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National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-2753,1 Max Planck Institute of Psychiatry, Munich D-80804, Germany,2 The Cancer Institute and Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213,3 The Fels Institute for Cancer Research and Molecular Biology and Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 191404
Received 29 August 2003/ Accepted 21 November 2003
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ABSTRACT |
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INTRODUCTION |
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The Sgy/Tead2 locus provides not only an example of two closely spaced, divergently transcribed genes but a unique paradigm for differential regulation of gene expression during mammalian development. Mammals contain four highly conserved genes that were originally named transcription enhancer factor or TEF genes but which have been redesignated TEA domain or Tead genes by the mouse genome project (www.informatics.jax.org; references 14, 15, and 18 and references therein). These genes encode site-specific DNA binding proteins that, in the company of the transcriptional coactivator YAP65, can activate expression of genes in a variety of embryonic and adult cells (47). Tead2 (TEF-4), like other Tead genes, is expressed to various extents in many cells and tissues (references 17 and 18 and references therein). However, Tead2 is the only Tead gene expressed in mouse embryos during the first 7 days of development (17, 50), suggesting that it plays a unique role at the beginning of mammalian development by allowing preimplantation mouse embryos to utilize Tead-dependent promoters and enhancers (17, 24, 26). Sgy is a novel single-copy gene whose mRNA start site is located only 3.8 kb upstream of the Tead2 mRNA start site (19). The function of Sgy is unknown, but it is presumed important because of its restricted expression pattern in the adult and its partial homology to the Dickkopf gene family, potential effectors of the Wnt signaling pathway (19). Since Tead2 and Sgy are transcribed in opposite directions, their regulatory elements lie in close proximity. In fact, the same locus is found in humans on chromosome 19q13.3, except that the two mRNA start sites are separated by only 1.5 kb. Both mRNA start sites lie within CpG islands (this report). Moreover, the Sgy/Tead2 locus (chromosome 7, 23.0 cM) lies adjacent to the imprinted region of chromosome 7 corresponding to Prader-Willi/Angelmann syndromes (20), suggesting that one or both genes may be imprinted.
In the examples reported so far, Sgy and Tead2 appear to be differentially expressed; either Tead2 or Sgy is expressed in a particular cell type, but the two are never expressed together. In adult mice, Tead2 is expressed strongly in heart and lung tissues and the granulosa cells of the ovary and weakly in several other tissues. Furthermore, Tead2 and its transcriptional coactivator YAP65 are enriched in embryonic, neural, and hematopoietic stem cells (30). In contrast, Sgy appears to be expressed only in the developing spermatocytes of seminiferous tubules and in lymphocytes.
In principle, DNA methylation could govern differential gene expression at bidirectional loci by preventing expression of one gene while allowing expression of the other. Clearly, differentiated cells first appear during blastocyst formation with a separation of embryonic stem (ES) cells (inner cell mass [ICM]) from trophoblasts (cells forming the outer layer). After implantation, most CpG sequences are progressively methylated, except for those located in the promoter region of active genes (32). Thus, active promoters are frequently associated with CpG islands (44). Methylation of CpG dinucleotides is commonly correlated with loss of gene expression both in vivo and in vitro (6, 29, 40). However, hypermethylation of CpG islands in some cell lines appears to be an intrinsic property of the cell line rather than the tissue from which it originated (41). Furthermore, the absence of a change in the DNA methylation pattern of several tissue-specific genes during development either of wild-type or of DNA methyltransferase-deficient mouse embryos suggests that CpG methylation is a consequence rather than a cause of the transcription repression seen (reference 49 and references therein). In this capacity, DNA methylation may serve primarily to ensure that repressed genes remain silent. Moreover, while DNA methylation has been linked directly to X chromosome inactivation, genomic imprinting, and silencing of transposable elements, a direct role for DNA methylation in regulating gene expression during animal development has yet to be demonstrated (33, 42, 49).
