CFP1 Interacts with DNMT1 Independently of Association
with the Setd1 Histone H3K4 Methyltransferase Complexes
Jill Sergesketter Butler, Jeong-Heon Lee, and David G. Skalnik
CXXC finger protein 1 (CFP1) is a component of the Setd1A and Setd1B methyltransferase complexes, localizes to
euchromatic regions of the genome, and specifically binds unmethylated CpG dinucleotides in DNA. Murine
embryos lacking CFP1 exhibit peri-implantation lethality, a developmental time that correlates with global epi-
genetic reprogramming. CFP1-deficient embryonic stem (ES) cells exhibit a 70% reduction in global cytosine
methylation and a 60% decrease in maintenance DNA methyltransferase (DNMT1) activity. DNMT1 protein level
is reduced 50% in CFP1-deficient ES cells. Experiments were performed to investigate the role of CFP1 in regu-
lating maintenance cytosine methylation. Coimmunoprecipitation experiments reveal that endogenous DNMT1
and CFP1 interact in vivo. Protein regions required for the interaction between DNMT1 and CFP1 were mapped.
Amino acids 169–493 and 970–1617 of DNMT1 are each sufficient for interaction with CFP1. Three regions
spanning the CFP1 protein, amino acids 1–123, 103–367, and 361–656, are each sufficient for interaction with
DNMT1. Interestingly, a single-point mutation (C375A) within CFP1 that abolishes the interaction with the
DNMT1. This result indicates that CFP1 intersects the cytosine methylation machinery independently of its
association with the Setd1 complexes.
quence. Epigenetic modifications that facilitate changes in
chromatin structure and thus influence transcription include
posttranslational covalent modifications of histone tails and
CXXC finger protein 1 (CFP1) is a unique DNA-binding
protein that contains a conserved CXXC domain both neces-
sary and sufficient for specific binding to unmethylated CpG
dinucleotides (Voo et al., 2000; Lee et al., 2001). CFP1 also
contains two plant homeodomain (PHD) fingers. Recently, a
global proteomic screen of Saccharomyces cerevisiae proteins
containing PHD fingers revealed that the PHD of Spp1, the
yeast homolog of CFP1, binds histone H3K4me2=3(Shi et al.,
2007). Additionally, the PHD within the NURF nucleosome
remodeling factor binds histone H3K4me3and targets the
NURF complex to Hox promoters during development to
activate transcription (Wysocka et al., 2006), thus demon-
strating a biological effect of PHD finger interaction with
methylated histone H3.
CFP1 is a component of the mammalian Setd1A (Lee and
Skalnik, 2005) and Setd1B (Lee et al., 2007) histone H3K4
pigenetics refers to heritable patterns of gene ex-
pression that occur without alteration of nucleotide se-
methyltransferase complexes, which are analogous to the
Set1=COMPASS complex in S. cerevisiae (Miller et al., 2001;
Nagy et al., 2002; Roguev et al., 2003). Spp1 is required for
Set1=COMPASS histone methyltransferase activity in Schi-
zosaccharomyces pombe, but only for appropriate histone
H3K4me3in S. cerevisiae (Roguev et al., 2003; Schneider et al.,
2005). While the mammalian Setd1A and Setd1B complexes
both localize to euchromatic regions of the genome, they
exhibit nonoverlapping subnuclear localization patterns (Lee
et al., 2007). This suggests that the Setd1A and Setd1B com-
plexes function at distinct genomic targets.
Mice lacking CFP1 exhibit peri-implantation lethality (4.5–
6.5 days postcoitus) (Carlone and Skalnik, 2001). CFP1 is ad-
ditionally required for postgastrulation development and
survival, because zebrafish embryos injected with antisense
morpholino oligonucleotides directed against CFP1 exhibit
defective primitive hematopoiesis and decreased survival
(Young et al., 2006). In addition, siRNA-mediated depletion of
CFP1 in human leukemia cell lines leads to defects in cell
proliferation and terminal myeloid differentiation (Young
and Skalnik, 2007). Lastly, CFP1-deficient murine embry-
onic stem (ES) cells are viable; however, they are unable to
differentiate upon removal of leukemia inhibitory factor
from the culture medium and exhibit a threefold increase in
Herman B Wells Center for Pediatric Research, Section of Pediatric Hematology=Oncology, Departments of Pediatrics and Biochemistry
and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana.
DNA AND CELL BIOLOGY
Volume 27, Number 10, 2008
ª Mary Ann Liebert, Inc.
apoptosis under normal culture conditions (Carlone et al.,
CFP1-deficient ES cells exhibit a *43% increase in histone
H3K4me2and *28% reduction in histone H3K9me2under
normal conditions, and an approximately fourfold induction
of histone H3K4me3upon induction of differentiation (Lee
and Skalnik, 2005). CFP1-deficient ES cells also exhibit a 70%
deficiency in global cytosine methylation that affects single-
copy genes, imprinted loci, and repetitive elements (Carlone
et al., 2005). Taken together, these data indicate that ES cells
lacking CFP1 contain reduced heterochromatin. A 60% re-
duction in maintenance DNA methyltransferase (DNMT1)
activity appears to contribute to the global cytosine methyla-
tion deficiency, because de novo DNMT activity is not de-
creased in ES cells lacking CFP1 (Carlone et al., 2005). DNMT1
protein expression is reduced 50% in the absence of CFP1
(Carlone et al., 2005) due to decreased DNMT1 protein sta-
bility and reduced translation (Jill Butler, unpublished data).
Recent reports reveal intricate interrelationships linking
cytosine methylation and histone modifications, thus pro-
viding a unifying framework for the control of chromatin
structure and gene regulation (Burgers et al., 2002). For ex-
ample, DNMT proteins associate with histone deacetylase
(HDAC) complexes (Fuks et al., 2000, 2001; Robertson et al.,
2000); cytosine methylation in Neurospora is dependent on
methylation of histone H3 and the presence of heterochro-
matin protein 1 (HP1) (Freitag et al., 2004); human HP1 re-
cruits DNMT1 to methylate DNA and silence gene expression
(Smallwood et al., 2007), and inhibition of HDAC activity by
trichostatin A results in a loss of cytosine methylation (Selker,
1998; Tamaru and Selker, 2001; Tamaru et al., 2003). Further,
the chromatin remodeling protein DDM1 in Arabidopsis and
the related factor LSH in mammals are required for normal
cytosine methylation (Jeddeloh et al., 1998, 1999; Dennis et al.,
2001). Disruption of the Suv39h1 histone H3K9 methyl-
transferase gene in murine ES cells leads to altered locali-
zation of DNMT3b and decreased cytosine methylation at
peri-centric satellite repeats (Lehnertz et al., 2003), and loss of
DNMT1 leads to perturbations in the histone code consistent
with reduced heterochromatin (Espada et al., 2004). Hence,
DNA methylation and covalent histone modifications are in-
tegrated and mutually reinforcing mechanisms to establish
and maintain heterochromatin.
