MOLECULAR AND CELLULAR BIOLOGY, June 2005, p. 4881–4891
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 25, No. 12
Reduced Genomic Cytosine Methylation and Defective Cellular
Differentiation in Embryonic Stem Cells Lacking
CpG Binding Protein
Diana L. Carlone,† Jeong-Heon Lee,† Suzanne R. L. Young,† Erika Dobrota,†
Jill Sergesketter Butler,† Joseph Ruiz, 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, Indiana 46202
Received 11 February 2005/Returned for modification 11 March 2005/Accepted 21 March 2005
Cytosine methylation at CpG dinucleotides is a critical epigenetic modification of mammalian genomes. CpG
binding protein (CGBP) exhibits a unique DNA-binding specificity for unmethylated CpG motifs and is
essential for early murine development. Embryonic stem cell lines deficient for CGBP were generated to further
examine CGBP function. CGBP?/?cells are viable but show an increased rate of apoptosis and are unable to
achieve in vitro differentiation following removal of leukemia inhibitory factor from the growth media. Instead,
CGBP?/?embryonic stem cells remain undifferentiated as revealed by persistent expression of the pluripotent
markers Oct4 and alkaline phosphatase. CGBP?/?cells exhibit a 60 to 80% decrease in global cytosine
methylation, including hypo-methylation of repetitive elements, single-copy genes, and imprinted genes. Total
DNA methyltransferase activity is reduced by 30 to 60% in CGBP?/?cells, and expression of the maintenance
DNA methyltransferase 1 protein is similarly reduced. However, de novo DNA methyltransferase activity is
normal. Nearly all aspects of the pleiotropic CGBP?/?phenotype are rescued by introduction of a CGBP
expression vector. Hence, CGBP is essential for normal epigenetic modification of the genome by cytosine
methylation and for cellular differentiation, consistent with the requirement for CGBP during early mamma-
The CpG dinucleotide is an important regulatory compo-
nent of mammalian genomes. The cytosine of this dinucleotide
serves as the target for methylation by DNA methyltransferase
(Dnmt) enzymes, which functions as a critical epigenetic mod-
ification of DNA. Methylated DNA is correlated with hetero-
chromatin and transcriptionally inactive genes, while actively
expressed genes are generally hypomethylated (58). Cytosine
methylation may also represent a defense mechanism to si-
lence parasitic repetitive DNA elements present in mammalian
genomes (72, 78). In addition, cytosine methylation is involved
in the processes of genomic imprinting, in which paternal and
maternal alleles of a gene exhibit distinct patterns of cytosine
methylation and expression (65), and X chromosome inactiva-
tion, in which one X chromosome in each cell of a female
becomes irreversibly inactivated during early development
(52). The CpG dinucleotide is underrepresented in mamma-
lian genomes (5 to 10% of the expected frequency), presum-
ably due to the propensity of 5-methylcytosine to undergo
spontaneous deamination to form thymine (8). Approximately
50% of human and mouse genes reside near unmethylated
CpG islands, which contain the statistically expected frequency
of CpG dinucleotides.
Global cytosine methylation patterns inherited from ga-
metes are erased during early embryogenesis (morula), fol-
lowed by a wave of de novo DNA methylation in the blastocyst
upon implantation (44). Dnmt3a and Dnmt3b are de novo
methyltransferases that preferentially recognize unmethylated
CpG motifs (49), while Dnmt1 is a maintenance methyltrans-
ferase that recognizes hemimethylated DNA (5), the immedi-
ate product of DNA replication. Appropriate cytosine meth-
ylation in mammals is essential for normal development.
Individual ablation of the Dnmt1 or Dnmt3b gene leads to a
lethal disruption of murine embryonic development (38, 49).
Mice lacking Dnmt3a develop to birth but become runted and
die within 4 weeks of age (49). Furthermore, mutations that
are predicted to partially inhibit Dnmt3b function are associ-
ated with the ICF (immunodeficiency, centromere instability,
and facial anomalies) syndrome in humans (77). Overexpres-
sion of Dnmt1 in mice leads to global hypermethylation, loss of
genomic imprinting, and embryonic lethality (7).
A number of DNA-binding factors interact with methylated
CpG motifs (27). These include MeCP2, methyl binding domain
protein 1 (MBD1) and MBD2, which are involved in repression
of gene expression, and MBD4, which functions in DNA repair.
Each of these factors contains a conserved methyl-CpG binding
domain, but otherwise they exhibit little sequence similarly. Mu-
tations in the methyl-CpG binding protein MeCP2 lead to Rett
syndrome, a progressive neurodegenerative disorder (2).
Recent reports reveal intricate interrelationships linking cy-
tosine methylation and histone modifications, thus providing a
unifying framework for the control of chromatin structure and
gene regulation (9). For example, MBD2 and MBD3 are com-
ponents of the histone deacetylase (HDAC) complexes
* Corresponding author. Mailing address: Cancer Research Building,
Room W327, 1044 West Walnut St., Indianapolis, IN 46202. Phone: (317)
274-8977. Fax: (317) 274-8679. E-mail: email@example.com.
† These authors contributed equally to this work.
MeCP1 and Mi-2, respectively (46, 81), and Dnmt proteins also
associate with HDAC complexes (20, 21, 55). Furthermore, the
chromatin remodeling protein DDM1 in Arabidopsis and the
related factor LSH in mammals are required for normal cyto-
sine methylation (15, 29, 30). Disruption of the Suv39h histone
methyltransferase gene in murine embryonic stem (ES) cells
leads to altered localization of Dnmt3b and decreased cytosine
methylation at pericentric satellite repeats (35). Hence, DNA
methylation and histone modifications appear to be highly
integrated and mutually reinforcing mechanisms that serve to
maintain heterochromatin structure and repress gene expres-
CpG binding protein (CGBP) exhibits a unique DNA-bind-
ing specificity for unmethylated CpG motifs and acts as a
transcriptional activator (71). Originally identified in humans,
homologues of CGBP have been detected in Drosophila, Cae-
norhabditis elegans, and both Saccharomyces cerevisiae and
Schizosaccharomyces pombe (41, 71). CGBP contains a cys-
teine-rich CXXC DNA-binding domain (34, 71) which is
present in several other proteins, including Dnmt1 (6); human
trithorax (HRX) (also known as ALL-1 or MLL), a histone
methyltransferase encoded by a gene frequently involved in
chromosomal translocations in leukemia (17, 25, 39, 53, 66,
80); MBD1 (14, 27); leukemia-associated protein LCX (50);
and MLL-2, which is often amplified in solid tumors (19).
