Reprogramming of the paternal genome upon
fertilization involves genome-wide oxidation
Khursheed Iqbala,1, Seung-Gi Jinb,1, Gerd P. Pfeiferb,2, and Piroska E. Szabóa,2
Departments ofaMolecular and Cellular Biology andbCancer Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010
Edited by Peter A. Jones, University of Southern California, Los Angeles, CA, and accepted by the Editorial Board January 28, 2011 (received for review
September 17, 2010)
Genome-wide erasure of DNA cytosine-5 methylation has been
reportedtooccuralongthe paternal pronucleusinfertilizedoocytes
in an apparently replication-independent manner, but the mecha-
nism of this reprogramming process has remained enigmatic. Re-
cently, considerable amounts of 5-hydroxymethylcytosine (5hmC),
most likely derived from enzymatic oxidation of 5-methylcytosine
(5mC) by TET proteins, have been detected in certain mammalian
tissues. 5hmC has been proposed as a potential intermediate in
active DNA demethylation. Here, we show that in advanced pro-
nuclear-stage zygotes the paternal pronucleus contains substantial
amounts of 5hmC but lacks 5mC. The converse is true for the
later cleavage-stage embryos, suggesting that 5mC oxidation is not
followed immediately by genome-wide removal of 5hmC through
excision repair pathways or other mechanisms. This conclusion is
supported by bisulfite sequencing data, which shows only limited
conversion of modified cytosines to cytosines at several gene loci.
but not Tet1 or Tet2, was expressed at high levels in oocytes
and zygotes, with rapidly declining levels at the two-cell stage.
Our results show that 5mC oxidation is part of the early life cycle
tion, X chromosome inactivation, reprogramming, and malig-
nant transformation are major events characterized by remark-
able changes in the epigenome and involve remodeling of DNA
methylation patterns (3–10). Despite the relatively stable and
heritable features of DNA methylation in somatic cells, genome-
wide DNA demethylation occurs both in developing primordial
germ cells and in fertilized oocytes (zygotes) (11, 12). In zygotes,
a striking asymmetric DNA demethylation of the two parental
genomes seems to occur within the same oocyte cytoplasm, be-
ginning as early as 6 h after fertilization, when the paternal ge-
nome undergoes active DNA demethylation but the maternal
genome resists demethylation (13–15). This process appears to
be largely independent of DNA replication. The maternal ge-
nome later on undergoes passive demethylation in the absence of
maintenance methyltransferase DNMT1 during DNA replica-
tion in cleavage-stage embryos (11, 13, 16).
The replication-independent DNA demethylation of the pa-
ternal genome points to the existence of a mammalian DNA
demethylase activity. However, the identity of such an activity
has remained enigmatic and controversial for over a decade (17,
18). Activation-induced cytidine deaminases or related activities
may work in conjunction with DNA glycosylases to remove 5-
methylcytosine (5mC) from DNA. After deamination of 5mC to
thymine has been catalyzed by the deaminase, the mismatched
thymine will be excised from the resulting G:T base pairs (19–
26). The base excision repair pathway can then be further en-
gaged to incorporate cytosine bases, resulting in replacement of
5mC with C (20). In plants, a demethylase pathway involving
ethylation at the 5-position of cytosines is an important
component of the epigenetic code (1, 2). Cell differentia-
direct removal of 5mC by DNA glycosylase activity has been
identified (27, 28), but these proteins do not have mammalian
homologs. Furthermore, it was reported that the protein
GADD45A promotes demethylation of CpG-methylated DNA
(29), perhaps in conjunction with excision repair activities (23,
30). However, a role of GADD45A in DNA demethylation has
not been confirmed (31, 32). Specifically addressing active de-
methylation of the paternal genome in zygotes, Okada et al. have
used a siRNA knockdown strategy in oocytes followed by in-
tracytoplasmic sperm injection to screen for candidate DNA
demethylase genes. Okada et al. identified the elongator com-
plex, and in particular its subunit Elp3, as a component required
for zygotic DNA demethylation in the paternal pronucleus (33).
Taking allavailable information intoaccount, perhaps themost
considerable evidence suggests that cytidine deaminases work in
conjunction with DNA glycosylases to remove 5mC in a DNA
repair pathway (19–26). However, if not strand-specifically co-
ordinated, excision repair would put the genome at risk for DNA
double-strand breakage, and this is expected to be detrimental at
those critical stages of development when the reprogramming
events take place.
