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Genomics. Author manuscript; available in PMC Mar 1, 2008.
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PMCID: PMC1847344
NIHMSID: NIHMS18545

Analysis of tissue-specific differentially methylated regions (TDMs) in humans

Abstract

Alterations in DNA methylation have been implicated in mammalian development. Hence the identification of the tissue-specific differentially methylated regions (TDMs) is indispensable for understanding its role. Using restriction landmark genomic scanning (RLGS) of 6 mouse tissues, 150 putative TDMs were identified and 14 further analyzed. The DNA sequences of the 14 mouse TDMs are analyzed in this study. Six of the human homologous regions show TDMs to both mouse and human, and genes in five of these regions have conserved tissue-specific expression; preferential expression in testis. A TDM, DDX4, are further analyzed in 9 testis tissues. An increase in methylation of the promoter region is significantly associated with a marked reduction of the gene expression and defects in spermatogenesis, suggesting that hypomethylation of the DDX4 promoter region regulates DDX4 gene expression in spermatogenic cells. Our results indicate that some genomic regions with tissue-specific methylation and expression are conserved between mouse and human, and suggest that DNA methylation may have an important role in regulating differentiation and tissue/cell-specific gene expression of some genes.

Keywords: DNA methylation, tissues, CpG Islands, bisulfites, DDX4, Gene Expression, testis, spermatocytes, Gene Expression Regulation, Human Genome

INTRODUCTION

DNA methylation of cytosine at position C5 in CpG dinucleotides in the mammalian genome is an inheritable modification of DNA that does not alter the nucleotide sequence [1, 2]. It is important to understand the global genome-wide DNA methylation patterns and to understand the role of methylation in diverse genomic processes such as chromosomal stability, gene regulation, and parental imprinting. Many of these processes and their relationship to genome wide methylation patterns are still unknown. This is partly due to a lack of high-throughput methods for scanning quantitative changes in DNA methylation status at each CpG dinucleotide in the genome. Generally, cytosine residues in the CpGs are methylated in the genome, especially within non-coding DNA, introns, repetitive sequences, and potentially active transposable elements resulting in long-term silencing [3, 4]. Most CpG clusters, called CpG islands, frequently found in the proximal promoter regions of many genes are unmethylated during normal cell development, with the exception of imprinted genes, genes on the inactive X chromosome, and tissue-specific differentially methylated genes [1, 57]. Studies indicate that aberrant DNA methylation patterns of gene promoters impair normal transcription, causing abnormal development associated with various diseases such as cancer [811].

A comparative genomic approach is valuable for studying the role of transcriptional regulation in diverse biological processes. Several studies suggest epigenetic modifications including DNA methylation are conserved between mouse and human. Histone modifications between human and mouse, are strongly conserved even though many histone H3 lysine 4 methylated sites, do not show sequence conservation [12]. The H3 lysine 4 methylation is associated chromatin remodeling through the plant homeodomain (PDH) finger proteins [13, 14]. Evolutionary conservation of tissue specific methylation in human tissue was recently identified among brain, keratinocyte and peripheral blood lymphocyte by a genome wide epigenome analysis using BAC microarrays. These studies indicated that the tissue specific methylation pattern of the SHANK3 gene was conserved in human, mice, and rats while the Arhgef17 was not [15]. Although several human genome wide methylation studies have been published [1619], comparative approach were also successfully applied for identification of human epigenetic alterations using mouse models for human cancers and mammalian development [2022]. One of the major problems of genome wide approach in the human genome is a high frequency of sequence polymorphisms in the population [23, 24]. However, a comparative approach that first identifies TDMs in inbred mice, will help to identify evolutionary conserved human TDMs and critical genes that are methylated during development and therefore avoid the problem of SNPs in humans.

Recently, 150 tissue-specific differentially methylated regions (TDMs) were predicted among tissues of C57BL/6J mice using restriction landmark genomic scanning (RLGS) in conjunction with virtual RLGS [25]. These results suggest that at least 5% of 15,500 CpG islands in the mouse are differentially methylated. In that study 14 mouse TDMs were further analyzed by methylation-specific PCR and by bisulfite genomic sequencing to confirm that the regions identified by RLGS are differentially methylated in a tissue-specific manner. These studies also indicated the TDMs within CpG island 5′ promoter regions correlate to tissue- specific gene expression patterns [25].