CGBP additionally contains two PHD domains, which are
characteristic of chromatin-associated proteins and/or regula-
tors of gene expression (1, 71) and often mediate protein-
protein interactions (22, 24, 48). CGBP is a component of the
nuclear matrix and localizes to nuclear speckles associated with
euchromatin (33). Targeted disruption of the CGBP gene re-
sults in peri-implantation embryonic lethality in mice (11), a
developmental stage associated with global remodeling of
chromatin structure and cytosine methylation patterns (32, 37,
The molecular mechanisms involved in targeting methyl-
ation to specific CpG motifs during development, as well as
maintaining hypomethylation of CpG islands, are not well un-
derstood. The binding specificity of CGBP for unmethylated
CpG motifs suggests a possible role in these events. The early
death of embryos lacking CGBP establishes the importance of
this protein for mammalian development. However, the sever-
ity of this phenotype makes further analysis of this mutant
difficult. In the study reported here, murine ES cells lacking
CGBP were isolated to permit a more detailed analysis of the
CGBP?/?phenotype and provide further insight into CGBP
function. The results presented implicate CGBP as a critical
regulator of DNA methylation and cellular differentiation.
MATERIALS AND METHODS
Generation and analysis of ES cell lines. Six- to eight-week-old CGBP?/?
females were superovulated (7.5 to 10 IU pregnant mare serum gonadotropin
followed 48 h later with 7.5 to 10 IU of human chorionic gonadotropin [Sigma-
Aldrich Co., St. Louis, MO]) and mated with heterozygous males. Blastocysts
(3.5 days postcoitum [dpc]) were collected and cultured for 4 days on mitomycin-
treated STO feeder layers in ES culture media containing leukemia inhibitory
factor (LIF). The inner cell mass was manually isolated, trypsinized, and replated
onto mitomycin-treated STO feeder layers. ES cell clusters were expanded on
mitomycin-treated STO feeder layers and maintained as ES cell lines. Approx-
imately 20% of the blastocysts gave rise to ES cell lines. For subsequent studies,
ES cells were cultured on gelatin-coated dishes in ES cell media containing LIF.
The genotypes of ES cell lines were determined by Southern blot analysis.
Twenty micrograms of isolated genomic DNA was digested with NcoI, subjected
to electrophoresis and transferred to nylon membrane. The blots were then
probed with a 500-bp KpnI-EcoRI fragment corresponding to the 3? region of
the CGBP gene locus, as previously described (10, 11). To rescue the CGBP?/?
ES cell phenotype, murine CGBP cDNA (10) was subcloned into pcDNA 3.1/
Zeo (Invitrogen). CGBP?/?ES cells were transfected with linearized plasmid by
electroporation (11), and zeomycin-resistant clones were recovered. Cell dou-
bling times were determined by seeding 1 ? 104cells in six-well dishes (in
triplicate) and collecting and counting cells at various times. To induce in vitro
differentiation, ES cells were trypsinized and single cell suspensions (1 ? 106
cells) were transferred to bacterial petri dishes containing 10 ml of ES cell media
lacking LIF (73) and cultured for up to 10 days. ES cells differentiated in culture
for 10 days were harvested, dispersed, and reseeded, and alkaline phosphatase
activity was histochemically detected using an alkaline phosphatase leukocyte
detection kit (Sigma).
Analysis of cytosine methylation. Analysis of global 5-methylcytosine in the
context of the sequence CCGG was analyzed by thin-layer chromatography as
described previously (38). Briefly, genomic DNA was digested with the restric-
tion enzyme MspI or the methyl-sensitive isoschizomer HpaII, labeled with T4
polynucleotide kinase and [?-32P]ATP, and digested with nuclease P1. Five-
methylcytosine monophosphate and cytosine monophosphate were separated by
thin-layer chromatography, visualized by autoradiography, and quantitated by
densitometry. Alternatively, global cytosine methylation was assessed utilizing a
methyl acceptance assay as described (4). Briefly, 500 ng of genomic DNA was
incubated with 2 ?Ci of3H-methyl-S-adenosyl L-methionine (Perkin-Elmer; 15
Ci/mmol), and 3 units of SssI methylase (New England Biolabs) in 120 mM NaCl,
10 mM Tris-HCl (pH 7.9), 10 mM EDTA, and 1 mM dithiothreitol (DTT) for 1 h
at 30°C. In vitro methylated DNA was isolated by filtration through Whatman
DE-81 ion-exchange filter, and incorporated radioactivity was measured by scin-
To analyze cytosine methylation at specific loci, genomic DNA was prepared
from various ES cell lines, subjected to restriction enzyme digestion and elec-
trophoresis, and transferred to nylon membrane for Southern blot analysis. Blots
were probed and washed as previously described (10, 71). Minor satellite and
intracisternal A-particle (IAP) probes were provided by En Li (Novartis Insti-
tutes for Biomedical Research, Cambridge, MA). The Rac2 probe was generated
from a 300-bp NcoI/KpnI fragment of the proximal murine promoter (51).
Additional probes were generated by PCR amplification. A 330-bp fragment was
used to analyze the MluI site of region 2 of the Igf2r gene (62). The H19
imprinted region was analyzed by using a 1.5-kb probe corresponding to 1,282 to
2,808 bp (GenBank accession no. U19619) (67). The Pgk-2 probe corresponded
to region III within the 3?-untranslated region (3).
Total DNA methyltransferase activity in ES cells was measured as described by
Li et al. (38). Exponentially growing ES cells were lysed and sonicated on ice in
1 to 2 ml of lysis buffer (20 mM Tris-HCl [pH 7.4], 0.4 M NaCl, 25% glycerol, 5
mM EDTA, 0.1% Nonidet P-40, 1 mM dithiothreitol [DTT]) containing a pro-
teinase inhibitor cocktail (Sigma). An equal volume of 50% DEAE-Sephace l
slurry equilibrated with lysis buffer was added and incubated for 10 min with
shaking at 4°C. The mixture was subjected to centrifugation, the supernatant was
collected, and protein concentration was determined by the Bradford method.
