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DOI: 10.1126/science.opms.p0900038

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Epigenetics: The Final Frontier?

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Epigenetics is hot. In recent years, researchers in fields as diverse as cell biology, development, and even microbial pathogenesis have become very interested in heritable traits that don't rely on DNA. The idea itself is not new—cancer biologists have known for decades that mechanisms such as DNA methylation and chromatin modification can transmit changes to subsequent generations of cells without changing DNA sequences—but studying these phenomena has recently been increasing in popularity, aided by new technological innovations.

By Alan Dove



Inclusion of companies in this article does not indicate endorsement by either AAAS or Science, nor is it meant to imply that their products or services are superior to those of other companies.

Scientists who had never pondered epigenetics are now anxious to understand how it affects their favorite processes. Ironically, one of the major drivers of this newfound interest in non-DNA mechanisms was the complete sequencg of human DNA.

"It became clear from the human genome project that humans have about 25,000 or 26,000 genes, and that clearly didn't account for all the complexity that was present; they expected maybe 100,000 genes based on different types of proteins," says John Archdeacon, director of research and development for antibodies and amino acids at Millipore in Billerica, Massachusetts. Epigenetics is one way biology generates so many outcomes with so few genes.

Researchers have discovered three major ways organisms alter their DNA's inherited messages: small RNAs can modify the expression of specific genes, enzymes can methylate DNA to modulate transcription, and histone modification can induce or repress target sequences.

Small RNAs spawned an entire industry of user-friendly assays almost immediately after their discovery, but understanding DNA methylation or protein-DNA interactions requires more specialized techniques that can be tricky for the uninitiated. As a small sampling of tool makers reveals, companies are now trying to address these problems with a new generation of kits, reagents, and equipment to simplify epigenetic assays.

Challenges and Solutions

One way or another, researchers who want to study DNA methylation patterns must confront a technique called bisulfite conversion. Bisulfite treatment of DNA changes unmethylated cytosine residues to uracil, but leaves methylated cytosines unchanged. Assays such as sequencing, quantitative PCR, and microarrays can then compare the sequences of treated and untreated DNA to determine which bases were methylated.

Though conceptually simple, bisulfite treatment has a reputation for difficulty. "If you interview everyone who has done it, they all say it's not easy to do," says Xiao Zeng, director of R&D at SA Biosciences in Frederick, Maryland. He adds that "part of the problem we have encountered is the recovery; essentially you're going to lose about 50 percent of your input DNA after bisulfite treatment."

The DNA that remains after the procedure isn't in great shape, either. "Another problem that goes along with bisulfite conversion is the degradation of DNA due to the harsh conditions during the bisulfite treatment. Fragmentation of DNA lowers the sensitivity in PCR and subsequent analytical techniques," says Gerald Schock, senior global product manager for epigenetics and whole genome amplification at Qiagen in Hilden, Germany. Schock adds that incomplete removal of the reagents after conversion makes things even worse, as the DNA continues to break down during storage.

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Unsurprisingly, Qiagen is one of the companies trying to address these problems. The company now offers a series of kits and reagents under the EpiTect name, covering every step of the bisulfite conversion process. The product line also includes important controls. "For example, when performing methylation-specific PCR, one must ensure that the PCR primers are specific for the detection of bisulfite-converted methylated or unmethylated DNA only," says Schock, adding that "EpiTect Control DNAs offer quality controlled pre-bisulfite-converted DNA derived from completely unmethylated or completely methylated DNA, which is suited for the analysis of any gene."

While every manufacturer claims its own bisulfite-conversion products are the best, Schock recommends asking pointed questions when shopping. For example, efficient bisulfite conversion is crucial. "Reliable results can only be achieved if complete conversion of all unmethylated CpG sites is ensured. One should ask for data on the conversion efficiency to be sure that the method chosen can accomplish that," says Schock.

Besides efficient CpG conversion, investigators may want to aim for efficient time management. Traditional bisulfite conversion involves overnight incubations, which add days to the already time-intensive procedure. Some reagent kits, such as the Methylcode product from Life Technologies (previously Invitrogen) in Carlsbad, California, can shorten these long incubations and speed the procedure considerably.

