Genetic Studies on Mammalian DNA Methyltransferases

Abstract

Cytosine methylation at the C5-position, generating 5-methylcytosine (5mC), is a DNA modification found in many eukaryotic organisms, including fungi, plants, invertebrates, and vertebrates, albeit its levels vary greatly in different organisms. In mammals, cytosine methylation occurs predominantly in the context of CpG dinucleotides, with the majority (60–80 %) of CpG sites in their genomes being methylated. DNA methylation plays crucial roles in the regulation of chromatin structure and gene expression and is essential for mammalian development. Aberrant changes in DNA methylation levels and patterns are associated with various human diseases, including cancer and developmental disorders. DNA methylation is mediated by three active DNA methyltransferases (Dnmts), namely, Dnmt1, Dnmt3a, and Dnmt3b, in mammals. Over the last two decades, genetic manipulations of these enzymes, as well as their regulators, in mice have greatly contributed to our understanding of the biological functions of DNA methylation in mammals. In this chapter, we discuss genetic studies on mammalian Dnmts, focusing on their roles in embryogenesis, cellular differentiation, genomic imprinting, and X-chromosome inactivation.

Full text

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© Springer International Publishing Switzerland 2016
A. Jeltsch, R.Z. Jurkowska (eds.), DNA Methyltransferases - Role and Function,
Advances in Experimental Medicine and Biology 945,
DOI 10.1007/978-3-319-43624-1_6
J. Dan
Department of Epigenetics and Molecular Carcinogenesis , The University of Texas MD
Anderson Cancer Center , 1808 Park Road 1C , Smithville , TX 78957 , USA
Center for Cancer Epigenetics , The University of Texas MD Anderson Cancer Center ,
1808 Park Road 1C , Smithville , TX 78957 , USA
T. Chen (*)
Department of Epigenetics and Molecular Carcinogenesis , The University of Texas MD
Anderson Cancer Center , 1808 Park Road 1C , Smithville , TX 78957 , USA
Center for Cancer Epigenetics , The University of Texas MD Anderson Cancer Center ,
1808 Park Road 1C , Smithville , TX 78957 , USA
Graduate School of Biomedical Sciences at Houston , Houston , TX 77030 , USA
e-mail: tchen2@mdanderson.org
Genetic Studies on Mammalian DNA
Methyltransferases
Jiameng Dan and Taiping Chen
Abstract
Cytosine methylation at the C5-position, generating 5-methylcytosine (5mC), is
a DNA modifi cation found in many eukaryotic organisms, including fungi,
plants, invertebrates, and vertebrates, albeit its levels vary greatly in different
organisms. In mammals, cytosine methylation occurs predominantly in the con-
text of CpG dinucleotides, with the majority (60–80 %) of CpG sites in their
genomes being methylated. DNA methylation plays crucial roles in the regula-
tion of chromatin structure and gene expression and is essential for mammalian
development. Aberrant changes in DNA methylation levels and patterns are asso-
ciated with various human diseases, including cancer and developmental disor-
ders. DNA methylation is mediated by three active DNA methyltransferases
(Dnmts), namely, Dnmt1, Dnmt3a, and Dnmt3b, in mammals. Over the last two
decades, genetic manipulations of these enzymes, as well as their regulators, in
mice have greatly contributed to our understanding of the biological functions of
DNA methylation in mammals. In this chapter, we discuss genetic studies on

124
mammalian Dnmts, focusing on their roles in embryogenesis, cellular differen-
tiation, genomic imprinting, and X-chromosome inactivation.
Abbreviations
5caC 5-Carboxylcytosine
5fC 5-Formylcytosine
5hmC 5-Hydroxymethylcytosine
5mC 5-Methylcytosine
ADCA-DN Autosomal dominant cerebellar ataxia deafness and narcolepsy
ADD ATRX-Dnmt3-Dnmt3L
AML Acute myeloid leukemia
BAH Bromo-adjacent homology
DKO Double knockout
DMR Differentially methylated region
DNMT DNA methyltransferase
ES Embryonic stem
EST Expressed sequence tag
HP1 Heterochromatin protein 1
HSAN IE Hereditary sensory and autonomic neuropathy with dementia and
hearing loss type IE
ICF Immunodefi ciency centromeric instability and facial anomalies
ICM Inner cell mass
ICR Imprinting control region
KAP1 KRAB-associated protein 1
KRAB Krüppel -associated box
lncRNA Long non-coding RNA
MBD3 Methyl CpG-binding domain protein-3
MEF Mouse embryonic fi broblast
MTA2 Metastasis tumor antigen 2
NLS Nuclear localization signal
NuRD Nuclear remodeling and histone deacetylation
PBD PCNA-binding domain
PCNA Proliferating cell nuclear antigen
PGC Primordial germ cell
PHD Plant homeodomain
PRC2 Polycomb repressive complex 2
PWWP Proline-tryptophan-tryptophan-proline
RFTS Replication foci-targeting sequence
RING Really Interesting New Gene
SRA SET- and RING-associated
TDG Thymine DNA glycosylase
TTD Tandem tudor domain
UBL Ubiquitin-like
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Uhrf1 Ubiquitin-like with PHD and RING fi nger domains 1
Xa Active X chromosome
XCI X-chromosome inactivation
Xi Inactive X chromosome
Xic X-inactivation center
Xist X-inactive-specifi c transcript
Xm Maternal X chromosome
Xp Paternal X chromosome
1 Distinct Roles of Dnmt1 and Dnmt3 Families
in DNA Methylation
In 1975, long before the identifi cation of any mammalian DNA methyltransferase,
Holliday and Pugh and Riggs independently proposed a theory that DNA methyla-
tion could serve as a heritable epigenetic mark for cellular memory. Recognizing that
the CpG dinucleotide is self-complementary, they postulated that methylated and
unmethylated CpG sites could be copied when cells divide so that DNA methylation
patterns would be replicated semiconservatively like the base sequence of DNA itself
(Holliday and Pugh 1975 ; Riggs 1975 ). A prediction of the theory was the existence
of at least two DNA methyltransferase activities: de novo methyltransferase(s) would
methylate unmodifi ed DNA and establish DNA methylation patterns, and mainte-
nance methyltransferase(s) would recognize hemimethylated sites and “copy” the
methylation patterns from the parental strand onto the daughter strand at each round
of DNA replication.
1.1 Dnmt1: The Major Maintenance Methyltransferase
The fi rst mammalian DNA methyltransferase gene, Dnmt1 , was cloned from murine
cells (Bestor et al. 1988 ). The Dnmt1 locus has several transcription start sites and
produces two major protein products (Mertineit et al. 1998 ; Rouleau et al. 1992 ).
Transcription initiation within a somatic cell-specifi c exon (exon 1 s) results in the
Dnmt1s isoform (generally referred to as Dnmt1) which consists of 1620 amino
acids. Transcription initiation within an oocyte-specifi c exon (exon 1o) produces a
transcript that utilizes a downstream AUG as the translation initiation codon. As a
result, the oocyte-specifi c isoform, Dnmt1o, lacks the N-terminal 118 amino acids
of Dnmt1s (Mertineit et al. 1998 ). Although Dnmt1o is more stable than Dnmt1s,
genetic evidence suggests no functional difference between these isoforms (Ding
and Chaillet 2002 ). Human DNMT1, consisting of 1616 amino acids, is nearly 80 %
identical to the mouse Dnmt1 at the amino acid level.
Dnmt1 contains a C-terminal catalytic domain containing characteristic amino
acid sequence motifs that are homologous to bacterial DNA methyltransferases and
an N-terminal regulatory region that is not present in bacterial enzymes (Bestor
Genetic Studies on Mammalian DNA Methyltransferases