Thirty micrograms of protein was incubated in DNA methyltransferase assay
buffer (20 mM Tris-HCl [pH 7.4], 5 mM EDTA, 25% glycerol, 5 ?Ci of [3H]
methyl-S-adenosyl L-methionine, 4 ?g poly(dI-dC), 1 mM DTT, and 200 ?g/ml
bovine serum albumin) in a 200-?l reaction volume at 37°C for 2 h and extracted
twice with phenol-chloroform. The aqueous phase was adjusted to 0.1 M NaOH
and incubated at 50°C for 2 h. The solution was neutralized with HCl, and
radioactivity that incorporated into DNA was measured by scintillation counting
after trichloroacetic acid precipitation. Control reactions lacked poly(dI-dC).
Alternatively, DNA methyltransferase activity was assessed using hemimethy-
lated or unmethylated 33-bp double-stranded oligonucleotide substrates: 5?-GA
TCGCCGATGCGCGAATCGCGATCGATGCGAT-3? (methylated cytosine
are underlined) (61). Nuclear extracts were prepared as described previously
(16) with DNA methyltransferase lysis buffer and quantitated for protein con-
centration by the Bradford method. Twenty-five micrograms of nuclear extract
was incubated at 37°C for 2 h in DNA methyltransferase assay buffer (20 mM
Tris-HCl [pH 7.4], 5 mM EDTA, 25% glycerol) containing 5 ?Ci of [3H]methyl-
S-adenosyl L-methionine, 8 ?g oligonucleotide, 1 mM DTT, and 200 ?g/ml
bovine serum albumin in a total reaction volume of 200 ?l. Reactions were
extracted twice with phenol-chloroform, and DNA was precipitated with ethanol.
Reaction products were analyzed by 9% polyacrylamide gel electrophoresis, and
autoradiography was performed following fluorography. Band intensities were
4882 CARLONE ET AL.MOL. CELL. BIOL.
determined by densitometry. As a positive control, SssI methylase was substi-
tuted for nuclear extract.
De novo DNA methyltransferase activity was also measured following retro-
viral transduction of ES cells as described previously (36). Briefly, 1 ? 106
CGBP?/?or CGBP?/?ES cells were seeded in six-well dishes and then trans-
duced with Moloney murine leukemia virus retrovirus (generously provided by
En Li, Novartis) the following day. Cells were incubated with virus in the pres-
ence of polybrene (3.2 ?g/ml) for 7 h. Cells were then grown in normal ES cell
media and harvested at various times following transduction for isolation of
genomic DNA. The cytosine methylation status of the provirus was assessed by
digestion of isolated genomic DNA with KpnI and HpaII and Southern blot
analysis using a retroviral probe (36).
Northern and Western analyses. Total RNA was isolated from exponentially
growing wild-type (CCE916), CGBP?/?, and CGBP?/?ES cells using Tri-re-
agent solution per the manufacturer’s recommended protocol (Life Technolo-
gies, Carlsbad, CA). Twenty micrograms of total RNA was fractionated by
formaldehyde agarose gel electrophoresis and transferred to a nylon membrane
as described previously (71). The blot was hybridized with a 347-bp BamHI
fragment probe derived from the murine CGBP cDNA (10), washed as previ-
ously described (71), and exposed to X-ray film.
Whole-cell lysates were prepared in 8 M urea or Laemmli sample buffers.
Samples were subjected to electrophoresis and Western blot analysis as previ-
ously described (33). Membranes were incubated with antibody directed against
actin (Sigma), CGBP (71), Dnmt1, Dnmt3a (Santa Cruz Biotechnology), or
Dnmt3b (Imgenex, Inc.), followed by horseradish peroxidase-labeled secondary
antibody. Signal was detected with an ECL kit (Amersham) and autoradiography
and was quantitated by densitometry.
Reverse transcription-PCR analysis. For detection of developmental and lin-
eage-specific mRNAs expressed during in vitro ES cell differentiation, total RNA
was isolated from undifferentiated (t ? 0) ES cells and differentiated embryoid
bodies (5 and 10 days following the removal of LIF) using Tri-reagent. Total
RNA (1 ?g) was reverse transcribed using avian myeloblastosis virus reverse
transcriptase and random hexamers (Roche, Inc., Indianapolis, IN) at 42°C for
60 min. Single-stranded cDNA (0.1 ?g) was amplified in a 25-?l reaction mixture
that included 0.2 mM of each deoxynucleoside triphosphate, 50 pmol of sense
and antisense primers, and 1 U of Taq DNA polymerase (Roche) in buffer
supplied by the manufacturer. Samples were heat denatured at 94°C for 2 min,
followed by 25 to 30 cycles at 94°C for 30 s, 60°C for 30 s, 72°C for 30 s, and finally
10 min at 72°C. PCR was performed for HPRT to monitor the integrity of the
cDNA produced by reverse transcription. Ten microliters of amplified DNA was
subjected to electrophoresis on a 1.5% agarose gel in 0.5? Tris-borate-EDTA.
Primer pairs used were the following: Brachyury, 5?-ATCAAGGAAGGCTTTA
GCAAATGGG-3? and 5?-GAACCTCGGATTCACATCGTGAGA-3? (76);
GATA-4, 5?-CACTATGGGCACAGCAGCTCC-3? and 5?-TTGGAGCTGGCC
TGCGATGTC-3? (76); c-fms, 5?-CTGAGTCAGAAGCCCTTCGACAAAG-3?
and 5?-CTTTGCCCAGACCAAAGGCTGTAGC-3? (40); gp-IIB, 5?-AGGC
AGAGAAGACTCCGGTA-3? and 5?-TACCGAATATCCCCGGTAAC (70);
MHC-?, 5?-TGCAAAGGCTCCAGGTCTGAGGGC-3? and 5?-GCCAACACCA
ACCTGTCCAAGTTC-3? (42); HPRT, 5?-CACAGGACTAGAACACCTGC-3?
and 5?-GCTGGTGAAAAGGACCTCT-3? (31); Oct4, 5?-GGCGTTCTCTTTGGA
AAGGTGTTC-3? and 5?-CTCGAACCACATCCTTCTCT-3? (generously pro-
vided by Rebecca Chan, Indiana University).
Statistical analysis. Statistical significance was assessed by one-tailed t tests,
with a P value of ?0.05 interpreted as statistical significance.
