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Differentiation of Trophoblast Lineage Is Associated with DNA Methylation and Demethylation
Biochemical and Biophysical Research Communications 290, 701–706 (2002) doi:10.1006/bbrc.2001.6258, available online at http://www.idealibrary.com on
Differentiation of Trophoblast Lineage Is Associated with DNA Methylation and Demethylation Jun Ohgane,* Naka Hattori,* ,† Mayumi Oda,* Satoshi Tanaka,* and Kunio Shiota* ,1 *Cellular Biochemistry, Animal Resource Sciences/Veterinary Medical Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan; and †Bio-oriented Technology Research Advancement Institution, 1-40-2 Nisshin-cho, Omiya, Saitama, Japan
Received November 11, 2001
Our previous study has shown that the placenta and kidney had different genomic methylation patterns regarding CpG island loci detected by restriction landmark genomic scanning (RLGS). To investigate whether differentiation involves changes in DNA methylation, we analyzed the rat Rcho-1 cell line, which retains trophoblast cell features and differentiates from stem cells into trophoblast giant cells in vitro. By RLGS, a total of 1,232 spots were identified in the Rcho-1 stem and differentiated giant cells. Four spots (0.3%) were detected only in giant cells, implying that the loci were originally methylated, but became demethylated during differentiation. Another four spots (0.3%) were detected only in stem cells, implying that these loci, originally unmethylated, became methylated during differentiation. DNAs from three loci that became methylated during differentiation were cloned and sequenced. All showed high homologies with expressed sequence tags (ESTs) or with genomic DNA of other species, suggesting that these loci are biologically important. Thus, the eight differentially methylated loci should be good tools to study epigenetic modification specific to differentiation of trophoblast giant cells. © 2002 Elsevier Science Key Words: DNA methylation; differentiation; trophoblast; CpG island; placenta.
Most mammalian cells differentiate without detectable changes in DNA sequence. Instead, epigenetic changes such as DNA methylation occur (1, 2). DNA methylation is involved in tissue-specific gene expression including placental hormones (3) and alternate use of tissue-specific exons (4). Therefore, DNA methylation may be a form of “cellular memory” for the gene expression pattern, which is established during development. To establish the tissue-specific pattern of DNA 1
To whom correspondence and reprint requests should be addressed. Fax: ⫹81-3-5841-8189. E-mail: [email protected].
methylation and gene expression, de novo methylation as well as demethylation need to occur in each cell lineage. The treatment of some cell types with demethylating reagents induces differentiation, and in some transcription factor genes, demethylation is associated with differentiation (5–7). In contrast, genomewide methylation occurs at organogenesis in embryonic development (8 –10), and this may explain why null mutations of DNA cytosine methyltransferase genes (Dnmt) 1 and 3b are embryonic lethal (11, 12). CpG dinucleotides are the main sites of DNA methylation, and are unevenly distributed in the mammalian genome: they appear at 10 –20 times their average density in selected regions, known as CpG islands. The mammalian genome contains 30,000 – 40,000 CpG islands, which are located around the promoters of housekeeping genes, and are associated with tissuespecific genes (13). One of the characteristics of CpG islands is that most cytosine residues are unmethylated. However, we found some CpG islands where some areas are differentially methylated, depending on tissue type (4). In addition, when more than 1,000 loci were analyzed by the genome-wide analysis by restriction landmark genomic scanning (RLGS) using NotI as a landmark enzyme (1), we found that the placental junctional zone had a distinct DNA methylation pattern compared with the placental labyrinth zone, suggesting that in the placenta differentiation involves changing the methylation pattern. The rat Rcho-1 cell line is derived from spontaneous choriocarcinoma cells, and can be induced to differentiate in vitro (14, 15). These cells express specific markers of trophoblast giant cells (15, 16). The stem cell state can proliferate in growth medium, but the differentiated state is not proliferative. After differentiation, the size of cells and nuclei increases, as does the DNA content (17, 18). Here, we compared methylation throughout the genome in Rcho-1 stem cells and differentiated cells in
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order to determine whether differentiation involves both methylation and demethylation. MATERIALS AND METHODS Culture conditions. Rcho-1 stem cells were cultured in NCTC-135 medium (GIBCO BRL Life Technology Inc., New York, NY) supplemented with 20% fetal bovine serum (FBS, Cansera, Ontario, Canada), 110 mg/liter sodium pyruvate, 100 mg/ml penicillin, 100 U/ml streptomycin and 50 mM 2-mercaptoethanol in a humidified atmosphere of 95% air–5% CO 2 at 37°C. The cells were routinely maintained in subconfluent conditions, and the culture media was changed every two days. To induce differentiation, the serum in the media was changed to 10% horse serum (HS, Starrate, Melbourne, Australia), and the cells were cultured for 8 days. Nuclear staining. Rcho-1 cells were fixed in 4% paraformaldehyde for 20 min. The fixed cells were incubated in 0.5% Triton X-100 in phosphate buffered saline (PBS) for 5 min, followed by an incubation in 0.05% Tween 20 in PBS for 5 