Placenta
Volume 29, Supplement , Pages 29-35, March 2008

Epigenetics: The DNA Methylation Profile of Tissue-Dependent and Differentially Methylated Regions in Cells

  • J. Ohgane

      Affiliations

    • Laboratory of Cellular Biochemistry, Animal Resource Sciences/Veterinary Medical Sciences, Graduate School of Agriculture and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
    • ,
  • S. Yagi

      Affiliations

    • Laboratory of Cellular Biochemistry, Animal Resource Sciences/Veterinary Medical Sciences, Graduate School of Agriculture and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
    • ,
  • K. Shiota

      Affiliations

    • Laboratory of Cellular Biochemistry, Animal Resource Sciences/Veterinary Medical Sciences, Graduate School of Agriculture and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
    • Organ Development Research Laboratory, National Institute of Advanced Industrial Science and Technology (AIST), Higashi, Tsukuba-city, Ibaraki 305-3962, Japan
    • Corresponding Author InformationCorresponding author: Laboratory of Cellular Biochemistry, Animal Resource Sciences/Veterinary Medical Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Tel.: +81 3 5841 5472; fax: +81 3 5841 8189.

Accepted 26 September 2007. published online 22 November 2007.

Article Outline

Abstract 

Methylation of DNA, which occurs at cytosines of CpG sequences, is a unique chemical modification of the vertebrate genome. Methylation patterns can be copied to daughter DNA after mitosis; thus DNA methylation has been suggested to act as a “cellular memory of the genome function”. Genome-wide analysis of DNA methylation revealed that there are numerous tissue-dependent differentially methylated regions (T-DMRs) in unique sequences of the mammalian genome. There are T-DMRs in both CpG-rich and -poor sequences. Methylation of T-DMRs is responsible for gene-silencing and chromatin structure change. Each tissue/cell type has a unique DNA methylation profile that consists of methylation patterns of numerous loci in the genome. DNA methylation profiles are not associated with bulk DNA, which is mainly comprised of repetitive sequences. Disruption of DNA methylation profiles putatively produce abnormal cells and tissues. Cloned mice produced by somatic nuclear transfer are associated with aberrant DNA methylation profiles. Tissue/cell type-specific DNA methylation profiles can provide a novel viewpoint for understanding normal and aberrant development, in terms of both differentiation and reproduction.

Keywords: Epigenetics, DNA methylation, Tissue-dependent and differentially methylated regions, T-DMRs, DNA methylation profile

 

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1. Introduction: what is epigenetics? 

A single fertilized egg gives rise to a complex multi-cellular organism consisting of various differentiated cell types. Based on a variety of physiological and morphological criteria, there are at least 200 differentiated cell types in mammals. In general, cellular differentiation occurs without changes in DNA sequence, although there are a few exceptions, such as lymphoid cells. Cell phenotypes may be maintained even after mitosis when the cell is equipped with epigenetic mechanisms that enable the inheritance of gene function. Initially, epigenetics was defined as “the study of mitotically and/or miotically heritable changes in gene function that cannot be explained by changes in DNA sequence” [1]. At present, epigenetics is more broadly defined as “the study of processes that produce a heritable phenotype that does not strictly depend on DNA sequence” [2].

DNA methylation occurs at the 5′-position of cytosine mainly in a 5′-CG-3′ dinucleotide pattern (CpG: C paired G) [3], [4]. Methyl-cytosine is the only chemical modification of mammalian genomic DNA. The status of methylated CpGs is maintained after DNA replication by DNA methyltransferases [5], and therefore the DNA methylation signal is heritable beyond cell generations. As shown in Fig. 1, DNA methylation is generally associated with chromatin condensation through histone modifications. Thus, DNA methylation and histone modification play crucial roles in gene silencing, stabilizing chromosomal structure, and suppressing the mobility of retrotransposons [6], [7]. The epigenetic mechanisms underlying various biological phenomena include differentiation, development, X-inactivation and imprinting.