However, the molecular mechanisms linking cytosine
methylation and histone modifications are not well under-
stood. Interestingly, organisms that lack cytosine methyla-
tion, such as Caenorhabditis elegans and S. cerevisiae, contain
CFP1 orthologs that lack the CXXC DNA-binding domain
(Voo et al., 2000). This suggests that CFP1 served an ances-
tral role in the regulation of covalent histone modifications,
and acquired DNA-binding capacity in organisms that uti-
lize cytosine methylation as an additional epigenetic modi-
fication. The influence of CFP1 on both cytosine methylation
and histone methylation suggests a role in mediating cross
talk between these epigenetic modifications.
Studies were performed to gain insight into the molecular
mechanism of how CFP1 affects cytosine methylation. The re-
sults presented in this report demonstrate that CFP1 and
DNMT1 physically interact. The minimal regions sufficient for
the interaction between DNMT1 and CFP1 were mapped and
include conserved domains involved in chromatin targeting.
Experiments were also performed to map the minimal region
required for CFP1 interaction with Setd1A and Setd1B. In-
terestingly, CFP1 interaction with Setd1A or Setd1B is not re-
quired for its interaction with DNMT1, strongly suggesting
Materials and Methods
Cell culture and transient transfections
Human embryonic kidney (HEK-293) cells were cultured
as previously described (Lee and Skalnik, 2002; Carlone et al.,
2005). Transient cotransfections in HEK-293cells were per-
formed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA)
per the manufacturer’s protocol. Briefly, cells were grown to
90–95% confluence as a monolayer in 10cm tissue culture
dishes. Ten micrograms of plasmid DNA was added to Li-
pofectamine 2000 reagent in OPTI-MEM serum-free medium
(Invitrogen). The mixture was added drop-wise to the cells,
which were then incubated for *40h at 378C with 5% CO2.
Construction of DNMT1 and CFP1 expression vectors
The human DNMT1 cDNA (generously provided by Dr.
Sriharsa Pradhan; New England Biolabs, Ipswich, MA) or
human CFP1 cDNA was ligated to the pcDNA3-FLAG vec-
tor that encodes an amino terminal FLAG epitope, as pre-
viously described (Lee et al., 2000). The CFP1 cDNA was
ligated to the pcDNA3-Myc vector that encodes an amino-
terminal Myc epitope. Constructs carrying truncations of the
DNMT1 or CFP1 cDNAs were generated using a combina-
tion of polymerase chain reaction (PCR) and restriction en-
zyme digestion methods, and then subcloned into the
pcDNA3-FLAG or pcDNA3-Myc expression vectors. Primer
sequences for PCR amplification are available upon request.
An exogenous nuclear localization signal (NLS) was made by
annealing complementary oligonucleotides corresponding to
the endogenous DNMT1 NLS that extends from amino acids
193–213. These oligonucleotides were designed to create a 50
EcoRI restriction enzyme site and a 30ClaI restriction enzyme
site when annealed. DNMT1 N-terminal deletion constructs
lacking the endogenous NLS were subcloned downstream of
the exogenous NLS in the pcDNA3-FLAG vector using the
EcoRI and ClaI restriction enzyme sites. Site-directed muta-
genesis was carried out using a QuikChange site-directed
mutagenesis kit (Stratagene, La Jolla, CA) per the manufac-
turer’s protocol. The sequences of the oligonucleotide prim-
ers used to generate amino acid substitutions within the
human CFP1 cDNA are as follows: C375A, 50-cctgcgtcactgcc
ccaggcgctggggcccggctgtgtg-30; Y390A, C391A, S392A, 50-
C580A, 50-gagctcacgggtgacttcgcccgcctgcccaagcgccag-30. The
nucleotide substitutions within the CFP1 primer sequences
are underlined. The nucleotide sequences of all constructs
were confirmed by automated DNA sequencing.
Coimmunoprecipitation and western blot analysis
Nuclear extracts were prepared from HEK-293cells tran-
siently expressing Myc and FLAG epitope-tagged proteins.
Cells were washed with 1? PBS, and then with hypotonic
acid [pH 7.9 at 48C], 1.5mM MgCl2, and 10mM KCl). Cells
were then swollen on ice for 10min in hypotonic buffer before
534BUTLER ET AL.
being lysed by Dounce homogenization 10 times. Nuclei were
pelleted by centrifugation at 48C and resuspended in lysis
buffer (20mM Tris HCl [pH 7.4], 400mM NaCl, 25% glycerol,
5mM ethylenediaminetetraacetic acid, 0.1% NP-40, 1mM
dithiothreitol, and 3% protease inhibitor cocktail [Sigma, St.
Louis, MO]). Nuclei were lysed by Dounce homogenization
30 times on ice, and debris was pelleted by centrifugation at
Either anti-Myc–conjugated agarose (Sigma) or FLAG M2–
conjugated agarose (Sigma) was incubated with soluble
nuclear extracts for 3h at 48C. Alternatively, the soluble
nuclear extracts were precleared with protein G agarose
(Millipore, Billerica, MA), and then incubated with CFP1 an-
tiserum (Lee et al., 2007) or a custom DNMT1 antiserum di-
the murine cDNA for 1h at 48C, followed by incubation with
with buffer containing 300mM NaCl, 0.1% NP-40, and then
gels (Lonza Group, Basel, Switzerland) by sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and
transferred to nitrocellulose membranes (Amersham, GE
Healthcare, Piscataway, NJ). Western blot analysis was per-
formed using primary antisera directed against the Myc epi-
tope (Santa Cruz Biotechnology, Santa Cruz, CA), FLAG
epitope (Sigma), or the Setd1 complex components Setd1A
(Lee et al., 2007), Setd1B (Lee et al., 2007), Wdr5 (Lee et al.,
2007), Wdr82 (Lee et al., 2007), Ash2 (Bethyl Laboratories,
Montgomery, TX), and Rbbp5 (Bethyl Laboratories), followed
by horseradish peroxidase–conjugated secondary antibodies.
Proteins were detected using an ECL detection kit (Amer-
sham, GE Healthcare, Montgomery, TX).