126
et al. 1988 ). The N-terminal regulatory region contains several functional domains,
including a proliferating cell nuclear antigen (PCNA)-binding domain (PBD)
responsible for the interaction with the DNA replication machinery, a nuclear local-
ization signal (NLS), a replication foci-targeting sequence (RFTS) that mediates the
association with late replicating heterochromatin, a zinc-fi nger CXXC domain that
recognizes unmethylated CpG-containing DNA, and a pair of bromo-adjacent
homology (BAH) domains (Fig. 1a ). Recent structural data revealed that the RFTS
domain binds to the catalytic domain and blocks the catalytic center, suggesting an
autoinhibitory role in the regulation of enzymatic activity (Takeshita et al. 2011 ).
In vitro biochemical assays revealed that, although Dnmt1 is capable of methyl-
ating both unmethylated and hemimethylated CpG dinucleotides, its activity toward
hemimethylated substrates is far more effi cient (Pradhan et al. 1999 ). Dnmt1 is
ubiquitously expressed through development, with high levels in proliferating cells.
Dnmt1 associates with the DNA replication machinery at S phase and with hetero-
chromatin at late S and G2 phases (Chuang et al. 1997 ; Easwaran et al. 2004 ;
Leonhardt et al. 1992 ; Schneider et al. 2013 ), suggesting that Dnmt1-mediated
methylation is coupled to DNA replication. These fi ndings supported the notion that
Dnmt1 functions as a maintenance enzyme (Fig. 1b ). However, because Dnmt1, the
only known DNA methyltransferase at the time, also has de novo methylation activ-
ity in vitro , it was initially debated whether de novo methylation and maintenance
methylation are carried out by Dnmt1 alone or by two or more distinct enzymes
(Bestor 1992 ).
Genetic studies in mouse models and murine cells helped settling the debate.
Several Dnmt1 mutant alleles were generated by gene targeting. The Dnmt1 n
allele
(n stands for N-terminal disruption) was reported in 1992 (Li et al. 1992 ). This
allele, in which a genomic region coding 60 amino acids near the N-terminal end
was replaced by a neomycin resistance cassette, is a partial loss-of-function (hypo-
morphic) mutation. Dnmt1 n / n
embryos have a ~70 % reduction in global DNA meth-
ylation and show mid-gestation lethality (Li et al. 1992 ). Subsequently, the Dnmt1 s
allele (s stands for Sal I site) was reported, which had a neomycin resistance cassette
inserted into a Sal I site in exon 17, disrupting the RFTS (Li et al. 1993 ). The Dnmt1 s
allele is functionally more severe than the Dnmt1 n
allele, as Dnmt1 s / s
embryos show
lower levels of DNA methylation and earlier lethality (Lei et al. 1996 ). However, it
was unclear whether the Dnmt1 s
allele was a null mutation, because the C-terminal
catalytic domain was intact. To completely inactivate Dnmt1 , Lei et al. generated
the Dnmt1 c
allele (c stands for C-terminal disruption) by disrupting the catalytic
domain, including the highly conserved PC and ENV motifs that are essential for
enzymatic activity (Lei et al. 1996 ). The development of Dnmt1 c / c
embryos is
arrested prior to the 8-somite stage, signifi cantly earlier than the developmental
phenotype of Dnmt1 n / n
embryos, while the viability and proliferation of Dnmt1 null
embryonic stem (ES) cells are not affected (Lei et al. 1996 ). Inactivation of Dnmt1
by mutating the cysteine (C1229) residue at the catalytic center (PC motif) results
in similar developmental defects (Takebayashi et al. 2007 ), suggesting that the phe-
notype is largely due to the loss of catalytic activity. DNA methylation analyses
revealed that Dnmt1 null embryos and ES cells contain low but stable levels of
J. Dan and T. Chen

127
F i g . 1 DNA methyltransferases and major regulatory proteins involved in DNA methylation. ( a )
Schematic diagrams of Dnmt1, Dnmt3a, Dnmt3b, Dnmt3L, and Uhrf1. The C-terminal catalytic
domains of the Dnmt1 and Dnmt3 families are conserved (the highly conserved signature motifs I,
IV, VI, IX, and X are shown), but their N-terminal regulatory regions are distinct. Functional
domains of the proteins are indicated. PBD PCNA-binding domain, NLS nuclear localization sig-
nal, RFTS replication foci-targeting sequence, CXXC a cysteine-rich domain implicated in binding
CpG-containing DNA sequences, BAH bromo-adjacent homology domain, PWWP proline-
tryptophan- tryptophan-proline domain, ADD ATRX-Dnmt3-Dnmt3L domain, and UBL ubiquitin-
like domain; TTD tandem tudor domain, PHD plant homeodomain, SRA SET- and RING-associated
domain, and RING Really Interesting New Gene domain. ( b ) De novo and maintenance methyl-
transferase activities. The de novo methyltransferases Dnmt3a and Dnmt3b, in complex with their
accessory factor Dnmt3L, methylate unmodifi ed DNA and establish methylation patterns. At each
round of DNA replication, the maintenance methyltransferase Dnmt1, aided by its accessory factor
Uhrf1, “copies” the methylation pattern from the parental strand onto the daughter strand. Open
circles represent unmethylated CpG dinucleotides, and fi lled circles represent methylated CpG
dinucleotides
Genetic Studies on Mammalian DNA Methyltransferases

