Enzymatic approaches for profiling cytosine methylation and hydroxymethylation

Abstract

Background: In mammals, modifications to cytosine bases, particularly in cytosine-guanine (CpG) dinucleotide contexts, play a major role in shaping the epigenome. The canonical epigenetic mark is 5-methylcytosine (5mC), but oxidized versions of 5mC, including 5-hydroxymethylcytosine (5hmC), are now known to be important players in epigenomic dynamics. Understanding the functional role of these modifications in gene regulation, normal development, and pathological conditions requires the ability to localize these modifications in genomic DNA. The classical approach for sequencing cytosine modifications has involved differential deamination via the chemical sodium bisulfite; however, bisulfite is destructive, limiting its utility in important biological or clinical settings where detection of low frequency populations is critical. Additionally, bisulfite fails to resolve 5mC from 5hmC.

Scope of review: To summarize how enzymatic rather than chemical approaches can be leveraged to localize and resolve different cytosine modifications in a non-destructive manner.

Major conclusions: Nature offers a suite of enzymes with biological roles in cytosine modification in organisms spanning from bacteriophages to mammals. These enzymatic activities include methylation by DNA methyltransferases, oxidation of 5mC by TET family enzymes, hypermodification of 5hmC by glucosyltransferases, and the generation of transition mutations from cytosine to uracil by DNA deaminases. Here, we describe how insights into the natural reactivities of these DNA-modifying enzymes can be leveraged to convert them into powerful biotechnological tools. Application of these enzymes in sequencing can be accomplished by relying on their natural activity, exploiting their ability to discriminate between cytosine modification states, reacting them with functionalized substrate analogs to introduce chemical handles, or engineering the DNA-modifying enzymes to take on new reactivities. We describe how these enzymatic reactions have been combined and permuted to localize DNA modifications with high specificity and without the destructive limitations posed by chemical methods for epigenetic sequencing.

Keywords: 5-Hydroxymethylcytosine; 5-Methylcytosine; AID/APOBEC DNA deaminases; Bisulfite; DNA cytosine methyltransferases; Epigenetics; Glucosyltransferases; TET dioxygenases.

Figure 1

Bisulfite sequencing and its limitations. Bisulfite selectively deaminates various cytosines, which can aid in localizing modifications upon PCR amplification and sequencing. Problematically, sodium bisulfite is both destructive and unable to distinguish between the two most common modifications in mammalian genomes, 5mC and 5hmC.

Figure 2

βGT activity and applications in sequencing. A) Canonical βGT reaction. βGT catalyzes the transfer of glucose from UDP-glucose to the hydroxymethyl group of 5hmC, generating 5ghmC. The dotted orange circle highlights a position on glucose that can be derivatized to transfer chemical groups of interest, such as azides. B) Various applications of the βGT enzyme in sequencing workflows.

Figure 3

TET activity and applications in sequencing. A) Canonical TET reaction. TET enzymes iteratively oxidize 5mC to produce the oxidized methylcytosines 5hmC, 5fC, and 5caC. B) Various applications of TET enzymes in sequencing workflows, with traditional bisulfite sequencing and oxBS-Seq for comparison on the top rows.

Figure 4

AID/APOBEC activity and applications in sequencing. A) Canonical AID/APOBEC reaction. AID/APOBECs, including A3A, deaminate either cytosines to generate uracils or 5mCs to generate thymines. B) Various applications of the A3A enzyme in sequencing workflows, with traditional bisulfite sequencing for comparison on the top row.

Figure 5

MTase activity and applications in sequencing. A) Canonical MTase reaction. MTases catalyze the addition of a methyl group (from the methyl donor SAM) to cytosine. The dotted orange circle highlights the position on SAM that can be derivatized to transfer chemical groups of interest such as azides. B) Various applications of MTase enzymes in sequencing workflows, with traditional bisulfite sequencing for comparison in the top row of base resolution techniques.