Methyl binding domain protein 2 mediates ?-globin
gene silencing in adult human ?YAC transgenic mice
Jeremy W. Rupon*†, Shou Zhen Wang*, Karin Gaensler‡, Joyce Lloyd*§, and Gordon D. Ginder*†§¶?
*Massey Cancer Center and Departments of¶Internal Medicine,†Microbiology and Immunology, and§Human Genetics, Virginia Commonwealth University,
Richmond, VA 23298; and‡Department of Medicine, University of California, San Francisco, CA 94143
Edited by Gary Felsenfeld, National Institutes of Health, Bethesda, MD, and approved March 6, 2006 (received for review October 27, 2005)
The genes of the vertebrate ?-globin locus undergo a switch in
expression during erythroid development whereby embryonic?
fetal genes of the cluster are sequentially silenced and adult genes
are activated. We describe here a role for DNA methylation and
MBD2 in the silencing of the human fetal ?-globin gene. The
?-globin gene is reactivated upon treatment with the DNA meth-
yltransferase inhibitor 5-azacytidine in the context of a mouse
containing the entire human ?-globin locus as a yeast artificial
chromosome (?YAC) transgene. To elucidate the mechanism
through which DNA methylation represses the ?-globin gene in
adult erythroid cells, ?YAC?MBD2?/? mice were generated by
breeding ?YAC mice with MBD2??? mice. Adult ?YAC?MBD2?/?
mice continue to express the ?-globin gene at a level commensu-
rate with 5-azacytidine treatment, 10- to 20-fold over that ob-
served with 1-acetyl-2-phenylhydrazine treatment alone. In addi-
tion, the level of ?-globin expression is consistently higher in
MBD2??? mice in 14.5- and 16.5-days postcoitus fetal liver eryth-
roblasts suggesting a role for MBD2 in embryonic?fetal erythroid
development. DNA methylation levels are modestly decreased in
MBD2??? mice. MBD2 does not bind to the ?-globin promoter
region to maintain ?-globin silencing. Finally, treatment of MBD2-
null mice with 5-azacytidine induces only a small, nonadditive
induction of ?-globin mRNA, signifying that DNA methylation acts
primarily through MBD2 to maintain ?-globin suppression in adult
DNA methylation ? epigenetics ? transcription
tional repression. A group of proteins, the methyl-CpG binding
proteins (MCBP), specifically recognizes and binds to DNA
sequences containing methylated cytosines. Most of these pro-
teins belong to a subgroup based on the common features of a
characteristic domain, the methyl-CpG binding domain (MBD),
which is necessary and sufficient for binding of the proteins to
methylated DNA (1). The MBD protein family consists of
MeCP2, MBD1, MBD2, MBD3, and MBD4. MBD2 and MeCP2
have been found to bind methylated DNA as large multiprotein
complexes (2–4). MBD4 differs from the other MBD proteins in
that its primary function is as a DNA repair enzyme (5). DNA
methylation-mediated transcriptional repression is thought to be
due to the recruitment of MCBPs and repressive protein com-
plexes to methylated DNA. These complexes are thought to
mediate transcriptional repression by recruiting histone deacety-
lases and transcriptional repressor proteins to methylated DNA
(6–10). MBD2 is part of the methyl-CpG binding protein
complex 1 (MeCP1), which contains Mi-2, MTA1, MTA2,
MBD3, HDAC1, HDAC2, RbAp46, and RbAp48 (4). Early
studies showed an interaction between MeCP2 and the SIN3A
transcriptional repression complex. However, recent evidence
suggests that this interaction may not be stable (11). Brahma, a
component of the SWI?SNF nucleosome remodeling complex,
does form a stable complex with MeCP2 (12). The Kaiso factor
represents another type of MCBP that contains a transcriptional
repressor domain as well as a zinc finger domain that confers
ethylation of the 5? position of cytosine in the CpG
dinucleotide in vertebrates is associated with transcrip-
sequence specificity, but lacks an MBD (13). The zinc fingers of
Kaiso can bind methylated CGCGs or the Kaiso binding site
Targeted deletions of MCBPs have brought insight into the
in adult neurogenesis and hippocampal function (15). Mice lacking
functional MeCP2 display a phenotype similar to Rett syndrome, a
human neurodevelopmental disorder leading to loss of voluntary
movements (16). Loss of MBD2 has a milder phenotype with the
chief manifestation being decreased maternal nurturing of pups
IL-4 gene in mouse Th1 and Th2 lymphocytes indicating that
MBD2 can contribute to the regulation of genes in a tissue-specific
manner (9). However, no other mammalian genes have been
Unlike the embryonic lethal phenotype of DNA methyltransferase
(DNMT) knockout mice, loss of individual MBDs is not cata-
strophic in mammals, indicating that MBDs may compensate for
each or that only a small number of genes required for normal cell
function are regulated by any specific MBD (18, 19). Interestingly,
Kaiso, a MCBP not in the family of MBD-containing genes, has
been shown to be essential for Xenopus development but not for
mouse development (20, 21).
