Linking site-specific loss of histone acetylation to repression of gene expression by the mycotoxin ochratoxin A

Abstract

Ochratoxin A (OTA) is a potent renal carcinogen but its mechanism has not been fully resolved. In vitro and in vivo gene expression studies consistently revealed down-regulation of gene expression as the predominant transcriptional response to OTA. Based on the importance of specific histone acetylation marks in regulating gene transcription and our recent finding that OTA inhibits histone acetyltransferases (HATs), leading to loss of acetylation of histones and non-histone proteins, we hypothesized that OTA-mediated repression of gene expression may be causally linked to HAT inhibition and loss of histone acetylation. In this study, we used a novel mass spectrometry approach employing chemical C-acetylation of unmodified lysine residues for quantification of post-translational acetylation sites to identify site-specific alterations in histone acetylation in human kidney epithelial cells (HK-2) exposed to OTA. These results showed OTA-mediated hypoacetylation at almost all lysine residues of core histones, including loss of acetylation at H3K9 and H3K14, which are hallmarks of gene activation. ChIP-qPCR used to establish a possible link between H3K9 or H3K14 hypoacetylation and OTA-mediated down-regulation of selected genes (AMIGO2, CLASP2, CTNND1) confirmed OTA-mediated H3K9 hypoacetylation at promoter regions of these genes. Integrated analysis of OTA-mediated genome-wide changes in H3K9 acetylation identified by ChIP-Seq with published gene expression data further demonstrated that among OTA-responsive genes almost 80% of hypoacetylated genes were down-regulated, thus confirming an association between H3K9 acetylation status and gene expression of these genes. However, only 7% of OTA repressed genes showed loss of H3K9 acetylation within promoter regions. Interestingly, however, GO analysis and functional enrichment of down-regulated genes showing loss of H3K9 acetylation at their respective promoter regions revealed enrichment of genes involved in the regulation of transcription, including a number of transcription factors that are predicted to directly or indirectly regulate the expression of 98% of OTA repressed genes. Thus, it is possible that histone acetylation changes in a fairly small set of genes but with key function in transcriptional regulation may trigger a cascade of events that may lead to overall repression of gene expression. Taken together, our data provide evidence for a mechanistic link between loss of H3K9 acetylation as a consequence of OTA-mediated inhibition of HATs and repression of gene expression by OTA, thereby affecting cellular processes critical to tumorigenesis.

An in vitro attempt at precision toxicology reveals the involvement of DNA methylation alteration in ochratoxin A-induced G0/G1 phase arrest

“…Therefore, after a 12 hour treatment with 10 nM OTA, the relative mRNA expression levels of two effectors in the Notch pathway, Hes1, and Hes5, were significantly decreased in rat brain cell cultures. Through ChIP-Seq analysis, Limbeck and colleagues identified H3K9 acetylation at promoter regions of NOTCH1 in human proximal tubular cells after treatment with 25 µM OTA for 24 h. This indicated a novel epigenetic mechanism of Notch signaling suppression, contributing to its renal toxicity [48].…”

Toxicodynamics of Mycotoxins in the Framework of Food Risk Assessment—An In Silico Perspective

“…The International Agency for Research of Cancer (IARC) classified ochratoxin A as a possible carcinogen for humans (group 2B) [ 131 ]. The mechanisms of carcinogenic action are not still fully understood yet [ 132 ]. However, along with the DNA damage due to an enhanced production of reactive species of oxygen and nitrogen, it has been hypothesized a prominent role of its direct genotoxic properties via the formation of DNA-adducts [ 133 , 134 ].…”

“…The prevalent expression of the toxins transporters in male in comparison to female may explain the gender-dependent effects found [ 137 , 139 ]. In addition, ochratoxin A is thought to alter a cascade of molecular events via inhibition of histone acetyltransferases [ 132 ], which may cause, inter alia, alterations in the cell-cycles progression during mitosis. This may lead to apoptotic death or to the generation of genetically unstable polyploid cells that may re-enter the cell cycle [ 137 ].…”

Mechanisms of Rodent Renal Carcinogenesis Revisited

“…Other research groups also have not been able to demonstrate ochratoxin A–derived DNA adducts using modern analytical chemistry techniques (Gautier et al 2001; Schlatter, Studer-Rohr, and Rasonyi 1996). On the other hand, gene expression studies consistently demonstrate downregulation of gene expression as the predominant transcriptional response to ochratoxin A (reviewed by Limbeck et al 2018).…”

“…These enzymes play an important role in regulating a wide range of cellular processes including progression through mitosis, gene transcription, and DNA damage and repair (reviewed by Mally 2012). Recent data suggest a mechanistic link between loss of histone acetylation and the repression of gene expression (Limbeck et al 2018). Interference with mitosis (observed by live-cell microscopy, Czakai et al 2011) associated with induction of various mitotic and chromosome aberrations in ochratoxin A exposed tubule cells, such as perturbation of genes involved in mitosis, perturbation of centrosome and spindle checkpoint function (Adler et al 2009), karyomegaly, asymmetric spindles, aberrant chromosome alignment (Rached et al 2006), and highly condensed metaphase plates with separated chromatids. Sustained stimulus for attempted compensatory tubule cell proliferation, specifically in the pars recta tubule cells (Rached et al 2007; Qi et al 2014).…”