Here we describe the analysis of the human genomic regions homologous to the 14 confirmed TDMs found in the C57BL/6J mouse to investigate whether the tissue-specific methylation is conserved in human. The expression of genes located nearby or overlapping with the homologous regions of the mouse TDMs were analyzed to assess whether the expression is associated with methylation patterns of the human homologous region.

RESULTS

Searching for human conserved regions of mouse TDMs

We searched for the human orthologs of the 14 TDMs identified in mice [25] using the Human Mar. 2006 (hg18) assembly, UCSC Genome Bioinformatics browser. The DNA sequences of 13 of the 14 confirmed mouse TDMs are conserved in the human genome and are located near homologous human genes. One TDM on mouse chromosome 17 (Pst46) is not conserved (Table 1), but the mouse TDM sequence overlaps an LTR repeat (96.65% similarity of ERV class II LTR sequence by RepeatMasker version open-3.1.5) (Smit, AFA, Hubley, R & Green, P. RepeatMasker Open-3.0. 1996–2004 http://www.repeatmasker.org). One of 13 conserved loci is duplicated in the human genome, one within the ZNF324 gene and another within the FLJ45850 gene (94% identities).

Table 1
Conserved regions of TDMs in human

Interestingly, the NotI sites, which were used for RLGS as a restriction landmark in the mouse, are conserved in two human homologous regions, one on chromosome 6 (hPvu6) and the other on chromosome 5 (hPvu66), located within the USP49 and ADRA1B genes, respectively (Table 1). To examine the methylation status of the NotI site in human, southern blot analysis was carried out for the region hPvu6, using one of autopsy cases (Figure 1A). For this experiment, EcoRI and NotI were employed as methylation insensitive enzyme and methylation sensitive enzyme, respectively. A southern blot of a double digestion with EcoRI and NotI produced a single 8-kb band signal observed in colon, heart, kidney, and muscle tissues, indicating that the NotI site is completely methylated in these tissues (Figure 1B). On the other hand, two bands signals, 8 kb and 5 kb in length, were detected in testis. The methylation state of the testis sample was estimated by the signal intensity of the Southern Blot bands. According to the (P-B)/mm2 values; (P-B)/mm2 value of 5kb band is 2.84 and that of 8kb is 2.12 measured by FLA5100, 57% of the NotI site was unmethylated (2.82/(2.84+2.12) × 100 = 57). This reflects either cell or allelic heterogeneity in tissue materials. Bisulfite sequence of the region was performed using the same sample to confirm the methylation level of the NotI site (Figure 1C). There are 9 CpGs in the 182-bp fragments and 2 of them are located within the NotI site. The 2 CpGs are methylated in kidney, heart, and colon. In contrast, they are unmethylated in 7 out of 12 clones in testis, indicating 58% hypomethylation in testis. This confirms that the region containing the NotI site is a TDM in humans. According to UCSC Genome Bioinformatics, there is a predicted cpgi 46 in the vicinity of hPvu6 (Figure 1A). We therefore analyzed the methylation pattern of the cpgi 46 by bisulfite sequence to see whether the CpG island is also differentially methylated. Both autopsy cases examined indicate that this region is largely hypomethylated in testis (Figure 1D). Thus, the sequence homologous to the mouse Pvu6 is also a TDM in human.

Figure 1Figure 1
Analysis of a human homologous region, hPvu6, and the corresponding human gene, USP49, which is associated with the conserved region. A. Chromosomal location of the hPvu6. An asterisk represents the position of hPvu6, which is corresponding to mouse ...

Analysis of human TDMs by bisulfite sequencing

Since we obtained a good correlation between the southern blot results and bisulfite sequencing of hPvu6, we carried out bisulfite sequencing on the remaining 12 conserved regions to confirm the methylation patterns (see supplement figures). For this experiment, we used 2 autopsy cases, 102 or 114 depending on the availability of tissue. The results are summarized in Table 2. For example, the bisulfite sequencing of hPst3 using case 102 is shown in Figure 2. Five of the human homologs to mouse TDMs (hPvu6, hPst3, hPvu4-1, hPvu4-2 and hPst32) are primarily hypomethylated in human testis, the same pattern as observed in the mouse (Figures 1, ,2,2, S2 and S8). In addition, testis-specific hypomethylation was observed in cpgi 93 which is located in the vicinity of hPst33. The human homologous region of Pst33, itself is out side of CpG island (cpgi 93) and methylated in all 6 tissues examined (supplement figure S9). In contrast to their mouse orthologs, five of the human loci do not exhibit tissue specific differences in methylation (hPvu2, hPvu8, hPvu29, hPvu42 and hPst6) (supplement figure S1, S3, S4, S5, and S7). Another autopsy case, 114, in which liver is available, was employed for the analysis of hPvu66, hPst61 and hPst21 since the corresponding loci in mice revealed either liver-specific methylation or hypomethylation [25]. Although there are some apparent differences in tissue-specific methylation, none of these loci had liver-specific differences in methylation as was observed in the mouse [25] (supplement figure S6, S10, and S11).