128
5-methylcytosine (5mC) and methyltransferase activity. Moreover, the de novo
methylation activity is not impaired by Dnmt1 loss, as integrated provirus DNA in
MoMuLV-infected Dnmt1 null ES cells becomes methylated at a similar rate as in
wild-type ES cells (Lei et al. 1996 ). Taken together, these studies provided compel-
ling evidence for the existence of one or more DNA methyltransferases that are
important for de novo methylation.
1.2 Dnmt2/Trdmt1: A tRNA Methyltransferase
Results from genetic studies of Dnmt1 prompted the search for more DNA meth-
yltransferase genes. In 1998, several groups reported the identifi cation of a second
putative DNA methyltransferase gene, named Dnmt2 , which encodes a protein of
391 amino acids in human or 415 amino acids in mouse (Okano et al. 1998b ; Van
den Wyngaert et al. 1998 ; Yoder and Bestor 1998 ). Despite the presence of all the
conserved motifs shared by known prokaryotic and eukaryotic DNA cytosine
methyltransferases, Dnmt2 has no detectable DNA methyltransferase activity in
standard in vitro assays. Furthermore, inactivation of Dnmt2 in mouse ES cells by
gene targeting has no effect on preexisting genomic methylation patterns or on the
ability to methylate newly integrated retrovirus DNA de novo (Okano et al.
1998b ). Indeed, a subsequent study demonstrated that Dnmt2 is a tRNA methyl-
transferase, specifi c for cytosine 38 in the anticodon loop of aspartic acid tRNA,
and has been renamed tRNA aspartic acid (D) methyltransferase 1 (Trdmt1) (Goll
et al. 2006 ).
1.3 Dnmt3a and Dnmt3b: The De Novo Methyltransferases
By searching an expressed sequence tag (EST) database using full-length bacterial
type II cytosine-C5 methyltransferase sequences as queries, Okano et al . identifi ed
two additional homologous genes, Dnmt3a and Dnmt3b , in both mouse and human.
Their protein products contain the highly conserved DNA methyltransferase motifs
in their C-terminal regions, but their N-terminal regulatory regions are unrelated to
that of Dnmt1 (Okano et al. 1998a ). The N-terminal regions of Dnmt3a and Dnmt3b
contain a variable region and two conserved domains, the proline-tryptophan-
tryptophan- proline (PWWP) domain and the ATRX-Dnmt3-Dnmt3L (ADD)
domain (Fig. 1a ). Both domains are implicated in chromatin binding. The PWWP
domain is required for heterochromatin localization and mediates H3K36me3 bind-
ing (Baubec et al. 2015 ; Chen et al. 2004 ; Dhayalan et al. 2010 ). The ADD domain
interacts with the N-terminal tail of histone H3, and the interaction is disrupted by
various posttranslational modifi cations of H3, including di- and trimethylation of
K4, acetylation of K4, and phosphorylation of T3, S10, or T11 (Otani et al. 2009 ;
Noh et al. 2015 ; Zhang et al. 2010 ).
Dnmt3a produces two major isoforms, Dnmt3a and Dnmt3a2, driven by differ-
ent promoters (Chen et al. 2002 ). The full-length Dnmt3a protein, consisting of
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129
908 amino acids in mouse and 912 amino acids in human, is expressed ubiqui-
tously at relatively low levels. The Dnmt3a2 transcript is initiated in intron 6 of the
Dnmt3a gene and encodes a protein that lacks the N-terminal 219 (in mouse) or
223 (in human) amino acids of Dnmt3a. Dnmt3a2, which is catalytically active, is
the predominant form in mouse ES cells, early embryos, and developing germ
cells, as well as human embryonal carcinoma cells, and is also detectable in spleen
and thymus (Chen et al. 2002 ). The Dnmt3b gene produces multiple alternatively
spliced isoforms, some of which encode catalytically inactive protein products.
The longest isoform, Dnmt3b1, consists of 859 amino acids in mouse and 853
amino acids in human, respectively. Both active and inactive Dnmt3b isoforms
appear to co-express in most, if not all, cell types. For example, Dnmt3b1, an active
form, and Dnmt3b6, an inactive form, are the predominant forms in mouse ES
cells, whereas Dnmt3b2, an active form, and Dnmt3b3, an inactive form, are
expressed at low levels in many somatic cells (Chen et al. 2002 ). There is evidence
that catalytically inactive Dnmt3b protein products may play regulatory roles in
DNA methylation. For example, overexpression of human DNMT3B7, a truncated
isoform frequently found in cancer cells, leads to higher levels of total genomic
methylation and altered gene expression in both transgenic mice and human cancer
cells (Ostler et al. 2012 ; Shah et al. 2010 ).
Several lines of evidence suggest the involvement of Dnmt3a and Dnmt3b in de
novo DNA methylation (Fig. 1b ). First, Dnmt3a and Dnmt3b are highly expressed
in early embryos (and ES cells) and developing germ cells, where an active de novo
methylation takes place, but are downregulated in somatic tissues and when ES
cells are induced to differentiate (Okano et al. 1998a ). Second, recombinant
Dnmt3a and Dnmt3b proteins methylate unmethylated and hemimethylated DNA
with equal effi ciency (Okano et al. 1998a ). Genetic studies provided defi nitive
evidence that Dnmt3a and Dnmt3b were the long-sought de novo methyltransfer-
ases. Inactivation of both Dnmt3a and Dnmt3b by gene targeting blocks de novo
methylation in ES cells and early embryos but has no effect on maintenance of
imprinted methylation patterns (Okano et al. 1999 ). Dnmt3a defi ciency also leads
to failure to establish DNA methylation imprints in developing germ cells (Kaneda
et al. 2004 ).
It is worth noting that the de novo DNA methyltransferase activity of Dnmt3a
and Dnmt3b is not only essential for the establishment of new DNA methylation
patterns but also important for the faithful maintenance of these patterns. In cul-
ture, Dnmt3a / 3b double knockout (DKO) ES cells exhibit gradual loss of global
DNA methylation and, after multiple passages, show severe hypomethylation
(Chen et al. 2003 ), suggesting that Dnmt1 and Dnmt3 enzymes have distinct and
nonredundant functions but act cooperatively in the maintenance of global DNA
methylation. Based on the kinetics of DNA methylation loss, it was proposed that
Dnmt1 is the major maintenance methyltransferase that, upon DNA replication,
methylates hemimethylated CpG sites with high effi ciency but not absolute fi del-
ity, whereas Dnmt3a and Dnmt3b, as de novo methyltransferases, act as “proof-
reading” enzymes that methylate the hemimethylated CpG sites missed by Dnmt1
(Chen et al.
2003 ).
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130
1.4 Dnmt3L: A Regulator of De Novo Methylation
A third member of the Dnmt3 family, Dnmt3 -like ( Dnmt3L ), was originally isolated
by database analysis of the human genome sequence (Aapola et al. 2000 ). Its murine
homolog was subsequently identifi ed (Aapola et al. 2001 ; Hata et al. 2002 ). The
human and mouse Dnmt3L proteins consist of 387 and 421 amino acids, respec-
tively. Dnmt3L contains an ADD domain, but lacks a PWWP domain, in the
N-terminal region. Its C-terminal region is highly related to the catalytic domains of
Dnmt3a and Dnmt3b, but lacks some motifs essential for enzymatic activity, includ-
ing the PC dipeptide at the active site and the sequence motif involved in binding of
the methyl donor S -adenosyl-L-methionine (Aapola et al. 2000 , 2001 ; Hata et al.
2002 ) (Fig. 1a ). Therefore, Dnmt3L has no methyltransferase activity. However,
Dnmt3L has been shown to interact with Dnmt3a and Dnmt3b, stimulate their enzy-
matic activities, and target them to chromatin (Hata et al. 2002 ; Jia et al. 2007 ; Ooi
et al. 2007 ; Suetake et al. 2004 ). The expression pattern of Dnmt3L during develop-
ment is also strikingly similar to that of Dnmt3a and Dnmt3b, including high expres-
sion in developing germ cells, early embryos, and ES cells (Hata et al. 2002 ). These
fi ndings indicate that Dnmt3L may regulate Dnmt3a/3b functions (Fig. 1b ). Genetic
studies indeed demonstrate that Dnmt3L is an essential accessory factor of Dnmt3a
in the germ line. Dnmt3L homozygous null mice are viable and grossly normal, but
both male and female mice are infertile (Bourc’his et al. 2001 ; Hata et al. 2002 ).
Male mice show activation of retrotransposons in spermatogonia and spermato-
cytes, due to failure to establish methylation at these elements, and are azoospermic
(Bourc’his and Bestor 2004 ). Female mice fail to establish maternal methylation
imprints in oocytes, and, as a result, embryos derived from these oocytes cannot
survive beyond mid-gestation (Bourc’his et al. 2001 ; Hata et al. 2002 ). The pheno-
type is indistinguishable from that of mice with conditional Dnmt3a deletion in
germ cells (Kaneda et al. 2004 ). Recently, Dnmt3L was shown to antagonize DNA
methylation at H3K4me3/K27me3 bivalent promoters, which are often associated
with developmental genes, and favor DNA methylation at gene bodies in ES cells.
It was suggested that Dnmt3L, via its ADD domain, interacts with Polycomb repres-
sive complex 2 (PRC2) in competition with Dnmt3a and Dnmt3b to maintain low
methylation levels at regions with H3K27me3, thus maintaining hypomethylation at
promoters of bivalent developmental genes (Neri et al. 2013 ). The physiological
relevance of this fi nding remains to be determined, given that zygotic Dnmt3L is not
required for embryonic development and postnatal survival (Bourc’his et al. 2001 ;
Hata et al. 2002 ).
1.5 Uhrf1: A Regulator of Maintenance Methylation
Besides Dnmts, a number of DNA methylation regulators have been identifi ed,
including the multi-domain protein Uhrf1 (ubiquitin-like with PHD and RING fi n-
ger domains 1), also known as NP95 (mouse) and ICBP90 (human) (Fig. 1a ).
Genetic studies demonstrated an essential role for Uhrf1 in maintaining DNA
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methylation (Fig. 1b ). Uhrf1 defi ciency leads to embryonic lethality and global
DNA hypomethylation (Bostick et al. 2007 ; Muto et al. 2002 ; Sharif et al. 2007 ),
resembling the phenotype of Dnmt1 defi ciency. Cellular and biochemical evidence
suggested functional interactions between Uhrf1 and Dnmt1. Uhrf1 co-localizes
with Dnmt1 at DNA replication foci and heterochromatin, and Dnmt1 fails to enrich
at these regions in the absence of Uhrf1 (Bostick et al. 2007 ; Liu et al. 2013 ; Sharif
et al. 2007 ). These fi ndings suggest that Uhrf1 is a key accessory factor for directing
Dnmt1 to hemimethylated CpG sites. However, it remains somewhat controversial
as to whether Uhrf1 directly recruits Dnmt1 or indirectly controls Dnmt1 localiza-
tion by affecting chromatin structure. Uhrf1 harbors fi ve known functional domains:
a ubiquitin-like domain (UBL) at the N-terminus, followed by a tandem tudor
domain (TTD), a plant homeodomain (PHD), a SET- and RING-associated (SRA)
domain, and a Really Interesting New Gene (RING) domain (Fig. 1a ). All the
domains, with the exception of UBL, have been shown to be important for Dnmt1
subnuclear localization and maintenance of DNA methylation. Biochemical and
structural evidence revealed that the SRA domain preferentially binds hemimethyl-
ated DNA and is thought to play an important role in loading Dnmt1 onto newly
synthesized DNA substrates (Arita et al. 2008 ; Avvakumov et al. 2008 ; Bostick
et al. 2007 ; Hashimoto et al. 2008 ; Sharif et al. 2007 ). The association of Uhrf1 with
heterochromatin is also mediated by TTD, which contains an aromatic cage for
binding of the heterochromatic H3K9me3 mark. The PHD acts in combination with
TTD to read the H3K9me3 mark and, additionally, interacts with histone H3 tails
with unmethylated R2 (H3R2me0) (Cheng et al. 2013 ; Liu et al. 2013 ; Rothbart
et al. 2012 , 2013 ; Rottach et al. 2010 ). Recent studies suggested that Uhrf1, via the
E3 ligase activity of its RING domain, mediates ubiquitylation of H3K23 and
H3K18, creating binding sites for Dnmt1 (Nishiyama et al. 2013 ; Qin et al. 2015 ).
It is worth noting that Uhrf1 has also been shown to control Dnmt1 ubiquitylation
and stability (Du et al. 2010 ; Qin et al. 2011 ). Indeed, a recent study revealed that
Uhrf1 overexpression results in DNA hypomethylation, due to destabilization and
delocalization of Dnmt1, which led the authors to propose that Uhrf1 overexpres-
sion, which is frequently observed in cancer cells, is a mechanism underlying global
DNA hypomethylation in cancer (Mudbhary et al. 2014 ).
2 Dnmts in Embryonic Development and Cellular
Differentiation
2.1 Dynamic Changes of DNA Methylation During Early
Embryogenesis
DNA methylation is relatively stable in somatic tissues but exhibits dynamic
changes in early embryos. During preimplantation development, both the maternal
and paternal genomes undergo global DNA demethylation, albeit the mechanisms
involved are distinct. The maternal genome is demethylated mainly through DNA
replication-dependent passive dilution because of defi cient maintenance
Genetic Studies on Mammalian DNA Methyltransferases