CFP1 and DNMT1 interact in vivo
To determine if DNMT1 and CFP1 interact in vivo, full-
length Myc-CFP1 and full-length FLAG-DNMT1 were tran-
siently expressed in HEK-293cells, and Myc-CFP1 was
expressing Myc-CFP1 (full length) and FLAG-DNMT1 (full length), as indicated above the lanes, and were subjected to
immunoprecipitation using anti-Myc–conjugated agarose. Western blot analysis with anti-FLAG antiserum was used to detect
FLAG-DNMT1. The membrane was re-probed with anti-Myc antiserum to verify Myc-CFP1 protein expression and Myc
immunoprecipitation efficiency. Transfection and immunoprecipitation of the empty vector serves as a negative control for the
Myc immunoprecipitation. (B) Nuclear extracts were prepared from HEK-293 cells transiently expressing Myc-CFP1 (full
length) and FLAG-DNMT1 (full length), as indicated above each lane,and were subjected toimmunoprecipitation using FLAG
M2–conjugated agarose. Western blot analysis with anti-Myc antiserum was used to detect Myc-CFP1. The membrane was re-
probed with anti-FLAG antiserum to verify FLAG-DNMT1 protein expression and FLAG immunoprecipitation efficiency.
Transfection and immunoprecipitation of the empty vector serves as a negative control for the FLAG immunoprecipitation.
(C) Endogenous CFP1 was immunoprecipitated from HEK-293 nuclear extracts using CFP1 antiserum and protein G agarose.
Western blot analysis was performed using anti-DNMT1 antiserum. The signal for DNMT1 is marked with an arrow. Im-
munoprecipitation reactions containing control serum (IgG control) or lacking primary antibody (No Ab) serve as negative
controls for the CFP1 immunoprecipitation. (D) Endogenous DNMT1 wasimmunoprecipitated from HEK-293 nuclear extracts
using antiserum against DNMT1 and protein G agarose. Western blot analysis was performed using anti-CFP1 antiserum. The
signal for CFP1 is marked with an arrow. An immunoprecipitation reaction lacking primary antibody (No Ab) serves as a
negative control for the DNMT1 immunoprecipitation.
CFP1 and DNMT1 physically interact in vivo. (A) Nuclear extracts were prepared from HEK-293 cells transiently
CFP1 AND DNMT1 INTERACT IN VIVO
immunoprecipitated from nuclear extracts using anti-Myc–
conjugated agarose. The immunoprecipitated material was
subjected to western blot analysis using antiserum against the
FLAG epitope. These experiments demonstrate that FLAG-
DNMT1 coimmunoprecipitates with Myc-CFP1 (Fig. 1A).
Empty vector was cotransfected individually and with each
construct to serve as a negative control for Myc immunopre-
cipitation reactions. The reciprocal experiment demonstrated
that Myc-CFP1 coimmunoprecipitates with FLAG-DNMT1
upon immunoprecipitation with FLAG M2–conjugated aga-
rose, thus confirming the interaction (Fig. 1B). These re-
sults indicate that full length CFP1 and DNMT1 interact
To examine whether CFP1 and DNMT1 interact at phys-
iologic concentrations in vivo, endogenous CFP1 was im-
munoprecipitated from HEK-293 nuclear extracts using
CFP1 antiserum. The immunoprecipitated material was
subjected to western blot analysis using antiserum against
in HEK-293 nuclear extracts (Fig. 1C). Addition of protein G
agarose to nuclear extracts without antibody or with control
serum served as negative controls. The reciprocal experiment
the conserved domains within DNMT1. DMAP, DNMT1-associated protein 1 binding region; PCNA, proliferating cell nuclear
antigen–binding region; NLS, nuclear localization signal; TS, region required to target DNMT1 to chromatin independently of
the replication fork; CXXC, conserved cysteine-rich DNA-binding domain; BAH, bromo-adjacent homology domain. The
amino acid position of each domain is denoted below the diagram. (B) A series of FLAG-DNMT1 C-terminal deletion mutants
encoding amino acids 1–650, 1–489, 1–173, and 169–493 were coexpressed with Myc-CFP1 (aa 1–367) in HEK-293 cells. Nuclear
extracts were prepared and subjected to Myc immunoprecipitation, followed by western blot analysis using anti-FLAG
antiserum. The FLAG-DNMT1 truncation mutants analyzed for interaction are denoted above each lane. The membranes were
re-probed withMyc antiserum to verifyMyc-CFP1 (aa 1–367) protein expression and Myc immunoprecipitation efficiency. The
empty vector was transfected alone or with Myc-CFP1 (aa 1–367) into HEK-293 cells to serve as negative controls for the Myc
tested for interaction with Myc-CFP1 (aa 1–367). FLAG-DNMT1 proteins were immunoprecipitated with Myc-conjugated
denoted above each lane. The membranes were re-probed with Myc antiserum to verify Myc-CFP1 (aa 1–367) protein ex-
pression and Myc immunoprecipitation efficiency. The empty vector was cotransfected with FLAG-DNMT1 (aa 637–1102) or
FLAG-DNMT1 (aa 970–1617) to serve as negative controls for the Myc immunoprecipitation.
Two independent domains of DNMT1 are each sufficient for interaction with CFP1. (A) A schematic representation of
536BUTLER ET AL.
was performed using DNMT1 antiserum to coimmunopre-
cipitate CFP1 from HEK-293 nuclear extracts. Western blot
analysis using CFP1 antiserum confirmed the interaction
with endogenous DNMT1 (Fig. 1D). These results demon-
strate a physical interaction between endogenous DNMT1
Independent domains within the amino and carboxyl
termini of DNMT1 are each sufficient for interaction
The amino terminus of DNMT1 interacts with a number of
chromatin-associated proteins, such as DNMT3a and 3b (Kim
et al., 2002), HDAC1 (Fuks et al., 2000; Robertson et al., 2000)
and HDAC2 (Rountree et al., 2000), the methyl CpG-binding
proteins, MeCP2 (Kimura and Shiota, 2003) and MBD2 and
MBD3 (Tatematsu et al., 2000), HP1 (Fuks et al., 2003; Small-
wood et al., 2007), and the histone H3K9 methyltransferases,
SUV39H1 (Fuks et al., 2003) and G9a (Esteve et al., 2006). A
diagram illustrating conserved domains within DNMT1 is
shown in Figure 2A. To further characterize the minimal re-
gion of DNMT1 required for interaction with CFP1, FLAG-
expressed in HEK-293cells along with Myc-CFP1 (aa 1–367),
followed by Myc immunoprecipitation. The C-terminal
truncated proteins, FLAG-DNMT1 (aa 1–650) and (aa 1–489),
interact with Myc-CFP1 (Fig. 2B). Analysis of the N-terminus
of DNMT1 revealed that the first 173 amino acids, encoding
the DNMT1-associated protein 1 (DMAP) and proliferating
cell nuclear antigen (PCNA)–binding regions, are not suffi-
cient for interaction with Myc-CFP1 (Fig. 2B). The FLAG-
large portion of the targeting sequence (TS) that directs
DNMT1 to late-replicating heterochromatin (Easwaran et al.,
2004). This portion of DNMT1 is sufficient for the interaction
with Myc-CFP1 (Fig. 2B).
