NATURE|Vol 454|7 August 2008 TECHNOLOGY FEATURE Detailed analysis Researchers now have access to an burgeoning collection of tools for unravelling the , which could lead to new drug targets and ways to track disease. Laura Bonetta reports.

Just as DNA mutations can cause disease, so too can changes in gene activity that don’t depend on DNA sequence. For example, COON J. the addition of methyl groups to cytosines within the regions of a gene, a proc- ess known as DNA methylation, typically represses the gene’s activity. Similarly, the methylation, acetylation or ubiquitination of certain amino acids on — the main protein components of — can change the way DNA is wrapped around the histones and can affect which nearby genes are available for activation by regulatory proteins such as transcription factors. Such ‘epigenetic’ processes can have dire consequences. Many cancers and other dis- eases have aberrant DNA methylation at particular sites in the . And certain patterns of modification are key to bio- logical events such as embryonic development, ageing and cell-cycle regulation. “We have found a gene that is highly meth- ylated in more than 90% of colorectal can- cers, regardless of stage and histology,” says Achim Plum, senior vice-president for cor- Mass spectrometry analysis of histone H4 isoforms in human stem cells. When differentiation was porate development at Epigenomics in Berlin, induced in the cells, levels of unmethylated isoforms fell and dimethylated isoforms rose.

TACKLING THE EPIGENOME So far, the approach to the followed by a more thorough The Epigenome Network of in Los Angeles, AHEAD would epigenome has been somewhat analysis of the methylation Excellence, headed by Thomas coordinate an international piecemeal, with disparate groups patterns on three chromosomes. Jenuwein of the Research Institute interdisciplinary project to map cataloguing just the modifications After showing the feasibility of of Molecular Pathology in Vienna, a defined subset of epigenetic in their cells or organisms such an approach “the consortium Austria, brings together 81 markers in a limited number of interest. Technological has decided to leave it to others laboratories from 10 European of human tissues at different advances that allow faster and to continue the endeavour”, says countries under a €12.5-million stages of development (see cheaper mapping of epigenetic Achim Plum, senior vice-president (US$19.7-million) grant from page 711). This would provide a modifications are now helping to for corporate development at the European Union to advance reference epigenome to which unite these efforts. Epigenomics. epigenetic research over the next samples from various diseases The Human Epigenome Project, ENCODE, or the Encyclopedia five years. The network, which could be compared. The alliance for example, was established of DNA Elements, was launched officially began in June 2004, would also try to develop a in 1999, when researchers in in September 2003 by the supports members and non- infrastructure Europe teamed up to identify, US National Human Genome members so that they can attend to support the collection of catalogue and interpret genome- Research Institute (NHGRI) in conferences, workshops and epigenomic data. wide DNA methylation patterns Bethesda, Maryland. The project training visits, as well as offering AHEAD’s planned efforts in human genes. The project aims to identify all the functional them shared resources. will be helped by January’s is a collaboration between the elements in the human genome The US American Association announcement from the National Wellcome Trust Sanger Institute sequence. Following initial for Cancer Research is Institutes of Health that it will in Hinxton, UK, the National success, the NHGRI last October championing the formation invest more than $190 million over Centre for Genotyping near pledged $80 million over four of the Alliance for the Human the next five years to accelerate Paris, France, and the German years to scale up the project. Epigenome and Disease epigenomics research. Grant company Epigenomics in Berlin. Many of the functional elements (AHEAD) project. Under the applications are now being The consortium completed so far identified by ENCODE leadership of Peter Jones, accepted for epigenome mapping a pilot study of methylation are being studied for changes director of the University of centres, epigenomics data analysis patterns within the major in methylation under a range of Southern California’s Norris and coordination, and technology histocompatibility complex conditions. Comprehensive Cancer Center development. L.B.