Figure 2
Bisulfite sequencing of hPst3. A. Chromosomal location of hPst3 and DDX4. This map is based on UCSC Genome Browser on Human Mar. 2006 Assembly (http://genome.ucsc.edu). The scale is represented below the diagram in base pair. DDX4 is shown as solid bars ...
Table 2
Hypomethylationmethylation level of CpGs determined by bisulfite sequencing

Expression analysis of genes

To determine whether the gene expression pattern is associated with the methylation patterns, we prepared RNA from the same tissue samples used in the bisulfite sequencing and analyzed the mRNA levels by quantitative RT-PCR (Figure 3). The genes, DDX4, DACT1, ZNF324, and FLJ45850, associated with TDMs (hPst3, hPst32, hPvu4-1 and hPvu4-2) that were unmethylated only in testis showed a notably high level of expression in testis and no or a low level of expression in other tissues (Figure 3A). There is a relative testis specific expression of USP49 and some expression of LMTK3 in testis, though the expression levels are low (Figure 3A). These results indicate that some of the hTDMs that were unmethylated only in testis had testis specific expression. Some genes expressed in tissue-specific fashion even though we did not detect tissue-specific methylation. HSPA1L is known for exclusive expression in testis as shown in Figure 3A. SLC16A5 and GATA2 show a high expression in kidney (Figure 3A and 3B). LMTK3 and NRXN2 express relatively high in brain compared to other tissues (Figure 3B). HSMPP8, bA16L21.2.1, and UBE2E express highly in testis of the samples examined (Figure 3A). However in these cases there is no clear relationship between the expression of these genes and methylation patterns of the conserved regions (Figures 3A, S3, S4 and S5).

Figure 3Figure 3
Gene expression. Quantitative real-time PCR was performed to measure the mRNA expression of genes. Sample 102 and 114 were used for Figures 3A and 3B, respectively. Y-axis represents normalized quantity determined by standard curve of each gene. The expression ...

The association of methylation patterns of DDX4 with pathological findings in human testis

We have shown that hypomethylation of the DDX4 CpG island promoter region (hPst3) is associated with testis specific expression (Figure 2, ,3).3). In order to characterize the effect of methylation changes on DDX4 expression in humans, a total of 9 testis samples (4 samples from individuals in which histology indicated no or low spermatocyte count, 2 from moderate and 3 from relatively high spermatocyte count) were examined. Figure 4A and B show three representative histological and corresponding methylation patterns. We could not measure the DDX4 expression level of one of cases, 106 because of RNA degradation. The DDX4 expression has a significant linear correlation with the number of spermatocytes of 8 testes (p-value = 0.0002) and inverse linear correlation with cpgi 30 methylation (p-value = 0.0248) (Figure 4C, D). There was an inverse correlation between the promoter methylation and the gene expression. An increase in methylation of the TDM region is accompanied by suppression of the gene expression and spermatogenesis. Thus, DDX4 expression is clearly important to the production of spermatocytes.

Figure 4Figure 4
Association of methylation status of DDX4 promoter region with the gene expression and pathological findings in testis samples. A: HE sections of testis (x 400) from three autopsy cases 114, 106, and 130. Case 114 represents preserved spermatogenesis, ...