We reasoned that if DNA methylation is the primary factor in determining which of two closely spaced genes is expressed in a particular cell type, there should exist a strict correlation between the methylation status of a gene's regulatory region and its expression. This correlation should exist in normal cells such as germ cells, preimplantation embryos, ES cells, splenocytes, and tissues, as well as in established cell lines. Moreover, changes in DNA methylation should accompany changes in gene expression in normal cells. We found that while DNA methylation can differentially regulate the expression of two closely linked genes such as Sgy and Tead2 in established mouse cell lines, DNA methylation is not the primary determinant of Sgy/Tead2 expression patterns during mouse development. Nevertheless, DNA methylation of downstream regulatory sequences did appear to restrict expression of the Sgy gene to basal levels in both normal cells and established cell lines.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Methylation-sensitive restriction enzyme assays. Fifteen micrograms of genomic DNA from either EL4 or TM3 cells was digested with SacI (40 to 80 U, 37°C, 16 h; Roche), precipitated with ethanol in the presence of 2.5 M ammonium acetate, resuspended in water, and combined with 100 pg of pBluescript-NE. This plasmid DNA consisted of Bluescript KS plasmid (Stratagene) modified by inserting double-stranded oligonucleotides with restriction sites for NsbI and Eco47III between the SacI and KspI sites. The appropriate buffer and enzyme were added, and the following mixture was incubated at 37°C (SmaI at 25°C) for 16 h: 5 U of Cfr10I, 12.5 U of KspI, 5 U of SmaI, 12.5 U of XhoI, 10 U of Bsh1285I, 10 U of NsbI, 2.5 U of Eco47III, and 5 U of Psp1406. The buffer recommended by the enzyme manufacturer was used. NsbI was from Fermentis, and all others were from Roche Molecular Biochemicals. DNA was then precipitated with ethanol-ammonium acetate, resuspended in Tris-EDTA (pH 8.0), fractionated by electrophoresis in 0.7% agarose (Tris-borate-EDTA buffer), transferred to Nytran N membrane (Schleicher & Schuell), and hybridized with a 32P-labeled 2.1-kb SacI/XhoI DNA fragment (see Fig. 6A) (19). The blot was stripped and reprobed with 32P-labeled plasmid DNA.
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Bisulfite genomic sequencing.
Bisulfite genomic sequencing was carried out as previously described (25, 35), with modifications. pGEM 3Zf(+) (100 ng; Applied Biosystems) and 20 U of SacI (Roche) were added to lysates (prepared as previously described [25]) of 80 to 100 oocytes, 75 to 375 two-cell embryos, or 20 to 200 morulae and then incubated overnight at 37°C. For all other samples, 100 µg of genomic DNA was digested with SacI in the presence of 100 ng of pGEM 3Zf(+). Bisulfite treatment and subsequent purification were carried out as already described, except that the digested genomic DNA was frozen and thawed twice (-80°C and room temperature) and then heated at 100°C for 10 min before addition of NaOH to ensure complete denaturation. One-fifth of the bisulfite-treated DNA was resuspended in 20 µl of H2O, amplified by PCR (5 min at 94°C and then 30 to 40 cycles of 30 s at 94°C, 30 s at 48°C, 1 min at 72°C, and then finally 7 min at 72°C). Except for amplicons D and F, two sets of primers were used to amplify the indicated region (Table 2). After the first round of amplification (outer primers), products were purified over a PCR purification spin column (Qiagen), and 1 to 4 µl of a 50-µl eluate was used for a second round of amplification (inner primers). PCR products were purified with a PCR purification spin column and eluted with 30 to 40 µl of H2O. The amount of DNA was estimated by fractionating a sample by agarose gel electrophoresis. A portion of the total PCR product (
100 ng) was sequenced directly with the inner primer set and an ABI 373 or 310 sequencer.
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RESULTS |
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-amanitin (a specific inhibitor of RNA polymerase II), most of this mRNA was inherited from the egg. Tead2 and Sgy mRNAs accumulated dramatically from the eight-cell stage to the blastocyst stage, consistent with their transcription from zygotic genes. In blastocysts, the level of both mRNAs was about 10,000 to 20,000 copies per embryo (Fig. 1B), about 1.5 to 3% of the level of ß-actin mRNA (31). Thus, the level of Tead2 in blastocysts was 20-fold greater than in oocytes or 50-fold greater than in two- and four-cell embryos. Comparisons of preimplantation embryos with oocytes (Fig. 1) and ICMs with blastocysts (Table 3) suggested that while the two genes are expressed at similar levels in blastocysts, Tead2 is expressed preferentially in the ICM while Sgy is expressed preferentially in the trophectoderm. This suggested that Sgy and Tead2 became differentially expressed as totipotent embryonic cells differentiated into specific cell types.