132
methylation, presumably due to the exclusion of Dnmt1 from the nucleus (Hirasawa
et al. 2008 ; Howell et al. 2001 ). In contrast, demethylation of the paternal genome
involves both active and passive mechanisms. Shortly after fertilization and before
the fi rst cell division, the 5mC dioxygenase Tet3 converts the majority of 5mC in the
male pronucleus to 5-hydroxymethylcytosine (5hmC) (Gu et al. 2011 ; Wossidlo
et al. 2011 ). 5hmC can be further oxidized to 5-formylcytosine (5fC) and
5- carboxylcytosine (5caC), which can be excised by thymine DNA glycosylase
(TDG) and replaced by unmodifi ed cytosine (He et al. 2011 ; Ito et al. 2011 ). 5hmC,
5fC, and 5caC can also be passively diluted during cleavage divisions (Inoue et al.
2011 ; Inoue and Zhang 2011 ). As a result of these processes, DNA methylation
marks inherited from gametes are largely erased by the blastocyst stage, with the
exception of imprinting control regions (ICRs) and some retroelements, which
resist this wave of global demethylation (Borgel et al. 2010 ; Smith et al. 2012 ).
Around the time of implantation, de novo methylation takes place when the inner
cell mass (ICM) starts to differentiate to form the embryonic ectoderm. Lineage-
specifi c DNA methylation patterns are then stably maintained.
2.2 Embryonic and Adult Phenotypes of Dnmt Mutant Mice
Most of our knowledge about the signifi cance of DNA methylation in mammalian
development came from genetic manipulations of Dnmt genes in mice. Results from
characterization of Dnmt mutant mice demonstrated that the establishment of
embryonic methylation patterns requires both de novo and maintenance Dnmts and
that maintaining genomic methylation above a threshold level is essential for embry-
onic development (Lei et al. 1996 ; Li et al. 1992 ; Okano et al. 1999 ). Complete
inactivation of Dnmt1 results in the arrest of embryonic development between pre-
somite and 8-somite stage around E9.5 (Lei et al. 1996 ). DNA methylation analysis
showed that embryos defi cient for Dnmt1 undergo dramatic decreases in global
DNA methylation (Lei et al. 1996 ; Li et al. 1992 ), in agreement with its role as the
major maintenance Dnmt. Disruption of Dnmt3b also leads to embryonic lethality
after E12.5, with multiple defects, including growth impairment and rostral neural
tube defects (Okano et al. 1999 ). In contrast, Dnmt3a -defi cient mice develop to term
and appear to be normal at birth but become runted and die at about 4 weeks (Okano
et al. 1999 ). Consistent with the developmental phenotypes, DNA methylation anal-
ysis of E9.5 embryos revealed that germ line-specifi c genes, pluripotency genes,
hematopoietic genes, and eye genes are severely hypomethylated in the absence of
Dnmt3b but not of Dnmt3a (Borgel et al. 2010 ). This suggested that Dnmt3b is the
main enzyme responsible for de novo methylation during embryogenesis. Dnmt3b
shows a dynamic expression change during pre- and early postimplantation devel-
opment, with preferential expression in the trophectoderm at the mid-blastocyst
stage and subsequent transition of expression in the embryonic lineage (Hirasawa
and Sasaki 2009 ). Notably, DNA methylation at certain genes such as Brdt , Dpep3 ,
Cytip , and Crygd is only partially reduced in Dnmt3b -/-
embryos (Borgel et al. 2010 ),
suggesting that Dnmt3a cooperates with Dnmt3b to methylate some loci. Indeed,
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133
Dnmt3a / 3b DKO embryos exhibit more severe defects than Dnmt3b -/-
embryos.
Specifi cally, DKO embryos show smaller size, lack somites, do not undergo embry-
onic turning, and die before E11.5, indicating that their growth and morphogenesis
are arrested shortly after gastrulation (Okano et al. 1999 ).
Conditional knockout (KO) studies have also demonstrated that Dnmts and DNA
methylation are essential in various organs and tissues. For example, disruption of
both Dnmt1 and Dnmt3a in forebrain excitatory neurons leads to abnormal synaptic
plasticity and defi cits in learning and memory (Feng et al. 2010 ). Conditional dele-
tion of Dnmt1 at sequential stages of T cell development has also revealed a critical
role for DNA methylation in T cell development, function, and survival. Specifi cally,
deletion of Dnmt1 in early double-negative thymocytes leads to an impaired sur-
vival of TCRαβ(+) cells and the generation of atypical CD8(+) TCRγδ(+) cells and
deletion of Dnmt1 in double-positive thymocytes impairs activation-induced prolif-
eration but differentially enhanced cytokine mRNA expression by naive peripheral
T cells (Lee et al. 2001 ).
2.3 Cellular Defects of Dnmt Mutations
The mechanisms underlying the developmental defects observed in Dnmt mutant
mice are not fully understood. Dnmt1, Dnmt3a, and Dnmt3b are all highly expressed
in pluripotent ES cells, but disruption of these genes individually, both Dnmt3a and
Dnmt3b , or even all three Dnmts , has no deleterious effects on mouse ES cells in the
undifferentiated state (Lei et al. 1996 ; Li et al. 1992 ; Okano et al. 1999 ; Tsumura
et al. 2006 ). However, Dnmt1 -/-
and Dnmt3a / 3b DKO ES cells die upon induction of
differentiation (Chen et al. 2003 ; Lei et al. 1996 ; Tucker et al. 1996 ). Interestingly,
a recent study showed that, in contrast to mouse ES cells, human ES cells require
DNMT1 , but not DNMT3A and DNMT3B , for survival (Liao et al. 2015 ). It is well
established that mouse and human ES cells represent different pluripotent states,
with human ES cells resembling the more mature epiblast state (Tesar et al. 2007 ),
which may explain the sensitivity of human ES cells to loss of DNA methylation.
During development, the effects of DNA methylation defi ciency become apparent
during or after gastrulation, when the embryo differentiates to form the three germ
layers (Lei et al. 1996 ; Li et al. 1992 ; Okano et al. 1999 ). Conditional inactivation
of Dnmt1 in mouse embryonic fi broblasts (MEFs) leads to severe hypomethylation
and cell death, and Dnmt3b -defi cient MEFs show modest hypomethylation, chro-
mosomal instability, and abnormal cell proliferation (Dodge et al. 2005 ; Jackson-
Grusby et al. 2001 ). Furthermore, although a hypomorphic mutation affecting the
N-terminal region of human DNMT1 has no effect on the survival and proliferation
of the colon cancer cell line HCT116 (Rhee et al. 2000 ), disruption of the DNMT1
catalytic domain in HCT116 leads to mitotic catastrophe and cell death (Chen et al.
2007 ). Taken together, these results suggest crucial roles for DNA methylation in
cellular differentiation and in the viability and proper functioning of differentiated
cells. Deregulation of gene expression likely plays a major role in the developmen-
tal and cellular defects associated with Dnmt mutations.
Genetic Studies on Mammalian DNA Methyltransferases

