Processive DNA Demethylation via DNA Deaminase-Induced Lesion Resolution

<div><p>Base modifications of cytosine are an important aspect of chromatin biology, as they can directly regulate gene expression, while DNA repair ensures that those modifications retain genome integrity. Here we characterize how cytosine DNA deaminase AID can initiate DNA demethylation. <i>In vitro</i>, AID initiated targeted DNA demethylation of methyl CpGs when in combination with DNA repair competent extracts. Mechanistically, this is achieved by inducing base alterations at or near methyl-cytosine, with the lesion being resolved either via single base substitution or a more efficient processive polymerase dependent repair. The biochemical findings are recapitulated in an <i>in vivo</i> transgenic targeting assay, and provide the genetic support of the molecular insight into DNA demethylation. This targeting approach supports the hypothesis that mCpG DNA demethylation can proceed via various pathways and mCpGs do not have to be targeted to be demethylated.</p></div

AID-induced demethylation in Embryo and Placenta.

<p>GAL4-AID wt (TG 5) or mutant (TG 8) transgenic females were crossed with H19 DMR-UAS homozygous males, and methylation was analyzed in embryos (E) and placentas (P) at E12.5 of transgene positive offspring. Bi-2 region was amplified and analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097754#pone-0097754-g005" target="_blank">Figure 5</a>. Filled circles represent methylated CpGs, open circles unmethylated ones.</p

Structure of GAL4-AID transgenes and their expression.

<p>(<b>A</b>) GAL4-AID fusion cDNAs were inserted into a CMV promoter containing vector resulting in three transgene constructs (GAL4-AID, GAL4-ΔAID1, and GAL4-ΔAID2), which were excised from the plasmid backbone with <i>Nru</i>I and <i>Dra</i>III and microinjected into zygotes, resulting in transgenic strains TG 4 and 5, TG 7, and TG 8. Lower panel: The GAL4 DNA binding domain was fused to the AID cDNA lacking the C terminal nuclear export signal (NES). In addition to wild-type AID two mutant forms of AID cDNA, harbouring amino acid exchanges D89G and C147R, and E58G, respectively, were fused. Numbers refer to amino acids position in AID. (<b>B</b>) GAL4-AID expression in transgenic strains TG 5 (GAL4-AID), TG 7 (GAL4-ΔAID1), and TG 8 (CMV GAL4-ΔAID2) was monitored by RT-PCR. RNA samples from embryo, placenta and various adult tissues were analyzed. <i>Hprt</i> mRNA served as loading control. (<b>C</b>) Transgenic AID localizes in pronuclei of zygotes in TG 5 (Gal-4-AID) and TG 7 (GAL4-ΔAID1). GAL4-AID expression in zygotes was analyzed by immunofluorescence using an anti-GAL4 antibody. DNA was stained with DAPI. TG 5 and TG 7 resulted from crossing of transgenic mother with H19 DMR-UAS father, as a control crosses between C57Bl6 mother and H19 DMR-UAS father was used.</p

Model for lesion-induced DNA demethylation.

<p>Active DNA demethylation dependent on 5mC targeting. 5mC (filled circle) can be hydroxymethylated (5hmC - small grey circle) and further processed by the TET protein family (top right panel - grey). 5mC can be deaminated by the cytosine deaminase AID (bottom left panel - red). Deamination of 5mC results in thymine (dT - in red) and creates a dT:G mismatch that can be recognized by multiple DNA repair pathways, including BER or processive polymerase dependent repair. The processing of the dT:G mismatch by BER produces a single cytosine demethylation event, while processive polymerase dependent repair, in either direction, replaces long stretches of all bases, including 5mC, leading to multiple cytosine-demethylation events. Active DNA demethylation independent of 5mC targeting (central panel - green). AID targets and deaminates cytosine (white C:G), forming uracil (dU – in green) and leading to a dU:G mismatch. BER processing leads to the status quo (intact methylated DNA), while activation of a processive polymerase dependent repair, in either direction, replaces long stretches of all bases, including 5mC, leading to multiple cytosine-demethylation events. After AID and DNA repair induced demethylation on one strand, the complementary strand can be targeted as well and/or DNA replication can take place in the absence of DNMT1 activity. Active DNA demethylation of TET enzyme induced 5hmC can be independent of targeting (right panel - blue). AID targets and deaminates cytosine (white C:G), forming uracil (dU – in green) and leading to a dU:G mismatch. Processing as for untargeted 5mC demethylation with the outcome of replacing 5hmC with dC.</p

AID-induced lesion repair results in demethylation.

<p>(<b>A</b>) AID induces demethylation of 5mCpG <i>in vitro</i>. Methylated plasmids from IVR reactions in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097754#pone-0097754-g001" target="_blank">Figure 1</a> were subjected to bisulfite sequence analysis of regions BS, 3' of the GAL4 binding site. Methylation was monitored from IVR-samples performed with GAL4-AID (G-AID + FE) or without (FE), and in the presence of Ugi (G-AID + FE (Ugi)). White and black circles represent unmethylated and methylated cytosines, respectively. (<b>B</b>) Quantification of 5mC demethylated after the IVR assay from (A). Error bars indicate ± SD (n = 3); a t-test showed a significant difference between the two groups (p<0.05). (<b>C</b>) Graphical representation of those CpGs that were significantly inhibited by Ugi treatment for resolving AID-induced demethylation in the IVR. Each CpG (1 - 29) was analyzed using chi-squared analysis, and red circles indicate p<0.01.</p

AID induces demethylation independent of the nucleotide context.

