AID/APOBEC deaminases disfavor modified cytosines implicated in DNA demethylation

Department of Medicine, Raymond and Ruth Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
Nature Chemical Biology (Impact Factor: 13). 07/2012; 8(9):751-8. DOI: 10.1038/nchembio.1042
Source: PubMed


Activation-induced deaminase (AID)/APOBEC-family cytosine deaminases, known to function in diverse cellular processes from antibody diversification to mRNA editing, have also been implicated in DNA demethylation, a major process for transcriptional activation. Although oxidation-dependent pathways for demethylation have been described, pathways involving deamination of either 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC) have emerged as alternatives. Here we address the biochemical plausibility of deamination-coupled demethylation. We found that purified AID/APOBECs have substantially reduced activity on 5mC relative to cytosine, their canonical substrate, and no detectable deamination of 5hmC. This finding was explained by the reactivity of a series of modified substrates, where steric bulk was increasingly detrimental to deamination. Further, upon AID/APOBEC overexpression, the deamination product of 5hmC was undetectable in genomic DNA, whereas oxidation intermediates remained detectable. Our results indicate that the steric requirements for cytosine deamination are one intrinsic barrier to the proposed function of deaminases in DNA demethylation.

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    • "We and others have previously shown that AID is unable to deaminate 5hmC containing substrates [32], [53], and hence the proposed direct genetic links [54], [55] between AID and 5hmC were only speculative. Our discovery that AID-induced lesions at non-methylated cytosines can lead to active DNA demethylation provides an alternative indirect pathway for AID to resolve TET induced 5hmC modifications. "
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    ABSTRACT: 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. In vitro, 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 in vivo 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.
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    • "In the following step, hmU might be excised either by TDG, methyl-CpG-binding domain protein 4 (MBD4) or the single-strand-specific monofunctional uracil-DNA glycosylase 1 (SMUG1) (31–33). However, there is evidence that AID/APOBEC members are less active on modified cytosines in vitro or in vivo, challenging the prominence of the proposed deamination-linked demethylation pathway in living cells (34). Furthermore, a direct decarboxylation of caC to unmodified cytosine has been detected in ESC lysates, but no specific decarboxylase has been identified so far (35). "
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    ABSTRACT: The discovery of hydroxymethyl-, formyl- and carboxylcytosine, generated through oxidation of methylcytosine by TET dioxygenases, raised the question how these modifications contribute to epigenetic regulation. As they are subjected to complex regulation in vivo, we dissected links to gene expression with in vitro modified reporter constructs. We used an Oct4 promoter-driven reporter gene and demonstrated that in vitro methylation causes gene silencing while subsequent oxidation with purified catalytic domain of TET1 leads to gene reactivation. To identify proteins involved in this pathway we screened for TET interacting factors and identified TDG, PARP1, XRCC1 and LIG3 that are involved in base-excision repair. Knockout and rescue experiments demonstrated that gene reactivation depended on the glycosylase TDG, but not MBD4, while NEIL1, 2 and 3 could partially rescue the loss of TDG. These results clearly show that oxidation of methylcytosine by TET dioxygenases and subsequent removal by TDG or NEIL glycosylases and the BER pathway results in reactivation of epigenetically silenced genes.
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    • "In fact, oxidation of 5mC to 5hmC by TET1 has been shown to promote DNA demethylation in the adult brain (Guo et al, 2011b). This study suggests the involvement of the AID/APOBEC family of cytidine deaminases in 5hmC demethylation, but more recent studies suggest that these deaminases favor unmodified cytosine (Nabel et al, 2012). Nevertheless , knockdown of endogenous TET1 in the dentate gyrus of the hippocampus can reverse the demethylation observed in the promoter region of Bdnf and Fgf1 (Guo et al, 2011a) in animals that receive synchronous electroconvulsive therapy (Ma et al, 2009). "
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    ABSTRACT: Epigenetic regulation of cellular identity and function is at least partly achieved through changes in covalent modifications on DNA and histones. Much progress has been made in recent years to understand how these covalent modifications affect cell identity and function. Despite the advances, whether and how epigenetic factors contribute to memory formation is still poorly understood. In this review, we discuss recent progress in elucidating epigenetic mechanisms of learning and memory, primarily at the DNA level, and look ahead to discuss their potential implications in reward memory and development of drug addiction.
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