DISCUSSION

In this report, we show that the nucleotide sequences of most confirmed mouse TDMs (13 out of 14) are conserved in human. However the tissue-specific methylation of these regions is conserved in only a subset of TDMs (hPst3, hPst32, hPvu4, and hPvu6). These regions are hypomethylated in testis and methylated in all other tissues tested in both mouse and human. Importantly, the genes associated with these regions (DDX4, DACT1, ZNF324/FLJ45850, and USP49) exhibit testis specific expression. However, the tissue specific methylation of several other mouse TDMs does not appear to be conserved in human. The methylation patterns of 3 mouse TDMs that are methylated (Pst21) or unmethylated (Pvu66, Pst61) in liver are not conserved and not correlated the gene expression in human. Currently, we do not know whether this reflects differences between tissues and/or whether there are additional functional consequences of tissue-specific methylation. Some of the genes associated with TDMs exhibited testis-specific expression in humans even though the orthologs were not hypomethylated exclusively in testis (HSPA1L, bA16L21.2.1, HSMPP8, UBE2W). The precise boundaries of the TDMs have not been clearly defined in either mouse or human. Thus, the region identified in mouse may not be the critical region for determining testis specific expression. For examples, the hPst33 within the LMTK3 gene was methylated in all tissues examined but the LMTK3 cpgi93 was TDM in humans (Supplementary Figure S9). The number of methylated clones of hPvu4-1 and hPvu4-2 (Supplementary Figure S2) is also much higher than those in the downstream regions examined for CpG islands within the ZNF324 and FLJ45850 genes in testis. It also appears likely that promoter methylation is not the only determinant for tissue specific expression, as seen in exonic methylation of the PAX6 and hypomethylation of Tact1/Actl7b genes, which associate with the specific gene expression in cancer and testis, respectively [26, 27]. Moreover, hypomethylation of the promoter CpG island regions may be necessary but not sufficient for expression.

We identified five TDMs in human that correlated with gene expression patterns, providing further evidences that human gene expression could be regulated by DNA methylation-mediated gene silencing. Analysis of the other 136 mouse TDMs predicted by RLGS [25] could allow us to identify additional unknown tissue-specific expressing genes regulated by DNA methylation in humans. Human TDM regions are located within exons of the 4 genes, DACT1, FLJ45850, ZNF324, and USP49 and their methylation repressed gene expressions. Although we have not analyzed promoter methylation of those genes, intragenic DNA methylation is known to be capable of altering chromatin structure and elongation efficiency in mammalian cells depleting RNA polymerase II exclusively in the methylated region [28]. By searching the sequence database (Repeat Masker program), there are some Alu elements found within 2-kb downstream or upstream of some TDMs. It has been suggested that methylation of Alu elements could suppress transcription and contribute to differential expressions of genes [2931]. A mouse TDM, Pst46 that was not conserved in human was located within the endogenous retrovirus LTR sequence in the mouse. A study of transgenic mice demonstrated that epigenetic modification of transgenes under the control of the mouse mammary tumor virus LTR conferred tissue-dependent influence on transcription of the transgenes [32]. Endogenous LTR sequences may be differentially methylated as seen in mouse Pst46, the LTR transgene and human repeat sequences. Thus repeat sequences flanking each TDM might be involved with gene silencing, though its mechanism remains to be elucidated.

HSPA1L encodes a heat shock 70 kDa protein and is located in the major histocompatibility complex (MHC) class III region [33]. There is a duplicated gene, HSPA1A, located 4-kb downstream to HSPA1L. It is well known that HSPA1L RNA expression is restricted to testis (Fig. 3)[34, 35]. The RNA expression of HSPA1A is significantly different from that of HSPA1L, showing a variable level of expression in several tissues [36]. The bisulfite sequencing of the conserved region, hPst6, which is within in coding sequence of HSPA1L, showed hypermethylation in all tissues we examined (supplement Figure S7). We also analyzed a CpG island that is localized at the 5′ region of HSPA1L and also overlaps with the duplicated gene, HSPA1A. The CpG island is hypomethylated (supplement Figure S7), indicating that neither the conserved region nor the CpG island is associated with the RNA expression of HSPA1L. In contrast, other duplicated genes, ZNF324 and FLJ45850 for hPvu4 have CpG islands in the coding regions, respectively (supplement Figure S2). Both CpG islands for hPvu4 are hypomethylated in testis and hypermethylated in other tissues and the expression of both genes are associated with the hypomethylation pattern (supplement Figure S2). It is of interest that the both genome duplication events in human evolution, are not in the Chimpanzee (Pan troglodytes) Genome, and that transcriptional regulation associated with DNA methylation was conserved in one at hPvu4, but not the other at hPst6 and cpgi185.

