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. 2012 Sep;8(9):751-8.
doi: 10.1038/nchembio.1042. Epub 2012 Jul 8.

AID/APOBEC deaminases disfavor modified cytosines implicated in DNA demethylation

Christopher S Nabel  1 , Huijue Jia, Yu Ye, Li Shen, Hana L Goldschmidt, James T Stivers, Yi Zhang, Rahul M Kohli
Affiliations

AID/APOBEC deaminases disfavor modified cytosines implicated in DNA demethylation

Christopher S Nabel et al. Nat Chem Biol. 2012 Sep.

VSports - Abstract

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 VSports手机版. .

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Conflict of interest statement

Competing Financial Interests

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Proposed non-canonical role for AID/APOBEC enzymes acting on modified cytosine substrates in DNA
(a) Deamination of cytosine plays known physiological roles in adaptive immunity (AID), innate immunity against retroviruses (APOBEC3 enzymes), and mRNA editing (APOBEC1). These canonical roles involve deamination of cytosine to generate uracil. (b) By contrast, the proposed function of AID/APOBEC family members on modified cytosine residues remains poorly understood despite their implication in potential pathways for active DNA demethylation. (c) Proposed pathways for DNA demethylation. Deamination of 5-methylcytosine (mC) or 5-hydroxymethylcytosine (hmC), the product of TET-mediated oxidation, could generate thymidine or 5-hydroxymethyluracil (hmU), respectively. Base excision repair (BER) could subsequently excise the deaminated bases and replace them with unmodified cytosine. An alternative deamination-independent pathway involves iterative oxidation, generating 5-formylcytosine (fC) or 5-carboxylcytosine (caC). BER-mediated excision of the oxidized cytosine would result in reversion to unmodified cytosine.
Figure 2
Figure 2. AID/APOBEC enzymes preferentially deaminate unmodified cytosine
(a) Fluorophore(FAM)-labeled oligonucleotides (S30) synthesized with a single internal modified cytosine (red) embedded in a CpG motif were incubated with AID/APOBEC family members. After incubation, oligonucleotides were duplexed with a complementary strand generating U:G, T:G, or hmU:G mismatches with the deaminated substrates (blue).Treatment with UDG (reactive with U:G), TDG (reactive with T:G) or SMUG (reactive with hmU:G), respectively, followed by base-mediated cleavage fragments the deaminated products to a 15-mer (P15). (b) The reaction products resulting from incubation of S30-TGX substrates with mA1, mA2, mA3 or hAID are shown separated on a denaturing gel. The substrate and product controls, without incubation with AID/APOBEC enzymes, are shown on the left. Gels are shown without cropping in Supplementary Figure 2. (c) The fraction of deaminated substrates are plotted as a function of increasing concentrations of enzyme for mA1 and hAID-ΔC assayed against substrates containing a cytosine (magenta), mC (blue) or hmC (green). hAID-ΔC was incubated with the S30-TGX series of substrates, while mA1 was incubated with the S30-ATX substrates. Error bars represent standard deviation from the mean of at least three independent replicates. For relative comparison of substrates, the slope of each plot in the region where product formation is linear with enzyme (dashed line) is listed. The standard error for the measurement of enzyme dependent product formation is ±20% of the reported value for all measurements. The values given for hmC substrates are the lower limits of detection with these substrates.
Figure 3
Figure 3. DNA deamination decreases as a function of increasing steric bulk at the 5-position of cytosine
(a) Schematic of the deamination assay with DNA oligonucleotides containing unnatural modifications at C5. (b) At left, the denaturing gel for hAID-ΔC with S30-TGX substrate series is shown. At right, the denaturing gel with mAPOBEC1 assayed against S30-ATX substrates. Samples were run in order of increasing steric bulk of the 5-substituent. Gels are shown without cropping in Supplementary Figure 7 (c) Shown are the electrostatic potential maps of each of the modified cytosine bases as determined using the SPARTAN program (6–31G* basis set). The electrostatic potential is colored from maximal negative (red) to positive (blue). The volume (*) is determined based on linking the 5-position substituent to a single hydrogen atom and calculating the total volume. The hydrophobic substituent constant (**) is derived from partitioning studies of substituted benzenes between octanol and water, where negative values for hydroxyl and hydroxymethyl substituents represent less hydrophobicity . Reported are the values for enzyme dependent product formation (nM product/μM enzyme) for each substrate examined with each active deaminases (†). The data for C, mC and hmC deamination are consolidated from Fig. 2c and Supplementary Fig. 4; data for unnaturally modified substrates are consolidated from Supplementary Fig. 8. The relative activity for each modified substrate when compared to unmodified cytosine for each deaminase is reported parenthetically, allowing for comparisons across a row with each enzyme.
Figure 4
Figure 4. AID/APOBEC enzymes do not perturb levels of mC oxidation intermediates in genomic DNA
(a) Genomic DNA was extracted from HEK 293T cells co-expressing TET2 along with an empty vector control, TDG, mA1 or hAID, digested to single nucleosides, and analyzed via mass spectrometry for the presence of C, mC, hmC, fC, caC, and hmU. The left axis depicts the absolute fmol of nucleoside on a logarithmic scale, while the right axis represents the approximate conversion from absolute fmol to the genomic prevalence of modified bases for every 105 dC bases. The dashed line demonstrates the lowest examined amount of fC, caC, and hmU standards. Error bars indicate the standard deviation from the mean for two to three biological replicates. Asterisks: p ≤ 10−3 for fC and caC in samples with TDG in comparison to plasmid only control. (b) To confirm that overexpressed hAID and mA1 are catalytically active, nuclear extracts were tested for deaminase activity against unmodified cytosine. At left, TET2-hAID nuclear extracts and negative controls (no extract, nuclear extract from untransfected 293T cells, and TET2-hAID E58A) were incubated with S30-TGC substrate; At right, TET2-mA1 nuclear extracts and negative controls (no extract and nuclear extract from untransfected 293T cells) were incubated with S30-ATC substrate. The lanes demonstrating deamination of S30-TGC by TET2-hAID and S30-ATC substrate by TET2-mA1 extracts are highlighted (red). Nuclear extracts were also incubated with S30-TGU or S30-ATU to verify robust uracil excision activity. Gels are shown without cropping in Supplementary Fig. 13.
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References

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