Báo cáo khoa học: CXXC finger protein 1 restricts the Setd1A histone H3K4 methyltransferase complex to euchromatin doc

CXXC finger protein 1 restricts the Setd1A histone H3K4
methyltransferase complex to euchromatin
Courtney M. Tate, Jeong-Heon Lee and David G. Skalnik
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, IN, USA
Introduction
DNA in eukaryotic cells is complexed with histones and
other proteins in the form of chromatin. The core histone
tails are subject to a variety of covalent modifications,
including acetylation, phosphorylation, methylation,
ubiquitination, sumoylation, and ADP-ribosylation
[1,2]. Histone methylation plays critical roles in gene
expression, epigenetic regulation, and disease [3].
Histone methylation is catalyzed by a family of histone
methyltransferase (HMT) enzymes, many of which are
characterized by an evolutionarily conserved catalytic
SET [Su(var)3–9, Enhancer of Zeste, Trithorax] domain
[4]. A major function of the SET domain-containing
proteins is to modulate gene activity [5]. Lys residues
of histones can be monomethylated, dimethylated,
Keywords
chromatin; epigenetics; histone methylation;
subnuclear targeting
Correspondence
D. Skalnik, Cancer Research Building, 1044
West Walnut Street, Indianapolis, IN 46202,
USA
Fax: +1 317 278 9298
Tel: +1 317 274 8977
E-mail:
(Received 21 September 2009, revised 28

October 2009, accepted 4 November 2009)
doi:10.1111/j.1742-4658.2009.07475.x
CXXC finger protein 1 (Cfp1), encoded by the CXXC1 gene, is a compo-
nent of the euchromatic Setd1A histone H3K4 methyltransferase complex,
and is a critical regulator of histone methylation, cytosine methylation, cel-
lular differentiation, and vertebrate development. Murine embryonic stem
(ES) cells lacking Cfp1 (CXXC1
) ⁄ )
) are viable but show increased levels of
global histone H3K4 methylation, suggesting that Cfp1 functions to inhibit
or restrict the activity of the Setd1A histone H3K4 methyltransferase com-
plex. The studies reported here reveal that ES cells lacking Cfp1 contain
decreased levels of Setd1A and show subnuclear mislocalization of both
Setd1A and trimethylation of histone H3K4 with regions of heterochroma-
tin. Remarkably, structure–function studies reveal that expression of either
the N-terminal fragment of Cfp1 (amino acids 1–367) or the C-terminal
fragment of Cfp1 (amino acids 361–656) is sufficient to restore appropriate
levels of Setd1A in CXXC1
) ⁄ )
ES cells. Furthermore, functional analysis
of various Cfp1 point mutations reveals that retention of either Cfp1
DNA-binding activity or association with the Setd1 histone H3K4 methyl-
transferase complex is required to restore normal Setd1A levels. In con-
trast, expression of full-length Cfp1 in CXXC1
) ⁄ )
ES cells is required to
restrict Setd1A and histone H3K4 trimethylation to euchromatin, indicat-
ing that both Cfp1 DNA-binding activity and interaction with the Setd1A
complex are required for appropriate genomic targeting of the Setd1A
complex. These studies illustrate the complexity of Cfp1 function, and

identify Cfp1 as a regulator of Setd1A genomic targeting.
Abbreviations
CTD, C-terminal repeat domain; DAPI, 4¢,6-diaminidino-2-phenylindone; Dnmt1, DNA methyltransferase 1; ES, embryonic stem; FITC,
fluorescein isothiocyanate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; H3K4me3, trimethylated histone H3K4; HMT, histone
methyltransferase; PHD, plant homeodomain; RNAP, RNA polymerase; Ser5-P CTD, C-terminal repeat domain phosphorylated at Ser5; SID,
Set1 interaction domain.
210 FEBS Journal 277 (2010) 210–223 ª 2009 The Authors Journal compilation ª 2009 FEBS
or trimethylated, and the functional relevance of these
modifications depends on the position. For example,
dimethylated and trimethylated histone H3K4 is found
associated with promoters and 5¢-regions of active genes
[6], whereas dimethylated and trimethylated his-
tone H3K9 is present at transcriptionally inactive chro-
matin sites [7–9]. Yeast express a single H3K4 HMT,
Set1, which associates with a complex known as COM-
PASS (Complex Proteins Associated with Set1) [10] and
is required for telomeric and rDNA silencing [11,12]. In
contrast, mammalian cells contain numerous HMTs that
show specificity for histone H3K4, including Setd1A,
Setd1B, Mll1, Mll2, Mll3 ⁄ Halr, Mll4 ⁄ Alr, Ash1L,
Smyd1, Smyd2, Smyd3, and Set7 ⁄ 9, which are present as
distinct multiprotein complexes and play critical roles in
gene expression and development [4,13–16].
The molecular mechanisms that control the targeting
and activity of HMT complexes are not well under-
stood. Methylation at histone H3K4 correlates with
transcriptional activation and is directly coupled to the
transcription process [17]. In yeast and mammals, Set1
and Setd1A localize to the 5¢-end of actively tran-
scribed genes and interact with the RNA polymerase

(RNAP) II C-terminal domain (CTD) phosphorylated
at Ser5 (Ser5-P CTD), a repeat marker associated with
transcription initiation [18–20]. In yeast, Paf1C interac-
tion with RNAP II is required for recruitment of the
Set1–COMPASS H3K4 HMT complex to actively
transcribed genes [19]. In mammals, Setd1A is tethered
to RNAP II by Wdr82, an integral component of the
Setd1A complex [18]. Wdr82 associates with the RNA
recognition motif within Setd1A, and directly recog-
nizes Ser5-P CTD of RNAP II [18]. In mammals, Mll1
interacts with RNAP II containing Ser5-P CTD and
mediates histone H3K4 methylation at a subset of
transcriptionally active genes [21]. In addition, menin,
a component of the Mll2 H3K4 HMT complex, associ-
ates with RNAP II containing Ser5-P CTD [22]. In
yeast and mammals, the Setd2 H3K36 HMT primarily
associates with the elongating hyperphosphorylated
form of RNAP II [23,24]. Therefore, histone methyla-
tion mediated by HMTs is involved in regulating both
transcription initiation and elongation.
Although generally widely expressed, mammalian
H3K4 HMTs have nonredundant functions. For exam-
ple, Mll2 is important for expression of the HOXB
gene cluster, but not the HOXA cluster [13], whereas
HOXA9 and HOXC8 are exclusive Mll1 targets
[22,25]. The HMTs Ash1L and Mll1 occupy the
5¢-regions of active genes, and their localization is
nearly indistinguishable, which suggests redundancy of
function [14]. However, in vivo depletion of either
enzyme results in diminished methylation of histone

