Jeffrey Perkel has been a scientific writer and editor since 2000. He holds a PhD in Cell and Molecular Biology from the University of Pennsylvania, and did postdoctoral work at the University of Pennsylvania and at Harvard Medical School.
If the experience of the Epigenomics Core Facility at the University of Michigan Medical School is any indication, an awful lot of researchers are interested in DNA methylation.
According to lab manager Claudia Lalancette, the facility has been up and running since June 2015. In the first year, the facility processed 402 samples.
“There is a big demand to have services to help researchers, because the assays can be a bit tricky,” Lalancette says. “You need some expertise.”
DNA methylation—most commonly methyl marks on the 5-position of cytosine bases (5-methylcytosine, or 5-mC) in the context of CpG dinucleotides—suppresses gene expression, among other consequences.
As described in one recent review, 5-mC “has been shown to participate in genomic imprinting, X‐chromosome inactivation, transposon suppression, gene regulation and epigenetic memory maintenance” [1]. (Recent work indicates that the well-known prokaryotic methylation mark, N6-methyladenosine, also is present in eukaryotic DNA—which was the subject of the recent review [1].)
Naturally, researchers are keen to detect 5-mC and its derivatives, and tools providers have created a number of reagents and kits to assist scientists.
A core menu
At the Epigenetics Core Facility at Michigan, Lalancette says, most customers order one of a few services. The most common request is for ERRBS (enhanced reduced representation bisulfite sequencing), a method for detecting 5-mC with single-nucleotide resolution. Genomic DNA is fragmented with a restriction enzyme whose cut site is enriched in CpG islands and gene regulatory regions, and it is then resolved on a gel. By selecting fragments in the 150- to 400-base-pair range, users can sample most of the genome’s CpG-rich regions and thus maximize the efficiency of downstream sequencing.
Critically, the method includes bisulfite conversion, a process that converts nonmethylated cytosines to uracil while leaving methylated cytosines unchanged. By comparing bisulfite-treated and untreated sequencing reads, researchers can determine which bases were actually methylated. (Bisulfite conversion kits are widely available, for example from Active Motif, MilliporeSigma and Zymo Research. Zymo also offers a menu of epigenetic fee-for-service options, including whole-genome bisulfite sequencing and downstream bioinformatics.)
For those who require a more localized view of methylation status, Lalancette’s facility offers targeted MassARRAY sequencing. Here, methylated bases are detected following bisulfite conversion, PCR amplification and transcription to RNA based on size of cleavage products resulting from cleavage at uracil residues. The results are read out on a dedicated time-of-flight mass spectrometer, available from Agena Biosciences. “MassARRAY is seen as the gold standard for quantification of methylation,” according to Lalancette.
The other popular offering on the facility’s menu, she adds, is a method for determining the location of 5-hydroxymethylcytosine.
5-hmC is a variant of 5-mC that appears to have a distinct epigenetic function.
However, as bisulfite conversion cannot distinguish between the two marks, researchers have developed additional methods to do so. Lalancette’s facility, for instance, offers antibody-based enrichment of 5-hmC-containing DNA followed by DNA sequencing, a strategy that provides regional, but not nucleotide-level resolution akin to ChIP-seq data, she says.
Deeper look into modifications
Additional options include TAB-seq [2] and OxBS-seq [3], both of which treat the DNA prior to bisulfite conversion to alter its response to methylated and hydroxymethylated bases. In TAB-seq, 5-hmC is enzymatically protected via glucosylation with beta-glucosyltransferase, and 5-mC is converted to 5-carboxylcysteine with a Tet enzyme. Subsequent bisulfite conversion reads the 5-caC as T and 5-hmC as C. OxBS-seq uses chemical oxidation to convert 5-hmC to 5-formylcytosine, which reads as T after bisulfite conversion, whereas 5-mC reads as C. In both cases, comparing the resulting sequences with standard bisulfite sequencing reveals the modified bases.