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  • Fig. 1 

    Epigenetic system of DNA methylation and chromatin structure change in mammalian cells. Methylation of CpGs occurring in the regulatory region of genes causes gene silencing. Methylation of DNA and chromatin structure is coordinated through modification of histones, including by acetylation and methylation. In general, DNA methylation and chromatin condensation are associated with gene silencing. The epigenetic state consisting of DNA methylation and chromatin configuration results in changes in the differentiation of cells. Once established, the epigenetic state is maintained during the proliferation of cells.

It is estimated that the human and mouse genomes consist of ∼3×109bp/haploid DNA. A large part of the genome is composed of non-genic repetitive elements; ∼41% and 48% in the human and mouse, respectively, including interspersed repeats and satellites [8], [9], [10]. There are numerous tissue-dependent and differentially methylated regions (T-DMRs) in the unique sequences of the genic area widely distributed in the mammalian genome. In this article, we will describe: 1) examples of genes regulated by DNA methylation; 2) cell- and tissue-specific DNA methylation profiles consisting of methylated or unmethylated T-DMRs; and 3) normal and abnormal DNA methylation profiles.

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2. Gene silencing by DNA methylation 

Most CpGs are methylated in the mammalian genome; the percentage varies between 60% and 90% depending on the source reports [4], [11]. Information on whole genome levels of DNA methylation reflects the hypermethylated status of the non-genic repetitive sequences rather than the gene-coding areas. Much interest has been focused on the unique sequences of the genic area; there are 30,000–40,000 genes in only a small part of the genome. Changes in the genic area contain T-DMRs. Epigenetic dynamics is not associated with bulk DNA, which is comprised mainly of non-genic repetitive sequences.

2.1. Genes with T-DMRs containing CpG-poor sequences 

The placenta is characterized by various support functions of fetal growth, including the production of placental hormones, transport of nutrition to the fetus and export of waste into the maternal blood, and protection of the fetus from the maternal immune system. Placental lactogen is an essential hormone for the maintenance of pregnancy in the rodent [12]. Expression of the rat placental lactogen-I (rPL-I) gene is specific to the placenta and never expressed in other tissues. The rPL-I gene contains a TATA box, and there are only a few CpG sites in the promoter region, i.e., 17 CpGs within the 3.4kb of the 5′-flanking region. There are several transcription factor binding sites including GATA and AP-1. These CpGs are restrictedly hypomethylated in the placenta [13], and heavily methylated in other tissues, including the kidney, germ cells and ES cells, indicating that the PL-I gene has a T-DMR within the promoter region. Thus, tissue-specific restricted expression of rPL-I is achieved by tissue-specifically hypomethylated T-DMR in the placenta. Similarly, some prolactin and growth hormone family members that are specific to the anterior pituitary have T-DMRs similar to the rPL-I gene [14]. Other family members expressed in the placenta are under investigation. An important aspect of the epigenetic regulation of the PL-I gene is that there are only a few CpGs in the regulatory region and placenta-non-specific transcription factors such as GATA and AP-1 can regulate the placenta-specific genes in combination with the epigenetic mechanism of DNA methylation [13]. Treatment of PL-I-non-expressing BRL cells with an inhibitor of DNA methyltransferase, 5-aza-deoxycitidine (5-aza-dC), or an inhibitor of histone deacetylase, Trichostain A (TSA), can induce ectopic rPL-I expression [13]. Therefore, DNA methylation and histone modification are responsible for gene silencing in PL-I-non-expressing cells. Another example of T-DMR with few CpGs is Sry, which encodes a transcription factor of the master protein for initiating testis differentiation in mammals [15]. There is a T-DMR with only eight CpGs within the 2kb upstream region of the linear form of the mouse Sry gene [16]. Thus, demethylation of a small number of CpGs is critical for the spatio-temporal expression of PL-I and Sry, while the T-DMR is highly methylated in other cell types and tissues.

2.2. Genes with T-DMRs containing CpG-rich sequences 

In mammalian embryogenesis, the first differentiation event that determines the lineage of the trophectoderm and inner cell mass (ICM) occurs at the blastocyst stage. Embryonic stem (ES) cells established from ICM have a pluripotent ability to contribute to all embryonic lineages [17], [18]. Trophoblast stem (TS) cells established from trophectoderm have the ability to differentiate into the trophoblast lineage in vitro [19]. The Oct-4 gene is a POU family transcription factor and has a CG-rich and TATA-less promoter [20], [21]. Oct-4 is expressed in ES cells but not in TS cells [19], and reduction in Oct-4 gene expression induces the transdifferentiation of ES cells into TS-like cells under certain culture conditions [22]. There is a T-DMR in the promoter and enhancer region of the Oct-4 gene and the T-DMR is hypomethylated in ES cells but hypermethylated in TS cells [23].