min, after which the cells were washed with PBS and incubated with 10 ng/ml DAPI in PBS for 30 min. The samples were washed twice with PBS before observation. All operations were performed at room temperature. Preparation of genomic DNA. Genomic DNA was extracted as described previously (1, 19). Briefly, Rcho-1 cells (5 ⫻ 10 7 cells) were suspended in 400 l of PBS to which we added 4 ml of lysis buffer (150 mM EDTA, 10 mM Tris–HCl pH 8.0, 1% SDS) containing 30 l of proteinase K (10 mg/ml; Merck, Darmstadt, Germany). The mixture was incubated at 55°C for 20 min. Following phenol/chloroform/ isoamyl alcohol (50:49:1) extraction twice, the genomic DNA was precipitated in ethanol, pelleted, and redissolved in 50 l of TE (10 mM Tris–HCl, 1 mM EDTA pH 7.6). Restriction landmark genomic scanning (RLGS). RLGS was performed as described previously (1, 19) using NotI, PvuII and PstI. Briefly, 3.5 g of genomic DNA in 7 l of TE was treated with 10 units of Klenow fragment (TaKaRa, Kyoto, Japan) in the presence of 0.4 M dGTP␣S, 0.2 M dCTP␣S (Amersham, Tokyo, Japan), 0.4 M ddATP and 0.4 M ddTTP (TaKaRa, Kyoto, Japan). The DNA was first digested with 20 units of NotI as a landmark enzyme (Nippon Gene, Toyama, Japan) and the cohesive ends were isotopically labeled with 1.3 units of Sequenase Ver 2.0 (USB Co. Ltd., NE) in the presence of 0.33 M [␣- 32P]dCTP and 0.33 M [␣- 32P]dGTP (Amersham). Labeled DNA (1 g) was then treated with 20 units of PvuII (Nippon Gene) and subjected to first dimension electrophoresis in a 0.9% agarose disc gel for about 23 h at 230V. DNA fragments in the gel were then treated with 1,000 units of PstI (Nippon Gene). The second dimension electrophoresis in a 5% polyacrylamide gel was carried out for 20 h at 150V. The gel was dried and exposed to X-ray film (Kodak XAR5, Eastman Kodak, NY) at ⫺80°C for 2–3 weeks. Spot cloning method. Three of the four RLGS spots, which disappeared after differentiation of Rcho-1 cells, were cloned using a NotI trapper (1, 20). In this study, eight parallel samples of 100 g of genomic DNA from the Rcho-1 stem cells were digested sequentially by 150 units of NotI and 120 units of PvuII, and then purified by phenol/chloroform/isoamyl alcohol extraction, followed by ethanol precipitation. DNA fragments containing NotI ends were collected using the DNA trapper R-Not I (Japan Synthetic Rubber Co., Ltd., Tokyo, Japan). Purified DNA fragments (6 g) were re-dissolved in 15 l of TE. Of these, one-fifth was labeled at the NotI site using 1.3 units of Sequenase Ver. 2.0 in the presence of 10 Ci each of [␣- 32P]dGTP (3,000 Ci/mmol) and [␣- 32P]dCTP (6,000 Ci/mmol). The labeled portion was mixed with the remaining four-fifths and analyzed by two-dimensional electrophoresis as described above. After exposure to X-ray film for two days, small pieces of the gel was cut out at targeted spots identical to the spot positions on the X-ray film. The DNA fragments were electroeluted and purified by phenol/ chloroform/isoamyl alcohol extraction followed by ethanol precipita-
TABLE 1
Characterization of the DNA Specifically Demethylated in Rcho-1 Stem Cells Spot No.
Length (bp)
GC content (%)
CpG frequency a
Homologue (species) b
s1 s2
610 436
53.0 53.9
0.58 0.52
s3
525
56.4
0.33
HTGS AC068650 (M) Ste1 upstream (R) HTGS AC084240 (R) EST AW494700 (M)
a CpG frequency was calculated from the formula of GardinerGarden and Frommer (21). b M, mouse; R, rat.
tion. The eluted DNA was initially ligated into the NotI and PstI sites of pBluescript II (Stratagene, CA). The inserted fragments were amplified by PCR using the M4-RV primer set that pBluescript II contains at both sides of its multicloning site. PCR conditions were: 91°C for 1 min, 55°C for 1 min and 72°C for 2 min; for 30 cycles. The PCR products were cloned into the pGEM-T vector (Promega, WI). Sequence analysis. Cloned DNA was sequenced using a Shimadzu autosequencer system (Shimadzu, Kyoto, Japan) following the manufacturer’s indications. In Table 1, the CpG frequency was calculated using the formula of Gardiner-Garden and Frommer (21), for each nucleotide sequence between the NotI site and the PstI site.
RESULTS Differentiation of Rcho-1 Cells Rcho-1 cells were induced to differentiate as described. The differentiated cells showed distinct morphological features with an increase of cellular and nuclear sizes (Fig. 1A). Placental lactogen-I (PL-I), a marker for trophoblast giant cells in early/mid pregnancy (16, 22), was expressed only in the differentiated cells (Fig. 1B). The intensity of DAPI staining indicated that the differentiated cells have about 35-fold more nuclear DNA than the stem cells (Fig. 1C). These findings confirm that the differentiated Rcho-1 cells resemble trophoblast giant cells (16, 17, 22). Figure 2A shows whole RLGS profile of differentiated Rcho-1 giant cells. In the Rcho-1 cells of both states, a total of 1,232 spots were detected. The spot patterns were reproducible in two parallel samples. Of these, 1,224 spots (99.4%) were identical in positions and intensities, regardless of the differentiation state. During differentiation, no individual spot became more intense relative to the others. Since the intensity of each spot reflects the number of copies of the restriction landmark in the genome, this indicated that the differentiated cells had become polyploid by endoreduplication of the whole genome. This suggest that the differentiated Rcho-1 cells become polyploid in the same way as do trophoblast giant cells (1). Thus, the Rcho-1 cell line shows irreversible phenotypic changes, and is a useful model for terminal differentiation of trophoblast.
