NATURE|Vol 454|7 August 2008 TECHNOLOGY FEATURE EPIGENOMICS Detailed analysis Researchers now have access to an burgeoning collection of tools for unravelling the epigenome, 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 promoter 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 histones — the main protein components of chromatin — 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 genome. And certain patterns of histone 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- bioinformatics 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. 795 TECHNOLOGY FEATURE EPIGENOMICS NATURE|Vol 454|7 August 2008 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