Newcomers to bisulfite conversion may also want to limit the number of variables by buying the key reagents rather than homebrewing them. "I would encourage customers to start with kits as much as possible for sample preparation as well as for sample analysis. The value to a customer in doing that is that they will be using validated reagents that have been optimized for these protocols and will have more consistent results," says Sallie Cassel, director of marketing for antibodies and immunoassays at Millipore.

Cassel also reiterates a simple but often ignored bit of laboratory wisdom: "Use controls." She adds that "a lot of times people think that they don't need them, but they can be very helpful."

Evasive Maneuvers

Given the inconvenience of bisulfite conversion, it's not surprising that many groups have developed ways to reduce or eliminate the need for it. At Life Technologies, for example, product developers have focused on simplifying the population of DNA in a sample to improve bisulfite conversion efficiency. The approach is especially useful for researchers doing genomewide methylation profiling.

"For these genomewide studies, you can actually pare down the number of sequences you need to look at—we've taken an approach of using a methyl binding domain protein," says Amy Cuneo, product manager for epigenetics at Life Technologies. The protein specifically binds methylated double-stranded DNA, separating it from the rest of the sample so it can be bisulfite-treated more efficiently.

Life Technologies' Carlsbad neighbor, Active Motif, takes the concept a step further. The company's MethylCollector Ultra kit uses two different methyl binding domain proteins to enrich for methylated DNA from samples, while its UnMethylCollector kit uses another isoform to pull out DNA with unmethylated CpG islands.

Active Motif says that the efficiency of the two kits is enough to get detailed methylation profiles from samples as small as 1,600 cells. Even better, researchers can perform PCR directly on the enriched samples to determine which sites are methlyated, without bisulfite conversion. Bart Challis, Active Motif's director of operations says, "Because of their ease of use and high specificities for methylated and unmethylated DNA, these complementary technologies can be used in combination to quickly and accurately perform DNA methylation analysis before proceeding with other, more costly downstream techniques."

Others are also keen to drive around the bisulfite problem. "We started with the pain," says SA Biosciences' Zeng. He explains that "we ourselves tried bisulfite treatment of DNA, and we realized it was very difficult, so then we looked at other technologies, such as enzymes."

In SA Biosciences' Methyl Profiler PCR system, the first step is not bisulfite conversion, but enzyme digestion. Splitting a DNA sample into two batches, then treating one with methylation-sensitive enzymes and the other with methylation-resistant enzymes, yields different fragment profiles that identify the methylation pattern in the original sample. "So essentially those two enzymes can recognize similar restriction sites, for example CGCG, but one of the enzymes will not cut if the C is methylated, but the other will," Zeng explains.

The Methyl Profiler system is designed for medium throughput, not whole-genome assays. Researchers who want to study the methylation status of specific subsets of promoters are the target market. "If they know their biology and they know there's a certain biological pathway involved in whatever system they study, they can do a panel of 24 genes or 96 genes in one shot," says Zeng, adding that "we want our customers to focus on their biology instead of the technology itself."

Not everyone is convinced that bisulfite conversion is so tough, though. "Just like swimming or bicycling, if you know how to do that, it's very normal, but if you're new, you need to learn. It's like this in epigenetics," says Larry Jia, founder and director of research and development at Zymo Research in Orange, California. He adds, "If you get into it and use it, it's pretty robust actually, so it depends on who defines whether it's easy or hard. Once you pick it up, it's not that bad at all."

Jia advocates starting with a kit and plenty of practice to learn traditional bisulfite conversion, rather than trying to work around it. "Antibodies, enzymes, other things—you can use them, but it's only indirect," he says.

Regardless of whether they choose practice or avoidance, researchers should plan their methylation studies with the entire procedure in mind, including the final assay. "There [are] performance differences and protocol differences, but you know looking at an entire workflow is probably one of the biggest questions that researchers would have when they're starting out. How are they going to read out their information and [make] sure that the tools work with their workflow?" says Cuneo.

Application of ChIP

While methylation is an important component of epigenetic regulation, it certainly isn't the only way genomic information gets modified. To probe one of the other major epigenetic mechanisms, histone modification, researchers often turn to chromatin immunoprecipitation, or ChIP.

In a typical ChIP experiment, an investigator uses antibodies to precipitate modified and unmodified histones, along with all of the chromatin pieces that associate with them. Gene chips or PCR can then reveal the sequences in the precipitate, identifying the target histone modification sites in the genome. The technique can also coprecipitate histone-modifying enzymes for further analysis.

