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¹ Laboratory of Cellular Biochemistry, Veterinary Medical Sciences/Animal Resource Sciences, The University of Tokyo, Tokyo 113-8657, Japan
² Department of Medicine (Hematology), Albert Einstein College of Medicine, Bronx, New York 10461, USA
³ Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan
⁴ Department of Molecular Genetics, Albert Einstein College of Medicine, Bronx, New York 10461, USA

	Abstract

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CpG islands, which have higher GC content and CpG frequencies compared to the genome as a whole, are generally believed to be unmethylated in tissues except at promoters of genes undergoing X chromosome inactivation or genomic imprinting. Recent studies, however, have shown that CpG islands at promoters of a number of genes contain tissue-dependent, differentially methylated regions (T-DMRs). In general, the tissue-specific methylation is restricted to a part of the promoter CpG island, with hypomethylation of the remaining sequence. In the current study, using comparison between Restriction Landmark Genomic Scanning (RLGS) and in silico RLGS, we identified ten sperm-specific unmethylated NotI sites, T-DMRs located in CpG islands that were hypomethylated in sperm but near-completely methylated in the kidney and brain. Unusually, these T-DMRs involve the whole CpG island at each of these loci. We characterized one of these genes, adenine nucleotide translocator 4 (Ant4), which is expressed in germ cells. Using a promoter assay, we demonstrated that expression of Ant4 gene is controlled by DNA methylation at the CpG island sequences within the promoter region. Ant4 and other sperm-specific hypomethylated loci represent a new class of CpG islands that become completely methylated in different cell lineages. T-DMRs at CpG islands are functionally important gene regulatory elements that may now be categorized into two classes: T-DMRs involving a subregion of the CpG island and those that occupy the whole CpG island.

	Introduction

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DNA methylation occurs mainly at the cytosine of CpG dinucleotides in mammals and is involved in the differentiation of cells and development through gene silencing in phenomena such as X inactivation and genomic imprinting. Recent studies have shown that promoters of many genes contain tissue-dependent, differentially methylated regions (T-DMRs), in which DNA methylation is involved in gene silencing, regardless of the richness of CpG dinucleotides. For example, there are T-DMRs at the upstream regulatory regions of the PL-I (Cho et al. 2001), Sry (Nishino et al. 2004), Ddah2 (Tomikawa et al. 2006), Oct4 (Hattori et al. 2004b) and Dnmt1o genes (Ko et al. 2005). While PL-I and Sry have only a small number of CpG dinucleotides in their T-DMRs, those of Oct4 and Dnmt1o are relatively rich in CpGs.

CpG islands have long been recognized as unmethylated regions in normal cells and tissues, except at loci undergoing X inactivation or genomic imprinting. CpG islands are defined as sequences where the length is > 200 bp, the (G+C) content (%GC) is > 50% and the ratio of observed to expected CpG dinucleotide frequencies (CpG_obs/CpG_exp) is > 0.6 (Gardiner-Garden & Frommer 1987). Most housekeeping genes and almost half of tissue-specifically expressed genes have CpG islands at their promoters (Gardiner-Garden & Frommer 1987; Larsen et al. 1992).

The frequent observation of T-DMRs in tissues (Futscher et al. 2002; Shiota 2004; Kitamura et al. 2007) indicates that the dogma that CpG islands are always unmethylated fails to represent the complexity of cytosine methylation in mammals. In addition to systematic searches for T-DMRs, there are many reports of DNA methylation at CpG-rich regions including CpG islands (De Smet et al. 1999; Strichman-Almashanu et al. 2002; Rakyan et al. 2004; Song et al. 2005). In general, T-DMRs in CpG islands are restricted to subregions within the CpG island sequences. For example, the T-DMR of the Sphk1 gene, which is conserved in mouse, rat and human, occupies ~200 bp at the edge of a 3.7-kb CpG island, a proportion of less than 10% of the entire CpG island (Imamura et al. 2001, 2004). Such T-DMRs restricted to a fraction of CpG island have also been found in genes for the endothelin receptor B (Pao et al. 2001) and proopiomelanocortin (Newell-Price et al. 2001). Interestingly, Hisano et al. found that all CpGs in the CpG island of the Tact1/Actl7b gene are methylated in somatic tissues whereas they are unmethylated in germ cells (Hisano et al. 2003). Therefore, another class of CpG islands is likely to exist in which the T-DMR extends throughout the whole CpG island.

