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. 2006 Jul 25;103(30):11112-7.
doi: 10.1073/pnas.0601793103. Epub 2006 Jul 13.

Active cytosine demethylation triggered by a nuclear receptor involves DNA strand breaks

Clémence Kress  1 , Hélène Thomassin, Thierry Grange
Affiliations

Active cytosine demethylation triggered by a nuclear receptor involves DNA strand breaks

Clémence Kress et al. Proc Natl Acad Sci U S A. .

Abstract

Cytosine methylation at CpG dinucleotides contributes to the epigenetic maintenance of gene silencing. Dynamic reprogramming of DNA methylation patterns is believed to play a key role during development and differentiation in vertebrates. The mechanisms of DNA demethylation remain unclear and controversial. Here, we present a detailed characterization of the demethylation of an endogenous gene in cultured cells. This demethylation is triggered in a regulatory region by a transcriptional activator, the glucocorticoid receptor. We show that DNA demethylation is an active process, occurring independently of DNA replication, and in a distributive manner without concerted demethylation of cytosines on both strands. We demonstrate that the DNA backbone is cleaved 3' to the methyl cytidine during demethylation, and we suggest that a DNA repair pathway may therefore be involved in this demethylation.

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Figures

Fig. 1.
Fig. 1.
Kinetics of glucocorticoid-induced DNA demethylation at the −2.5 Tat GRU in hepatoma cells. (A) The four MeCpGs within the Tat GRU (−2,425 to −2,336) that are demethylated upon glucocorticoid treatment are numbered from 1 to 4 (upper strand, U1 to U4; lower strand, L1 to L4). (B) Demethylation kinetics of the three neighboring Cs of the upper strand. Rat hepatoma cells were grown with 10−7 M dexamethasone (+Dex) for the indicated time. The corresponding genomic DNA was treated with hydrazine and piperidine and analyzed by LM-PCR (refs. and ; Fig. 7). Genomic DNA cleaved at A+G and C+T was analyzed simultaneously.
Fig. 2.
Fig. 2.
Demethylation of the −2.5 Tat GRU is not restricted to a particular phase of the cell cycle. Hepatoma cells were synchronized in early S phase in two steps. They were first presynchronized in G1 by isoleucine and serum deprivation and then released from this block in the presence of aphidicolin. They were then released from the early S block in the presence of 10−7 M dexamethasone for the indicated time (0 h, arrested cells). The demethylation kinetics in nonsynchronized cells was analyzed in parallel. (Upper) The proportion of unmethylated U3 during glucocorticoid treatment as determined by using the MethylQuant assay (22). (Lower) The proportion of cells in the various phases of the cell cycle in the synchronized culture (determined by using propidium iodide staining and flow cytometry) and the deduced time frames of the phases of the cell cycle.
Fig. 3.
Fig. 3.
Distribution of the unmethylated cytosines during demethylation of the −2.5 Tat GRU. Methylation patterns of individual double-stranded molecules were assessed by using the hairpin-bisulfite PCR method (5). DNA from the synchronized cells described in Fig. 2 was analyzed 0, 10, and 16 h after release from the aphidicolin block in the presence of dexamethasone (Dex). (A) Frequencies of the various methylation patterns obtained during demethylation of the −2.5 Tat GRU. The patterns are represented by a succession of circles symbolizing the methylation status of cytosines U1/L1, U2/L2, U3/L3, U4/L4: filled, methylated; open, unmethylated. The patterns are organized in subgroups according to the number of unmethylated Cs as indicated on the left. The frequency of each pattern is indicated (%). Because the total numbers of clones analyzed were 43, 77, and 54 for the dexamethasone treatments of 0, 10, and 16 h, respectively, the frequencies corresponding to one event are 2.3, 1.3, and 1.85%, respectively. (B) Time course of the demethylation of each individual cytosine. The percentages of unmethylated cytosines, belonging to either hemimethylated or fully demethylated CpGs as indicated, are plotted for the three analyzed times of dexamethasone induction.
Fig. 4.
Fig. 4.
DNA is cleaved 3′ to the methylated cytosines during glucocorticoid-induced demethylation. Hepatocytes isolated from E15 fetuses were cultured for the indicated time (hours) without (control or −) or with (+Dex) 10−7 M dexamethasone. (A) Kinetics of glucocorticoid-induced DNA demethylation in hepatocytes analyzed by LM-PCR by using genomic DNA treated with hydrazine-piperidine as described in Fig. 1B. (B) Native genomic DNA was analyzed by LM-PCR to visualize strand breaks in the upper strand of the −2.5 Tat GRU. The arrows indicate the bands revealing cleavage at MeCpG. (C) Analysis of the lower strand as in B. (D) Analysis of the upper strand in an upstream region where there are two MeCpGs that are not demethylated. The arrows indicate the location of the corresponding Cs. (E) Competitive PCR analysis of the abundance of the glucocorticoid-induced cleavage products. A fixed amount of genomic DNA from fetal hepatocytes treated for 5 h with dexamethasone was diluted with varying amounts of rat genomic DNA digested with AvaII that cleaves just upstream the −2.5 Tat GRU and analyzed by LM-PCR. The relative amount of the competing liver DNA corresponded to either the same amount (1/1) or serial 5-fold dilutions (1/5 to 1/625). The regions of the gel showing the bands corresponding to the AvaII cleavages and the dexamethasone-induced cleavages at the MeCpG are shown. (F) Representation of the location of the strand break detected relative to the CpG dinucleotide. Note that LM-PCR allows only analysis of the fragment downstream the break and thus that the location and nature of the 3′ end of the upstream fragment (represented in gray) cannot be assessed.
Fig. 5.
Fig. 5.
Scheme depicting the possible DNA demethylation pathways. (A) Location of the cleavages expected for the published putative demethylation mechanisms. The curved arrows indicate the cleavages expected for the methyl excision pathway (16), the base excision pathway (26), and the dinucleotide excision pathway (25). The cleavage observed herein is indicated by the arrow labeled with an asterisk. (B) The short patch repair pathway involving a monofunctional DNA glycosylase that could generate the cleavage product observed (41). (C) The action of a bifunctional DNA glycosylase/AP lyase that could generate the cleavage product observed (41). The opening of the sugar ring that normally occurs has not been represented.
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