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Laboratory of Cellular Biochemistry, Animal Resource Sciences/Veterinary Medical Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan
Address all correspondence and requests for reprints to: Dr. Kunio Shiota, Laboratory of Cellular Biochemistry, Animal Resource Sciences/Veterinary Medical Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan. E-mail: ashiota{at}mail.ecc.u-tokyo.ac.jp
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several elements responsible for the gene expression of placental PRL family members have been identified. By analyzing the promoter region of mouse PL-I (mPL-I) and rat PL-II (rPL-II), sites for transcription factors activating protein-1 (AP1), GATA2, GATA3, and ETS have been found to be responsible for placental gene expression (7, 8, 9, 10). HAND1, previously termed Hxt, was also found to be involved in PL-I expression in trophoblast cells (11). Duckworth and colleagues (12) reported that the 5'-flanking region responsible for rPL-I expression was homologous to that of rPL-II, but there is no report to date identifying the regulatory elements of rPL-I.
The placenta is one of the tissues that strongly expresses DNA methyltransferase-1 (13). In vertebrates, methylation of DNA occurs at the 5'-position of cytosine in the CpG dinucleotides, forming 5-methylcytosine, and approximately 60–90% of the CpG in the genome of adult mammals is reported to be methylated (14, 15). An inverse correlation between DNA methylation in the promoter region and gene expression has been documented (15), and some endogenous genes are activated from their repressed status by treatment with demethylating agents such as 5-azacytidine (5-azaC) and 5-aza-2'-deoxycytidine (5-aza-dC) (16, 17). For example, treatment with 5-azaC induced expression of collagen IV in F9 teratocarcinoma cells (18) and PRL in GH3CDL cells (19). Involvement of DNA methylation in the regulation of chromatin structure has recently been postulated in view of findings indicating that the methyl-CpG-binding protein (MeCP2), which is one of the methyl-binding domain (MBD) proteins, forms a complex with proteins such as histone deacetylase 1 (HDAC1) that affects the chromatin architecture and a repressor protein (Sin3) (20, 21, 22, 23). In addition, another MBD protein (MBD1) is likely to associate with the other HDAC than HDAC1 to form a complex (24). Therefore, DNA methylation is thought to be one of the fundamental mechanisms in gene silencing (for reviews, see Ref. 25).
We have analyzed the methylation status of CpG islands in tissues including the placenta by restriction landmark genomic scanning, which enables us to analyze the genome-wide methylation pattern (26). We found that there are placenta-specific methylated or unmethylated loci in genomic DNA, and that the differentiation process is accompanied by changes in the methylation of CpG islands (26), implying that the formation of a DNA methylation pattern contributes to placental development, and that the establishment of a unique CpG methylation pattern in each cell is an essential event in development. Sequences in the promoter region upstream of the placental PRL/GH family members, however, contain relatively few CpG sites with a TATA box (27). In the present study we determined the methylation pattern of rPL-I promoter and provided evidence that DNA methylation is important for the tissue-specific expression of the rPL-I gene.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rcho-1 trophoblast cells, which were derived from a rat choriocarcinoma (28) and are capable of differentiating into trophoblast giant cells (29), were cultured in NCTC-135 medium (Sigma, St. Louis, MO) supplemented with 20% FBS, 50 µM 2-mercaptoethanol, and 1 mM sodium pyruvate (29, 30). The cells were induced to differentiate by growing to near confluence in FBS-supplemented medium and then replacing the FBS with 10% horse serum (30). BRL cells, which were derived from Buffalo rat liver (31), were maintained in DMEM supplemented with 10% FBS.
For treatment with 5-aza-dC (Sigma), cells were plated (3 x 104 cells/100-mm dish) and incubated for 24 h, and then cultured for 3 d in medium containing 10 µM 5-aza-dC. TSA, a gift from Dr. M. Yoshida, is a potent and specific inhibitor of histone deacetylase both in vivo and in vitro (32, 33). For treatment with TSA, cells were plated at 5 x 105 cells/100-mm dish, incubated for 24 h, and exposed to 400 nM TSA for 48 h.