Flow cytometric analysis. For cell cycle analysis, exponentially growing asyn-
chronous cells were harvested, washed twice with phosphate-buffered saline
(PBS), and incubated in PBS containing 0.3% NP-40, 0.5-mg/ml RNase A, and
50-?g/ml propidium iodide for 30 min on ice. Following staining, samples were
analyzed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA) and
ModFit LT software (Verity Software, Topsham, ME). Apoptotic cells were
detected using an Annexin V-FLUOS and propidium iodide staining kit
(Roche). One million exponentially growing asynchronous cells were harvested,
washed once with PBS, and incubated with 100 ?l of 1% Annexin V-FLUOS and
0.5 ?g/ml propidium iodide in 10 mM HEPES (pH 7.5), 140 mM NaCl, and 5
mM CaCl2at room temperature for 15 min. Binding buffer (400 ?l) was then
added prior to flow cytometric analysis. Apoptotic cells were defined as the
fraction of Annexin V-positive and propidium iodide-negative cells.
ES cells lacking CGBP are viable. The absence of murine
CGBP results in embryonic lethality at the peri-implantation
stage of development (11), indicating a critical role for this
DNA-binding protein in early mammalian embryogenesis. Due
to thedifficulty ofstudying
CGBP?/?ES cell lines were generated to permit more-de-
tailed studies on the function of CGBP. ES cell lines were
derived from blastocysts (3.5 dpc) resulting from matings be-
tween CGBP?/?mice. Several CGBP?/?(null) and CGBP?/?
(heterozygous) ES cell lines were obtained (Fig. 1A). Loss of
CGBP expression in CGBP?/?cells was confirmed by North-
ern (Fig. 1B) and Western (Fig. 1C) blot analyses. Western
blot analysis also revealed that CGBP?/?ES cells express
approximately 50% of the wild-type level of CGBP. A rescued
cell line, the CGBP?/cDNAcell line, was produced by stable
transfection of CGBP?/?ES cells with a CGBP cDNA expres-
sion vector. Western blot analysis confirmed the expression of
CGBP in the CGBP?/cDNAES cell line (approximately 50% of
wild type) but not in CGBP?/?ES cells transfected with the
parental vector (CGBP?/vector) (Fig. 1C).
CGBP-deficient ES cells exhibit an extended doubling time
due to increased apoptosis. Initial observations indicated that
CGBP?/?ES cells grow slower than wild-type ES cells.
CGBP?/?ES cells exhibit a doubling time of approximately
14.5 h, compared to 10.7 h for CGBP?/?ES cells (Fig. 2A).
Similar growth characteristics were observed for a second in-
dependent CGBP?/?ES cell line (data not shown). CGBP?/?
cells exhibit a growth rate indistinguishable from that of wild-
FIG. 1. Generation of ES cell lines lacking CGBP. (A) Southern blot analysis was performed on genomic DNA isolated from ES cell clones
derived from blastocysts resulting from heterozygous crosses. DNA was digested with NcoI and hybridized with a 500-bp KpnI/EcoRI probe
downstream of the CGBP gene (10, 11). The arrows indicate the wild-type and disrupted alleles, and the deduced genotype at the CGBP locus is
indicated above each lane. (B) Northern blot analysis was performed using total RNA isolated from either CGBP?/?or CGBP?/?ES cells and
a 347-bp BamHI fragment probe derived from the murine CGBP cDNA (10). The arrow indicates the murine CGBP transcript of approximately
2.6 kb. As a control for loading, 28S and 18S rRNA bands were visualized by ethidium bromide staining. (C) Western blot analysis was performed
on protein extracts isolated from ES cells carrying the indicated CGBP alleles using antiserum raised against CGBP (71). The blot was also
incubated with an antiactin antibody as a loading control.
VOL. 25, 2005CGBP REGULATES CYTOSINE METHYLATION 4883
type cells (data not shown). In addition, normal doubling time
was restored in rescued CGBP?/cDNAES cells (Fig. 2A), indi-
cating that this phenotype is a consequence of CGBP defi-
ciency. Despite an extended doubling time, CGBP?/?ES cells
exhibit a normal cell cycle distribution (Fig. 2B). Instead, stain-
ing for Annexin V reveals that approximately 33% of expo-
nentially growing CGBP?/?ES cells are dead or undergoing
apoptosis, in contrast to approximately 11% of CGBP?/?ES
cells (Fig. 2C). Hence, the extended doubling time of
CGBP?/?ES cells results from an increased rate of apoptosis.
Importantly, apoptosis rates return to near-normal levels in
rescued CGBP?/cDNAES cells, since 83% of CGBP?/cDNAES
cells are viable, compared to 67% of CGBP?/?ES cells and
88% of wild-type ES cells.
CGBP is required for in vitro ES cell differentiation. Be-
cause murine embryos lacking CGBP fail to gastrulate (11), in
vitro differentiation assays were performed to determine the
developmental potential of CGBP?/?ES cells. Both CGBP?/?
and CGBP?/?ES cells formed embryoid bodies at 2 days
following removal of LIF from the growth medium (data not
shown). However, after further culture under differentiation
conditions, CGBP?/?colonies grew in size and produced a
prominent outgrowth, while CGBP?/?colonies remained
small and failed to produce outgrowths (Fig. 3A). Gross mor-
phological evidence of in vitro differentiation was restored in
Molecular markers of differentiation were also examined to
assess the ability of CGBP?/?cells to achieve in vitro differ-
entiation. Alkaline phosphatase is a marker of pluripotency in
undifferentiated ES cells and is down-regulated upon cellular
differentiation (74). As expected, very few CGBP?/?ES cells
(2 to 3%) express alkaline phosphatase activity 10 days after
FIG. 2. CGBP?/?ES cells exhibit an extended doubling time due
to an elevated rate of apoptosis. (A) Growth rates were measured for
ES cells carrying the indicated CGBP alleles. The curves for the
CGBP?/?and rescued CGBP?/cDNAcells are overlapping. (B) Expo-
nentially growing ES cells of the indicated CGBP alleles were analyzed
for cell cycle distribution by propidium iodide (PI) staining and flow
cytometry. Numerical values represent the summary of data from three
experiments. 2N and 4N represent diploid (G0-G1) and tetraploid
(G2-M) genome content, respectively. (C) Exponentially growing ES
cells of the indicated CGBP alleles were analyzed for apoptosis using
Annexin V and propidium iodide staining and flow cytometry. UR,
upper right panel corresponding to dead cells; LL, lower left panel
corresponding to healthy cells; LR, lower right panel corresponding to
apoptotic cells. Numerical values represent the summary of data from
FIG. 3. Failure of in vitro cellular differentiation in the absence of
CGBP. (A) Colony morphology following induction of differentiation.
