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Furthermore, a recent study suggested that the kinetics of methylation loss in
mouse primordial germ cells between E10.5 and E13.5 is slow enough as to
not require a requisite active demethylation process ( Kagiwada et al., 2013 ).
Rather, a passive or replication-dependent loss of genomic methylation
would suffice.
5.3. Active versus passive demethylation
The aforementioned discussion leads us to consider alternatives to active
demethylation processes in mammals. With the known exceptions of pachy-
tene spermatocytes and pronuclear-stage embryos, DNMT1 appears to be a
ubiquitously expressed nuclear protein, including in all other stages of pre-
implantation development and in primordial germ cells. Therefore, a mech-
anism of passive demethylation (loss of methylation due to lack of
maintenance methylation following DNA replication) would require sup-
pression of nuclear DNMT1 catalytic activity. In principle, this process
would be very pronounced in primordial germ cells and preimplantation-
stage embryos, where levels of genomic methylation are very much less than
those found in adult somatic cells. In fact, the enduring expression of
DNMT1 in nuclei of cells undergoing demethylation raises the distinct pos-
sibility that regulation of DNMT1 activity is a key feature of a passive
demethylation process. Viewed from another perspective, if passive demeth-
ylation is indeed responsible for the significant decline in genomic methyl-
ation during preimplantation development, the DNMT1-dependent
maintenance of genomic imprints in this developmental window indicates
that DNMT1 enzymatic activity must be highly regulated, not simply
completely inactive.
5.4. Regulating DNMT1 and demethylation
Recent evidence suggests that DNA methyltransferases (including
DNMT1, DNMT3A, and DNMT3B) possess demethylase activity
( Chen et al., 2013 ). Incubation of methylated DNA constructs that were
incubated with catalytically active methyltransferases resulted in
30%
reduction in methylation levels. The demethylase activity required the pres-
ence of Ca 2 รพ ions and was inhibited in the presence of reducing agents such
as dithiothreitol. Interestingly, amino acid substitutions in the known cata-
lytic (methyltransferase) domain also caused a significant reduction in the
demethylase activity of all three methyltransferases. This raises the possibility
that DNMTs mediate the reversible reactions of methylation and
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