Move over, Mendel. The age of epigenomics is here, and it's provoking a lot of interesting questions. To answer them –– whether they concern chromatin remodeling, gene silencing pathways, or nuclear organization and assembly –– scientists are combining tools and customizing methods to get a handle on epigenomic change.
Epigenetics, or the transmission of information not encoded in a genome's sequence alone, has been years in the making. More than 60 years ago, Conrad Waddington formulated ideas about ‘causal interactions between genes and their products,' which were quietly set aside during the days of the genetic revolution. In these post-genomic days, however, you can't flip through a top-tier journal without chancing on a new epigenetic study.
Because of the proliferation of high-thoughput technologies, genome-wide approaches to study epigenetic modifications have emerged. Either DNA or the proteins that govern chromatin packaging can be affected by epigenetic chemical modifications, such as methylation. When methyl groups latch on to CpG sites in DNA or protein histone tails, gene expression is altered. DNA methylation has been linked to everything from development to cancer, placing it squarely in the domains of basic and clinical researchers alike.
In the Lab
Epigenomics research touches on a bit of everything in terms of biology. Because the phenomenon is common to virtually all eukaryotes, basic scientists are searching for methylation marks across the model system spectrum. Clinical researchers are also mining epigenomes for clues to understand human disease progression and eventually design effective drug and diagnostics.
At Rockefeller University, Dave Allis' lab focuses on how chemical changes to histone proteins affect downstream gene expression. His lab may be best known for accessing acetylation enzymes, but Allis is no stranger to methylation. A couple of years ago, researchers working with a mold system discovered that mutations in the histone methylation pathway could lead to loss of DNA methylation. This prompted Allis to think about how histone marks may guide DNA methylation, which is exactly what his lab has started exploring.
New research questions generally spur new techniques, which in turn shed light on novel molecular processes. "It's dizzying, really," Allis says. "A lot of [molecular] machinery has been accessed by the whole field in the last 10 years or so, and now it's creeping into every corner of biology."
According to Allis, his lab's research relies on "brute force biochemistry," antibodies directed to histone modifications, and mass spec. Allis himself thinks that "the combination of new antibody reagents with mass spec approaches are starting to make a lot of headway."
University of Leeds professor Peter Meyer agrees with this assessment. His lab's work aims to uncover new DNA methylation targets, as well as chromo-domain complexes and mechanisms for gene expression by non-coding RNAs in plant systems. To those ends, Meyer says that available technologies are proving powerful, especially large-scale sequencers for RNA screens, highly specific antibodies to detect histone methylation patterns, and CHiP-on-chip assays for mapping such patterns on chromosomes.
Jörg Tost of the Centre National de Génotypage is concentrating on more clinical topics as he gets the center's new epigenetics research and technology group off to a start. The four-member team collaborates with external researchers on the discovery of methylation-based biomarkers and profiles for cancer and diabetes. To find candidate genes in these studies, he uses MALDI to screen large cohorts and bisulfite-based pyrosequencing to monitor the methylation status of multiple CpG sites. Tost's group is currently working to combine pyrosequencing with methylation-specific PCR to increase sensitivity and eliminate false positives.
High-tech tools in expert hands have propelled epigenomics forward, but the field may owe more to good old communication. The field's discoveries started heating up in the years following completion of the Human Genome Project. Technology was ripe, researchers were ready, and new horizons were begging for exploration. It's no surprise, then, that there are now several coordinated, multi-national efforts striving to unravel epigenetic modifications, especially at the whole-genome level.
The Human Epigenome Consortium was one of the first ventures aiming to crack the human epigenome. Scientists at the Wellcome Trust Sanger Institute, the Centre National de Génotypage, and Epigenomics launched a pilot project in October 2003 to analyze methylation patterns in a region of chromosome 6. This was a successful prelude to the group's Human Epigenome Project, which set itself the larger task of identifying, cataloging, and interpreting genome-wide methyl-ation patterns in major tissues. Last December, the HEP published results for chromosomes 6, 20, and 22 that detailed methylation-variable positions associated with 873 genes.
One year after HEP's pilot project kicked off, research groups at some of Europe's top labs joined together to form the Epigenome Network of Excellence. Funded by the EU, the Epigenome NoE is working to establish a research area in epigenetics and a virtual core institute by 2009. The network is structured around eight key topics, such as chromatin modification and epigenomic mapping. Meyer, an associate member, says that the network has been especially valuable for forging contact with labs focusing on different, albeit complementary, research questions. It also doesn't hurt that investigators can share tips and protocols via a common website.
Europe doesn't have a monopoly on coordinated epigenomic programs, though it may have seemed that way for a while. The American Association for Cancer Research has formed the Alliance for Human Epigenomics and Disease, an international research effort to integrate ongoing epigenome projects.
Technologies are already on the scene for studying epigenetic mechanisms and epigenomic patterns. While no means exhaustive, here's a short list of essential tools for methylation-minded scientists.
First, the basics: One key to efficient methylation detection lies in bisulfite conversion, which is a very sensitive method used to detect hypermethylated genes. PCR alone won't pick up on chemical changes to DNA, so some tweaking of the starting material is necessary. By treating DNA with bisulfite, unmethylated cytosine is converted into uracil. Over the course of amplification, uracils are then replaced with thymines, paving the way to detecting methylated cytosines.
Developed by Stephen Baylin's team a decade ago, MSP relies on bisulfite conversion to look at CpG methylation. Primers specific to one or more CpG sites are used to detect methylated and unmethylated DNA after bisulfite treatment and amplification. OncoMethylome Sciences holds the IP for this tool, which was developed at Johns Hopkins, and researchers can access MSP technology and reagents via Chemicon.
For those interested in higher throughput than single gene PCR, bisulfite conversion can also be used in conjunction with sequencing. This was the tack taken by the Human Epigenome Project in the recently completed whole-genome methylation study, says Achim Plum, Epigenomics' vice president of communications. The protocols themselves were designed by Epigenomics and they formed the basis of the EpiTect Kit launched by Qiagen last year.
With development help from Affymetrix, Plum says that Epigenomics developed its own microarray platform to profile methylation in very small amounts of human samples. Known as differential methylation hybridization, the technique works by chopping up DNA with restriction enzymes, thereby preserving CpG-rich sites, followed by hybridization to an array. DMH is available to researchers as a biomarker discovery service administered by Epigenomics.
Another tool on the array front is Illumina's Golden Gate Assay for Methylation Detection, which launched last month. The assay covers 800 genes, and is capable of surveying up to 1,505 CpG sites across 96 samples in one go. Two centers leading the epigenetics efforts of the Cancer Genome Atlas Pilot Project will be using this tool to detect epigenetic modifications associated with cancer, according to Illumina's methylation product manager, Vivian Zhang.
Methylation Detection Service
For those on the hunt for methylation biomarkers, but lacking the time or lab hardware, BioServe Biotechnologies' recently launched its DNA Methylation Analysis Service may fit the bill. The service uses Sequenom's MassArray system, by which the company can process up to six different genes simultaneously, says BioServe CEO Rama Modali. Clinical researchers need only send in their samples; BioServe will do the rest of the dirty work, from DNA extraction to analysis.