RESEARCH HIGHLIGHTS EPIGENETICS The sixth base and counting Enzymatic glycosylation and oxidation C mC hmC NH NH OH NH of methylated DNA together with high- 2 2 2 H3C throughput sequencing allows genome- N N N N O N O N O wide base-resolution of 5-hydroxymethyl H H H cytosine distribution. 5-Hydroxymethyl cytosine (5-hmC) is O NH2 O NH2 increasingly being recognized as a base in its own right rather than an intermediate HO N H N N O N O of methyl cytosine (5-mC) on its way to H H demethylation. 5-hmC, touted as the sixth caC fC base, is produced by ten-eleven translocation Enzymatic steps of cytosine methylation and (TET) enzymes as they oxidize 5-mC. demethylation. Whereas the role of 5-mC in epigenetic regulation is undisputed, 5-hmC’s func- still being read as Cs, thus providing a direct tion is less clear, but it putatively includes readout of their positions. the regulation of gene expression and DNA He and his colleagues Bing Ren at the demethylation, particularly in embryonic University of California, San Diego, and stem cells and mammalian brain cells, where Peng Jin at Emory University found almost 5-hmC levels are high. half of all abundant 5-hmC sites to be in To understand what 5-hmC is doing, one distal regulatory regions, areas with low needs to know where it is. In the last 3 years, overall methylation levels. The researchers researchers have developed various tools and also found 5-hmC to be enriched around methods to enrich 5-hmC–bearing regions. transcription factor (TF) binding sites but In 2010, Chuang He from the University depleted in the binding motif itself. When of Chicago used glycosyltransferase to tag asked what this might say about the func- 5-hmC with an azido glucose analog to tion of 5-hmC, He is quick to point out that which he appended biotin via click chemis- these findings need further scrutiny, but he try, making for an easy pulldown. nonetheless speculates, “It is my sense that All of these enrichment methods led to specific complexes, including the TF, may valuable insights about the distribution of recruit TET1 proteins, which demethylate © 2012 Nature America, Inc. All rights reserved. America, Inc. © 2012 Nature 5-hmC, but, says He, they all have a substan- the neighborhood [and may thereby influ- tial drawback in their inability to assess abun- ence gene expression]. This agrees very well dance. “A genome-wide look at relative abun- with the correlation of 5-hmC with regula- npg dance is very important to get a real view of tory elements.” the biological function of 5-hmC,” he says. What is clear is that the distribution of Such a genome-wide look is only possible 5-hmC in human embryonic stem cells is through sequencing. But although sequenc- not random, which would be expected if it ing of 5-mC is done by bisulfite conversion were simply a byproduct of demethylation, of unmethylated Cs to Us, bisulfite treatment but instead is generated and maintained at does not distinguish between 5-mC and specific loci involved in the regulation of 5-hmC, neither is converted to U and both gene expression. are read as C in bisulfite sequencing. Thus And if 5-hmC has specific roles, this raises He needed a method to distinguish the fifth the question of whether the downstream oxi- from the sixth base. dation products 5-fC and 5-caC do as well. Is His team revisited the glucose-tagging there a ‘methylation code’, a carefully orches- strategy and found that glycosyl-5-hmC is trated combination of 5-mC, 5-hmC, 5-fC protected from further oxidation, whereas and 5-caC, to fine tune cellular function? unprotected 5-hmC gets oxidized to form- Methods specifically geared toward exam- ylcytosine (fC) and then to carboxylcytosine ining each of these nucleotides are needed (caC). After protecting 5-hmC in human to answer this question. embryonic stem cells, the researchers sim- Nicole Rusk ply let the TET enzyme do its job overnight RESEARCH PAPERS of converting most 5-mCs to caCs, which are Yu, M. et al. Base-resolution analysis of read as Ts after bisulfite conversion. Glucose- 5-hydroxymethylcytosine in the mammalian genome. protected 5-hmCs, on the other hand, were Cell 149, 1368–1380 (2012). 646 | VOL.9 NO.7 | JULY 2012 | NATURE METHODS.