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EPIGENETICS Dynamics of DNA demethylation

Locating the final oxidation products of prior method for detecting 5-hmC by tag- methylated by enrichment and ging it with biotin via an enzymatic conver- sequencing reveals that DNA demethyl- sion and ‘click’ chemistry step. In the new ation is common across the . approach, dubbed fC-Seal, 5-hmC sites are Until recently, methylation was viewed as enzymatically blocked, 5-fC is chemically a stable DNA mark, one that disappears only converted to 5-hmC, and only newly con- during dramatic episodes of global repro- verted 5-hmC sites are then tagged with gramming such as at fertilization. Work by biotin. Enriching DNA by biotin pulldown a few labs, including those of Yi Zhang of is efficient and is not biased by the density of Harvard and Chuan He of the University of tagged sites in the genome. Chicago, has changed that perception. The researchers discovered 5-fC in regula- “We now come to realize that DNA meth- tory regions and ‘poised’ enhancers bearing ylation is not static,” says He. Rather, much epigenetic marks that indicate they can be of the genome exists as “a dynamic equilib- rapidly activated. They also found that 5-fC rium of methylation and demethylation.” may coordinate gene activation by recruiting DNA methylation is linked to repressed gene the transcriptional activator p300. expression. The discovery that ten-eleven Challenges remain. Direct precipitation translocation (TET) proteins can oxidize by antibodies can suffer density-dependent methylated cytosine to 5-hydroxymethylcy- biases and fail when marks are sparse, where- tosine (5-hmC) prompted questions about as chemical conversions mitigate these prob- whether this effect can be actively reversed. lems but add steps that may introduce arti- Zhang’s group and others showed that the facts. Both methods offer limited resolution. TET proteins further oxidize 5-hmC to 5-for- To achieve single-base resolution, He’s mylcytosine (5-fC) and 5-carboxylcytosine team developed chemically assisted bisul- (5-caC). The low abundance of these marks fite sequencing (fCAB-seq), in which 5-fC makes profiling their location in the genome is found by comparing sequence from a difficult. Highly specific antibodies can be sample in which the mark is converted used to enrich genomic fragments bearing to uracil by bisulfite to another sample in cytosine modifications such as 5-hmC for which the mark is chemically protected sequencing, but its two derivatives are much from bisulfite. The rarity of 5-fC requires

© 2013 Nature America, Inc. All rights reserved. America, Inc. © 2013 Nature rarer. Nonetheless, Zhang and colleagues sur- very deep sequencing, which makes mounted the challenge using existing anti- genome-wide applications expensive, so He bodies that recognize 5-fC and 5-caC, and recommends fC-Seal for genomic profiling

npg they showed that the modified bases occur and fCAB-seq to follow up on interesting mainly in repeat regions in mouse embryonic loci. Another option described in He’s work stem cells (Shen et al., 2013). is to first enrich for 5-fC and then profile The researchers also used a clever form of with fCAB-seq. genetic enrichment. Thymine-DNA glyco- These studies show that methylation is sylase (TDG) replaces 5-fC and 5-caC with dynamic and widespread. Profiling demeth- unmodified cytosine through base excision ylation intermediates provides rich informa- repair, an event suggestive of what Zhang tion about gene regulation and reveals that calls a methylation-demethylation cycle run derepression serves as an activating mecha- by DNA methylase, TET and TDG proteins. nism—as He notes, many regions annotated “We found the best way to demonstrate that as ‘unmethylated’ are actually ‘constantly this hypothesis is correct is to block one of demethylated’. To explore deeper, economi- the steps to prevent the cycling,” he says. In cal methods are needed to profile 5-fC and cells depleted of TDG, the accumulation of 5-caC genome wide at single-base resolution 5-fC and 5-caC painted a dynamic picture of from limited starting material. demethylation in nonrepetitive and regula- Tal Nawy tory elements, suggesting a role for active RESEARCH PAPERS cycling in regulating gene expression. Shen, L. et al. Genome-wide analysis reveals TET- Similar discoveries came from He, who and TDG-dependent 5-methylcytosine oxidation dynamics. Cell 153, 692–706 (2013). worked with Peng Jin of Emory, and their Song, C.-X. et al. Genome-wide profiling of colleagues (Song et al., 2013). The selective 5-formylcytosine reveals its roles in epigenetic labeling approach they took builds on their priming. Cell 153, 678–691 (2013).

466 | VOL.10 NO.6 | JUNE 2013 | nature methods