Human Molecular Genetics

Human Molecular Genetics 2006 15(Review Issue 1):R95-R101; doi:10.1093/hmg/ddl095

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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

The emerging science of epigenomics

Pauline A. Callinan and Andrew P. Feinberg^*

Division of Molecular Medicine, Department of Medicine and Center for the Epigenetics of Common Human Disease, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA

^* To whom correspondence should be addressed at: Division of Molecular Medicine, Department of Medicine and Center for the Epigenetics of Common Human Disease, Johns Hopkins University School of Medicine, 1064 Ross, 720 Rutland Avenue, Baltimore, MD 21205, USA. Tel: +1 4106143489; Fax: +1 4106149819; Email: afeinberg{at}jhu.edu

Received March 2, 2006; Accepted April 3, 2006

	ABSTRACT

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One of the most exciting frontiers in both epigenetics and genome sciences is the new field of epigenomics. This new discipline promises novel insights into the genome because of its potential to detect quantitative alterations, multiplex modifications and regulatory sequences outside of genes. A number of new epigenomic strategies are emerging to exploit microarray formats with varying substrate choice, pre-processing and data analysis. These approaches are designed to detect large numbers of variations in DNA methylation and chromatin modification. Many groups are joining forces toward developing an organized Human Epigenome Project to exploit these new technologies to better understand the basis of normal development and human disease.

	Introduction

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One of the most exciting frontiers in both epigenetics and genome sciences is the new field of epigenomics or the study of epigenetic modification at a level much larger than a single gene. Epigenetics is the study of heritable changes other than those in the DNA sequence and encompasses two major modifications of DNA or chromatin: DNA methylation, the covalent modification of cytosine, and post-translational modification of histones including methylation, acetylation, phosphorylation and sumoylation (1). Functionally, epigenetic marks act to regulate gene expression (2), silence the activity of transposable elements and stabilize adjustments of gene dosage, as seen in X inactivation and genomic imprinting. Curiously, the term epigenetics has evolved from its original definition by Waddington, meaning essentially developmental biology, yet epigenetics in the current meaning of the word may in fact be critical to understanding developmental biology. This is because the DNA sequence is invariant across tissues with the exception of rearranged genes such as immunoglobulin family members, yet the epigenome shows tissue-specific variation. Given the novelty of this field, we do not yet know much about the epigenome. In this review, we will discuss the concept of epigenomics, emerging technology in its study and early examples of its application, emphasizing progress within the last 12–18 months.

	POTENTIALLY UNIQUE CONTRIBUTIONS OF EPIGENETICS TO GENOMIC SCIENCES

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Epigenomic data promises a number of unique contributions to the study of genomic sciences. First, epigenetic information is inherently multiplex, with hundreds of potentially methylated cytosines in a gene and dozens of known post-translational modifications of chromatin. Secondly, epigenetic information is quantitative, in contrast to the sequence itself, which is discrete. Tissues can maintain partial methylation at a locus, and the extent of methylation at affected sites can vary. Chromatin modifications are also inherently quantitative due to the opposing action of gene-activating trithorax proteins and gene-silencing polycomb complexes. Thirdly, epigenetics may aid in understanding the function of DNA regulatory sequence within the mammalian genome. Indeed, there is more constrained non-coding than coding sequence in most complex genomes (3), and much of this is methylated (4). Finally, the topological conformation of DNA within the nucleus is thought to be epigenetically controlled, and emerging studies suggest that its arrangement is important in gene regulation (5).

	EMERGING TECHNOLOGY OF EPIGENOMICS RESEARCH

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Historically, technology has limited large-scale approaches to epigenomics, but the emergence of highly reproducible quantitative high-throughput microarray technology will allow virtually all epigenomics research to be read on microarray platforms, although the substrates, pre-processing and data analysis will differ substantially depending on the modification that is being addressed (Tables 1 and 2). Early approaches to epigenomics used custom-made slide-based arrays of CpG rich regions corresponding to methylated or unmethylated DNA (6,7). However, in the last 18 months there has been a shift toward commercial high-density oligonucleotide arrays because of their greater precision and potential quantitative character. These include the photolithographic masked arrays of Affymetrix, photolithographic adaptive optics arrays of NimbleGen, inkjet arrays of Agilent and, recently, the adaptation of bead arrays for epigenetic applications of Illumina. Each of these approaches offers potential advantages and disadvantages but as yet, no direct comparison of epigenomic technology has been performed across platforms. However, comparisons of conventional gene expression analysis across platforms have been published and illustrate that commercial platforms perform similarly and that lab effect—variance introduced into the data through the operator—affects precision more than the platform type (8). An advantage of a flexible design for epigenomics is that one can tailor arrays to genomic targets of interest, such as imprinted genes, differentially methylated regions (DMRs) and imprinting control regions.