The genes of the human ?-globin locus are expressed sequen-
tially during development in the order that they appear on
chromosome 11: 5? ?-,G?-,A?-, and ?-3?. The individual genes
have been shown to be regulated by a complex interplay between
cis elements, trans factors, competition for an upstream en-
hancer, and epigenetics (22). Despite a tremendous amount of
progress in this field, the exact mechanism(s) of globin gene
silencing is still not completely understood. Among the initial
observations in vertebrates that DNA methylation is inversely
related to polymerase type II gene expression were those noted
in the globin locus (23, 52, 53). Furthermore, the globin genes
were the first group of genes for which the DNA methyltrans-
ferase inhibitor 5-azacytidine was shown to activate silenced
embryonic and fetal genes in vivo in both animal models and in
a therapeutic setting (24–27). Previously, we have shown a role
for DNA methylation and MBD2 in the normal developmental
regulation of the chicken embryonic ?-globin gene (25, 28–30).
The ?-globin gene becomes highly methylated at the same time
that the gene becomes transcriptionally silent. Furthermore, the
?-globin gene is enriched for MBD2 in adult erythrocytes when
the gene is silent but not when it is actively transcribed. Finally,
a protein complex containing MBD2 purified from primary
chicken erythrocytes binds to a methylated ?-globin gene 5?
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: ?YAC, human ?-globin locus yeast artificial chromosome; ChIP, chromatin
immunoprecipitation; dpc, days postcoitus.
?To whom correspondence should be addressed at: Departments of Internal Medicine,
Human Genetics, and Microbiology and Immunology, Massey Cancer Center, Virginia
Commonwealth University, 401 College Street, P.O. Box 980037, Richmond, VA 23298-
0037. E-mail: email@example.com.
© 2006 by The National Academy of Sciences of the USA
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sequence and differs in biochemical make-up from MeCP1
(unpublished data). These findings are evidence that MCBPs
may form tissue-restricted complexes that are targeted to spe-
cific sets of genes. Taken together, this evidence suggests MBD2
may be involved in the process of globin gene silencing by DNA
To determine the role of MBD2 in mammalian globin gene
silencing, mice containing the entire human globin locus as a
yeast artificial chromosome (?YAC) were bred with mice con-
taining a targeted deletion of MBD2. Adult ?YAC?MBD2?/?
mice inappropriately express the ?-globin gene at a level com-
mensurate to mice treated with 5-azacytidine, whereas adult
wild-type ?YAC mice do not express the ?-globin gene. This is,
to our knowledge, the first evidence of a specific MCBP regu-
lating ?-globin expression. In addition, loss of MBD2 delays the
silencing of the ?-globin gene during embryonic development.
greatly enhance the expression of the ?-globin gene, indicating
that the inductive effect of 5-azacytidine seen in knockout mice
does not largely depend on DNA methylation or is mediated by
a different MCBP.
?-Globin Expression Is Increased in Adult ?YAC?MBD2?/? Mice.
?YAC?MBD2?/? mice were generated by breeding ?YAC
(lines A20.1 and A85.68; Fig. 1A) mice with MBD2??? mice
and breeding the subsequent transgenic hemizygotes with
MBD2??? mice (17, 31). Mice were treated with 1-acetyl-2-
phenylhydrazine to induce hemolytic anemia for the purpose of
increasing the number of reticulocytes for RNA analysis and
converting the spleen primarily to a site of erythropoiesis (32).
Peripheral blood was collected on the third day after treatment.
For 5-azacytidine treatment, ?YAC?MBD2?/? mice were
treated for 2 days with 1-acetyl-2-phenylhydrazine followed by 5
days of 5-azacytidine treatment. Peripheral blood was collected
the day after the final treatment. Globin gene expression was
analyzed by using RNase protection assay. As seen in Fig. 1B,
?-globin mRNA was barely detectable by this assay in adult
?YAC?MBD2?/? mice and ?YAC?MBD2?/? mice. How-
ever, both ?YAC mice treated with 5-azacytidine and ?YAC?
MBD2?/? mice express the ?-globin gene at increased levels,
?2% per copy of mouse ?-globin (Fig. 1C). Similar results were
obtained when using nonanemic animals (data not shown). Only
trace levels of endogenous mouse embryonic globin genes or
human ?-globin gene transcripts were detectable by RNase
protection assay or real-time PCR (data not shown). However,
we have observed that a human epsilon gene transgene contain-
ing a full LCR and 12 kb of downstream flanking sequence but
no ?- or ?-globin gene is partially activated in the MBD2???
