Single cell epigenetic visualization assay.

Sam Kint,Wim Van Criekinge,Winnok H De Vos,Diane S Krause,Oleg Denisenko,Linos Vandekerckhove,Karol Bomsztyk

doi:10.1093/nar/gkab009

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.

Highlights

Epigenetic programs specify cell phenotypes through covalent modifications of histones and DNA, nucleosome position and density, and substitution by histone variants [1,2,3]
The assay takes advantage of the ability of 5 -3 ␭-exonuclease to selectively degrade 5 -phosphorylated strands of doublestranded DNA, as opposed to un-phosphorylated strands or single-stranded DNA. ␭-exonuclease activity in the assay is controlled by an alkaline phosphatase (AP) recruited as an antibody conjugate to the epigenetic mark of interest
In absence of AP, the probe remains phosphorylated at its 5 end, causing the ␭-exonuclease to degrade the 5 half of the detector oligo up to the nick region, thereby releasing the sensor oligo

Summary

Introduction

Epigenetic programs specify cell phenotypes through covalent modifications of histones and DNA, nucleosome position and density, and substitution by histone variants [1,2,3]. While epigenetic alterations drive normal organism development, aberrations in these processes have emerged as hallmarks of cancer and other diseases where particular cell types and individual cells may play critical roles. Conventional approaches used to study epigenetic events, such as chromatin immunoprecipitation (ChIP) and bisulfite sequencing (BS-seq), measure averaged gene epigenetic states in bulk cell populations or tissue fragments and can not be used to estimate the contribution of individual cells to the epigenetic profile of a specimen, let alone combined epigenetic and transcription analysis of gene alleles in a single cell. To assess epigenetic states in individual cells, sequencingbased and imaging-based techniques have recently been introduced for single cell epigenetic analyses [4]. A microscopy-based approach has been developed to measure histone modifications at individual genes in single cells using a proximity ligation assay (ISH-PLA [5]).

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Results

Conclusion