Another 5 genes (HSPA1L, SLC16A5, UBE2W, bA16L21.2.1, and HSMPP8) have no obvious TDMs within the conserved regions although the genes are expressed in tissue-specific manner. Although additional tissues from young individuals should be analyzed, expression of these genes may be regulated by some transcriptional factors or cis-elements, but not by DNA methylation-medicated gene silencing at least in the individuals examined. Alternatively, we may not have identified the critical region required for tissue specific methylation and expression. Various patterns of tissue-specific differential methylation are also observed in 3 genes, ADRA1B, GATA2, and NRXN2, showing small differences in methylation (Supplement Figures S6, S10 and S11). The TDM patterns of these genes, between human and mouse have some similarity, but are not completely the same. This might be attributed to the lack of identification and bisulfite sequence analysis of the critical target region of the TDMs, the mixture of various cell types in samples, the age effect of the cell population in humans or simply the difference of species. Analysis of characterized cells isolated from the tissues of various ages would be informative for understanding a mechanism of DNA methylation-mediated gene silencing of these genes.

DDX4 (also called VASA) is an RNA helicase, an essential enzyme involved in RNA metabolism and well known as a specific marker for germ cells [37]. The knock out of Ddx4 in mice results in infertility in males although primordial germ cells do form, suggesting an essential role in spermatogenesis but not in germ cell development [38]. Recent studies show that Ddx4/MVH interacts with Dicer and components of micro RNA-processing body in chromatoid bodies [39]. Ddx4 also interacts with a protein involved in microtubule nucleation, suggesting a functional relationship between translational control of Ddx4 and microtubule nucleation that occurs during meiosis [40]. Castrillon et al. [41] has reported that DDX4 protein expresses in cells within the seminiferous tubules. The expression is low in spermatogonia and high in spermatocytes. As seen in mice, the CpG island promoter region of the DDX4 gene was hypomethylated in human testis. We also demonstrated the association of DDX4 expression and spermatogenesis in addition to the association of the promoter methylation and DDX4 expression, suggesting promoter hypomethylation of DDX4 and possibly other testis specific expressed genes may play an important role in initiating spermatogenesis process in testis. However, at this time we can not determine whether hypomethylation is causally linked to spermatogenesis or is a consequence of spermatogenesis. It is also important that a DNA methylation at TDMs is associated with the gene expression in a specific cell population during spermatogenesis process, confirming previous hypothesis of that TDMs are cell-type-specific events [20, 25, 42]. It will, therefore, be important to determine the TDMs in specific cell types that make up a particular tissue.

In this study, we only analyzed 14 TDMs in humans. Additional analysis of the sequence and genomic features of additional TDM regions should help to elucidate the critical roles of DNA methylation in evolution, development and disease.

MATERIALS AND METHODS

Human samples

Normal tissues were obtained from organ donation of autopsy cases after receiving the informed, written consent of bereaved families or relatives at Pathology Division, School of Medicine, Nihon University, Tokyo, Japan. The samples were immediately frozen and stored at −80 C. At the same time, a part of each sample was fixed, paraffin slides prepared and stained by routine hematoxylin and eosin (H&E) staining to observe pathological features. Nine male autopsy cases age between 58 and 81 (average 68 years old) are used in this study. All experiments were conducted in accordance with the guidelines approved by the Ethics Committee of Nihon University, School of Medicine.

Bisulfite sequencing

The METHPRIMER program (http://www.urogene.org/methprimer/index1.html) [43] was used to design bisulfite PCR primers (Table 3). All primers were purchased from Sigma Genosys. Genomic DNA from each sample was prepared using QickGene-800 (FUJIFILM) and was subjected to bisulfite modification by using the EZ DNA Methylation kit (Zymo Research). The bisulfite-treated genomic DNA was amplified by Taq DNA polymerase (Roche) (2 min at 94C followed by 40 cycles of 15 s at 94C, 30 s at 50–60C and 30 s at 72C with a 4 min final extension at 72C) or Ampli Taq Gold (Applied Biosystems) (10 min at 95C followed by 40 cycles of 45 s at 95C and 45 s at 50–60C with a 10 min final extension at 72C) in a PTC-100 thermal cycler (MJ Research). The PCR products obtained were cloned into pGEM-easy T vector (Promega) or pCR2.1 TOPO vector (Invitrogen). Random white colonies were screened for the correct-size insert in the plasmid by colony PCR. Twelve positive clones were picked from each single bisulfite treated DNA and the clones were sequenced by Dragon Genomics Ctr, Takarabio Inc. (Mie, Japan).