background. Thus, MBD2 is necessary for maintaining complete
?-globin silencing in adult mice, and loss of MBD2 results in
markedly increased ?-globin expression but not expression of
mouse or human embryonic ?-type globin genes. This result is
likely due to the ability of the fetal ?-globin gene but not the
embryonic ?-type globin genes to partially compete with the
the transgenic mice used in this study. Expression analysis was performed with both lines; the rest of the experiments were performed with the A20.1 line. (B)
RNA was analyzed by RNase protection assay. Fifty times more RNA was analyzed by using the ?-probe than mouse ?-globin. Samples were mixed after
(RPA) results. The level of ?-globin mRNA is expressed per copy of the endogenous mouse ?-globin. Bands were quantitated by using a phosphorimager. The
results shown are the average of three separate RPAs from at least two mice per genotype or treatment. The black bars represent the A20.1 line, and the gray
bar represents the A85.68 line. (D) RPA showing levels ofG?- andA?-globin expression in ?YAC mice treated with 5-azacytidine and ?YAC?MBD2?/? mice. The
probe used in this RPA is able to distinguish between theG?- andA?-globin genes. (E) Quantitation of RPA results showing the level ofA?-globin expression
relative to total ?-globin expression.
www.pnas.org?cgi?doi?10.1073?pnas.0509322103Rupon et al.
adult ?-globin gene for the LCR because of more dominant
autonomous silencing of the embryonic genes. In addition, the
level of induction seen is similar to the level achieved after
treatment with 5-azacytidine, indicating that MBD2 and 5-aza-
cytidine possibly work through the same pathway. The percent-
age ofG?- vs.A?-globin mRNA was determined by using an
RNase protection assay probe that discriminates between the
two genes (33). The percentage ofA?-globin mRNA was 60% for
?YAC?MBD2?/? mice and 70% for ?YAC?MBD2?/? mice
similar to the levels seen in adult human F-cells and opposite to
theA?- toG?-globin ratio in K562 cells or fetal human erythroid
cells, suggesting that the ?-globin mRNA is from definitive cells
rather than from a reactivation of primitive erythropoiesis.
The ?-globin gene is also expressed in adult transgenic knock-
out mice derived from a different ?YAC transgene (A85.68; Fig.
because each single copy ?YAC transgene is located in a
different region of the mouse genome. In addition, each line is
derived from a different genetic background, and through
multiple rounds of breeding, heterogeneous backgrounds were
generated, indicating that the effect seen is not via an unknown
Loss of MBD2 Delays ?-Globin Gene Silencing During Development.
Adult erythroid cells from mice lacking MBD2 inappropriately
express the ?-globin gene. We next wanted to determine the
impact of MBD2 on globin gene expression during development.
Timed matings were performed and embryos were harvested
14.5 and 16.5 days postcoitus (dpc). At 14.5 dpc, embryos lacking
MBD2 expressed 2-fold more ?-globin mRNA than did wild-
type embryos (Fig. 2). This difference increased to 3.5-fold in
16.5-dpc fetal livers (Fig. 2). In addition, no human ?-globin
transcripts are detectable by RNase protection assay after 16.5
dpc in wild-type or knockout mice (data not shown). The effect
seen in adult knockout mice appears to be at least in part due to
an inability to fully silence the ?-globin gene during the switch
to definitive erythropoiesis. The effect is specific for the ?-globin
genes and not the embryonic ?-type globin genes.
DNA Methylation Around the ?-Globin Promoter Is Largely Unaffected
in MBD2??? Erythroblasts. Lack of MBD2 results in expression of
the ?-globin gene in adult ?YAC transgenic mice. MCBPs bind
to methylated DNA and recruit factors that lead to transcrip-
tional repression. In addition, MBD2 has been shown to colo-
calize with DNA methyl transferase 1 (DNMT1) (34). We
therefore postulated that if MBD2 binds at or near the ?-globin
gene promoter, then the effect seen in adult ?YAC?MBD2?/?
mice may be accompanied by a loss of DNA methylation around
the ?-globin promoter. Bisulfite sequencing was performed to
determine the methylation status in ?YAC?MBD2?/? mice,
?YAC?MBD2?/? mice treated with 5-azacytidine, and ?YAC?
MBD2?/? mice. A 70-bp region containing four CpGs around
the ?-globin promoter was analyzed. As shown in Fig. 3, wild-
type adult erythroblasts show a high level of methylation (75–
100%) at each CpG tested. Furthermore, no individual clone
(represented as a row in Fig. 3) was either completely or ?75%
unmethylated, indicating that all cells are highly methylated
around the ?-globin promoter. Treatment with 5-azacytidine led
to a decrease in methylation at all sites (40–60% methylated)
with four clones having no methylation at any site tested. This
result is similar to results seen in MEL cells containing human
chromosome 11 and primary erythroid cells of patients treated
with 5-azacytidine (35, 36). MBD2??? mice show an interme-
diate level of DNA methylation at each site (60–90% methyl-
was 75% demethylated. DNA methylation levels were modestly
decreased in the absence of MBD2 relative to the results seen in
animals treated with 5-azacytidine. The observed partial de-
methylation is more consistent with increased transcription of
the gene preventing DNA methylation by DNMT rather than
MBD2 recruiting DNMT activity to the ?-globin promoter.
