Abstract
Exposure to inorganic arsenic, a ubiquitous environmental toxic metalloid, leads to carcinogenesis. However, the mechanism is unknown. Several studies have shown that inorganic arsenic exposure alters specific gene expression patterns, possibly through alterations in chromatin structure. While most studies on understanding the mechanism of chromatin-mediated gene regulation have focused on histone post-translational modifications, the role of histone variants remains largely unknown. Incorporation of histone variants alters the functional properties of chromatin. To understand the global dynamics of chromatin structure and function in arsenic-mediated carcinogenesis, analysis of the histone variants incorporated into the nucleosome and their covalent modifications is required. Here we report the first global mass spectrometric analysis of histone H2B variants as cells undergo arsenic-mediated epithelial to mesenchymal transition. We used electron capture dissociation-based top-down tandem mass spectrometry analysis validated with quantitative reverse transcription real-time polymerase chain reaction to identify changes in the expression levels of H2B variants in inorganic arsenic-mediated epithelial-mesenchymal transition. We identified changes in the expression levels of specific histone H2B variants in two cell types, which are dependent on dose and length of exposure of inorganic arsenic. In particular, we found increases in H2B variants H2B1H/1K/1C/1J/1O and H2B2E/2F, and significant decreases in H2B1N/1D/1B as cells undergo inorganic arsenic-mediated epithelial-mesenchymal transition. The analysis of these histone variants provides a first step toward an understanding of the functional significance of the diversity of histone structures, especially in inorganic arsenic-mediated gene expression and carcinogenesis.
Highlights
From the ‡Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky 40536; §Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32306; ¶Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory, Florida State University, Tallahassee, Florida 32310
Millions of people worldwide are chronically exposed to inorganic arsenic1, a ubiquitous environmental carcinogen, through drinking water and food, with consequences ranging from acute toxicities to malignant transformations [1, 2]
We focused on the variants of histone H2B, in particular, as their expression changes during inorganic arsenic (iAs)-mediated epithelial-to-mesenchymal transition (EMT) [42]
Summary
BEAS-2B and HeLa Growth Conditions and Cell Transformation Through Sodium Arsenite Exposure—BEAS-2B and HeLa cells were grown in Dulbecco’s modified Eagle medium 9.4 T FT-ICR MS—Prepared histone samples were dissolved in 50% acetonitrile (H2O/CH3CN) (50/50) with 1.0% formic acid to reach a final concentration of 1.0 M and introduced into a custom-built 9.4 Tesla FT-ICR mass spectrometer via positive electrospray ionization [44]. Both precursor and product ion spectra were acquired by direct infusion at the flow rate of 0.35 l/min. A secondary antibody (␣-Rabbit or ␣-Mouse) was applied the day and developed by use of ECF (GE-Typhoon FLA9500)
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