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.
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This work was supported by NSF grant MCB 1517986 to YFN-M, and NIEHS grant R01-ES024478 to YNF-M. A portion of this work was performed at the National High Magnetic Field Laboratory, which is supported by National Science Foundation Cooperative Agreement # DMR-1157490 and the State of Florida.
This article contains supplemental material.
Raw mass spectrometry data was deposited into ProteomeXchange via the PRIDE database and the accession number is PXD003503.
Rea, Matthew; Jiang, Tingting; Eleazer, Rebekah; Eckstein, Meredith; Marshall, Alan G.; and Fondufe-Mittendorf, Yvonne N., "Quantitative Mass Spectrometry Reveals Changes in Histone H2B Variants as Cells Undergo Inorganic Arsenic-Mediated Cellular Transformation" (2016). Molecular and Cellular Biochemistry Faculty Publication. 110.
Supplemental Table 1: Primers used for qRT-PCR.
mcp.M116.058412-2.pdf (141 kB)
Supplemental Fig. 1. Comparative analyses of the identified histone variants.
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Supplemental Fig. 2. The full ECD spectrum of H2B1N.
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Supplemental Fig. 3. The full spectrum of H2B2E.
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Supplemental Fig. 4. The full ECD spectrum of H2B2F.
mcp.M116.058412-6.pdf (976 kB)
Supplemental Fig. 5. Fragmentation maps for H2B variants.
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Supplemental Fig. 6. Biological Replicates of HeLa and BEA S-2B Mass Spectrometry analysis.
mcp.M116.058412-8.pdf (28 kB)
Supplemental Fig. 7. Time-dependent changes in H2B variants as cells undergo iAs- mediated EMT.