MBD2 Does Not Bind Near the ?-Globin Promoter. To determine
directly whether MBD2 was mediating its effect at or near the
?-globin promoter, chromatin immunoprecipitation (ChIP) as-
gene during development in wild-type and MBD2??? ?YAC transgenic mice.
Fetal livers were dissected from 14.5- and 16.5-dpc embryos, and RNA was
isolated. The level of expression of the ?-globin relative to the endogenous
mouse ?-globin was determined by using RPA and quantitated by using a
phosphorimager. The results shown are the average of at least three livers
from a total of at least two litters.
Level of expression of the ?-globin gene per copy of mouse ?-globin
site. Each row represents one sequenced clone. A black bar indicates a meth-
ylated CpG, a white bar indicates an unmethylated CpG, and a gray bar
indicates an undetermined CpG.
Bisulfite analysis of 1-acetyl-2-phenylhydrazine-treated spleens. The
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says were performed. Both ?YAC?MBD2?/? and ?YAC?
MBD2?/? mice were treated with 1-acetyl-2-phenylhydrazine
to promote conversion of the spleen to a primarily erythroid
organ (32). Splenic erythroblasts were used for ChIP with
antibody directed against MBD2. As shown in Fig. 4, no enrich-
ment is seen for MBD2 near the ?-globin promoter in wild-type
?YAC transgenic mice. However, MBD2 does bind in vivo to the
CpG island region of the Ugt8 gene. This gene is induced
?4-fold in splenic erythroblasts in the absence of MBD2 (data
not shown). In addition, only baselines levels of enrichment are
seen at the Ugt8 CpG island region and the ?-globin promoter
region in MBD2-null mice, indicating that the MBD2 antibody
is very specific. As expected, no MBD2 enrichment is seen near
the highly transcribed human ?-globin gene. These results
indicate MBD2 is not acting directly at the ?-globin promoter
region to mediate ?-globin silencing in adult ?YAC transgenic
Treatment of ?YAC?MBD2?/? Mice with 5-Azacytidine Results in only
a Small Further Increase in ?-Globin Gene Expression. Treatment of
?YAC?MBD2?/? mice with 5-azacytidine leads to a 10- to
15-fold induction of the ?-globin gene versus mice treated with
1-acetyl-2-phenylhydrazine only (Fig. 5), which is similar to the
level of activation seen in ?YAC?MBD2?/? mice and as
reported by Pace et al. (37) in a different ?YAC line treated with
5-azacytidine. To determine whether 5-azacytidine and loss of
MBD2 induce ?-globin via the same mechanism through over-
coming DNA methylation, ?YAC?MBD2?/? mice were
treated with 5-azacytidine. Blood was collected before 5-azacy-
tidine treatment and again 5 days after treatment and analyzed
by both RNase protection assay and quantitative real-time PCR
(Figs. 1 and 5, respectively). 5-Azacytidine only induces the
?-globin by 2.5-fold in MBD2??? mice. The lack of an additive
effect of MBD2 deficiency upon 5-azacytidine-mediated induc-
tion strongly suggests that both are largely working through the
same pathway, which is mediated by DNA methylation. This
pathway regulates the ?-globin gene at the transcriptional level
because loss of MBD2 leads to a ?4-fold enrichment of Tri-
MeK4, a mark of active transcription, at the ?-globin gene region
(Fig. 4) (38). To confirm that mice deficient for MBD2 do not
display the hematological parameters observed in erythropoietic
stress, red blood cell indices and reticulocyte counts were
and MBD2??? mice are not statistically different except for a
slight microcytosis seen in MBD2-null mice. However, the
MBD2??? mice used in this study were all females, whereas the
wild-type group consisted of more male mice that were younger
in age. This age and sex difference may account for the slight
difference in red cell mean corpuscular volume observed. Re-
gardless, the reticulocyte counts did not differ significantly,
indicating that MBD2 loss does not result in erythropoietic
MBD2 is needed for complete suppression of ?-globin gene
expression in ?YAC transgenic mice. Loss of the protein leads
to a level of expression from a single copy of the ?-globin gene
commensurate with ?YAC mice treated with 5-azacytidine,
?2–4% of the endogenous mouse ?-globin RNA per gene copy
representing a ?10-fold increase. In addition, developmental
silencing of the transgene is delayed in the absence of MBD2
because these mice express the ?-globin gene at higher levels in
anti-MBD2, anti-TriMeK4, and control antibodies (IgG or normal serum) from
MBD2??? and MBD2??? splenic erythroblasts. The sequence-specific en-
richment was determined by real-time PCR and calculated by using the fol-
lowing formula: (experimental antibody-gene/amylase)?(control antibody-
gene/amylase). MBD2 and MBD2-control IPs were performed on three mice.