Genome-wide analysis of T-DMRs, performed by Restriction Landmark Genomic Scanning (RLGS) using NotI as landmark enzyme focusing on CpG islands, discovered that more than 10% of identified T-DMRs were unmethylated in sperm (Shiota et al. 2002). Based on this information, we identified and characterized several sperm-specific hypomethylated genes in this study. We found that CpG islands associated with these genes were totally unmethylated in the sperm but methylated in kidney and brain and that the T-DMR extended through the whole CpG island at each of these loci. DNA methylation of the CpG island regulates the expression of Ant4/Slc25a31, which belongs to adenine nucleotide translocator (Ant) gene family and is an example of this unique type of whole CpG island T-DMR.

	Results

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Genome scanning identifies sperm-specific hypomethylated loci

Genomic DNA of several tissues and cells had previously been analyzed by RLGS using NotI as a landmark enzyme (Shiota et al. 2002). Since NotI is a methylation-sensitive enzyme, the appearance of RLGS spots indicates hypomethylation at those sites in the tissue. Among 247 sites that were differentially methylated in these tissues and cells, 30 sites were hypomethylated in sperm but methylated in all other tissues and cells examined (Shiota et al. 2002). By matching RLGS profiles with virtual image (Vi)-RLGS images, followed by confirmation with genomic PCR, we could successfully identify the locations of ten sperm-specific hypomethylated sites which were characterized in the present study (Table 1). Nine of the ten sites were contained within CpG islands according to the definition of CpG island by Gardiner-Garden & Frommer 1987, the remaining T-DMR was located in a CpG-rich region that did not meet the CpG island criteria.

The question prompted by the results is whether DNA methylation at CpG sites other than at the NotI site within these CpG islands occurs in somatic tissues. To investigate further the methylation status of each of these regions, we analyzed flanking regions of the sperm-specific hypomethylated sites by sodium bisulfite sequencing. We quantified the methylation in sperm, kidney and brain at eight of the loci with sperm-specific hypomethylation (Fig. 1). Seven of the eight regions were methylated throughout the whole CpG island or CpG-rich region in the kidney and/or brain, while one CpG island was methylated only partially. Focusing on the CpG islands, the Stox1 CpG island was methylated at five of the 13 CpGs within the CpG island in kidney, but the other six CpG islands were hypomethylated in the sperm but methylated throughout the entire CpG island in the kidney and/or brain. From these data, we can classify sperm-specifically hypomethylated CpG islands into two categories: those in which part of the CpG island is hypermethylated in somatic tissues and those in which the entire CpG island is hypermethylated. The first one is typical of previously reported T-DMRs, but the second represents a new type of T-DMR involving entire CpG islands in tissues.

View larger version (48K): [in this window] [in a new window]   Figure 1  The methylation status of each CpG dinucleotide surrounding the sperm-specific hypomethylated NotI sites. The methylation status of each CpG dinucleotide surrounding the sperm-specific hypomethylated NotI sites was investigated by bisulfite genomic sequencing. Open circles indicate unmethylated CpGs and filled circles are methylated CpGs. Each row represents an independent clone. The methylation status of CpG islands are summarized in Table 1. In all CpG islands except for that in the Stox1 gene, almost all CpGs including NotI sites were unmethylated in sperm, while they were completely methylated in the kidney and/or brain. In the CpG island of Stox1, the T-DMR is located in a subregion of the CpG island.