Embryoid body outgrowths carrying the indicated CGBP alleles were
cultured for 5 days in the absence of LIF. Magnification, ?20. (B) ES
cells grown in the absence of LIF for 10 days were harvested, disag-
gregated, reseeded, and stained for alkaline phosphatase activity. One
hundred cells of each genotype were scored for staining. Magnifica-
tion, ?64. (C) ES cells carrying the indicated CGBP alleles were
cultured for various times (0, 5, or 10 days) in the absence of LIF to
induce differentiation. Total RNA was isolated, and reverse transcrip-
tion-PCR was performed to examine the expression of lineage and
development-specific gene markers. Oct4, marker of undifferentiated
ES cells; Brachyury, mesoderm; MHC-?, muscle; c-fms, myeloid; gp-
IIB, megakaryocyte; GATA-4, endoderm; HPRT, control for RNA
quantity and integrity.
4884CARLONE ET AL.MOL. CELL. BIOL.
induction of differentiation. In contrast, alkaline phosphatase
activity was detected in ?96% of similarly treated CGBP?/?
cells, indicating a failure to differentiate (Fig. 3B). However,
alkaline phosphatase expression was extinguished in ?96% of
rescued CGBP?/cDNAcells, indicating that restoration of
CGBP expression permits in vitro differentiation.
The expression patterns of additional developmental and
lineage-specific gene markers were assessed in undifferentiated
ES cells and 5 or 10 days following the removal of LIF (Fig.
3C). Consistent with the alkaline phosphatase results,
CGBP?/?ES cells fail to down-regulate Oct4, a marker of
pluripotent cells that is down-regulated upon cellular differen-
tiation (47). In contrast, CGBP?/?and CGBP?/?cells down-
regulate Oct4 levels by day 10 of differentiation, as do rescued
CGBP?/cDNAcells. Following induction of in vitro differentiation,
CGBP?/?ES cells also fail to normally induce the expression of
the cardiac lineage marker MHC-? (42), the hematopoietic mark-
ers gp-IIb (megakaryocyte) (70) and c-fms (myeloid) (40), and
markers of earlier stages of differentiation, such as GATA-4
(visceral/parietal endoderm) (31) and Brachyury (mesoderm) (73)
(Fig. 3C). CGBP?/?and CGBP?/?ES cells induce the expres-
sion of each of these markers following removal of LIF. Rescued
CGBP?/cDNAES cells induce expression of all differentiation
markers that were examined (Fig. 3C). These results indicate that
CGBP plays a critical role in the initiation and execution of in
vitro ES cell differentiation.
Loss of CGBP results in reduced levels of 5-methylcytosine
in the genome. Studies were performed to gain insight into the
molecular basis for the developmental defects exhibited by
CGBP?/?ES cells. CGBP has the unique property of specifi-
cally binding to DNA sequences containing unmethylated CpG
motifs (71). Hence, we reasoned that CGBP might function to
modulate the methylation state of the genome. Global cytosine
methylation levels were quantitated by thin-layer chromatog-
raphy following digestion with MspI to determine if loss of
CGBP affects DNA methylation in the context of the sequence
CCGG. As previously demonstrated (38), CGBP?/?ES cells
exhibit a high degree of cytosine methylation (approximately
55% of CpG dinucleotides) (Fig. 4A and B). Loss of a single
CGBP allele led to a slight but statistically significant reduction
(P ? 0.05) in the level of global cytosine methylation. How-
ever, a dramatic, approximately 60% reduction in global cyto-
sine methylation was observed in CGBP?/?ES cells. Absence
of signal for 5-methylcytosine following digestion with the
methyl-sensitive enzyme HpaII (Fig. 4A) demonstrates the
specificity of the assay. A similar decrease in global cytosine
methylation was observed in a second, independently derived
CGBP?/?ES cell line (data not shown). Cytosine methylation
levels were significantly increased in rescued CGBP?/cDNA
cells (compared to CGBP?/?cells) but not in CGBP?/vectorES
cells (Fig. 4A and B).
Global cytosine methylation was also assessed by determin-
ing the ability of genomic DNA to accept methyl groups (4).
These experiments confirm the involvement of CGBP in reg-
ulating global cytosine methylation (Fig. 4C). DNA derived
from CGBP?/?cells accepts fivefold more methyl groups than
do DNA samples isolated from CGBP?/?or CGBP?/?ES
cells, indicating an 80% decline in global cytosine methylation
in cells lacking CGBP. The capacity to accept methyl groups
was reduced to wild-type levels in CGBP?/cDNAES cells but
not CGBP?/vectorcells. These results indicate that CGBP is
required for normal global cytosine methylation.
Additional studies were conducted to examine cytosine
methylation at specific genomic loci. Southern blot analysis
reveals that minor satellite repetitive elements are highly
methylated in CGBP?/?and CGBP?/?ES cells, as indicated
by the absence of a low-molecular-weight ladder in HpaII-
digested genomic DNA (Fig. 5A). In the absence of CGBP,
these sequences become hypomethylated, as revealed by in-
creased HpaII digestion and appearance of a low-molecular-
weight ladder. A similar disruption in cytosine methylation is
observed at IAP retroviral sequences (Fig. 5B). Four distinct
HpaII-digested fragments were detected in DNA isolated from
CGBP?/?ES cells but not from CGBP?/?ES cells. However,
CGBP?/?ES cells did not exhibit a complete loss of cytosine
methylation at these repetitive elements, since the levels of
low-molecular-weight DNA fragments produced by HpaII di-
gestion were not comparable in intensity with those generated
by digestion with the methyl-insensitive isoschizomer MspI.
These results are consistent with the partial decrease of global
cytosine methylation observed in CGBP?/?ES cells (Fig. 4).
Methylation of repetitive sequences was restored in rescued
FIG. 4. CGBP is essential for proper DNA methylation in murine ES cells. (A) Global cytosine methylation levels in ES cells carrying the
indicated CGBP alleles were determined in the context of CCGG by thin-layer chromatography following digestion of genomic DNA with the
restriction enzymes MspI or HpaII. A representative experiment is shown. (B) Summary of thin-layer chromatography data from three experi-
ments. Error bars represent standard error, and asterisks denote a statistically significant (P ? 0.05) difference compared to the wild type. Double
asterisks denote a statistically significant (P ? 0.05) difference between CGBP?/?cells and CGBP?/cDNAcells. (C) Global cytosine methylation
levels in ES cells carrying the indicated CGBP alleles were determined by methyl acceptance assay. Error bars represent standard error, and
asterisks denote a statistically significant (P ? 0.05) difference compared to the wild type. Double asterisks denote a statistically significant (P ?