TriMeK4 and control IPs were performed on two mice.
Results from ChIP assay. Chromatin was immunoprecipitated with
wild-type and MBD2??? ?YAC transgenic mice. Data are shown as the level
before 5-azacytidine treatment. Mice were treated for 2 days with 1-acetyl-
2-phenylhydrazine, after which peripheral blood was collected and 5-aza-
cytidine treatment begun for 5 days. Peripheral blood was collect after
5-azacytidine treatment. The level of ?-globin RNA expression was compared
before and after 5-azacytidine treatment by using real-time PCR and SYBR
Real-time PCR analysis of ?-globin induction by 5-azacytidine in
Table 1. Red cell indices and reticulocyte counts
RBC, 106??l Hb, g?dlHct MCV, fl MCH, pg per cellMCHC, g?dlRetic, %
7.6 ? 1.1
6.7 ? 1.6
13.2 ? 1.2
11.7 ? 2.7
37.2 ? 5.1
30.3 ? 8.1
49.3 ? 1.5
44.6 ? 0.3
17.5 ? 0.9
17.2 ? 0.4
35.8 ? 1.8
38.6 ? 1.2
6.2 ? 0.9
5.9 ? 1.5
Red cell indices were determined using blood from three nonanemic mice of each genotype. Reticulocyte count was performed on
two MBD2??? mice. P values were determined by using a Student t test.
www.pnas.org?cgi?doi?10.1073?pnas.0509322103Rupon et al.
14.5- and 16.5-dpc fetal livers. Studies in human–mouse ery-
throid cell hybrids indicated that the ?-globin gene was meth-
ylated after it was silenced (39). The results in this transgenic
model show that silencing is at least delayed in the absence of the
methyl-CpG binding protein MBD2, suggesting that DNA meth-
ylation may contribute to ?- to ?-globin gene switching. The
results shown here are consistent with the requirement for DNA
methylation and MBD2-mediated silencing at a site other than
the ?-globin gene for complete switching to occur. In this case,
methylation at the ?-globin promoter might be expected to lag
behind silencing, thus reconciling the present results with those
in human–mouse erythroid cell hybrids.
DNA methylation levels are modestly reduced whereas Tri-
MeK4 levels are modestly increased around the ?-globin gene in
mice with 5-azacytidine leads to only a 2.5-fold induction of
?-globin mRNA relative to ?YAC mice treated with 5-azacyti-
dine. Together these results indicate that MBD2 and 5-azacyti-
dine both are influencing the same pathway to induce ?-globin
expression. In as much as the regulation of the ?-globin gene in
this transgenic model reflects the situation in human erythroid
cells, the results support the notion that most of the effect of
5-azacytidine on stimulating ?-globin gene expression in humans
is through its inhibitory effects on DNA methylation (27, 40, 41).
The additional stimulation of ?-globin gene expression in
?YAC?MBD2?/? mice may be due to cytotoxic or cell cycle
effects, such as those observed in primates treated with S-phase
active cytotoxic drugs that do not affect DNA methylation
Most MCBPs are thought to inhibit transcription by binding
near promoters and recruiting transcriptional repression com-
plexes. We have shown here that MBD2 does not bind near the
?-globin promoter and that only minor changes in DNA meth-
ylation status are present in ?YAC?MBD2?/? mice. This is not
completely surprising because the ?-globin promoter does not
possess a CpG island, a region of 200 bp or more that has a
CpG:GpC ratio of ?0.5 and that is required for MeCP1 complex
formation in vitro (45, 46). In addition, there are no significant
changes in global DNA methylation levels in MBD2??? mice
(17). While MBD2 has been shown to bind to a sequence
containing as few as three CpG in vitro, MBD2 has only been
shown to bind in vivo to methylated CpG island sequences (7, 9,
47). The lack of major enrichment in posttranslational histone
modifications in the absence of MBD2 may be due to the
relatively low level of ?-globin expression. The human ?-globin
gene is highly expressed in both MBD2??? and MBD2???
?YAC transgenic mice and shows a much higher enrichment for
H3 trimethyl K4 (data not shown) (31). The ?-globin gene may
not be expressed highly enough to impart large changes in
histone modifications that occur secondary to recruitment of
activating modifier enzymes by the RNA polymerase II complex.
Similar results have been seen in transgenic mice carrying a
mutant ?YAC expressing the human ?-globin gene at low levels
(48). Taken together, the results here show that the ?-globin
gene becomes actively transcribed either in the absence of
MBD2 or upon treatment with 5-azacytidine with definite but
not major changes in epigenetic modifications that most likely
reflect changes induced by transcription rather than by loss of
inactivating histone modifier enzymes recruited directly by
MBD2. In addition, because erythropoiesis in MBD2??? mice
is not globally perturbed, the effect is quite specific. Because
there are no CpG islands within 6 kb of the ?-globin gene, these
results are most consistent with a model in which loss of MBD2
results in transcriptional activation of a gene or genes that are
normally silent in adult erythroid cells. The products of this
gene(s) would, in turn, results in transcriptional activation of the
?-globin gene. Microarray analyses have identified numerous
genes that are activated in splenic erythroblasts of MBD2???
mice, including genes that reside in hematopoietic pathways
(data not shown).