H3K4 at active HOXA genes [14]. In addition, loss
of a single member of the H3K4 HMT family can lead
to disease or death [26,27]. MLL1 is frequently the
target of chromosomal translocations involved in
acute lymphoid and myeloid leukemias [28–31]. In
addition, genetic disruption of murine MLL1 or
MLL2 leads to embryonic lethality [13,32]. In addition,
Smyd3 expression is upregulated in colorectal and
hepatocellular carcinomas, and its H3K4 HMT activity
activates oncogenes and other genes associated with
the cell cycle, whereas depletion of Smyd3 by small
interfering RNA treatment leads to suppression of cell
growth [27].
With the exception of the enzymatic Setd1 compo-
nent, the subunit composition of the mammalian
Setd1A and Setd1B HMTase complexes are identical
[16], each containing CXXC finger protein 1 (Cfp1),
Rbbp5, Wdr5, Ash2, and Wdr82 [15,16]. Setd1A and
Setd1B mRNA are ubiquitously expressed in murine
tissues, and Setd1A and Setd1B do not show differen-
tial cell type expression [16]. However, confocal immu-
nofluorescence reveals that endogenous Setd1A and
Setd1B show largely nonoverlapping subnuclear locali-
zation [16]. This suggests that Setd1A and Setd1B are
targeted to unique sets of genomic sites, and that each
has unique functions in the regulation of chromatin
structure and gene expression. Consequently, it is
likely that the nonredundant function of each H3K4
HMT is a result of distinct target gene specificity [16].
Cfp1 is a critical epigenetic regulator of both

cytosine methylation and histone methylation, and
interacts with both the maintenance DNA methyltrans-
ferase [DNA methyltransferase 1 (Dnmt1)] [33] and
with the Setd1A H3K4 HMT complex [15]. Cfp1
localizes nearly exclusively to euchromatic nuclear
speckles, and associates with the nuclear matrix [34].
Cfp1 contains two Cys-rich plant homeodomains
(PHDs); a PHD is a Cys-rich CXXC DNA-binding
domain that shows specificity for unmethylated CpG
dinucleotides, an acidic domain, a basic domain, a
coiled-coil domain, and a Cys-rich Set1 interaction
domain (SID), which is required for interaction with
the Setd1A and Setd1B H3K4 HMT complexes
[33,35,36].
Disruption of murine CXXC1 results in embryonic
lethality shortly after implantation [37]. Murine embry-
onic stem (ES) cell lines lacking Cfp1 (CXXC1
) ⁄ )
) are
viable but show a variety of defects, including an
increased population doubling time due to increased
apoptosis, a  70% decrease in global cytosine methyl-
ation, decreased Dnmt1 protein expression and main-
tenance DNA methyltransferase activity, and an
inability to achieve in vitro differentiation [38]. In
C. M. Tate et al. Cfp1 restricts the Setd1A complex to euchromatin
FEBS Journal 277 (2010) 210–223 ª 2009 The Authors Journal compilation ª 2009 FEBS 211
addition, CXXC1
) ⁄ )
ES cells express elevated levels of

histone H3K4 dimethylation and trimethylation, and
reduced levels of histone H3K9 dimethylation [15].
Consequently, Cfp1 plays an important role in the reg-
ulation of cytosine methylation, histone methylation,
and cellular differentiation.
The purpose of this study was to obtain insights into
the molecular mechanisms regulating the activity and
targeting of the Setd1A H3K4 HMT complex. The
results reported here reveal that CXXC1
) ⁄ )
ES cells
contain reduced levels of Setd1A and show mislocal-
ization of both Setd1A protein and trimethylated
histone H3K4 (H3K4me3) to areas of heterochro-
matin. Surprisingly, expression in CXXC1
) ⁄ )
ES cells
of either the amino half of Cfp1 (amino acids 1–367)
or carboxyl half of Cfp1 (amino acids 361–656) is
sufficient to restore appropriate levels of Setd1A.
However, full-length Cfp1 is required to restrict the
subnuclear localization of both Setd1A and H3K4me3
to euchromatin.
Results
ES cells lacking Cfp1 contain decreased levels of
Setd1A
Exogenous expression of Setd1A fragments in HEK293
cells competes with endogenous Setd1A binding with
the Setd1A H3K4 HMT complex, resulting in decreased
stability of endogenous Setd1A [16]. To examine

whether loss of Cfp1 has a similar effect, western blot
analysis was performed to determine protein levels of
Setd1A complex components in wild-type ES cells
(CXXC1
+ ⁄ +
), ES cells heterozygous for the disrupted
CXXC1 allele (CXXC1
+ ⁄ )
), ES cells lacking Cfp1
(CXXC1
) ⁄ )
), CXXC1
) ⁄ )
ES cells transfected with a
full-length Cfp1 expression vector (Rescue), and
CXXC1
) ⁄ )
ES cells cells carrying the empty expression
vector (Vector). A significant decrease ( 50%) in the
level of Setd1A was observed in CXXC1
) ⁄ )
ES cells
(Fig. 1A). Appropriate levels of Setd1A were restored
upon introduction of a Cfp1 expression vector (Rescue),
but not in ES cells carrying the empty expression vector
(Vector). CXXC1
+ ⁄ )
ES cells express approximately
50% as much Cfp1 as CXXC1
+ ⁄ +

ES cells [38], and
show a slight decrease in Setd1A levels. In contrast, no
difference in protein levels was observed for the other
Setd1A HMT complex components (Rbbp5, Wdr5,
Wdr82, and Ash2) in CXXC1
) ⁄ )
ES cells (Fig. 1A).
Previous work demonstrated that Cfp1 functions as
a transcriptional activator in cotransfection assays
[34,36]. Thus, further studies were performed to exam-
ine whether reduced Setd1A levels in ES cells lacking
Cfp1 are due to reduced transcription of the cognate
gene. Surprisingly, quantitative real-time PCR analysis
demonstrated that Setd1A mRNA levels were elevated
four-fold to five-fold in CXXC1
) ⁄ )
ES cells as com-
pared with CXXC1
+ ⁄ +
and CXXC1
+ ⁄ )
ES cells, and
are restored to wild-type levels in rescued ES cells but
not in CXXC1
) ⁄ )
ES cells carrying the empty expres-
sion vector (Fig. 1B). Therefore, the decreased levels of
Setd1A observed in CXXC1
) ⁄ )
ES cells is not

explained by reduced transcription of SETD1A.
Previous work by our laboratory demonstrated that
disruption of the interaction between endogenous
Setd1A and other components of the intact HMT
complex led to a reduction of Setd1A levels as a conse-
quence of a reduced Setd1A half-life [16]. Additional
studies were therefore performed to assess the role of
protein stability in Setd1A levels in CXXC1
) ⁄ )
ES
cells. These experiments revealed that treatment of
CXXC1
) ⁄ )
ES cells with the proteosome inhibitor
MG132 led to an elevation of Setd1A levels to near
wild-type levels (Fig. 1C).
Cfp1 is required to restrict Setd1A and H3K4me3
to euchromatin
The molecular mechanisms regulating HMT activity
and genomic targeting remain largely unknown. Previ-
ous studies revealed the paradoxical finding that ES
cells lacking the Cfp1 component of the Setd1A H3K4
HMT complex have increased levels of histone H3K4
methylation. These findings suggest that Cfp1 may
inhibit or restrict the activity of the Setd1A HMT
complex. To examine this issue further, subnuclear
localization of Setd1A relative to 4¢,6-diaminidino-
2-phenylindone (DAPI) staining was examined by
confocal immunofluorescence. DAPI is a fluorescent
DNA stain that preferentially binds to the condensed

structure of pericentromeric heterochromatin [39].
Quantification of colocalization revealed that Setd1A
showed only a slight ( 4%) overlap with DAPI-
bright heterochromatin in wild-type ES cells. However,
a significant (four-fold to five-fold) increase in colocal-
ization of Setd1A with DAPI-bright heterochromatin
was observed in CXXC1
) ⁄ )
ES cells (Fig. 2A). Rescue
of appropriate restriction of Setd1A to euchromatin
was observed in CXXC1
) ⁄ )
ES cells expressing
full-length Cfp1 (1–656), but not in cells carrying the
empty expression vector (Fig. 2A).
The subnuclear localization of H3K4me3, a product
of Setd1A HMT activity, was similarly analyzed
by confocal immunofluorescence. Consistent with the
findings of Setd1A mislocalization in CXXC1
) ⁄ )
ES
cells, quantification of overlap between H3K4me3 and
Cfp1 restricts the Setd1A complex to euchromatin C. M. Tate et al.
212 FEBS Journal 277 (2010) 210–223 ª 2009 The Authors Journal compilation ª 2009 FEBS
DAPI-bright heterochromatin indicated that H3K4me3
showed only a slight overlap with DAPI-bright heteo-
chromatin in wild-type ES cells. However, a significant
(five-fold to six-fold) increase in colocalization of
H3K4me3 with DAPI-bright heterochromatin regions
was observed in CXXC1