Analysis of rPL-I expression by RT-PCR
Total RNA was prepared from cells and tissues with TRIzol
reagent (Life Technologies, Inc., Gaithersburg, MD). One
microgram of total RNA was subjected to first strand cDNA synthesis
with oligo(deoxythymidine) primers and Superscript II reverse
transcriptase (Life Technologies, Inc.). PCR was
performed with forward (5'-ATGCAGCTGACTTTGACTCTT-3') and reverse
(5'-TCAAAAGGTGGACACTCCA-3') primers to detect the full-length open
reading frame of rPL-I cDNA under the following conditions: 94 C for 2
min, 30 cycles of 94 C (40 sec), 62 C (45 sec), and 72 C (1 min), and
then 72 C for 7 min. Control detection of ß-actin was performed with
forward (5'-GACAACGGCTCCGGCATGTGCAAAG-3') and reverse
(5'-TTCACGGTTGGCCTTAGGGTTCAG-3') primers under the same
conditions.
Identification of the 5'-flanking region of rPL-I gene
Selective amplification of the genome segment was performed by
cassette ligation-mediated PCR (34), in which the
5'-flanking region of the gene can be obtained. Genomic DNA containing
approximately 3.5 kb upstream of the rPL-I-coding region was cloned by
this PCR-based method with the primer sets, which were designed in the
first exon and intron.
Briefly, genomic DNA was digested with restriction enzymes, EcoRI, PstI, and SalI, respectively. Each digested fragment was ligated with oligonucleotide-adapters that have specific cohesive ends corresponding to the restriction enzymes described above. The constructed cassettes were amplified by a series of PCR reactions with two sets of primers. One was primer C1 (5'-GTACATATTGTCGTTAGAACGCG-3'), which was presented in all of the adapters, and primer rPL-I IntA(R) (5'-TAAATGTTTACTAAGAGGTACATG -3') corresponding to the 5'-terminal sequence in the first intron of rPL-I. The other primer set was cassette primer C2 (5'-ACGACTCACTATAGGGAGAAG-3') and specific primer rPL-I Ex 1(R) corresponding to the sequence in the rPL-I first exon (5'-AGAGCCCGAAAGAGTCAAAGTCAGCTG-3'). The resulting cassettes were cloned into pUC118 vector (Takara, Kyoto, Japan) and analyzed by sequencing.
Southern blotting
Genomic DNA from the dissected tissues was extracted as
previously described (26). Genomic DNA was digested with
HindIII and a methyl-sensitive enzyme, HhaI, that
digests only unmethylated GCGC sites. The resulting fragments were
size-fractionated on a 0.8% agarose gel, transferred to nylon filters,
and hybridized with
[
-32P]deoxy-CTP (Amersham Pharmacia Biotech, NJ)-labeled probes corresponding to the rPL-I
5'-flanking region. The probes used were probe A (-3407 to -2520) or
probe B (-1006 to +30; Fig. 2A
). The signals were visualized by a
Fujix BAS-2000 analyzer (Fuji Photo Film Co., Ltd., Tokyo,
Japan).
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Reporter gene constructs
The 5'-flanking fragment of the rPL-I gene was used for
generating a series of reporter constructs to evaluate the promoter
activity. The fragment from -3365 to -5 was cloned into pGL2-Basic
vector (Promega Corp., Madison, WI; the translation start
site was designated +1) and subjected to PCR with specific forward
primers and a common reverse primer (5'-GGGCTAGCAGATCCAGGACAGTGTAG-3')
to generate genome fragments of various lengths. Amplified fragments
were subcloned into pGL-Luciferase vector (Promega Corp.),
which expresses firefly luciferase. The resultant vectors were
designated according to the positions of the fragments as
-3365Luc, -2445Luc, -1402Luc, -1069Luc, and -498Luc, which were
generated by using each specific forward primer of the following
sequencesm respectively: 5'-AGGAGCTCACAACATAAGATGCCTGA-3' (-3365 to
-3340), 5'-CCGAGCTCATCTGCATAGTGATATTTTGA-3' (-2445 to -2417),
5'-TAGAGCTCAATATTCATAAGGGAAATTGG-3' (-1402 to -1374),
5'-CTGAGCTCAGCCCTGAGTTTGATTATTTC-3' (-1069 to -1061), and
5'-ACGAGCTCATTTTACAGTGTTTGTGGTTA-3' (-498 to -470; Fig. 3A).
