The curious chemical biology of cytosine: deamination, methylation, and oxidation as modulators of genomic potential

Abstract

A multitude of functions have evolved around cytosine within DNA, endowing the base with physiological significance beyond simple information storage. This versatility arises from enzymes that chemically modify cytosine to expand the potential of the genome. Some modifications alter coding sequences, such as deamination of cytosine by AID/APOBEC enzymes to generate immunologic or virologic diversity. Other modifications are critical to epigenetic control, altering gene expression or cellular identity. Of these, cytosine methylation is well understood, in contrast to recently discovered modifications, such as oxidation by TET enzymes to 5-hydroxymethylcytosine. Further complexity results from cytosine demethylation, an enigmatic process that impacts cellular pluripotency. Recent insights help us to propose an integrated DNA demethylation model, accounting for contributions from cytosine oxidation, deamination, and base excision repair. Taken together, this rich medley of alterations renders cytosine a genomic "wild card", whose context-dependent functions make the base far more than a static letter in the code of life.

Figure 1. Cytosine as the Genomic “Wild Card”

Within the context of the genome, cytosine can be modified by deamination, methylation, oxidation or demethylation to generate a series of analogs. In turn, these cytosine modifications influence coding sequences, gene expression and cellular identity. Amongst these analogs, enzymatic modifications can generate 5-methylcytosine (mC), 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), 5-carboxycytosine (caC), 5-hydroxymethyluracil (hmU) and uracil (U) and thymine (T).

Figure 2. The Toolbox for Enzymatic Modification or Excision of Cytosine and Uracil Analogs

(A) The cytosine nucleobase and its numbering are shown. DNA modifying enzymes target numerous positions for modification, exploiting the susceptibility of C4 or C6 to nucleophilic attack, the accessibility of C5 for alkylation or oxidation, and the cleavable sugar/base linkage for base excision repair. (B) The modifying enzymes include deaminases of the AID/APOBEC family, DNA methyltransferases and TET family oxidases. Y represents variable substitution at the 5-position of cytosine (unmodified, methyl or hydroxymethyl groups) in deamination, while X represents the variable oxidation state of the 5-methyl group in oxidation (hydroxymethyl, formyl or carboxyl groups). (C) DNA glycosylase enzymes can recognize uracil analogs and some modified cytosine bases, catalyzing hydrolysis of the N-glycosidic bond and excision of the base.

Figure 3. Cytosine modifications generate variety

Cytosine typically serves as a stable reservoir of information, permitting gene expression and providing coding information. Deamination, methylation and oxidation all can alter the phenotype that results from the same starting genome. (A) Cytosine deamination in the immunoglobulin locus generates uracil. Error-prone repair of uracil results in localized mutations that increase antibody affinity in somatic hypermutation. Clustering of uracil bases leads to DNA breaks which are recombined, ultimately altering the antibody isotype. (B) Cytosine deamination of viral genomes by APOBEC3G. At high levels of deamination, retroviral restriction is achieved, while low-level mutagenesis can promote viral evolution and escape. (C) Cytosine methylation and hydroxymethylation regulate transcription. While methylation typically represses gene expression, the epigenetic role of hydroxymethylation is still being explored.

Figure 4. Integrated Model for Cytosine Demethylation

Numerous mechanisms have been proposed for DNA demethylation, in which 5-methylcytosine (bold, top right) is converted to cytosine (bold, bottom right). Current evidence supports the existence of an iterative oxidation, BER-coupled pathway (orange) in embryonic stem cells. Though some evidence exists in favor of deamination-initiated, BER-coupled repair (green) and oxidation-initiated, deamination/BER-coupled (purple) pathways, important shortcomings of these routes make them more likely to serve accessory or tissue-specific roles. Enzymes which might directly removed the oxidized 5-substituent from intermediates in demethylation are possible but none have yet been clearly identified (pink).