Each cell in the body has the same genetic code, yet different cells interpret the code differently. Muscle cells express different genes than nerve cells and blood cells, and when they divide, their daughter cells “know” to become muscle cells–they inherit their parents’ particular spin on the genome. Scientists believe that this cellular memory is carried in the way the genome is packaged, including how DNA is wrapped around proteins called histones. Some scientists believe that chemical decorations on histones might even follow a “histone code” passed from one cell to another. But researchers have debated whether the code exists and what it would look like: a strict set of rules like DNA, or a looser system?

Scientists from the University of Illinois, Urbana-Champaign, have now taken the first step in determining whether there is a histone code by creating an inventory of the chemicals added to histones, using a novel combination of proteomics tools. The findings suggest that “there’s going to be enormous amounts of complexity to wade through” in searching for the code, says Brian Strahl, a histone biologist at the University of North Carolina at Chapel Hill who was not involved in the research.

Research on histone modifications is part of the emerging field of epigenetics, which is trying to understand how cells inherit their specific interpretations of the genome. Epigenetic changes are now thought to contribute to certain psychiatric disorders, autoimmune diseases, and cancer.

Histone proteins control DNA by forming spools around which DNA winds. Modifications to the histone tails can force the DNA to wind tighter, making it inaccessible, or loosen it, making that piece of DNA available for translation. Since each histone tail can carry multiple modifications, some scientists have suggested that different combinations of modifications might constitute a “code” that directs how DNA is accessed by the cell.

Studying these modifications in context has been a challenge: scientists traditionally analyze proteins by chopping them up, thus fragmenting the modified tails. And some of the rarer (or low-level) chemical modifications employed in epigenetics can’t be detected with traditional approaches.

The Illinois researchers overcame these problems by combining two different techniques, creating one of the most detailed catalogues of histone modifications to date. To identify modifications that occur together but in different places on the same histone, the researchers used a mass spectrometry technique called “top down,” in which a large piece of a protein is first analyzed directly, rather than digested into small pieces, as in the “bottom up” approach common in biology. They then used a specialized type of chromatography, which can separate different histone proteins according to the number of modifications they carry. Together, the two techniques provided much better resolution than other methods had.

An analysis of the most actively modified portion of histone H3, one of four histone types, uncovered more than 150 different patterns of modifications from the sample, most of which contain multiple modifications. Because cells also have two other variants of histone H3 that differ slightly, the researchers suggest that there may be hundreds of possible modification patterns for each H3 variant.

The findings are “opening up a way for other investigators to determine what are the unique patterns that exist on histone tails,” says Strahl. With this information, scientists can start to address the question of whether certain modifications are linked together, which would suggest some kind of code. For instance, the analysis showed that the attachment of a methyl group at a particular site on the histone occurs in concert with the attachment of acetyl groups, a known sign of gene activation. The technique can also be used to compare patterns of histone modifications in different cell types, such as cancer cells versus normal cells, says Neil Kelleher, senior author of the paper and a chemical biologist.