MINIREVIEW Epigenetics: differential DNA methylation in mammalian somatic tissues Hiroki Nagase 1,2 and Srimoyee Ghosh 2 1 Advanced Research Institute for the Sciences and Humanities, Nihon University, Tokyo, Japan 2 Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, NY, USA Cytosine methylation of CpG dinucleotides is an important epigenetic modification that has profound roles in gene regulation, development and carcinogene- sis [1,2]. Methylation of CpG clusters or CpG islands within gene promoters can silence gene expression [3,4]. Therefore, identifying changes in DNA methyla- tion at CpG islands is expected to lead to a clearer understanding of the differentiation of normal tissues and the development of complex diseases including cancer [5]. The DNA methylation pattern is somati- cally heritable via the effect of the maintenance DNA methyltransferase, DNMT1 [6]. The error rate of maintaining DNA methylation is low ( 1% per divi- sion) at human CpG sites [7]. During embryonic devel- opment, both somatic and germ-cell DNA methylation patterns are erased and then re-established during cell differentiation. Once established, DNA methytlation patterns are thought to be stable. Although it has been reported that DNA methylation may play a role in the regulation of tissue-specific gene expression [8,9], differential DNA methylation patterns among adult tissues were not confirmed until recently. The flowering plant Arabidopsis thaliana, with muta- tions in the cytosine–DNA-methyltransferase gene, MET1, which led to a global reduction in cytosine methylation, is viable, and the delay in its flowering Keywords cancer; CpG islands; differentially methylated region; differentiation; DNA methylation; epigenetics; mouse; restriction landmark genomic scanning (RLGS); tissue- specific DMR; Vi-RLGS Correspondence H. Nagase, Advanced Research Institute for the Sciences and Humanities, Nihon University, Nihon University Kaikan Daini Bekkan, 12-5, Goban-cho, Chiyoda-ku, Tokyo 102-8251, Japan Fax: +81 3 3972 8337 Tel: +81 3 3972 8337 E-mail: nagase-hiroki@arish.nihon-u.ac.jp (Received 30 November 2007, revised 28 January 2008, accepted 11 February 2008) doi:10.1111/j.1742-4658.2008.06330.x Epigenetics refers to heritable phenotypic alterations in the absence of DNA sequence changes, and DNA methylation is one of the extensively studied epigenetic alterations. DNA methylation is an evolutionally con- served mechanism to regulate gene expression in mammals. Because DNA methyation is preserved during DNA replication it can be inherited. Thus, DNA methylation could be a major mechanism by which to produce semi- stable changes in gene expression in somatic tissues. Although it remains controversial whether germ-line DNA methylation in mammalian genomes is stably heritable, frequent tissue-specific and disease-specific de novo meth- ylation events are observed during somatic cell development ⁄ differentia- tion. In this minireview, we discuss the use of restriction landmark genomic scanning, together with in silico analysis, to identify differentially methylat- ed regions in the mammalian genome. We then present a rough overview of quantitative DNA methylation patterns at 4600 NotI sites and more than 150 differentially methylated regions in several C57BL ⁄ 6J mouse tis- sues. Comparative analysis between mice and humans suggests that some, but not all, tissue-specific differentially methylated regions are conserved. A deeper understanding of cell-type-specific differences in DNA methylation might lead to a better illustration of the mechanisms behind tissue-specific differentiation in mammals. Abbreviations DMR, differentially methylated region; RLGS, restriction landmark genomic scanning; T-DMR, tissue-specific differentially methylated region; Vi-RLGS, virtual-image restriction landmark genomic scanning; V-RLGS, virtual restriction landmark genomic scanning. FEBS Journal 275 (2008) 1617–1623 ª 2008 The Authors Journal compilation ª 2008 FEBS 1617 time is observed only after several generations [10]. Embryos from DNA methyltransferase gene-deficient mice, which have reduced levels of cytosine methyla- tion, develop until the stage of 8.5 days, when many tissues are already differentiated [11]. Furthermore, analysis of DNA methylation patterns in genes known to be expressed in a tissue-specific manner failed to confirm a major role for DNA methylation in differen- tiation [12,13]. Using microarray analysis, only five genes expressed in a tissue-specific manner showed a significant increase in expression level in an in vivo system lacking DNA methylation [14]. These data suggested that DNA methylation had no role in regulating gene expression during development. Song et al. [15] reported 150 tissue-specific differen- tially methylated regions (T-DMRs) in the mouse genome using restriction landmark genomic scanning (RLGS) in conjunction with virtual-image restriction landmark genomic scanning (Vi-RLGS) and confirmed at least 14 T-DMRs by bisulfite sequencing. Some of the confirmed T-DMRs exhibited a tissue-specific expression pattern that is consistent with methylation status and may play a role in tissue differentiation [15]. Subsequent studies reported the existence of fre- quent tissue-specific methylation events in mice [16,17] and humans [9,18–20]. Thus, the extent of DNA meth- ylation appears to change in a systematic way during mammalian development. DNA methylation status may be influenced by environmental exposure [2,20,21]. In gastric mucosa, Helicobacter pylori infection potently induces the meth- ylation of several CpG islands [21]. Young monozy- gotic twins are essentially indistinguishable in their epigenetic markings, whereas older monozygous twins exhibit remarkable differences in overall content and genomic distribution of 5-methylcytosine DNA and histone acetylation [22]. Thus, the previous hypothesis that DNA methylation patterns acquired during devel- opment in mammals were stable in adult somatic cells can be discarded in favor of the accumulated evidence of frequent appearances of differentially methylated genomic regions in various tissue environments. RLGS method for the mammalian genome RLGS is a method for the 2D display of end-labeled DNA restriction fragments [23]. This method involves digestion of high molecular mass genomic DNA with a ‘landmark’ enzyme. The landmark enzyme, such as the methylation-sensitive enzyme NotIorAscI, determines the sites of the genome that will be labeled by filling in enzyme half-sites with radioactive nucleotides. Because the NotI recognition site contains two CpGs, and > 90% of the NotI sites are thought to lie within CpG islands, RLGS (with NotI and similar restriction enzymes) displays the DNA methylation status of the CpG islands and associated regions [23]. For example, when comparing normal and cancer RLGS profiles, spot loss due to methylation occurs because the methy- lated NotI site is not cut by the enzyme and is there- fore not labeled. By contrast, spot gain in cancer indicates ‘demethylation’ of a NotI site which was methylated in normal tissues. Using methylation- sensitive NotI as a landmark,  1500–2000 spots (end-labeled restriction fragments) can be resolved on a single gel. These methods have been used to identify imprinted sites, aberrant methylated sites in cancers and epigenetic remodeling of mammalian tissues [23–25]. The lack of a PCR step and hybridization in the RLGS procedure provides an important advantage over other methods in identifying aberrant DNA meth- ylation. RLGS profiles are quantitative, and the sensi- tivity is such that methylation can be reliably detected when > 40% of the alleles are methylated. This level of sensitivity ensures that the major demethylation events present in the sample could be detected. By con- trast, other approaches, such as bisulfite sequencing or chromatin ⁄ methyl-cytosine precipitation, would allow the detection of very rare methylation events (false positive) or the omission of a partial DNA methyla- tion (false negative), due to the involvement of array hybridization, PCR amplification and affinity precipi- tation. Computational approaches for RLGS (Vi-RLGS) Despite its clear importance and successes, RLGS has some potential drawbacks, the most significant of which is the difficulty of cloning individual spots [26,27]. This is critically important because the sequence of the altered RLGS spot must be deter- mined in order to identify the affected gene. Clearly, with the availability of the genome sequence for many organisms, it has become possible to use this informa- tion to identify specific restriction fragments within ge- nomes and produce in silico size fractionations [28–30]. Several in silico analyses that have been made in this sense include the Virtual Genome Scan (http://dot. ped.med.umich.edu:2000/VGS/index.html) [30], in silico digests [28], Vi-RLGS [29] and virtual restriction land- mark genomic scanning (Conime by R. Wenger; http:// www.cse.ohio-state.edu/wenger/research/conime/contact. html). These tools help to create a more complete map DNA methylation in the mammalian genome H. Nagase and S. Ghosh 1618 FEBS Journal 275 (2008) 1617–1623 ª 2008 The Authors Journal compilation ª 2008 FEBS of genomic sites that are either methylated or demethy- lated in current human and mouse genomic DNA sequence data. Application of Vi-RLGS to the C57BL ⁄ 6J mouse genome We applied the Vi-RLGS software directly to the mouse genome using a NotI–PstI–PvuII combination. Examin- ations of a sample field from C57BL ⁄ 6J liver DNA identified  1460 unmethylated spots in real RLGS, compared with 2170 spots in the same field of the vir- tual pattern. The vast majority of the ‘extra spots’ in the virtual profile in the mouse are, in fact, derived from repetitive sequences, which would be expected to be methylated and absent in the real profile [15]. This method