View this table: [in this window] [in a new window]   Table 1. Glossary of terms in epigenomics

 

View this table: [in this window] [in a new window]   Table 2. Emerging new technologies in epigenomic science

 

	DNA METHYLATION STUDIES

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The aberration of DNA methylation is now well established in cancer (9,10) and is associated with a number of genome-wide alterations. For example, global hypomethylation leads to oncogene activation (11) and chromosomal rearrangement. In addition, chromatin modification and hypermethylation are observed in cancer and are associated with tumor suppressor gene silencing (12). Loss of imprinting, or loss of parent of origin-specific gene silencing, appears to increase colorectal cancer risk in humans and mouse models (13–16). Altered DNA methylation may also play an important role in psychiatric and other complex disorders. For example, Rett syndrome, which causes autistic-like features and eventual global mental deterioration, is caused by a mutation in methyl CpG binding protein 2 and involves loss of imprinting of DLX5 (17,18). Dietary methylation can also affect the expression of mutant gene phenotypes in mice (19).

The detection of DNA methylation is based on the ability to distinguish cytosine from 5-methylcytosine in the DNA sequence. There are three main strategies for methylation detection: the digestion of DNA by a methylation-sensitive or -insensitive restriction endonuclease; the chemical modification of DNA by sodium bisulfite or metabisulfite; and immunoprecipitation of 5-methylcytosine to separate directly the unmethylated and methylated fractions of the genome. Recently, all three approaches have been coupled to high-throughput technologies. One of the challenges of these approaches is to detect methylation variation on a large genomic scale, without completely sacrificing the ability to resolve subtle changes in individual genes.

The first methylation-based technologies we discuss are restriction enzyme-based. An adaptation of restriction endonuclease-based approaches to high-throughput genome analysis is restriction landmark genome scanning (RLGS) (20), a two-dimensional approach combining restriction enzyme polymorphisms and DNA methylation-sensitive sites and originally developed for tumor clonality assays (21). RLGS has been used to successfully identify a number of imprinted genes but is time consuming and the resolution is low. More recently, some restriction endonuclease-based assays have been coupled with high-throughput microarray technology, leading to significant improvements in genomic resolution and detection sensitivity. An example illustrates the utility of BAC clone arrays, which can be hybridized with biotin-labeled DNA generated from end-filling of genomic DNA samples digested with the rare-cutting methylation-sensitive restriction endonuclease NotI (22). BAC clones are a useful resource from which to gather CpG methylation data, as they contain CpG islands. In addition, NotI cuts ~75% of all CpG islands in the human genome. By sampling two types of human cell, astrocytes and peripheral blood lymphocytes, the technique was able to resolve a number of differentially methylated CpG islands, including tissue-specific methylation of SHANK3, a gene involved in post-synaptic density. The tissue-specific nature of the methylation mark was found to be conserved in human, rat and mouse (22). Further comparative genomics studies of this nature may enable the discovery of other functionally important but, as yet, unannotated genes in the mammalian genome.

Another technique combining both restriction enzyme digestion and microarray technology uses differential hybridization of unmethylated and total genomic DNA. Known as HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR) (23), the assay is a quantitative restriction enzyme-based assay that interrogates both CpG islands and other unmethylated CpG-rich genomic regions at high resolution (200 bp). This is achieved by co-hybridizing HpaII digestion fragments (unmethylated DNA enrichment) with digestion fragments from a methylation-insensitive isoschizomer (MspI) onto a customized array.

An exception to our suggestion that microarrays are the converging platform for epigenomics involves a modification of digital karyotyping which embodies SAGE technology, a general high-throughput sequencing alternative to microarrays. Methylation-specific digital karyotyping (MSDK), like the methods described earlier, uses methylcytosine-sensitive restriction endonucleases to discriminate methylated from unmethylated DNA, but generates short-tagged DNA segments, approximately 7000, through restriction digestion of the genome and maps their position in the genome using SAGE. Although this technology has a relatively low resolution when compared with others, the tagged DNA is identified through simple direct sequencing rather than through microarray hybridization, potentially eliminating some problems associated with statistical interpretation of microarray data. Using breast epithelial, myoepithelial and stromal cells in both normal and tumor states, significant global hypomethylation in tumor epithelial cells was discovered when compared with normal tissue, in addition to significant differences in the methylation status of normal and tumor states in all cell types (24).