Many compounds currently available to treat patients with
hemoglobinopathies carry short- or long-term potential risks of
toxicity, and, in addition, the response to the agents is variable.
Here, we show that loss of MBD2 leads to expression of the
normally silent ?-globin gene in adult ?YAC transgenic mice.
Given the mild phenotype of MBD2-null mice, MBD2 may
represent an attractive potential target for the treatment of
?-thalassemia or sickle cell anemia, conditions in which in-
creased ?-globin gene expression has an ameliorating effect.
Generation of ?YAC?MBD2?/? Mice.MBD2??? mice (a generous
gift from Adrian Bird, University of Edinburgh, Edinburgh,
U.K.) were mated with ?YAC mice. The subsequent ?YAC?
MBD2?/? mice. To maintain the line, ?YAC?MBD2?/? were
bred with MBD2??? mice. All progeny were screened for the
presence of the transgene and absence of MBD2 by using PCR
of DNA from tail snips. Two lines of ?YAC mice were used to
generate transgenic knockout mice, the A20.1 and A85.68 lines
(Fig. 1A). Both contain a single copy of the ?YAC transgene in
the C57 BL?6 background (A20.1) or the FVB?N background
(A85.68). The MBD2??? mice were in the BALB?C back-
ground. Through multiple rounds of breeding to establish and
maintain lines, the final transgenic knockout mice were in a
heterogeneous genetic background.
Timed Matings. For MBD2 wild-type embryos, ?YAC males were
bred with nontransgenic females. For MBD2??? embryos,
?YAC?MBD2?/? males were bred with nontransgenic
MBD2??? females. The presence of a vaginal plug was desig-
nated day 0.5. Fetal livers were dissected from day-14.5 and
Treatments. Mice were treated for 2 days with i.p. injection of
1-acetyl-2-phenylhydrazine (10 mg?ml; Sigma) at a dose of 0.4
mg?10 g. On the third day, mice were treated with 5-azacytidine
(0.5 mg?ml; Sigma) at a dose of 2 mg?kg for 5 days via i.p.
RNA Isolation. RNA was extracted from fetal livers or peripheral
blood by using TRIzol (Invitrogen) according to the manufac-
turer’s protocol. Fetal liver RNA was further purified by LiCl
RNase Protection Assay. The assay was performed as described in
ref. 49. The probe for mouse ?-globin has been described (50).
Probes for ?-globin analysis were from a 1.7-kb fragment of the
A?-globin gene protecting two bands (205 and 144 bp) when cut
with EcoRI or one band when cut with NcoI (188 bp). To
discriminate betweenG?- andA?-globin mRNA, a probe iden-
tical to one used previously was generated by PCR (33).
Bisulfite Conversion. Mice were treated with 1-acetyl-2-
phenylhydrazine for 2 days and harvested on the fifth or seventh
day. Genomic DNA was isolated from spleens by using the Zymo
Research genomic isolation kit. The DNA was bisulfite-
converted by using the Zymo Research EZ methylation kit.
Bisulfite converted DNA was amplified by using nested PCR
with primers specific for the ?-globin promoter region. PCR
products were cloned, screened, and sequenced.
ChIP. Mice were treated for 2 days with 1-acetyl-2-phenylhy-
drazine. On the fifth day, spleens were harvested and gently
brushed into single-cell suspension in ice-cold RPMI containing
2% FBS and 5 mM butyrate, PMSF, aprotinin, leupeptin, and
Rupon et al.
April 25, 2006 ?
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no. 17 ?
pepstatin. Protease inhibitors were included in all steps until the Download full-text
to remove debris. Cells were spun and washed with ice-cold PBS
containing 2% FBS. Cells were then resuspended in room-
temperature PBS?FBS. Formaldehyde was added dropwise to a
final concentration of 0.4% (TriMeK4) or 1% (MBD2), and cells
were crosslinked for 10 min at room temperature. The reaction
was terminated by adding glycine to 0.125 M and incubating at
room temperature for 5 min. Cells were washed twice with
PBS?FBS. Cells were resuspended in SDS?lysis buffer at a
concentration of 25–30 mg?ml and incubated on ice for 10 min.
Chromatin was sonicated six times (TriMeK4) or eight times
(MBD2) for 20 s. Twenty-five milligrams of spleen was used per
IP with anti-trimethyl H3 K4 (Abcam or Upstate), rabbit IgG
(Upstate), normal rabbit serum (Santa Cruz Biotechnology),
anti-MBD2 (Upstate), and sheep IgG (Upstate). The rest of the
procedure was performed as described previously with slight
modifications for MBD2 ChIPs (51). For MBD2 ChIP, protein
G salmon sperm agarose beads (Upstate) were used and were
washed three times with low-salt buffer and one time with
medium-salt buffer (250 mM NaCl).
