rich promoters. Surprisingly, demethylated retrotransposons remain transcriptionally silent in G9a(-/-) cells, and show only a modest decrease in H3K9me2 and no decrease in H3K9me3 or HP1alpha binding, indicating that H3K9 methylation per se is not the relevant trigger for DNA methylation. Indeed, introduction of catalytically inactive G9a transgenes partially 'rescues' the DNA methylation defect observed in G9a(-/-) cells. Taken together, these observations reveal that H3K9me3 and HP1alpha recruitment to retrotransposons occurs independent of DNA methylation in ES cells and that G9a promotes DNA methylation independent of its HMTase activity.

Abstract 557: Aging and DNA methylation in Alu and LINE-1 repeated elements

Abstract DNA methylation exhibits a gradual genome-wide decrease in association with aging and the rate of decrease appears to be inversely proportional to life expectancy. The observed age-related increase in cancer incidence cannot be explained entirely by genetic mutation rates. Accumulation of epigenetic alterations during aging may contribute to determine the malignant transformation. In particular, global DNA hypomethylation, which has been associated with repetitive DNA demethylation may promote chromosomal instability. About 55% of the human genome consists of repetitive elements, which are for the most part made up of approximately 500,000 long interspersed nucleotide elements (LINE elements) and 1.4 million Alu repetitive elements, which are normally heavily methylated. DNA methylation of LINE-1 and Alu elements has shown to correlate with total 5mC content and thus used to estimate global DNA methylation. In humans, LINEs are the major source of insertional mutagenesis, being involved in both germinal and somatic mutant phenotypes. Alu retrotransposition has also been associated with human disease. However, whether LINE/Alu methylation decreases with age has never been established. We examined the epigenetic effect of aging on DNA methylation in repetitive elements Alu and LINE-1, measured in peripheral blood DNA from 693 subjects evaluated between 1999-2006 as part of the Normative Aging Study (NAS), a longitudinal study established in 1963 by the U.S. Veterans Administration. We measured DNA methylation through quantitative analysis by PCR-Pyrosequencing. Two different blood samples, taken approximately three years apart, were available for each subject. Repeated element methylation was negatively associated with age. In linear regression, we found a decrease equal to 0.016% for LINE-1 (p=0.07) and 0.015% for Alu (p=0.007), for each year increase. We did not find significant changes between repeated measures taken three years apart from each other on the same subjects. The two repeated measures showed high correlation for both Alu (n=300, r=0.46, p<0.001) for Alu and LINE-1 (n=303, r=0.59, p<0.001). DNA methylation of LINE-1 and Alu repetitive elements is inversely correlated with age, although is not captured in repeated measurements taken over a three years period. Such age-related decrease in methylation may increase the risk of mutational events potentially leading to cancer development.

Effect of Cytarabine and Decitabine in Combination in Human Leukemic Cell Lines

1-beta-D-Arabinofuranosylcytosine (cytarabine; ara-C) is the most active agent in myeloid leukemia. 5-Aza-2'-deoxycytidine (DAC) is a cytosine analogue that inhibits DNA methylation and also has activity in myeloid leukemia. Therefore, we investigated combining these two drugs in human leukemia cell lines in vitro. We initially examined the effects of ara-C and DAC on human leukemia cell lines HL60, ML-1, RAji, and Jurkat. We measured IC(50) of DAC and ara-C in these cell lines and calculated a combination index of these two drugs given either simultaneously or sequentially. In searching for mechanisms relative to epigenetic regulation for this effect, we examined DNA methylation of LINE and Alu repetitive elements as a surrogate for global genomic DNA methylation. In addition, we sorted Annexin V positive and negative cells and measured differences in LINE methylation between them. The combination of DAC and ara-C showed additive induction of cell death in ML-1 and synergistic induction in HL60, Raji, and Jurkat. Sequentially, DAC followed by ara-C was a synergistic combination in all cell lines. Low-dose DAC induced more hypomethylation than high doses of the drug, whereas ara-C had no effects on methylation. The combination of ara-C with DAC either together or DAC followed by ara-C resulted in inhibition of LINE demethylation in HL60. The RIL gene, which is silenced by DNA hypermethylation, was activated by DAC, but the addition of ara-C to DAC reduced RIL gene activation. DAC treatment increased H3 Lys(9) acetylation of Alu elements, whereas ara-C had no effect, and the addition of ara-C to DAC inhibited this effect. Finally, we showed that after DAC exposure, Annexin V positive cells were more hypomethylated than Annexin V negative cells. The combination of DAC and ara-C showed additive or synergistic effects on cell death in four human leukemia cell lines in vitro, but antagonism in terms of epigenetic effects. One possible explanation for these paradoxical observations is that hypomethylated cells are sensitized to cell killing by ara-C. These data suggest that DAC used in combination with ara-C has clinical potential in the treatment of acute myeloid leukemia.

