Epigenetics: interaction of DNA methylation and chromatin

Gene 278 (2001) 25–31 www.elsevier.com/locate/gene

Review

Epigenetics: interaction of DNA methylation and chromatin q Mitsuyoshi Nakao* Department of Tumor Genetics and Biology, Kumamoto University School of Medicine, 2-2-1 Honjo, Kumamoto 860-0811, Japan Received 25 June 2001; received in revised form 28 August 2001; accepted 14 September 2001 Received by A.J. van Wijnen

Abstract Epigenetic regulation is the mechanism by which gene function is selectively activated or inactivated in the cells. It provides higherordered and more specified genetic information, compared with the whole genome itself. Recently, a variety of regulatory proteins including DNA methyltransferases, methyl-CpG binding proteins, histone-modifying enzymes, chromatin remodeling factors, and their multimolecular complexes have been identified. These facilitate our understanding of the molecular basis for transcription, DNA replication, mutation and repair, DNA recombination, and chromosome dynamics, which are crucial for normal cell regulation. Abnormalities in the epigenetic states represent human disease phenotypes, especially developmental defects and tumorigenesis. Therefore, epigenetics will become the focus and a major target for emerging biological and medical discoveries. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Epigenetics; DNA methylation; Chromatin; Post-translational modification; Histone; Transcription

1. Introduction The twenty-first century, heralded as the century of life science, is coming to a point of reflecting significant intellectual properties of the genome projects, databases, and modern science and technology upon the next generation of research. Whole genome sequences have been determined for some living species including the human being; thus, we can now clearly recognize what kinds of genes exist in the genome, as if we know all kinds of cards for a metaphorical card game. However, it is essential to understand when, where and how these cards will be used. Somatic cells in an individual multicellular organism have basically identical genomes, but each of these cells has a distinct structure and function. This is due to the different uses of genes on the genome, that is, epigenetics. Epigenetic modification of the genome may involve cytosine methylation and chromatin, and therefore produce alterations in gene expression without any differences in DNA sequence. Looking back over the last few years, issues such as DNA q For further information, a special number featuring articles on the epigenetics was published by Science, volume 293, 10 August 2001. Abbreviations: DNMT, DNA methyltransferase; HAT, histone acetyltransferase; HDAC, histone deacetylase; MBD, methylated DNA-binding domain; MeCP, methyl-CpG binding protein; NuRD, nucleosome-remodeling histone deacetylase * Tel.: 181-96-373-5118; fax: 181-96-373-5120. E-mail address: [email protected] (M. Nakao).

methylation, histone acetylation, chromatin and chromosomes, transcriptional control and genome dynamics, which have been discussed separately, have turned out to be closely interrelated, as is discussed below. In order to understand cellular and biological phenomena such as development, aging and tumorigenesis, we need an overall view of epigenetics. In this special issue, I wish to provide recent information regarding biochemical aspects of the epigenetic system and its implication in human diseases. 2. Concept of epigenetics Dr Alan Wolffe defined the term epigenetics as “heritable changes in gene expression that occur without a change in DNA sequence” (Wolffe and Matzke, 1999). The definition agrees with the central theme of this review that epigenetics is achieved by DNA methylation and chromatin. Wolffe further placed most emphasis on the transcriptional repression mechanism, as seen in the subtitle ‘regulation through repression’. When we observe a single cell, inactivated genes are widely known to outnumber actively transcribed genes, suggesting that repression of unnecessary genes may be a basic rule of transcriptional control. Hence, epigenetics can be understood as an ingenious system to selectively utilize genome information, through activating or inactivating functional genes. At a molecular level, DNA methyltransferases, methyl-CpG binding proteins, histonemodifying enzymes, chromatin remodeling factors, tran-

0378-1119/01/$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0378-111 9(01)00721-1

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scriptional factors and chromosomal proteins cooperate together (Fig. 1B). Additionally, chromosome structures such as centromere, kinetochore and telomere come under the category of epigenetics even though they are or are not connected directly to gene function. With reference to

morphological features of epigenetics, euchromatin contains a lot of actively transcribed genes in an expanded and open structure. Meanwhile, heterochromatin shows a contracted and transcriptionally inactive condition. During DNA replication followed by somatic cell division, such

