Nearly 10 years ago, C. David Allis and Brian Strahl, then at the University of Virginia Health Science Center, proposed a novel way of thinking about histones.
Histones, the authors surmised, were not merely the scaffolds about which nuclear DNA winds. Rather, they were active participants in gene expression, their many post-translational modifications serving as complex semaphores to control DNA and regulate nearby genes.
“We will refer to the hypothesis—that multiple histone modifications, acting in a combinatorial or sequential fashion on one or multiple histone tails, specify unique downstream functions—as the histone code hypothesis,” the authors wrote (1).
The suggestion was controversial. Scientists already knew that histones (the building blocks of nucleosomes and in turn the building blocks of chromatin) could be extensively modified, their N-terminal tails bristling with acetyl, methyl, and phosphate groups, ADP-ribosylation, and ubiquitin. What they didn't know was why.
Conventional thinking at the time was that the modifications served to strengthen or loosen the nucleosomes' grip on DNA, altering gene expression accordingly. But Allis and Strahl proposed instead that these epigenetic modifications represented “a histone ‘language’” that other proteins could read, write, erase, and modify. If deciphered, this histone code could predict events such as transcription, chromatin remodeling, and silencing.
Though all agree that histones are extensively modified and that those modifications have functional consequences, not everyone believes these modifications rise to the strict definition of a “code.”
“It's like the Bible,” says Neil Kelleher, professor of chemistry at the University of Illinois at Urbana-Champaign. “There are strict constructionists—they are more literal, more bound by dogma. And then there are those who say, yes there's a God, but it's a bit more loose.”
Epigenetic theology aside, the idea of a histone code has legs: Allis and Strahl's paper has been cited nearly 2500 times, according to the ISI Web of Science. The concept “is really quite exciting,” says Bryan Turner of the University of Birmingham in the UK, a self-described histone-code agnostic. “To try and crack that code is just an enormous challenge, much more difficult than the genetic code—and we forget how hard that was to crack.”
Learning the LanguageKelleher is a mass spectrometry expert who focuses on the histone modification lexicon using top-down proteomics.
Most proteomics studies are “bottom-up,” first digesting protein samples to peptides prior to fractionation and sequencing. The technique is simple and works well if only protein identification is desired. But if a researcher is concerned with complex post-translational modifications, and how those patterns change under different conditions—say, during oncogenesis or stem cell development—a problem emerges. Since the proteins are diced into bite-sized pieces, it's impossible to tell if two modifications are physically linked, or if they are, instead, mutually exclusive.
“Once you cleave with a protease between two sites of modification, it becomes incredibly difficult to quantify these things going up and down,” says Kelleher.
Top-down proteomics, on the other hand, is a protein cataloging strategy in which intact proteins, complete with modifications, are broken into smaller and smaller pieces until the whole has been fully characterized. Because it starts with intact protein, the approach ensures that all detected modifications coexist.
Kelleher is something of a top-down evangelist, but the approach isn't for everyone: excellent chromatography and top-of-the-line hardware, such as with Fourier transform mass spectrometry (FT-MS), is necessary to achieve the resolution and mass accuracy required to distinguish large proteins that differ by just a few Daltons.
In fact, histones don't even need the full top-down treatment because most modifications are confined to the proteins' N-terminal tails. So Kelleher and then-postdoc Benjamin Garcia applied a stripped-down “middle-down” approach—or, in Kelleher's words, “Goldilocks proteomics—these peptides are too small, those proteins are too big, but these fragments are just right.”