) ⁄ )
ES cells (Fig. 2B). Rescue
of appropriate subnuclear localization of H3K4me3
was observed in CXXC1
) ⁄ )
ES cells expressing full-
length Cfp1 (1–656), but not in cells carrying the
empty expression vector (Fig. 2B). These results
demonstrate that ES cells lacking Cfp1 show partial
mislocalization of both Setd1A and H3K4me3 to
DAPI-bright regions of heterochromatin, and reveal
that Cfp1 restricts the Setd1A H3K4 HMT complex to
euchromatin.
Retention of either Cfp1 DNA-binding activity or
association with the Setd1A HMT complex is
required to restore appropriate levels of Setd1A
The defects in Setd1A level and localization observed
in CXXC1
) ⁄ )
ES cells were corrected upon introduc-
tion of a full-length Cfp1 expression vector (Figs 1 and
2), thus providing a convenient method for assessment
of the structure–function relationships of Cfp1. Vari-
ous cDNA expression constructs encoding FLAG-
tagged Cfp1 truncations and mutations were stably
expressed in CXXC1
) ⁄ )
ES cells to identify the
functional domains of Cfp1 that are necessary and suf-
ficient to restore normal levels of Setd1A (Fig. 3A).

Isolated ES cell lines were screened for protein
Fig. 1. ES cells lacking Cfp1 contain decreased levels of Setd1A. (A) Whole cell protein extracts were isolated from the ES cell lines
CXXC1
+ ⁄ +
, CXXC1
+ ⁄ )
, CXXC1
) ⁄ )
, and CXXC1
) ⁄ )
, expressing full-length Cfp1 (Rescue), and CXXC1
) ⁄ )
, carrying the empty expression vector
(Vector). Extracts were subjected to western blot analysis, using antisera directed against the Setd1A HMT complex components Setd1A,
Cfp1, Ash2, Rbbp5, Wdr5, and Wdr82. The graph presents the relative level of Setd1A normalized to b-actin expression from at least three
independent experiments, and error bars indicate standard error. Asterisks denote statistically significant (P < 0.05) differences as compared
with CXXC1
+ ⁄ +
ES cells. (B) Quantitative RT-PCR was performed to assess Setd1A mRNA levels in the indicated ES cell lines. The graph
presents Setd1A transcript levels relative to those for GAPDH from three independent experiments, and error bars indicate standard error.
Asterisks denote statistically significant differences (P < 0.05) as compared with CXXC1
+ ⁄ +
ES cells. (C) Western blot analysis was
performed as described in (A) to assess Setd1A levels in CXXC1
) ⁄ )
ES cells following treatment with 5 lM MG132 for 6 h.
C. M. Tate et al. Cfp1 restricts the Setd1A complex to euchromatin
FEBS Journal 277 (2010) 210–223 ª 2009 The Authors Journal compilation ª 2009 FEBS 213
expression by western blot analysis, using an antibody
against Cfp1. CXXC1

+ ⁄ )
ES cells express  50% as
much Cfp1 as CXXC1
+ ⁄ +
ES cells, but show normal
levels of cytosine methylation and histone methylation,
and are able to differentiate in vitro [38]. Consequently,
clones were selected for analysis that have at least
50% of the level of Cfp1 observed in CXXC1
+ ⁄ +
ES
cells [44].
Expression of a C-terminal deletion fragment of
Cfp1 that lacks PHD2 (amino acids 1–481), or an
N-terminal deletion fragment that lacks PHD1, the
CXXC domain and the acidic domain (amino
acids 302–656), leads to restoration of normal levels of
Setd1A, indicating that none of these Cfp1 domains
are necessary for this rescue activity (Fig. 3B). Surpris-
ingly, expression of either the amino half of Cfp1
(amino acids 1–367, containing PHD1, and the CXXC,
acidic and basic domains) or the carboxyl half of Cfp1
(amino acids 361–656, containing the coiled-coil
domain, SID, and PHD2) is sufficient to restore
appropriate levels of Setd1A, indicating that Cfp1
contains redundant functional domains that support
Setd1A levels, and that no single Cfp1 domain is
essential for this function (Fig. 3B).
The N-terminal fragment of Cfp1 (amino acids
1–367) contains the CXXC DNA-binding domain, and

the C-terminal Cfp1 fragment (amino acids 361–656)
contains the SID [33]. Previous work determined that
mutation of a conserved Cys residue (C169A) within
the CXXC domain ablates Cfp1 DNA-binding activity
[35], and mutation of a conserved Cys residue within
the SID (C375A) ablates the interaction of Cfp1 with
Fig. 2. Cfp1 is required to restrict Setd1A
and H3K4me3 to euchromatin. (A) The sub-
nuclear distribution of endogenous Setd1A
was determined in CXXC1
+ ⁄ +
, CXXC1
) ⁄ )
and CXXC1
) ⁄ )
ES cells expressing full-
length Cfp1 (amino acids 1–656) or carrying
the empty expression vector, using rabbit
antibody against Setd1A and FITC-conju-
gated bovine anti-rabbit IgG as secondary
antibody. Nuclei were counterstained with
DAPI and observed by confocal microscopy.
Colocalization is indicated by yellow in the
merged and colocalization image. The num-
bers inside the colocalization image indicate
the percentage colocalized signal for the
presented nucleus. The numbers outside of
the image summarize the average percent-
age overlap of Setd1A with DAPI-bright het-
erochromatin and standard error for at least

30 nuclei. Asterisks denote a statistically
significant difference (P < 0.05) as com-
pared with CXXC1
+ ⁄ +
ES cells. (B) Subnu-
clear distribution of endogenous H3K4me3
was detected in the indicated ES cell lines,
using rabbit antibody against H3K4me3 and
FITC-conjugated bovine anti-rabbit IgG as
secondary antibody, as described above.
The asterisks denote a statistically signifi-
cant difference (P < 0.05) as compared with
CXXC1
+ ⁄ +
ES cells.
Cfp1 restricts the Setd1A complex to euchromatin C. M. Tate et al.
214 FEBS Journal 277 (2010) 210–223 ª 2009 The Authors Journal compilation ª 2009 FEBS
Fig. 3. Cfp1 DNA-binding activity or associa-
tion with the Setd1A complex is required for
appropriate levels of Setd1A. (A) Schematic
representation of full-length Cfp1 (amino
acids 1–656) and Cfp1 truncations and
mutations that were stably expressed in
CXXC1
) ⁄ )
ES cells. The filled circle at the
N-terminus of Cfp1 represents the FLAG
epitope, and NLS represents a nuclear locali-
zation signal. Mutations that ablate DNA-
binding activity (C169A) or interaction with