One plausible mechanism for demethylation of 5mC, without
the need for a DNA repair process, is oxidation of the methyl
group followed by secondary reactions that eventually lead to
restoration of cytosine. Recently, Kriaucionis and Heintz and
Tahiliani et al. made the important discovery that substantial
amounts of 5-hydroxymethylcytosine (5hmC), initially thought to
be only a rare DNA damage product (34), are present in mouse
Purkinje and granule neurons and in embryonic stem cells (35,
36). An enzymatic activity involved in producing 5hmC from
5mC by oxidation was identified as TET1 (36). The two other
mammalian homologs of TET1, TET2, and TET3, all containing
a dioxygenase motif involved in Fe(II) and α-ketoglutarate
binding and catalytic activity, were shown to posses similar ac-
tivities as well (37).
The goal of our study was to investigate if 5mC oxidation
occurs in fertilized oocytes and is part of the apparent DNA
demethylation process that takes place during this early de-
Author contributions: G.P.P. and P.E.S. designed research; K.I. and S.-G.J. performed re-
search; K.I., S.-G.J., G.P.P., and P.E.S. analyzed data; and G.P.P. and P.E.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. P.A.J. is a guest editor invited by the Editorial
1K.I. and S.-G.J. contributed equally to this work.
2To whom correspondence may be addressed. E-mail: email@example.com or pszabo@coh.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| March 1, 2011
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Using specific antibodies, we determined the levels of 5mC and
5hmC in male and female pronuclei in zygotes and in early
cleavage-stage embryos. We used a recently available commer-
cial polyclonal antibody directed against 5hmC. Initially, we
verified the specificity of this antibody toward 5hmCs versus
5mCs or unmodified cytosines placed at identical positions
within CpG sequences in synthetic single-stranded 76-mer oli-
gonucleotide substrates (38). In immuno-dot blot assays, we
observed that this antibody is specific for 5hmC and does not
react with substrates containing only unmodified cytosines, nor
does it react with substrates containing 5mC (Fig. S1A). We then
tested the suitability of the anti-5hmC antibody for immunos-
taining experiments using human 293T cells. As initially deter-
mined by immuno-dot blot assays, this cell line contains de-
tectable levels of 5hmC (Fig. S1B). A nuclear staining pattern
was observed with the anti-5hmC antibody (Fig. S1C). To test for
specificity of the staining reaction, we preincubated the antibody
with synthetic oligonucleotides containing C, 5mC, or 5hmC. As
shown in Fig. S1C, the nuclear staining was completely elimi-
nated by preincubation of the antibody with 5hmC-containing
oligonucleotides but not by competition with the other oligo-
nucleotides attesting to the suitability of the antibody for im-
We hypothesized that 5hmC might be detectable as a potential
intermediate during DNA demethylation in zygotes. Using the
anti-5hmC antibody, we observed intense staining of the paternal
pronucleus in mouse zygotes (Fig. 1A), whereas 5hmC staining
was almost completely absent from the maternal pronucleus. To
further test the specificity of the staining pattern, we carried out
competition experiments with synthetic oligonucleotides and
observed that the staining of zygotes for 5hmC is specific (Fig.
S2). Simultaneous double-staining with an established anti-5mC
antibody (16), which does not react with 5hmC (38), detected
5mC in the maternal but not in the paternal pronucleus (Fig. 1 A
and B). Thus, the two staining patterns are mutually exclusive,
suggesting that 5mC has been converted to 5hmC specifically in
the paternal pronucleus. Fig. 1 A and B show late pronuclear
stages (PN4–PN5). In vitro fertilized zygotes have similar stain-
ing patterns. Bispermic zygotes exhibit 5hmC staining in both
paternal pronuclei (Fig. 1C). The maternally and paternally
inherited chromosomes are localized in separate compartments
at metaphase and are marked by 5mC and 5hmC staining, re-
spectively (Fig. 1D). The condensed chromosomes at anaphase
stain differentially for 5hmC or 5mC, suggesting that they are
paternally or maternally derived (Fig. 1E).
