news
Revealing the secrets of histone H3 modification
determine the abundances of the histones in the tissues. One clear trend was that H3.2 was the most abundant If you were to take the DNA from one form of H3 in all tissues, except in tesof your cells, shake off the proteins tes. Overall, the H3.2 and H3.3 levels bound to it, and line each strand end varied among the analyzed tissues. to end, the whole thing would measure Because H3.1 was present in some ∼2-3 m. And that’s the DNA from just tissues at levels that were too low for a single cell. Fortunately, DNA does the analysis, the researchers focused not lie in your cells like loose strands on H3.2 and H3.3. These forms were of spaghetti. To conserve space, digested, and the N-terminal fragyour genetic material is wound ments were separated with HILIC tightly around proteins called according to their modifications histones. and then analyzed with FTMS. The Histones compact DNA into patterns of H3.2 and H3.3 modifimanageable lengths, and they also cations varied. For example, the appear to have regulatory funcH3.2 and H3.3 from liver, heart, tions. Access to certain genes can and testes were less modified than be granted or denied, depending H3.2 and H3.3 isolated from other on which posttranslational moditissues. Next, a multiplexed ECD fications (PTMs) are present on method was applied to identify particular histones. For example, PTMs on the N-terminal fragments trimethylation of lysine 4 on hisof histone H3 forms present in tone H3 is associated with gene each sample. The most abundant activation. In addition, combinamodifications were methylations tions of PTMs (known as histone and acetylations on six lysine resiModifications abound. A model of the nucleosome core codes) may work together to dues. The levels of these specific particle is shown with the superhelical DNA axis modulate the degree of activation perpendicular to the page. Major acetylation and modifications were reproducibly or repression. Thus, histones play methylation sites in histone N-terminal tails of human cells different depending on the tissue. important roles in cell-cycle proare indicated with stars and filled circles, respectively. Finally, the investigators folgression and development. To get (Other types of PTMs have been omitted for clarity.) lowed up on a strange form of a handle on the multiple PTMs on H3.1 that they observed in testes. histone H3, Craig Mizzen, Neil Mizzen says that once this histone Pesavento developed a middle-down Kelleher, and co-workers at the Univerform was analyzed by MS, the reapproach for the analysis of histone sity of Illinois used a “middle-down” searchers knew that they had isolated a H3.2 from HeLa cells in which the proproteolytic method and a multiplexed novel histone. “This resembles the tein was cut into large pieces with electron capture dissociation (ECD) standard H3.1, except that it has two GluC (Nat. Methods 2007, 4, 487-489). procedure, and they report their results amino acid substitutions,” he explains. The resulting N-terminal fragment inin JPR (DOI 10.1021/pr800044q). Because H3 histones have only ∼130 cluded the first 50 amino acids and A conventional approach to the residues to begin with, two changes are most of the known PTM sites. These study of histone PTMs is to examine significant, he points out. Other organdifferentially modified fragments were them individually with antibodies diisms have testes-specific H3 forms, so separated by hydrophilic interaction chrorected against a single type of PTM. this finding was not unprecedented. matography (HILIC) and analyzed by “The antibodies are intentionally made The biological significance of this new FTMS. With this method, the scientists to see a single modification, and they H3.1 is unknown. could tease apart the various multiply will bind to a molecule that has that According to Mizzen, this work is modified forms of HeLa histone H3.2. PTM, but that molecule may also have just the beginning. “The problem has Mizzen and Garcia, a postdoc who modifications elsewhere; this happens been that no one has had good tools to worked jointly in the Mizzen and Kellevery frequently with H3,” explains Mizstudy combinations of modifications, her labs and is now an assistant prozen. With this strategy, PTMs that are and this paper represents one of the fessor at Princeton University, applied not recognized by the antibody esseninitial successes in dealing with combithis procedure to the investigation of tially are invisible. Mizzen and Kellenations on the histone with the most histone H3 variants in 10 rat organs. her’s groups, however, have been complex pattern of modification,” he Reversed-phase LC separated the hisstudying PTMs on various histones says. tones from one another, and the rewith top-down MS. Although this —Katie Cottingham searchers used these chromatograms to CRAIG MIZZEN
method worked well for histones H4, H2A, and H2B, the results it provided for H3 were overly complicated. So many mixtures of multiple PTMs were present that these forms could not be resolved routinely with the top-down approach alone, says Mizzen. Clearly, H3 required a different approach. In a previous study, Mizzen, Kelleher, Benjamin Garcia, and James
10.1021/pr800556r
© 2008 American Chemical Society
Journal of Proteome Research • Vol. 7, No. 10, 2008 4211
