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that CpG island DNA methylation at many genes is a late event in the process
of X inactivation (
Gendrel et al., 2012; Lock, Takagi, & Martin, 1987
). In
addition, mutant mouse embryos with defective DNAmethylation only show
modest effects on the silencing of X-linked genes (
Blewitt et al., 2008; Sado
et al., 2000; Sado, Okano, Li, & Sasaki, 2004
). Altogether, this suggests that
DNA methylation is not the initiating event in X-chromosome inactivation
but might be required to maintain long-term silencing of the inactive X copy.
4.2. Regulation of lineage-specific gene expression
The role of DNA methylation in orchestrating gene expression in the
embryo has been debated for a long time after initial experiments indicated
that CpG island promoters remain constitutively unmethylated irrespective
of gene expression. This dogma has been challenged by genome-scale map-
ping experiments that identified examples of nonimprinted genes with
methylated CpG-rich promoters in mouse and human somatic cells. Many
of these genes are pluripotency and germ line-specific genes that have con-
stitutively methylated promoters in somatic lineages (
Farthing et al., 2008;
Mohn et al., 2008; Shen et al., 2007; Weber et al., 2007
). These promoters
undergo
de novo
methylation at the time of implantation, and reduced meth-
ylation at these genes leads to incomplete gene silencing in the embryo
(
Borgel et al., 2010; Velasco et al., 2010
). Using ES cells as an experimental
system, it has been possible to dissect the process of
de novo
methylation at
pluripotency genes. One well-studied example is
Oct4
, a pluripotency
marker that has a promoter with moderate CpG richness. Interestingly
DNA methylation in this promoter does not initiate silencing in differenti-
ating ES cells but is a late event that follows histone deacetylation, recruit-
ment of the histone methyltransferase G9a, methylation of H3K9, and
binding of HP1 (
Feldman et al., 2006
). Once DNA methylation has
occurred, the
Oct4
gene can no longer be reversed to an active state in cul-
ture, suggesting that DNA methylation serves to stabilize the silent state
(
Fig. 2.5
;
Epsztejn-Litman et al., 2008; Feldman et al., 2006
). From these
experiments, one can postulate that DNA methylation has evolved as a sec-
ondary safeguarding event that prevents the potentially deleterious reac-
tivation of germ line and pluripotency genes in differentiated somatic
cells. An illustration of the role of DNA methylation in repressing
pluripotency is that changes in DNA methylation are necessary for the suc-
cessful reprogramming of differentiated cells into induced pluripotent stem
cells. In particular, reprogramming entails the erasure of promoter DNA
