Functions of DNA Methylation and Hydroxymethylation in Mammalian Development - Current Topics in Developmental Biology - page 57

possibility is supported by the fact that nucleosome insertion precedes DNA
methylation at the OCT4 and NANOG loci during cellular differentiation
( You et al., 2011 ).
3.2. Distribution of 5hmC in mammalian genomes
In 2009, two groundbreaking studies discovered that TET proteins convert
5mC into 5hmC and that 5hmC is abundant in ES cells and Purkinje neurons
( Kriaucionis & Heintz, 2009; Tahiliani et al., 2009 ). 5hmC was estimated to
account for 0.59% and 0.03% of all nucleotides in Purkinje neurons and
mouse ES cells, respectively (in comparison 5mC constitutes around 1% of
all nucleotides). Other estimates confirmed that 5hmC levels are around 10
times higher in the mouse brain compared to ES cells ( Song, Szulwach,
et al., 2011 ). Intriguingly, in contrast to 5mC levels that are relatively equal
between cell types, the amount of 5hmC in DNA is variable between cell
types and generally below the one observed in ES cells and the nervous system
( Globisch et al., 2010; Nestor et al., 2012; Ruzov et al., 2011; Szwagierczak,
Bultmann, Schmidt, Spada, & Leonhardt, 2010 ). The observation that 5hmC
is highest in the brain suggests a link with active DNA demethylation because
most adult neurons do not divide, and therefore, any demethylation of DNA
would require a replication-independent process.
After this discovery, efforts have been concentrated on studying the dis-
tribution of 5hmC in the genome. Because standard approaches such as
sodium bisulfite sequencing or methylation-sensitive restriction enzymes can-
not discriminate 5hmC from 5mC, novel experimental strategies have been
developed, which are detailed in some recent reviews ( Wu & Zhang, 2011 ).
Using affinity purification, several groups reported that 5hmC in ES cells is
present in gene bodies, transcription start sites (TSS), and cis -regulatory ele-
ments, albeit with no information on the absolute amounts ( Ficz et al.,
2011; Pastor et al., 2011; Song, Szulwach, et al., 2011; Wu, D'Alessio, Ito,
Wang, et al., 2011 ). 5hmC was also profiled by affinity purification in the
brain and found to be enriched in gene bodies but to a lesser extent at TSS
( Jin, Wu, Li, & Pfeifer, 2011; Szulwach, Li, Li, Song, Wu, et al., 2011 ).
Recently, two modified bisulfite sequencing protocols have been developed
to generate for the first time single-base resolution maps of the absolute abun-
dance of 5hmC in mammalian cells ( Booth et al., 2012; Yu et al., 2012 ). One
of these studies generated the first genome-wide maps of 5hmC in mouse and
human ES cells, which revealed that 5hmC occurs nearly exclusively in CpGs
( Yu et al., 2012 ). Interestingly, 5hmC is most enriched at CpG-poor distal-
regulatory regions corresponding to enhancers, CTCF-binding sites or