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Luciferase assays
Luciferase assays were carried out according to the method
described previously (37) with a slight modification.
Rcho-I cells (4 x 104) were transfected
with 430 nmol reporter constructs with Lipofectamine (Life Technologies, Inc.) on d 0 (undifferentiated state), 4, and 8 of
differentiation, and cells were harvested after 48 h. To normalize
the luciferase activity driven by the rPL-I promoters, 21.5 nmol (34
ng) control plasmid expressing Renilla luciferase, which
requires a different substrate from firefly luciferase, were
cointroduced to cells, and the activities of both luciferases were
determined by means of a Dual-Luciferase Reporter System (Promega Corp.).
To examine the function of the MeCP2 in promoter activity in the rPL-I 5'-flanking region, full-length rat MeCP2 cDNA was cloned by PCR according to a previous report (38) and subcloned into pFLAG-CMV-2 expression vector (Sigma). We confirmed the protein expression with the MeCP2 expression construct by Western blotting (data not shown). A mixture of the promoter constructs containing luciferase reporters and 2 µg MeCP2 expression vectors was cotransfected into Rcho-I cells. To analyze the action of TSA on the reporters, 50 nM TSA was added to the medium at 3 h after transfection, and the cells were incubated for 45 h. All luciferase assay experiments were performed twice independently in triplicate. All results are shown as the mean ± SE.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, a single PCR fragment corresponding to
the rPL-I open reading frame was detected only in the placenta. The PCR
product was confirmed to be derived from rPL-I mRNA by nucleotide
sequencing (data not shown) and Southern blotting of the PCR products
(Fig. 1, lower panel). RT-PCR products corresponding to
rPL-I were not detected in any of the other tissues examined (brain,
heart, kidney, liver, skeletal muscle, ovary, pituitary, and spleen;
Fig. 1). These results indicate that the expression of rPL-I mRNA was
restricted to the placenta and severely suppressed in all other
tissues.
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and 3A). We found 17 CpG dinucleotides
that are potential targets of cytosine methylation in the 3.4-kb rPL-I
upstream region (Fig. 2A). There is no region of the clustered CpG
dinucleotides characteristic of a CpG island.
We performed Southern blot analysis to study the methylation
status of the rPL-I gene in the various tissues (placenta, brain,
liver, muscle, ovary, pituitary, and spleen) by using
methylation-sensitive (HhaI) and insensitive
(HindIII) enzymes. There are two HhaI sites
identified at -1279 and -2543 in the rPL-I gene (Fig. 2A). We used
two probes (probe A from -3407 to -2450 and probe B from -1006 to
+30) to study the methylation status of these HhaI sites
(Fig. 2A). With probe A, we obtained two resulting bands by
HindIII digestion at 3.9 and 0.2 kb in all tissues examined
(Fig. 2B, left panel). When the genomic DNA was digested by
HhaI in addition to HindIII, a novel band
appeared at 2.0 kb in all tissues (Fig. 2B, right panel).
When the resulting bands were normalized by the 0.2-kb band in each
tissue from both methylated and unmethylated DNA, the relative
densities of bands at 2.0 kb in the brain, liver, skeletal muscle,
ovary, pituitary, spleen, and d 12 and 20 of placenta were 0.33, 0.20,
0.38, 0.32, 0.15, 0.43, 1.46, and 1.55, respectively. In the placenta,
the 2.0-kb band was much more intense than those in any other tissues,
including anterior pituitary and liver, suggesting that the
HhaI site at -1279 was hypomethylated in the placenta.
Similarly with probe B, a band generated by HhaI digestion
at 1.9 kb was the most intense in the placenta (Fig. 2C, right
panel), whereas digestion with HindIII alone resulted
in a single band at 3.9 kb in all tissues (Fig. 2C, left
panel). We did not obtain the resulting fragments of a
HhaI digestion at -2543 in all tissues. These data
indicated that the 5'-flanking region in the rPL-I gene is
hypomethylated at the HhaI site at -1279 in the placenta,
whereas the HhaI site at -2543 is hypermethylated in all
tissues.