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FIG. 1. Differentiation of Rcho-1 cells. (A) Morphological changes during the differentiation of Rcho-1 cells. (B) Differentiation-dependent transcription of placental lactogen I (PL-I), a marker specific to trophoblast giant cells. Total RNA (10 g) was separated on a 1% agarose gel, blotted onto a nylon membrane, and hybridized with a PL-I cDNA probe. As an internal control, the membrane was stripped and rehybridized with a GAPDH probe. (C) Nuclear volume of stem and differentiated Rcho-1 cells. The intensity of DAPI staining was measured by NIH Image software. Values were standardized to those of Rcho-1 stem cells. Bars indicate the mean value for each treatment, and horizontal lines indicate the standard error of the mean (n ⫽ 20).
Differences in DNA Methylation between Stem and Differentiated Rcho-1 Cells
Genomic DNA Loci That Become Methylated during Differentiation
The RLGS profiles show the methylation status of CpG islands. Of 1,232 spots, 1,224 (99.4%) always appeared on the RLGS autoradiography, showing that there are constitutively unmethylated CpG islands regardless of differentiation status (Fig. 2A). There were eight spots (0.6%) detected either in stem cells or in differentiated cells. As shown in left panel of Fig. 2A, four spots (d1-4) were observed only in differentiated cells. These four loci were presumably methylated in the stem cells, and became unmethylated during differentiation. In contrast, four other spots (s1– 4) were observed only in stem cells (Fig. 2A, right panel). They were presumably unmethylated in the stem cells, and became methylated during differentiation. Therefore, differentiation of Rcho-1 cells involved bi-directional changes; both demethylation and de novo methylation occurred.
Demethylation of certain known loci is important for cellular differentiation (5–7), but the loci that become methylated in differentiation are still ill defined. It is, therefore, of interest to investigate the loci that become methylated in differentiation of Rcho-1 cells. The four spots (s1– 4) observed only in the stem cells were candidates for loci that become methylated during differentiation. Of the four spots, three DNA fragments were cloned and sequenced (Table 1, Spots s1–3). Two DNA sequences had no significant homologies with known rat genes, but had homologies with a mouse sequence in the HTGS database(AC068650: spot s1) and a mouse EST (AW494700: spot s3). The third sequence was identical to part of a BAC clone of rat chromosome 4 (AC084240: spot s2). The clone had the Estm 25 gene and an L1-like transposon transcribed to RNA. The L1-like sequence was also highly
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FIG. 2. RLGS profiles of Rcho-1 cells and schematic representation for methylation changes during differentiation. (A) Typical RLGS profiles of DNA from Rcho-1 cells, showing the locations and appearances of spots specific to differentiated cells (D) and stem cells (S). Genomic DNA of Rcho-1 cells (1 g) was subjected to RLGS analysis. In the autoradiograms of stem and differentiated Rcho-1 cells, a total of 1,232 spots were detected reproducibly in two independent samples each. Center panel: A representative RLGS profile from differentiated Rcho-1 cells, and locations of the spots detected in either stem or differentiated cells. Left panels: Enlarged views of the spot positions detected only in the differentiated cells (D) but not in the stem cells. Right panels: Enlarged views of the spot positions detected only in stem cells (S). Arrows indicate the differently detected spots. (B) Schematic diagram of the differentiation of the rat trophoblast cell line, Rcho-1, with de novo methylation and demethylation. Open circles: unmethylated CpG. Closed circles: methylated CpG.
homologous with many genes such as estrogensulfotransferase 1 and insulin 1, both of which contain L1-like elements in their intron or 5⬘ upstream region. The results that all three differentially methylated DNA fragments had similarities to transcript or genomic sequences of other species suggest that these genomic regions are biologically important loci, including transcriptional units around CpG islands. All three DNAs had 53.3–56.4% GC content, which is higher than the genome-wide average of about 40%, suggesting that these loci are within or adjacent to CpG islands. The CpG frequencies ranged between 0.33 and 0.58 (Table 1). Since loci a possessing CpG frequency greater than 0.6 are designated as CpG islands (21), s3 is out of the criteria for CpG island. The loci corresponding s1 and s2 are thought to be adjacent to CpG islands taking into account our previous data (4). In fact, s1 and s2 were located at the edge of CpG islands (data not shown).