Table 3
Primers used for Bisulfite sequencing and quantitative RT-PCR

Southern Blot analysis

The probe for hPvu6 (the USP49 gene) was prepared by PCR using the primer sets as follows: USP49.probe-F: 5′-CAATGTTCCTTAGTCACGT-3′/USP49.probe-R: 5′-TGGTGGCACACTCTAAGCA-3′ (product size; 553 bp). Genomic DNA was double-digested with the combination of methylation sensitive enzyme, NotI, and methylation insensitive enzyme EcoRI. The digested DNA was separated by electrophoresis in 0.8% agarose gels and transferred to Hybond-N+ membranes (GE Healthcare). The probe used for hybridization was labeled with [α-32P]dCTP using MegaprimeTM DNA Labeling Systems (GE Healthcare). The signals were detected and measured using FLA-5100 (FUJIFILM). The IP (Image Plate) method allowed to generate PSL units as an intensity of signals using MultiGauge software (FUJIFILM). PSL units were background subtracted and then divided by square millimeters. This value is designated (P-B)/mm2 and was used for the analysis of the band signal intensity.

Real-time quantitative RT-PCR

mRNA expressions were analyzed by real-time RT-PCR. Total RNAs were isolated from human tissues using TRIzol Reagent (Invitrogen) and then treated with TURBO DNA-free (Ambion) to remove the contaminating genomic DNA. Single-stranded cDNA was synthesized using High-Capacity cDNA Archive Kit (Applied Biosystems). The generated cDNA was amplified on the ABI 7300 System (Applied Biosystems) using Power SYBR Green PCR Master Mix (Applied Biosystems) or SYBR Premix Ex Taq (TaKaRa Bio Inc.). The resulting PCR cycle time (Ct) values were collected by using the software provided for the ABI 7300 systems and the data were analyzed in Microsoft EXCEL. The expression level of the GAPDH gene was used to normalize for differences in input cDNA. Primers were chosen from exons separated by introns except ZNF324 and FLJ45850, and the PCR quality and specificity were verified by dissociation curve analysis and sequencing the amplified products. The primer sets used are shown in Table 3. Experiments were done in triplicate.

Sequence analysis

The databases used for sequence analysis, BLAT can be found on UCSC Genome Bioinformatics website (http://genome.brc.mcw.edu/). The mouse TDM sequence is the sequence of a NotI-PvuII or NotI-PstI Restriction Landmark fragment (6). The UCSC Genome Browser, Human Mar. 2006 assembly was used for homology searches. The Primer 3 program (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3-www.cgi) and Primer Express (Applied Biosystems) were used to design primers for RT-PCR.

Histological evaluation

A part of normal tissue sample was subjected to paraffin slides and stained by routine hematoxylin and eosin (H&E) staining to observe pathological features. The number of spermatocytes was counted from 30 seminiferous tubules of each testis by the pathologist. Average number of spermatocytes per a seminiferous tube is used to evaluate spermatogenesis.

Supplementary Material

Supplementary Figure S1-11

Bisulfite genomic sequences of 11 human genomic regions homologous to mouse TDMs. A. The location of each homologous region on human genome. The maps are drawn based on UCSC Genome Browser on Human Mar. 2006 Assembly (http://genome.ucsc.edu). The scale is indicated below the diagram in base pair. The genes located nearby or overlapping with the homologous regions are shown as solid bars with arrowhead indicating the orientation of genes. CpG islands (cpgi) are shown with the number of CpGs within the CpG island sequence. The positions correspoinding to mouse NotI sites are indicated as asterisks with the locus names. The human sequences corresponding to mouse NotI site are shown in uppercase characters. B. Bisulfite genomic sequences of the homologous regions including the sequences corresponding to mouse NotI sites marked by asterisks above. Arrowheads point the CpG corresponding to the CpG underlined above. Black and white circles represent methylated and unmethylated CpGs, respectively. N means undetectable. Figure S1: hPvu2, Figure S2: hPvu4-1 and hPvu4-2 (To distinguish the duplicated highly homologous regions, sequence specific bisulfite sequencing PCR primers were designed. The regions sequenced are indicated by the solid bars; 241-652 bp downstream of hPvu4-1 and 82-490 bp downstream of hPvu4-2 were examined), Figure S3: hPvu8, Figure S4: hPvu29, Figure S5: hPvu42, Figure S6: hPvu66 (25 of 75 CpGs examined are shown), Figure S7: hPst6 (bisulfite genomic sequence analysis of two regions are shown), Figure S8: hPst32, Figure S9: hPst33(bisulfite genomic sequence analysis of two regions are shown), Figure S10: hPst61, S11: hPst21.