The DNA methylation status of a CpG island, when located at a promoter region, usually corresponds to the gene expression status. We focused on a CpG island located near the transcription start site of the 1700034J06Rik gene (also known as Slc25a31), consisting of six exons encoding a protein with 320 amino acids (Fig. 2A). The gene maps to chromosome 3B in the Mammalian Gene Collection Program (Strausberg et al. 2002) and is identified as NM_178386. The gene is highly homologous to adenine nucleotide translocator 1 (Ant1, mRNA; 63% identity, amino acid; 87% identity and similarity) and is ontologically annotated with transporter activity (GO:0005215) and mitochondrial inner membrane localization (GO:005743). We refer to this Ant family member as Ant4. At the transcription start site of the Ant4 gene predicted from RefSeq of mRNA (NM_ 178386), we found a dense cluster of CpG dinucleotides of 543 bp in length (–233~+311 relative to TSS), %GC = 63.4 and CpG_obs/CpG_exp = 0.88. All of the 43 CpGs in the CpG island were completely methylated also in the liver. In addition, nine CpGs further upstream of the CpG island (–343~–556) were also hypermethylated in somatic tissues. In contrast, the same CpGs in the CpG islands and the upstream region were completely unmethylated in the sperm, indicating that the boundary of methylated and unmethylated regions at the Ant4 promoter is located outside of the CpG island.

View larger version (56K): [in this window] [in a new window]   Figure 2  The expression and methylation status of Ant4. (A) In the upper panel, the genomic structure of Ant4, which is located on the chromosome 3B, is illustrated with local CpG island structure. Ant4 has six exons extending over 16.38 kb. Open boxes indicate the untranslated regions and closed boxes indicate the coding regions. CG% (line) and CpG frequency (histogram) of exon 1 and flanking regions are shown. In the lower panel, the methylation status of each CpG dinucleotide in the Ant4 CpG island was investigated by bisulfite genomic sequencing. In both regions of the CpG island itself and upstream sequences, the cytosine residues are heavily methylated in the kidney, brain and liver but unmethylated in sperm. (B) Expression of Ant4 detected by RT-PCR. Ant4 was expressed only in the testis but not in the other somatic tissues. (C) Expression of Ant4 in the developing gonads. Ant4 was detected faintly in the 12.5 dpc male gonad and abundantly at 15.5 and 18.5 dpc. It was also detected in the female gonad constantly from 12.5 to 18.5 dpc. (D) In situ hybridization analysis of Ant4 expression in the testis. The upper panel shows the signals of Ant4 transcripts detected with antisense probes and the lower panel is a control staining with sense probes. Ant4 expression was detected in spermatocyte layer and spermatogonia indicated by a bar and arrowheads, respectively. No signal was observed in spermatids and spermatozoa. (E) DNA methylation analyses of spermatogonia, spermatocytes and spermatids by bisulfite genomic sequencing in the Ant4 CpG island. In all these cell types, CpG dinucleotides were unmethylated throughout the CpG island except for one clone in the spermatogonia sample.

Ant4 mRNA was not detected by RT-PCR in somatic tissues (kidney, liver and brain), indicating a complete suppression of gene expression (Fig. 2B). The all or none expression observed in the Ant4 gene is suggestive of tissue-specific epigenetic regulation of gene expression. Expression of Ant4 was detectable also in the male gonad of 15.5 dpc and 18.5 dpc fetuses (Fig. 2C). Unexpectedly, expression was also observed in the female gonad at 12.5, 15.5 and 18.5 dpc. The expression studies indicate that the expression of Ant4 is specific to the developing male and female gonadal cells (Fig. 2C). In situ hybridization was performed to identify the cell types expressing Ant4 in the testis (Fig. 2D). Signals for Ant4 mRNA were strong in spermatocytes, while they were weakly detectable in round spermatids. The signals were detected only in a minor population of spermatogonia. Ant4 was expressed neither in Sertoli cells nor Leydig cells.

The T-DMR of the Ant4 CpG island is unmethylated in testicular germ cells

From in situ hybridization results, the expression signals were detected in spermatocytes, round spermatids and minor population of spermatogonia; however, the signals did not show uniform distribution. We tested whether DNA methylation status varied among the testicular germ cells. The testicular germ cell samples were collected with the laser micro-dissection of spermatogonia, spermatocytes and spermatids from adult mouse testis. All 43 CpGs in the Ant4 CpG island were completely unmethylated in these three cell types (Fig. 2E). The single clone of spermatogonia in which CpGs were methylated may possibly represent contamination of genomic DNA from a somatic cell. Overall, while Ant4 expression is generally correlated with the methylation status of its promoter CpG island, we also find that demethylation alone is not sufficient to allow expression of the gene in spermatogenic cells.