DISCUSSION In vitro differentiation of the placental Rcho-1 cell line was accompanied by a change in the methylation status of genomic DNA. During differentiation, four loci become unmethylated and another four loci become methylated, implying that not only demethylation but also de novo methylation are involved in the differentiation of these cells. In a previous study by Taylor and Jones (23), forced demethylation using inhibitors such as 5-aza deoxycytidine caused cultured mouse cells to differentiate into myoblasts. Similarly, treatment of bone marrow stromal cells with the demethylating reagent induced transformation into cardiomyocytes (24). From these observations, it is generally believed that differentiation involves overall demethylation of genomic DNA. However, DNA methylation level is lowest in the blastocyst and increases after implantation (8 –10),
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suggesting that development also involves de novo methylation. Here we present direct evidence that differentiation involves bi-directional changes in DNA methylation. As shown schematically in Fig. 2B, proliferating Rcho-1 stem cells have specific methylation patterns, presumably associated with that cellular type. Induction of differentiation alters the methylation pattern to one which is presumably specific for giant cells. This is followed by changes in gene expression and morphology. Since DNA methylation regulates gene activity (25), formation of cell type-specific patterns of DNA methylation must be one of the crucial factors to define cellular phenotypes. CpG islands are located around promoter regions of many genes (26). In CpG islands, cytosine residues are generally unmethylated, regardless of richness of methylatable sequences. However, a genome-wide survey of methylation status indicated that in several CpG islands, cytosine residues were unmethylated in some normal tissues but not others (4). Indeed, some of the spots which became methylated in the differentiated cells (spot s1 and 2) showed high GC content and high CpG frequency. Of the 1,232 spots/CpG islands we detected, 0.6% changed their methylation status after differentiation. Since there are 30,000-40,000 CpG islands in the mammalian genome (13), it can be estimated that during differentiation a total of 180-240 CpG islands could change their methylation status. Genes without CpG islands can also show tissuespecific methylation. For instance, expression of PL-I is completely specific to the placenta (3), but as there are only 17 CpG dinucleotides in the 3.4 kb of DNA upstream of the transcription start site, this promoter region does not contain a CpG island. Nevertheless, we have found that methylation of these CpG sequences repressed PL-I transcription (3). Taken together, the data suggest that changes in the methylation status are not confined to genes with CpG islands. In rodents, the placenta contains trophoblast giant cells that have several hundred times more DNA than diploid cells (27). The DNA content of Rcho-1 giant cells reached 64 N by day 6 of differentiation, and the cells continue to endo-reduplicate their genome until at least day 12 (17, 28). In the present study, we showed that differentiated cells have polyploid DNA similar to that of placental trophoblast giant cells. Thus, Rcho-1 differentiation involves changing the pattern of DNA methylation, and also endo-reduplication. In conclusion, we showed that both demethylation and de novo methylation occurred during the differentiation of Rcho-1 cells. This is the first data obtained by a genome-wide analysis technique, RLGS, indicating that both types of change occurred simultaneously during differentiation of a cell lineage. Since CpG methylation is important for appropriate embryonic development, the differentially methylated loci and the genes
around them could have important roles in the differentiation of trophoblast giant cells. ACKNOWLEDGMENT This work was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (K.S.).
REFERENCES 1. Ohgane, J., Aikawa, J., Ogura, A., Hattori, N., Ogawa, T., and Shiota, K. (1998) Analysis of CpG islands of trophoblast giant cells by restriction landmark genomic scanning. Dev. Genet. 22, 132–140. 2. Ohgane, J., Wakayama, T., Kogo, Y., Senda, S., Hattori, N., Tanaka, S., Yanagimachi, R., and Shiota, K. (2001) DNA methylation variation in cloned mice. Genesis 30, 45–50. 3. Cho, J.-H., Kimura, H., Minami, T., Ohgane, J., Hattori, N., Tanaka, S., and Shiota, K. (2001) DNA methylation regulates placental lactogen I gene expression. Endocrinology 142, 3389 –3396. 4. Imamura, T. Ohgane, J., Ito, S., Ogawa, T., Hattori, N., Tanaka, S., and Shiota, K. (2001) CpG island of rat sphingosine kinase-1 gene: Tissue-dependent DNA methylation status and multiple alternative exons. Genomics 76, 117–125. 5. Constantinides, P. G., Jones, P. A., and Gevers, W. (1977) Functional striated muscle cells from non-myoblast precursors following 5-azacytidine treatment. Nature 267, 364 –366. 6. Davis, R. L., Weintraub, H., and Lassar, A. B. (1987) Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell 51, 987–1000. 7. Brunk, B. P., Goldhamer, D. J., and Emerson, C. P., Jr. (1996) Regulated demethylation of the myoD distal enhancer during skeletal myogenesis. Dev. Biol. 177, 490 –503. 8. Monk, M., Boubelik, M., and Lehnert, S. (1987) Temporal and regional changes in DNA methylation in the embryonic, extraembryonic and germ cell lineages during mouse embryo development. Development 99, 371–382. 9. Kafri, T., Ariel, M., Brandeis, M., Shemer, R., Urven, L., McCarrey, J., Cedar, H., and Razin, A. (1992) Developmental pattern of gene-specific DNA methylation in the mouse embryo and germ line. Genes Dev 6, 705–714. 10. Razin, A., and Shemer, R. (1995) DNA methylation in early development. Hum. Mol. Genet. 4, 1751–1755. 11. Li, E., Bestor, T. H., and Jaenisch, R. (1992) Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69, 915–926. 12. Okano, M., Bell, D. W., Haber, D. A., and Li, E. (1999) DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99, 247–257. 13. Antequera, F., and Bird, A. (1993) Number of CpG islands and genes in human and mouse. Proc. Natl. Acad. Sci. USA 90, 11995–11999. 14. Teshima, S., Sakashita, S., Kikuchi, Y., and Aizawa, M. (1981) Histogenesis of yolk sac carcinoma induced by fetectomy in the rat. Gann 72, 524 –530. 15. Faria, T. N., and Soares, M. J. (1991) Trophoblast cell differentiation: Establishment, characterization, and modulation of a rat trophoblast cell line expressing members of the placental prolactin family. Endocrinology 129, 2895–2906. 16. Iwatsuki, K., Shinozaki, M., Hattori, N., Hirasawa, K., Itagaki, S., Shiota, K., and Ogawa, T. (1996) Molecular cloning and characterization of a new member of the rat placental prolactin (PRL) family, PRL-like protein D (PLP-D). Endocrinology 137, 3849 –3855.