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Germany. “You will have a hard time What we are looking for are regions with finding a single somatic mutation that is higher densities of methylation,” says Mary as prevalent in this particular cancer.” Harper, chief scientific officer at Genpath- Because these epigenetic changes way in San Diego, California. “These can can potentially be reversed by drugs, be regulatory regions and there are tens they are good targets for the preven- of thousands of these regions across the AGILENT TECHNOLOGIES AGILENT tion and treatment of disease. To assess genome.” the effect of epigenetic regulation on ChIP-on-chip has grown in popularity health and disease, researchers are with the availability of high-density arrays now cataloguing epigenetic variations of contiguously tiled oligonucleotide across the genome — or epigenome probes. Affymetrix of Santa Clara, Califor- — in different tissues and at various nia, offers whole-genome tiling yeast arrays stages of development. with a resolution of 5 base pairs (a total Agilent’s DNA microscanner has a resolution of 2 micrometres. of 3.2 million probes). The company also Chips with everything makes human and mouse whole-genome Cytosine is typically methylated when it is immunoprecipitation, or ChIP. This involves tiling arrays, each as a set of 14 arrays contain- next to guanine in what is known as a CpG crosslinking DNA with its associated proteins ing around 45 million probes at a spacing of dinucleotide. Although most CpGs are meth- and then shearing the DNA. The fragments 35 base pairs. ylated in mammals, some are not methylated that contain methylated cytosine are extracted Along with other companies such as Nimble- and these are usually grouped in clusters called by immunoprecipitation with antibodies spe- Gen in Madison, Wisconsin, and Agilent Tech- CpG islands. These tend to be located in the 5΄ cific for 5-methylcytosine or fragments associ- nologies in Santa Clara, California, Affymetrix regulatory regions of genes. In many cancers, ated with other proteins such as transcription provides several array formats for ChIP-on- these CpG islands become hypermethylated, factors or histones. The immunoprecipitated chip experiments. In addition to whole genome resulting in the heritable silencing of transcrip- DNA is purified, amplified and labelled with arrays, these companies sell CpG islands arrays, tion of downstream genes. a fluorescent tag. This is then applied to the promoter arrays, ENCODE arrays (see ‘Tack- DNA methylation can be detected in several surface of a DNA microarray containing a set ling the epigenome’) or custom-made arrays. ways. One method compares what happens of probes — a procedure commonly referred The choice of the array depends mainly on the when genomic DNA is digested by enzymes to as ChIP-on-chip. type of experiment being done, the resolution that are either sensitive or insensitive to meth- “The genome is methylated at a low level needed and cost. ylation. Another approach uses chromatin everywhere, but it is more of a sprinkling. “Different arrays are not that different,”

TOOLS OF THE TRADE For researchers tackling the PCR methylation Epigentek’s Methylamp kit takes two epigenome, there are two key analysis or hours to convert cytosines to uracil.

