Fusaric acid-induced promoter methylation of DNA methyltransferases triggers DNA hypomethylation in human hepatocellular carcinoma (HepG2) cells

ABSTRACT

Fusaric acid (FA), a mycotoxin contaminant of maize, displays toxicity in plants and animals; however, its epigenetic mechanism is unknown. DNA methylation, an epigenetic modification that regulates gene expression, is mediated by DNA methyltransferases (DNMTs; DNMT1, DNMT3A, and DNMT3B) and demethylases (MBD2). The expression of DNMTs and demethylases are regulated by promoter methylation, microRNAs (miR-29b) and post-translational modifications (ubiquitination). Alterations in these DNA methylation modifying enzymes affect DNA methylation patterns and offer novel mechanisms of FA toxicity. We determined the effect of FA on global DNA methylation as well as a mechanism of FA-induced changes in DNA methylation by transcriptional (promoter methylation), post-transcriptional (miR-29b) and post-translational (ubiquitination) regulation of DNMTs and MBD2 in the human hepatocellular carcinoma (HepG2) cell line. FA induced global DNA hypomethylation (p < 0.0001) in HepG2 cells. FA decreased the mRNA and protein expression of DNMT1 (p < 0.0001), DNMT3A (p < 0.0001), and DNMT3B (p < 0.0001) by upregulating miR-29b (p < 0.0001) and inducing promoter hypermethylation of DNMT1 (p < 0.0001) and DNMT3B (p < 0.0001). FA decreased the ubiquitination of DNMT1 (p = 0.0753), DNMT3A (p = 0.0008), and DNMT3B (p < 0.0001) by decreasing UHRF1 (p < 0.0001) and USP7 (p < 0.0001). FA also induced MBD2 promoter hypomethylation (p < 0.0001) and increased MBD2 expression (p < 0.0001). Together these results indicate that FA induces global DNA hypomethylation by altering DNMT promoter methylation, upregulating miR-29b, and increasing MBD2 in HepG2 cells.

KEYWORDS:

• Fusaric acid
• global DNA hypomethylation
• promoter methylation
• DNA methyltransferases
• miR-29b
• DNMT ubiquitination
• MBD2

Author contributions

TG, SN, PN, and AC conceptualized and designed the study. TG conducted all laboratory experiments, analyzed the data, and wrote the manuscript. PN assisted in conducting laboratory experiments. SN, PN, and AC revised the manuscript. All authors have read the manuscript prior to submission.

Disclosure statement

No potential conflict of interest was reported by the authors.

Data availability

All datasets generated in this study are available in Supplementary Information and from the corresponding author on reasonable request.

Supplementary material

Supplemental data for this article can be accessed here.

Additional information

Funding

This work was supported by the National Research Foundation Innovation Doctoral Scholarship (Grant no.: SFH160703175722) and the College of Health Sciences (University of Kwa-Zulu Natal; Grant no.: 570869).