Quantitation of nucleosome acetylation and other histone posttranslational modifications using microscale NU-ELISA.
Methods in molecular biology (Clifton, N.J.)
2013; 981: 167-176
Detection of post-translational modifications on native intact nucleosomes by ELISA.
Journal of visualized experiments : JoVE
Histone posttranslational modifications (PTMs) are highly important molecular determinants of epigenetic regulatory mechanisms. Histone PTMs associated with nucleosomes are intimately tied to the transcriptional activity or silence of genes. In addition, nucleosomal PTMs participate in the organization of chromatin into higher-order structures and the progression through mitosis. Changes in histone PTMs are also regulated during the course of mammalian development and are altered in pathological states including cancer. Histone acetyl modifications (and also methylation and phosphorylation) are frequently assayed by western blotting (WB), mass spectrometry (MS), and chromatin immunoprecipitation (ChIP). Here we show that an enzyme-linked immunosorbent assay performed on nucleosomes (NU-ELISA) can quickly and effectively yield quantitative detection of global levels of histone acetylation on small samples such as single human embryonic stem cell colonies. The microscale NU-ELISA method presented here can be performed in most laboratories equipped with basic instrumentation for molecular and cellular biology.
View details for DOI 10.1007/978-1-62703-305-3_13
View details for PubMedID 23381861
Progressive accumulation of epigenetic heterogeneity during human ES cell culture
2009; 4 (5): 330-338
The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 base pairs of DNA associated with two each of histones H2A, H2B, H3, and H4. The N-terminal tails of histones are rich in lysine and arginine and are modified post-transcriptionally by acetylation, methylation, and other post-translational modifications (PTMs). The PTM configuration of nucleosomes can affect the transcriptional activity of associated DNA, thus providing a mode of gene regulation that is epigenetic in nature. We developed a method called nucleosome ELISA (NU-ELISA) to quantitatively determine global PTM signatures of nucleosomes extracted from cells. NU-ELISA is more sensitive and quantitative than western blotting, and is useful to interrogate the epiproteomic state of specific cell types. This video journal article shows detailed procedures to perform NU-ELISA analysis.
View details for DOI 10.3791/2593
View details for PubMedID 21540828
Global epiproteomic signatures distinguish embryonic stem cells from differentiated cells
2007; 25 (10): 2567-2574
Human embryonic stem cells (hESCs) can be maintained in culture over a large number of passages while maintaining apparently normal colony morphology. However, recent reports describe variability in epigenetic states in comparisons among different human ES cell lines. These epigenetic differences include changes in CpG methylation, expression of imprinted genes, and the status of X chromosome inactivation (XCI). We report here that the status of XCI in the female hESC line H9 (WA09) is hypervariable. We find that XIST expression can differ between individual culture isolates of H9. In addition, we find that XIST expression status can vary even between different colonies present within the same H9 culture, effectively rendering the culture mosaic. H9 cultures that lack XIST expression, but have cytological evidence of completed XCI, can also exhibit altered response to BMP4, a growth factor known to induce differentiation of hESCs to a trophectodermal lineage. In the same cultures we find biallelic expression of X-linked genes suggesting that these lines consist of mixtures of cells that retain inactivation of one of two X chromosomes following random choice. Prolonged culture of the XIST-negative isolates to high passage numbers did not result in changes in global epiproteomic signatures, demonstrating rather stable levels of post-translational nucleosome modifications within the culture-adapted hESC lines. The results show that epigenetic variants arise within human ES cell cultures after cell line derivation. In addition, the results indicate that apparently normal cultures of hESCs may contain mixtures of cells with differing epigenetic states. Assays of epigenetic integrity are warranted as quality control measures for the culture of hESCs.
View details for Web of Science ID 000269588300010
View details for PubMedID 19571681
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.
View details for DOI 10.1634/stemcells.2007-0131
View details for Web of Science ID 000249929900019
View details for PubMedID 17641388