has been applied to the mouse genome using six different tissues (testis, brain, colon, kidney, liver and muscle) [15]. The methylation status of  4600 genomic sites fell into one of the following three categories: constitutively methylated, constitu- tively demethylated and methylated in a tissue-specific manner. The frequency of T-DMRs is estimated to be  5% (836 ⁄ 15 500 CpG islands) in the mouse genome [15]. This estimate may be different because RLGS is strongly biased by the genomic location of the NotI sites. DNA methylation profiling of human chromo- somes 6, 20 and 22 suggested that many T-DMRs are not in CpG islands [31], but recent global human genome searches have identified  700 T-DMRs for human promoter regions, many of which are included in CpG islands [9,19,20]. However, when the two recent human global analyses are compared, 283 gene promoters are identified as ‘testis-specific’ DMRs in one analysis [19] and 104 in another [20]. Among these gene promoters, only 18 were concurrently identified as ‘testis-specific’ DMRs by both studies [19,20]. Although potential sources of contradictions in these two publications may be the differences in type of tis- sue, DNA purification and methodology used, most of recent methodologies are not accurately quantitative. A reproducible method for quantitative DNA methyla- tion detection is needed for the in vivo study, in which DNAs are often prepared from a mixed cell popula- tion. The application of a quantitative whole-genome methylation analysis by RLGS to the mouse genome provides evidence for specific differences in the DNA methylation patterns during development, differentia- tion, aging and in diseases such as cancer. The Vi-RLGS application, together with a sophisticated image-matching and registration program and spot intensity analysis program may provide a new analyti- cal tool to measure the global methylation patterns of cell population in specific tissue environments [32,33] (G. E. Bove and P. Rogersen, University at Buffalo, NY, USA; unpublished data). Analysis of deposited numbers of previously performed RLGS images may prove to be a treasure chest for understanding the methylation patterns of each tissue or disease state. Based on those efforts and a considerable number of RLGS experiments, a rough draft overview of quantita- tive DNA methylation patterns with a quantitative DNA measurement of the C57BL ⁄ 6J mouse genome has been created by using the Vi-RLGS in silico analy- sis. Figure 1 shows a draft NotI methylation map of C57BL ⁄ 6J strain based on two RLGS profiles of NotI– PstI–PvuII and NotI–PvuII–PstI enzyme combinations. Table 1 is a preliminary distribution pattern on each chromosome of T-DMRs, constitutively methylated and constitutively non- or partially methylated NotI sites using a virtual-image RLGS analysis (note that for most NotI sites the methylation pattern has yet to be confirmed by other methods). Interestingly, consid- ering the gene-poor regions [34], a significantly high number of T-DMRs are located in gene-rich genomic region, while non-T-DMRs are located in both (Fig. 2). In addition, a relatively high percentage of NotI sites in T-DMRs are located in the non-promoter region (exons, introns and 3’ regions). This may suggest that T-DMRs are likely to modify gene expression through transcriptional regulation or may have other functions that are unrelated to transcription. However, the func- tional relationships between T-DMRs and regulation mechanisms involved in tissue differentiation are unknown. Intragenic DNA methylation is known to be capable of altering the chromatin structure and elonga- tion efficiency in mammalian cells, depleting RNA polymerase II exclusively in the methylated region [35]. It has been suggested that the methylation of Alu ele- ments could suppress transcription and contribute to differential gene expression [36–38]. A study of trans- genic mice demonstrated that the epigenetic modifica- tion of transgenes under the control of the mouse mammary tumor virus LTR conferred a tissue-depen- dent influence on the transcription of the transgenes [39]. Recent evidence suggested that there are related regulation mechanisms between micro RNA and epige- netics [40]. Although it has been reported that 95% of mammalian genomes are transcribed and have some functional means [41], the localization bias of T-DMRs may suggest that DNA methylation is a critical tissue- specific regulation mechanism and that it modifies RNA transcription. The T-DMR located in gene-poor regions may also facilitate the identification of previ- ously unidentified regulatory mechanisms. H. Nagase and S. Ghosh DNA methylation in the mammalian genome FEBS Journal 275 (2008) 1617–1623 ª 2008 The Authors Journal compilation ª 2008 FEBS 1619 RLGS for cancer study RLGS has been used to study the degree of hypome- thylation as well as to identify the targets of hyperme- thylation in many different types of