Genome-wide loss of 5mC signal by antibody staining is known
to occur beginning around PN3 (15, 39), which is consistent with
our observations. To determine if the timing of 5hmC appear-
ance coincides with the loss of 5mC staining, we looked at earlier
pronuclear stages, including PN1 to PN3 (Fig. 2A). The distance
of the two pronuclei decreases and the size of both pronuclei
increases with advancing pronuclear stages. Staining with anti-
5mC antibody detected 5mC in both maternal and paternal nu-
clei at the earliest pronuclear stages, and this signal was de-
creasing in the paternal pronucleus as the zygote developed. The
signal for 5hmC was visible at low levels in both pronuclei at the
early stages. The level of 5hmC staining of the paternal pronu-
cleus increased relative to the maternal pronucleus with devel-
opmental stage. At the same time, the paternal to maternal pro-
nucleus signal decreased for 5mC staining (Fig. 2B).
The asymmetrical staining pattern is still observed in two-cell–
stage embryos (Fig. 3 A and B), in which different compartments
of the nuclei are strikingly enriched for 5mC or 5hmC, re-
spectively. These nuclear compartments are derived from pa-
ternal and maternal chromosomes, respectively, which occupy
distinct territories (13, 40). Confocal microscopy images give
a clear example of the asymmetric distribution of 5hmC and 5mC
along the two-chromosome sets in two-cell–stage embryos en-
tering mitosis (Fig. 3C). We further observed that the asym-
metrical 5hmC signal persists toward the four- and eight-cell
stages (Fig. 3D). These findings suggest that 5hmC is maintained
for a considerable amount of time after it was initially formed in
the paternal pronucleus at the one-cell stage by 5mC oxidation.
There are three mammalian proteins with known 5mC oxidase
activities: Tet1, Tet2, and Tet3 (36, 37). One strategy employed
previously in the search for mammalian DNA demethylases is
that this activity should be expressed at high levels and specifi-
cally in oocytes and zygotes (31, 41). Therefore, we examined the
expression of the three Tet genes in mouse oocytes, zygotes, two-,
four-, and eight-cell–stage embryos by quantitative real-time
PCR (Fig. 4) using primers, as indicated in Fig. S3. We found
that Tet3 is expressed at high levels in oocytes and zygotes, but its
expression is drastically down-regulated at the two-cell stage and
at later cleavage stages. On the other hand, Tet1 and Tet2 were
not expressed at substantial levels in oocytes and zygotes. Tet1
was expressed at moderate to low levels at the two- and four-cell
stages. As a control, we measured the expression of the Stella/
Dppa3 transcript encoding a protein that protects the maternal
genome from active DNA demethylation (42). As expected,
Stella/Dppa3 was expressed at high levels in oocytes and zygotes,
and its level of expression gradually declined toward the eight-
cell stage (Fig. 4). Although expression of Tet3 has been dem-
mouse zygote was double-stained with anti-5hmC antibody (green) and
anti-5mC antibody (red). The smaller maternal pronucleus is closer to the
polar body (pb). A bright-field image is shown on the far left. (B) Additional
zygotes were double-stained with anti-5hmC antibody (green) and anti-5mC
antibody (red). Merged images are shown. (C) Zygotes obtained by in vitro
fertilization were double-stained similarly. Two polyspermic zygotes (to the
right) exhibit 5hmC staining in two paternal pronuclei. (D) 5mC and 5hmC
staining reveal two separate chromosome sets at metaphase of zygote di-
vision. A confocal image is shown. (E) Individual chromosomes are largely
stained for either 5mC (likely originated from the maternal pronucleus) or
5hmC (likely from the paternal pronucleus) at anaphase of zygote division.
Two Z sections of the same zygote are shown.