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epigenetic states in a parent cell are inherited identically by daughter cells, as DNA sequences are conserved during mitosis. Therefore, memories of epigenetic states, methylation patterns and chromatin organization are maintained during the cell cycle (Rakyan et al., 2001). Traditional depiction of gene expression shows that ‘DNA produces RNA produces protein’, the two steps being called transcription and translation. In such molecular conversion of DNA–RNA–protein, we occasionally regard the whole process except for the DNA sequence itself as epigenetics in a broad sense. In this case, it is an extensive concept that includes cytosine methylation and chromatin formation, RNA synthesis and degradation, polypeptide synthesis and post-translational modification, and turnover of protein. Thus, the DNA sequence is a program of life, and gene function is inherently controlled at DNA–RNA– protein levels. However, there will be no doubt that transcription is the most important and substantial process of epigenetic regulation.

mic methylation patterns in these somatic cells are generally stable and heritable. However, genome-wide methylation patterns are fully reprogrammed in mammalian germ cells and in pre-implantation embryos. In genomic imprinting and X-chromosome inactivation, which are major epigenetic phenomena in mammals, methylation is believed to be indispensable. Abnormal methylation patterns in many cancers induce the inactivation of tumor suppressor genes and the instability of the whole genome. In addition, 5-methyl-cytosine on the genome is spontaneously converted to thymine in the deamination reaction. This is the main cause for generating gene mutations in hereditary diseases and cancers when the mismatched T-G is inherited in the cell division without correcting it by the repair system. Foreign genes introduced into the cells for gene therapy, research and industry purposes tend to be methylated and suppressed as a host defense mechanism. The DNA methylation system mentioned below will give important clues to reveal the molecular basis of these biological phenomena.

3. System of DNA methylation

3.2. DNA methyltransferases and demethylase

3.1. DNA methylation

Mammalian DNA methyltransferases are classified into two groups: maintenance DNA methyltransferase (DNMT1 or maintenance methylase) and de novo methylase (Bestor, 2000). Maintenance methylase is highly active to methylate a hemi-methylated DNA that is methylated in one strand and unmethylated in the other of double-stranded DNA. It provides the methylation pattern to the newly replicated daughter strand, based on the parent strand. In addition to the enzymatic activities, DNMT1 was reported to repress transcription directly in cooperation with histone deacetylases (HDAC). On the other hand, recently identified de novo methylases (DNMT3a, DNMT3b) add a methyl group to unmethylated CpG base pairs, resulting in the creation of a new hemi-methylated and then fully methylated CpG. For this reason, de novo methylation is considered to be implicated in cell growth and differentiation, and in altered methylation in tumorigenesis. Further, DNMT3b was found to be mutated in patients with ICF syndrome (immunodeficiency in association with centromere instability of chromosomes 1, 9 and 16, and facial anomalies) (Okano et al., 1999; Xu et al., 1999). Demethylation activity still remains uncertain (Wolffe et al., 1999; Kress et al., 2001). There are two possible processes for removing a methyl group from methylated

In vertebrate genomes, 5-positioned carbon of cytosine in 5 0 -CpG-3 0 dinucleotide is usually modified by a methyl group. Cytosine residue in complementary 3 0 -GpC-5 0 that makes the base pairs is also methylated symmetrically, and these two methyl groups show a three-dimensional structure prominent in the major groove of the double-stranded DNA (Ohki et al., 2001). Approximately 60–90% of all CpG sequences in the genome are methylated, while unmethylated CpG dinucleotides are mainly clustered in the CpGrich sequence, termed CpG island, of the gene promoter region (Ng and Bird, 1999). Normally, both core promoter and transcription start site are included within the CpG island, and gene expression is completely repressed when this region becomes hypermethylated. DNA methylation, either reversibly or irreversibly, regulates genome functions through affecting gene transcription and chromatin formation (Bird and Wolffe, 1999). During cell differentiation, for example muscle and T-cell differentiation, genome methylation may specifically change to activate or inactivate genes that affect the determination of cell fate, and cell type-specific gene expression is also controlled by methylation in the differentiated cells. Geno-

Fig. 1. Overview of epigenetics. (A) Epigenetics is an advanced biological system that selectively utilizes genomic information and is involved in various fundamental phenomena. Specifically, it puts emphasis on the regulation of gene expression, through DNA methylation, chromatin, and post-translational modification of proteins such as histones. Arrows indicate possible functional interactions between them. DNA hypermethylation, histone hypoacetylation and inactive chromatin repress transcription. In contrast, a transcriptionally active condition may encourage DNA hypomethylation, histone hyperacetylation and active chromatin. Also, a particular chromatin structure may be required for establishing DNA methylation (see text). (B) DNA methylation and histone modification play key roles in transcriptional control. The figure shows transcriptional factors (TF), RNA polymerase (Pol II), general transcription factors (GTF), acetylated histone (Ac) and methylated cytosine (mC). Either HAT or HDAC recruited by TF and other DNA-binding proteins induce transcriptional activation or repression, respectively. Modifications (acetylation, phosphorylation, methylation) of histone tail domains are described in the text. Chromatin remodeling factors convert the chromatin to active (upper) and inactive (lower) states.