A second approach to high-throughput DNA methylation analysis takes advantage of the bisulfite treatment of DNA, which converts cytosine to uracil. After PCR the sequence is read as thymine by DNA sequencing unless the base is methylated (25), allowing one to discriminate methylated from unmethylated DNA. The method is based on the fact that the methyl group at the C5 position of the pyrimidine ring inhibits the hydrolytic deamination of the 5,6-dihydrocytosine-6-sulphonate product at the C4 position (26,27). One application is automated high-throughput sequencing of treated material, which requires many PCR reactions on a setting of a reduced complexity genome after treatment and thus creates more problematical PCR than in conventional DNA sequencing. An alternative detection platform is MALDI-TOF mass spectrometry, which can read a single nucleotide inexpensively at very high throughput (28). Another approach involves hybridizing PCR-amplified bisulfite-treated DNA to microarrays. This method has been used to predict the tumor class of samples on the basis of large-scale analysis of CpG methylation patterns (7) and to determine that brain-specific methylation differences exist between human and chimpanzees (29). High-throughput analysis of bisulfite-treated DNA can also be performed using MethyLight, a methodology that utilizes real-time PCR (30), and has been used to investigate DNA methylation marks shown to be associated with patient prognosis in colorectal (31,32), bladder (33) and cervical (34,35) cancers.

All of the bisulfite-based approaches are dependent upon complete chemical conversion of unmethylated DNA and robust amplification of the treated material, banes in the laboratories of even the most dedicated epigenomicists. The hybridization-based applications of this method also depend upon complete methylation or unmethylation of the template. A further limitation of sequencing is cost, although emerging very high-throughput sequencing technologies may change this equation.

A third epigenomic approach to methylation quantification is taking a lead from chromatin immunoprecipitation (ChIP) assays by using anti-methylcytosine antibodies to isolate methylated DNA directly from the genome. A 5-methylcytosine-specific antibody is used to enrich for methylated sequences. This method, termed MeDIP (methylated DNA immunoprecipitation), was used in a high-resolution analysis of methylated sequences coupled to a comparative genomic hybridization (CGH) microarray (36). One of the most common alterations in cancer is genomic instability, which leads to the gain and/or loss of genomic material. In lung adenocarcinoma, the MeDIP/CGH array detected a simultaneous copy number gain and hypomethylation of locus 1q21–q23 containing Mcl-1, a gene known to be overexpressed in non-small cell lung carcinoma. Thus, both genetic and epigenetic factors appear to play a role in the overexpression of Mcl-1, although it is not certain which change occurs first. Additional data documented large regions of hypomethylation in transformed cells in gene-poor regions, with surprisingly little hypermethylation found in CpG island promoters when compared with normal primary cells (36). Loss of DNA methylation may be a major factor inducing chromosomal rearrangements in cancer (reviewed in 37). Indeed, an assay that uses CGH coupled with methylation screening capabilities will shed light on the complex molecular etiology of common cancer.

	ChIP STUDIES

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In addition to direct DNA methylation, epigenetic modifications to the histone proteins can influence gene expression by regulating the accessibility and recruitment of transcriptional regulatory proteins to the DNA. The regulation of chromatin is accomplished in part via the chemical modification of the histone N-terminal tails that protrude from the nucleosome. For example, gene silencing is associated with the methylation of the ninth amino acid, lysine, from the N-terminal end of histone H3 (H3K9). Acetylation at the same lysine residue, on the other hand, is associated with transcriptional activation. Interestingly, methylation is not always tied to gene silencing. For example, the methylation of histone H3K4 is a well-characterized modification of active genes. Owing to the complex nature of the histone code, defined as the pattern of chemical modifications read by proteins to stimulate downstream events (1), much remains to be learned about how the eukaryotic genome is epigenetically regulated.

ChIP is commonly used to study the chromatin state of a living cell (38). ChIP involves the crosslinking of DNA and its associated binding proteins in vivo by chemical treatment. Following shearing, the DNA–protein complex is immunoprecipitated by antibodies specific for a protein or histone modification of interest. The DNA sequence located in the vicinity of the binding protein can then be analyzed, providing investigators with location information and, depending on the histone modification or protein of interest, insight into the transcriptional activity level of captured nearby genes. The development of ChIP on chip (39), a microarray platform upon which immunoprecipitated DNA is hybridized against known probes, is enabling high-throughput mapping of chromatin marks and protein binding sites.