5hmC is present in the male pronucleus of mouse zygotes. (A) A
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onstrated in other tissues by RT-PCR (37), our data for oocyte-
and zygote-specific expression of Tet3 are consistent with a set of
published microarray data, which show almost complete absence
of the Tet3 transcript in all somatic mouse tissues tested but high
expression of Tet3 in oocytes and fertilized eggs, in a pattern
similar to that of Stella/Dppa3 (Fig. S4). Likely, there are dif-
ferentially spliced isoforms of Tet3, which give rise to the dif-
ferent expression patterns. To determine if 5hmC in zygotes is
further converted to cytosine, we conducted sodium bisulfite
sequencing analysis of DNA from mouse sperm, oocytes, and
zygotes (PN4–PN5). We analyzed the methylation pattern of the
Line1 (long interspersed element-1) 5′ region and of the ETn
(early transposon) repetitive elements (Fig. 5 A and B). The
Line1 sequences were highly methylated in sperm DNA (98%)
and in oocytes (87%). This level was 85% in zygotes, indicating
only a rather limited conversion of 5mC or 5hmC to C, although
the difference between sperm and oocyte combined and zygotes
is statistically significant (P = 0.0016; Fisher’s exact test, two-
tailed). Our data are showing less demethylation than reported
in previous studies in which the same sequences were analyzed
Zygotes at pronuclear stages PN1, PN2, and PN3 were double-
stained with anti-5hmC antibody (green) and anti-5mC anti-
body (red). Merged images are shown. (B) The levels of 5hmC
and 5mC in paternal and maternal pronuclei were quanti-
tated. The ratio of staining signal between the paternal and
maternal pronucleus is plotted. The number of zgotes ana-
lyzed in PN1/PN2 (Early), in PN3 (Mid), and in PN4/PN5 (Late)
are indicated with n values. The median value is indicated by
a horizontal line and a number. The difference between each
two datasets is statistically significant, as seen in the P values
5hmC and 5mC in early pronuclear stage zygotes. (A)
Two-cell stage embryos were double-stained with anti-5hmC
antibody (green) and anti-5mC antibody (red). pb, polar body.
A bright-field image is shown on the far left. (B) Two-cell–stage
embryos double-stained with anti-5hmC antibody (green) and
anti-5mC antibody (red). These images were obtained by
confocal microscopy. (C) Confocal microscopy image of a two-
cell (2c) stage embryo entering mitosis. The condensed chro-
mosomes are labeled with anti-5mC antibody (red) and anti-
5hmC antibody (green). (D) 5hmC and 5mC in four- (4c) and
eight-cell (8c) –stage embryos. Four-cell (Upper Left) and eight-
cell (remaining images) embryos were double-stained with
anti-5hmC antibody (green) and anti-5mC antibody (red). A
confocal image is shown in the upper right image.
5hmC and 5mC in early cleavage-stage embryos. (A)
| www.pnas.org/cgi/doi/10.1073/pnas.1014033108 Iqbal et al.
(33, 39). ETn sequences were methylated at a level of 82% in
sperm DNA. The level of ETn methylation in the paternal ge-
nome, identified by sequence polymorphism of the mouse strains
used, was 69% in zygotes (P = 0.0001; Fisher’s exact test, two-
tailed), again indicating limited conversion of modified cyto-
sines to cytosines relative to sperm DNA. We then analyzed two
single-copy genes, Myl3 and Acta1, coding for myosin light-chain-
C and α-actin, respectively, by bisulfite sequencing (Fig. 5 C and
D). Both genes were highly methylated in sperm DNA (96–
97%). Myl3 was methylated at a level of 20% in oocytes and 23%
in zygotes indicating significant demethylation (i.e., conversion
of 5mC/5hmC to C). Acta1 was methylated at a level of 24% in
oocytes and 48% in zygotes, indicating some demethylation
Our data explain previous observations of asymmetrical staining
of maternal and paternal pronuclei by anti-5mC antibodies (13,
15, 39). This antibody does not recognize 5hmC (38), which is
formed in the paternal pronucleus by genome-wide 5mC oxida-
tion, leading to lack of staining of the male pronucleus by anti-
5mC antibody. Sodium bisulfite sequencing, which cannot distin-
guish between 5mC and 5hmC (38, 43), has been used by several
laboratories to demonstrate active DNA demethylation—that is,
conversion of 5mC to C—of certain sequences in zygotes. Based
on this technique, active DNA demethylation has been inferred
for a few genomic loci, including the repetitive Line1 and ETn
elements (14, 33, 39), although ETn demethylation in the zygote
occurred only to a very small extent or not at all (33, 39). Our
in oocytes, zygotes, and early cleavage-stage em-
bryos. RNA was isolated from oocytes, zygotes,
two-, four-, and eight-cell–stage embryos. Real-
time PCR was used to assess the expression of the
three Tet genes and Stella/Dppa3. Data were nor-
malized relative to expression of β-actin. N.D., no
detectable signal in real-time PCR. Expression of
Tet1 in the zygote and of Tet3 at the two-cell stage
has a detectable signal, which is close to zero.