2.3. Genes with T-DMRs associated with CpG islands 

In mammals, CpG sequences appear at low frequency, based on the estimation of the average base-pair composition. The sequences are unevenly distributed in the mammalian genome, and CpG-rich regions are called CpG islands [24]. The human genome project has uncovered 30,000–40,000 genes with approximately 29,000 CpG islands [8], [9]. There are 30,000 genes and 16,100 CpG islands in the mouse genome (March 2005/mm6 mouse genome assembly, UCSC Genome Browser, http://genome.ucsc.edu) [10]. The CpG islands are approximately 1–2kb in length. Gardiner-Garden and Frommer [24] originally defined CpG islands as regions of greater than 200 base pairs with a GC content of >50% and an observed/expected CpG ratio of >0.6. CpG islands have been used as landmarks to find gene coding regions in the genome [24], [25], since CpG islands are frequently located near promoters and the first exons of genes. To exclude small repetitive sequences from the CpG islands, the definition of CpG islands was recently modified [26]. Most housekeeping genes and about half of the tissue-specifically expressed genes have CpG islands at their promoters [27].

Based on the HpaII/MspI digestion experiment, CpG islands had long been believed to be unmethylated regions in normal tissues [28], [29] except for those under the control of X-inactivation [30], [31], and genomic imprinting [32], [33]. However, T-DMRs in which the methylation status is correlated with gene expression have also been reported in several CpG island-associated genes such as Sphingosine kinase 1 (Sphk1) [34], Endothelin receptor B (EDNRB) [35] and Proopiomelanocortin (POMC) [36].

The Sphk1 gene has six mRNA subtypes (Sphk1a, -b, -c, -d, -e, and -f) [34]. In the upstream region of the Sphk1 gene, there is a CpG island with a T-DMR. Sphk1 catalyzes the production of sphingosine 1-phosphate, an intracellular signal messenger, and plays a critical role in signal transduction, cell growth and differentiation [37]. The methylation status of the T-DMR is closely correlated with the expression of Sphk1a. When Sphk1a is expressed in the adult rat brain, the T-DMR is hypomethylated, whereas it is hypermethylated and silenced in other tissues. Therefore, the genes associated with CpG islands should not be excluded from the list of candidate genes that are regulated by DNA methylation.

2.4. Classification of genes with CpG island-associated T-DMRs 

Systematic analysis of the DNA methylation levels of approximately 250 NotI sites has been performed, and approximately 70% of the NotI sites were located within CpG islands. Of these, CpG islands having T-DMRs exhibited a relatively small size [38]. In general, T-DMRs within CpG islands are restricted to subregions of the CpG islands. For example, the T-DMR of the Sphk1 gene, which is conserved in mouse, rat and human, occupies ∼200bp at the edge of a 3.7kb CpG island, a proportion of less than 10% of the entire CpG island [34]. This type of T-DMR that is restricted to a small region of CpG islands has also been found in the EDNRB [35] and POMC [36] genes.

Interestingly, a new class of CpG islands characterized by tissue-dependent methylation of all of the CpGs in certain CpG islands has now been discovered. Hisano et al. [39] 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. Therefore, another class of CpG islands is likely to exist in which the T-DMR extends throughout the whole CpG island. Indeed, there are unique germ-cell-specific T-DMRs, including Ant4/Slc25a31. In the CpG island of the Ant4/Slc25a31 gene, all CpGs are methylated in somatic cells, but unmethylated in the sperm. Therefore, gene loci with CpG islands should not be eliminated from the list of those having T-DMRs.

Cell-type-specific genes with T-DMRs include receptors, hormones, intracellular signaling molecules and so on. Thus, genes with various functions have T-DMRs regardless of being poor/rich in CpG sequences. In addition, another target of DNA methylation is transcription factors, and T-DMRs are thought to be epigenetic marks to distinctively determine the silenced/activated status of transcription factors. In addition, Dnmt1o, a subtype of DNA methyltransferase specific to oocytes, has a T-DMR [40]. Therefore, there is epigenetic regulation to control the factors that are involved in the epigenetic gene regulation of development.