DNA methylation inhibits Ant4 promoter activity

To test whether DNA methylation is involved in transcriptional repression of the Ant4 gene, we performed a promoter assay. By PCR amplification, Ant4 mRNA was detected neither in embryonic stem (ES) cells nor in embryonic germ (EG) cells (Supplementary Fig. S1A), indicating that the Ant4 gene is repressed in these pluripotent stem cells. However, in multi-potent germ-line stem (mGS) cells, which were established from the testis of neonatal mice (Kanatsu-Shinohara et al. 2004), a weak signal for Ant4 expression could be detected by RT-PCR performed using 35 cycles (Supplementary Fig. S1A). Various levels of DNA methylation at CpGs in the Ant4 CpG island were observed in the ES, EG and mGS cells (Supplementary Fig. S1B). The 5'-region of the CpG island (region 1: –258~+69) showed partial methylation in all three cell types. By contrast, CpGs in the 3'-region of the CpG island (region 2: +101~+247) showed relatively higher methylation in ES and EG cells than those in mGS cells. Although no pattern of DNA methylation distinguishing stem cell lines appears to exist, several CpGs (CpGs nos. 29–31) in region 2 tended to be hypomethylated in mGS cells. Taken together with the result that mGS cells showed weak gene expression, we chose mGS cells as the most suitable for a promoter assay.

To test the effect of DNA methylation on Ant4 transcription, reporter constructs were created consisting of a luciferase gene fused to the 5'-upstream region (–935 to –5 bp) of the Ant4 gene with and without in vitro methylation (pGL3-Ant4-met and pGL3-Ant4-unmet). The reporter construct with the unmethylated 5'-upstream region had promoter activity, with a 16-fold increase over the empty vector construct, while methylation of the same region exhibited only a threefold increase in activity compared to the empty vector (Fig. 3). We conclude that the transcription of the Ant4 gene is regulated by DNA methylation at CpGs in its promoter region.

View larger version (12K): [in this window] [in a new window]   Figure 3  Promoter activity of Ant4 gene is repressed by DNA methylation. The 5' upstream regions of Ant4 gene (–5 to –935 bp) were treated with/without methylase in vitro and ligated to an empty vector (pGL3-Ant4-met and pGL3-Ant4-unmet). The luciferase activities were measured after transfection into mGS cells. Data are represented as mean ± S.E. of three independent experiments, each of which was performed in duplicate. Note that the promoter activity of the Ant4 upstream region was suppressed by in vitro methylation.

	Discussion

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Focusing on the CpG island of the Ant4 gene, we find the locus to be distinctive in terms of DNA methylation, which involves all of the CpG sites throughout the promoter CpG island in somatic tissues but completely spares the CpG island in germ cells. Thus, the entire Ant4 CpG island is the T-DMR, in contrast with other somatic tissue-specific T-DMRs at the CpG islands of promoters, where the tissue-specific methylation involves only part of the promoter CpG islands (Imamura et al. 2001, 2004; Newell-Price et al. 2001; Pao et al. 2001). Given the fact that DNA methylation levels in Ant4 T-DMR were nearly 100% in the somatic tissues, precluding allelic differences, and that Ant4 gene is located at chromosome 3B, a region not known to harbor imprinted genes, Ant4 is unlikely to be imprinted and is obviously not subject to X chromosome inactivation. Since the CpG island is located at the transcription start site and at the first exon of the Ant4 gene, it is plausible that the expression of Ant4 is controlled by DNA methylation.

