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Recognition and potential mechanisms for replication and erasure of cytosine hydroxymethylation.

Hashimoto H, Liu Y, Upadhyay AK, Chang Y, Howerton SB, Vertino PM, Zhang X, Cheng X - Nucleic Acids Res. (2012)

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA.

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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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.
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gks155-F1: 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.

Mentions: In mammals, DNA methyltransferases (DNMTs) include three members, in two families that are structurally and functionally distinct (17). The DNMT3A and DNMT3B (18,19), coupled with regulatory factor Dnmt3-Like (DNMT3L) protein (20,21), establish the initial methylation pattern de novo, while DNMT1 and its accessory protein UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) (22,23) maintain this pattern during chromosome replication. We first asked which Dnmts methylate the newly synthesized cytosine in the context of hemi-hydroxymethylated CpG site (H/C) (Figure 1). Using a 32-bp DNA oligonucleotide containing a single CpG site, either unmodified (C/C), hemi-methylated (M/C) or hemi-hydroxymethylated (H/C), we find that whereas the known maintenance methyltransferase DNMT1 (24) has high intrinsic activity for the M/C substrate (Kcat = 6.6 h−1; Figure 1b and Supplementary Figure S1), it has measurable but greatly reduced activity for the H/C substrate (Kcat ∼ 0.1 h−1) and no detectable activity on C/C (Figure 1b). Thus, unlike M/C, H/C is not a preferred substrate of DNMT1 and is unlikely to be methylated by DNMT1 after replication, in agreement with previous findings (25). Consistent with this notion, UHRF1, which is essential for DNMT1 function and selectivity for hemi-methylated CpG (M/C) sites in vivo (22,23), loses its intrinsic preference for hemi-methylated DNA when 5mC is replaced by 5hmC. UHRF1 (residues 124–628) shows a >10-fold reduced binding affinity for H/C DNA as compared to M/C DNA, that was similar in magnitude to its affinity for fully methylated (M/M), fully hydroxymethylated (H/H) and H/M DNA (Figure 2a and Supplementary Discussion). Therefore, neither DNMT1 nor UHRF1 are likely to be involved in post-replicative maintenance of H/C methylation, which, without the involvement of DNMT3 (see below), would lead to ‘passive demethylation’ of 5hmC.Figure 1.


Recognition and potential mechanisms for replication and erasure of cytosine hydroxymethylation.

Hashimoto H, Liu Y, Upadhyay AK, Chang Y, Howerton SB, Vertino PM, Zhang X, Cheng X - Nucleic Acids Res. (2012)

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.
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getmorefigures.php?uid=PMC3367191&req=5

gks155-F1: 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.
Mentions: In mammals, DNA methyltransferases (DNMTs) include three members, in two families that are structurally and functionally distinct (17). The DNMT3A and DNMT3B (18,19), coupled with regulatory factor Dnmt3-Like (DNMT3L) protein (20,21), establish the initial methylation pattern de novo, while DNMT1 and its accessory protein UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) (22,23) maintain this pattern during chromosome replication. We first asked which Dnmts methylate the newly synthesized cytosine in the context of hemi-hydroxymethylated CpG site (H/C) (Figure 1). Using a 32-bp DNA oligonucleotide containing a single CpG site, either unmodified (C/C), hemi-methylated (M/C) or hemi-hydroxymethylated (H/C), we find that whereas the known maintenance methyltransferase DNMT1 (24) has high intrinsic activity for the M/C substrate (Kcat = 6.6 h−1; Figure 1b and Supplementary Figure S1), it has measurable but greatly reduced activity for the H/C substrate (Kcat ∼ 0.1 h−1) and no detectable activity on C/C (Figure 1b). Thus, unlike M/C, H/C is not a preferred substrate of DNMT1 and is unlikely to be methylated by DNMT1 after replication, in agreement with previous findings (25). Consistent with this notion, UHRF1, which is essential for DNMT1 function and selectivity for hemi-methylated CpG (M/C) sites in vivo (22,23), loses its intrinsic preference for hemi-methylated DNA when 5mC is replaced by 5hmC. UHRF1 (residues 124–628) shows a >10-fold reduced binding affinity for H/C DNA as compared to M/C DNA, that was similar in magnitude to its affinity for fully methylated (M/M), fully hydroxymethylated (H/H) and H/M DNA (Figure 2a and Supplementary Discussion). Therefore, neither DNMT1 nor UHRF1 are likely to be involved in post-replicative maintenance of H/C methylation, which, without the involvement of DNMT3 (see below), would lead to ‘passive demethylation’ of 5hmC.Figure 1.

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA.

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.

Show MeSH