tools: bisulphite conversion kits sequencing. EPIGENTEK and ChIP-grade antibodies. Epigentek target while it is bound to DNA, Getting complete conversion in Brooklyn, the epitope that the antibody of unmethylated cytosine to New York, also recognizes must be available uracil has been one of the main sells epigenetic in a spatial conformation challenges for the bisulphite products and that is not obscured by the treatment of DNA, not least kits for DNA shape the protein takes while because the reaction conditions methylation and bound to DNA,” explains Dana can cause the DNA to degrade. A histone modification. Meents, a product manager number of companies have tackled Its Methylamp DNA at Active Motif in Carlsbad, the problem. modification kit takes Madison, California. Active Motif is one “There are incredibly good kits two hours to convert Wisconsin; and of several companies now selling for bisulphite conversion,” says cytosine to uracil with more Zymo Research of products that have been ChIP Achim Plum, senior vice-president than 99% accuracy and requires Orange, California. validated. for corporate development at only 50 picograms of starting When it comes to antibodies that Abcam in Cambridge, UK, Epigenomics in Berlin. “If you use DNA, according to Adam Li, the can be used in ChIP experiments, sells just over 200 ChIP-grade home brews you can make many company’s chief scientific officer. a fresh set of problems presents antibodies, many of them specific mistakes, but with kits this is no The sample can then be applied itself. “For most transcription to histone post-translational longer an issue.” QIAGEN of Venlo, for methylation-specific PCR or a factors there are not that many modifications. the Netherlands, sells EpiTect, a kit methylation array. antibodies,” says Kevin Struhl, a As an alternative to antibodies, based on Epigenomics’ technology In June last year, Sigma-Aldrich ChIP pioneer at Harvard Medical the HaloCHIP system developed that Plum says offers nearly of St Louis, Missouri, launched a School in Boston , Massachusetts. by Promega allows researchers to 100% conversion without DNA number of products for epigenetic “And a lot of antibodies don’t work perform ChIP-on-chip experiments degradation. In June, QIAGEN research based on Epigentek’s well on ChIP.” by cross-linking DNA to a protein expanded the EpiTect product line technologies. Other providers ChIP-on-chip requires highly of interest fused to a modified to include a standardized workflow of bisulphite conversion kits and specific antibodies that recognize haloalkane dehalogenase tag for methylation analysis from related products a protein’s epitope in free solution (HaloTag). The protein–DNA sample collection, stabilization include Human Genetic Signatures and under fixed conditions. complex can then be captured on a and purification to bisulphite in Sydney, Australia; Invitrogen of “Because an antibody that works resin that recognizes the HaloTag conversion, and real-time endpoint Carlsbad, California; Promega of well in ChIP must recognize the for further microarray analysis. L.B.