Similar to Ant4, the expression of Tact1/Actl7b is restricted to germ cells and is regulated by DNA methylation (Hisano et al. 2003). Since Tact1/Actl7b is an intronless gene, it is likely to have been generated by a retroposition event during gene evolution. By contrast, the Ant4 gene has six exons and five introns and is not a retroposed locus. This indicates that generation of the class of T-DMRs that occupy the whole CpG island is not dependent on an evolutionary history of retrotransposition. To date, several germ cell-specific genes, in addition to Ant4 and Tact1/Actl7b, have been reported to be under the control of DNA methylation; testis-specific H2B histone, TH2B (Choi & Chae 1991); TFIIA/β-like factor, ALF (Xie et al. 2002); and testis-specific phosphoglycerate kinase, Pgk2 (Geyer et al. 2004); although these genes do not have CpG islands. In germ cells, these genes are expressed after loss of DNA methylation (Xie et al. 2002; Geyer et al. 2004), suggesting that DNA demethylation is important for the regulation of these genes and is involved in germ cell differentiation. In other words, the loss of DNA methylation in germ cell differentiation may be involved in not only the regulation of imprinted genes but also that of non-imprinted genes.

A gene promoter assay using mGS cells indicated that in vitro DNA methylation of the construct suppressed the Ant4 promoter activity, supporting the conclusion that DNA methylation observed in somatic cells represses Ant4 gene expression. The Ant4 T-DMR was unmethylated in the spermatogonia, spermatocytes, spermatids and mature sperm. In the present study, in situ hybridization study showed that Ant4 mRNA was detectable in a minor population of spermatogonia and the expression became strongest in spermatocytes. Given the nearly complete demethylation of the Ant4 T-DMR in spermatogonia, where Ant4 mRNA was barely detectable, we presume that DNA demethylation at the Ant4 T-DMR precedes the onset of Ant4 expression during spermatogenesis.

Male gonads of 12.5 dpc embryos showed a weak expression of Ant4, but the expression levels in the later developmental stages were greater, suggesting that Ant4 expression starts in these later stages of male germ cell development. Interestingly, the expression of Ant4 was also observed in female gonads. Therefore, demethylation of the Ant4 T-DMR appears to occur at the early stages of germ cell differentiation in the fetal period. From this context, it was unexpected that mGS cells as well as EG cells showed hypermethylation at the Ant4 T-DMR, because mGS cells were established from neonatal testicular cells (Kanatsu-Shinohara et al. 2004). Further studies will be needed to identify the cell types expressing Ant4 in fetal gonads and to identify when demethylation is initiated.

Previously, Rodic and colleagues investigated the DNA methylation status of the Ant4 gene locus in ES cells (129/SvJ), embryoid bodies developed from the ES cells, testis and several somatic tissues of BALB/c mice and found that the gene locus was unmethylated specifically in the stem cells and testis (Rodic et al. 2005). Interestingly, they found an increase in DNA methylation levels during differentiation from ES cells into embryoid bodies. In the present study, however, ES cells (C57BL/6Ncrj) showed DNA hypermethylation even at the stem cell stage and undetectable Ant4 gene expression. It is possible that these discordant results are due to the different strains of mice used, or that the culture history of the ES cells used in each case introduced epigenetic variability. Both in the study by Rodic et al. (2005) and in the present study, the Ant4 gene was found to be unmethylated in germ cells, in which it is expressed. The epigenetic characteristics of the Ant4 locus in germ cells are therefore independent of the genetic backgrounds used in these studies.

In conclusion, we identified and characterized a number of loci undergoing germ cell-specific hypomethylation. We identified the Ant4 gene as a locus for which gene expression is under the control of DNA methylation, leading to silencing in somatic cells and possibly contributing to germ cell differentiation during development. The sperm-specific hypomethylated loci define a new class of CpG islands that are hypermethylated at all CpGs in non-expressed somatic tissues. We propose that there are two classes of T-DMRs: T-DMRs involving only part of promoter CpG islands and those that occupy the whole CpG island.

	Experimental procedures

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Cells and tissues

Embryonic stem (ES) and embryonic germ (EG) cells derived from mice of the same genetic background (C57BL/6NCrj) (Kawase et al. 1994; Labosky et al. 1994) were maintained in DMEM with 15% fetal calf serum (FCS) on a feeder layer as described previously (Shiota et al. 2002). Multi-potent germ-line stem (mGS) cells derived from DBA/2 mouse were cultured as described in a previous study (Kanatsu-Shinohara et al. 2004).

