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Covisualization of Global DNA Methylation/Hydroxymethylation and Protein Biomarkers for Ultrahigh-Definition Epigenetic Phenotyping of Stem Cells

  • Jian TajbakhshEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2150)

Abstract

DNA methylation and DNA hydroxymethylation are genomic-scale key regulatory modifications in cellular differentiation and are skewed in complex diseases. Therefore, analyzing the nuclear distribution of globally methylated and hydroxymethylated DNA in conjunction with relevant cellular components, such as protein biomarkers, may well add cell-by-cell-specific spatial and temporal information to quantitative molecular data for the discovery of signaling networks in stem cell differentiation and their exploitation in the therapeutic reprogramming of cells. Fluorescence imaging provides an optical approach that has become an essential tool in this context. The in situ fluorescent covisualization of globally methylated and hydroxymethylated DNA (5-methylcytosine = 5mC, 5-hydroxymethylcytosine = 5hmC), global DNA (gDNA), and proteins can be challenging, as the immunofluorescence detection of 5mC and 5hmC sites requires thorough denaturing of double-stranded DNA for antigen retrieval. The protocol we present overcomes this obstacle through optimization of the necessary cell processing to delineate cytosine variants and gDNA while preserving the three-dimensional (3-D) structure of the cells and in connection the immunostaining of protein biomarkers and DNA counterstaining, making it suitable for ultrahigh definition (UHD) imaging of single cells by confocal and super-resolution microscopy, 3-D visualization, and high-content cytometry.

Keywords

Confocal DNA methylation Hydroxymethylcytosine Single cell Stem cell Super-resolution Ultrahigh definition imaging 

Notes

Acknowledgments

The author thanks Kolja Wawrowsky (Cedars-Sinai) and Carlos Alonso (Leica Microsystems Inc.) for advice and assistance with confocal and 3D-GSD imaging.

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Copyright information

© Springer Science+Business Media New York 2019

Authors and Affiliations

  1. 1.Cedars-Sinai Medical CenterLos AngelesUSA

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