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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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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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gks155-F3: 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.

Mentions: In the pathway shown in Figure 3a, Tet-mediated production of 5hmC becomes the substrate for AID, which converts 5hmC to 5hmU. We thus tested whether 5hmU can be excised by MBD4 and TDG glycosylase domains (15,39). We used the same 32-bp DNA duplexes containing a G:X mismatch within the CpG sequence context (where X = 5hmU, T or U) as the substrate. The X-containing strand was FAM labeled, and the excision of the mismatched base was monitored by denaturing gel electrophoresis following NaOH hydrolysis (Supplementary Figure S4). As expected, no glycosylase activity was observed on oligonucleotides bearing the ‘natural’ G:C and G:M base pairs, and there was efficient cleavage of substrates bearing G:T and G:U mismatches (Figure 3b and c). We extend these findings to show here that the TDG and MBD4 glycosylases are also inactive on G:H (which preserves Watson–Crick base-pair hydrogen bonds; see Figure 3a), but act on G:5hmU mismatched substrates, and further, that the modification status (methyl or hydroxymethyl) of the C in the opposite strand of neighboring G had no impact on the ability to remove G:T, G:U or G:5hmU mismatches (Figure 3b and c). Therefore, TDG and MBD4 are capable of acting on AID-generated 5hmU and completing the ‘demethylation’ of 5hmC.Figure 3.


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)

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3367191&req=5

gks155-F3: 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.
Mentions: In the pathway shown in Figure 3a, Tet-mediated production of 5hmC becomes the substrate for AID, which converts 5hmC to 5hmU. We thus tested whether 5hmU can be excised by MBD4 and TDG glycosylase domains (15,39). We used the same 32-bp DNA duplexes containing a G:X mismatch within the CpG sequence context (where X = 5hmU, T or U) as the substrate. The X-containing strand was FAM labeled, and the excision of the mismatched base was monitored by denaturing gel electrophoresis following NaOH hydrolysis (Supplementary Figure S4). As expected, no glycosylase activity was observed on oligonucleotides bearing the ‘natural’ G:C and G:M base pairs, and there was efficient cleavage of substrates bearing G:T and G:U mismatches (Figure 3b and c). We extend these findings to show here that the TDG and MBD4 glycosylases are also inactive on G:H (which preserves Watson–Crick base-pair hydrogen bonds; see Figure 3a), but act on G:5hmU mismatched substrates, and further, that the modification status (methyl or hydroxymethyl) of the C in the opposite strand of neighboring G had no impact on the ability to remove G:T, G:U or G:5hmU mismatches (Figure 3b and c). Therefore, TDG and MBD4 are capable of acting on AID-generated 5hmU and completing the ‘demethylation’ of 5hmC.Figure 3.

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