Another method for distinguishing the two modifications uses Pacific Biosciences SMRT sequencing. SMRT sequencing can automatically distinguish cytosine from 5-mC and 5-hmC based on the kinetics of nucleotide incorporation, says Jonas Korlach, the company’s chief scientific officer—a parameter called the “interpulse duration.” But by enzymatically modifying 5-hmC either via glucosylation and subsequent biotinylation or by using diglucosylation, that IPD signal becomes far easier to see, he adds [4,5].
ELISA solutions
For researchers who do not need nucleotide-level resolution, Active Motif, MilliporeSigma and Zymo Research all offer ELISA assays for surveying global methylation changes, for instance following drug treatment. Active Motif uses biotinylated capture oligonucleotides complementary to repetitive LINE sequences (which are rich in CpG dinucleotides) to pull down a substantial fraction of the cell’s methylated regions. The pulled-down DNA is then affixed to microtiter plates, incubated with an anti-5-mC antibody and detected using a colorigenic reagent. “You can detect differences of 0.5% to 1%” between samples, says Active Motif product manager Kyle Hondorp.
MilliporeSigma offers ELISAs that can detect global levels of 5-mC or 5-hmC.
Other reagents available to researchers for examining methylated DNA include specially methylated DNA standards, enzymes, antibodies and more. Those looking to enrich methylated or hydroxymethylated DNA from genomic DNA samples, for instance, can use kits based on the so-called MeDIP/hMeDIP (methylated DNA immunoprecipitation) and MIRA (methylated CpG island recovery assay) formats. In the former assay, methylated DNA is pulled down using a 5-mC- or 5-hmC-directed antibody, which (in the case of 5-mC) requires single-stranded DNA; MIRA instead uses methyl-DNA-binding proteins, which recognize double-stranded DNA.
Active Motif’s MethylCollector™ Ultra kit, for instance, is based on MIRA, and its Hydroxymethyl Collector™ kits modify 5-hmC by glucosylation and biotinylation to effect purification. Zymo Research’s Quest 5-hmC™ DNA Enrichment Kit uses a J-base binding protein (JBP) to purify glucosylated 5-hmC residues directly.
And still more tools are coming. At least three research groups have reported independently coupling a dead form of the Cas9 enzyme used in genome editing to a DNA methyltransferase or a DNA demethylase to control methylation status and gene expression at specific genomic locations [6,7,8]; some of those reagents are available on Addgene. But given the continued excitement in the DNA methylation field, they surely won’t be the last of their type. Bottom line: Keep an eye on those product catalogs.
References
[1] Luo, G-Z, et al., “DNA N6-methyladenine: A new epigenetic mark in eukaryotes?” Nat Rev Mol Cell Biol, 16:705-10, 2015. [PMID: 26507168]
[2] Yu, M, et al., “Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine,” Nat Protocols, 7:2159-70, 2012. [PMID: 23196972]
[3] Booth, MJ, et al., “Quantitative sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution,” Science, 336:934-7, 2012. [PMID: 22539555]
[4] Song, C-X, et al., “Sensitive and specific single-molecule sequencing of 5-hydromethylcytosine,” Nat Methods, 2011. DOI:10.1038/nmeth.1779 [PMID: 22101853]
[5] Chavez, L, et al., “Simultaneous sequencing of oxidized methylcytosines produced by TET/JBP dioxygenases in Coprinopsis cinerea,” PNAS, E5154-8, November 18, 2014. [PMID: 25406324]
[6] Vojta, A, et al., “Repurposing the CRISPR-Cas9 system for targeted DNA methylation,” Nucleic Acids Research, 44:5615-28, 2016.[PMID: 26969735]
[7] McDonald, JI, et al., “Reprogrammable CRISPR/Cas9-based system for inducing site-specific DNA methylation,” Biology Open, 0:1-9, 2016. DOI:10.1242/bio.019067 [PMID: 27170255]
[8] Xu, X, et al., “A CRISPR-based approach for targeted DNA demethylation,” Cell Discovery, 2:16009, 2016. [PMID: 27462456]
Image: Shutterstock Images