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Culling the Epigenetic Herd

09/26/2012
Ashley Yeager

Bisulfite conversion and ChIP, it’s time to saddle up. Single-cell epigenetics is the new wrangler in this one-cell town, riding in on nanofluidic technology. Ashley Yeager telegraphs from the leading edge of the epigenetics frontier.


Epigenetics is a field on the frontier of biomedical research. Right now, pioneering researchers have ventured into these parts trying to round up and study epigenetic marks, which are chemical tags—such as methyl groups—that regulate gene expression when attached to DNA and histones.

Fluorescent methylation mapped in nanochannels. DNA is marked in green, and methylation markers are red. Source: Robert Riehn, North Carolina State University

Historically, their lassos haven’t been very tight. Right now, scientists track methylation using techniques like bisulfite conversion and chromatin immunoprecipitation (ChIP). To perform these methods, researchers need herds of cells that number in the thousands or billions. So, their conclusions are based on these large groups and not individual cells.

“Ensemble analyses of a population are useful, and they will report average states, but knowing the variation within a population provides another layer of information, and that information can only come from single-cell analysis,” says Paul Soloway, a molecular geneticist at Cornell.

But now, Soloway and other like-minded epigenetic wranglers are developing nanofluidic devices that can sort individual cells based on their methylation states. In the end, their hope is that these single-cell epigenetic techniques will provide a guiding light for others, a light that might help uncover the potential gold mines of treatments for diseases like cancer that are hidden within the epigenetic frontier.

Epigenetics by the Herd

When it comes down to it, bisulfite sequencing is just plain old harsh. The process converts cytosine bases in the genome into uracil, while leaving methylated cytosine alone. From there, researchers can then sequence the bisulfite-converted DNA, inferring that any remaining cytosine bases were methylated in the original DNA. But the bisulfite conversion degrades DNA, leading to significant sample loss.

Similarly, ChIP is also not efficient when it comes to epigenetic studies. Relying on antibodies to identify the interactions between methylated histones and specific DNA sequences in a cell, the technique does not precipitate all of the chromatin with the methylated histones of interest, leading to missing data.

Because these methods only probe one epigenetic mark at a time, scientists must then superimpose the data from several of these single-mark analyses. When peaks from separate analyses overlap, the scientists can infer that the epigenetic marks occur on the same piece of DNA from different cells.

“While this inference might be correct many times, there are many times it is likely to be misleading, especially for heterogeneous populations of cells,” says Soloway.

In addition, both of these methods require thousands of cells. So, scientists perform their analyses with populations of cells, making it a challenge to determine how the molecules and DNA are interacting in individual cells. Individual cells in a herd are rarely, if ever, all the same type. Even cultures that scientists derive from single-cell cloning have subpopulations at different stages of the cell cycle, and stem cell cultures might have subpopulations with various differentiation states.

But getting to single-cell level of analysis using these techniques has its problems. When scientists analyze a cell population, the abundance of cells overcomes the inefficiencies of these methods, but when doing single-cell analysis, “this means of compensating is just not there.”

And, that’s not the only burr under the saddle, according to Shuichi Takayama, a biomedical engineer at the University of Michigan. “Unlike DNA, chromatin cannot be amplified. Thus, you have to manipulate very small amounts of very delicate, complex, multi-biopolymer structures,” says Takayama.

Single-cell epigenetics also provides an “astronomical” amount of data and costs $1000 to $2000 dollars per cell, says Robert Riehn, a physicist at North Carolina State University. This could quickly become more than $100,000 for just a small set of cells. “Scaling-up is a big obstacle,” says Riehn.

A mix of DNA molecules is pushed through a nanofluidic channel. Molecules given a fluorescent tag trigger an electric field that sorts them to one side, allowing scientists to single out epigenetic states of interest in DNA of individual cells. Source: Craighead Lab

Nanofluidic Cowboys

Because of these issues, epigenetic researchers and bioengineers alike are developing alternatives. One technique of interest is the use of fluorescently labeled methyl-CpG binding domain proteins (MBD) that bind to methylated DNA. In this approach, the scientists feed methylated DNA bound with fluorescently labeled MBD into nanofluidic channels to straighten and elongate DNA sections of interest in order to provide scientists a “bird’s-eye view” of the spatial patterns of fluorescence on the DNA, which correspond to the sites of DNA methylation.