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17. Kimura, H., Takeda, T., Tanaka, S., Ogawa, T., and Shiota, K. (1998) Expression of rat DNA (cytosine-5) methyltransferase (DNA MTase) in rodent trophoblast giant cells: Molecular cloning and characterization of rat DNA MTase. Biochem. Biophys. Res. Commun. 253, 495–501. 18. Shirai, T., Itonori, S., Tai, T., Soares, M. J., Shiota, K., and Ogawa, T. (1996) Ganglioside composition of the rat choriocarcinoma cell line, Rcho-1. Glycoconj. J. 13, 415– 421. 19. Okazaki, Y., Okuizumi, H., Sasaki, N., Ohsumi, T., Kuromitsu, J., Hirota, N., Muramatsu, M., and Hayashizaki, Y. (1995) An expanded system of restriction landmark genomic scanning (RLGS Ver. 1.8). Electrophoresis 16, 197–202. 20. Ohsumi, T., Okazaki, Y., Hirotsune, S., Shibata, H., Muramatsu, M., Suzuki, H., Taga, C., Watanabe, S., and Hayashizaki, Y. (1995) A spot cloning method for restriction landmark genomic scanning. Electrophoresis 16, 203–209. 21. Gardiner-Garden, M., and Frommer, M. (1987) CpG islands in vertebrate genomes. J. Mol. Biol. 196, 261–282. 22. Hirosawa, M., Miura, R., Min, K. S., Hattori, N., Shiota, K., and Ogawa, T. (1994). A cDNA encoding a new member of the rat
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Differentiation of Trophoblast Lineage Is Associated with DNA Methylation and Demethylation Jun Ohgane,* Naka Hattori,* ,† Mayumi Oda,* Satoshi Tanaka,* and Kunio Shiota* ,1 *Cellular Biochemistry, Animal Resource Sciences/Veterinary Medical Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan; and †Bio-oriented Technology Research Advancement Institution, 1-40-2 Nisshin-cho, Omiya, Saitama, Japan
Received November 11, 2001
Our previous study has shown that the placenta and kidney had different genomic methylation patterns regarding CpG island loci detected by restriction landmark genomic scanning (RLGS). To investigate whether differentiation involves changes in DNA methylation, we analyzed the rat Rcho-1 cell line, which retains trophoblast cell features and differentiates from stem cells into trophoblast giant cells in vitro. By RLGS, a total of 1,232 spots were identified in the Rcho-1 stem and differentiated giant cells. Four spots (0.3%) were detected only in giant cells, implying that the loci were originally methylated, but became demethylated during differentiation. Another four spots (0.3%) were detected only in stem cells, implying that these loci, originally unmethylated, became methylated during differentiation. DNAs from three loci that became methylated during differentiation were cloned and sequenced. All showed high homologies with expressed sequence tags (ESTs) or with genomic DNA of other species, suggesting that these loci are biologically important. Thus, the eight differentially methylated loci should be good tools to study epigenetic modification specific to differentiation of trophoblast giant cells. © 2002 Elsevier Science Key Words: DNA methylation; differentiation; trophoblast; CpG island; placenta.
Most mammalian cells differentiate without detectable changes in DNA sequence. Instead, epigenetic changes such as DNA methylation occur (1, 2). DNA methylation is involved in tissue-specific gene expression including placental hormones (3) and alternate use of tissue-specific exons (4). Therefore, DNA methylation may be a form of “cellular memory” for the gene expression pattern, which is established during development. To establish the tissue-specific pattern of DNA 1
To whom correspondence and reprint requests should be addressed. Fax: ⫹81-3-5841-8189. E-mail: [email protected].