It was also necessary to analyze the C-terminus of DNMT1
for possible interaction with CFP1. The highly conserved C-
terminus of DNMT1 has previously been found to interact
with chaperone 23 (p23) (Zhang and Verdine, 1996), the tu-
mor suppressor p53 (Esteve et al., 2005), and the histone
H3K27 methyltransferase, EZH2 (Vire et al., 2006). The highly
conserved C-terminal catalytic domain contained within the
FLAG-DNMT1 (aa 970–1617) protein fragment is sufficient to
interact with Myc-CFP1 (aa 1–367) (Fig. 2C). Bromo-adjacent
homology (BAH) domains are found in transcriptional regu-
(Callebaut et al., 1999). However, the BAH domains of
DNMT1 are not sufficient for interaction with Myc-CFP1 (Fig.
2C), as the FLAG-DNMT1 (aa 637–1102) protein fragment
fails to interact with Myc-CFP1 (aa 1–367). Lastly, central re-
gions of DNMT1, including amino acids 392–661, 169–330,
and 169–392, failed to interact with CFP1 (aa 1–367) (data not
shown). These results demonstrate a complex pattern of in-
teractions, in which either of two nonoverlapping domains of
DNMT1 is sufficient for interaction with CFP1.
CFP1 interacts with DNMT1 through several
CFP1 contains multiple highly conserved domains that
contribute to CFP1 subcellular localization and function (Voo
et al., 2000). For example, the acidic, basic, and coiled-coil
domains are necessary and sufficient to target CFP1 to the
nuclear matrix (Lee and Skalnik, 2002), while the CXXC do-
main is required for DNA binding (Lee et al., 2001). To de-
termine if any ofthese conserved domains arerequired for the
interaction with DNMT1, various CFP1 protein fragments
were expressed in HEK-293cells and tested for interaction
with DNMT1. A schematic diagram representing the organi-
zation of conserved domains within the CFP1 protein is
shown in Figure 3A. Truncations encoding Myc-tagged C-
terminal deletion mutants of CFP1 were transiently expressed
in HEK-293cells along with FLAG-DNMT1 (aa 1–489).
Immunoprecipitation was carried out using FLAG M2-
conjugated agarose. Western blots probed with anti-Myc
antiserum demonstrate that Myc-CFP1 (aa 1–123), which
contains the PHD1 domain, is sufficient for interaction with
analyzed for interaction with DNMT1. Western blot analysis
demonstrates thatMyc-CFP1(aa103–367),which containsthe
CXXC, acidic, and basic domains, is sufficient for interaction
with FLAG-DNMT1 (Fig. 3C). However, Myc-CFP1 (aa 213–
367), which lacks the CXXC domain, fails to interact with
FLAG-DNMT1, indicating that the acidic and basic domains
of CFP1 are not sufficient for interaction with DNMT1 (Fig.
3C). Taken together, these results indicate that multiple re-
gions within CFP1 are sufficient, but no individual domain is
necessary, for interaction with DNMT1.
The carboxyl terminus of CFP1 mediates
the interaction with the Setd1A and Setd1B
histone methyltransferase complexes
CFP1 is a component of the Setd1A and Setd1B histone
H3K4 methyltransferase complexes (Lee and Skalnik, 2005;
Lee et al., 2007). The Setd1A and Setd1B complex–interacting
region within CFP1 was mapped using truncation analysis to
determine whether CFP1 interacts with the cytosine methyl-
ation machinery while associated with the Setd1 complexes.
A series of FLAG-epitope–tagged CFP1 truncation mutants
were expressed in HEK-293cells. Nuclear extracts were pre-
pared and subjected to immunoprecipitation using FLAG
M2–conjugated agarose. Western blot analysis was then per-
formed using antisera directed against components of the
Setd1A and Setd1B histone methyltransferase complexes—
Setd1A, Setd1B, Ash2, Rbbp5, Wdr5, and Wdr82. The Setd1A
and Setd1B complexes consist of identical components with
exception of the catalytic (Setd1) subunits (Lee and Skalnik,
2005; Lee et al., 2007). Coimmunoprecipitation studies re-
vealed that a Setd1-interacting domain (SID) lies between the
conserved basic and coiled-coil domains of CFP1 (Fig. 4A, B).
The SID of CFP1 is required for CFP1 interaction with the
Setd1A and Setd1B complexes, because the FLAG-CFP1 (aa
423–656) mutant that lacks this region fails to interact with
any components of the Setd1A or Setd1B complexes (Fig. 4B).
Further, the C-terminus of CFP1, which contains the PHD2
domain, is also required for interaction with the Setd1A and
that contains the SID, but lacks the C-terminus, also fails to
interact with any Setd1A or Setd1B complex components
(Fig. 4). Finally, FLAG-CFP1 (aa 360–597) fails to interact
with the Setd1 complexes, demonstrating that the SID and
PHD2 domains are not sufficient for this interaction (Fig. 4).
CFP1 AND DNMT1 INTERACT IN VIVO
A multispecies alignment of CFP1 revealed several highly
conserved amino acids within the SID and PHD2 domains
(Fig. 4C). Single– or triple–amino acid substitutions were
made within the SID or PHD2 domains in the context of full-
length CFP1 to determine the functional significance of these
amino acids in mediating the interaction between CFP1 and
the Setd1A and Setd1B complexes. Full-length FLAG-CFP1
encoding single-alanine substitutions at cysteine 375 (C375A)
within the SID, or 580 (C580A) within the PHD2 domain, or a
triple mutant encoding alanine substitutions at tyrosine 390,
cysteine 391, and serine 392 (YCS?AAA) within the SID all
failed to interact with any of the components of the Setd1A
C-terminus of CFP1 is important for the interaction with the
Setd1A and Setd1B complexes, and identify subtle mutations
within CFP1 that ablate interaction with the Setd1 histone
CFP1 interaction with the Setd1A complex
is not required for interaction with DNMT1
The C-terminus of CFP1 (aa 361–656) is necessary and
sufficient for the interaction with the Setd1A and Setd1B
complexes (Fig. 4B). To determine if the C-terminus of CFP1
is also sufficient for the interaction with DNMT1, the Myc-
CFP1 (aa 361–656) truncation mutant was coexpressed with
FLAG-DNMT1 (aa 1–489) in HEK-293 cells and subjected to
FLAG immunoprecipitation analysis. Myc-CFP1 (aa 361–
656) interacts with FLAG-DNMT1 (aa 1–489) (Fig. 5), thus
revealing another DNMT1 interaction domain with CFP1.
Importantly, introduction of the C375A point mutation in
Myc-CFP1 (aa 361–656), which ablates interaction with the
Setd1 histone methyltransferase complexes (Fig. 4D), does
not disrupt the interaction with FLAG-DNMT1 (Fig. 5).
Thus, CFP1 association with the Setd1A and Setd1B com-
plexes is not required for its interaction with DNMT1.
