Demystifying DNA Demethylation

Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Science (Impact Factor: 31.48). 09/2011; 333(6047):1229-30. DOI: 10.1126/science.1211917
Source: PubMed

ABSTRACT Variability and adaptability are necessary for overcoming the challenges of multicellular life. To address this need, nature
has evolved a substantial enzymatic toolbox for altering cytosine within the genome. Methylation of the nucleotide cytosine
(C) at the 5-position of the base has profound impacts on gene expression and cellular identity. The reverse of this process,
DNA demethylation, is equally important for cleaning the genomic slate during embryogenesis or achieving rapid reactivation
of previously silenced genes. Although the mechanism of DNA methylation has been rigorously established, active DNA demethylation
in mammals has remained enigmatic, as disparate observations have failed to coalesce into a consistent model. Cytosine deamination,
oxidation, and base excision repair enzymes have been proposed in a dizzying variety of combinations (1). Against this backdrop, two reports in this issue, by Ito et al. (2) on page 1300 and He et al. on page 1303 (3), help bring new clarity to the mechanistic model for DNA demethylation.

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    ABSTRACT: Increasing evidence indicates that active DNA demethylation is involved in several processes in mammals, resulting in developmental stage-specificity and cell lineage-specificity. The recently discovered Ten-Eleven Translocation (TET) dioxygenases are accepted to be involved in DNA demethylation by initiating 5-mC oxidation. Aberrant DNA methylation profiles are associated with many diseases. For example in cancer, hypermethylation results in silencing of tumor suppressor genes. Such silenced genes can be re-expressed by epigenetic drugs, but this approach has genome-wide effects. In this study, fusions of designer DNA binding domains to TET dioxygenase family members (TET1, -2 or -3) were engineered to target epigenetically silenced genes (ICAM-1, EpCAM). The effects on targeted CpGs' methylation and on expression levels of the target genes were assessed. The results indicated demethylation of targeted CpG sites in both promoters for targeted TET2 and to a lesser extent for TET1, but not for TET3. Interestingly, we observed re-activation of transcription of ICAM-1. Thus, our work suggests that we provided a mechanism to induce targeted DNA demethylation, which facilitates re-activation of expression of the target genes. Furthermore, this Epigenetic Editing approach is a powerful tool to investigate functions of epigenetic writers and erasers and to elucidate consequences of epigenetic marks.
    Nucleic Acids Research 11/2013; 42(3). DOI:10.1093/nar/gkt1019 · 8.81 Impact Factor
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    ABSTRACT: Age-related cataracts are a leading cause of blindness. Previously, we have demonstrated the association of unfolded protein response with various cataractogenic stressors. However, DNA methylation alterations leading to suppression of lenticular antioxidant protection remains unclear. Here, we report the methylglyoxal-mediated sequential events responsible for Keap1 promoter DNA demethylation in human lens epithelial cells, because Keap1 is a negative regulatory protein that regulates the Nrf2 antioxidant protein. Methylglyoxal induces the ER stress and activates the unfolded protein response leading to overproduction of ROS prior to human lens epithelial cells death. Methylglyoxal also suppresses the Nrf2 and DNA methyltransferases but activates the DNA demethylation pathway enzyme, TET1. Bisulfite genomic DNA sequencing confirms the methylglyoxal-mediated Keap1 promoter DNA demethylation leading to over-expression of Keap1 mRNA and protein. Similarly, bisulfite genomic DNA sequencing of human clear lenses (n=15) slowly lose 5-methylcytosine in the Keap1 promoter throughout life, at a rate of 1% per year. By contrast, diabetic cataractous lenses (n=21) lose an average of 90% of the 5-methylcytosine regardless of the age. Over-expressed Keap1 protein is responsible for decreasing the Nrf2 by proteasomal degradation, thereby suppressing the Nrf2 dependent stress protection. This study demonstrates for the first time about the associations of unfolded protein response activation, Nrf2 dependent antioxidant system failure and loss of Keap1 promoter methylation because of altered active and passive DNA demethylation pathway enzymes in human lens epithelial cells by methylglyoxal. As an outcome, cellular redox balance is altered towards lens oxidation and cataract formation.
    Free Radical Biology and Medicine 04/2014; 72. DOI:10.1016/j.freeradbiomed.2014.04.010 · 5.71 Impact Factor
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    ABSTRACT: During gene transcription, proteins appear to cycle on and off some gene promoters with both long (tens of minutes) and short periods (no more than several minutes). The essence of these phenomena still remains unclear. Here, we propose a stochastic model for the state evolution of promoters in terms of DNA-protein interactions. The model associates the characteristics of microscopic molecular interactions with macroscopic measurable quantities. Through theoretical derivation, we reconcile the contradictory viewpoints on the concurrent fast and slow cycling; both the cycling phenomena are further reproduced by fitting simulation results to the experimental data on the pS2 gene. Our results suggest that the fast cycling dictates how the proteins behave on the promoter and that stable binding hardly occurs. Different kinds of proteins rapidly bind/unbind the promoter at distinct transcriptional stages fulfilling specific functions; this feature is essentially manifested as the slow cycling of proteins when detected by chromatin immunoprecipitation assays. Thus, the slow cycling represents neither stable binding of proteins nor external modulation of the fast cycling. This work also reveals the relationship between the essence and measurement of transcriptional dynamics.
    Journal of The Royal Society Interface 04/2014; 11(96):20140253. DOI:10.1098/rsif.2014.0253 · 3.86 Impact Factor


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