0.05) difference between CGBP?/?cells and CGBP?/cDNAcells. The experiment was performed three times.
VOL. 25, 2005 CGBP REGULATES CYTOSINE METHYLATION4885
CGBP?/cDNAES cells, further demonstrating a functional role
for CGBP in the regulation of DNA methylation.
The pattern of cytosine methylation at single-copy genes was
also assessed. A probe derived from the hematopoietic cell-
restricted Rac2 promoter (51) was used to analyze the meth-
ylation status of a 2-kb NcoI genomic fragment that contains
several HpaII restriction sites. Analysis of genomic DNA iso-
lated from CGBP?/?or CGBP?/?ES cells reveals a dominant
2-kb band following digestion with NcoI and HpaII, indicating
heavy cytosine methylation throughout this region (Fig. 6A).
The intensity of this band is dramatically reduced in CGBP?/?
ES cells, indicating reduced cytosine methylation, although
partial methylation persists because a band representing a fully
demethylated region (300 bp) is not apparent. Similar results
were found for the Pgk-2 gene. A probe derived from the
3?-flanking region detects three dominant high-molecular-
weight bands when genomic DNA isolated from CGBP?/?or
CGBP?/?ES cells is digested with BamHI and the methyl-
sensitive enzyme HhaI (Fig. 6B). However, the intensity of
these bands is reduced when DNA isolated from CGBP?/?ES
cells is similarly analyzed, and four low-molecular-weight
bands become more abundant. Introduction of the CGBP
cDNA into CGBP?/?ES cells (CGBP?/cDNA) restores cyto-
sine methylation at both of these gene loci.
The methylation status of imprinted genes in CGBP?/?ES
cells was next examined (Fig. 7). The upstream region of the
paternally imprinted H19 gene, which contains the differen-
tially methylated domain that controls expression of the Igf2r
and H19 genes (64), was examined by Southern blot analysis. A
large DNA fragment corresponding to the paternally methyl-
ated H19 allele and a smaller fragment representing the hy-
pomethylated maternal allele were observed in CGBP?/?and
CGBP?/?ES cells, while a loss of methylation at the paternal
allele was observed in CGBP?/?ES cells (Fig. 7A). Methyl-
ation at the H19 locus is increased following restoration of
CGBP expression (CGBP?/cDNA), suggesting that epigenetic
marks persist elsewhere within this locus in CGBP?/?ES cells.
Analysis of region 2 of the maternally methylated Igf2r gene
(60) revealed methylated and unmethylated DNA fragments
corresponding to the maternal and paternal alleles, respec-
tively, in CGBP?/?and CGBP?/?ES cells. In CGBP?/?ES
cells, however, only the unmethylated fragment was detected,
indicating a loss of maternal imprinting (Fig. 7B). In contrast
to the H19 locus, the Igf2r locus was not remethylated in
CGBP?/?ES cells exhibit decreased DNA methyltrans-
ferase activity. Additional experiments were performed to di-
rectly assess the DNA methylation machinery in CGBP?/?ES
cells. Protein extracts isolated from these cells exhibited a 30%
reduction in total DNA methyltransferase activity towards a
poly(dI-dC) substrate (Fig. 8A). Activity was restored in
CGBP?/cDNAES cells. Similar experiments were performed
using hemimethylated or unmethylated oligonucleotide sub-
strates to distinguish between maintenance and de novo DNA
methyltransferase activity (Fig. 8A). Extracts derived from
CGBP?/?cells exhibit a 60% decline in DNA methyltrans-
ferase activity towards the hemimethylated oligonucleotide
substrate but normal activity towards the unmethylated sub-
strate. These results indicate a deficiency in maintenance cy-
tosine methylation in the absence of CGBP. As an indepen-
dent method to determine whether CGBP is necessary for de
novo cytosine methylation, CGBP?/?ES cells were assessed
for their ability to methylate a newly introduced retroviral
transgene. The retroviral provirus begins to acquire cytosine
methylation within 48 h of transduction in both CGBP?/?and
CGBP?/?ES cells (Fig. 8B), indicating normal de novo DNA
methyltransferase activity. However, in contrast to CGBP?/?
cells, significant levels of unmethylated provirus persist in
CGBP?/?ES cells. The intensity of the unmethylated band is
70% of that of the fully methylated band in CGBP?/?ES cells
4 days following transduction, compared to 30% for CGBP?/?
ES cells, consistent with a defect in maintenance DNA methyl-
transferase activity in the absence of CGBP. A similar pattern
of proviral cytosine methylation was previously found in ES
cells lacking Dnmt1 (36).
Western blot analysis was performed to determine the ex-
pression level of Dnmt enzymes (Fig. 9). These studies reveal
a 50% reduction of the Dnmt1 protein in CGBP?/?ES cells.
Dnmt1 levels recover in CGBP?/cDNAES cells but not in
CGBP?/vectorcells. Significantly reduced levels of Dnmt3a are
also observed in CGBP?/?cells, but this does not correlate
with the absence of CGBP, since similarly reduced levels of
Dnmt3a are observed in CGBP?/?and CGBP?/cDNAES cells.
However, heterozygous and rescued ES cells contain only ap-
proximately 50% of wild-type CGBP levels (Fig. 1C), so it
remains possible that this partial deficiency leads to decreased
Dnmt3a expression. The significance of this finding is unclear,
since CGBP?/?and CGBP?/cDNAES cells contain normal
genomic cytosine methylation and CGBP?/?cells exhibit nor-
mal de novo methyltransferase activity. However, Dnmt3a has
FIG. 5. CGBP?/?ES cells exhibit reduced cytosine methylation at
repetitive genomic elements. (A) Genomic DNA was isolated from ES
cells carrying the indicated CGBP alleles, digested with MspI or HpaII,
and Southern blot analysis was performed using a probe for the minor
satellite repetitive element. The bracket indicates the region of low-
molecular-weight bands that reflect cytosine hypomethylation. The
ethidium bromide-stained gel is shown below to illustrate relative
DNA loading. (B) Same as in panel A, except the probe corresponds
to the IAP retrovirus repetitive element. Arrows indicate bands that
reflect hypomethylation. The ethidium bromide-stained gel is shown
below to illustrate relative DNA loading.