The results reported in this study demonstrate that CFP1
and DNMT1 physically interact in vivo. Immunoprecipitation
analysis of Myc-CFP1 protein fragments revealed three re-
gions, amino acids 1–123, 103–367, and 361–656, that are
sufficient for the interaction with FLAG-DNMT1. These re-
gions all contain conserved domains involved in Zn2þbind-
ing and=or targeting to euchromatic regions of the genome—
namely, the PHD1, CXXC, and PHD2 domains. The CXXC
domain of CFP1 is the sole DNA-binding domain within the
within CFP1. The amino acid position of each domain is denoted below the schematic. (B) A series of C-terminal Myc-CFP1
293 cells. Nuclear extracts were prepared and subjected to FLAG immunoprecipitation. Western blot analysis was performed
expression and FLAG immunoprecipitation efficiency. (C) Myc-CFP1 truncation mutants encoding amino acids 103–367 or
213–367 were coexpressed with FLAG-DNMT1 (aa 1–489) in HEK-293 cells. Nuclear extracts were prepared and subjected to
FLAGimmunoprecipitation followedbywesternblot analysiswithanti-Mycantiserum.Membranes werere-probedwithanti-
FLAG antiserum to verify FLAG-DNMT1 (aa 1–489) protein expression and FLAG immunoprecipitation efficiency. Empty
vector was transfected alone or with FLAG-DNMT1 (aa 1–489) or Myc-CFP1 (aa 213–367) to serve as negative controls for the
Identification of CFP1 domains that interact with DNMT1. (A) A schematic representation of conserved domains
538 BUTLER ET AL.
protein and specifically recognizes unmethylated CpG dinu-
chromatin-associated proteins have recently been identified
as binding modules for methylated histone H3K4 or histone
H3K36 (Martin et al., 2006; Shi et al., 2006; Taverna et al., 2006;
Shi et al., 2007). Further, the PHD within the yeast homolog of
CFP1, Spp1, binds histone H3K4me2=3(Shi et al., 2007). It will
be of interest to determine if the PHDs within mammalian
CFP1 bind methylated histone H3K4. Importantly, a point
mutation (C375A) that abolishes the interaction between
CFP1 and the Setd1A and Setd1B complexes does not ablate
the interaction between CFP1 and DNMT1, thus illustrating
histone methyltransferase complexes. (A) A schematic representation of CFP1 is shown. Various CFP1 protein fragments were
generated and are shown in schematic form. The filled circle at the N-terminus of each CFP1 truncation mutant represents the
FLAG epitope. NLS, nuclear localization signal derived from the CFP1 protein (Lee and Skalnik, 2002). (B) A series of FLAG-
CFP1 truncation mutants were expressed in HEK-293 cells and analyzed for interaction with endogenous components of
the Setd1A and Setd1B histone H3K4 methyltransferase complexes. The FLAG-CFP1 truncation mutants were im-
munoprecipitated from nuclear extracts using FLAG M2–conjugated agarose. Western blot analysis was performed using
antisera directed against the following Setd1A and B complex components: Setd1A, Setd1B, Ash2, Rbbp5, Wdr5, and Wdr82.
The membrane was re-probed with anti-FLAG antiserum to verify FLAG-CFP1 mutant protein expression and FLAG im-
munoprecipitation efficiency. Asterisks denote the position of each FLAG-tagged fusion protein. (C) A multispecies alignment
of the C-terminus of CFP1 is shown. Asterisks mark identical amino acid residues conserved between species. A double filled
circle marks a conserved amino acid substitution, while a single filled circle denotes a semi-conserved amino acid substitution.
Amino acids chosen for site-directed mutagenesis are underlined. (D) Site-directed mutagenesis was performed to change
cysteine 375 within the SID of CFP1 to alanine (C375A). A triple mutant within the SID of FLAG-CFP1 was generated that
encoded the following substitutions: tyrosine 390 to alanine, cysteine 391 to alanine, and serine 392 to alanine (YCS?AAA).
Finally, cysteine 580 within the PHD2 domain of CFP1 was mutated to alanine (C580A). The full-length FLAG-CFP1 mutants
encoding the single– or triple–amino acid point mutations were expressed in HEK-293 cells and analyzed for interaction with
the Setd1A and Setd1B complexes, as described above for (A).
Severalconserved aminoacidswithintheC-terminusofCFP1arerequiredfor interaction withtheSetd1AandSetd1B
CFP1 AND DNMT1 INTERACT IN VIVO
that CFP1 interacts independently with enzymes responsible
for cytosine methylation and histone methylation. These re-
sults are consistent with previously published studies that
failed to detect DNMT1 interaction with either the Setd1A or
Setd1B methyltransferase complexes (Lee and Skalnik, 2005;
Lee et al., 2007).
Immunoprecipitation analysis of truncated FLAG-DNMT1
proteins revealed two regions (amino acids 169–493 and 970–
1617) sufficient for interaction with Myc-CFP1. A similar re-
sult was recently observed for the interaction between
DNMT1 and the EZH2 histone H3K27 methyltransferase
(Vire et al., 2006). Two regions within the N-terminus of
DNMT1, amino acids 1–343 and 305–609, as well as the C-
terminal region encoding amino acids 1124–1620 are each
sufficient for interaction with EZH2 (Vire et al., 2006). The C-
terminus of DNMT1 requires interaction with the N-terminus
for catalytic activity (Fatemi et al., 2001; Margot et al., 2003).
Perhaps CFP1 binding to both regions stabilizes the in-
tramolecular interaction within DNMT1 to enhance enzy-
Direct interaction between CFP1 and DNMT1 fragments
was not observed using an in vitro histidine pull-down assay
(data not shown). However, these studies utilized protein
fragments isolated from Escherichia coli. Therefore, the in-
teraction would not have occurred if posttranslational
modifications of DNMT1 or CFP1 play a role in mediating
their interaction. DNMT1 interacts with a number of chro-
matin-associated proteins (Fuks et al., 2000, 2003; Robertson
et al., 2000; Rountree et al., 2000; Tatematsu et al., 2000; Kim
et al., 2002; Kimura and Shiota, 2003; Esteve et al., 2006;
Vire et al., 2006; Smallwood et al., 2007), and CFP1 is a
component of the *450kDa Setd1A and Setd1B complexes
(Lee and Skalnik, 2005; Lee et al., 2007). Therefore, it is also
possible that additional molecules or proteins mediate the
interaction between DNMT1 and CFP1.
While cytosine methylation is critical for embryonic de-
velopment and survival, the differentiation and survival de-
fects observed in ES cells lacking CFP1 are more severe than
those observed in cells deficient solely in global cytosine
methylation. Mouse embryos lacking DNMT1 exhibit a 70%
reduction in global cytosine methylation and exhibit embry-
onic lethality between embryonic days 9.5–11 (Li et al., 1992).