Adult male mice of the C57BL/6NCrj strain (Charles River Japan, Yokohama, Japan) were dissected for tissue preparation under the guidelines for the care and use of laboratory animals (Graduate School of Agriculture and Life Sciences, The University of Tokyo). Sperm sample was collected from vas deferens. All samples were frozen in liquid nitrogen and stored at –80 °C until use.

Micro-dissection from paraffin-embedded testis sections

Paraffin-embedded testes that were prepared from 8-week-old C57BL/6NCrj male mice were sliced into 6-µm sections and stained with Mayer's hematoxylin. Isolation of each spermatogenic cell from the sections was performed using the P.A.L.M. MicroBeam system (P.A.L.M. Microlaser Technologies AG, Bernried, Germany) according to the manufacturer's instructions. Spermatogenic cells were collected (spermatocytes 300 cells, spermatogonia 500 cells and spermatids 1000 cells) and stored at –80 °C until extraction of the genomic DNA.

Genomic DNA and RNA preparation

Genomic DNA was extracted from frozen samples as described previously (Ohgane et al. 1998). Total RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Following the treatment of extracted total RNA with DNase I (TAKARA, Kyoto, Japan), complementary DNA (cDNA) was generated using Superscript III reverse transcriptase (Invitrogen) with random hexamers.

Identification of the sperm-specific unmethylated NotI site by Vi-RLGS and genomic PCR

The sperm-specific unmethylated NotI sites in the mouse genome were explored by comparing the genomic profiles of cells and tissues by the RLGS method (Shiota et al. 2002). The genomic locus was identified by matching the profiles of RLGS and Vi-RLGS as previously described (Matsuyama et al. 2003; Hattori et al. 2004a). Using the sequence information from Vi-RLGS, the genomic location of the NotI site was obtained using BLAST <http://www.ncbi.nlm.nih.gov/genome/seq/MmBlast.html> and the UCSC genome browser <http://genome.ucsc.edu/>. The G+C content and CpG frequency of the genomic DNA was analyzed computationally (CpG-View version 1.5, provided by the National Institute of Infectious Diseases <http://www.nih.go.jp/yoken/genebank/>). The methylation status of the NotI site in the mouse tissues was confirmed by the combination of restriction digestion of genomic DNA and quantitative real-time PCR as described previously (Hattori et al. 2004a). Briefly, genomic DNA extracted from the sperm, kidney and brain was digested with EcoRI and the aliquots were further digested with NotI. DNA methylation at the NotI site is estimated by the amount of PCR fragments amplified from NotI-digested genomic DNA compared with that from NotI-undigested DNA. The amount of initial genomic DNA in the reaction is corrected by the amount of the internal control. Primer sets for quantitative real-time PCR used in this study are listed in Supplementary Table S1. For all samples, independent PCRs in duplicate were performed at least 3 times.

Bisulfite genomic sequencing

Bisulfite genomic sequencing was performed as previously described (Hattori et al. 2004b). Briefly, genomic DNA was digested with EcoRI and denatured with NaOH for 15 min at 37 °C. After the incubation, sodium metabisulfite, pH 5.0, and hydroquinone were added to final concentrations of 2.0 M and 0.5 mM, respectively, and the mixture was further incubated in the dark for 16 h at 55 °C. The modified DNA was purified using the Wizard DNA Clean-Up system (Promega, Madison, WI), and the bisulfite reaction was terminated with NaOH at a final concentration of 0.3 M for 15 min at 37 °C. The solution was then neutralized by adding NH₄OAc, pH 7.0, to a final concentration of 3.0 M. The ethanol-precipitated DNA was resuspended in water. The primer sets that we used in this study were described in the Supplementary Table S2. The amplified PCR fragments were cloned into pGEM-T easy vector (Promega) and analyzed by sequencing.

RT-PCR

To detect the expression of Ant4 and β-actin, PCR was performed using rTaq polymerase (TOYOBO, Osaka, Japan). Primer sequences are as follows: 5'-TCCAACATGTCGAACGAATCC-3' and 5'-GAACAGAGACACCAAACCCTT-3' for Ant4 and 5'-GACAACGTCTCCTGCATGTGCAAAG-3' and 5'-TTCACGGTTGGCCTTAGGGTTCAG-3' for β-actin. PCR reactions were performed at 94 °C, 4 min; 30 cycles of 94 °C, 30 s; 58 °C, 30 s; 72 °C, 1 min; final extension 72 °C, 10 min.

