학술논문
Single cell epigenetic visualization assay.
Document Type
Academic Journal
Author
Kint S; Department of Data Analysis and Mathematical Modeling, Ghent University, Ghent, Belgium.; Department of Internal Medicine and Pediatrics, Ghent University, Ghent, Belgium.; Van Criekinge W; Department of Data Analysis and Mathematical Modeling, Ghent University, Ghent, Belgium.; Vandekerckhove L; Department of Internal Medicine and Pediatrics, Ghent University, Ghent, Belgium.; De Vos WH; Department of Veterinary Sciences, University of Antwerp, Antwerp, Belgium.; Bomsztyk K; Department of Medicine, University of Washington, Seattle, WA 98109, USA.; Krause DS; Depts. of Laboratory Medicine, Pathology and Cell Biology; Yale Stem Cell Center, Yale University, New Haven, CT 06520, USA.; Denisenko O; Department of Medicine, University of Washington, Seattle, WA 98109, USA.
Source
Publisher: Oxford University Press Country of Publication: England NLM ID: 0411011 Publication Model: Print Cited Medium: Internet ISSN: 1362-4962 (Electronic) Linking ISSN: 03051048 NLM ISO Abbreviation: Nucleic Acids Res Subsets: MEDLINE
Subject
Language
English
Abstract
Characterization of the epigenetic status of individual cells remains a challenge. Current sequencing approaches have limited coverage, and it is difficult to assign an epigenetic status to the transcription state of individual gene alleles in the same cell. To address these limitations, a targeted microscopy-based epigenetic visualization assay (EVA) was developed for detection and quantification of epigenetic marks at genes of interest in single cells. The assay is based on an in situ biochemical reaction between an antibody-conjugated alkaline phosphatase bound to the epigenetic mark of interest, and a 5'-phosphorylated fluorophore-labeled DNA oligo tethered to a target gene by gene-specific oligonucleotides. When the epigenetic mark is present at the gene, phosphate group removal by the phosphatase protects the oligo from λ-exonuclease activity providing a quantitative fluorescent readout. We applied EVA to measure 5-methylcytosine (5mC) and H3K9Ac levels at different genes and the HIV-1 provirus in human cell lines. To link epigenetic marks to gene transcription, EVA was combined with RNA-FISH. Higher 5mC levels at the silenced compared to transcribed XIST gene alleles in female somatic cells validated this approach and demonstrated that EVA can be used to relate epigenetic marks to the transcription status of individual gene alleles.
(© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)
(© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)