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Sgy and Tead2 were expressed coordinately in ES cells, but Tead2 was expressed to a greater extent than Sgy (Fig. 2, zero time point), suggesting that differential expression of these two genes begins when the two distinct cell lineages of the blastocyst, trophectoderm and ICM, are produced (Table 3). In addition, within 1 to 2 days after induction of ES cell differentiation, Sgy expression was selectively repressed, and within 5 days, Tead2 expression was selectively stimulated (Fig. 2). These changes were accompanied by repression of Rex-1 expression (Fig. 2) and by morphological transformation of ES cells into embryoid bodies (data not shown), changes that have been reported previously (37, 38). The ubiquitous GAPDH gene was expressed continuously at high levels during this period of time, as previously reported (27), and was therefore used as a standard reference throughout subsequent studies.
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Differential expression of Sgy and Tead2 in differentiated cells. PCR-based assays [RT-PCR and poly(A)+ PCR] revealed three levels of gene expression in mouse cells and tissues (summarized in Table 4): off, basal level, and on. Cells in which RNA was not detected by PCR were considered not to express the gene. For example, Sgy was not expressed in either oocytes (Fig. 1) or TM3 cells (Fig. 3B), and Tead2 was not expressed in either MPC-11 or EL4 cells (Fig. 3B). Cells in which RNA could only be detected by RT followed by >25 cycles of PCR, and in which expression was not detected by Northern analysis, were considered to express the gene at basal levels. For example, Sgy was expressed at basal levels in MPC-11, F9, and ES cells (Fig. 3). Cells in which RNA could be detected by both PCR and Northern analyses were considered to fully express the gene. For example, Sgy was fully expressed in EL4 cells and testis tissue (Fig. 3).
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Differential methylation of Sgy and Tead2 genes in established cell lines. To determine whether or not differential gene expression is accompanied by differential DNA methylation, the methylation status of the Sgy/Tead2 locus was determined by measuring its sensitivity to methylation sensitive restriction endonucleases (34). Eleven specific sites within a 7.9-kb SacI DNA fragment that encompassed the Sgy/Tead2 locus (Fig. 6A) were examined in TM3 and EL4 cells. TM3 cells expressed Tead2 but not Sgy, whereas EL4 cells expressed Sgy but not Tead2. Genomic DNA was mixed with an unmethylated plasmid DNA control and then digested with the indicated endonuclease. The DNA products were fractionated by gel electrophoresis, attached to a membrane, and then hybridized either with an Sgy-specific 32P-labeled DNA probe (Fig. 6A) or with a plasmid-specific probe. In each sample, the unmethylated plasmid DNA was cleaved completely by the indicated enzyme (Fig. 7B), confirming that digestion was complete. Therefore, in those samples where the cellular DNA was digested completely (Fig. 7A), the indicated restriction site was not methylated (Cfr10I, KspI, and SmaI in TM3 cells [Fig. 6B]; XhoI and Bsh1285I in EL4 cells [Fig. 6C]). In those samples where genomic DNA was either not digested or partially digested (Psp1406 in TM3 cells), the indicated site was either completely or partially methylated, respectively. Partial methylation meant that only a fraction of the genomes (i.e., cells) in the population was methylated at this site.
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DNase I-hypersensitive sites were associated only with the active gene. Regulatory sequences for transcriptionally active genes commonly contain nuclease-hypersensitive sites that result from the presence of site-specific DNA binding proteins (9). To determine whether or not such sites exist in the Sgy/Tead2 locus, nuclei were isolated and digested with increasing concentrations of pancreatic DNase I. The results (Fig. 8A) revealed three hypersensitive sites (S1, S2, and S3) at the Sgy gene locus in cells that expressed Sgy at high levels (e.g., EL4) and two hypersensitive sites (S1 and S2) in cells that expressed Sgy at basal levels (e.g., F9 and MPC-11 cells). Conversely, cells that did not express Sgy did not exhibit DNase I-hypersensitive sites in the Sgy gene region (e.g., TM3 cells). Similarly, cells that expressed Tead2 (TM3 and F9 cells) contained at least one DNase I-hypersensitive site (T1) just upstream of the Tead2 mRNA start site, whereas cells that did not express Tead2 (EL4 and MPC-11 cells) did not exhibit any hypersensitive sites in the Tead2 gene region. These data are consistent with the presence of site-specific DNA binding proteins in the promoters of active genes but not in the promoters of silent genes. Moreover, the S3 site in EL4 cells in the Sgy locus suggests the presence of a regulator element downstream of the Sgy mRNA start that is required for full Sgy expression.