CFP1-deficient ES cells exhibit a 70% reduction in global cy-
tosine methylation, yet mouse embryos lacking CFP1 die
prior to implantation (E4.5–6.5) (Carlone and Skalnik, 2001;
Carlone et al., 2005). ES cells lacking CFP1 exhibit a threefold
increase in apoptosis in culture, whereas DNMT1?=?ES cells
do not undergo apoptosis until induction of differentiation
(Lei et al., 1996). Lastly, DNMT1þ=?ES cells express DNMT1
at 50% of wild-type levels, similar to what is observed in ES
cells lacking CFP1, yet do not exhibit a deficiency in global
cytosine methylation or methyltransferase activity (Li et al.,
1992, 1996). These observations suggest that a mechanism in
addition to a 50% reduction in DNMT1 protein levels con-
tributes to the epigenetic and differentiation defects observed
in ES cells lacking CFP1.
DNMT1 maintenance methylation activity at the DNA
replication fork is well characterized. However, the kinetics of
the DNMT1 methylation reaction are too slow to efficiently
fork (Pradhan et al., 1999; Easwaran et al., 2004). DNMT1
additionally methylates nucleosomal DNA (Okuwaki and
Verreault, 2004), and maintenance methyltransferase activ-
ity of DNMT1 is independent of its association with PCNA
(Spada et al., 2007). These results imply that additional
mechanisms exist to target DNMT1 to chromatin indepen-
dently ofprotein interactions at the DNAreplication fork. The
TS region of DNMT1 (amino acids 324–628) is responsible for
targeting DNMT1 to chromatin independent of DNA repli-
cation (Easwaran et al., 2004). A portion of the DNMT1 N-
terminal CFP1-interacting region, amino acids 169–493, is
contained within the TS region. This raises the possibility that
CFP1 could be involved in targeting DNMT1 to chromatin
independently of DNA replication. It will be important to
identify CFP1 target genes and determine if DNMT1 cooc-
cupies these loci.
Why does CFP1, a protein associated with euchromatic
regions of the genome, interact with DNMT1, a protein in-
volved in modifying heterochromatic regions? It is possible
that the interaction between these proteins occurs at times of
coexpressed with Myc-CFP1 (aa 361–656) or Myc-CFP1 (aa 361–656; C375A) in HEK-293 cells. Nuclear extracts were pre-
pared, and FLAG M2–conjugated agarose was used to immunoprecipitate FLAG-DNMT1 (aa 1–489). Western blot analysis
using anti-Myc antiserum was used to detect Myc-CFP1 (aa 361–656) or Myc-CFP1 (aa 361–656; C375A). Membranes were re-
probed with anti-FLAG antiserum to verify FLAG-DNMT1 (aa 1–489) protein expression and FLAG immunoprecipitation
efficiency. Transfection and immunoprecipitation of Myc-CFP1 (aa 361–656) or Myc-CFP1 (aa 361–656; C375A) with empty
vector served as negative controls for the FLAG immunoprecipitation.
Interaction with Setd1A or Setd1B is not required for CFP1 interaction with DNMT1. FLAG-DNMT1 (aa 1–489) was
540BUTLER ET AL.
transition from euchromatin to heterochromatin states. For
example, after transcription the open chromatin template
requires repackaging into a transcriptionally inactive chro-
matin context to suppress aberrant transcription reinitiation
(Kaplan et al., 2003; Mason and Struhl, 2003; Carrozza et al.,
2005). The presence of DNA methylation at the junction of
transcription initiation and coding regions is sufficient for
exclusion of histone H3K4me2, and may play a role in pro-
tecting transcriptionally repressed regions from aberrant
transcription initiation (Okitsu and Hsieh, 2007). An alter-
native explanation would be a mechanism to specifically si-
lence euchromatic genes. For example, the PHD contained in
the inhibitor of growth 2 protein binds histone H3K4me3
within the cyclin D1 and c-Myc promoters and directly leads
to transcriptional repression via recruitment of the mSin3a–
HDAC1 complex upon DNA damage (Shi et al., 2006). Per-
haps CFP1 recruits DNMT1 to specific euchromatic targets in
a similar manner to initiate specific gene repression.
A recent study provides evidence that DNA methylation
by DNMT1 is required for G9a and HP1 recruitment in order
to initiate heterochromatin formation required to silence eu-
chromatic genes (Smallwood et al., 2007). Perhaps decreased
heterochromatin formation via reduced cytosine methylation,
combined with increased histone H3K4 methylation activity
of the Setd1A and Setd1B complexes, results in the global
euchromatin architecture observed in CFP1-deficient ES cells.
These observations suggest that the function of CFP1 within
the Setd1A and Setd1B histone H3K4 methyltransferase
complexes in conjunction with its role in regulating cytosine
methylation may explain the differentiation and survival de-
fects in CFP1-deficient ES cells.
This work was supported by the Riley Children’s Foun-
dation, the Lilly Endowment, a Showalter Trust award (to J.-
H.L.), and National Science Foundation Grants MCB-0344870
and MCB-0641851 (to D.G.S.). J.S.B. was supported by a
predoctoral fellowship from National Institutes of Health
Grant T32 CA111198. We thank Dr. Sriharsa Pradhan (New
Burgers, W.A., Fuks, F., and Kouzarides, T. (2002). DNA me-
thyltransferases get connected to chromatin. Trends Genet 18,
Callebaut, I., Courvalin, J.-C., and Mornon, J.-P. (1999). The BAH
(bromo-adjacent homology) domain: a link between DNA
methylation, replication, and transcriptional regulation. FEBS
Lett 446, 189–193.
Carlone, D.L., Lee, J.-H., Young, S.R.L., Dobrota, E., Butler, J.S.,
Ruiz, J., and Skalnik, D.G. (2005). Reduced genomic cytosine
methylation and defective cellular differentiation in embryonic
stem cells lacking CpG binding protein. Mol Cell Biol 25, 4881–
Carlone, D.L., and Skalnik, D.G. (2001). CpG binding protein is
crucial for early embryonic development. Mol Cell Biol 21,
Carrozza, M.J., Li, B., Florens, L., Suganuma, T., Swanson, S.K.,
Lee, K.K., Shia, W.J., Anderson, S., Yates, J., Washburn, M.P.,
and Workman, J.L. (2005). Histone H3 methylation by Set2
directs deacetylation of coding regions by Rpd3S to suppress
spurious intragenic transcription. Cell 123, 581–592.
Dennis, K., Fan, T., Geiman, T., Yan, Q., and Muegge, K. (2001).
Lsh, a member of the SNF2 family, is required for genome-
wide methylation. Genes Dev 15, 2940–2944.
Easwaran, H.P., Schermelleh, L., Leonhardt, H., and Cardoso,
M.C. (2004). Replication-independent chromatin loading of
DNMT1 during G2 and M phases. EMBO Rep 5, 1181–1186.