cancerous tissues [24,42]. These studies reveal that CpG island methyla- tion in cancers is non-random and shows both inter- and intratumor heterogeneity. Furthermore, certain Fig. 1. NotI methylation map of the C57BL ⁄ 6J genome. The methylation status of NotI sites is plotted on 19 mouse autosomal chromo- somes. Constitutively methylated, constitutively unmethylated or tissue-specific methylated sites are indicated by red bars (left-hand side of the chromosome), green bars (right-hand side of the chromosome) and blue bars (left-hand side of the chromosome), respectively. Vi-RLGS analysis was performed in conjunction with duplicate RLGS analyses of six tissues (liver, muscle, kidney, colon, testis and brain) using Mouse Aug. 2005 (mm7) assembly, and then the methylation pattern of each spot of RLGS autographs were analyzed. DNA methylation in the mammalian genome H. Nagase and S. Ghosh 1620 FEBS Journal 275 (2008) 1617–1623 ª 2008 The Authors Journal compilation ª 2008 FEBS RLGS fragments were found to be methylated in many different cancers, whereas others were methylated exclusively in one. In the set of tumors described by Costello et al. [24], gliomas had 34 RLGS loci methy- lated in > 40% of the samples (n = 14), colon tumors had 23 (n = 8) and medulloblastomas had eight (n = 22). Even if the associated gene is not known or ever found to be associated with cancer, given that they are methylated at such high frequency, methyla- tion of these loci could be used as biomarkers. Rarely known tumor-suppressor genes have been shown to be methylated at a frequency of > 40% of tumors, even using highly sensitive PCR-based techniques [43]. One remarkable exception to this is the GSTP1 gene, which was shown to be hypermethylated in 40 ⁄ 42 human prostate cancers [44] and shows promising signs of becoming an excellent biomarker for prostate cancer [45]. A quantitative analysis such as RLGS provides an opportunity to scan the genome for frequent targets of methylation in a mixed cell population such as tumor DNA. This may have even more potential than existing DNA methylation biomarkers. In addition, studying DNA methylation for loci whose primary route of inactivation is through CpG island methyla- tion, rather than through genetic disruption, may be more likely to result in the identification of effective therapeutic targets. Conclusion The recent technical revolution in epigenetic detection is providing a clearer understanding of epigenetic Table 1. Preliminary distribution pattern of T-DMRs, on each chromosome showing constitutively methylated and constitutively non- or par- tially methylated NotI sites identified by virtual-image RLGS analysis system. Chromosome 12345678910111213141516171819Total Constitutively unmethylated regions 224 342 184 275 271 174 252 218 228 207 316 154 171 129 143 138 174 125 124 3849 % 78.0 88.4 81.1 91.1 86.9 80.2 86.0 85.8 86.0 83.8 89.0 86.5 81.4 79.1 85.6 81.7 84.1 82.2 87.3 85.9 Constitutively methylated regions 58 37 35 17 29 32 28 25 31 36 23 15 35 29 18 25 25 22 14 534 % 20.2 9.6 15.4 5.6 9.3 14.7 9.6 9.8 11.7 14.6 6.5 8.4 16.7 17.8 10.8 14.8 12.1 14.5 9.9 11.8 T-DMRs 5881012111311641694566854151 % 1.7 2.1 3.5 3.3 3.8 5.1 4.4 4.3 2.3 1.6 4.5 5.1 1.9 3.1 3.6 3.6 3.9 3.3 2.8 3.3 Total 287 387 227 302 312 217 293 254 265 247 355 178 210 163 167 169 207 152 142 4534 % 6.2 8.4 4.9 6.6 6.8 4.7 6.4 5.5 5.8 5.4 7.7 3.9 4.6 3.5 3.6 3.7 4.5 3.3 3.1 Physical length Mb 195 181 158 154 150 150 139 127 124 130 121 115 114 118 103 97 92 90 60 2596 % 7.5 7.0 6.1 5.9 5.8 5.8 5.4 4.9 4.8 5.0 4.7 4.4 4.4 4.5 4.0 3.7 3.5 3.5 2.3 10 0 20 30 40 50 60 70 80 90 100 non-CpG promoter non-CpG exon non-CpG intron non-CpG 3’ non-CpG junk CpG promoter CpG exon CpG Intron CpG 3’ CpG junk CpG islands non-CpG islands Junk non-junk % NotI unmethylated regions NotI T -DMRs Fig. 2. Genomic regions of unmethylated NotI sites and NotI sites showing tissue- specific differentially methylated regions (T-DMRs). A bar graph indicates the percent of genomic distribution of NotI sites sepa- rately analyzed between those located within constitutively unmethylated regions (white bars) and those in T-DMRs (gray bars) at each indicated genomic region. Intergenic region (Junk), intragenic region (promoter, exon, 3¢ regions; non-Junk), CpG island and non-CpG island were evaluated by UCSC genome browser (mm8). H. Nagase and S. Ghosh DNA methylation in the mammalian genome FEBS Journal 275 (2008) 1617–1623 ª 2008 The Authors Journal compilation ª 2008 FEBS 1621 marks in the genomes of every mammalian cell type, even though genome-wide quantitative DNA methyla- tion analysis is yet to be completely established. RLGS, together with in silico analysis, is a useful tech- nique for comparing genome-wide DNA methylation patterns between tissues or disease states. 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Although it remains controversial whether germ-line DNA methylation in mammalian genomes is