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      5-Aza-2′-deoxycytidine-induced genome rearrangements are mediated by DNMT1

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          Abstract

          Observations that genome-wide DNA hypomethylation induces genomic instability and tumors in animals caution against the indiscriminate use of demethylating agents, such as 5-aza-2′-deoxycytidine (5-Aza-dC). Using primary mouse embryonic fibroblasts harboring a lacZ mutational reporter construct that allows the quantification and characterization of a wide range of mutational events, we found that in addition to demethylation, treatment with 5-Aza-dC induces γ-H2AX expression, a marker for DNA breaks, and both point mutations and genome rearrangements. To gain insight into the source of these mutations we first tested the hypothesis that the mutagenic effect of 5-Aza-dC may be directly mediated through the DNA methyltransferase 1 (DNMT1) covalently trapped in 5-Aza-dC-substituted DNA. Knock-down of DNMT1 resulted in increased resistance to the cytostatic effects of 5-Aza-dC, delayed onset of γ-H2AX expression and a significant reduction in the frequency of genome rearrangements. There was no effect on the 5-Aza-dC-induced point mutations. An alternative mechanism for 5-Aza-dC-induced demethylation and genome rearrangements via activation-induced cytidine deaminase (AID) followed by base excision repair (BER) was found not to be involved. That is, 5-Aza-dC treatment did not significantly induce AID expression and inhibition of BER did not reduce the frequency of genome rearrangements. Thus, our results indicate that the formation of DNMT1 adducts is the prevalent mechanism of 5-Aza-dC-induced genome rearrangements, although hypomethylation per se may still contribute. Since the therapeutic effects of 5-Aza-dC greatly depend on the presence of DNMT1, the expression level of DNA methyltransferases in tumors may serve as a prognostic factor for the efficacy of 5-Aza-dC treatment.

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          Most cited references 40

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          Quantitative high-throughput analysis of DNA methylation patterns by base-specific cleavage and mass spectrometry.

          Methylation is one of the major epigenetic processes pivotal to our understanding of carcinogenesis. It is now widely accepted that there is a relationship between DNA methylation, chromatin structure, and human malignancies. DNA methylation is potentially an important clinical marker in cancer molecular diagnostics. Understanding epigenetic modifications in their biological context involves several aspects of DNA methylation analysis. These aspects include the de novo discovery of differentially methylated genes, the analysis of methylation patterns, and the determination of differences in the degree of methylation. Here we present a previously uncharacterized method for high-throughput DNA methylation analysis that utilizes MALDI-TOF mass spectrometry (MS) analysis of base-specifically cleaved amplification products. We use the IGF2/H19 region to show that a single base-specific cleavage reaction is sufficient to discover methylation sites and to determine methylation ratios within a selected target region. A combination of cleavage reactions enables the complete evaluation of all relevant aspects of DNA methylation, with most CpGs represented in multiple reactions. We successfully applied this technology under high-throughput conditions to quantitatively assess methylation differences between normal and neoplastic lung cancer tissue samples from 48 patients in 47 genes and demonstrate that the quantitative methylation results allow accurate classification of samples according to their histopathology.
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            Mechanism and regulation of class switch recombination.

            Antibody class switching occurs in mature B cells in response to antigen stimulation and costimulatory signals. It occurs by a unique type of intrachromosomal deletional recombination within special G-rich tandem repeated DNA sequences [called switch, or S, regions located upstream of each of the heavy chain constant (C(H)) region genes, except Cdelta]. The recombination is initiated by the B cell-specific activation-induced cytidine deaminase (AID), which deaminates cytosines in both the donor and acceptor S regions. AID activity converts several dC bases to dU bases in each S region, and the dU bases are then excised by the uracil DNA glycosylase UNG; the resulting abasic sites are nicked by apurinic/apyrimidinic endonuclease (APE). AID attacks both strands of transcriptionally active S regions, but how transcription promotes AID targeting is not entirely clear. Mismatch repair proteins are then involved in converting the resulting single-strand DNA breaks to double-strand breaks with DNA ends appropriate for end-joining recombination. Proteins required for the subsequent S-S recombination include DNA-PK, ATM, Mre11-Rad50-Nbs1, gammaH2AX, 53BP1, Mdc1, and XRCC4-ligase IV. These proteins are important for faithful joining of S regions, and in their absence aberrant recombination and chromosomal translocations involving S regions occur.
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              DNA demethylation dynamics.

              The discovery of cytosine hydroxymethylation (5hmC) suggested a simple means of demethylating DNA and activating genes. Further experiments, however, unearthed an unexpectedly complex process, entailing both passive and active mechanisms of DNA demethylation by the ten-eleven translocation (TET) and AID/APOBEC families of enzymes. The consensus emerging from these studies is that removal of cytosine methylation in mammalian cells can occur by DNA repair. These reports highlight that in certain contexts, DNA methylation is not fixed but dynamic, requiring continuous regulation. Copyright © 2011 Elsevier Inc. All rights reserved.
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                Author and article information

                Journal
                8711562
                6325
                Oncogene
                Oncogene
                Oncogene
                0950-9232
                1476-5594
                18 January 2012
                20 February 2012
                13 December 2012
                13 June 2013
                : 31
                : 50
                : 5172-5179
                Affiliations
                [1 ]Department of Genetics, Albert Einstein College of Medicine, Bronx, NY, USA
                [2 ]Department of Medicine, Albert Einstein College of Medicine, Bronx, NY, USA
                [3 ]Institute of Aging Research, Guangdong Medical College, Dongguan, China
                Author notes
                [* ]Corresponding authors: Alexander Y. Maslov Albert Einstein College of Medicine 1301 Morris Park Avenue Bronx, NY 10461 Phone: (718) 678-1153 alex.maslov@ 123456einstein.yu.edu Yousin Suh Albert Einstein College of Medicine 1301 Morris Park Avenue Bronx, NY 10461 Phone: (718) 678-1111 yousin.suh@ 123456einstein.yu.edu Jan Vijg Albert Einstein College of Medicine 1301 Morris Park Avenue Bronx, NY 10461 Phone: (718) 678-1151 jan.vijg@ 123456einstein.yu.edu
                Article
                NIHMS347163
                10.1038/onc.2012.9
                3381073
                22349820

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                Funding
                Funded by: National Institute on Aging : NIA
                Award ID: R01 AG024391-05 || AG
                Funded by: National Institute on Aging : NIA
                Award ID: P01 AG017242-15 || AG
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