<p>(<b>A</b>) The table summarizes whether methylation of the adenosine (<i>m6</i>A) in GATC sites influences cutting by the restriction enzymes <i>Mbo</i>I, <i>Sau3A</i>I or <i>Dpn</i>I. On the left is the preference of cutting depending on the state of adenosine-methylation. In the middle the absolute PCR recovery after digestion is indicated. The outcome of the PCR recovery of digested DNA after AID-induced demethylating <i>m6</i>A is indicated on the right. (<b>B</b>) (<b>C</b>) AID induces demethylation of methyl-adenosine. Supercoiled plasmids (<i>m6A(+</i>) as isolated from standard <i>E. coli</i>) were subjected to IVR assays containing GAL4-AID as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097754#pone-0097754-g001" target="_blank">Figure 1</a>. Streptavidin-isolated plasmids were either undigested (mock) or incubated with the restriction enzymes <i>Mbo</i>I, <i>Dpn</i>I, and <i>Sau3A</i>I (NEB, USA) prior to qPCR amplification. The different GATC sites (positions: 468, 1287, 2001 and 3230) are indicated by colors and marked on the plasmid map; there are 37 GATC sites within the plasmid substrate. IVR results were quantitated by setting the uncut G-AID treated samples to 100%. Correction factors based on FE alone activity (for <i>Mbo</i>I) or Fe + <i>Sau3A</i>I activity (for <i>Dpn</i>I) was determined from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097754#pone.0097754.s003" target="_blank">Figure S3</a> and applied to the G-AID induced recoveries. Loss of <i>m6</i>A is shown in (<b>B</b>) by a decrease in recovery after <i>Mbo</i>I cutting versus uncut, and by an increase in recovery after <i>Dpn</i>I digest (<b>C</b>). A standard t-test indicated that after G-AID treatment all sites showed a significant difference between <i>Sau3A</i>I and <i>Dpn</i>I cutting; (p<0.05 - indicated as *), (n≥3).</p

AID-induced lesions on a methylated substrate are resolved by DNA repair.

<p>(<b>A</b>) AID-induced lesions result in bio-dC incorporation. G-AID was incubated with a pre-methylated substrate (<i>M.Sss</i>I - <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097754#pone.0097754.s002" target="_blank">Figure S2</a>) for 30 min and then added to FE. Repaired plasmids (bio-dC labelled) were isolated and quantified by qPCR. The bars represent the ratio (Fold change) of the amount of recovered plasmids from reactions carried out in the presence of G-AID, untagged AID (AID), the GAL4 DNA binding domain (G-DBD), or the mutant G-AID C87R versus levels of plasmids recovered from reactions that did not contain G-AID (FE, set to 1). Error bars indicate ± standard deviation (SD, n = 3). Line schematics for all IVR assays are shown above the graphs, and they indicate the order of addition of substrates/proteins/nucleotides/extract/etc. or treatments. (<b>B</b>) AID-induced lesion resolution on methylated targets involves BER and processive DNA polymerases-dependent repair. IVR reactions were performed as in (A), containing either bio-dC or bio-dA during the resolution phase of the IVR. Where indicated, FE was treated with Ugi prior to addition of the plasmids (FE (Ugi)). The bars depict the levels of plasmids (bio-dC or bio-dA labelled) that were recovered from the individual reactions. Samples were normalized to FE reactions without G-AID (FE) and were set to 1. Error bars indicate ± standard deviation (SD, n = 3).</p

Correlation between naturally acquired IgG immune response against PvRMC-RBP1 and exposure to malaria.

<p>(A) Spearman's rank correlation between age and IgG reactivity index against the chimeric recombinant protein in a population naturally exposed to malaria (r = 0.3613; p<0.0001). (B) Spearman's rank correlation between time of residence in malaria endemic area and IgG reactivity index against the chimeric recombinant protein in a population naturally exposed to malaria (r = 0.2781; p<0.0001).</p

Multivariate regression analysis of independent variables (PMI, TREA and TLI) associated with the magnitude of IgG immune response against both studied antigens.

<p>PMI (Past malaria infections); TREA (Time of residence in endemic area); (Time since the last malaria infection)</p

Frequency (F) and number (n) of IgG responders and non-responders to the PvRMC-RBP1and Pv-RBP1_23-751 recombinant proteins tested by HLA-DRB1* and HLA-DQB1*allelic groups from individuals naturally exposed to malaria.

<p>Frequency (F) and number (n) of IgG responders and non-responders to the PvRMC-RBP1and Pv-RBP1_23-751 recombinant proteins tested by HLA-DRB1* and HLA-DQB1*allelic groups from individuals naturally exposed to malaria.</p