Setd1A (C375A) are indicated by ‘X’. (B)
Western blot analysis was performed
on whole cell extracts collected from
CXXC1
+ ⁄ +
, CXXC1
) ⁄ )
and CXXC1
) ⁄ )
ES
cells expressing full-length Cfp1 (amino
acids 1–656) or the indicated Cfp1 muta-
tions (or carrying the empty expression vec-
tor), using antisera directed against Setd1A
[16]. The level of b-actin serves as a loading
control. The graph represents relative
Setd1A levels normalized to b-actin from at
least three independent experiments, and
error bars indicate standard error. Asterisks
denote statistically significant (P < 0.05)
differences as compared with CXXC1
) ⁄ )
ES
cells expressing full-length Cfp1 (amino
acids 1–656).
C. M. Tate et al. Cfp1 restricts the Setd1A complex to euchromatin
FEBS Journal 277 (2010) 210–223 ª 2009 The Authors Journal compilation ª 2009 FEBS 215
the Setd1A HMT complex [33]. Additional studies
were performed to assess the functional significance of
these Cfp1 properties for the ability to restore normal

levels of Setd1A. CXXC1
) ⁄ )
ES cells expressing full-
length Cfp1 that lacks DNA-binding activity (amino
acids 1–656, C169A) or interaction with the Setd1A
H3K4 HMT complex (amino acids 1–656, C375A)
contain normal levels of Setd1A. This was expected,
given that expression of either half of Cfp1 is sufficient
to restore normal Setd1A levels. However, ablation of
DNA-binding activity within the N-terminal fragment
of Cfp1 (amino acids 1–367, C169A), or disruption of
Setd1A interaction with the C-terminal Cfp1 fragment
(amino acids 361–656, C375A), results in the loss of
Setd1A rescue activity (Fig. 3B). Finally, rescue activ-
ity was lost upon introduction of both point mutations
into full-length Cfp1 (amino acids 1–656, C169A ⁄
C375A). These data indicate that retention of either
Cfp1 DNA-binding activity or interaction with the
Setd1A H3K4 HMT complex is required to restore
appropriate Setd1A levels in CXXC1
) ⁄ )
ES cells.
Full-length Cfp1 is required to restrict Setd1A
and H3K4me3 to euchromatin
CXXC1
) ⁄ )
ES cells expressing various Cfp1 trunca-
tions and mutations were analyzed by confocal immu-
nofluorescence to determine the functional domains of
Cfp1 required to restrict the subnuclear localization

of Setd1A and H3K4me3 to euchromatin. The vast
majority of Setd1A and H3K4me3 was localized to
DAPI-dim euchromatic regions in CXXC1
) ⁄ )
ES cells
expressing full-length Cfp1 (amino acids 1–656)
(Figs 4 and 5). In contrast to the pattern of Cfp1 res-
cue activity seen for Setd1A levels, however, expres-
sion of the N-terminal (amino acids 1–481 or 1–367)
or C-terminal (amino acids 302–656 or 361–656) frag-
ments of Cfp1 in CXXC1
) ⁄ )
ES cells is not sufficient
to exclude Setd1A and H3K4me3 from DAPI-bright
heterochromatin (Figs 4 and 5). In addition,
CXXC1
) ⁄ )
ES cells expressing full-length Cfp1 that
lacks DNA-binding activity (amino acids 1–656,
C169A) or fails to interact with the Setd1A H3K4
HMT complex (amino acids 1–656, C375A) also fail
to restrict Setd1A and H3K4me3 to euchromatin
(Figs 4 and 5). As expected, ablation of the DNA-
binding activity within the N-terminal fragment of
Cfp1 (amino acids 1–367, C169A), disruption of the
Setd1A interaction with the C-terminal fragment of
Cfp1 (amino acids 361–656, C375A) or introduction
of both mutations within full-length Cfp1 (1-656
C169A, C375A) also results in a failure to exclude
Setd1A and H3K4me3 from DAPI-bright heterochro-

matin (Figs 4 and 5). Therefore, full-length Cfp1 is
required to restrict Setd1A and H3K4me3 localization
to euchromatin, and Cfp1 DNA-binding activity and
interaction with the Setd1A H3K4 HMT complex are
both required for proper restriction of Setd1A and
H3K4me3 to euchromatin.
Discussion
The results of the studies reported here reveal that ES
cells lacking the epigenetic regulator Cfp1 contain
decreased levels of the histone H3K4 methyltransferase
Setd1A. Yeast cells lacking Spp1, the Cfp1 homolog,
also express reduced amounts of Set1 [40], and Spp1 is
thought to stabilize Set1 [40]. Furthermore, expression
of Cfp1-interacting Setd1A fragments in human cells
disrupts the association of endogenous Setd1A with
the intact HMT complex, resulting in reduced Setd1A
levels as a consequence of reduced Setd1A half-life
[16]. Thus, the reduced levels of Setd1A observed in
ES cells lacking Cfp1 may be due to decreased Setd1A
stability. The observed increase of Setd1A level in
CXXC1
) ⁄ )
ES cells following treatment with the prote-
osome inhibitor MG132 supports this hypothesis. In
contrast, the levels of the other components of the
Setd1A complex (Ash2, Rbbp5, Wdr5, and Wdr82) are
not altered in CXXC1
) ⁄ )
ES cells, which may be due
to their association with additional H3K4 HMT com-

plexes (Setd1B, Mll1, Mll2, and Mll3) [16,18,22,28,
41–43]. Despite reduced Setd1A levels, CXXC1
) ⁄ )
ES
cells express an approximately five-fold increased level
of Setd1A mRNA, suggesting that these cells increase
transcription of the SETD1A gene to compensate for
reduced levels of Setd1A.
Expression of either an N-terminal fragment (amino
acids 1-367) or C-terminal fragment (amino acids
361–656) of Cfp1 is sufficient to restore normal levels
of Setd1A in CXXC1
) ⁄ )
ES cells. These results are
consistent with previous findings that expression
in CXXC1
) ⁄ )
ES cells of either Cfp1(1–367) or
Cfp1(361–656) is sufficient to rescue defects in ES cell
plating efficiency, cytosine methylation, and in vitro
differentiation [44]. Interestingly, Cfp1(1–367) fails to
interact with the Setd1A complex [33], but still restores
appropriate levels of Setd1A, indicating that a physical
interaction of Cfp1 with the Setd1A complex is not
required for appropriate levels of Setd1A. In addi-
tion, analysis of point mutations within the CXXC
domain (C169A) or SID (C375A) reveals that reten-
tion of either Cfp1 DNA-binding activity or interac-
tion with the Setd1A H3K4 HMT complex is
necessary to restore normal levels of Setd1A in

CXXC1
) ⁄ )
ES cells.
Cfp1 restricts the Setd1A complex to euchromatin C. M. Tate et al.
216 FEBS Journal 277 (2010) 210–223 ª 2009 The Authors Journal compilation ª 2009 FEBS
ES cells that lack Cfp1 show increased levels of
histone H3K4 dimethylation and trimethylation [15],
despite expressing decreased levels of Setd1A, suggest-
ing that Cfp1 restricts the activity of the Setd1A
H3K4 HMT complex. Consistent with this model,
confocal immunofluorescence reveals that both
Setd1A and H3K4me3 are partially mislocalized to
DAPI-bright regions of heterochromatin in
CXXC1
) ⁄ )
ES cells. In contrast to the pattern of
Cfp1 rescue activity observed for Setd1A levels,
expression of full-length Cfp1 in CXXC1
) ⁄ )
ES cells
is required to properly restrict subnuclear localization
Fig. 4. Full-length Cfp1 is required to
restrict Setd1A to euchromatin. The subnu-
clear distribution of endogenous Setd1A
was detected in CXXC1
) ⁄ )
ES cells
expressing full-length Cfp1 (amino
acids 1–656) or the indicated Cfp1 trunca-
tions and mutations, using rabbit antibody