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DNA. One is a passive mechanism whereby methylation is not maintained during the DNA replication, and the other is an active mechanism catalyzed by an as yet unidentified DNA demethylase(s). Although MBD2 noted below was reported to have demethylase activities, this result has not been reproduced by other research institutions. In addition, 5-methylcytosine DNA glycosylase is noted as a candidate for demethylase in vivo (Zhu et al., 2001). Demethylation of the genome may have great consequences for the regulation of transposons, imprinted genes, and genes on the inactive X-chromosome. At present, we need to focus on the fundamental problems, that is, how DNA methylases and demethylation are functionally regulated, and how methylation patterns are established, maintained and eliminated.

3.3. Methyl-CpG binding proteins DNA methylation is known as an epigenetic mark identifying the template strand in DNA replication and the parental origin in imprinted regions of the genome. It is also known that methylation alone hinders DNA-binding activities of methylation-sensitive transcriptional factors including E2F, CREB, AP2, cMyc/Myn, NF-kB, cMyb, and ETS. In addition to methylated DNA, methyl-CpG binding proteins are theoretically required to inhibit transcription by methylation-insensitive transcriptional factors such as Sp1, CTF and YY1. Methyl-CpG binding proteins are deciphering epigenetic methylation patterns and moreover mediate interactions between DNA methylation, histone deacetylation, and chromatin components. Currently, five family members with a conserved methylated DNA-binding domain (MBD) have been described. Among them, MeCP2, MBD1, MBD2 and MBD3 can be involved in methylationmediated transcriptional repression, and MBD4 has a DNA glycosylase activity for removing a thymine from T-G mismatch sites (Bird and Wolffe, 1999; Ballestar and Wolffe, 2001). The MeCP1 complex, first noted by Dr Adrian Bird’s group, was reported to consist of MBD2 and other proteins, and some components may be different in cell types. The MeCP2 gene locates on the X-chromosome and was mutated in the Rett syndrome patients (Amir et al., 1999). This syndrome is the most frequent of the female neurodevelopmental disorders, with loss of speech, autism, ataxia, erratic hand movements and mental retardation, being recognized from 6–18 months after birth. The MBD4 gene is also altered in tumors with microsatellite instabilities. A recent gene knockout strategy in mice found that MBD3 is required for embryonic development, whereas MBD2-deficient mice are viable (Hendrich et al., 2001). MBD2-deficient cells lacked MeCP1 complex and can not efficiently repress exogenous methylated promoter. However, it is further necessary to analyze the inactivation of the endogenous methylated gene by MBD-containing proteins, and their functional interrelationship and redundancy in vivo.