methylation and gene expression, de novo methylation as well as demethylation need to occur in each cell lineage. The treatment of some cell types with demethylating reagents induces differentiation, and in some transcription factor genes, demethylation is associated with differentiation (5–7). In contrast, genomewide methylation occurs at organogenesis in embryonic development (8 –10), and this may explain why null mutations of DNA cytosine methyltransferase genes (Dnmt) 1 and 3b are embryonic lethal (11, 12). CpG dinucleotides are the main sites of DNA methylation, and are unevenly distributed in the mammalian genome: they appear at 10 –20 times their average density in selected regions, known as CpG islands. The mammalian genome contains 30,000 – 40,000 CpG islands, which are located around the promoters of housekeeping genes, and are associated with tissuespecific genes (13). One of the characteristics of CpG islands is that most cytosine residues are unmethylated. However, we found some CpG islands where some areas are differentially methylated, depending on tissue type (4). In addition, when more than 1,000 loci were analyzed by the genome-wide analysis by restriction landmark genomic scanning (RLGS) using NotI as a landmark enzyme (1), we found that the placental junctional zone had a distinct DNA methylation pattern compared with the placental labyrinth zone, suggesting that in the placenta differentiation involves changing the methylation pattern. The rat Rcho-1 cell line is derived from spontaneous choriocarcinoma cells, and can be induced to differentiate in vitro (14, 15). These cells express specific markers of trophoblast giant cells (15, 16). The stem cell state can proliferate in growth medium, but the differentiated state is not proliferative. After differentiation, the size of cells and nuclei increases, as does the DNA content (17, 18). Here, we compared methylation throughout the genome in Rcho-1 stem cells and differentiated cells in
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0006-291X/02 $35.00 © 2002 Elsevier Science All rights reserved.
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order to determine whether differentiation involves both methylation and demethylation. MATERIALS AND METHODS Culture conditions. Rcho-1 stem cells were cultured in NCTC-135 medium (GIBCO BRL Life Technology Inc., New York, NY) supplemented with 20% fetal bovine serum (FBS, Cansera, Ontario, Canada), 110 mg/liter sodium pyruvate, 100 mg/ml penicillin, 100 U/ml streptomycin and 50 mM 2-mercaptoethanol in a humidified atmosphere of 95% air–5% CO 2 at 37°C. The cells were routinely maintained in subconfluent conditions, and the culture media was changed every two days. To induce differentiation, the serum in the media was changed to 10% horse serum (HS, Starrate, Melbourne, Australia), and the cells were cultured for 8 days. Nuclear staining. Rcho-1 cells were fixed in 4% paraformaldehyde for 20 min. The fixed cells were incubated in 0.5% Triton X-100 in phosphate buffered saline (PBS) for 5 min, followed by an incubation in 0.05% Tween 20 in PBS for 5 min, after which the cells were washed with PBS and incubated with 10 ng/ml DAPI in PBS for 30 min. The samples were washed twice with PBS before observation. All operations were performed at room temperature. Preparation of genomic DNA. Genomic DNA was extracted as described previously (1, 19). Briefly, Rcho-1 cells (5 ⫻ 10 7 cells) were suspended in 400 l of PBS to which we added 4 ml of lysis buffer (150 mM EDTA, 10 mM Tris–HCl pH 8.0, 1% SDS) containing 30 l of proteinase K (10 mg/ml; Merck, Darmstadt, Germany). The mixture was incubated at 55°C for 20 min. Following phenol/chloroform/ isoamyl alcohol (50:49:1) extraction twice, the genomic DNA was precipitated in ethanol, pelleted, and redissolved in 50 l of TE (10 mM Tris–HCl, 1 mM EDTA pH 7.6). Restriction landmark genomic scanning (RLGS). RLGS was performed as described previously (1, 19) using NotI, PvuII and PstI. Briefly, 3.5 g of genomic DNA in 7 l of TE was treated with 10 units of Klenow fragment (TaKaRa, Kyoto, Japan) in the presence of 0.4 M dGTP␣S, 0.2 M dCTP␣S (Amersham, Tokyo, Japan), 0.4 M ddATP and 0.4 M ddTTP (TaKaRa, Kyoto, Japan). The DNA was first digested with 20 units of NotI as a landmark enzyme (Nippon Gene, Toyama, Japan) and the cohesive ends were isotopically labeled with 1.3 units of Sequenase Ver 2.0 (USB Co. Ltd., NE) in the presence of 0.33 M [␣- 32P]dCTP and 0.33 M [␣- 32P]dGTP (Amersham). Labeled DNA (1 g) was then treated with 20 units of PvuII (Nippon Gene) and subjected to first dimension electrophoresis in a 0.9% agarose disc gel for about 23 h at 230V. DNA fragments in the gel were then treated with 1,000 units of PstI (Nippon Gene). The second dimension electrophoresis in a 5% polyacrylamide gel was carried out for 20 h at 150V. The gel was dried and exposed to X-ray film (Kodak XAR5, Eastman Kodak, NY) at ⫺80°C for 2–3 weeks. Spot cloning method. Three of the four RLGS spots, which disappeared after differentiation of Rcho-1 cells, were cloned using a NotI trapper (1, 20). In this study, eight parallel samples of 100 g of genomic DNA from the Rcho-1 stem cells were digested sequentially by 150 units of NotI and 120 units of PvuII, and then purified by phenol/chloroform/isoamyl alcohol extraction, followed by ethanol precipitation. DNA fragments containing NotI ends were collected using the DNA trapper R-Not I (Japan Synthetic Rubber Co., Ltd., Tokyo, Japan). Purified DNA fragments (6 g) were re-dissolved in 15 l of TE. Of these, one-fifth was labeled at the NotI site using 1.3 units of Sequenase Ver. 2.0 in the presence of 10 Ci each of [␣- 32P]dGTP (3,000 Ci/mmol) and [␣- 32P]dCTP (6,000 Ci/mmol). The labeled portion was mixed with the remaining four-fifths and analyzed by two-dimensional electrophoresis as described above. After exposure to X-ray film for two days, small pieces of the gel was cut out at targeted spots identical to the spot positions on the X-ray film. The DNA fragments were electroeluted and purified by phenol/ chloroform/isoamyl alcohol extraction followed by ethanol precipita-
TABLE 1
Characterization of the DNA Specifically Demethylated in Rcho-1 Stem Cells Spot No.