Changes in DNA Methylation Patterns in Subjects Exposed to Low-Dose Benzene

Aberrant DNA methylation patterns, including global hypomethylation, gene-specific hypermethylation/hypomethylation, and loss of imprinting (LOI), are common in acute myelogenous leukemia (AML) and other cancer tissues. We investigated for the first time whether such epigenetic changes are induced in healthy subjects by low-level exposure to benzene, a widespread pollutant associated with AML risk. Blood DNA samples and exposure data were obtained from subjects with different levels of benzene exposure, including 78 gas station attendants, 77 traffic police officers, and 58 unexposed referents in Milan, Italy (personal airborne benzene range, < 6-478 microg/m(3)). Bisulfite-PCR pyrosequencing was used to quantitate DNA methylation in long interspersed nuclear element-1 (LINE-1) and AluI repetitive elements as a surrogate of genome-wide methylation and examine gene-specific methylation of MAGE-1 and p15. Allele-specific pyrosequencing of the H19 gene was used to detect LOI in 96 subjects heterozygous for the H19 imprinting center G/A single-nucleotide polymorphism. Airborne benzene was associated with a significant reduction in LINE-1 (-2.33% for a 10-fold increase in airborne benzene levels; P = 0.009) and AluI (-1.00%; P = 0.027) methylation. Hypermethylation in p15 (+0.35%; P = 0.018) and hypomethylation in MAGE-1 (-0.49%; P = 0.049) were associated with increasing airborne benzene levels. LOI was found only in exposed subjects (4 of 73, 5.5%) and not in referents (0 of 23, 0.0%). However, LOI was not significantly associated with airborne benzene (P > 0.20). This is the first human study to link altered DNA methylation, reproducing the aberrant epigenetic patterns found in malignant cells, to low-level carcinogen exposure.

Changes in DNA Methylation of Repetitive Elements during the Progression of Chronic Myelogenous Leukemia.