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3. DNA methylation profiles specific to tissue/cell types 

3.1. Normal DNA methylation profile with a genome-wide pattern of methylated and unmethylated T-DMRs 

More than 90% of NotI sites were thought to represent CpG islands in the mammalian genome [41], and completion of mouse genome sequencing has revealed that about 70% of NotI sites are in CpG islands with the remaining sites distributed in non-CpG islands [42]. Using NotI as a landmark enzyme, genome-wide analysis of T-DMRs was performed by Restriction Landmark Genomic Scanning (RLGS) [43]. A genome wide analysis of ES cells, EG cells, TS cells, germ cells and several somatic tissues focusing on 1500 CpG islands and CpG-rich regions identified 247 T-DMRs, which are methylated or unmethylated depending on the cell or tissue type [43], [44]. The analysis has indicated that each cell/tissue type has a specific methylation profile of T-DMRs, and the profiles can distinguish between cell/tissue types (Fig. 2).

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  • Fig. 2 

    Tissue and cell type specific DNA methylation profiles comprising DNA methylation patterns of T-DMRs. A given cell type has a unique pattern of DNA methylation status at each T-DMR (lower panel). These patterns constitute cell type-specific DNA methylation profiles (upper panel). The DNA methylation profile of a cell type is unique and can be a novel tool to define and characterize the cell type.

There are T-DMRs both in CpG-poor and CpG-rich loci, as described above. Considering that there are 16,100 CpG islands in the mouse genome, RLGS can sample only a subset. The number of T-DMRs will expand with additional studies in the near future. Therefore, the DNA methylation profile in the whole genome must be more complex, and the profile is specific to cell and tissue type much like a fingerprint is distinct, and can be used as an identification (ID) tag for cells. The profiles clearly show that differentiation involves massive alterations of the genome-wide methylation status of multiple gene loci with changes in both de novo methylation and demethylation.

Since DNA methylation is a unique ID of cells, a change in the DNA methylation profile will cause an alteration in the properties of the cells. DNA methylation analysis has been employed to evaluate cells, including human ES cells [45] and mouse ES cells produced by somatic nuclear transfer technology [46]. The DNA methylation profile also provides a novel method to evaluate the similarities of cells which reflect the similarities of differentiation lineages.

3.2. Aberrant DNA methylation patterns in cloned animals 

Placentomegaly is quite a common phenomenon in cloned mice regardless of sex, strain, and cell type of the somatic donor nucleius [47], [48], [49]. Placentomegaly involves expansion of the spongiotrophoblast layer with an increased incidence of glycogen cell differentiation [50]. Since the formation of DNA methylation profiles is involved in development and differentiation, disruptions in DNA methylation profiles may cause aberrant cellular conditions that lead to severe symptoms. Indeed, the Spalt-like gene 3 (Sall3) locus showed frequent aberrant methylation in placentas of cloned mice [51]. The Sall3 gene is located at the telomeric E3 subregion of mouse chromosome 18, and the region is suggested to be involved in the human 18q deletion syndrome [52]. Aberrant DNA methylation may cause abnormal phenotypes of cloned animals. Genome-wide studies revealed that the DNA methylation profiles of cloned mice that developed to full term are 99% identical to those of naturally produced mice, implying that they have an almost normal epigenetic status. However, all of the cloned mice we observed had a small number of aberrant DNA methylation profiles, i.e. 1–2% of RLGS spots [51]. Thus, the DNA methylation profile is almost identical to normal mice, although there is a slightly aberrant DNA methylation status in a small number of T-DMRs.

The process of epigenetic change during the development of cloned embryos must be quite different from that in animals produced by normal fertilization. In cloned animals, the DNA methylation profile must be reestablished from donor somatic cells to become different cell types through zygotic and early embryonic cell types following nuclear transfer. Indeed, in contrast to the fully developed cloned animals, the developing cloned animals exhibit more severe abnormality in their DNA methylation profile. For example, a cloned cow at 59 fetal days displayed numerous T-DMRs with aberrant DNA methylation status, and some of these were caused by failure of the reprogramming that results in retention of the methylation patterns of the donor cell type [53].