134
3 Dnmts in Genomic Imprinting
In early 1980s, elegant nuclear transplantation experiments using pronuclear stage
embryos showed that mouse embryos constructed to contain only maternal or pater-
nal diploid genome complements fail to develop beyond mid-gestation. This sug-
gested that the parental genomes are functionally nonequivalent and marked or
“imprinted” differently during male and female gametogenesis (Barton et al. 1984 ;
McGrath and Solter 1984 ; Surani et al. 1984 ). Separate experiments using chromo-
some translocations in mice showed that specifi c chromosome segments function
differently depending on the parental origin (Cattanach and Kirk 1985 ). In early
1990s, the fi rst murine imprinted genes, Igf2r , Igf2 , and H19 , were discovered,
which are expressed only from one parental allele (Barlow et al. 1991 ; Bartolomei
et al. 1991 ; DeChiara et al. 1991 ). To date, approximately 150 imprinted genes,
which exhibit monoallelic expression strictly according to the parental origin,
have been identifi ed in mouse ( http://www.mousebook.org/mousebook-catalogs/
imprinting- resource ), and many of them are also imprinted in human. Imprinted
genes are involved in diverse biological processes, including embryonic develop-
ment, placental formation, fetal and postnatal growth, and adult behavior (Frontera
et al. 2008 ; Reik and Walter 2001 ). In human, altered expression of imprinted genes,
due to genetic and epigenetic changes, has been linked to infertility, molar preg-
nancy, and various congenital disorders such as Prader-Willi syndrome, Angelman
syndrome, Beckwith-Wiedemann syndrome, and Silver-Russell syndrome (Butler
2009 ; Tomizawa and Sasaki 2012 ; Walter and Paulsen 2003 ). Loss of imprinting
(biallelic expression or silencing of imprinted genes) is also frequently observed in
cancer (Jelinic and Shaw 2007 ).
The majority of imprinted genes are arranged in chromosomal clusters, which
usually span hundreds to thousands of kilobases. Each of the imprinting clusters is
controlled by an ICR, an essential regulatory sequence that contains one or more
differentially methylated regions (DMRs) between the two alleles. Thus, allele-
specifi c DNA methylation is believed to be the primary epigenetic mark that con-
trols the monoallelic expression of imprinted genes.
The life cycle of DNA methylation imprints consists of three major steps: estab-
lishment, maintenance, and erasure (Fig. 2 ).
3.1 Establishment of Methylation Imprints
DNA methylation imprints are acquired in the germ line, with the majority being
established during oogenesis (maternally imprinted) and only four known loci ( H19 ,
Dlk1 - Gtl2 , Rasgrf1 , and Zdbf2 ) being established during spermatogenesis (pater-
nally imprinted). Conditional deletion of Dnmt3a in primordial germ cells (PGCs)
disrupts both maternal and paternal imprinting. Embryos from crosses between con-
ditional Dnmt3a mutant females and wild-type males die around E10.5, and condi-
tional Dnmt3a mutant males are sterile due to impaired spermatogenesis (Kaneda
et al. 2004 ). Dnmt3L KO mice show an identical phenotype, with the exception of
J. Dan and T. Chen