4. Chromatin conversion system Genomic DNA is folded in the nucleus as a multimolecular complex with proteins, called chromatin. Nucleosome is a fundamental unit of chromatin and consists of core histones bound to DNA. Dr David Allis and colleagues proposed the concept of ‘histone code’ whereby combinations of N-terminal modifications on histones, including acetylation, methylation, phosphorylation, ubiquitination and ADP-ribosylation, have an influence on gene expression, DNA replication and chromatin-dependent processes (Strahl and Allis, 2000). Phosphorylation at serine 10 of histone H3 is important for chromosome condensation in mitosis and for an initial response to mitogens, and it is also suggested that this phosphorylation induces acetylation of neighboring lysine residues by histone acetylases. Recently, H3-specific methylase was identified, and methylation at lysine 9 has been proved to inhibit phosphorylation at serine 10 by the Ipl-1/aurora kinase. The H3 methylation at lysine 9 generates a binding site for heterochromatinassociated protein HP1 (Jenuwein, 2001). In contrast, H3 methylated at lysine 4 is specific to the euchromatic regions and correlates with H3 acetylation. Thus, it can be assumed that the different modifications of one histone are functionally related to each other. Further, Drosophila TAFII250, a major subunit of the basic transcription factor TFIID, was reported to be a ubiquitinating enzyme for histone H1, and mono-ubiquitination of H1 promoted transcriptional activation (Pham and Sauer, 2000). A unique intranuclear structure, PML body or nuclear dot 10 (ND10), widely modulates histone-modifying enzymes and transcriptional factors (Zhong et al., 2000). Interestingly, some molecules connected to this nuclear body are conjugated with the ubiquitin-like protein SUMO-1 or sentrin. Thus, the details of these post-translational modifications are the subject of a number of studies. Here, I focused on histone-modifying enzymes and chromatin-remodeling complexes as the chromatin conversion systems. 4.1. Histone-modifying enzymes Acetylation of histones H3 and H4 normally increases gene expression by promoting an open chromatin structure. Transcriptional co-activators such as CBP/p300 and PCAF are intrinsic histone acetyltransferase (HAT) (Marmorstein and Roth, 2001). Conversely, HDAC contribute to form transcriptional co-repressor complexes. Two complexes, SIN3 and Mi2-NuRD (nucleosome-remodelling histone deacetylase), are known to take HDAC1/HDAC2 as component molecules (Ahringer, 2000; Knoepfler and Eisenman, 1999; Wade, 2001). As shown in Fig. 2, HDAC1/HDAC2 and RbAp46/RbAp48 are core proteins common to these complexes. Sin3A/Sin3B, SAP30, and SAP18 participate in the SIN3 complex, while Mi2, MTA1/MTA2, and MBD3 are specific subunits for the

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Fig. 2. HDAC complexes. Both SIN3 and Mi2-NuRD complexes contain HDAC1/HDAC2 and RbAp46/RbAp48 as core molecules. The SIN3 complex additionally has Sin3A/Sin3B, SAP18, and SAP30, while the Mi2-NuRD complex contains Mi2, MTA1/MTA2, p66, and MBD3. The SIN3 complex is recruited to methylated DNA by interaction with MBD2 and MeCP2. The Mi2-NuRD complex seems to be localized in the methylated region by MBD3MBD2 interaction. Many other factors interacting with these complexes are known but not shown for simplicity.

Mi2-NuRD complex. The SIN3 complex has an effect on chromatin through interacting with sequence-specific transcription factors or co-repressors, including Mad-Max, nuclear hormone receptor and N-CoR/SMRT, and methyl-CpG binding proteins such as MeCP2 and MBD2. On the other hand, MBD3, one of components in the Mi2NuRD complex, has a MBD-like sequence but very weak binding affinity to methylated DNA. NMR analysis suggested that some amino acids important for methylCpG binding of the MBD are substituted to another residue in MBD3 (Ohki et al., 2001). It is postulated that the MBD3-MBD2 interaction recruits the Mi2-NuRD complex to methylated DNA regions. Thus, DNA methylation and histone deacetylation are cooperatively involved in transcriptional repression. The SIN3 and Mi2-NuRD complexes may be effective in long-term and short-term transcriptional repression, respectively, since the Mi2NuRD has additional chromatin remodeling activity as seen below. It is of great interest how they share and split their functions in transcriptional repression. Until now, human HDACs have been roughly divided into three classes: HDAC1, HDAC2, HDAC3 and HDAC8 in class I, and HDAC4, HDAC5, HDAC6 and HDAC7 in class II. Based on the protein structure, classes I and II correspond to Rpd3 and Hda1 in yeast, respectively. Recently, Yeast Sir2 has been reported to be a new class of HDAC, as a NAD-dependent HDAC enzyme (Guarente, 2000). 4.2. Chromatin remodeling and assembly factors Transcription, DNA replication, repair and recombina-

tion are dynamically carried out at the chromatin level. Chromatin remodeling represents a change of nucleosome position and conformation, leading to chromatin assembly and disassembly. ATP-dependent chromatin remodeling complexes, especially the SWI/SNF and ISWI families, were initially found in yeast and Drosophila (Kingston and Narlikar, 1999; Workman and Kingston, 1998). As an ATPase subunit of chromatin remodeling complexes in human, BRG1 and hBRM for SWI/SNF and hSNF2L/ hSNF2H for ISWI are well known (Fig. 3). The hSWI/ SNF complex, which is a huge multimolecular structure including either BRG1 or hBRM and tumor suppressor protein hSNF5/Ini-1, mainly activates gene transcription (though gene inactivation was also reported). These complexes are also associated with the cell cycle and the assembly of immunoglobulin and TCR genes by V(D)J recombination. The RSF heterodimer complex including hSNF2H is involved in the initiation of transcription. The above-mentioned Mi2-NuRD complex has been reported to convert active to inactive chromatin due to both ATPase of Mi2 and HDAC activities (Fig. 2). In addition, the HuCHRAC complex including hSNF2H and chromatin assembly factor hACF1 is considered to associate with the replication and maintenance of heterochromatin. There is a possibility that topoisomerase II may be included in the HuCHRAC. As a chromatin assembly complex, the CAF1 complex has a function in the maintenance of chromatin coupled to DNA replication. In some interesting analyses of mutants in arabidopsis, mutations of chromatin remodeling factor termed DDM1 were found to cause hypomethylation of the genome (Mittelsten Scheid and Paszkowski, 2000). It is thus suggested that DDM1-mediated chromatin