Promoter activity of the 5'-flanking region of rPL-I gene
It was previously reported that some transcription factors, such
as GATA2, GATA3, AP1, and HAND1, seemed to be implicated in PL-I
expression in the mouse (7, 8, 9, 10, 11). The proximal region
upstream of the ATG site of the rPL-I gene is homologous to that of
mPL-I (data not shown), in which cis-acting sites for these
transcription factors exist (Fig. 3A).
Nevertheless, the result of Southern blot analysis showed that the
methylation status at the more distal region around the -1279
HhaI site was different in the placenta. To assess the
possibility that the differentially methylated region is involved in
the regulation of rPL-I expression, we constructed a series of reporter
plasmids, each containing various lengths of the 5'-flanking region of
rPL-I (Fig. 3A). We confirmed the promoter activity of -498Luc
construct, which contained cis-elements for the
transcription factors described above, in the trophoblastic cell line,
Rcho-1 (Fig. 3B). In Rcho-1 cells on d 8 of differentiation, the
-498Luc construct demonstrated an activity approximately 8 times as
potent as in the no promoter control, 0Luc (Fig. 3C). We further
analyzed the activity of the distal region of the promoter and found
that -1402Luc and -2445Luc reporters had higher activity than
-498Luc, whereas -1069Luc had comparable activity to -498Luc. The
activity of -2445Luc was approximately 20 times as potent as that of
the control (Fig. 3C). These data suggest that the 5'-flanking region
between -1069 and -2445 may contain cis-acting elements
that positively regulate rPL-I transcription in addition to those
reported previously. The hypomethylated HhaI site in the
placenta is located in this region, implying that methylation in the
rPL-I 5'-flanking region is involved in the regulation of rPL-I
transcription. The promoter activity of -3365Luc declined from the
level of -2445Luc to that of -498Luc, suggesting that the region from
-3365 to -2445 may contain negative regulatory elements for rPL-I
transcription.
Differences in the methylation of CpG sites among the tissues
revealed by bisulfite sequencing
We further analyzed all CpG sites between -2545 and -189 (12 CpG
sites) by the bisulfite sequencing method in the placenta, pituitary,
and liver and compared the total methylation status of these tissues
(Fig. 4).
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Among the 12 CpG sites in the rPL-I 5'-flanking region, the ratios of methylated CpGs were less than 50% at 7 sites (CpG-2309, CpG-1807, CpG-1790, CpG-1309, CpG-1279, CpG-1181, and CpG-189) in the placenta (d 12 and 20), whereas the methylation score at these sites was more than 60% in anterior pituitary and liver, indicating that these 7 CpG dinucleotides might be involved in the repression of rPL-I transcription. Taken together with the results of the promoter activity assay, it is likely that hypomethylation at 7 CpG sites is important for rPL-I transcription.
The methylation pattern in the placenta was almost the same on d 12 and 20 of pregnancy, which is in agreement with the results of Southern blotting. This suggests that the methylation pattern of rPL-I 5'-flanking region is maintained in the placenta throughout gestation.
Repression of PL-I promoter activity by DNA methylation
To investigate the possibility that rPL-I transcription is
repressed by methylation of the 5'-flanking region, we applied in
vitro methylation to the reporter constructs for the luciferase
assay. Methylation of the -498Luc reporter did not show a significant
decrease in rPL-I promoter activity compared with the unmethylated
reporter (Fig. 5A), suggesting that
methylation does not have an inhibitory effect on luciferase activity
itself (24). In contrast, methylation of -2445Luc and
-3365Luc dramatically decreased their promoter activities in Rcho-1
cells (Fig. 5A), indicating that methylation of the rPL-I regulatory
region can contribute to the repression of rPL-I transcription.
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). We also investigated the effect of the histone deacetylase
inhibitor, TSA. Recent studies have indicated that in gene silencing a
relationship may exist between methylation and chromatin remodeling
through histone deacetylation (43, 44). In the present
experiment the treatment of Rcho-1 cells with TSA restored the reporter
activity to 50% of unmethylated -2445Luc activity (Fig. 5C),
suggesting that the inhibitory effect of methylation occurs through
such chromatin remodeling mechanisms. It is to be noted that MeCP2
itself could interfere with the promoter activity without methylation
of the reporter (Fig. 5C). This effect of MeCP2 could also be relieved
by TSA treatment. Because it was reported that MeCP2 has an ability of
DNA binding distinct from that of the MBD and binds weakly to
nonmethylated DNA (45), it is possible that MeCP2, which
was expressed with a relatively excess amount due to the transfection,
might cause a nonspecific effect without methylation at the transfected
reporters.