Real-Time PCR. For expression analysis, RNA was reverse-
transcribed by using iScript (Bio-Rad) and quantitated by using
SYBR green chemistry on an Applied Biosystems 7900HT PCR
system. For ChIP, real-time PCR was performed as described in
ref. 51. Primer sequences are available upon request.
Red Cell Indices. Peripheral blood was collected into citrate-
coated tubes from nonanemic mice. Red cell indices were
measured on a Coulter MicroDiff 1600. Reticulocyte counts
were determined by manually counting methylene blue-stained
Basu for thoughtful insights. This work was supported by National
Institutes of Health Grant DK29902 and the Massey Cancer Center.
J.W.R. was supported by the Virginia Commonwealth University M.D.?
1. Nan, X., Meehan, R. R. & Bird, A. (1993) Nucleic Acids Res. 21, 4886–4892.
2. Nan, X., Ng, H. H., Johnson, C. A., Laherty, C. D., Turner, B. M., Eisenman,
R. N. & Bird, A. (1998) Nature 393, 386–389.
3. Jones, P. L., Veenstra, G. J., Wade, P. A., Vermaak, D., Kass, S. U.,
4. Feng, Q. & Zhang, Y. (2001) Genes Dev. 15, 827–832.
6. Sarraf, S. A. & Stancheva, I. (2004) Mol. Cell 15, 595–605.
7. Magdinier, F. & Wolffe, A. P. (2001) Proc. Natl. Acad. Sci. USA 98, 4990–4995.
8. Lin, X. & Nelson, W. G. (2003) Cancer Res. 63, 498–504.
9. Hutchins, A. S., Mullen, A. C., Lee, H. W., Sykes, K. J., High, F. A., Hendrich,
B. D., Bird, A. P. & Reiner, S. L. (2002) Mol. Cell 10, 81–91.
10. Chen, W. G., Chang, Q., Lin, Y., Meissner, A., West, A. E., Griffith, E. C.,
Jaenisch, R. & Greenberg, M. E. (2003) Science 302, 885–889.
11. Klose, R. J. & Bird, A. P. (2004) J. Biol. Chem. 279, 46490–46496.
12. Harikrishnan, K. N., Chow, M. Z., Baker, E. K., Pal, S., Bassal, S., Brasacchio,
D., Wang, L., Craig, J. M., Jones, P. L., Sif, S., et al. (2005) Nat. Genet. 37,
13. Prokhortchouk, A., Hendrich, B., Jorgensen, H., Ruzov, A., Wilm, M., Geor-
giev, G., Bird, A. & Prokhortchouk, E. (2001) Genes Dev. 15, 1613–1618.
14. Daniel, J. M., Spring, C. M., Crawford, H. C., Reynolds, A. B. & Baig, A. (2002)
Nucleic Acids Res. 30, 2911–2919.
15. Zhao, X., Ueba, T., Christie, B. R., Barkho, B., McConnell, M. J., Nakashima,
K., Lein, E. S., Eadie, B. D., Willhoite, A. R., Muotri, A. R., et al. (2003) Proc.
Natl. Acad. Sci. USA 100, 6777–6782.
16. Guy, J., Hendrich, B., Holmes, M., Martin, J. E. & Bird, A. (2001) Nat. Genet.
17. Hendrich, B., Guy, J., Ramsahoye, B., Wilson, V. A. & Bird, A. (2001) Genes
Dev. 15, 710–723.
18. Okano, M., Bell, D. W., Haber, D. A. & Li, E. (1999) Cell 99, 247–257.
19. Li, E., Bestor, T. H. & Jaenisch, R. (1992) Cell 69, 915–926.
20. Ruzov, A., Dunican, D. S., Prokhortchouk, A., Pennings, S., Stancheva, I.,
Prokhortchouk, E. & Meehan, R. R. (2004) Development (Cambridge, U.K.)
21. Prokhortchouk, A., Sansom, O., Selfridge, J., Caballero, I. M., Salozhin, S.,
Aithozhina, D., Cerchietti, L., Meng, F. G., Augenlicht, L. H., Mariadason,
J. M., et al. (2006) Mol. Cell. Biol. 26, 199–208.
22. Stamatoyannopoulos, G. & Grosveld, F. (2001) in The Molecular Basis of Blood
Diseases, eds. Stamatoyannopoulos, G., Majerus, P. W., Perlmutter, R. M. &
Varmus, H. (Saunders, Philadelphia), pp. 135–182.
23. McGhee, J. D. & Ginder, G. D. (1979) Nature 280, 419–420.
24. Ley, T. J., DeSimone, J., Anagnou, N. P., Keller, G. H., Humphries, R. K.,
25. Ginder, G. D., Whitters, M. J. & Pohlman, J. K. (1984) Proc. Natl. Acad. Sci.
USA 81, 3954–3958.