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Fig. 3. Chromatin remodeling and assembly factors. The hSWI/SNF complex, which is a large multimolecular structure containing either BRG1 or hBRM and tumor repressor protein hSNF5/Ini-1, mainly activates gene transcription. The heterodimer RSF complex containing hSNF2H is involved in initiation of transcription. The CAF1 complex is related to chromatin assembly in DNA replication. The HuCHRAC complex containing hSNF2H and hACF1 maintains heterochromatin. The Mi2-NuRD complex represses transcription through Mi2 and HDAC activities (Fig. 2). BRG1, hBRM, hSNF2H, and Mi2 show ATPase activities. Variant forms of these complexes may occur, but are not all indicated here.

formation may be a requisite for the maintenance of genome methylation. We expect that information on chromatin remodeling will rapidly increase in the near future.

5. Conclusions The ‘shape’ of chromatin effectively supports the ‘language’ of genetic information. Epigenetics is a physiological system that enables ‘motion’ of the genome, leading to the conducting of all the biological activities of the genome. Replicated DNA is immediately methylated in somatic cells, and methyl-CpG binding proteins recognize the methylation patterns. Histone-modifying enzymes, chromatin remodeling factors, transcriptional factors and co-regulators, and chromosomal proteins such as Polycomb-group enter chromatin complexes to establish and maintain the epigenetic states (Muller and Leutz, 2001). Looking at the processes for gametogenesis or early embryogenesis, both DNA methylation and chromatin are dynamically reconstructed in the whole genome. In addition to such global roles, local epigenetics in a single chromosome, a chromosomal subdomain, or a specific gene locus also

plays an important role. We will be able to understand the overall concept of epigenetics by investigating relationships between DNA methylation, chromatin, and post-translational modification of histones and other proteins, and many biological phenomena (Fig. 1A). It is of note that human diseases related to abnormal epigenetic conditions are dramatically increasing (Table 1), showing that the molecules and protein complexes discussed here can be appropriate targets for new medical therapies and drug discoveries. In fact, HDAC inhibitors are extensively proposed as a new anti-cancer drug (Marks et al., 2000). Epigenetics is responsible for clarifying mechanisms for selective utilization of genes on the genome. Accordingly, the universal significance of epigenetics will ensure it is one of the central fields of life science in the future.

Acknowledgements Drs Naoyuki Fujita, Kazuhito Matsuzaki, Masahide Tojo, Sugiko Watanabe, Hideyuki Saya, and Masahiro Shirakawa are thanked for their help. I apologize that all works were not cited here due to space limitations. Our work is

M. Nakao / Gene 278 (2001) 25–31 Table 1 Epigenetics and human diseases Gene/protein DNA methylation system MeCP2 MBD2 MBD4 DNMT3b Epigenetic regulation of genes FMR-1 IGF2 Imprinted genes

Tumor suppressor genes X-Inactivation center Histone acetylation system CBP p300 MOZ-CBP MLL-CBP Histone modification Phosphorylation defect of histone H3 Chromatin remodeling system Mi2 MTA1 hSNF5/Ini-1 BRG1 ATRX Transcriptional control PML-RARa

Disease

Rett syndrome Colon cancer antigen Tumors with microsatellite instability ICF syndrome Fragile X mental retardation Wilms’ tumor Prader–Willi & Angelman syndromes, Beckwith– Wiedemann syndrome Many tumors Functional disomy of X-linked genes Rubinstein–Taybi syndrome Gastric cancer, colon cancer, brain tumor Acute myelocytic leukemia Leukemias Coffin–Lowry syndrome

Autoantibody in dermatomyositis Metastatic potential of cancer Rhabdoid tumor Tumors a-Thalassemia/mental retardation syndrome, X-linked Acute promyelocytic leukemia

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