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. 2017 Sep 15:331:6-17.
doi: 10.1016/j.taap.2017.03.017. Epub 2017 Mar 21.

Transient and permanent changes in DNA methylation patterns in inorganic arsenic-mediated epithelial-to-mesenchymal transition

Meredith Eckstein  1 , Matthew Rea  1 , Yvonne N Fondufe-Mittendorf  2
Affiliations

Transient and permanent changes in DNA methylation patterns in inorganic arsenic-mediated epithelial-to-mesenchymal transition

Meredith Eckstein et al. Toxicol Appl Pharmacol. .

Abstract

Chronic low dose inorganic arsenic exposure causes cells to take on an epithelial-to-mesenchymal phenotype, which is a crucial process in carcinogenesis. Inorganic arsenic is not a mutagen and thus epigenetic alterations have been implicated in this process. Indeed, during the epithelial-to-mesenchymal transition, morphologic changes to cells correlate with changes in chromatin structure and gene expression, ultimately driving this process. However, studies on the effects of inorganic arsenic exposure/withdrawal on the epithelial-to-mesenchymal transition and the impact of epigenetic alterations in this process are limited. In this study we used high-resolution microarray analysis to measure the changes in DNA methylation in cells undergoing inorganic arsenic-induced epithelial-to-mesenchymal transition, and on the reversal of this process, after removal of the inorganic arsenic exposure. We found that cells exposed to chronic, low-dose inorganic arsenic exposure showed 30,530 sites were differentially methylated, and with inorganic arsenic withdrawal several differential methylated sites were reversed, albeit not completely. Furthermore, these changes in DNA methylation mainly correlated with changes in gene expression at most sites tested but not at all. This study suggests that DNA methylation changes on gene expression are not clear-cut and provide a platform to begin to uncover the relationship between DNA methylation and gene expression, specifically within the context of inorganic arsenic treatment.

Keywords: DNA methylation; EMT; Epigenetics; Infinium MethylationEPIC BeadChip; Inorganic arsenic.

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Conflict of interest statement

Conflict of interest statement

The authors state no conflicts of interest pertaining to this work.

Figures

Fig. 1
Fig. 1
Chronic low-dose exposure to iAs and subsequent reversal causes cells to undergo EMT. (A). Diagram of experimental design. HeLa cells were treated with 0.5 µM sodium arsenite (iAsT) while some were mock treated with dIH2O (NT). After 36 days of iAs treatment, sodium arsenite was removed from growth media for some treated cells and replaced with dIH2O (iAs-rev); (B). western blot analysis of EMT markers show HeLa cells undergoing iAs-induced EMT. Claudin-1 decreased with treatment, and remained decreased in the iAs-rev condition. Some markers, like Snail, increased in the iAsT, but did not return towards NT levels in iAs-rev cells while N-cadherin and Slug increased in iAsT and returned towards NT levels.
Fig. 2
Fig. 2
Global DNA methylation levels are increased with 0.5 µM arsenite exposure. Results from the 5mC-ELISA show that 0.5 µM sodium arsenite exposure significantly increased global DNA methylation levels in HeLa cells. iAsT cells that had exposure removed (iAs-rev) showed a significant decrease from the iAsT DNA methylation levels; however, these are still significantly increased from NT levels. ELISA was performed in triplicate and error bars reflect the SEM of these replicates. Students t-test was performed for significance (p < 0.05); * denotes difference from NT † denotes difference from iAsT to iAs-rev.
Fig. 3
Fig. 3
Methylation EPIC BeadChip Assay reveals global DNA methylation changes between NT, iAsT, and iAs-rev. Comparison of loci from NT v. iAsT shows that 67% of loci on the chip are hypermethylated in iAsT, while only 33% are hypomethylated. Comparison of methylation loci between NT and iAs-rev shows that 77% of loci are hypermethylated in iAs-rev while only 23% are hypomethylated. Comparison of iAsT and iAs-rev reveals that the loci are approximately equal for hyper- and hypomethylation, suggesting that when iAs is removed many loci return towards NT levels.
Fig. 4
Fig. 4
DNA methylation patterns show changes both within CGIs and outside of CGIs during iAs-mediated EMT. (A). Diagram showing the relationship of CGIs and neighboring regions. CGI is defined as >50% CG content in at least a 200 base pair region. The north shore is 0–2000 bp upstream of the CGI and north shelf is 2001–4000 bp upstream of the CGI. The south shore is 0–2000 bp downstream of the CGI and the south shelf is 2001–4000 bp downstream of the CGI. Open sea regions are >4000 bp outside of the CGI. (B). Regional methylation changes between iAsT v. NT (C). Regional methylation changes between iAs-rev v. NT (D). Regional methylation changes between iAs-rev v. iAsT. Levels of methylation in shelves and shores stay relatively unchanged between groups and hyper- or hypomethylation. Prominent methylation changes occur at the CGIs and out in the open seas.
Fig. 5
Fig. 5
DNA methylation patterns within gene regulatory regions reveal large changes within gene bodies, at the 1st exon, and 200–1500 bp upstream of start of transcription. (A). Diagram mapping gene regulatory regions. TSS1500 is 200–1500 bp upstream of transcription start site (TSS), TSS200 is 1–200 bp upstream of TSS, 5′UTR is the 5′ untranslated region, 1st exon is the first translated region, gene body includes all other exons and all introns, and 3′UTR is the 3′ untranslated region where translation ends. Outside of the TSS1500 and 3′UTR is considered intergenic. (B). Gene regulatory methylation differences between iAsT and NT. (C). Gene regulatory methylation differences between iAs-rev and NT. (D). Gene regulatory methylation differences between iAs-rev and iAsT. Methylation within gene regulatory regions is generally stable throughout the treatment groups. Gene body and TSS1500 methylation shows the most variation between hyper- and hypomethylated DMRs.
Fig. 6
Fig. 6
DNA methylation level changes at promoter associated regions reveal large changes within the promoter and unclassified regions. Comparison of DMRs within different promoter types. (A). Regional promoter-associated methylation differences between iAsT and NT. (B). Regional promoter-associated methylation differences between iAs-rev and NT. (C). Regional promoter-associated methylation differences between iAs-rev and iAsT. Promoter methylation changes are most apparent at promoter associated (dark green) and unclassified regions (purple), which suggests that iAs targets distinct promoters and regions in methylation changes.
Fig. 7
Fig. 7
Correlation of differential methylation and gene expression. (A). Three genes were upregulated with iAs treatment. CORO1B and PPME1 both increased in expression with iAsT and a further increase was observed in iAs-rev. PPM1L increased in iAsT, but shifted towards NT levels with iAs-rev. (B). Seven genes were downregulated with iAs treatment. CDH12 and ARID5B decreased expression levels with iAsT as well as with iAs-rev. DYNC12, PRDX1 and EPC1 all decreased expression levels with iAsT, but with iAs-rev the expression levels increased closer to or even above NT levels. qRT-PCR reactions were performed in triplicate. * indicates a significant change when iAsT or iAs-rev is compared to NT and † indicates a significant change between iAsT and iAs-rev with p-value < 0.05. Error bars are from triplicate experiments and represent SEM.
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