135
one paternally methylated locus, Dlk1 - Gtl2 , which is methylated in Dnmt3L KO but
not in Dnmt3a mutant spermatogonia (Bourc’his et al. 2001 ; Hata et al. 2002 ;
Kaneda et al. 2004 ). In contrast, conditional deletion of Dnmt3b in PGCs shows no
apparent phenotype (Kaneda et al. 2004 ). These results provide compelling genetic
evidence that Dnmt3a is responsible for the establishment of germ line imprints,
and Dnmt3L is an essential cofactor for Dnmt3a in this regard.
How Dnmt3L facilitates Dnmt3a function in the germ line is not fully under-
stood. Dnmt3L, via its C-terminal domain, forms a tetrameric complex with Dnmt3a
and, via its ADD domain, interacts with the N-terminal tail of histone H3 (Hata
et al. 2002 ; Jia et al. 2007 ; Ooi et al. 2007 ; Suetake et al. 2004 ). These fi ndings led
to the hypothesis that Dnmt3L plays a critical role in targeting Dnmt3a to specifi c
chromatin regions, including imprinted loci. A recent study showed that, similar to
Dnmt3L null mutant mice, mice homozygous for an engineered point mutation
(D124A) in the Dnmt3L ADD domain exhibit DNA methylation and spermatogen-
esis defects (Vlachogiannis et al. 2015 ), supporting a critical role of the ADD
domain in Dnmt3L function in the male germ line. It would be interesting to
F i g . 2 Life cycle of DNA methylation imprints. The paternal ( blue ) and maternal ( red ) methyla-
tion imprints are established during gametogenesis and transmitted to the offspring through fertil-
ization. These marks are maintained and control monoallelic expression of imprinted genes during
embryogenesis and in somatic cells throughout adult life. However, they are erased in primordial
germ cells (PGCs) before sex-specifi c methylation imprints are reestablished in later stages of
germ cell development
Genetic Studies on Mammalian DNA Methyltransferases

136
determine whether female mice homozygous for the D1124A mutation show defects
in the establishment of maternal imprints. However, the Dnmt3a ADD domain also
binds H3K4-unmethylated histone H3 (Otani et al. 2009 ; Zhang et al. 2010 ), which
raises the question of the specifi c role of the Dnmt3L ADD domain. It is possible
that Dnmt3L interacts with one or more proteins or histone marks that are not rec-
ognized by Dnmt3a.
The observation that H3K4 modifi cations disrupts the interaction between
Dnmt3 proteins and histone H3 (Ooi et al. 2007 ; Otani et al. 2009 ; Zhang et al.
2010 ) suggests that chromatin organization may be an important determinant of the
sites of de novo DNA methylation in the germ line. Indeed, genetic evidence indi-
cated that the H3K4 demethylase KDM1B (also known as LSD2 and AOF1) is
essential for the establishment of a subset of maternal imprints (Ciccone et al. 2009 ).
KDM1B is highly expressed in growing oocytes, where maternal imprints are
acquired, but shows little expression in most somatic tissues. Kdm1b KO mice are
viable and show no defects in spermatogenesis and oogenesis, and male mice are
fertile. However, oocytes from KDM1B-defi cient females exhibit global accumula-
tion of H3K4me2 and fail to establish DNA methylation imprints at a subset of
imprinted loci. Consequently, embryos derived from these oocytes die around mid-
gestation (Ciccone et al. 2009 ), similar to embryos derived from Dnmt3L- or
Dnmt3a-defi cient female mice (Bourc’his et al. 2001 ; Hata et al. 2002 ; Kaneda
et al. 2004 ). These results strongly suggested that removal of H3K4me2 is a prereq-
uisite for de novo DNA methylation. There is also evidence that transcription is an
additional requirement in specifying DNA methylation, at least at some maternally
imprinted loci. In the mouse Gnas locus, transcription initiated at the promoter of
Nesp55 , a gene upstream of the DMRs of the Gnas locus, occurs in growing oocytes,
placing a large genomic region, including the DMRs, within an active transcription
unit. Deletion of the Nesp55 promoter or insertion of a transcription termination
cassette downstream of Nesp55 to ablate transcription results in failure to establish
DNA methylation at the ICR of the Gnas locus (Chotalia et al. 2009 ; Frohlich et al.
2010 ; Williamson et al. 2011 ). Methylation of the DMR at the Snrpn locus has also
been shown to depend upon transcription (Smith et al. 2011 ).
3.2 Maintenance of Methylation Imprints
The paternal and maternal imprints are transmitted to the zygote through fertiliza-
tion, and despite extensive demethylation during preimplantation development (as
described above), parental allele-specifi c DNA methylation imprints are faithfully
maintained through development and adult life. Notably, recent genome-wide DNA
methylation analyses revealed far more differentially methylated loci in oocytes and
sperm than the number of imprinted genes (Kobayashi et al. 2012 ; Smallwood et al.
2011 ; Smith et al. 2012 ). Thus, the previous notion that imprinted loci are deter-
mined by distinct methylation patterns in gametes has been revised to the current
view that genomic imprinting results from selective maintenance of germ line-
derived allele-specifi c methylation. Genetic studies using conditional KO mice
J. Dan and T. Chen

137
demonstrated that Dnmt1, but not Dnmt3a or Dnmt3b, is responsible for maintain-
ing methylation marks at imprinted loci during preimplantation development
(Hirasawa et al. 2008 ). The oocyte-specifi c variant, Dnmt1o, is the predominant
Dnmt1 isoform in preimplantation embryos (Hirasawa et al. 2008 ; Kurihara et al.
2008 ). However, offspring of females lacking Dnmt1o exhibit only a ~50 %
reduction of methylation at certain imprinted loci (Howell et al. 2001 ). While initial
evidence suggested that the somatic form, Dnmt1s, does not express until the blas-
tocyst stage (Ratnam et al. 2002 ), subsequent work showed that Dnmt1s is present
at very low levels in the nucleus of oocytes and preimplantation embryos (Hirasawa
et al. 2008 ; Kurihara et al. 2008 ). Conditional deletion of Dnmt1 (both Dnmt1o and
Dnmt1s) in growing oocytes leads to a partial loss of methylation imprints in the
offspring (Hirasawa et al. 2008 ), resembling the effect of Dnmt1o loss (Howell
et al. 2001 ). However, ablation of both maternal and zygotic Dnmt1 results in a
complete loss of methylation at paternally and maternally methylated DMRs in
embryos (Hirasawa et al. 2008 ). Therefore, both maternal and zygotic Dnmt1 pro-
teins are necessary for the maintenance of methylation imprints in preimplantation
embryos. Dnmt1 is also responsible for the maintenance of methylation imprints in
postimplantation embryos (Li et al. 1993 ) and likely in adult somatic cells as well.
It is not well understood what confers the specifi city of Dnmt1, such that meth-
ylation is maintained at imprinted genes but not at other sequences in preimplanta-
tion embryos. Genetic and epigenetic features may distinguish imprinted loci from
other regions. Several other factors have been shown to be essential for the main-
tenance of DNA methylation imprints. PGC7 (also known as Stella and Dppa3), a
DNA-binding protein, is highly expressed in oocytes and persists in preimplanta-
tion embryos. Genetic evidence suggested that, in early embryos, maternal PGC7
plays a crucial role in protecting the maternal genome against DNA demethylation.
PGC7 also protects the paternally imprinted H19 and Rasgrf1 against demethyl-
ation (Nakamura et al. 2007 ). While the mechanisms involved remain to be deter-
mined, PGC7 has been shown to play a role in chromatin condensation during
oogenesis and to protect the maternal genome against Tet3-mediated conversion of
5mC to 5hmC in early embryos (Bian and Yu 2014 ; Liu et al. 2012 ; Nakamura
et al. 2012 ). Gene targeting experiments in mice have also implicated the involve-
ment of the Krüppel -associated box (KRAB)-containing zinc-fi nger protein
ZFP57 in the maintenance of genomic imprints (Li et al. 2008 ), and human ZFP57
mutations are associated with hypomethylation at multiple imprinted loci in
patients with transient neonatal diabetes (Mackay et al. 2008 ). ZFP57 specifi cally
binds the methylated allele of ICRs, recognizing a hexanucleotide sequence
(TGCCGC) shared by all murine ICRs and some human ICRs (Quenneville et al.
2011 ). ZFP57 interacts with KRAB-associated protein 1 (KAP1, also known as
TRIM28), which acts as a scaffold protein for various heterochromatin proteins,
including heterochromatin protein 1 (HP1), the histone H3K9 methyltransferase
Setdb1 (also known as ESET and KMT1E), the nuclear remodeling and histone
deacetylation (NuRD) complex, and Dnmt proteins and Uhrf1 (Nielsen et al. 1999 ;
Quenneville et al.
2011 ; Ryan et al. 1999 ; Schultz et al. 2001 , 2002 ; Zuo et al.
2012 ). Ablation of either maternal or zygotic KAP1 causes partial loss of DNA
Genetic Studies on Mammalian DNA Methyltransferases

