Decoding the Histone

To fit inside the cell nucleus, DNA molecules wrap around tiny protein spindles known as histones. These histones carry an intriguing biochemical code that helps decide a cell’s destiny—whether it turns into a neuron or a lymphocyte, or turns cancerous, for instance. Decoding the so-called histone code is now faster and easier, thanks to a new system that combines innovative chromatographic techniques with advanced computer algorithms.


Spectrometric heat map showing relative amounts of various modified forms measured from a human histone sample. The vertical axis indicates the chromatographic separation time, and the horizontal axis shows the mass/charge ratio of ionized histone forms. The relative amount of each form is color-coded in ascending value from blue to red. Forms separate out in 2D by degree of acetylations (0ac, 1ac, ...) and methylations (0me, 1me, ...), and then by the positions of these mod- ifications. The new method identified more than 200 distinct histone forms from the sample, includ- ing ones never before associated with human cells. The method is sensitive enough to distinguish between an acetylation and a trimethylation—two modifications that differ in mass by only a few parts per million. Reprinted from Young, N.L. et al., High throughput characterization of combina- torial histone codes, Molecular & Cellular Proteomics, 8(10):2266 - 2284 (2009).“What previously took a year, we can now do in a three-hour run, and get better results” says Benjamin Garcia, PhD, assistant professor of molecular biology at Princeton University, who co-led the effort with Christodoulos Floudas, PhD, of the chemical engineering department. The study appeared in the October 2009 issue of Molecular & Cellular Proteomics. The new system could advance our understanding of cell differentiation, stem cells, cancer, and other key problems in biology.


Each histone’s tail region typically sports several chemical modifications such as methylations, acetylations, or phosphorylations. Individual modifications are known to activate or silence nearby genes, but their net effect—the histone code—remains unknown. This is partly because distinguishing the various histone “forms”—each carrying a distinct pattern of modifications—in a sample is very tricky; despite vastly different biological effects, they have very similar mass and structure.


The new system tackles this with an advanced chromatography process that induces different histone forms in a sample to separate out over a remarkably short 2 to 3 hour period. The emerging histone molecules are then analyzed by tandem mass spectrometry. A typical histone sample might yield thousands of spectra, each carrying contributions from one to three histone forms. To unscramble this, a computer algorithm finds the optimal mix of forms that best matches each spectrum. Combining these results from all the spectra yields an accurate tally of the identities and relative amounts of the forms present in the sample, says Floudas.


The approach successfully identified nearly 200 distinct forms in a histone sample, including some never before seen in human cells. It is sensitive enough to distinguish between modifications with nearly equal masses; indeed, it even teases apart forms that differ merely by the position of a single modification.


“If you want to characterize histone modifications on a large scale, and do it very quickly, this is the way to do it,” says University of Wisconsin chemist Joshua Coon, PhD. The method is an important technical and methodological advance, agrees Michael Washburn, PhD, of the Stowers Institute for Medical Research in Kansas City, Missouri. Washburn cautions, however, that the method will have a real impact only if other researchers succeed in implementing it. Due to their complexity, proteomics techniques are hard to replicate, he notes.


Next, Garcia says, his team will use the approach to unravel the histone code governing cellular phenomena such as stem cell differentiation and cancer. “We’ve shown we can measure modified histone forms, but there’s so much to do now,” says Garcia. “This is really the beginning of some true biological breakthroughs.”

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