Global epiproteomic signatures distinguish embryonic stem cells from differentiated cells

Complex organisms contain a variety of distinct cell types but only a single genome. Therefore, cellular identity must be specified by the developmentally regulated expression of a subset of genes from an otherwise static genome. In mammals, genomic DNA is modified by cytosine methylation, resulting in a pattern that is distinctive for each cell type (the epigenome). Because nucleosomal histones are subject to a wide variety of post-translational modifications (PTMs), we reasoned that an analogous "epiproteome" might exist that could also be correlated with cellular identity. Here, we show that the quantitative evaluation of nucleosome PTMs yields epiproteomic signatures that are useful for the investigation of stem cell differentiation, chromatin function, cellular identity, and epigenetic responses to pharmacologic agents. We have developed a novel enzyme-linked immunosorbent assay-based method for the quantitative evaluation of the steady-state levels of PTMs and histone variants in preparations of native intact nucleosomes. We show that epiproteomic responses to the histone deacetylase inhibitor trichostatin A trigger changes in histone methylation as well as acetylation, and that the epiproteomic responses differ between mouse embryonic stem cells and mouse embryonic fibroblasts (MEFs). ESCs subjected to retinoic acid-induced differentiation contain reconfigured nucleosomes that include increased content of the histone variant macroH2A and other changes. Furthermore, ESCs can be distinguished from embryonal carcinoma cells and MEFs based purely on their epiproteomic signatures. These results indicate that epiproteomic nucleosomal signatures are useful for the investigation of stem cell identity and differentiation, nuclear reprogramming, epigenetic regulation, chromatin dynamics, and assays for compounds with epigenetic activities. Disclosure of potential conflicts of interest is found at the end of this article.