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says ChIP pioneer Kevin Struhl at compared with microarrays. Harvard Medical School in Boston, One of the first companies to Massachusetts. He notes that the perform faster ‘next generation’ N. KELLEHER greatest source of variation among sequencing was 454 Life Sciences of ChIP-on-chip experiments comes Branford, Connecticut, but its tech- from the sample preparation and nology is not well suited to ChIP immunoprecipitation steps. He sequencing. “For ChIP sequencing and his colleagues have found that you don’t need long read runs and although most commercially avail- super accuracy like with the 454 able arrays perform similarly, those technology,” says Struhl. “Right now carrying longer oligonucleotides, Illumina [of San Diego, California] such as the ones from NimbleGen has a better technology for ChIP (50–75-mer) or Agilent (60-mer), sequencing and others are being are slightly more sensitive than developed.” shorter ones for lower levels of Other sequencing technolo- enrichment1. gies that can be applied to ChIP Agilent make its arrays by inkjet sequencing include the SOLiD printing, which allows it to respond system from Applied Biosystems quickly to design changes, says in Foster City, California, and the Kevin Meldrum, manager of the single-molecule sequencing process company’s genomic collaborations. from Helicos BioSciences in Cam- “Once an array design has been bridge, Massachusetts. completed, the sequences are sim- ply sent to the printer and the chip Bisulphite treatment is produced.” This allows Agilent Another way to detect methylation to focus on custom arrays. “Right is to modify DNA using sodium now many scientists are conducting bisulphite, which converts unmeth- broad, whole genome scans,” Mel- ylated cytosine into uracil. This drum says. “But once they identify technique used to be fraught with regions of interest, it will be more difficulties, but that is no longer cost effective to do more targeted The mass range profile of possible histone H4 modifications. the case, says Plum. In fact, the studies.” technique is growing in popularity “Compared with expression profiling there is thanks to the number of effective kits available All kinds of arrays better signal-to-noise,” says Meldrum. “Gener- (see ‘Tools of the trade’). DNA microarrays have many applications in ally what you are measuring is ‘have I or have I Bisulphite-treated DNA is analysed in dif- epigenomics. SwitchGear in Menlo not had any binding?’.” ferent ways depending on the resolution and Park, California, uses custom-designed oligo- throughput required. Methylation-sensi- nucleotide arrays from Affymetrix to profile In sequence tive PCR uses primers that anneal only those DNA methylation patterns in genomic DNA As well as using microarrays to analyse ChIP sequences that contain 5-methyl cytosines, treated with methylation-sensitive enzymes. results, the method can also be subjected to which don’t get converted by bisulphite. For The array design covers nearly all of the sequencing. This technique uses sequencing higher-throughput screens, bisulphite-treated annotated CpG islands in the genome and to localize the methylation sites or interaction DNA is hybridized to microarrays containing about 20,000 additional CpG-rich regions points of proteins or histones. two sets of oligonucleotides, one of which is the company has identified as potential ChIP sequencing offers a couple of advan- complementary to the unaltered methyl- regulatory regions. “We can profile 50,000 tages over ChIP-on-chip. It is better suited for ated sequence and the other to the converted distinct 200–800-base-pair regions in the regions of DNA close to repetitive sequences, unmethylated sequence. For a more detailed genome and quickly determine which can create ‘noise’ in a look at the cytosines that are methylated and whether they are methylated or microarray experiment, and their precise location, bisulphite-treated DNA not,” says Nathan Trinklein, co- it can cover a larger portion can be sequenced directly. Earlier this year, this founder and chief executive of of the genome. “Tiling arrays ‘bisulphite sequencing’ was used to decipher EPIGENOMICS the company. require masking of the repeti- DNA methylation patterns in the Arabidopsis Epigenomics uses a slightly tive regions of the genome,” genome at nucleotide resolution2. different microarray design to says Harper. “With sequencing, Epigenomics combines these analytical tech- offer a similar service, which it genome coverage is greater. We niques to identify biomarkers for cancer. The calls differential methylation are evaluating the next-genera- company has so far identified several genes hybridization (DMH). “The tion sequencing in combination that show changes in methylation in breast, data we produce with DMH are with our methylated DNA assays colon, prostate and lung cancers. “We believe highly reproducible and compa- to determine the potential for that there are some technical advantages to rable across different projects,” attaining the same high level of using epigenetic markers,” says Plum. “For one says Plum. “We are building a Achim Plum is working information with sequencing thing, we are looking at a signal on the DNA. database of profiles for healthy to identify epigenetic that we do with arrays.” We can analyse the DNA in paraffin sections. and disease tissues.” changes associated with The main drawback of ChIP We can look for fragments shed by tumours in One of the advantages of using various cancers. sequencing is that the throughput the blood stream.” arrays for methylation studies, is much lower than is obtained Although methylation can change in dif- rather than for monitoring the expression of with microarray screens. Although this will ferent tissues or as a result of ageing and envi- thousands of mRNAs at once (expression pro- probably increase, sequencing studies are ronmental factors, it is not as dynamic as gene filing), is that the signal is much easier to score. likely to remain a more expensive proposition expression. As a result, a single methylation