Site-specific transition from unmethylated to methylated DNA. To define accurately the transitions from unmethylated to methylated DNA, the methylation status of each cytosine in the transition loci was determined by bisulfite genomic sequencing (34). Bisulfite-induced deamination converts C to U in single-stranded DNA. Subsequent amplification by PCR translates each uracil into thymidine. Thus, CpG dinucleotides are converted into TpG dinucleotides on one strand and CpA dinucleotides on the complementary strand. Cytosines are not converted by bisulfite if they are either methylated or reside in double-stranded DNA (34). In the work described here, the possibility that unconverted cytosines resulted from regions of undenatured DNA was eliminated in two ways. First, only cytosines within CpG dinucleotides were resistant to bisulfite; all of the cytosines in CpC, CpA, and CpT dinucleotides were converted to U. Second, PCR primers were designed to select against any unconverted DNA that may have been present as a result of incomplete denaturation (34).
Bisulfite genomic sequencing can be analyzed in two ways: either individual DNA molecules from the PCR amplification product are cloned and sequenced (Fig. 9A), or the entire PCR amplification product is sequenced (Fig. 9B). The first method reveals the methylation status of individual genomes, whereas the second method determines the average methylation status of a cell population. In addition, it avoids pitfalls inherent in the cloning and sequencing of individual genomes (5, 34). Therefore, since the results of the two approaches were comparable, the second method was used routinely in order to directly observe the average methylation status of thousands of individual genomes.
With a single DNA fragment, a sharp transition between unmethylated and methylated DNA was detected 646 bp upstream of the Sgy transcription start site in EL4 cells (Fig. 9). Here, the last CpG and the first mCpG were separated by only 64 bp. Upstream of this transition site, all of the CpGs were methylated (Fig. 9, 10A, and 11A). Downstream of this transition site, all of the CpGs were unmethylated, at least to +691 (Fig. 9, and 10A). These data were consistent with those gathered at methylation-sensitive restriction endonuclease sites (Fig. 6C).
The CpG-to-mCpG transition in the Tead2 locus was not mapped with the same accuracy, because primers were not found that would amplify bisulfite-treated DNA in the transition site. Nevertheless, the available bisulfite data (Fig. 11A, amplicons D and E), together with restriction endonuclease data (Fig. 6B), indicate that a sharp transition also exists somewhere within a 167-bp region that encompasses the Tead2 mRNA start site (Fig. 11). These transitions presumably mark the upstream boundary of the active promoter.
In normal cells, gene activity was restricted, but not determined, by DNA methylation. To determine whether or not the three levels of Sgy gene transcription described above (off, basal, and on) are related to DNA methylation, the methylation status of all 52 CpG dinucleotides within a 1,337-bp region encompassing the Sgy gene regulatory region was determined by bisulfite genomic sequencing of DNA from a variety of cells and tissues, and the data were related to Sgy gene expression (Fig. 10). The same analysis was also carried out for 36 of the 53 CpG dinucleotides within a 1,726-bp region encompassing the Tead2 locus (Fig. 11). Unfortunately, despite numerous attempts, we were not able to amplify the bisulfite-treated DNA product from the 167-bp segment containing exon 1 and 17 CpGs. Fortunately, we were able to analyze the 30 CpGs in the remaining 387-bp portion of the Tead2 CpG island, and this served to clearly distinguish methylated from unmethylated promoter regions. These results revealed that DNA methylation may restrict Sgy gene expression by limiting it to basal levels but that DNA methylation could not be the primary mechanism for preventing either Sgy or Tead2 expression during animal development, because the promoter regions in normal cells and tissues were unmethylated, even when the gene was silent.