against Setd1A and FITC-conjugated bovine
anti-rabbit IgG as secondary antibody, as
described for Fig. 2. Asterisks denote a
statistically significant difference (P < 0.05)
as compared with CXXC1
) ⁄ )
ES cells
expressing full-length Cfp1 (amino
acids 1–656).
C. M. Tate et al. Cfp1 restricts the Setd1A complex to euchromatin
FEBS Journal 277 (2010) 210–223 ª 2009 The Authors Journal compilation ª 2009 FEBS 217
of Setd1A and H3K4me3 to euchromatin. These
studies further indicate that Cfp1 DNA-binding acti-
vity and interaction with the Setd1A H3K4 HMT
complex are both required for proper subnuclear
localization of Setd1A. The requirement for an intact
Cfp1 CXXC domain for proper genomic localization
may indicate that Cfp1 DNA-binding activity restricts
the Setd1A H3K4 HMT complex to euchromatin by
binding to unmethylated CpG dinucleotides in
euchromatin.
Individual CXXC1
) ⁄ )
ES cell nuclei show a range
(5–30%) of colocalization between Setd1A and
H3K4me3 with DAPI-bright heterochromatin, and
20–30% mislocalization of Setd1A and H3K4me3 is
observed in 35–40% of CXXC1
) ⁄ )
ES cell nuclei. It is

possible that cell-to-cell variation in the degree of
Fig. 5. Full-length Cfp1 is required to
restrict H3K4me3 to euchromatin. The
subnuclear distribution of H3K4me3 was
detected in CXXC1
) ⁄ )
ES cells expressing
full-length Cfp1 (amino acids 1–656) or the
indicated Cfp1 truncations and mutations,
using rabbit antibody against H3K4me3 and
FITC-conjugated bovine anti-rabbit IgG as
secondary antibody, as described for Fig. 2.
Asterisks denote statistically significant
differences (P < 0.05) as compared with
CXXC1
) ⁄ )
ES cells expressing full-length
Cfp1 (amino acids 1–656).
Cfp1 restricts the Setd1A complex to euchromatin C. M. Tate et al.
218 FEBS Journal 277 (2010) 210–223 ª 2009 The Authors Journal compilation ª 2009 FEBS
colocalization may be cell cycle-dependent. However,
significant mislocalization of Setd1A and H3K4me3 is
never observed in wild-type ES cells or in rescued
CXXC1
) ⁄ )
ES cells expressing full-length Cfp1. The
persistence of DAPI-bright staining colocalizing with
H3K4me3 indicates that deposition of this euchroma-
tin epigenetic mark is insufficient to induce general
chromatin remodeling in these heterochromatin

regions.
Little is known regarding the relative contributions
of each mammalian histone H3K4 HMT complex.
However, Cfp1 has been shown to be an integral com-
ponent of only the Setd1A and Setd1B HMT com-
plexes [15,16]. The localization of Setd1B in the
absence of Cfp1 has not been determined, but the find-
ing that the extent of Setd1A mislocalization is similar
to that of H3K4me3 localization suggests that the
Setd1 HMT complexes are responsible for the bulk of
histone H3K4 trimethylation. This conclusion is con-
sistent with a recent report that small interfering
RNA-mediated depletion of Setd1A and Setd1B leads
to a dramatic global reduction in histone H3K4 trime-
thylation [45].
The full-length Cfp1 that is required to restrict
subnuclear localization of Setd1A and H3K4me3 to
euchromatin contains two PHDs. PHDs are thought
to be involved in chromatin-mediated transcriptional
control [46], and can serve as binding modules for
unmodified and methylated histone H3K4 and methy-
lated histone H3K36 [17,47–51]. For example, PHD1
of Spp1, the yeast homolog of Cfp1, binds dimethylat-
ed and trimethylated histone H3K4 [51]. In addition,
the PHD finger of the tumor suppressor Ing2 directly
associates with H3K4me3, and this interaction is criti-
cal for proper occupancy of the Ing2–HDAC1 complex
at target promoters during the DNA damage response
and active transcriptional repression [48]. Therefore,
the PHDs of Cfp1 may be important for binding mod-

ified histone H3K4 and targeting the Cfp1–Setd1A
complex to specific genomic sites.
The mechanisms responsible for appropriate subnu-
clear localization of histone H3K4 HMTs are complex,
and involve gene-specific recruitment by DNA-binding
factors. For example, the insulator DNA-binding pro-
tein Boris recruits Setd1A to the MYC and BRCA1
genes [52]; NF-E2 recruits Mll2 to the b-globin locus
[53]; the Ap2d transcription factor recruits Ash2L and
Mll2 to the HOXC8 locus [54]; and the paired-box
transcription factor Pax7 recruits Mll2 to the MYF5
gene [55].
In addition, several integral components of the
mammalian Set1-like histone H3K4 HMT complexes
have been implicated in genomic targeting. Wdr5,
which is common to each member of the mammalian
Set1-like HMT complex family, has been reported to
bind directly to histone H3 [56–59]. In addition, the
Wdr82 component of the Setd1A and Setd1B HMT
complexes binds to RNAP II containing Ser5-phos-
phorylated CTD, thus recruiting these complexes to
sites of transciption initiation [18]. Furthermore, the
compositions of the Setd1A and Setd1B HMT com-
plexes are identical, except for the identity of the enzy-
matic (Setd1) component [15,16], but confocal
microscopy reveals that these complexes show a nearly
nonoverlapping euchromatic subnuclear localization
[16]. This finding strongly suggests that these closely
related complexes regulate distinct sets of target genes,
and that this specificity is mediated by each Setd1 pro-

tein, presumably through interactions with distinct tar-
geting effector molecules. The data reported here
reveal that Cfp1 plays a novel role in restricting the
subnuclear localization of Setd1A and H3K4me3 to
euchromatin, thus identifying Cfp1 as another critical
regulator of histone H3K4 HMT genomic targeting.
Experimental procedures
Cell culture
Generation of murine CXXC1
) ⁄ )
ES cell lines was as previ-
ously described [38]. ES cells were cultured on 0.1% gela-
tin-coated tissue culture dishes in high-glucose DMEM
(Gibco BRL, Life Technologies, Grand Island, NY, USA)
supplemented with 20% fetal bovine serum (Gibco BRL),
100 unitsÆmL
)1
penicillin ⁄ streptomycin (Invitrogen, Carls-
bad, CA, USA), 2 mml-glutamine (Invitrogen), 1% nones-
sential amino acids (Invitrogen), 0.2% leukemia inhibitory
factor-conditioned medium, 100 nm b-mercaptoethanol,
0.025% Hepes (pH 7.5) (Invitrogen), and 1% Hank’s
balanced salt solution (Invitrogen).
Plasmid construction and transfection of ES cells
Murine Cfp1 cDNA [38,60] was subcloned into
pcDNA3.1 ⁄ Zeo (Invitrogen). The Cfp1 expression vector or
the empty expression vector was electroporated into
CXXC1
) ⁄ )
ES cells as previously described [38]. Single