Expression of Tet and Stella/Dppa3 genes
ETn, Mylc, and Acta1 sequences in sperm,
oocytes, and zygotes. DNA was isolated from
mouse oocytes, sperm, or zygotes (PN4–PN5)
andsubjected tosodium bisulfite conversion.
(A) Line1 5′ end sequences were amplified,
cloned, and sequenced. Open squares, un-
methylated CpGs; black squares, methylated
CpGs; gray squares, not analyzable/mutated
CpG site. Each row represents an individual
sequenced DNA strand. (B) ETn sequences
were amplified, cloned, and sequenced. The
sequences from zygotes represent the pa-
ternal allele distinguishable by a sequence
polymorphism. (C) Acta1 sequences. (D)Myl3
sequences. The percentage of methylated
CpGs is indicated.
Sodium bisulfite sequencing of Line1,
Iqbal et al. PNAS
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bisulfite sequencing data confirmed very limited demethylation
(that is, conversion of 5mC or 5hmC to C) for ETn and Line1
sequences, as well as for the single-copy gene Acta1. We did see
substantial conversion of modified to unmodified cytosines for
the Myl3 gene in zygotes. However, there remains the possibility
that the apparent conversion of 5hmC to C may have occurred
during DNA replication, which begins in the late PN3 stage (39).
Some DNA demethylation (conversion to C) may occur in the
prereplicative phase, but it is not very pronounced (39). In any
event, beyond the few loci examined by us and by others to date,
we lack information about the fate of 5mC and 5hmC in most
sequences of the zygotic genome. Clearly, our data show that
conversion of 5mC to C in the zygote cannot be a genome-wide
event because considerable amounts of 5hmC are formed and
this base modification would be indistinguishable from 5mC us-
ing bisulfite conversion-based techniques, consistent with our
results. Furthermore, 5hmC is formed in the zygote and persists
into the two-cell stage and later cleavage stages in an asym-
metrical manner (Figs. 1 D and E and 3), suggesting that it is not
formed de novo by 5mC oxidase activity at the two-, four-, and
eight-cell stages. Such an activity should operate on all (paternal
and maternal) chromosomes at these stages. Combined, our data
on 5hmC levels in zygotes and in early cleavage stage embryos
and data from sodium bisulfite sequencing suggest that 5hmC
conversion to C may occur only to a limited extent and perhaps
at specific sequences. Our data are thus arguing against the
possibility that 5hmC is efficiently removed by DNA repair-me-
diated processes at a genome-wide level. Although initially
reported in 1988 (44), the nature of a protein or enzymatic ac-
tivity that would excise 5hmC from DNA has not been de-
termined. It is of note, however, that excision repair processes do
take place in paternal pronuclei in mammalian zygotes, as in-
dicated by the occurrence of γ-H2AX–marked DNA strand
breaks and base excision repair proteins at this developmental
stage (39, 45), although it is not clear what DNA base or lesion is
being removed. Our results also argue against the possibility that
most 5hmC may be further oxidized and potentially decarboxy-
lated to form C, this being one possible mechanism for 5hmC
processing and demethylation that has been proposed (18).
However, our data do not exclude the possibility that 5hmC is
processed into C at certain sequences. Alternatively, multiple
mechanisms may be at work to reprogram paternal genome
methylation patterns that include, for example, deamination of
5mC followed by excision repair, in addition to 5mC oxidation.
The role of 5mC oxidation in the paternal pronucleus is cur-
rently unknown. One immediate effect of this oxidation step
should be the neutralization of the functional role of 5mC in gene
suppression. Embryonic genome activation in the mouse takes
place at the two-cell stage and it is expected that many genes that
are methylation-suppressed during spermatogenesis (e.g., Oct4
and Nanog) will need to be activated to allow development to
proceed. Afteroxidation of5mC,the 5hmC-containing sequences
will no longer be capable of interacting with repressor proteins
that are known to bind to 5mC (34, 38). DNA sequences con-
taining 5hmC in place of 5mC are not substrates for the mainte-
nance methyltransferase activity of DNMT1 (46). This finding
means that the formation of 5hmC may serve to dilute DNA CpG
methylation during replication in early embryos, even in the
presence of any nuclear DNMT activity. Interestingly, we did not
observe much signal for 5hmC in the presumably maternally de-
rived chromosome domains of two-, four-, and eight-cell nuclei
(Fig. 3). This finding is consistent with the assumption that the
maternal genome undergoes passive, replication-dependent de-
methylation in early cleavage-stage embryos in a manner that is
not dependent on 5mC oxidation but may simply the conse-
quence of replication in absence of DNMT1 maintenance meth-
The most likely candidate for 5mC oxidation in the paternal
pronucleus is Tet3, which is specifically expressed in oocytes and
zygotes but not in two-cell–stage embryos (Fig. 4). We attempted
to knock-down Tet3 expression in oocytes by siRNA before in
vitro fertilization but were unable to achieve efficient knock-
down. Mouse models are under construction to prove that Tet3
is the activity that converts 5mC to 5hmC in fertilized oocytes. In
conclusion, our data show that 5mC oxidation is one initial step
in reprogramming of the paternal genome upon fertilization,
suggesting that this event is an important part of the early
mammalian life cycle.