The Sgy and Tead2 promoter regions were unmethylated in all cells that expressed the gene, consistent with the hypothesis that DNA methylation would repress gene expression. This was true for normal cells, as well as for established cell lines. For example, in both splenocytes (a mixture of T and B lymphocytes isolated from spleen tissue; Fig. 10D) and EL4 cells (a cell line derived from a T-cell lymphoma; Fig. 10A), the Sgy gene locus was unmethylated and the Sgy gene was expressed to similar levels (Fig. 4). In fact, all cells that expressed Sgy, at either basal or fully activated levels, contained an Sgy promoter region that was unmethylated. Several of these genomes also exhibited a sharp transition between unmethylated and methylated DNA at positions -385 to -450, similar to EL4 cells (Fig. 10; data not shown). Similar, but less complete, data were obtained for the Tead2 promoter region (Fig. 6 and 11).
In surprising contrast, neither the Sgy nor the Tead2 promoter region was methylated in any cells that did not express the gene, revealing that DNA methylation is not the primary determinant in silencing these genes. While established cell lines that did not express Sgy contained fully methylated Sgy gene regions (TM3 and CA51 cells, Fig. 10A and B), normal cells that did not express Sgy did not contain a methylated Sgy promoter (oocytes, Fig. 10C). Similarly, established cell lines that did not express Tead2 contained fully methylated Tead2 gene regions (EL4 and MPC-11 cells, Fig. 11A). However, splenocytes also did not express Tead2, even though their Tead2 promoter was unmethylated (Fig. 11C). Therefore, while all methylated promoters were silenced, DNA methylation was not required to silence either gene. This conclusion was confirmed by differentiation of ES cells into embryoid bodies. Sgy expression was completely repressed by day 2 and then again expressed by day 7 (Fig. 2), but the Sgy promoter remained unmethylated during the entire period (Fig. 10C).
DNA methylation did appear to restrict Sgy expression in both normal cells (ES cells and uterus, lung, and liver tissues [Fig. 10C and D]) and established cell lines (MPC11, F9, and CA51* cells [Fig. 10A and B]) because Sgy expression was inversely related to the extent of DNA methylation downstream of the promoter region. Only a basal level of Sgy expression was detected when sequences downstream of the promoter were methylated. These sequences included about half of the CpG island encompassing the Sgy mRNA start site. The DNase I-hypersensitive site at +615 (S3) was absent from MPC-11 and F9 cells, although the sites at -120 (S1) and -430 (S2) were present (Fig. 8). These data suggest that the site near +615 marks the location of an Sgy gene regulatory element that is sensitive to DNA methylation. Thus, cells containing low levels of Sgy transcripts always contained an unmethylated Sgy gene promoter, whereas in cells that contained high levels of Sgy transcripts, the unmethylated region was extended downstream of exon 2 and exhibited an additional DNase I-hypersensitive site.
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DISCUSSION |
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Moreover, a direct role for DNA methylation in regulating gene expression during animal development has yet to be demonstrated (2, 33, 42). On the one hand, methylation of promoter sequences can repress gene expression by interfering with binding of proteins required for transcription through recruitment of histone deacetylase and other transcription repressors (16, 28). On the other hand, the promoters of several tissue-specific genes are not methylated in some tissues in which they are inactive, and they remain inactive under conditions in which global demethylation causes up regulation of imprinted loci (49). These results suggest that while DNA methylation can repress gene expression, DNA methylation is not the primary mechanism that regulates gene expression during animal development.
The studies described here addressed this conundrum by determining whether or not there is a strict correlation between the methylation status of a gene's regulatory region and its expression in both normal mouse cells and established cell lines. The Sgy/Tead2 locus was chosen for this study, because these two closely linked genes each contain a CpG island and became differentially expressed concurrent with the onset of cell differentiation and DNA methylation. The results described here, however, reveal that while DNA methylation can repress Sgy expression in established cell lines and restrict its expression to basal levels during mouse development, DNA methylation per se is not the mechanism primarily responsible for repressing either Sgy or Tead2 expression during development.
