Tracking epigenetic markers at the single-molecule level

April 24, 2013 § Leave a comment

The crystal structure of the nucleosome with the core histones and DNA. Image from the PDB; Released under the GNU Free Documentation License (http://en.wikipedia.org/wiki/File:Nucleosome_1KX5_colour_coded.png)

The crystal structure of the nucleosome with the core histones and DNA. Image from the PDB; Released under the GNU Free Documentation License (http://en.wikipedia.org/wiki/File:Nucleosome_1KX5_colour_coded.png)

Chemical modifications to DNA and histones are the cornerstone of epigenetics. But current methods to track these modifications are cumbersome and not sensitive enough to bring out the nuanced differences and connections between the various modifications. In a paper just out in the Proceedings of the National Academy of Sciences, a group of researchers describe a method that can quickly detect and quantify two different modifications on histones and DNA at the single-molecule levels.

Information about disease processes can be found in the chemical modifications of the chromosomes of cells. “These modifications of the DNA and histone proteins, along with the underlying genetic sequence, have influence on the fate of cells in the body,” explains Harold Craighead of Cornell University. “Particular combinations of epigenentic marks may provide indicators that could be used to diagnose or track the effectiveness of therapies in diseases, such as cancer.”

But current methods to analyze combinations of epigenetic marks can lose this information; chromosomal material from heterogeneous cell samples get mixed together to give out a bulk measurement. So Craighead, along with Paul Soloway who is also at Cornell, explored approaches that could “analyze material from very small numbers of cells, perhaps even a single cell,” says Craighead.

The Cornell team based their method on nanofluidics, an analytical approach that uses channels that are as narrow as a strand of hair. These tiny channels force molecules to flow through one at a time. The chromosomes, which consist of DNA packaged around complexes of histones, are cut into small fragments. Each fragment contains one or a few histone complexes with the wrapped DNA; these travel down the channels one by one. If the DNA and histones are tagged with different fluorescent markers that go after the various chemical epigenetic modifications, then researchers can track the type and amount of each fluorescent marker on single histone and DNA molecules and measure the epigenetic information contained in those molecules.

That’s exactly what the Cornell team did. The investigators tracked a gene-silencing mechanism on histone H3 that involves a trimethylation on lysine number 9  (H3K9me3 in shorthand form) as well as methylated cytosine (mC), known as 5-methylcytosine, on DNA. They demonstrated that in normal mouse fibroblasts, the effects of mC are reversed. This finding suggests that improper cooperation between mC and H3K27me3 placement could cause incorrect gene silencing in cancerous cells.

The investigators are now figuring out how to incorporate information from other kinds of epigenetic markers and look at more than two markers at once. Because of the underlying technology for the approach,  Soloway and Craighead have established a spin-off company called Odyssey Molecular to commercialize the method.

“Our experiments are based on integrated fluidic/optical devices that have the potential for scaling up to allow many such devices to operate in parallel for higher speed or higher throughput operation,” explains Craighead. “We envision, in the future, more compact and automated systems that could augment  the currently available epigenetic research tools and possibly evolve into new types of diagnostic devices.”

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