Length (bp)
GC content (%)
CpG frequency a
Homologue (species) b
s1 s2
610 436
53.0 53.9
0.58 0.52
s3
525
56.4
0.33
HTGS AC068650 (M) Ste1 upstream (R) HTGS AC084240 (R) EST AW494700 (M)
a CpG frequency was calculated from the formula of GardinerGarden and Frommer (21). b M, mouse; R, rat.
tion. The eluted DNA was initially ligated into the NotI and PstI sites of pBluescript II (Stratagene, CA). The inserted fragments were amplified by PCR using the M4-RV primer set that pBluescript II contains at both sides of its multicloning site. PCR conditions were: 91°C for 1 min, 55°C for 1 min and 72°C for 2 min; for 30 cycles. The PCR products were cloned into the pGEM-T vector (Promega, WI). Sequence analysis. Cloned DNA was sequenced using a Shimadzu autosequencer system (Shimadzu, Kyoto, Japan) following the manufacturer’s indications. In Table 1, the CpG frequency was calculated using the formula of Gardiner-Garden and Frommer (21), for each nucleotide sequence between the NotI site and the PstI site.
RESULTS Differentiation of Rcho-1 Cells Rcho-1 cells were induced to differentiate as described. The differentiated cells showed distinct morphological features with an increase of cellular and nuclear sizes (Fig. 1A). Placental lactogen-I (PL-I), a marker for trophoblast giant cells in early/mid pregnancy (16, 22), was expressed only in the differentiated cells (Fig. 1B). The intensity of DAPI staining indicated that the differentiated cells have about 35-fold more nuclear DNA than the stem cells (Fig. 1C). These findings confirm that the differentiated Rcho-1 cells resemble trophoblast giant cells (16, 17, 22). Figure 2A shows whole RLGS profile of differentiated Rcho-1 giant cells. In the Rcho-1 cells of both states, a total of 1,232 spots were detected. The spot patterns were reproducible in two parallel samples. Of these, 1,224 spots (99.4%) were identical in positions and intensities, regardless of the differentiation state. During differentiation, no individual spot became more intense relative to the others. Since the intensity of each spot reflects the number of copies of the restriction landmark in the genome, this indicated that the differentiated cells had become polyploid by endoreduplication of the whole genome. This suggest that the differentiated Rcho-1 cells become polyploid in the same way as do trophoblast giant cells (1). Thus, the Rcho-1 cell line shows irreversible phenotypic changes, and is a useful model for terminal differentiation of trophoblast.
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FIG. 1. Differentiation of Rcho-1 cells. (A) Morphological changes during the differentiation of Rcho-1 cells. (B) Differentiation-dependent transcription of placental lactogen I (PL-I), a marker specific to trophoblast giant cells. Total RNA (10 g) was separated on a 1% agarose gel, blotted onto a nylon membrane, and hybridized with a PL-I cDNA probe. As an internal control, the membrane was stripped and rehybridized with a GAPDH probe. (C) Nuclear volume of stem and differentiated Rcho-1 cells. The intensity of DAPI staining was measured by NIH Image software. Values were standardized to those of Rcho-1 stem cells. Bars indicate the mean value for each treatment, and horizontal lines indicate the standard error of the mean (n ⫽ 20).
Differences in DNA Methylation between Stem and Differentiated Rcho-1 Cells
Genomic DNA Loci That Become Methylated during Differentiation
The RLGS profiles show the methylation status of CpG islands. Of 1,232 spots, 1,224 (99.4%) always appeared on the RLGS autoradiography, showing that there are constitutively unmethylated CpG islands regardless of differentiation status (Fig. 2A). There were eight spots (0.6%) detected either in stem cells or in differentiated cells. As shown in left panel of Fig. 2A, four spots (d1-4) were observed only in differentiated cells. These four loci were presumably methylated in the stem cells, and became unmethylated during differentiation. In contrast, four other spots (s1– 4) were observed only in stem cells (Fig. 2A, right panel). They were presumably unmethylated in the stem cells, and became methylated during differentiation. Therefore, differentiation of Rcho-1 cells involved bi-directional changes; both demethylation and de novo methylation occurred.