amino acid substitutions within Cfp1 were performed using
the QuikChange II site-directed mutagenesis kit (Strata-
gene, La Jolla, CA, USA) according to the manufacturer’s
protocol, as previously described [33,35]. For structure–
function studies, cDNA constructs encoding full-length
FLAG epitope-tagged human Cfp1 (amino acids 1–656)
and various Cfp1 truncations and ⁄ or mutations were
subcloned into the pcDNA3.1 ⁄ Hygro mammalian expres-
sion vector (Invitrogen). The N-terminal bipartite nuclear
localization signal of Cfp1 (amino acids 109–121) was
C. M. Tate et al. Cfp1 restricts the Setd1A complex to euchromatin
FEBS Journal 277 (2010) 210–223 ª 2009 The Authors Journal compilation ª 2009 FEBS 219
inserted between the FLAG epitope and Cfp1 sequence for
constructs containing amino acids 302–656 and 361–656.
Linearized DNA (25 lg) was electroporated into
CXXC1
) ⁄ )
ES cells at 300 V and 500 lF capacitance. Cells
were grown in selection medium containing 200 lgÆmL
)1
hygromycin B (Sigma-Aldrich, St Louis, MO, USA) for
approximately 2 weeks before single colonies were isolated
for expansion; these were subsequently maintained in med-
ium containing 50 lgÆmL
)1
hygromycin B (Sigma-Aldrich).
Expression of FLAG–Cfp1 was verified by western blot
analysis using antibodies against FLAG (Sigma-Aldrich)
and Cfp1 [16]. Two or three independent clones carrying
each construct were selected for analysis on the basis of the

protein expression level of FLAG–Cfp1, and data for a
representative clone are presented.
Analysis of Setd1A mRNA expression
Total RNA was isolated from ES cells using TriReagent
solution (Molecular Research Center, Cincinnati, OH,
USA) and reverse transcribed as previously described [16].
Relative Setd1A gene expression was determined by quanti-
tative RT-PCR (qRT-PCR) using TaqMan gene expression
assays containing a primer set and probe (FAM fluorescent
reporter dye) (purchased from PE Applied Biosystems) spe-
cific for Setd1A (exon 6–7, catalog no. Mm00626143_m1).
Mouse glyceraldehyde-3-phosphate dehydrogenase (GAP-
DH) (catalog no. 4352932E) served as an endogenous
control. An Applied Biosystems 7500 Real-Time PCR
System was used to detect PCR products following a stan-
dard amplification protocol recommended by the manu-
facturer. The comparative C
T
method was used to
determine the gene expression level of Setd1A relative to the
GAPDH control, which was averaged over three indepen-
dent experiments.
Western blot analysis
Levels of Cfp1, Setd1A, Ash2, Wdr5, Wdr82, and Rbbp5
were assessed by isolating whole cell protein extracts as pre-
viously described [15,16]. Protein extracts were quantified
using the Bradford method [61], solubilized with 1·
Laemmli sample buffer, boiled for 5 min, separated by elec-
trophoresis on 7% (Cfp1) or 4–12% (Setd1A, Ash2, Wdr5,
Wdr82, and Rbbp5) PAGEr-Gold precast Tris ⁄ glycine gels

(Lonza Group, Ltd, Switzerland), and then transferred to
nitrocellulose membranes (GE Healthcare, Amersham,
UK). Membranes were probed with primary antibodies,
washed, and incubated with appropriate horseradish peroxi-
dase-linked secondary antibodies as previously described
[15,16]. Signal was detected by electrochemiluminescence
detection reagents (GE Healthcare), and quantified by den-
sitometry (image j; NIH). The following antibodies were
used for immunoblotting: mouse monoclonal antibodies
against b-actin and FLAG M2 (Sigma-Aldrich), rabbit
polyclonal antibodies against Setd1A, Wdr82, Cfp1, and
Wdr5 [16], and rabbit polyclonal antibodies against Ash2
and Rbbp5 (Bethyl Laboratories, Montgomery, TX, USA).
Confocal immunofluorescence
Exponentially growing ES cells were seeded onto sterilized
glass coverslips at a density of 3 · 10
4
cells per well in 24-
well tissue culture dishes. After 48 h of culture, cells were
washed with ice-cold NaCl ⁄ P
i
, fixed with 4% paraformalde-
hyde, and permeabilized with 0.2% Triton X-100 in
NaCl ⁄ P
i
. Cells were incubated with blocking solution con-
taining 5% normal donkey serum (Santa Cruz Biotechnol-
ogy) in NaCl ⁄ P
i
containing 0.2% Tween-20 before being

incubated with 1 : 500 dilutions of primary antibodies
against H3K4me3 (Abcam, Cambridge, MA, USA) or
Setd1A [16]. Coverslips were washed with NaCl ⁄ P
i
contain-
ing 0.2% Tween-20, and incubated with 1 : 200 dilutions of
fluorescein isothiocyanate (FITC)-conjugated secondary
antibodies (Santa Cruz Biotechnology, Santa Cruz, CA,
USA). Coverslips were then incubated with a solution of
0.1 lgmL
)1
DAPI. Images were captured on a Zeiss UV
LSM-510 confocal microscope (Carl Zeiss Inc., Thorn-
wood, NY, USA) at the Indiana University Center for Bio-
logical Microscopy, using a UV argon laser (364 nm
excitation) for DAPI, and a visible argon laser (488 nm
excitation) for FITC. The immunofluorescent images were
analyzed with metamorph 6.0 (Universal Imaging Corpo-
ration, West Chester, PA, USA). The threshold for FITC
was adjusted to exclude background and nonspecific stain-
ing, and the threshold for DAPI was adjusted to include
DAPI-bright regions. The same threshold values were used
to analyze each image. Quantification of colocalization
of positive fluorescent signals was analyzed using the
metamorph colocalization module. At least 30 nuclei were
analyzed for each cell line.
Acknowledgements
We thank S. Atkinson for helpful discussions regard-
ing confocal microscopy. This work was supported by
the Riley Children’s Foundation, the Lilly Endow-

ment, and National Science Foundation Grants NSF
MCB-0344870 and MCB-0641851 (D. G. Skalnik). C.
M. Tate was supported by a predoctoral fellowship
from National Institutes of Health Grant T32
AI060519 and a Department of Education training
grant in Graduate Assistance in Areas of National
Need (GAANN).
References
1 Peterson CL & Laniel MA (2004) Histones and histone
modifications. Curr Biol 14, R546–R551.
Cfp1 restricts the Setd1A complex to euchromatin C. M. Tate et al.
220 FEBS Journal 277 (2010) 210–223 ª 2009 The Authors Journal compilation ª 2009 FEBS
2 Zhang Y & Reinberg D (2001) Transcription regulation
by histone methylation: interplay between different
covalent modifications of core histone tails. Genes Dev
15, 2343–2360.
3 Kouzarides T (2007) Chromatin modifications and their
functions. Cell 128, 693–705.
4 Dillon SC, Zhang X, Trievel RC & Cheng X (2005)
The SET-domain protein superfamily: protein lysine
methyltransferases. Genome Biol 6, 227.
5 Jenuwein T, Laible G, Dorn R & Reuter G (1998) SET
domain proteins modulate chromatin domains in
eu- and heterochromatin. Cell Mol Life Sci 54, 80–93.
6 Santos-Rosa H, Schneider R, Bannister AJ, Sherriff J,
Bernstein BE, Emre NCC, Schreiber SL, Mellor J &
Kouzarides T (2002) Active genes are tri-methylated at
K4 of histone 3 Nature 419, 407–411.
7 Boggs BA, Cheung P, Heard E, Spector DL, Chinault
AC & Allis CD (2002) Differentially methylated forms