Derivation and Immunostaining of Oocytes, Zygotes, and Early Embryos. Ani-
mal handling was done in accordance with institutional guidelines and was
approved by the City of Hope Institutional Animal Care and Use Committee.
Preimplantation embryonic stages (one to eight cells) were collected from
6- to 8-wk-old female FVB mice. Pronuclear stages were identified as de-
scribed (15). Cumulus cells were removed from zygotes with 1% hyaluroni-
dase treatment. The zona pellucida was removed by using acidic tyrode
solution. After washing in M2 medium + 0.3% BSA, zygotes or embryos were
fixed in 3.7% paraformaldehyde in PBS at room temperature for 20 min.
Embryos were permeabilized in 0.2% Triton-X 100 in PBS at room temper-
ature for 10 min. Permeabilized embryos were incubated in 4 N HCl solution
at room temperature for 10 min followed by neutralization in Tris-Cl, pH
8.0, for 10 min. The embryos were blocked overnight at 4 °C in 1% BSA,
0.2% Triton X-100 in PBS. Embryos were incubated with anti-5hmC (rabbit
polyclonal; Active Motif) and anti-5mC antibodies (mouse monoclonal;
Eurogentec) in blocking solution for 1 h at room temperature. The embryos
were washed several times in 0.05% Tween 20 in PBS (PBST), transferred to
secondary antibody mixture of Alexa Fluor 568 goat anti-mouse and Alexa
Fluor 488 goat anti-rabbit (Molecular Probes), and incubated at room tem-
perature for 1 h. The embryos were washed several times with PBST before
mounting on slides with ProLong Gold antifade reagent with DAPI (Molec-
ular Probes). Fluorescence images were acquired using an Olympus AX70
upright microscope with Image Pro Plus version 6.3 software. Confocal
images were acquired using a Zeiss LSM 510 upright microscope and pro-
cessed using the Zeiss LSM image browser. Quantitative analysis of pronuclei
was done using Image-pro plus version 6.3 (Media Cybernetics Inc.). All
software settings for intensity and saturation were maintained constant
across all experimental groups. A region of interest was drawn around each
pronucleus in zygotes and the mean optical density was calculated within
the region of interest. The median 5hmC intensity in the male pronucleus
was divided by the median 5hmC intensity in the female pronucleus to ob-
tain the PAT/MAT ratio for 5hmC. The PAT/MAT ratio for the control 5mC
was obtained from the respective 5mC values.
Antibody Specificity Test. Synthetic oligonucleotides containing cytosine,
5mC, or 5hmC bases were prepared as described previously (38) and were
used in antibody dot-blot assays. The 76-mer oligonucleotide sequence was
TAATATTGAGGGAGAAGTGGTGA-3′, where X is 5hmC, C, or 5mC. For com-
petition immunocytochemistry, we preincubated the anti-5hmC antibody
(1:6,000; Active Motif) with 0.5 μg/mL of single-stranded 38-mer oligonu-
cleotides (C38R for normal cytosine; 5mC38R for 5mC; 5hmC38R for 5hmC)
in 0.05% PBST at room temperature for 1 h, then incubated with the fixed
cells for immunostaining. The sequence of the 38-mers was 5′-ATTATAA-
XGXGAAATAXGXGATATAXGXGTAATATAAT-3′ where X is either 5hmC
(5hmC38R), C (C38R), or 5mC (5mC38R).
