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Archives of Toxicology

, Volume 92, Issue 2, pp 995–1014 | Cite as

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

  • Elisabeth Limbeck
  • Jens T. Vanselow
  • Julian Hofmann
  • Andreas Schlosser
  • Angela Mally
Biologics

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 13C-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.

Keywords

Ochratoxin A Histone hypoacetylation Repression of gene expression Renal carcinogenicity 

Abbreviations

ac

Acetylation

AMIGO2

Adhesion molecule with Ig-like domain 2

ChIP-qPCR

Chromatin immunoprecipitation followed by quantitative real-time PCR

ChIP-Seq

Chromatin immunoprecipitation combined with DNA sequencing

CLASP2

Cytoplasmic linker associated protein 2

CTNND1

Catenin delta 1

H3K9

Histone 3 lysine 9

H3K14

Histone 3 lysine 14

HAT

Histone acetyltransferase

LC–MS/MS

Liquid chromatography–tandem mass spectrometry

OTA

Ochratoxin A

Notes

Acknowledgements

The authors thank Tanja Röder and Elisabeth Rüb-Spiegel for excellent technical support.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Funding

Elisabeth Limbeck was supported by the Carcinogenesis Specialty Section Postdoctoral Fellowship Award received in March 2016 during the 55th Annual Meeting of the Society of Toxicology.

Supplementary material

204_2017_2107_MOESM1_ESM.docx (326 kb)
Supplementary material 1 (DOCX 325 kb)

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Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  1. 1.Department of ToxicologyUniversity of WürzburgWürzburgGermany
  2. 2.Rudolf Virchow Center for Experimental BiomedicineUniversity of WürzburgWürzburgGermany

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