Espada, J., Ballestar, E., Fraga, M.F., Villar-Garea, A., Juarranz,
A., Stockert, J.C., Robertson, K.D., Fuks, F., and Esteller, M.
(2004). Human DNA methyltransferase 1 is required for
maintenance of the histone H3 modification pattern. J Biol
Chem 279, 37175–37184.
Esteve, P.-O., Chin, H.G., and Pradhan, S. (2005). Human
maintenance DNA (cytosine-5)-methyltransferase and p53
modulate expression of p53-repressed promoters. Proc Natl
Acad Sci USA 102, 1000–1005.
Esteve, P.-O., Chin, H.G., Smallwood, A., Feehery, G.R., Gang-
isetty, O., Karpf, A.R., Carey, M.F., and Pradhan, S. (2006).
Direct interaction between DNMT1 and G9a coordinates DNA
and histone methylation during replication. Genes Dev 20,
Fatemi, M., Hermann, A., Pradhan, S., and Jeltsch, A. (2001).
The activity of the murine DNA methyltransferase Dnmt1 is
controlled by interaction of the catalytic domain with the
N-terminal part of the enzyme leading to an allosteric acti-
vation of the enzyme after binding to methylated DNA. J Mol
Biol 309, 1189–1199.
Freitag, M., Hickey, P.C., Khlafallah, T.K., Read, N.D., and
Selker, E.U. (2004). HP1 is essential for DNA methylation in
Neurospora. Mol Cell 13, 427–434.
Fuks, F., Burgers, W.A., Brehm, A., Hughes-Davies, L., and
Kouzarides, T. (2000). DNA methyltransferase Dnmt1 associ-
ates with histone deacetylase activity. Nat Genet 24, 88–91.
Fuks, F., Burgers, W.A., Godin, N., Kasai, M., and Kouzarides, T.
(2001). Dnmt3a binds deacetylases and is recruited by a
sequence-specific repressor to silence transcription. EMBO J 20,
Fuks, F., Hurd, P.J., Deplus, R., and Kouzarides, T. (2003).
The DNA methyltransferases associate with HP1 and the
SUV39H1 histone methyltransferase. Nucleic Acids Res 31,
Jeddeloh, J.A., Bender, J., and Richards, E.J. (1998). The DNA
methylation locus DDM1 is required for maintenance of gene
silencing in Arabidopsis. Genes Dev 12, 1714–1725.
Jeddeloh, J.A., Stokes, T.L., and Richards, E.J. (1999). Main-
tenance of genomic methylation requires a SWI2=SNF-like
protein. Nat Genet 22, 94–97.
Kaplan, C.D., Laprade, L., and Winston, F. (2003). Transcription
elongation factors repress transcription initiation from cryptic
sites. Science 310, 1096–1099.
Kim, G.-D., Ni, J., Kelesoglu, N., Roberts, R.J., and Pradhan, S.
(2002). Co-operation and communication between the human
maintenance and de novo DNA (cytosine-5) methyltrans-
ferases. EMBO J 21, 4183–4195.
Kimura, H., and Shiota, K. (2003). Methyl-CpG binding protein,
MeCP1, is a target molecule for maintenance DNA methyl-
transferase, Dnmt1. J Biol Chem 278, 4806–4812.
Lee, J.-H., Shimojo, M., Chai, Y.-G., and Hersh, L.B. (2000).
Studies on the interaction of REST4 with the cholinergic re-
pressor element-1=neuron restrictive silencer element. Mol
Brain Res 80, 88–98.
Lee, J.-H., and Skalnik, D.G. (2002). CpG binding protein is a
nuclear matrix- and euchromatin-associated protein localized
CFP1 AND DNMT1 INTERACT IN VIVO
to nuclear speckles containing human trithorax: identification
of nuclear matrix targeting signals. J Biol Chem 277, 42259–
Lee, J.-H., and Skalnik, D.G. (2005). CpG binding protein (CXXC
finger protein 1) is a component of the mammalian Set1 his-
tone H3-Lys4 methyltransferase complex, the analogue of
the yeast Set1=COMPASS complex. J Biol Chem 280, 41725–
Lee, J.-H., Tate, C.M., You, J.-S., and Skalnik, D.G. (2007).
Identification and characterization of the human Set1B histone
H3-Lys4 methyltransferase complex. J Biol Chem 282, 13419–
Lee, J.-H., Voo, K.S., and Skalnik, D.G. (2001). Identification and
characterization of the DNA binding domain of CpG-binding
protein. J Biol Chem 276, 44669–44676.
Lehnertz, B., Ueda, Y., Derijck, A.A.H.A., Braunschweig, U.,
Perez-Burgos, L., Kubicek, S., Chen, T., Li, E., Jenuwein, T.,
and Peters, A. (2003). Suv39h-mediated histone H3 lysine
9 methylation directs DNA methylation to major satellite
repeats at pericentric heterochromatin. Curr Biol 13, 1192–
Lei, H., Oh, S.P., Okano, M., Juttermann, R., Goss, K.A., Jaenisch,
R., and Li, E. (1996). De novo DNA cytosine methyltransferase
activities in mouse embryonic stem cells. Development 122,
Li, E., Bestor, T.H., and Jaenisch, R. (1992). Targeted mutation of
the DNA methyltransferase gene results in embryonic lethal-
ity. Cell 69, 915–926.
Margot, J.G., Ehrenhofer-Murray, A.E., and Leonhardt, H.
(2003). Interactions within the mammalian DNA methyltrans-
ferase family. BMC Mol Biol 4, 7.
Martin, D.G., Baetx, K., Shi, X., Walter, K.L., MacDonald, V.E.,
Wlodarski, M.J., Gozani, O., Hieter, P., and Howe, L. (2006).
The Yng1p plant homeodomain finger is a methyl-histone
binding module that recognizes lysine 4-methylated histone
H3. Mol Cell Biol 26, 7871–7879.
Mason, P.B., and Struhl, K. (2003). The FACT complex travels
with elongating RNA polymerase II and is important for the
fidelity of transcriptional initiation in vivo. Mol Cell Biol 23,
Miller, T., Krogan, N.J., Dover, J., Erdjument-Bromage, H.,
Tempst, P., Johnston, M., Greenblatt, J.F., and Shilatifard, A.
(2001). COMPASS: a complex of proteins associated with a
trithorax-related SET domain protein. Proc Natl Acad Sci USA
Nagy, P.L., Griesenbeck, J., Kornberg, R.D., and Cleary, M.L.
(2002). A trithorax-group complex purified from Saccharomyces
cerevisiae is required for methylation of histone H3. Proc Natl
Acad Sci USA 99, 90–94.
Okitsu, C.Y., and Hsieh, C.-L. (2007). DNA methylation dictates
histone H3K4 methylation. Mol Cell Biol 27, 2746–2757.