The popularity of ChIP on chip to assess chromatin states has grown with the availability of high-density arrays that enable the binding signal to be captured across contiguously tiled oligonucleotide probes. Until the arrival of high-density genomic tiling arrays, ChIP on chip experiments relied upon single probes within and between genes, likely missing much of the important epigenetic information. A recent study utilized a whole genome tiling array of Saccharomyces cerevisiae to map genes with transcriptional activity using antibodies specific for epigenetic marks of activation (acetylated H3K9 and H3K14, hyperacetylated histone H4K5, 8, 12, 16 and mono-, di- and tri-methylated H3K4, 36, 79) (40). Data showed that the transcriptional activities of yeast genes were generally positively correlated with the presence of the activating marks at the promoter regions, with few exceptions. It was noted that active transcription is always accompanied by specific chromatin modifications, such as trimethylated H3K4 at the promoter and H3K4 dimethylation and monomethylation further downstream within the transcriptional unit of the gene (40). A whole genome approach such as this is fundamental to understanding the role that chromatin plays in gene expression, and in chromatin maintenance in a cell, and should be useful in deciphering the histone code in more complex genomes.

ChIP on chip provides the researcher with flexibility in experimental design. Enriching for a specific transcriptional protein allows for the study of protein networks, or a specific gene class, whereas the study of histone modifications allows the researcher to study generalized chromatin behavior. In well-defined model organisms, it is possible to investigate the binding sites of all known transcription factors that allow for the construction of complete genome-wide maps. ChIP on chip was recently used in this way to investigate the transcriptional map of yeast (41). A total of 203 strains were generated by integrating a myc-tag into the endogenous gene of regulator proteins chosen using a bioinformatic interrogation of yeast DNA binding sites, phylogenetic analysis and prior knowledge. Alteration of the yeast growth conditions enabled the different patterns of organization of specific transcription factors along the DNA sequence to be determined and further facilitated the localization of hitherto unknown transcription factor binding sites.

An alternative approach to mapping transcription factor binding proteins is serial analysis of chromatin occupancy (SACO) (42). Similar to the methylation assay MSDK described earlier, SACO is based on the SAGE technique of identifying mRNA transcripts, a quantitative technique in the sense that the number of tagged fragments recovered from a particular locus represents the frequency of protein binding to that locus (42). Regulator protein binding frequency may provide data on the transcriptional activity of the locus in question. In addition, SACO is able to identify the binding sites of regulatory proteins that are not well defined or are located variably within and around the gene. A similar use of ChIP and SAGE has been adopted using a genome-wide mapping technique (GMAT) to map histone modifications across the yeast genome (43). Another related and clever approach involves ChIP and paired-end ditag sequencing (ChIP-PET) (44). Techniques such as SACO, ChIP-PET and GMAT will prove useful for the unbiased identification of transcriptional activity in eukaryotic genomes of high complexity.

Unlike in yeast, where many individual transcriptional regulatory proteins have been genetically tagged, allowing direct immunoprecipitation of the target sequences, studies of mammalian chromatin complexes have relied on indirect methods. Nevertheless, recent progress has been made examining promoter complexes. The pre-initiation complex (PIC), a complex that is common to all promoters of active genes transcribed by RNA polymerase II, was recently used to this end (45,46). This study, utilizing ChIP on chip on human fetal lung cells, determined the location of 252 PIC binding sites on a tiling array format with ~1% genomic coverage (45). Although approximately three quarters of the PIC sites found were located at the 5' end of known genes, the remainder represented unknown promoter sites within the human genome, half of which were shown to produce messenger RNA transcripts. The other half of the unannotated promoter sites were assayed for transcriptional enhancing properties in transient transfection assays using a luciferase reporter gene. From this group, an additional six were found to enhance transcription over controls (45). Further genome-wide studies by the same group determined the existence of over 1000 previously un-annotated promoter sites and illustrated the complex multi-promoter nature of human genes (46). In addition, the study highlighted the clustering of transcriptionally active promoters, a phenomenon that is likely influenced by long range epigenomic regulators, boundary elements and higher order chromatin configuration.