Differential gene expression is developmentally acquired. Both Sgy and Tead2 were expressed coordinately and in equivalent amounts during the activation of zygotic genes from the 2-cell stage to the morula stage (compacted 8- to 32-cell embryos) in preimplantation mouse embryos (Fig. 1), consistent with the lack of DNA methylation in their promoter regions (Fig. 10 and 11). Differential expression appeared only with the onset of cell differentiation and DNA methylation. Sgy was overexpressed in the trophoblasts but underexpressed in the ICM (Table 3), in totipotent ES cells (Fig. 2) derived from the ICM, and in pluripotent F9 cells (Fig. 3) derived from an embryonic carcinoma. Moreover, ES cells induced to undergo differentiation into embryoid bodies rapidly repressed Sgy expression and then stimulated Tead2 expression (Fig. 2). Only one of the two genes was expressed in 10 different tissues (testis, ovary, uterus, kidney, muscle, liver, lung, spleen, brain, and heart [15, 17, 19, 54, 55]), in 4 normal cell types (spermatocytes, splenocytes, oocytes, and embryoid bodies), as well as in 15 different established cell lines (summarized in Table 4). Moreover, the gene chosen for expression was independent of cell immortalization or cell transformation. For example, normal lymphocytes (splenocytes), immortalized lymphocytes (EL4 cells), and transformed lymphocytes (YAC-1, A20, MPC-11, and CH1) all expressed the Sgy gene exclusively. Thus, Sgy and Tead2 are differentially expressed in most mammalian cells, but differential expression of these two genes is developmentally acquired when cell differentiation begins.
DNA methylation can repress gene expression at the Sgy/Tead locus in established cell lines. One mechanism that could account for the developmental acquisition of differential gene expression (i.e., repressing one of two closely linked but divergently transcribed genes) during cell differentiation is DNA methylation. Only two CpG islands exist within a 10-kb region encompassing the Sgy/Tead2 locus, one at the Sgy mRNA start site and one at the Tead2 mRNA start site (Fig. 6A). Analyses of these two regions and the intervening sequences by methylation-sensitive restriction endonucleases (Fig. 6 and 7) and by bisulfite genomic sequencing (Fig. 9 to 11) in established cell lines TM3 and EL4 revealed DNA methylation patterns consistent with the hypothesis that DNA methylation can differentially repress expression of Sgy and Tead2 during mouse development.
In fact, none of the cells in which either the Sgy or the Tead2 promoter region was methylated expressed that gene. In these cases, gene-specific RNA was not detected either by Northern analysis or by RT-PCR. In those cases, such as CA51 cells, in which Sgy expression could be restored by treatment with 5AC alone (Fig. 4), recovery of Sgy expression was accompanied by demethylation of its promoter region (Fig. 10B). In those cases, such as TM3 cells, in which recovery of Sgy expression required treatment with both 5AC and TSA (Fig. 5), Sgy expression was accompanied by only partial demethylation of its promoter region (data not shown), as previously reported for other genes that could be reactivated by the same regimen (4). Taken together, these results revealed an inverse correlation between the extent of DNA methylation in the promoter region and the extent of gene expression, consistent with a role for DNA methylation in repressing gene activity during development.
DNA methylation restricts Sgy expression to basal levels during mouse development. Methylation of downstream sequences appeared to restrict Sgy expression to basal levels during mouse development. Full Sgy expression was observed only when both the promoter region and the downstream hypersensitive site (S3 in Fig. 8) were unmethylated in either normal cells such as splenocytes, two-cell embryos, and morulae or in established cell lines such as EL4 cells (Fig. 10). However, expression was restricted to basal levels when the promoter region was unmethylated but the S3 site was methylated in either normal cells such as ES cells, embryoid bodies, and uterus, lung, and liver cells or in established cell lines such as MPC-11, F9, and CA51 cells following treatment with 5AC (Fig. 10).
A similar result occurs in the skeletal alpha-actin gene promoter, where a subset of CpG dinucleotides are preferentially methylated in nonexpressing tissues (51). These data are consistent with previous studies showing that the extent of transcriptional suppression by DNA methylation in a plasmid-encoded reporter gene depends on the density of mCpG dinucleotides (12) and on the location of the methylated region (13). Maximum repression occurs when both the promoter and downstream regions of the gene are methylated.
The sequences downstream of the Sgy mRNA start site that contain hypersensitive site S3 appear to contain an enhancer or other regulatory element. Sequences from this region stimulated expression of a plasmid-encoded reporter gene driven by a viral promoter and bound a protein(s) present in EL4 cells but not in MPC-11 cells (data not shown). Interestingly, S3 corresponds to a repetitive SINE/B4 element located within intron 2. Such repetitive Alu elements are associated with insulator activity (53), and insulator activity can be regulated by DNA methylation (11). Therefore, sequences within intron 2 and their methylation status may determine whether Sgy is expressed at basal levels or fully activated. An analogous situation may exist with the Tead2 gene, where an enhancer has been identified within intron 1 of the Tead2 gene (45; data not shown).
