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: dskalnik@iupui.edu
(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 ª2009 The Authors Journal compilation ª2009 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 ª2009 The Authors Journal compilation ª2009 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 ª2009 The Authors Journal compilation ª2009 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 lMMG132 for 6 h.
C. M. Tate et al. Cfp1 restricts the Setd1A complex to euchromatin
FEBS Journal 277 (2010) 210–223 ª2009 The Authors Journal compilation ª2009 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 ª2009 The Authors Journal compilation ª2009 FEBS