797 TECHNOLOGY FEATURE EPIGENOMICS NATURE|Vol 454|7 August 2008 THERMO FISHER SCIENTIFIC

Thermo Fisher Scientific’s Proteome Discoverer software (left) along with its LTQ Orbitrap Xl ETD system are being used to analyse histone modifications. marker can often be used to detect a disease, mass spectrometry is an unbiased approach. It Coon’s group has used an ETD-enabled says Plum, whereas several markers would be also avoids the problem caused by one bound linear ion trap mass spectrometer to map needed for expression profiling. antibody inhibiting another from binding a all the modifications that occur in the first Sequenom, based in San Diego, California, nearby modification. 23 residues of the N-terminal tail of the his- provides genomic services and has taken a dif- To determine the modification patterns, tone H4 in differentiating human embryonic ferent approach to analysing bisulphite-treated intact histones — or at least entire N-termini stem cells3. Similarly, Neil Kelleher, a chemist DNA. The company’s EpiTYPER platform peptides — have to be analysed. Such proteins at the University of Illinois at Urbana-Cham- identifies and quantifies methylated sequences are much larger than those usually examined in paign, has used ECD to identify combinations by gene-specific amplification of bisulphite- conventional ‘bottom-up’ mass spectrometry. of modifications on the first 50 amino acids treated DNA followed by matrix-assisted laser It also requires highly sensitive and precise of the histone H3 protein, finding more than desorption/ionization time-of-flight (MALDI- mass measurements: the difference in mass 150 forms of the protein4. TOF) mass spectrometry. “Although it does not between a trimethylation and acetylation is The next step for mass spectrometry is the provide genome-wide analysis, the value of our only 36 millidaltons, for example. This issue is development of methods to determine levels technique is fine mapping of sample cohorts,” being successfully addressed by hybrid mass of modifications and the ability to track pat- says Mathias Ehrich, Sequenom’s group leader spectrometers, which bring two types of ana- terns of change under different conditions. for epigenetics. lyser into one instrument. Waters, a liquid chromatography and mass The two main advantages of the technique For example, a combination of a very-high- spectrometry firm in Milford, Massachusetts, are its quantitative capabilities — allowing a resolution Fourier-transform ion cyclotron is working with researchers at the University researcher to determine whether, for exam- resonance (FT-ICR) mass spectrometer with of Southern Denmark in Odense to determine ple, methylation at a specific gene increased a relatively low-resolution ion trap mass spec- histone modifications in normal cells and cells from 20% to 40% in a tumour sample — and trometer has produced some spectacular undergoing senescence. “We have combined its long read lengths. “If you have a 2,000-base- results, says Coon. “The hybridization with ion mobility and mass spectrometry,” says pair CpG island, you want to look at the entire ion traps resulted in significant gains in routine James Langridge, director of the island for changes in methylation. If you only mass accuracy and resolution, because the ion mass spectrometry business at Waters. “We look at single CpG sites using PCR or a micro- trap regulates the ion population going to the are also pushing the boundaries to understand array you will not have enough information,” FT-ICR,” he explains. where the limits are and how to improve the says Ehrich. “You can get much more detailed informa- technology.” tion from hybrid mass specs,” says Andreas The next five years will see a boom in epi- Critical mass Hühmer, proteomics marketing director genomic research thanks to advances in mass When it comes to mapping the combinations at Thermo Fisher Scientific in Waltham, spectrometers, array design and sequencing of histone modifications, mass spectrometry Massachusetts. These instruments mean that technologies. Several groups and consortia offers a way to get at important details. “Indi- it should be possible to characterize every are taking advantage of these developments vidual modifications are like words in a sen- protein modification in different types of cells, to characterize the entire epigenome in both tence, but you have to know the context,” says he adds. healthy and disease states. The knowledge Joshua Coon, a biomolecular chemist at the Another component to analysis involves obtained will increase our understanding of University of Wisconsin, Madison. “For exam- breaking all the bonds between the amino- gene regulation and should yield new biomar- ple, in human embryonic stem cells, methyla- acid residues so that the mass spectrometer kers for disease. ■ tion at arginine 3 of histone H4 is found only can read their sequence. This used to be done Laura Bonetta is a science writer based in in the presence of dimethylation at lysine 20. by colliding ions in a neutral gas, but recent Garrett Park, Maryland. That is the context that mass spectrometry developments have introduced more effective can deliver. It is challenging to determine such techniques. These include electron capture 1. Johnson, D. S. et al. Genome Res. 18, 393–403 (2008). context with antibodies alone.” dissociation (ECD) and electron transfer dis- 2. Cokus, S. J. et al. Nature 452, 215–219 (2008). Unlike the use of antibodies, in which sociation (ETD), which induce fragmentation 3. Phanstiel, D. et al. Proc. Natl Acad. Sci. USA 105, 4093–4098 (2008). researchers select their antibodies based on by transferring electrons to positively charged 4. Garcia, B. A., Pesavento, J. J., Mizzen, C. A. & Kelleher, N. L. the particular modification they are seeking, peptides. Nature Methods 4, 487–489 (2007).

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