Acknowledgments

The authors thank Yukari Obana, Kunio Goto for their excellent technical assistance and Open Research Center for Genome and Infectious Disease Control for provision of instrument. This work is supported by Nihon University Multidisciplinary Research Grant for 2006 (to H.N.), Academic Frontier Project for 2006 Project for Private Universities: matching fund subsidy from MEXT (to H.N.), National Cancer Institute Grant CA102423 (to W.A.H.) and National Cancer Institute Center Support Grant CA16056 (to Roswell Park Cancer Institute).

Footnotes

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Reference List

1. Holliday R, Pugh JE. DNA modification mechanisms and gene activity during development. Science. 1975;187:226–232. [PubMed]
2. Bird AP. CpG-rich islands and the function of DNA methylation. Nature. 1986;321:209–213. [PubMed]
3. Jones PA. The DNA methylation paradox. Trends Genet. 1999;15:34–37. [PubMed]
4. Jaenisch R, Bird A. Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat Genet. 2003;33:245–254. Suppl. [PubMed]
5. Swain JL, Stewart TA, Leder P. Parental legacy determines methylation and expression of an autosomal transgene: a molecular mechanism for parental imprinting. Cell. 1987;50:719–727. [PubMed]
6. Riggs AD. X inactivation, differentiation, and DNA methylation. Cytogenet Cell Genet. 1975;14:9–25. [PubMed]
7. Shiota K, et al. Epigenetic marks by DNA methylation specific to stem, germ and somatic cells in mice. Genes Cells. 2002;7:961–969. [PubMed]
8. Ushijima T, Okochi-Takada E. Aberrant methylations in cancer cells: where do they come from? Cancer Sci. 2005;96:206–211. [PubMed]
9. Egger G, Liang G, Aparicio A, Jones PA. Epigenetics in human disease and prospects for epigenetic therapy. Nature. 2004;429:457–463. [PubMed]
10. Ushijima T. Detection and interpretation of altered methylation patterns in cancer cells. Nat Rev Cancer. 2005;5:223–231. [PubMed]
11. Laird PW. Cancer epigenetics. Hum Mol Genet. 2005;14(1):R65–R76. [PubMed]
12. Bernstein BE, et al. Genomic maps and comparative analysis of histone modifications in human and mouse. Cell. 2005;120:169–181. [PubMed]
13. Wysocka J, et al. A PHD finger of NURF couples histone H3 lysine 4 trimethylation with chromatin remodelling. Nature. 2006;442:86–90. [PubMed]
14. Shi X, et al. ING2 PHD domain links histone H3 lysine 4 methylation to active gene repression. Nature. 2006;442:96–99. [PMC free article] [PubMed]
15. Ching TT, et al. Epigenome analyses using BAC microarrays identify evolutionary conservation of tissue-specific methylation of SHANK3. Nat Genet. 2005;37:645–651. [PubMed]
16. Dai Z, et al. Global methylation profiling of lung cancer identifies novel methylated genes. Neoplasia. 2001;3:314–323. [PMC free article] [PubMed]
17. Kondo T, et al. Whole-genome methylation scan in ICF syndrome: hypomethylation of non- satellite DNA repeats D4Z4 and NBL2. Hum Mol Genet. 2000;9:597–604. [PubMed]
18. Costello JF, et al. Cyclin-dependent kinase 6 (CDK6) amplification in human gliomas identified using two-dimensional separation of genomic DNA. Cancer Res. 1997;57:1250–1254. [PubMed]
19. Rakyan VK, et al. DNA methylation profiling of the human major histocompatibility complex: a pilot study for the human epigenome project. PLoS Biol. 2004;2:e405. [PMC free article] [PubMed]
20. Futscher BW, et al. Role for DNA methylation in the control of cell type specific maspin expression. Nat Genet. 2002;31:175–179. [PubMed]
21. Okada H, et al. Frequent trefoil factor 3 (TFF3) overexpression and promoter hypomethylation in mouse and human hepatocellular carcinomas. Int J Oncol. 2005;26:369–377. [PMC free article] [PubMed]
22. Bernstein BE, et al. Genomic maps and comparative analysis of histone modifications in human and mouse. Cell. 2005;120:169–181. [PubMed]