Epigenetic changes are a common finding in cancer, and promoter hypermethylation of tumor suppressor genes leads to aberrant gene silencing. In contrast to gene promoter hypermethylation, global hypomethylation is also a well-described phenomenon in cancer. Measurement of total 5-methylcytosine in the DNA show decreases in cancer despite increases in gene specific methylation. This discrepancy can be accounted for by the decrease of DNA methylation found in DNA repetitive elements, which make up approximately over half of the human genome. Decreases in repetitive element DNA methylation are a sensitive surrogate marker of global methylation, and have been used to measure DNA methylation changes induced by chemotherapy, dietary folate and environmental exposure. Decreases in LINE1 methylation have been described in numerous human cancers. In CML decrease in LINE1 methylation has been associated with progression to blast crisis. We investigated the DNA methylation changes of DNA repetitive elements in CML during the progression of CML from chronic phase to accelerated phase and finally blast crisis. We used a previously described assay (Yang et al. 2004) employing Bisulfite-PCR Pyrosequencing to quantitatively study the DNA methylation changes associated with the LINE1, Alu, D4Z4, and NBL-2 repetitive elements (See table).LINE1 and Alu hypomethylation was significantly associated with the progression of CML. Surprisingly, we found that NBL-2 and D4Z4 hypermethylation significantly increased during the progression of CML. Interestingly, these two repetitive elements were found to be specifically hypomethylated in immunodeficiency, centromeric instability and facial abnormalities (ICF) syndrome which is caused by a germline mutation of DNA methyltransferase 3b (DNMT3b) (Kondo T et al, 2000). This result suggests that DNMT3b may be involved in the progression of CML.DNA methylation (Mean ± S.E.M)Repetitive ElementsNormalChronic PhaseAccelerated PhaseBlast CrisisLINE132.4 ± 1.329.3 ± 0.627.8 ± 0.828.3 ± 0.8Alu79.4 ± 1.177.9 ± 0.677.6 ± 0.977.5 ± 0.8D4Z447.9 ± 2.949.0 ± 1.152.9 ± 1.457.5 ± 2.0NBL-271.7 ± 2.271.7 ± 0.775.9 ± 1.377.2 ± 1.6

Analysis of repetitive element DNA methylation by MethyLight

Repetitive elements represent a large portion of the human genome and contain much of the CpG methylation found in normal human postnatal somatic tissues. Loss of DNA methylation in these sequences might account for most of the global hypomethylation that characterizes a large percentage of human cancers that have been studied. There is widespread interest in correlating the genomic 5-methylcytosine content with clinical outcome, dietary history, lifestyle, etc. However, a high-throughput, accurate and easily accessible technique that can be applied even to paraffin-embedded tissue DNA is not yet available. Here, we report the development of quantitative MethyLight assays to determine the levels of methylated and unmethylated repeats, namely, Alu and LINE-1 sequences and the centromeric satellite alpha (Satα) and juxtacentromeric satellite 2 (Sat2) DNA sequences. Methylation levels of Alu, Sat2 and LINE-1 repeats were significantly associated with global DNA methylation, as measured by high performance liquid chromatography, and the combined measurements of Alu and Sat2 methylation were highly correlative with global DNA methylation measurements. These MethyLight assays rely only on real-time PCR and provide surrogate markers for global DNA methylation analysis. We also describe a novel design strategy for the development of methylation-independent MethyLight control reactions based on Alu sequences depleted of CpG dinucleotides by evolutionary deamination on one strand. We show that one such Alu-based reaction provides a greatly improved detection of DNA for normalization in MethyLight applications and is less susceptible to normalization errors caused by cancer-associated aneuploidy and copy number changes.

Estimating the total mouse DNA methylation according to the B1 repetitive elements

Measuring the degree of methylation of the B1 element in mouse may represent the global DNA methylation status because about 30,000 copies of the B1 element are randomly dispersed in the total mouse genome. Six CpG dinucleotides are located within each 163 bp size of B1 element, and each CpG dinucleotide was partially methylated. We quantitated the DNA methylation of the B1 repetitive elements by performing PCR for the methylation specific PCR (MSP) and also by the pyrosequencing. Each CpG dinucleotide was methylated at an average of 9% in the mouse genome by the pyrosequencing and MSP. Especially, we checked whether CpG methylation of the B1 element could respond to a treatment of the DNA methylation inhibitor, 5-azacytidine (5-AzaC). Consequently, the calibration graph resulting from measuring the relative CpG methylation percentage of the B1 element is linearly decreased with the increasing amount of 5-AzaC (up to 50 ng/ml concentration) in the NIH3T3 cells with a standard deviation of only 1.73% between three independent assays. Our methods can be applied to the routine analysis of the global DNA methylation changes in mouse in vivo and in vitro in pharmaceuticals and basic epigenetic research with efforts being less labor-intensive.