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. 2012 Jun;40(11):4841-9.
doi: 10.1093/nar/gks155. Epub 2012 Feb 22.

Recognition and potential mechanisms for replication and erasure of cytosine hydroxymethylation

Hideharu Hashimoto  1 , Yiwei Liu, Anup K Upadhyay, Yanqi Chang, Shelley B Howerton, Paula M Vertino, Xing Zhang, Xiaodong Cheng
Affiliations

Recognition and potential mechanisms for replication and erasure of cytosine hydroxymethylation

Hideharu Hashimoto et al. Nucleic Acids Res. 2012 Jun.

Abstract

Cytosine residues in mammalian DNA occur in at least three forms, cytosine (C), 5-methylcytosine (M; 5mC) and 5-hydroxymethylcytosine (H; 5hmC). During semi-conservative DNA replication, hemi-methylated (M/C) and hemi-hydroxymethylated (H/C) CpG dinucleotides are transiently generated, where only the parental strand is modified and the daughter strand contains native cytosine. Here, we explore the role of DNA methyltransferases (DNMT) and ten eleven translocation (Tet) proteins in perpetuating these states after replication, and the molecular basis of their recognition by methyl-CpG-binding domain (MBD) proteins. Using recombinant proteins and modified double-stranded deoxyoligonucleotides, we show that DNMT1 prefers a hemi-methylated (M/C) substrate (by a factor of >60) over hemi-hydroxymethylated (H/C) and unmodified (C/C) sites, whereas both DNMT3A and DNMT3B have approximately equal activity on all three substrates (C/C, M/C and H/C). Binding of MBD proteins to methylated DNA inhibited Tet1 activity, suggesting that MBD binding may also play a role in regulating the levels of 5hmC. All five MBD proteins generally have reduced binding affinity for 5hmC relative to 5mC in the fully modified context (H/M versus M/M), though their relative abilities to distinguish the two varied considerably. We further show that the deamination product of 5hmC could be excised by thymine DNA glycosylase and MBD4 glycosylases regardless of context.

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Figures

Figure 1.
Figure 1.
DNMT3A and DNMT3B can methylate the cytosine in the context of hemi-hydroxylmethylated CpG site (H/C). (a) Diagram showing the potential fate of single CpG sites that are either unmodified (C/C), fully methylated (M/M) or fully hydroxymethylated (H/H) at DNA replication. After strand synthesis, unmodified (C/C), hemi-methylated (M/C) or hemi-hydroxymethylated (H/C) sites are transiently generated. MBD indicates DNA methyl-binding domain proteins, while Tet refers to ten–eleven translocation proteins. (b–d) Enzymatic activity of recombinant DNMT1, DNMT3A2/DNMT3L and DNMT3B2/DNMT3L against a 32-bp DNA containing a single unmodified (C/C), hemi-methylated (M/C) or hemi-hydroxymethylated (H/C) CpG site. Note that DNMT1 (panel b) has robust preference for maintenance methylation at M/C sites over H/C and C/C sites in naked oligonucleotide DNA, whereas DNMT3A2/3L (c) and DNMT3B2/3L (d) have approximately similar activities on all three substrates.
Figure 2.
Figure 2.
Effect of hydroxymethylation on DNA methylation ‘readers’. Binding affinities of UHRF1 (residues 124–628) (a) and five MBD proteins (b–f) for a double stranded oligonucleotide containing a single CpG site with one of the six different modification states: unmodified (C/C), fully modified (M/M, H/H), hemi-(hydroxy)methylated (M/C, H/C) or hemi-methylated/hemi-hydroxymethylated (H/M) CpG site. Binding was assessed by fluorescence polarization. (a) UHRF1 has a strong preference for binding the hemi-methylated CpG (M/C) site. (b and c) MeCP2 and MBD1 have the strongest binding to fully methylated CpG (M/M). Although there is significantly lower affinity among the MBDs for H/M CpG dinucleotides (5-fold decreased affinity for MeCP2 and 18-fold for MBD1), this substrate is still preferred over hemi-methylated DNA (M/C), and is bound by MeCP2 and MBD1 with an affinity similar in magnitude to MBD2 (d), and greater affinity than MBD3 (e) and MBD4 (f) bind to fully methylated DNA (M/M). (g) Summary of relative binding affinities (by factor of x) of five MBD proteins for M/M and H/M substrates (top two lines). Note that the binding affinities of MeCP2 (and MBD1) for H/M and H/H are in the same order of magnitude as that of MBD2 and MBD4 to M/M substrate, respectively (lines 3 and 4).
Figure 3.
Figure 3.
MBD4 and TDG are capable of excising 5-hydroxymethyluracil in the context of a double-stranded CpG dinucleotide. (a) A putative pathway of DNA demethylation involving DNA methylation by DNMTs, hydroxylation by Tet proteins, deamination by AID and glycosylation by MBD4 or TDG linked to base excision repair (BER). Double stranded 32-bp oligonucleotides bearing a single CpG dinucleotide and the indicated modification status (where M = 5mC and H = 5hmC) and labeled with FAM on the top strand were incubated with the glycosylase domain of MBD4 (b) or TDG (c) at 37°C for 1 h. The products of the reaction were separated on a denaturing polyacrylamide gel, and the FAM-labeled strand was excited by UV and photographed.
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