23. Rakyan VK, et al. DNA methylation profiling of the human major histocompatibility complex: a pilot study for the human epigenome project. PLoS Biol. 2004;2:e405. [PMC free article] [PubMed]
24. Ching TT, et al. Epigenome analyses using BAC microarrays identify evolutionary conservation of tissue-specific methylation of SHANK3. Nat Genet. 2005;37:645–651. [PubMed]
25. Song F, et al. Association of tissue-specific differentially methylated regions (TDMs) with differential gene expression. Proc Natl Acad Sci U S A. 2005;102:3336–3341. [PMC free article] [PubMed]
26. Salem CE, et al. PAX6 methylation and ectopic expression in human tumor cells. Int J Cancer. 2000;87:179–185. [PubMed]
27. Hisano M, Ohta H, Nishimune Y, Nozaki M. Methylation of CpG dinucleotides in the open reading frame of a testicular germ cell-specific intronless gene, Tact1/Actl7b, represses its expression in somatic cells. Nucleic Acids Res. 2003;31:4797–4804. [PMC free article] [PubMed]
28. Lorincz MC, Dickerson DR, Schmitt M, Groudine M. Intragenic DNA methylation alters chromatin structure and elongation efficiency in mammalian cells. Nat Struct Mol Biol. 2004;11:1068–1075. [PubMed]
29. Kochanek S, Renz D, Doerfler W. DNA methylation in the Alu sequences of diploid and haploid primary human cells. EMBO J. 1993;12:1141–1151. [PMC free article] [PubMed]
30. Hellmann-Blumberg U, Hintz MF, Gatewood JM, Schmid CW. Developmental differences in methylation of human Alu repeats. Mol Cell Biol. 1993;13:4523–4530. [PMC free article] [PubMed]
31. Rubin CM, VandeVoort CA, Teplitz RL, Schmid CW. Alu repeated DNAs are differentially methylated in primate germ cells. Nucleic Acids Res. 1994;22:5121–5127. [PMC free article] [PubMed]
32. Betzl G, Brem G, Weidle UH. Epigenetic modification of transgenes under the control of the mouse mammary tumor virus LTR: tissue-dependent influence on transcription of the transgenes. Biol Chem. 1996;377:711–719. [PubMed]
33. Milner CM, Campbell RD. Structure and expression of the three MHC-linked HSP70 genes. Immunogenetics. 1990;32:242–251. [PubMed]
34. Ito Y, et al. Genomic structure of the spermatid-specific hsp70 homolog gene located in the class III region of the major histocompatibility complex of mouse and man. J Biochem (Tokyo) 1998;124:347–353. [PubMed]
35. Fourie AM, Peterson PA, Yang Y. Characterization and regulation of the major histocompatibility complex-encoded proteins Hsp70-Hom and Hsp70-1/2. Cell Stress Chaperones. 2001;6:282–295. [PMC free article] [PubMed]
36. Fourie AM, Peterson PA, Yang Y. Characterization and regulation of the major histocompatibility complex-encoded proteins Hsp70-Hom and Hsp70-1/2. Cell Stress Chaperones. 2001;6:282–295. [PMC free article] [PubMed]
37. Noce T, Okamoto-Ito S, Tsunekawa N. Vasa homolog genes in mammalian germ cell development. Cell Struct Funct. 2001;26:131–136. [PubMed]
38. Tanaka SS, et al. The mouse homolog of Drosophila Vasa is required for the development of male germ cells. Genes Dev. 2000;14:841–853. [PMC free article] [PubMed]
39. Kotaja N, et al. The chromatoid body of male germ cells: similarity with processing bodies and presence of Dicer and microRNA pathway components. Proc Natl Acad Sci U S A. 2006;103:2647–2652. [PMC free article] [PubMed]
40. Shibata N, et al. Mouse RanBPM is a partner gene to a germline specific RNA helicase, mouse vasa homolog protein. Mol Reprod Dev. 2004;67:1–7. [PubMed]
41. Castrillon DH, Quade BJ, Wang TY, Quigley C, Crum CP. The human VASA gene is specifically expressed in the germ cell lineage. Proc Natl Acad Sci U S A. 2000;97:9585–9590. [PMC free article] [PubMed]
42. Jones PA, Taylor SM. Cellular differentiation, cytidine analogs and DNA methylation. Cell. 1980;20:85–93. [PubMed]
43. Li LC, Dahiya R. MethPrimer: designing primers for methylation PCRs. Bioinformatics. 2002;18:1427–1431. [PubMed]
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