4886 CARLONE ET AL.MOL. CELL. BIOL.
been found to be required for maintenance of cytosine meth-
ylation patterns in ES cells (13). No significant decrease in
Dnmt3b was detected in CGBP?/?ES cells. In summary, these
data indicate that CGBP?/?ES cells exhibit reduced levels of
genomic cytosine methylation and reduced maintenance DNA
CGBP is a transcriptional activator that exhibits a unique
DNA-binding specificity for sequences containing unmethyl-
ated CpG motifs (34, 71). Disruption of the CGBP gene in
mice leads to peri-implantation death (11). Although this find-
ing establishes the importance of CGBP for normal mamma-
lian development, the early time of death makes further anal-
ysis of this mutant difficult. In the study reported here, murine
ES cells lacking CGBP were generated to permit a more-
detailed analysis of the CGBP?/?phenotype and provide fur-
ther insight into normal CGBP function.
The ability to isolate CGBP?/?ES cell lines demonstrates
that this protein is not essential for ES cell viability. However,
consistent with the inability of CGBP?/?embryos to gastru-
late, CGBP?/?ES cells are unable to achieve in vitro differ-
entiation. Instead, they remain undifferentiated following re-
moval of LIF from the growth medium, as indicated by
persistent expression of Oct4 and alkaline phosphatase, mark-
ers of the undifferentiated state. These in vitro data suggest
that CGBP?/?mice exhibit a peri-implantation death due to
the requirement of CGBP for the execution of specific differ-
entiation programs, rather than exhaustion of maternally in-
herited stores of CGBP protein. Methylation of the Oct4 pro-
moter is essential for its down-regulation during ES cell
differentiation (23, 26). It remains to be determined whether
persistent Oct4 expression, possibly as a consequence of de-
FIG. 6. CGBP?/?ES cells exhibit reduced cytosine methylation at single-copy genes. (A) Genomic DNA was isolated from ES cells carrying
the indicated CGBP alleles and digested with NcoI and HpaII, and Southern blot analysis was performed using a probe for the Rac2 gene (shown
schematically). Arrows indicate a 2-kb band produced by HpaII and NcoI digestion of the fully methylated promoter and a 300-bp band produced
by MspI digestion or by HpaII digestion if the restriction sites are unmethylated. The bent arrow indicates site of transcription initiation. N, NcoI;
H, HpaII. The ethidium bromide-stained gel is shown below to illustrate relative DNA loading. (B) Same as in panel A, except the genomic DNA
was digested with HhaI and BamHI, and the probe corresponds to the Pgk-2 gene (shown schematically). The top three arrows indicate
high-molecular-weight bands that correspond to heavy cytosine methylation which are dominant in CGBP?/?, CGBP?/?, and CGBP?/cDNAcells.
The bottom four arrows indicate low-molecular-weight bands that reflect hypomethylation and increase in intensity in CGBP?/?and CGBP?/vector
cells. The bent arrow indicates site of transcription initiation. B, BamHI. HhaI sites are denoted by hatch marks, and the question mark indicates
a region of undetermined nucleotide sequence. The ethidium bromide-stained gel is shown below to illustrate relative DNA loading.
VOL. 25, 2005 CGBP REGULATES CYTOSINE METHYLATION4887
fective promoter methylation, is causally related to the inability
of CGBP?/?ES cells to achieve in vitro differentiation. Alter-
natively, persistent Oct4 expression could be a secondary con-
sequence of the failure of these cells to effectively initiate the
differentiation program, possibly due to a global derangement
of epigenetic modifications.
Given the binding specificity of CGBP for DNA sequences
containing unmethylated CpG motifs (34, 71), genomic cyto-
sine methylation patterns in CGBP?/?ES cells were analyzed.
CGBP?/?ES cells exhibit a 60 to 80% decrease in global
cytosine methylation, including reduced cytosine methylation
of repetitive elements, single-copy genes, and imprinted genes.
This deficiency is correlated with reduced maintenance DNA
methyltransferase activity, because CGBP?/?cell extracts ex-
hibit a 60% reduction in DNA methyltransferase activity to-
wards a hemimethylated DNA substrate and a 50% reduction
in the level of Dnmt1 protein. In contrast, de novo DNA
methyltransferase activity in CGBP?/?ES cells is normal. To
our knowledge, CGBP?/?ES cells represent the first example
of reduced maintenance DNA methyltransferase activity with-
out direct abrogation of Dnmt gene function.
Importantly, nearly all of the epigenetic perturbations de-
tected in CGBP?/?ES cells are corrected upon introduction of
the CGBP cDNA into these cells, illustrating the plasticity of
the epigenome. The exception is the maternally imprinted Igf2r
gene, which remains hypomethylated following restoration of
CGBP expression, DNA methyltransferase activity, and global
cytosine methylation. These results are consistent with previ-
ous reports of variable degrees of corrected genomic imprint-
ing upon restoration of Dnmt expression in DNA methyltrans-
ferase-deficient cells (7, 13, 68, 69). The ability of wild-type
CGBP to rescue defects in CGBP?/?ES cells offers an attrac-
tive system with which to probe structure/function relation-
ships of this novel factor.
The deficiencies observed for cytosine methylation in
CGBP?/?cells, although dramatic, cannot account for the
severity of the observed phenotype. For example, ES cells
lacking Dnmt1 exhibit a 90% reduction in DNA methyltrans-
ferase activity and cytosine methylation yet exhibit normal
growth prior to in vitro differentiation (36). In contrast, undif-
ferentiated CGBP?/?ES cells exhibit a 35% increase in dou-
bling time as a consequence of increased apoptosis. Others
have reported the existence of an epigenetic surveillance
mechanism which induces apoptosis or cell cycle arrest in re-
sponse to aberrations in cytosine methylation patterns or re-
duced levels of Dnmt1, respectively (28, 43, 59). In addition,
mouse embryos lacking Dnmt1 die later in gestation (8.5 to 9.5
dpc) (36) than CGBP?/?embryos (4.5 to 6.5 dpc) (11). Finally,
decreased DNA methyltransferase activity and Dnmt1 protein
levels detected in CGBP?/?ES cells are unlikely to fully ex-
plain the observed deficiency in genomic cytosine methylation,
since Dnmt1?/?ES cells expressing reduced Dnmt1 protein
retain normal levels of cytosine methylation (12, 38).