A major interest in current biology is the identification of regulatory sequences within the human genome sequence. Recently, a strategy that exploits the observed hypersensitivity of regulatory sequences to endonuclease DNase I has been employed to predict, via the analysis of chromatin structure, the location of regulatory sequences in complex genomes (47). Quantitative chromatin profiling (QCP) isolates intact nuclei, half of which are treated with DNase I. Primers are designed to amplify 250 bp fragments across candidate gene loci, and the relative number of intact amplicons is determined in the treated sample compared to control. By plotting the ratios of intact amplicons according to the genomic position, a baseline of sensitivity can be determined and outliers identified as hypersensitive sites and likely regulatory elements. As the majority of genetic variation resides within the non-coding portion of the genome, the identification of regulatory regions may be important in understanding the large amount of phenotypic variation observed in the human species.

	THE HUMAN EPIGENOME PROJECT

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Over the last 18 months, an international consensus has emerged in the epigenetic community for the need of an organized human epigenome project. Indeed, efforts toward that end have already been initiated in Europe. An international collaboration among the Sanger Center, Epigenomics AG and The Centre National de Génotypage established the Human Epigenome Consortium, which has examined 150 loci in the MHC region of chromosome 6. The approach uses automated bisulfite DNA sequencing on small aliquots of material on a panel of cell lines and is expanding to four chromosomes (48). A second group is the European Epigenome Network of Excellence led by Thomas Jenuwein, is a consortium of 25 established research groups, 12 newly established research teams and 26 associates involved in technology development, animal models, biochemistry and translational science. Third is ‘HEROIC’, or High Throughput Epigenetic Regulatory Organisation in Chromatin, an EU-funded collaboration among 11 academic centers and two companies, led by Henk Stunnenberg. Its two goals are to develop high-throughput tools for the analysis of chromatin–DNA interaction and to apply these tools to models of progenitor and differentiated cell types. Fourth is a private/public partnership involving eight centers funded by the German government, primarily for technology development and application, led by Joerg Hoheisel.

Last summer, the American Association for Cancer Research sponsored a workshop to formulate a proposal for a Human Epigenome Project with a working group tasked to define the number of epigenomes that should be analyzed and at what resolution and to define a series of tissues and pathological states which could be investigated relatively quickly at a lower level of resolution (49). A National Cancer Institute workshop that followed from that meeting and an earlier National Cancer Institute Think Tank proposed a long-term goal of 1 bp resolution of DNA methylation and a comprehensive analysis of chromatin, including modifications (such as H3K9 and H3K4 methylation), chromatin factor binding (such as polycomb, HP1) and structural change (such as DNase hypersensitivity). These investigations would be performed on normal human embryonic stem cells, fibroblasts and epithelial cells derived from newborns and a panel of 10 normal tissues corresponding to targets of common human disease to be selected by experts in the tissue biology. A short-term goal would be to examine the same targets at 1 kb resolution in pilot projects. The value of an organized and comprehensive effort is that it would reveal without bias the nature of the epigenome and its relationship to development and disease, as only a tiny fraction is currently amenable to analysis. It would also permit investigators outside of traditional epigenetics to ask novel questions based on the free availability of this epigenetic information and use standardized tools and reagents for its study.

An example of an early step in approaching the epigenome comes from a recent study by Fraga et al. (50), which addressed the relationship between epigenetics and age. They used samples from monozygotic (MZ) twins, a tactic commonly used to quantify the environmental component of complex human diseases. MZ twins, despite being genetically identical, have been observed to frequently display phenotypic discordance for complex diseases, especially psychiatric disorders (51). In order to examine whether epigenetic differences exist in the genomes of identical twins, Fraga et al. used a number of global- and locus-specific measurements of epigenetic modification, such as HPLC, AIMS, RLGS and microarray analysis. The study determined that epigenetic disparities are present in the identical genetic background of MZ twins and that the number of differences increases significantly with age (50). This study serves to highlight the important contribution of an individual's epigenotype to the phenotypic manifestation of the inherited genotype. By comparing the epigenotype of discordant MZ twins in disease studies, researchers may eventually determine the source of many complex common diseases.

Epigenomics, the merged science of epigenetics and genomics, has arisen as a new discipline with the aim of understanding genetic regulation and its contribution to cellular growth and differentiation, disease and aging. Multiple complementary technologies are emerging to analyze DNA methylation, protein binding patterns and chromatin regulation on a genome-wide level. Early efforts are providing glimpses into the epigenetics of gene regulation and the mechanism of cancer and aging. It is hoped that the development of high-throughput technologies will continue to unravel the enigma of the epigenome.

	ACKNOWLEDGEMENTS

 
We thank Carroll Davis for the critical reading of the manuscript. This work was supported by NIH Grant P50HG003233 (A.P.F.).

Conflict of Interest statement. None declared.

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