Activation of rPL-I transcription by the treatment with
5-aza-dC or TSA in a nonexpressing cell line
We treated BRL cells, which do not express rPL-I, with the reagent
for DNA demethylation (5-aza-dC) or inhibitor for histone deacetylation
(TSA) to investigate whether the repression of rPL-I could be relieved
in non-rPL-I-expressing cells. In BRL cells, the expression of rPL-I
mRNA could not be detected by RT-PCR under nontreated conditions (Fig. 5D), confirming that rPL-I is inactive in this cell line. It is
striking that 5-aza-dC treatment of BRL cells elicited de
novo expression of rPL-I mRNA (Fig. 5D). The conversion of
methylcytosine to unmodified cytosine seemed to cause the release of
rPL-I silencing in BRL cells. Activation of rPL-I expression was also
observed in BRL cells treated with TSA. These results strongly support
our hypothesis that expression of the rPL-I gene is regulated by
methylation and chromatin remodeling. The level of rPL-I mRNA evoked by
treatment with 5-aza-dC or TSA was not high compared with that in
Rcho-1 cells, which suggests that some transcription factors required
for maximal rPL-I expression, such as HAND1, are lacking or
insufficient in BRL cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The sequence within the Orange to -300 bp upstream region of the rPL-I gene contains cis-elements for AP1, GATA2, GATA3, and HAND1, which had been found to be important in the regulation of mPL-I and rPL-II genes (7, 8, 9, 10, 11). These elements were also found in the 5'-flanking region of rPL-I, and the -498 Luc reporter gene containing these elements had considerable promoter activity in the present study, supporting the possibility that these cis-elements are involved in rPL-I expression in the placenta. As a further increase in reporter activity was observed in the -2445Luc construct, there should be additional important regions responsible for rPL-I transcription within the region between -2445 and -1402 of the 5'-flanking region. In addition to the enhancer elements, there may be a negative regulatory element(s) between -2445 and -3365 of the rPL-I gene. Taken together, multiple elements for rPL-I transcription are located within 3.4 kb in the 5'-flanking region and seem to be coordinated in transcriptional regulation of the rPL-I gene.
In the region spanning the rPL-I promoter, the density of CpG dinucleotides is lower than 1 CpG/220 nucleotides, which was found to be the minimally required density for repression by MeCP2 (38, 45, 46). As the methylation of -498Luc did not affect the promoter activity, CpG-189, which is close to the AP1 element, is unlikely to be involved in the DNA methylation-mediated repression of rPL-I gene expression. The methylation of the -2445Luc construct, however, severely repressed the reporter gene activity. Several mechanisms for methylation-mediated repression of gene expression have been postulated. Methylation of CpG interferes with the binding of transcription factors to their recognition sequences by modifying cytosine nucleotides, or the binding of specific proteins, such as MeCP2, to the methylated DNA physically blocks basal transcription machinery to access the region (47). Moreover, recent studies have indicated that MeCP2 interacts with methylated DNA to recruit HDAC, which functions to close the chromatin structure and renders transcriptional machineries unable to associate with genes (25). In the present study coexpression of MeCP2 repressed the reporter activity, whereas TSA could recover reporter activity to up to 50% of the unmethylated control. In addition, the treatment of BRL cells with TSA resulted in rPL-I gene expression, implying that the mechanism for rPL-I repression is likely to involve deacetylation of histones. Considering our findings and previous reports, DNA methylation-mediated silencing of rPL-I requires MeCP2 and may be linked to the alteration of the chromatin structure through the process of histone deacetylation.