Okuwaki, M., and Verreault, A. (2004). Maintenance DNA
methylation of nucleosome core particles. J Biol Chem 279,
Pradhan, S., Bacolla, A., Wells, R.D., and Roberts, R.J. (1999).
Recombinant human DNA (cytosine-5) methyltransferase. I.
Expression, purification, and comparison of de novo and
maintenance methylation. J Biol Chem 274, 33002–33010.
Robertson, K.D., Ait-Si-Ali, S., Yokochi, T., Wade, P.A., Jones,
P.L., and Wolffe, A.P. (2000). DNMT1 forms a complex with
Rb, E2F1 and HDAC1 and represses transcription from E2F-
responsive promoters. Nat Genet 25, 338–342.
Roguev, A., Schaft, D., Shevchenko, A., Aasland, R., Shevchen-
ko, A., and Stewart, A.F. (2003). High conservation of the
Set1=Rad6 axis of histone 3 lysine 4 methylation in budding
and fission yeasts. J Biol Chem 278, 8487–8493.
Rountree, M.R., Bachman, K.E., and Baylin, S.B. (2000). DNMT1
binds HDAC2 and a new co-repressor, DMAP1, to form a
complex at replication foci. Nat Genet 25, 269–277.
Schneider, J., Wood, A., Lee, J.S., Schuster, R., Dueker, J.,
Maguire, C., Swanson, S.K., Florens, L., Washburn, M.P., and
Shilatifard, A. (2005). Molecular regulation of histone H3
trimethylation by COMPASS and the regulation of gene ex-
pression. Mol Cell 19, 849–856.
Selker, E.U. (1998). Trichostatin A causes selective loss of DNA
methylation in Neurospora. Proc Natl Acad Sci USA 95, 9430–
Shi, X., Hong, T., Walter, K.L., Ewalt, M., Michishita, E.,
Hung, T., Carney, D., Pena, P., Lan, F., Kaadige, M.R.,
Lacoste, N., Cayrou, C., Davrazou, F., Saha, A., Cairns,
B.R., Ayer, D.E., Kutateladze, T.G., Shi, Y., Cote, J., Chua,
K.F., and Gozani, O. (2006). ING2 PHD domain links histone
H3 lysine 4 methylation to active gene repression. Nature
Shi, X., Kachirskaia, I., Walter, K.L., Kui, J.-H.A., Lake, A.,
Davrazou, F., Chan, S.M., Martin, D.G.E., Fingerman, I.M.,
Briggs, S.D., Howe, L., Utz, P.J., Kutateladze, T.G., Lugovskoy,
A.A., Bedford, M.T., and Gozani, O. (2007). Proteome-wide
analysis in S. cerevisiae identifies several PHD fingers as
novel direct and selective binding modules of histone H3
methylated at either lysine 4 or lysine 36. J Biol Chem 282,
Smallwood, A., Esteve, P.-O., Pradhan, S., and Carey, M. (2007).
Functional cooperation between HP1 and DNMT1 mediates
gene silencing. Genes Dev 21, 1169–1178.
Spada, F., Haemmer, A., Kuch, D., Rothbauer, U., Schermelleh,
L., Kremmer, E., Carell, T., Langst, G., and Leonhardt, H.
(2007). DNMT1 but not its interaction with the replication
machinery is required for maintenance of DNA methylation in
human cells. J Cell Biol 176, 565–571.
Tamaru, H., and Selker, E.U. (2001). A histone H3 methyl-
transferase controls DNA methylation in Neurospora crassa.
Nature 414, 277–283.
Tamaru, H., Zhang, X., McMillen, D., Singh, P.B., Nakayama,
J.I., Grewal, S.I., Allis, C.D., Cheng, X., and Selker, E.U.
(2003). Trimethylated lysine 9 of histone H3 is a mark
for DNA methylation in Neurospora crassa. Nat Genet 34,
Tatematsu, K.-I., Yamazaki, T., and Ishikawa, F. (2000). MBD2-
MBD3 complex binds to hemi-methylated DNA and forms a
complex containing DNMT1 at the replication fork in late S
phase. Genes Cells 5, 677–688.
Taverna, S.D., Ilin, S., Rogers, R.S., Tanny, J.C., Lavender, H., Li,
H., Baker, L., Boyle, J., Blair, L.P., Chait, B.T., Patel, D.J.,
Aitchison, J.D., Tackett, A.J., and Allis, C.D. (2006). Yng1 PHD
finger binding to H3 trimethylated at K4 promotes NuA3
HAT activity at K14 of H3 and transcription at a subset of
targeted ORFs. Mol Cell 24, 785–796.
Vire, E., Brenner, C., Deplus, R., Blanchon, L., Fraga, M.F.,
Didelot, C., Morey, L., van Eynde, A., Bernard, D., Van-
derwinden, J.M., Bollen, M., Esteller, M., DiCroce, L., de
Launoit, Y., and Fuks, F. (2006). The polycomb group protein
EZH2 directly controls DNA methylation. Nature 439, 871–
Voo, K.S., Carlone, D.L., Jacobsen, B.M., Flodin, A., and Skalnik,
D.G. (2000). Cloning of a mammalian transcriptional activator
that binds unmethylated CpG motifs and shares a CXXC
domain with DNA methyltransferase, human trithorax, and
542BUTLER ET AL.
methyl-CpG binding domain protein 1. Mol Cell Biol 20,
Wysocka, J., Swigut, T., Xiao, H., Milne, T.A., Kwon, S.Y.,
Landry, J., Kauer, M., Tackett, A.J., Chait, B.T., Badenhorst, P.,
Wu, C.-L., and Allis, C.D. (2006). A PHD finger of NURF
couples histone H3 lysine 4 trimethylation with chromatin
remodelling. Nature 442, 86–90.
Young, S.R.L., Mumaw, C., Marrs, J.A., and Skalnik, D.G. (2006).
Antisense targeting of CXXC finger protein 1 inhibits genomic
cytosine methylation and primitive hematopoiesis in zebra-
fish. J Biol Chem 281, 37034–37044.
Young, S.R.L., and Skalnik, D.G. (2007). CXXC-finger protein 1 is
required for normal proliferation and differentiation of the
PLB-985 myeloid cell line. DNA Cell Biol 26, 80–90.
Zhang, X., and Verdine, G.L. (1996). Mammalian DNA cytosine-
5-methyltransferase interacts with p23 protein. FEBS Lett 392,
Address reprint requests to:
David G. Skalnik, Ph.D.
Cancer Research Building, Room W327
1044 West Walnut St.
Indianapolis, IN 46202
Received for publication December 12, 2007; received in
revised form May 9, 2008; accepted May 29, 2008.
CFP1 AND DNMT1 INTERACT IN VIVO