Gene promoter assays

The 5'-flanking region of the Ant4 was isolated by genomic PCR with specific primers: 5'-GCTAGCACCCAGCTATGAACCCTGTG-3' and 5'-AAGCTTGAACTGGAAAACCGCTTCAG-3' and ligated into pGEM-T easy vector for cloning. The resulting construct containing 5'-flanking region of the Ant4 was re-digested with NheI and HindIII and subcloned into pGL3-Basic vector (Promega) digested with the same enzymes. Amplification of the construct was carried out using dam–, dcm– bacterial strain, SCS110 (Stratagene, CA), to avoid methylation of the construct. The construct was dissected with BglII and HindIII whose restriction sites are located at the inside of the amplified 5'-flanking region by the genomic PCR and one primer sequence, respectively. The resultant fragment (–935 to –5) was isolated and methylated in vitro with SssI methylase (New England BioLabs, Ipswich, MA) as described previously (Tomikawa et al. 2006). The methylated fragment was re-ligated with the empty pGL3-Basic vector that had been digested with BglII and HindIII in advance. Unmethylated control vector was constructed by the same method without the in vitro methylation step. These methylated and unmethylated control vectors, designated pGL3-Ant4-met and pGL3-Ant4-unmet, respectively, were linearized by BamHI digestion and purified by size selection with agarose gel electrophoresis to exclude non-ligated pGL3-Basic vector and those ligated with concatemer fragments. These purified constructs were not amplified in bacteria after the ligation step in order to maintain the regional methylation status. Completion of in vitro methylation of Ant4 5'-flanking region in pGL3-Ant4-met was confirmed by the bisulfite sequencing method as described above.

The mGS cells plated at 5 x 10⁴ cells/well in a 24-well dish were incubated for 24 h, then transfected transiently with 373.8 ng of the luciferase reporter construct using Lipofectamine 2000 Reagent (Invitrogen). To normalize the luciferase activity, 26.2 ng of a control plasmid with Renilla luciferase sequence (pRL-TK; Promega) were co-transfected to each well. After culturing for 48 h, the activities of both luciferases were determined by means of a Dual Luciferase Reporter System (Promega) according to the manufacturer's instructions. Assays were performed 3 times each in duplicate.

In situ hybridization

In situ hybridization was performed as previously described (Hoshino et al. 1999) under contract by Genostaff (Tokyo, Japan). In brief, a 261-bp cDNA fragment corresponding to the nucleotide positions 672–932 of mouse 1700034J06Rik cDNA (GENBANK accession number NM_178386) was amplified by RT-PCR. The amplified product was cloned into pGEM-T easy vector (Promega), and was used for generation of sense or antisense digoxigenin (DIG)-labeled RNA probes by using DIG RNA labeling Mix (Roche Molecular Biochemicals, Tokyo, Japan). Paraffin-embedded testis sections (6 µm) of 8-week-old C57BL/6NCrj male mice were subjected to hybridization with the antisense or sense probes at the concentration of 100 ng/mL in the Probe Diluent (Genostaff) at 60 °C overnight. Hybridized probes were detected by alkaline phosphatase-conjugated anti-DIG IgG and visualized with NBT/BCIP solution (Roche Molecular Biochemicals). Then the sections were counterstained with Kernechtrot stain solution (Muto Chemical, Tokyo, Japan), dehydrated and mounted with Malinol (Muto Chemical).

	Acknowledgements

 
This work was supported by the Program for Promotion of Basis Research Activities for Innovative Biosciences and the Grant-in-aid for Scientific Research, Ministry of Education, Culture, Sports, Science and Technology of Japan (15080202) to K. S.

	Footnotes

 
Communicated by: Yo-ichi Nabeshima

^aThese authors contributed equally to this work.

^bPresent address: Department of Medicine (Hematology), Albert Einstein College of Medicine, Bronx, New York 10461, USA.

^* Correspondence: E-mail: ashiota{at}mail.ecc.u-tokyo.ac.jp

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Accepted: 9 August 2007

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