138
methylation imprints, and ablation of both maternal and zygotic KAP1 leads to a
complete loss of imprinting (Lorthongpanich et al. 2013 ; Messerschmidt et al.
2012 ; Quenneville et al. 2011 ). Depletion of the NuRD components methyl CpG-
binding domain protein-3 (MBD3) or metastasis tumor antigen 2 (MTA2) also
results in reduction of methylation at some imprinted loci in preimplantation
embryos (Ma et al. 2010 ; Reese et al. 2007 ). The current view is that the ZFP57/
KAP1 complex specifi cally recruits the DNA methylation machinery, as well as
other heterochromatin proteins, to the methylated allele of ICRs to maintain
genomic imprints and control monoallelic expression of imprinted genes.
3.3 Erasure of Methylation Imprints
The last step of the imprint life cycle is the erasure of methylation imprints in
PGCs, which ensures the establishment of sex-specifi c imprints in later stages of
germ cell development. In mice, PGCs are specifi ed around E7.25 in the epiblast
of the developing embryo. Shortly afterwards, PGCs begin migrating along the
embryonic- extraembryonic interface and eventually arrive at the genital ridge by
E12.5. Recent genome-wide DNA methylation analyses reveal that PGCs undergo
demethylation in two major phases (Guibert et al. 2012 ; Kobayashi et al. 2013 ;
Popp et al. 2010 ; Seisenberger et al. 2012 ). The fi rst phase takes place during PGC
expansion and migration from ~ E8.5, which leads to a global demethylation affect-
ing almost all genomic regions. Passive demethylation likely plays a major role in
this phase, as Dnmt3a, Dnmt3b, and Uhrf1 are repressed in PGCs (Kagiwada et al.
2013 ; Kurimoto et al. 2008 ). The second phase occurs from E9.5 to E13.5 and
affects specifi c loci including ICRs, germ line-specifi c genes, and CpG islands on
the X chromosome (Guibert et al. 2012 ; Hackett et al. 2013 ; Popp et al. 2010 ;
Seisenberger et al. 2012 ; Yamaguchi et al. 2013 ). Genetic studies suggested that
Tet1- and Tet2-mediated 5mC oxidation is important in the second phase of
demethylation (Zhao and Chen 2013 ).
4 Dnmts in X-Chromosome Inactivation
In mammals, sex determination is controlled by a pair of sex chromosomes, the X
and the Y. Whereas the Y chromosome harbors very few protein-coding genes com-
pared to other chromosomes, the X chromosome has a relatively high gene density.
The balance of X-linked gene dosage between XX females and XY males is
achieved through X-chromosome inactivation (XCI), whereby one of the two X
chromosomes present in female mammals is inactivated. Marsupial mammals show
imprinted XCI, with only the paternal X chromosome (Xp) being inactivated.
Eutherian mammals exhibit two forms of XCI: imprinted Xp inactivation in the
early embryo and extraembryonic tissues and random inactivation of either Xp or
the maternal X chromosome (Xm) in the embryonic (epiblast) lineage (Payer and
Lee
2008 ; Wutz 2011 ).
J. Dan and T. Chen

139
Our knowledge about XCI mostly came from studies in the mouse. In the female
zygote, both X chromosomes appear active. Soon thereafter, a series of events result
in the inactivation of Xp. This imprinted XCI occurs in a two-step manner, with Xp
repeat elements fi rst silenced at the two-cell stage followed by Xp genic silencing
emerging at the eight- to sixteen-cell stage. Imprinted XCI is complete by the blas-
tocyst stage. Whereas inactivation of Xp is maintained in extraembryonic tissues, it
is reversed in the ICM of the blastocyst, resulting in the biallelic expression of
X-linked genes. Shortly after implantation, epiblast cells undergo random XCI
(Payer and Lee 2008 ). Murine ES cells, derived from the ICM, represent a useful
model for the study of XCI. Undifferentiated murine ES cells, like ICM cells, have
two active X chromosomes, and random XCI can be recapitulated during in vitro
differentiation (Chaumeil et al. 2004 ).
The process of XCI, which converts an X chromosome from relatively open
euchromatin to highly condensed heterochromatin (known as the “Barr body”), can
be divided into three steps: initiation of X inactivation, spreading of heterochroma-
tin to the entire chromosome, and maintenance of the inactive state (Fig. 3 ). XCI is
controlled by the X-inactivation center ( Xic ), a complex locus on the X chromo-
some that determines how many (counting step) and which X chromosomes (choice
step) will be silenced. A critical gene in Xic encodes the “X-inactive-specifi c tran-
script” ( Xist ), a 17-kb long, non-coding RNA (lncRNA). Xist is expressed only from
the presumptive inactive X chromosome (Xi) and then coats the same chromosome
in cis (Clemson et al. 1996 ). This step is necessary and suffi cient for the initiation
of XCI, as targeted disruption of the Xist gene abrogates XCI (Marahrens et al.
1997 ; Penny et al. 1996 ), and Xist transgenes on autosomes can induce autosomal
gene inactivation (Jiang et al. 2013 ; Lee and Jaenisch 1997 ; Lee et al. 1996 ). The
Xic also harbors several other genes encoding proteins and non-coding RNAs,
including the Xist antisense RNA Tsix , the Jpx and Ftx RNAs, and the E3 ubiquitin
ligase RNF12, that act as part of a sophisticated regulatory network to modulate Xist
expression in cis and in trans (Gendrel and Heard 2014 ). Xist is also required for the
spreading of XCI from the Xic to the rest of the chromosome. Xist RNA is able to
recruit PRC2, a complex responsible for the deposition of H3K27me3, which con-
tributes to chromatin and transcriptional changes during the initiation and spreading
of XCI (Zhao et al. 2008 ; da Rocha et al. 2014 ; Cifuentes-Rojas et al. 2014 ). Once
established, the globally silent state and heterochromatin structure of Xi are trans-
mitted through somatic cell division and clonally inherited. Although Xist is not
required for the maintenance of gene silencing on the Xi (Brown and Willard 1994 ;
Csankovszki et al. 1999 ), it appears to be important for maintaining the heterochro-
matin structure of Xi, as deletion of Xist leads to refolding of the Xi into a structure
resembling the active X chromosome (Xa) (Splinter et al. 2011 ).
The link between DNA methylation and XCI has been well established. In
somatic tissues, the 5′ end of the Xist gene is fully methylated on Xa and completely
unmethylated on Xi. Similarly, in tissues that undergo imprinted Xp inactivation,
the paternal Xist allele is unmethylated, and the maternal allele is fully methylated
(Norris et al.
1994 ). Studies of Dnmt1 -defi cient ES cells and embryos revealed that
XCI can occur in the absence of DNA methylation, but maintenance of Xist
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140
promoter methylation is necessary for its stable repression in differentiated cells
(Beard et al. 1995 ; Panning and Jaenisch 1996 ; Sado et al. 2000 ). A recent study
showed that loss of Dnmt1o disrupts imprinted XCI and accentuates placental
defects in females (McGraw et al. 2013 ). De novo DNA methylation is also dispens-
able for the initiation and propagation of XCI, as Xist expression is appropriately
regulated and XCI occurs properly in female embryos defi cient for both Dnmt3a
and Dnmt3b (Sado et al.
2004 ). Interestingly, despite multiple mechanisms involved
Fig. 3 Major steps of
X-chromosome
inactivation. The process
of X-chromosome
inactivation can be divided
into three steps: (1)
initiation ( Xist RNA is
expressed from the
presumptive inactive X
(Xi), but not the active X
(Xa)), (2) spreading ( Xist
RNA coats the entire
Xi chromosome, which
recruits other factors
(e.g., PRC2 complex,
Dnmts) to induce
heterochromatinization),
and (3) maintenance (the
highly compacted
chromatin structure and
most of the genes on Xi are
stably maintained and
clonally transmitted
through somatic cell
divisions). DNA
methylation is required for
the stable maintenance of
Xi-linked gene silencing
J. Dan and T. Chen