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4. Molecular link between DNA methylation and histone modification 

There are several mechanisms of DNA methylation-mediated gene silencing. Methylation of DNA inhibits the direct binding of certain transcription factors to DNA [54], [55]. There are methyl-CpG binding proteins, which have a conserved methyl-CpG binding domain (MBD), such as MeCP2, MBD1, MBD2, MBD3, and MBD4 [56] and another type of protein known as KAISO, which lacks MBD but has the ability to bind the CpG via zinc finger motifs [57].

These methyl-CpG binding proteins constitute a chromatin modulation complex containing different sets of histone modifiers such as histone deacetylases (HDACs) and histone methyltransferases. The HDACs, which induce deacetylation of the lysine residues of histone tails, are responsible for the formation of condensed chromatin structures that represse gene expression. MeCP2 represses gene expression by organizing a transcriptional repressor complex together with HDAC1 via mSin3A [58], [59]. MBD3 comprises a component of a complex called Mi-2/NURD which consists of chromatin-remodeling ATPase and histone deacetylase [60]. Methylation of the histone H3–K9 also causes chromatin condensation. For example, a histone methyltransferase, Suv39h1, distributed in heterochromatin, interacts with methyl-CpG binding proteins as well [61]. G9a, which is another histone methyltransferase localized in euchromatin, induces DNA methylation at specific T-DMRs in mouse ES cells [62]. Chromatin configuration also affects DNA methylation; H3K9 methylation attracts heterochromatin protein HP1, which in turn recruits a DNA methyltransferase, DNMT3a [63]. Histone modification and DNA methylation are closely related to each other [62].

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5. Conclusion 

Genome-wide DNA methylation profiles are in a certain sense the epigenetic memory that is indispensable for cells and tissues to maintain their unique features. Cell and tissue-specific gene expression is a central issue in efforts to understand development and differentiation. Cell/tissue-specific expressions of various genes, including transcription factors, had been explained by the binding of cell/tissue specific transcription factors to their promoters. However, the cell/tissue specific transcription factors can themselves be regulated by different cell/tissue specific transcription factors. If such transcription factors are specific to cell types, what is the mechanism producing such transcription factors? Is it regulated by another, upstream cell/tissue-specific transcription factor? The initial triggering transcription factor of a “sequential cascade” of transcription factors surely requires another level of genetic control, since the genome sequence is identical among different cell types. As a consequence, the concept of a “sequential cascade” of transcription factors may run into difficulties when it comes to producing different cell types using the identical genome sequence.

In addition, regulation by transcription factors is generally temporally constrained and rapidly changeable in response to environmental and extracellular stimuli. In differentiated cells, the gene expression repertoire defining the phenotype must be maintained after proliferation of the cells. This is another reason why another level of gene control, other than transcription factors, is required to understand differentiation and development. The second key, then, is irreversible gene control, which may be heritable into the next cell generation. It is becoming clear that various genes are controlled by DNA methylation and histone modifications, as described above. Genome-wide DNA methylation profiles, which consist of information on the DNA methylation profile of numerous loci, store the “cellular memory” that governs tissue/cell-type features.

To understand the differentiation of cells, in the post genomic research era we need another layer of genetic information in addition to the genome sequences and their variations, including SNPs. Epigenetics connects the genome (DNA sequences) and transcriptome by introducing another layer of gene control, a stable memory of gene-set activity heritable to the next generation (Fig. 3). Thus, epigenetic codes of genome wide DNA methylation profiles and histone modifications will provide new insight into mammalian development and differentiation of cells. In addition, epigenetic analysis provides a new paradigm for understanding the normal and abnormal status of cells and tissues, including their pathogenic contribution in a variety of diseases.

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6. Conflict of interest 

The authors do not have any potential or actual personal, political, or financial interest in the material, information, or techniques described in this paper.

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PII: S0143-4004(07)00248-2

doi:10.1016/j.placenta.2007.09.011

Placenta
Volume 29, Supplement , Pages 29-35, March 2008

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