26. DeSimone, J., Heller, P., Hall, L. & Zwiers, D. (1982) Proc. Natl. Acad. Sci.
USA 79, 4428–4431.
27. Charache, S., Dover, G., Smith, K., Talbot, C. C., Jr., Moyer, M. & Boyer, S.
(1983) Proc. Natl. Acad. Sci. USA 80, 4842–4846.
28. Singal, R., Wang, S. Z., Sargent, T., Zhu, S. Z. & Ginder, G. D. (2002) J. Biol.
Chem. 277, 1897–1905.
29. Singal, R., Ferris, R., Little, J. A., Wang, S. Z. & Ginder, G. D. (1997) Proc.
Natl. Acad. Sci. USA 94, 13724–13729.
30. Burns, L. J., Glauber, J. G. & Ginder, G. D. (1988) Blood 72, 1536–1542.
31. Porcu, S., Kitamura, M., Witkowska, E., Zhang, Z., Mutero, A., Lin, C., Chang,
J. & Gaensler, K. M. (1997) Blood 90, 4602–4609.
32. Spivak, J. L., Toretti, D. & Dickerman, H. W. (1973) Blood 42, 257–266.
33. Morley, B. J., Abbott, C. A. & Wood, W. G. (1991) Blood 78, 1355–1363.
34. Tatematsu, K. I., Yamazaki, T. & Ishikawa, F. (2000) Genes Cells 5, 677–688.
35. Ley, T. J., Chiang, Y. L., Haidaris, D., Anagnou, N. P., Wilson, V. L. &
Anderson, W. F. (1984) Proc. Natl. Acad. Sci. USA 81, 6618–6622.
36. Humphries, R. K., Dover, G., Young, N. S., Moore, J. G., Charache, S., Ley,
T. & Nienhuis, A. W. (1985) J. Clin. Invest. 75, 547–557.
37. Pace, B., Li, Q., Peterson, K. & Stamatoyannopoulos, G. (1994) Blood 84,
38. Schneider, R., Bannister, A. J., Myers, F. A., Thorne, A. W., Crane-Robinson,
C. & Kouzarides, T. (2004) Nat. Cell Biol. 6, 73–77.
39. Enver, T., Zhang, J. W., Papayannopoulou, T. & Stamatoyannopoulos, G.
(1988) Genes Dev. 2, 698–706.
40. Ley, T. J., DeSimone, J., Noguchi, C. T., Turner, P. H., Schechter, A. N., Heller,
P. & Nienhuis, A. W. (1983) Blood 62, 370–380.
41. Koshy, M., Dorn, L., Bressler, L., Molokie, R., Lavelle, D., Talischy, N.,
Hoffman, R., van Overveld, W. & DeSimone, J. (2000) Blood 96, 2379–2384.
42. Veith, R., Galanello, R., Papayannopoulou, T. & Stamatoyannopoulos, G.
(1985) N. Engl. J. Med. 313, 1571–1575.
43. Papayannopoulou, T., Torrealba, d. R., Veith, R., Knitter, G. & Stamatoyan-
nopoulos, G. (1984) Science 224, 617–619.
44. Letvin, N. L., Linch, D. C., Beardsley, G. P., McIntyre, K. W. & Nathan, D. G.
(1984) N. Engl. J. Med. 310, 869–873.
45. Cross, S. H. & Bird, A. P. (1995) Curr. Opin. Genet. Dev. 5, 309–314.
46. Antequera, F. & Bird, A. (1993) Proc. Natl. Acad. Sci. USA 90, 11995–11999.
47. Fraga, M. F., Ballestar, E., Montoya, G., Taysavang, P., Wade, P. A. & Esteller,
M. (2003) Nucleic Acids Res. 31, 1765–1774.
48. Fang, X., Sun, J., Xiang, P., Yu, M., Navas, P. A., Peterson, K. R., Stamatoy-
annopoulos, G. & Li, Q. (2005) Mol. Cell. Biol. 25, 7033–7041.
49. Little, J. A., Dempsey, N. J., Tuchman, M. & Ginder, G. D. (1995) Blood 85,
50. Curtin, P. T., Liu, D. P., Liu, W., Chang, J. C. & Kan, Y. W. (1989) Proc. Natl.
Acad. Sci. USA 86, 7082–7086.
51. Barrett, D. M., Gustafson, K. S., Wang, J., Wang, S. Z. & Ginder, G. D. (2004)
Mol. Cell. Biol. 24, 6194–6204.
52. Van der Ploeg, L. H. T. & Flavell, R. A. (1980) Cell 19, 947–958.
53. Shen, C. K. & Maniatis, T. (1980) Proc. Natl. Acad. Sci. USA 77, 6634–
www.pnas.org?cgi?doi?10.1073?pnas.0509322103 Rupon et al.