The existence of CGBP homologues in lower eukaryotes
that lack CpG methylation, such as yeast and C. elegans, pro-
vides circumstantial evidence for a function of CGBP that is
independent of cytosine methylation. Interestingly, sequence
alignment reveals that CGBP homologues in organisms that
lack CpG methylation lack the CXXC DNA-binding domain
(data not shown). The yeast CGBP homologue, Spp1, is a
FIG. 7. CGBP?/?ES cells exhibit reduced cytosine methylation at imprinted genes. (A) Genomic DNA was isolated from ES cells carrying the
indicated CGBP alleles and digested with HhaI and SacI, and Southern blot analysis was performed using a probe for the H19 gene (shown
schematically). Arrows indicate position of paternally derived (imprinted) and maternally derived (nonimprinted) alleles. The bent arrow indicates
site of transcription initiation. S, SacI; H, HhaI. The ethidium bromide-stained gel is shown below to illustrate relative DNA loading. (B) Same
as in panel A, except the DNA was digested with MluI and PvuII and the probe corresponds to the Igf2r locus (shown schematically). Arrows
indicate position of maternally derived (imprinted) allele and paternally derived (nonimprinted) allele. P, PvuII. The ethidium bromide-stained gel
is shown below to illustrate relative DNA loading.
4888CARLONE ET AL.MOL. CELL. BIOL.
component of the megadalton Set1 histone methyltransferase
complex (41). Spp1 is dispensable for Set1 histone methyl-
transferase activity in Saccharomyces cerevisiae but is necessary
for histone methyltransferase activity in Schizosaccharomyces
pombe (56). In addition, human CGBP colocalizes to an iden-
tical set of nuclear speckles with the human trithorax (HRX,
ALL-1, MLL1) histone methyltransferase (33). The composition
of the megadalton HRX histone methyltransferase complex has
been reported, but CGBP was not detected (45, 79). Similarly,
CGBP was not detected as a component of the mammalian Set1/
Ash2 histone methyltransferase complex (75). However, these
studies reported the composition of soluble complexes. CGBP is
localized nearly exclusively in the nuclear matrix and hence might
not have been recovered by the extraction methods utilized.
Whether CGBP interacts with histone methyltransferase com-
plexes at the nuclear matrix remains to be determined. However,
given the presence of the yeast CGBP homologue in a histone-
modifying complex, it is tempting to speculate that mammalian
CGBP may also play a role in the control of histone modification
and chromatin structure.
Indeed, there are several examples of perturbed patterns of
cytosine methylation as a consequence of altered chromatin
structure. For example, cytosine methylation in Neurospora is
dependent on methylation of histone H3 (63), and inhibition of
HDAC activity by trichostatin A results in a loss of cytosine
methylation (57). Furthermore, the chromatin remodeling pro-
tein DDM1 in Arabidopsis and the related factor LSH in mam-
mals are required for normal cytosine methylation (15, 29, 30),
and disruption of the Suv39h histone methyltransferase gene in
murine ES cells leads to altered localization of Dnmt3b and
decreased cytosine methylation at pericentric satellite repeats
(35). Conversely, deficiency in Dnmt1 leads to increased his-
tone acetylation and decreased histone H3–Lys9 methylation
at pericentromeric sequences (18). It will therefore be inter-
esting to determine if CGBP?/?ES cells exhibit aberrations in
Many details regarding the mechanism of CGBP function
remain unknown. For example, how does CGBP influence the
expression of Dnmt1? What are the target genes to which
CGBP binds in vivo? Does CGBP also influence the activity of
histone-modifying complexes? And why is the action of this
transcriptional activator necessary for normal cytosine meth-
ylation, a modification usually associated with repressed gene
expression? The data reported here indicate that CGBP facil-
itates the activity of the DNA methylation machinery and im-
plicate CGBP as an important epigenetic regulator. A better
FIG. 8. CGBP?/?ES cells exhibit reduced maintenance DNA
methyltransferase activity. (A) Protein extracts were prepared from ES
cells carrying the indicated CGBP alleles and assayed for in vitro DNA
methyltransferase activity using poly(dI-dC), hemimethylated oligonu-
cleotide, or unmethylated oligonucleotide substrates. Error bars rep-
resent standard error, and asterisks denote a statistically significant (P
? 0.05) difference compared to the wild type. Double asterisks denote
a statistically significant (P ? 0.05) difference between CGBP?/?cells
and CGBP?/cDNAcells. Each experimental value represents a summary
from three experiments, with the exception of the analysis of CGBP?/?
and CGBP?/?extracts using the poly(dI-dC) substrate, which was
performed seven times. (B) CGBP?/?or CGBP?/?ES cells were
transduced with the murine Moloney leukemia virus retrovirus.
Genomic DNA was isolated at the indicated times following transduc-
tion and the provirus analyzed for cytosine methylation by Southern
blot analysis. The structure of the provirus is illustrated schematically
above the blot. H, HpaII; K, KpnI. Arrows indicate position of un-
methylated and fully methylated provirus fragments.
FIG. 9. CGBP?/?ES cells express reduced levels of Dnmt1 pro-
tein. Protein extracts isolated from ES cells carrying the indicated
CGBP alleles were subjected to Western blot analysis using antiserum
directed against Dnmt1, Dnmt3a, or Dnmt3b. An antiserum directed
against actin was utilized as an internal control for protein loading. A
representative experiment is presented, and the summary of data from
numerous experiments is presented below. The number of replicates
for each experimental value is indicated (N#). Error bars represent
standard errors, and asterisks denote a statistically significant (P ?
0.05) difference compared to the wild type. Double asterisks denote a
statistically significant (P ? 0.05) difference between CGBP?/?cells
VOL. 25, 2005CGBP REGULATES CYTOSINE METHYLATION 4889
understanding of CGBP function will provide important in-
sights into epigenetic regulation.
We thank En Li for the minor satellite and IAP probes, Rebecca
Chan for providing the Oct4 primers, and Merv Yoder and Loren
Field for helpful discussions.
This work is supported by the Riley Children’s Foundation, NIH
grant HL69974 (D.G.S.), a grant from the 21st Century Fund from the
State of Indiana (J.R.), an NRSA from the NIH (D.L.C.), an American
Heart Association postdoctoral fellowship (D.L.C.), American Heart
Association and GAANN predoctoral fellowships (S.R.L.Y.), and an
Indiana University Cancer Biology Training Fellowship to J.S.B.
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