Real-Time PCR. Poly(A) mRNA was isolated from MII oocytes (n = 8), zygotes
(n = 8), two-cell (n = 4), four-cell (n = 2), and eight-cell (n = 1) embryos by
using the Dynabeads mRNA DIRECT Micro Kit (Invitrogen). Oligo (dT)25-
coupled Dynabeads and mRNA complexes were immediately used for re-
verse transcription using the SuperScript III reverse transcriptase (Invitrogen),
according to the manufacturer’s instructions. Real-time quantitative PCR
reactions were performed at 50 °C for 2 min and 95 °C for 10 min followed
by 50 cycles at 95 °C for 15 s and 60 °C for 1 min using TaqMan Gene Ex-
pression Master Mix (Applied Biosystems) on an iQ5 real-time PCR cycler
(Biorad). PCR was performed with the TaqMan MGB primer with 6FAM-
based probes (Applied Biosystems) using the following assay ID numbers:
Tet1 (Mm01169088_m1), Tet2 (Mm01312907_m1), Tet3 (Mm00805754_m1),
and Stella/Dppa3 (Mm01184198_g1). The cDNA levels of target genes were
| www.pnas.org/cgi/doi/10.1073/pnas.1014033108Iqbal et al.
analyzed using comparative Ct methods and normalized to internal
Bisulfite Sequencing. For bisulfite sequencing, cells were directly subjected to
bisulfite conversion by using the EZ DNA Methylation Direct kit (Zymo Re-
search). Bisulfite-modified DNAs were amplified using the following PCR
primers: Line1-5′ region, for the first PCR, the forward primer 5′-GTTAGA-
GAATTTGATAGTTTTTGGAATAGG-3′ and reverse primer 5′-CCAAAACAAA-
ACCTTTCTCAAACACTATAT-3′, and for the second PCR, the forward primer
5′-TAGGAAATTAGTTTGAATAGGTGAGAGGT-3′ and reverse primer 5′-TCA-
AACACTATATTACTTTAACAATTCCCA-3′, were used.
For ETn elements, for the first PCR, the forward primer 5′-CTTAACTA-
CATTTCTTCTTTT-3′ and reverse primer 5′-AGTTAGYGTTAGTATGTGTATTT-
GTACC-3′, and for the second PCR, the forward primer 5′-TCTAAATTCCT-
CTCTTACAACT-3′ and reverse primer 5′-AGTTAGYGTTAGTATGTGTATTTGT-
ACC-3′ were used. For α-actin (Acta1) promoter amplification, for the first
PCR, the forward primer 5′-AAGTAGTGATTTTTGGTTTAGTATAGT- 3′ and
reverse primer 5′-ACTCAATAACTTTCTTTACTAAATCTCCAAA-3′, and for the
second PCR, the forward primer 5′-GGGGTAGATAGTTGGGGATATTTTT-3′
and reverse primer 5′-CCTACTACTCTAACTCTACCCTAAATA-3′ were used.
For Myl3 promoter amplification, for the first PCR, the forward primer 5′-
GTATAATAAATTTGGATAGGTAAAGGTTAG- 3′ and reverse primer 5′-AAA-
CCTAAAACACTAATCTTAAAAATTTTA′, and for the second PCR, the forward
primer 5′-ATATTATAGTAGGGGTTGGAATGATTAAAG-3′ and reverse primer
5′-CCTATTAAACTAATCTAAAAAACAATCCTC-3′ were used.
The reaction buffer contained dNTPs and 1.25 U of PfuTurbo Cx Hotstart
DNA Polymerase (Stratagene) and the samples were incubated at 95 °C for 3
min, and then 36 cycles of PCR at 95 °C for 20 s, 50 °C for 30 s, and 72 °C for
45 s were performed, followed by a final extension step at 72 °C for 5 min.
The second-round PCR was carried out with Platinum Taq polymerase
(Invitrogen), and the samples were incubated at 95 °C for 2 min, and then 36
cycles of PCR at 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 1 min were
performed, followed by a final extension step at 72 °C for 5 min. The PCR
products were then ligated into the pCR2.1 TA cloning vector (Invitrogen).
The cloned samples were sequenced using the M13 reverse sequencing
primer and analyzed.
ACKNOWLEDGMENTS. This work was supported by National Institutes
of Health Grants AG036041 (to G.P.P.) and ES015185 and GM064378
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| March 1, 2011
| vol. 108
| no. 9