DNA methylation is not the primary determinant of Sgy/Tead2 expression during mouse development. If DNA methylation is the primary mechanismdetermining differential expression at the Sgy/Tead2 locus, then the gene that is not expressed should always contain a methylated promoter region. Surprisingly, the promoter regions of both genes were unmethylated in all of the primary cells and tissues examined. These included sperm cells, oocytes, two-cell embryos, morulae, ES cells, primary embryo fibroblasts, embryoid bodies, and splenocytes and uterus, lung, and liver tissues. Furthermore, in three of these examples, one of the two genes was silent despite the fact that its promoter region was unmethylated. (i) Oocytes expressed Tead2 but not Sgy, although the Sgy promoter region was unmethylated. Sgy expression began with zygotic gene expression following fertilization. (ii) The mixture of T and B lymphocytes isolated from spleen tissue did not express Tead2, despite the fact that the Tead2 promoter region was unmethylated. (iii) ES cells expressed Sgy at basal levels, but when they were induced to differentiate into embryoid bodies, Sgy gene expression was rapidly repressed by day 2 without increasing the extent of DNA methylation in the Sgy promoter region. Therefore, DNA methylation is not the primary determinant of Sgy/Tead2 expression during mouse development. The absence of DNA methylation at promoter regions is required but not sufficient for gene activity. The only silent Sgy or Tead2 genes that were also methylated were found in established cell lines, consistent with previous observations that hypermethylation of CpG islands is an intrinsic property of cultured cell lines rather than a general mechanism for regulating gene activity during animal development (41).
Regulation of zygotic gene activation. The results described here suggest that the regulatory regions of genes such as Sgy and Tead2 that are destined to be transcribed during zygotic gene activation are not methylated in either sperm cells or oocytes. In oocytes, both the Sgy and Tead2 promoter regions were unmethylated. In sperm cells, the entire Sgy gene CpG island and sequences upstream to the Tead2 gene were largely unmethylated. This pattern was also found in two-cell embryos and in morulae, consistent with previous reports that, with the exception of imprinted genes (reference 52 and references therein), a global demethylation of the mouse genome occurs upon fertilization (32). Sequences downstream of exon 2 remained methylated, revealing that sequences in this region are immune to the global demethylation that occurs during preimplantation development (33). DNA methylation downstream of the Sgy mRNA start site increased as two-cell embryos underwent development to the ES cell stage and further increased as ES cells differentiated into embryoid bodies.
What is the purpose of these demethylation-remethylation events in preimplantation embryos? If the primary function of DNA methylation is to suppress expression of parasitic repeat sequences (49), then it is curious that oocytes and cleavage stage embryos contain an abundance of mRNA transcripts for B1 and B2 repeat elements (Alu repeats in humans) (46), consistent with the notion that global demethylation would result in an increase in the transcription of repeated sequences. Thus, oocytes and early embryos can tolerate expression of these potentially harmful sequences during the first 3 to 4 days of development. One general consequence of global demethylation is to remove one of the major obstacles to binding of proteins to DNA. These proteins may include repressor proteins whose binding is eliminated by methylation (7). With the exception of the five proteins known to bind specifically to mCpG dinucleotides, the affinity for DNA of all other DNA binding proteins appears to be reduced by methylation of their DNA binding sites. Thus, demethylation may facilitate remodeling of sperm chromatin into somatic cell chromatin in one-cell embryos, and it may allow more subtle remodeling to take place in both paternal and maternal genomes during the preimplantation period.
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ACKNOWLEDGMENTS |
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This work was supported in part by a grant from NIH/NCRR (RR15253) awarded to K.L.
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FOOTNOTES |
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, CpG). (C) Methylation status of 11 CpG dinucleotides in EL4 cells, which express Sgy but not Tead2. The transitions from unmethylated to methylated DNA (vertical shaded bars) determined from these analyses were 385 bp at the Sgy locus and 789 bp at the Tead2 locus.
). Some one-cell embryos were cultured in the presence of 
). RT-PCR was used to amplify the entire population of poly(A)+ mRNA from mouse ova and embryos under conditions that preserve the relative abundance of each mRNA in the cDNA population (
