Title

Nucleosome-ELISA: A novel method to uncover the epiproteomic signatures of mouse and human embryonic stem cells

Date of Completion

January 2009

Keywords

Biology, Molecular|Biology, Genetics

Degree

Ph.D.

Abstract

Many biological processes are profoundly controlled or influenced by epigenetic mechanisms, which involve heritable changes in gene expression that are independent of DNA sequences. At a molecular level, epigenetics is gene regulation by chromatin modifications, which include DNA methylations and histone modifications. In mammals, genomic DNA is modified by cytosine methylation, resulting in a pattern that is distinctive for each cell type (the epigenome). Since 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, epigenetic drift, chromatin function, cellular identity, and epigenetic responses to pharmacologic agents. We have developed a novel and sensitive enzyme-linked immunosorbent assay-based method nucleosome-ELISA (NU-ELISA), for the quantitative evaluation of the steady-state levels of PTMs and histone variants in preparations of native intact nucleosomes, which is thousands times more sensitive than western blotting. 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 (ESCs) and mouse embryonic fibroblasts (MEFs). ESCs can be distinguished from embryonal carcinoma cells (ECCs), MEFs, and even reprogrammed cells (MEF/ESC hybrids), based purely on their epiproteomic signatures. Furthermore, mouse ESCs subjected to retinoic acid-induced differentiation contain reconfigured nucleosomes that include increased content of the histone variant macroH2A and other PTM changes. We also found that specific histone methylations of mice can respond to monoamine oxidase inhibitor in vivo and in vitro. In addition, human embryonic stem cells (hESCs) H9 cultured at low passage or high passage did not show dramatic epigenetic drift as judged by their respective epiproteomic signatures. However, H9 hESCs at high passage showed heterogeneity in their colony morphology. Taken together, these results indicate that NU-ELISA is useful for the investigation of stem cell identity and differentiation, epigenetic drift, nuclear reprogramming, epigenetic regulation, chromatin dynamics and assays for compounds with epigenetic activities. ^