In the early 2000s, Cornell physicist Harold Craighead and Princeton University biophysicist Robert Austin pioneered the use of nanofluidic channels to manipulate individual, fluorescent DNA molecules. Soon after, Craighead asked colleague Stephen Levy, who is now a physicist at Binghamton University, to come up with a list of biological applications for the technology. In turn, Levy contacted Soloway, showing him videos of a device manipulating single, fluorescent DNA molecules in submicron fluidic channels.

“[Levy] asked if the technology could be used for epigenetic analysis. It wasn't obvious to me right away how it could, but within a week, I realized that if we substituted chromatin for DNA, and bound it to antibodies recognizing epigenetic marks with different flurophores, we'd have methods that are analogous to flow cytrometry,” says Soloway. Then, researchers could operate the device in an analytical mode to identify epigenetic markers or in a preparative mode to sort the cells based on their DNA methylation profiles.

Based on research and conversations with Soloway, Levy saw that single-molecule techniques could certainly advance epigenetics, especially in the measurement of multiple marks on single molecules and single-cell analysis. “The standard bulk methods being used to measure epigenetic marks were not well suited to tease out this information,” says Levy. Since those early conversations, the researchers have been developing and marketing those methods through their company, Odyssey Molecular.

Culling Based on Methylation

And now, those nanofluidic devices are reining in the open range of epigenetics. In a paper published by Proceedings of the National Academy of Sciences in May 2012, Soloway, Craighead, Levy, and colleagues reported on a proof-of-concept experiment for a nanofluidic device that uses fluorescence-activated single molecule sorting for methylation analysis (1). The method preserves the epigenetically marked DNA that has been sorted so that scientists can sequence and analyze the sample further.

The nanofluidic device identified methylated DNA molecules by their binding to fluorescently labeled MBD and then sorted those DNA molecules with a brief pulse, which redirected cell flow so that the methylated DNA were sorted into a different channel from non-methylated DNA. So far, the system has proven to be about 98% accurate.

“I know that they are attempting nanofluid targeting and possibly on-chip amplification. But a lot of the design is proprietary, so I don’t know exactly what they are up to,” says Riehn.

“Ensemble analyses of a population are useful, and they will report average states, but knowing the variation within a population provides another layer of information, and that information can only come from single-cell analysis,” says Paul Soloway. Source: Cornell University

Overall, Craighead’s device has three key features: the nanofluidics for flowing molecules, the electronics for controlling their flow, and the optics that detect molecules of interest and inform the electronics of their epigenetic features. Eventually, the group would like to use chromatin from cells rather than defined DNA mixtures, which would maintain the integrity of both the genetic and epigenetic features of the samples.

Likewise, other groups are also developing methods for studying epigenetics at the single-cell level. “There are several other groups in academia and industry trying to push this frontier. I think the field is wide open,” says Craighead.

Take, for example, Riehn’s group. In 2011, his group also showed that scientists could create a methylation map of DNA using fluorescently labeled MBD that binds to methylated cytosines in methylated DNA (2), a technique that Riehn says is similar to Craighead’s method.

Or Takayam’s group, that has developed DNA nanochannels to detect epigenetic changes on full-length DNA (3). Size-adjustable nanochannels and DNA linearization should allow scientists to better manipulate single strands of chromatin, according to Takayama. In the end, this could enable direct reading of epigenetic information from single strands of protein bound DNA. But sample preparation is “very important and difficult,” he says. There must be a balance between stretching out the chromosome versus maintaining the interesting and important higher-order structures, which is critical too.

“I think it is extremely likely that this technology can be scaled for clinical applications,” says Levy.

References

1. Cipriany, B. et al. 2012. Real-time analysis and selection of methylated DNA by fluorescence-activated single molecule sorting in a nanofluidic channel. PNAS 109: 22, 8477–8482.

2. Lim, S. et. al. 2011. DNA methylation profiling in nanochannels. Biomicrofluidics 5: 034106, 1-8.

3. Douville, N. et al. 2008. DNA linearization through confinement in nanofluidic channels. Analytical and Bioanalytical Chemistry 391: 7, 2395-2409.

Keywords:  epigenetics