HeLa TI cell-based assay as a new approach to screen for chemicals able to reactivate the expression of epigenetically silenced genes.

Varvara Maksimova,Gennady Belitsky,Anastasia Beizer,Natalya Shalginskikh,Olga Vlasova,Kirill Kirsanov,Richard Katz,Olga Lizogub,Marianna Yakubovskaya,Dmitry Fedorov,Polina Bugaeva,Olga Usalka,Ekaterina Lesovaya,Miguel Branco

doi:10.1371/journal.pone.0252504

Abstract

Chemicals reactivating epigenetically silenced genes target diverse classes of enzymes, including DNMTs, HDACs, HMTs and BET protein family members. They can strongly influence the expression of genes and endogenous retroviral elements with concomitant dsRNA synthesis and massive transcription of LTRs. Chemicals reactivating gene expression may cause both beneficial effects in cancer cells and may be hazardous by promoting carcinogenesis. Among chemicals used in medicine and commerce, only a small fraction has been studied with respect to their influence on epigenetic silencing. Screening of chemicals reactivating silent genes requires adequate systems mimicking whole-genome processes. We used a HeLa TSA-inducible cell population (HeLa TI cells) obtained by retroviral infection of a GFP-containing vector followed by several rounds of cell sorting for screening purposes. Previously, the details of GFP epigenetic silencing in HeLa TI cells were thoroughly described. Herein, we show that the epigenetically repressed gene GFP is reactivated by 15 agents, including HDAC inhibitors–vorinostat, sodium butyrate, valproic acid, depsipeptide, pomiferin, and entinostat; DNMT inhibitors–decitabine, 5-azacytidine, RG108; HMT inhibitors–UNC0638, BIX01294, DZNep; a chromatin remodeler–curaxin CBL0137; and BET inhibitors–JQ-1 and JQ-35. We demonstrate that combinations of epigenetic modulators caused a significant increase in cell number with reactivated GFP compared to the individual effects of each agent. HeLa TI cells are competent to metabolize xenobiotics and possess constitutively expressed and inducible cytochrome P450 mono-oxygenases involved in xenobiotic biotransformation. Thus, HeLa TI cells may be used as an adequate test system for the extensive screening of chemicals, including those that must be metabolically activated. Studying the additional metabolic activation of xenobiotics, we surprisingly found that the rat liver S9 fraction, which has been widely used for xenobiotic activation in genotoxicity tests, reactivated epigenetically silenced genes. Applying the HeLa TI system, we show that N-nitrosodiphenylamine and N-nitrosodimethylamine reactivate epigenetically silenced genes, probably by affecting DNA methylation.

Highlights

Epigenetic gene silencing is an important mechanism of genome regulation and includes DNA methylation, histone modification, ncRNA interference and chromatin remodeling [1]
trichostatin A (TSA), a histone deacetylase inhibitor, was added to the culture medium of HeLa TSA-inducible (HeLa TI) cells at different concentrations, while 0.1% dimethyl sulfoxide (DMSO) was used as the vehicle control (Fig 1A)
The treatment of HeLa TI cells with TSA was followed by an increase in the fraction of GFP-positive cells in a dose-dependent manner

Summary

Introduction

Epigenetic gene silencing is an important mechanism of genome regulation and includes DNA methylation, histone modification, ncRNA interference and chromatin remodeling [1]. Technologies appropriate for the visualization of molecular events could be very helpful for the elucidation of unknown or persistent effects of environmental chemicals In this respect, there is an urgent need to develop new screening methods that are scalable to medium and high throughput, have clear end points for different epigenetic events, produce low false positive and false negative rates and are cost effective [8,17]

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