Demethylation of certain known loci is important for cellular differentiation (5–7), but the loci that become methylated in differentiation are still ill defined. It is, therefore, of interest to investigate the loci that become methylated in differentiation of Rcho-1 cells. The four spots (s1– 4) observed only in the stem cells were candidates for loci that become methylated during differentiation. Of the four spots, three DNA fragments were cloned and sequenced (Table 1, Spots s1–3). Two DNA sequences had no significant homologies with known rat genes, but had homologies with a mouse sequence in the HTGS database(AC068650: spot s1) and a mouse EST (AW494700: spot s3). The third sequence was identical to part of a BAC clone of rat chromosome 4 (AC084240: spot s2). The clone had the Estm 25 gene and an L1-like transposon transcribed to RNA. The L1-like sequence was also highly
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FIG. 2. RLGS profiles of Rcho-1 cells and schematic representation for methylation changes during differentiation. (A) Typical RLGS profiles of DNA from Rcho-1 cells, showing the locations and appearances of spots specific to differentiated cells (D) and stem cells (S). Genomic DNA of Rcho-1 cells (1 g) was subjected to RLGS analysis. In the autoradiograms of stem and differentiated Rcho-1 cells, a total of 1,232 spots were detected reproducibly in two independent samples each. Center panel: A representative RLGS profile from differentiated Rcho-1 cells, and locations of the spots detected in either stem or differentiated cells. Left panels: Enlarged views of the spot positions detected only in the differentiated cells (D) but not in the stem cells. Right panels: Enlarged views of the spot positions detected only in stem cells (S). Arrows indicate the differently detected spots. (B) Schematic diagram of the differentiation of the rat trophoblast cell line, Rcho-1, with de novo methylation and demethylation. Open circles: unmethylated CpG. Closed circles: methylated CpG.
homologous with many genes such as estrogensulfotransferase 1 and insulin 1, both of which contain L1-like elements in their intron or 5⬘ upstream region. The results that all three differentially methylated DNA fragments had similarities to transcript or genomic sequences of other species suggest that these genomic regions are biologically important loci, including transcriptional units around CpG islands. All three DNAs had 53.3–56.4% GC content, which is higher than the genome-wide average of about 40%, suggesting that these loci are within or adjacent to CpG islands. The CpG frequencies ranged between 0.33 and 0.58 (Table 1). Since loci a possessing CpG frequency greater than 0.6 are designated as CpG islands (21), s3 is out of the criteria for CpG island. The loci corresponding s1 and s2 are thought to be adjacent to CpG islands taking into account our previous data (4). In fact, s1 and s2 were located at the edge of CpG islands (data not shown).
DISCUSSION In vitro differentiation of the placental Rcho-1 cell line was accompanied by a change in the methylation status of genomic DNA. During differentiation, four loci become unmethylated and another four loci become methylated, implying that not only demethylation but also de novo methylation are involved in the differentiation of these cells. In a previous study by Taylor and Jones (23), forced demethylation using inhibitors such as 5-aza deoxycytidine caused cultured mouse cells to differentiate into myoblasts. Similarly, treatment of bone marrow stromal cells with the demethylating reagent induced transformation into cardiomyocytes (24). From these observations, it is generally believed that differentiation involves overall demethylation of genomic DNA. However, DNA methylation level is lowest in the blastocyst and increases after implantation (8 –10),
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suggesting that development also involves de novo methylation. Here we present direct evidence that differentiation involves bi-directional changes in DNA methylation. As shown schematically in Fig. 2B, proliferating Rcho-1 stem cells have specific methylation patterns, presumably associated with that cellular type. Induction of differentiation alters the methylation pattern to one which is presumably specific for giant cells. This is followed by changes in gene expression and morphology. Since DNA methylation regulates gene activity (25), formation of cell type-specific patterns of DNA methylation must be one of the crucial factors to define cellular phenotypes. CpG islands are located around promoter regions of many genes (26). In CpG islands, cytosine residues are generally unmethylated, regardless of richness of methylatable sequences. However, a genome-wide survey of methylation status indicated that in several CpG islands, cytosine residues were unmethylated in some normal tissues but not others (4). Indeed, some of the spots which became methylated in the differentiated cells (spot s1 and 2) showed high GC content and high CpG frequency. Of the 1,232 spots/CpG islands we detected, 0.6% changed their methylation status after differentiation. Since there are 30,000-40,000 CpG islands in the mammalian genome (13), it can be estimated that during differentiation a total of 180-240 CpG islands could change their methylation status. Genes without CpG islands can also show tissuespecific methylation. For instance, expression of PL-I is completely specific to the placenta (3), but as there are only 17 CpG dinucleotides in the 3.4 kb of DNA upstream of the transcription start site, this promoter region does not contain a CpG island. Nevertheless, we have found that methylation of these CpG sequences repressed PL-I transcription (3). Taken together, the data suggest that changes in the methylation status are not confined to genes with CpG islands. In rodents, the placenta contains trophoblast giant cells that have several hundred times more DNA than diploid cells (27). The DNA content of Rcho-1 giant cells reached 64 N by day 6 of differentiation, and the cells continue to endo-reduplicate their genome until at least day 12 (17, 28). In the present study, we showed that differentiated cells have polyploid DNA similar to that of placental trophoblast giant cells. Thus, Rcho-1 differentiation involves changing the pattern of DNA methylation, and also endo-reduplication. In conclusion, we showed that both demethylation and de novo methylation occurred during the differentiation of Rcho-1 cells. This is the first data obtained by a genome-wide analysis technique, RLGS, indicating that both types of change occurred simultaneously during differentiation of a cell lineage. Since CpG methylation is important for appropriate embryonic development, the differentially methylated loci and the genes
around them could have important roles in the differentiation of trophoblast giant cells. ACKNOWLEDGMENT This work was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (K.S.).
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