of histone H3 show unique association patterns
with inactive human X chromosomes. Nat Genet 30,
73–76.
8 Noma K, Allis CD & Grewal SI (2001) Transitions in
distinct histone H3 methylation patterns at the hetero-
chromatin domain boundaries. Science 293, 1150–1155.
9 Peters AH, O’Carroll D, Scherthan H, Mechtler K,
Sauer S, Schofer C, Weipoltshammer K, Pagani M,
Lachner M, Kohlmaier A et al. (2001) Loss of the
Suv39h histone methyltransferase impairs mammalian
heterochromatin and genome stability. Cell 107, 323–
337.
10 Miller T, Krogan NJ, Dover J, Erdjument-Bromage H,
Tempst P, Johnston M, Greenblatt JF & Shilatifard A
(2001) COMPASS: a complex of proteins associated
with a trithorax-related SET domain protein. Proc Natl
Acad Sci USA 28, 12902–12907.
11 Bryk M, Briggs SD, Strahl BD, Curcio MJ, Allis CD &
Winston F (2002) Evidence that Set1, a factor required
for methylation of histone H3, regulates rDNA silenc-
ing in S. cerevisiae by a Sir2-independent mechanism.
Curr Biol 12, 165–170.
12 Krogan NJ, Dover J, Khorrami S, Greenblatt JF,
Schneider J, Johnston M & Shilatifard A (2002)
COMPASS, a histone H3 (lysine 4) methyltransferase
required for telomeric silencing of gene expression.
J Biol Chem 277, 10753–10755.
13 Glaser S, Schaft J, Lubitz S, Vinterten K, van der
Hoeven F, Tufteland KR, Asasland R, Anastassiadis
K, Ang S-L & Stewart AF (2006) Multiple epigenetic

maintenance factors implicated by the loss of Mll2 in
mouse development. Development 133, 1423–1432.
14 Gregory GD, Vakoc CR, Rozovskaia T, Zheng X, Patel
S, Nakamura T, Canaani E & Blobel GA (2007)
Mammalian ASH1L is a histone methyltransferase that
occupies the transcribed region of active genes. Mol Cell
Biol 27, 8466–8479.
15 Lee JH & Skalnik DG (2005) CpG-binding protein
(CXXC finger protein 1) is a component of the
mammalian Set1 histone H3-Lys4 methyltransferase
complex, the analogue of the yeast Set1 ⁄ COMPASS
complex. J Biol Chem 280, 41725–41731.
16 Lee JH, Tate CM, You JY & Skalnik DG (2007)
Identification and characterization of the human Set1B
histone H3-Lys4 methyltransferase complex. J Biol
Chem 282, 13419–13428.
17 Martin DGE, Baetz K, Shi X, Walter KL, MacDonald
VE, Wlodarski MJ, Gozani O, Hieter P & 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.
18 Lee JH & Skalnik DG (2008) Wdr82 is a C-terminal
domain-binding protein that recruits the Setd1A his-
tone H3-Lys4 methyltransferase complex to transcrip-
tion start sites of transcribed human genes. Mol Cell
Biol 28, 609–618.
19 Ng H-H, Robert F, Young RA & Struhl K (2003)
Targeted recruitment of Set1 histone methylase by elon-
gating Pol II provides a localized mark and memory of
recent transcriptional activity. Mol Cell 11, 709–719.

20 Palancade B & Bensaude O (2003) Investigating RNA
polymerase II carboxyl-terminal domain (CTD) phos-
phorylation. Eur J Biochem 270, 3859–3870.
21 Milne TA, Dou Y, Martin ME, Brock HW, Roeder
RG & Hess JL (2005) MLL associates specifically with
a subset of transcriptionally active target genes. Proc
Natl Acad Sci USA 102, 14765–14770.
22 Hughes CM, Rozenblatt-Rosen O, Milne TA, Copeland
TD, Levine SS, Lee JC, Hayes DN, Shanmugam KS,
Bhattacharjee A, Biondi CA et al. (2004) Menin associ-
ates with a trithorax family histone methyltransferase
complex and with the hoxc8 locus. Mol Cell 13, 587–
597.
23 Edmunds JW, Mahadevan LC & Clayton AL (2008)
Dynamic histone H3 methylation during gene induc-
tion: HYPB ⁄ Setd2 mediates all H3K36 trimethylation.
EMBO J 27, 406.
24 Li B, Howe L, Anderson S, Yates JR III & Work-
man JL (2003) The Set2 histone methyltransferase
functions through the phosphorylated carboxyl-termi-
nal domain of RNA polymerase II. J Biol Chem 278,
8897–8903.
25 Yokoyama A, Wang Z, Wysocka J, Sanyal M, Aufiero
DJ, Kitabayashi I, Herr W & Cleary ML (2004) Leuke-
mia proto-oncoprotein MLL forms a SET1-like histone
methyltransferase complex with menin to regulate Hox
gene expression. Mol Cell Biol 24 , 5639–5649.
26 Domer PH, Fakharzadeh SS, Chen CS, Jockel J,
Johansen L, Silverman GA, Kersey JH & Korsmeyer SJ
(2003) Acute mixed-lineage leukemia t(4;11)(q21;q23)

generates an MLL–AF4 fusion product. Proc Natl Acad
Sci USA 90, 7884–7888.
C. M. Tate et al. Cfp1 restricts the Setd1A complex to euchromatin
FEBS Journal 277 (2010) 210–223 ª 2009 The Authors Journal compilation ª 2009 FEBS 221
27 Hamamoto R, Furukawa Y, Morita M, Iimura Y, Silva
FP, Li M, Yagyu R & Nakamura Y (2004) SMYD3
encodes a histone methyltransferase involved in the pro-
liferation of cancer cells. Nat Cell Biol 6, 731–740.
28 Gu Y, Nakamura T, Alder H, Prasad R, Canaani O,
Cimino G, Croce CM & Canaani E (1992) The t(14;11)
chromosome translocation of human acute leukemias
fuses the ALL-1 gene, related to Drosophila trithorax,
to the AF-4 gene. Cell 71, 701–708.
29 Ma Q, Alder H, Nelson KK, Chatterjee D, Gu Y,
Nakamura T, Canaani E, Croce CM, Siracusa LD &
Buchberg AM (1993) Analysis of the murine All-1 gene
reveals conserved domains with human ALL-1 and
identifies a motif shared with DNA methyltransferases.
Proc Natl Acad Sci USA 90, 6350–6354.
30 Prasad R, Yano T, Sorio C, Nakamura T, Rallapalli R,
Gu Y, Leshkowitz D, Croce CM & Canaani E (1995)
Domains with transcriptional regulatory activity within
the ALL1 and AF4 proteins involved in acute leukemia.
Proc Natl Acad Sci USA 92, 12160–12164.
31 Tkachuk DC, Kohler S & Cleary ML (1992) Involve-
ment of a homolog of Drosophila trithorax by 11q23
chromosomal translocations in acute leukemias. Cell 71,
701–708.
32 Yu BD, Hell JL, Horning SE, Brown GAJ & Korsmeyer
SJ (1995) Altered Hox expression and segmental iden-

tity in MLL-mutant mice. Nature 378 , 505–508.
33 Butler JS, Lee JH & Skalnik DG (2008) CFP1 interacts
with DNMT1 independently of association with the
Setd1 histone H3K4 methyltransferase complexes. DNA
Cell Biol 27, 533–543.
34 Lee JH & Skalnik DG (2002) CpG-binding protein is a
nuclear matrix- and euchromatin-associated protein
localized to nuclear speckles containing human tritho-
rax. J Biol Chem 277, 42259–42267.
35 Lee JH, Voo KS & Skalnik DG (2001) Identification
and characterization of the DNA binding domain
of CpG-binding protein. J Biol Chem 276, 44669–
44676.
36 Voo KS, Carlone DL, Jacobsen BM, Flodin A &
Skalnik DG (2000) Cloning of a mammalian transcrip-
tional activator that binds unmethylated CpG motifs
and shares a CXXC domain with DNA methyltransfer-
ase, human trithorax, and methyl-CpG binding domain
protein 1. Mol Cell Biol 20, 2108–2121.
37 Carlone DL & Skalnik DG (2001) CpG binding protein
is crucial for early embryonic development. Mol Cell
Biol 21, 7601–7606.
38 Carlone DL, Lee JH, Young SRL, Dobrota E, Butler
JS, Ruiz J & Skalnik DG (2005) Reduced genomic cyto-
sine methylation and defective cellular differentiation in
embryonic stem cells lacking CpG binding protein. Mol
Cell Biol 25, 4881–4891.
39 Guenatri M, Bailly D, Maison C & Almouzni G (2004)
Mouse centric and pericentric satellite repeats form
distinct functional heterochromatin. J Cell Biol 166,

