Here we present APOBEC-Coupled Epigenetic Sequencing (ACE-Seq), a bisulfite-free method for localizing 5-hydroxymethylcytosine (5hmC) at single-base resolution with low DNA input. The method builds upon the observation that AID/APOBEC family DNA deaminase enzymes can potently discriminate between cytosine modification states, and exploits the non-destructive nature of enzymatic, rather than chemical, deamination. ACE-Seq yields high-confidence 5hmC profiles with at least 1000-fold less DNA input than conventional methods. Applying ACE-Seq to generate a base-resolution map of 5hmC in tissue-derived cortical excitatory neurons, we find that 5hmC is almost entirely confined to CG dinucleotides. The map permits cytosine, 5-methylcytosine (5mC) and 5hmC to be parsed and reveals genomic features that diverge from global patterns, including enhancers and imprinting control regions with high and low 5hmC/5mC ratios, respectively. Enzymatic deamination overcomes many challenges posed by bisulfite-based methods and expands the scope of epigenome profiling to include scarce samples and open new lines of inquiry regarding the role of cytosine modifications in genome biology.
Lysosomal enzymes catalyze the breakdown of macromolecules in the cell. In humans, loss of activity of a lysosomal enzyme leads to an inherited metabolic defect known as a lysosomal storage disorder. The human lysosomal enzyme galactosamine-6-sulfatase (GALNS, also known as N-acetylgalactosamine-6-sulfatase and GalN6S; E.C. 3.1.6.4) is deficient in patients with the lysosomal storage disease mucopolysaccharidosis IV A (also known as MPS IV A and Morquio A). Here we report the three-dimensional structure of human GALNS, determined by x-ray crystallography at 2.2 Å resolution. The structure reveals a catalytic gem diol nucleophile derived from modification of a cysteine side chain. The active site of GALNS is a large, positively charged trench suitable for binding polyanionic substrates such as keratan sulfate and chondroitin-6-sulfate. Enzymatic assays on the insect cell-expressed human GALNS indicate activity against synthetic substrates and inhibition by both substrate and product. Mapping 120 MPS IV A missense mutations onto the structure reveals that a majority of mutations affect the hydrophobic core of the structure, indicating that most MPS IV A cases result from misfolding of GALNS. Comparison of the structure of GALNS to paralogous sulfatases shows a wide variety of active site geometries in the family, but strict conservation of the catalytic machinery. Overall, the structure and the known mutations establish the molecular basis for MPS IV A and for the larger MPS family of diseases.
AID/APOBEC family enzymes are best known for deaminating cytosine bases to uracil in single-stranded DNA, with characteristic sequence preferences that can produce mutational signatures in targets such as retroviral and cancer cell genomes. These deaminases have also been proposed to function in DNA demethylation via deamination of either 5-methylcytosine (mC) or TET-oxidized mC bases (ox-mCs), which include 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine. One specific family member, APOBEC3A (A3A), has been shown to readily deaminate mC, raising the prospect of broader activity on ox-mCs. To investigate this claim, we developed a novel assay that allows for parallel profiling of activity on all modified cytosines. Our steady-state kinetic analysis reveals that A3A discriminates against all ox-mCs by >3700-fold, arguing that ox-mC deamination does not contribute substantially to demethylation. A3A is, by contrast, highly proficient at C/mC deamination. Under conditions of excess enzyme, C/mC bases can be deaminated to completion in long DNA segments, regardless of sequence context. Interestingly, under limiting A3A, the sequence preferences observed with targeting unmodified cytosine are further exaggerated when deaminating mC. Our study informs how methylation, oxidation, and deamination can interplay in the genome and suggests A3A's potential utility as a biotechnological tool to discriminate between cytosine modification states.
Chemical modifications to genomic DNA can expand and shape its coding potential. Cytosine methylation in particular has well-established roles in regulating gene expression and defining cellular identity. The discovery of TET family enzymes opened a major frontier beyond DNA methylation, revealing three oxidized forms of cytosine that could mediate DNA demethylation or encode independent epigenetic functions. Chemical biology has been instrumental in uncovering TET’s intricate reaction mechanisms and scope of reactivity on a surprising variety of substrates. Moreover, innovative chemoenzymatic strategies have enabled sensitive detection of oxidized cytosine products in vitro and in vivo. We highlight key recent developments that demonstrate how chemical biology is advancing our understanding of the extended, dynamic epigenome.
Human APOBEC3A (A3A) is a nucleic acid-modifying enzyme that belongs to the cytidine deaminase family. Canonically, A3A catalyzes the deamination of cytosine into uracil in single-stranded DNA, an activity that makes A3A both a critical antiviral defense factor and a useful tool for targeted genome editing. However, mutagenesis by A3A has also been readily detected in both cellular DNA and RNA, activities that have been implicated in cancer. Given the importance of substrate discrimination for the physiological, pathological, and biotechnological activities of A3A, here we explore the mechanistic basis for its preferential targeting of DNA over RNA. Using a chimeric substrate containing a target ribocytidine within an otherwise DNA backbone, we demonstrate that a single hydroxyl at the sugar of the target base acts as a major selectivity determinant for deamination. To assess the contribution of bases neighboring the target cytosine, we show that overall RNA deamination is greatly reduced relative to that of DNA but can be observed when ideal features are present, such as preferred sequence context and secondary structure. A strong dependence on idealized substrate features can also be observed with a mutant of A3A (eA3A, N57G), which has been employed for genome editing due to altered selectivity for DNA over RNA. Altogether, our work reveals a relationship between the overall decreased reactivity of A3A and increased substrate selectivity, and our results hold implications both for characterizing off-target mutagenesis and for engineering optimized DNA deaminases for base-editing technologies.
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