141
in X-chromosome gene silencing, approximately 25 % of genes on Xi escape inac-
tivation to some extent and exhibit biallelic expression in females (Carrel and
Willard 2005 ; Yang et al. 2010 ). The promoter regions of these escapee genes are
unmethylated (Weber et al. 2007 ). Furthermore, treatment of cells with the demeth-
ylating agents 5-azacytidine and 5-azadeoxycytidine has been shown to reactivate
some genes on Xi (Haaf 1995 ). Collectively, these fi ndings indicate that DNA meth-
ylation is not required for the initiation and propagation of XCI but is an essential
component of the epigenetic mechanisms that stably maintain the silent state of
Xi-linked genes.
5 Concluding Remarks
Since the discovery of mammalian Dnmts (Bestor et al. 1988 ; Okano et al. 1998a ),
great progress has been made in understanding the biological functions of DNA meth-
ylation in mammals. Genetic studies using Dnmt mutant mice and murine cells have
provided important insights into the roles of DNA methylation in various develop-
mental and cellular processes (Table 1 ). It is generally believed that DNA methyla-
tion, a relatively stable epigenetic mark, acts in concert with other epigenetic
mechanisms such as histone modifi cations to stably maintain gene silencing and chro-
matin structure. It is well documented that aberrant DNA methylation patterns are
associated with various human diseases. Studies in recent years have also identifi ed
genetic alterations affecting major components of the DNA methylation machinery,
including DNA methylation “writers” (DNMTs), “erasers” (e.g., TETs), and “read-
ers” (e.g., MeCP2), in cancer and developmental disorders (Hamidi et al. 2015 ). For
example, DNMT1 mutations are reported in two related neurodegenerative diseases
(hereditary sensory and autonomic neuropathy with dementia and hearing loss type IE
(HSAN IE), autosomal dominant cerebellar ataxia, deafness, and narcolepsy
(ADCA-DN)), DNMT3A mutations are frequently found in acute myeloid leukemia
(AML) and other hematologic malignancies, and DNMT3B mutations cause the
immunodefi ciency, centromeric instability, and facial anomalies (ICF) syndrome
(Hamidi et al. 2015 ). The mechanisms by which these mutations contribute to the
disease phenotypes are generally not well understood. Besides their values in eluci-
dating the fundamental functions of DNA methylation, Dnmt mutant mice and cells
provide important research tools for investigating the effects of DNMT mutations
found in human patients. For instance, by expressing a Dnmt3a protein harboring a
point mutation equivalent to human DNMT3A:R882H (the most prevalent DNMT3A
mutation in AML) in Dnmt3a and Dnmt3b mutant murine ES cells, we recently dem-
onstrated that this mutation, which occurs on only one allele in AML patients, not
only leads to haploinsuffi ciency of DNMT3A enzymatic activity but also exhibits
dominant-negative effect by forming functionally defi cient complexes with wild-type
DNMT3A and DNMT3B (Kim et al. 2013 ). Most of the DNMT mutations identifi ed
in patients are not null alleles, making Dnmt KO mice less ideal for modeling human
diseases. With the development of new technologies such as CRISPR/Cas9-mediated
gene editing, it now becomes more feasible to create genetically engineered animal
Genetic Studies on Mammalian DNA Methyltransferases

142
and cellular models that better recapitulate the major features of human diseases asso-
ciated with DNMT mutations. Genomic, epigenomic, transcriptomic, and proteomic
analyses of these models will be powerful approaches for defi ning the molecular
mechanisms and pathways involved in pathogenesis. Ultimately, such studies will
likely lead to novel therapeutic and preventive strategies.
Acknowledgments Work in the Chen laboratory is supported by a Rising Star Award from
Cancer Prevention and Research Institute of Texas (CPRIT, R1108) and a grant from the National
Institutes of Health (NIH, 1R01DK106418-01).
Table 1 Developmental phenotypes of Dnmt knockout mice
Gene Mutations Major developmental phenotypes References
Dnmt1 Dnmt1 n /n ~70 % reduction in global DNA
methylation, embryonic lethality at
E12.5–15.5
Li et al. (
1992 )
Dnmt1 c / c
~90 % reduction in global DNA
methylation, developmental arrest
at E8.5, embryonic lethality before
8-somite stage at ~ E9.5. Unstable
random XCI
Lei et al. (
1996 );
Sado et al. (
2000 )
Dnmt1 s / s
~90 % reduction in global DNA
methylation, developmental arrest
at E8.5, embryonic lethality
at ~ E9.5. Loss of methylation at
Xist locus and abnormal Xist
expression in male embryos
Beard et al.
(
1995 ); Lei et al.
(
1996 )
Dnmt1o -/-
Maternal-effect phenotype: partial
loss of DNA methylation imprints,
defects in imprinted XCI,
embryonic lethality at
mid-gestation
Howell et al.
(
2001 ); McGraw
et al. (
2013 )
Maternal
and zygotic Dnmt3a
-/-
Complete loss of paternal and
maternal methylation imprints,
embryonic lethality at
mid-gestation
Hirasawa et al.
(
2008 )
Dnmt3a Dnmt3a -/-
Gut malfunction, spermatogenesis
defects, death at ~4 weeks of age
Okano et al.
(
1999 )
Dnmt3a -/-
in PGCs Failure to establish maternal and
paternal methylation imprints,
spermatogenesis defects
Kaneda et al.
(
2004 )
Dnmt3b Dnmt3b -/-
Hypomethylation of minor satellite
DNA, neural tube defects,
embryonic lethality at E14.5–18.5
Okano et al.
(
1999 )
Dnmt3a -/- ,
Dnmt3b -/-
Failure to initiate de novo
methylation after implantation,
developmental arrest at E8.5
Okano et al.
(
1999 )
Dnmt3L Dnmt3L -/-
Failure to establish maternal and
paternal methylation imprints,
spermatogenesis defects
Bourc’his et al.
(
2001 ); Hata et al.
(
2002 )
J. Dan and T. Chen

143
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