493–505.
40 Dehe PM, Dichtl B, Schaft D, Roguev A, Pamblanco
M & Lebrun R (2006) Protein interactions within the
Set1 complex and their roles in the regulation of his-
tone 3 lysine 4 methylation and transcription termina-
tion. J Biol Chem 281, 35404–35412.
41 Goo YH, Sohn YC, Kim DH, Kim SW, Kang MJ,
Jung DJ, Kwak E, Barlev NA, Berger SL, Chow VT
et al. (2003) Activating signal cointegrator 2 belongs to
a novel steady-state complex that contains a subset of
trithorax group proteins. Mol Cell Biol 23, 140–149.
42 Wysocka J, Myers MP, Laherty CD, Eisenmann RN &
Herr W (2003) Human Sin3 deacetylase and trithorax-
related Set1 ⁄ Ash2 histone H3-K4 methyltransferase are
tethered together selectively by the cell-proliferation
factor HCF-1. Genes Dev 17, 896–911.
43 Nakamura T, Mori T, Tada S, Krajewski W, Rozovs-
kaia T, Wassell R, Dubois G, Mazo A, Croce CM &
Canaani E (2002) ALL-1 is a histone methyltransferase
that assembles a supercomplex of proteins involved in
transcriptional regulation. Mol Cell 10, 1119–1128.
44 Tate CM, Lee J-H & Skalnik DG (2009) CXXC finger
protein 1 contains redundant functional domains that
support embryonic stem cell cytosine methylation,
histone methylation, and differentiation. Mol Cell Biol
29, 3817–3831.
45 Wu M, Wang PF, Lee JS, Martin-Brown S, Florens L,
Washburn M & Shilatifard A (2008) Molecular regula-
tion of H3K4 trimethylation by Wdr82, a component of
human Set1 ⁄ COMPASS. Mol Cell Biol 28, 7337–7344.

46 Halbach T, Scheer N & Werr W (2000) Transcriptional
activation by the PHD finger is inhibited through an
adjacent leucine zipper that binds 14-3-3 proteins.
Nucleic Acids Res 28, 3542–3550.
47 Li H, Ilin S, Wqang W, Dunchan EM, Wsocka J, Allis
CD & Patel DJ (2006) Molecular basis for site-specific
read-out of histone H3K4Me3 recognition by the BPTF
PHD finger of NURF. Nature 442, 91–95.
48 Pena PV, Davroaou F, Shi X, Walter KL, Verkhusha
VV, Gozani O, Zhao R & Kutatladze TG (2006)
Molecular mechanism of histone H3K4me3 recognition
by plant homeodomain of ING2. Nature 442, 100–103.
49 Ruthenburg AJ, Li H, Patel DJ & Allis CD (2007)
Multivalent engagement of chromatin modifications by
linked binding modules. Nat Rev Mol Cell Biol 8,
983–994.
50 Shi X, Hong T, Walter KL, Ewalt M, Michishita E,
Hung T, Carney D, Pena P, Lan F, Kaadige MR et al.
(2006) ING2 PHD domain links histone H3 lysine 4
methylation to active gene respression. Nature 442,
96–99.
51 Shi X, Kachirskaia I, Walter KL, Kuo J-HA, Lake A,
Davrazou F, Chan SM, Martin DGE, Fingerman IM,
Briggs SD et al. (2007) Proteome-wide analysis in
Cfp1 restricts the Setd1A complex to euchromatin C. M. Tate et al.
222 FEBS Journal 277 (2010) 210–223 ª 2009 The Authors Journal compilation ª 2009 FEBS
Saccharomyces 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, 2450–2455.

52 Lee J, Saha PK, Yang Q-H, Lee S, Park JY, Suh Y,
Lee S-K, Chan L, Roeder RG & Lee JW (2008)
Targeted inactivation of MLL3 histone H3-Lys-4
methyltransferase activity in the mouse reveals vital
roles for MLL3 in adipogenesis. Proc Natl Acad Sci
USA 105, 19229–19234.
53 Demers C, Chaturvedi C-P, Ranish JA, Juban G, Lai
P, Morle F, Aebersold R, Dilworth FJ, Groudine M &
Brand M (2007) Activator-mediated recruitment of the
MLL2 methyltransferase complex to the beta-globin
locus. Mol Cell 27, 573–584.
54 Tan CC, Sindhu KV, Li S, Nishio H, Stoller JZ, Oishi
K, Puttreddy S, Lee TJ, Epstein JA, Walsh MJ et al.
(2008) Transcription factor AP2delta associates with
Ash2l and ALR, a trithorax family histone
methyltransferase to activate Hoxc8 transcription. Proc
Natl Acad Sci USA 105, 7472–7477.
55 McKinnel IW, Ishibahi J, Grand FL, Punch VGJ,
Addicks GC, Greenblatt JF, Dilworth FJ &
Rudnicki MA (2008) Pax7 activates myogenic genes by
recruitment of a histone methyltransferase complex. Nat
Cell Biol 10, 77–83.
56 Couture J-F, Collazo E & Trievel RC (2006) Molecular
recognition of histone H3 by the WD40 protein WDR5.
Nat Struct Mol Biol 13, 698–703.
57 Han Z, Guo L, Wang H, Shen Y, Deng XW & Chai J
(2006) Structural basis for the specific recognition of
methylated histone H3 lysine 4 by the WD-40 protein
WDR5. Mol Cell 22, 137–144.
58 Ruthenburg AJ, Wang W, Graybosch DM, Li H, Allis

CD, Patel DJ & Verdine GL (2006) Histone H3 recog-
nition and presentation by the WDR5 module of the
MLL1 complex. Nat Struct Mol Biol 13, 704–712.
59 Wysocka J, Swigut T, Milne TA, Dou Y, Zhang X,
Brurlingame AL, Roeder RG, Brivanlou AH & Allis
CD (2005) WDR5 associates with histone H3 methylat-
ed at K4 and is essential for H3 K4 methylation and
vertebrate development. Cell 121, 859–872.
60 Carlone DL, Hart SRL, Ladd PD & Skalnik DG
(2002) Cloning and characterization of the gene encod-
ing the mouse homologue of CpG binding protein. Gene
295, 71–77.
61 Bradford MM (1976) A rapid and sensitive method for
the quantitation of microgram quantities of protein
utilizing the principle of protein-dye binding. Anal
Biochem 72, 248–254.
C. M. Tate et al. Cfp1 restricts the Setd1A complex to euchromatin
FEBS Journal 277 (2010) 210–223 ª 2009 The Authors Journal compilation ª 2009 FEBS 223