To date, most data on methylation-mediated gene repression concern TATA-less and GC-rich promoters that are associated with the CpG islands (48, 49). It is striking that the expression of rPL-I seems to be controlled by DNA methylation even though there are only 17 CpG sites in the 5'-flanking region of the rPL-I gene. Similarly, there are only 14 CpG sites within 3.6 kb of the 5'-flanking region of the rPL-II gene, in which enhancer elements for expression exist within the region from -2828 to -1729 and negative regulatory elements between -3031 and -2838 (10), indicating that rPL-I and rPL-II share a common feature of gene regulation characterized by the location of enhancer and repressor elements. Placental PRL/GH genes are thought to have evolved from common ancestor genes (50), and PL/PRL genes are localized as a gene cluster in the genome. It was reported that a specific methylation pattern in the promoter regions of rGH and rPRL was implicated in the gene expression and that DNA methylation was inversely correlated with gene expression (19, 27), so that PL/PRL genes as well as the GH gene, which are characterized by possessing TATA boxes and poor CpG sites in their promoters, are likely to be good models for analysis of the methylation-mediated gene silencing mechanism.
In the PRL gene, methylation of CpG dinucleotides in the proximal region of the promoter decreases the activities of transcription factors such as cAMP response element-binding protein/activating transcription factor and AP2 (27). In the mPL-I and rPL-I gene, cis-elements for AP1, GATA2, and GATA3, which have been previously determined in the proximal promoter regions, do not contain any CpG sequences. Taken together with comparison of the methylation status within -2545 by bisulfite sequencing, only six of these CpGs (CpG-2309, CpG-1807, CpG-1790, CpG-1309, CpG-1279, and CpG-1181) are thought to be responsible for the repression of gene expression. Although the tissue-specific expression of rPL-I is likely to involve the change in chromatin structure as mentioned above, we cannot eliminate the possibility that the modification of CpG sites in a limited domain by methylation inhibits specialized functions of the DNA sequence by directly preventing the binding of particular transcription factors. In this context, the sequences close to the six CpG sites mentioned above will be useful in determining the gene control region in the near future.
The extraembryonic lineage develops into multiple placental cell types. Because the expression of the placental PRL/GH family is quite specific to the trophoblast cell lineage, analyses of the expression of these genes have been performed to characterize the genetic elements involved in the coordinated program of trophoblast gene expression and cellular differentiation. The rPL-I gene is predominantly expressed in midpregnancy around d 10–12 of gestation and declines toward the end of pregnancy, whereas rPL-II is mainly expressed in late pregnancy (6). Bisulfite sequencing analysis demonstrated that the methylation status in the 5'-flanking region of the rPL-I gene was similar on d 12 and 20 of pregnancy. Considering these results, it is likely that the methylation pattern of the rPL-I gene is established at an early stage of development, and once it is established, it does not change during the course of pregnancy. Considering that expression of the rPL-I gene is restricted to the trophoblast cells and that there is no leakage of expression in other tissues, DNA methylation may ensure the silencing of the rPL-I gene in nonexpressing cells, whereas a defined combination of transcription factors in trophoblast cells may dictate the stage specificity of rPL-I expression.
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Acknowledgments |
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Footnotes |
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1 Present address: Meiji Seika Kaisha Ltd., 760 Morooka-cho,
Kohoku-ku, Yokohama 222-8567, Japan. 
Abbreviations: AP1, Activating protein-1; 5-azaC, 5-aza-2'-deoxycytidine; 5-aza-dC, 5-aza-2'-deoxycytidine; MBD, methyl-binding domain; MeCP2, methyl-CpG-binding protein; mPL-II, mouse placental lactogen I; rPL-II, rat placental lactogen I; PL, placental lactogen; TSA, trichostatin A.
Received January 30, 2001.
Accepted for publication April 27, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and -ß subunit mRNAs in the rat
placenta. Endocr J 44:79–87[Medline]
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A. L. B. Cabral, K. S. Lee, and V. R. Martins Regulation of the Cellular Prion Protein Gene Expression Depends on Chromatin Conformation J. Biol. Chem., February 8, 2002; 277(7): 5675 - 5682. [Abstract] [Full Text] [PDF]
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, Probes. B and C, Southern blot analyses of
rPL-I gene in various tissues. Genomic DNA extracted from tissues was
digested by HindIII and methyl-sensitive
HhaI, and then hybridized with the probes indicated. Br,
Brain; Li, liver; Mu, skeletal muscle; Ov, ovary; Pit, anterior
pituitary; Sp, spleen and Pl, placenta on d 12 (d12) and d 20 (d20) of
pregnancy.
) is represented at
each CpG site. All data were calculated from the results for 10–12
independent genomic DNA clones.