ChIP is relatively straightforward, but it has a few pitfalls. In particular, precipitating enough DNA for detection often means using a lot of cells, which may not be possible for some studies. For example, investigators working with difficult-to-culture stem cells or precious human biopsy samples may not be able to spare enough to perform a ChIP assay.

In response, companies have developed reagents for ChIP on smaller samples. Life Technologies, for example, recently introduced the MAGnify kit, which uses a version of the popular Dynabead magnetic precipitation system. "What we've done is to take one of the most widely published products, the Dynabeads protein A/G that are used in ChIP, and really optimized a kit that includes all the buffers and other reagents that are needed," Cuneo explains.

Instead of requiring a million cells, MAGnify kits can produce useful results from as few as 10,000 cells. The protocol also shortens the ChIP procedure by a day, and eliminates the use of blocking DNA that can contaminate the sample. "Using the Dynabeads eliminates any need for blockers that contain, for example, salmon sperm DNA [for assays where] you really need to avoid background at all cost," says Cuneo.

Other manufacturers also cater to the burgeoning ChIP market, of course, but nearly everyone cautions that no kit is foolproof. In particular, researchers need to be sure that they use suitable antibodies. Even a highly specific antibody may not be enough, if it hasn't been validated specifically for ChIP. Antibodies that work well on a denatured protein in a Western blot may not recognize their target at all in its native chromatin-binding conformation.

Worse, not every antibody that has been tested in one lab's ChIP assay will work in another lab. "Oftentimes [researchers] may have an antibody and somewhere in some paper somebody said that it works in ChIP," says Millipore's Cassel, adding that "that antibody, that lot, that vial that a customer gets may be very remotely related at best to that paper, and so the results may not be directly correlative." In response, companies such as Millipore now validate every lot of ChIP antibodies before selling it.

With a proven protocol and a validated antibody in hand, the next challenge is to figure out whether a target protein is abundant enough to precipitate. According to Millipore's Archdeacon, unmodified histones or those with common modifications may be easy to detect, but rarer proteins might not produce enough material to detect, even with a good antibody.

After the ChIP procedure, investigators need to identify the precipitated sequences, which usually means analyzing the sample with either a DNA microarray or quantitative PCR. Each approach has its adherents.

At Millipore, which recently announced a collaboration with chip maker Agilent, the emphasis is now on ChIP followed by DNA microarray analysis, or ChIP-chip. "We will have products in which we have partnered with Agilent to provide Agilent arrays and reagents in the kit for a total solution," says Cassel, adding that the new Millipore/Agilent kits should be on the market by late 2009.

For researchers who don't need a whole-genome view of a micro-array, ChIP-qPCR may make more sense, especially for studying rare binding proteins. "The dynamic linear range for [microarrays] is about 5 logs less than real time PCR," says Jeffrey Hung, director of marketing at SA Biosciences, adding that "you can imagine that if you have a low abundance transcript, the real-time PCR-based technology can detect both the low abundance and the high abundance DNA fragments that associate with certain ChIP samples."

Regardless of the specific techniques they choose, researchers will find plenty of interesting questions to answer in epigenetics. "I think it might be the last great frontier in biology," says Zymo's Jia. He adds, "We understand genes and proteins and we can sequence genomes. The next thing in biology we ask is how do genomes organize. How is it working?"


Featured Participants



Active Motif
www.activemotif.com

Life Technologies
www.lifetechnologies.com

Millipore
www.millipore.com

Qiagen
www.qiagen.com

SA Biosciences
www.sabiosciences.com

Zymo Research
www.zymoresearch.com



Note: Readers can find out more about the companies and organizations listed by accessing their sites on the World Wide Web (WWW). If the listed organization does not have a site on the WWW or if it is under construction, we have substituted its main telephone number. Every effort has been made to ensure the accuracy of this information. Inclusion of companies in this article does not indicate endorsement by either AAAS or Science, nor is it meant to imply that their products or services are superior to those of other companies.

Alan Dove is a science writer and editor based in Massachusetts.

DOI: 10.1126/science.opms.p0900038



This article was published as a special advertising feature in the 9 October 2009 issue of Science Magazine.


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