Quantitative Mass Spectrometry Reveals Changes in Histone H2B Variants as Cells Undergo Inorganic Arsenic-Mediated Cellular Transformation

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.

Fig. 1.

Chronic low-dose exposure to iAs causes cells to undergo EMT. A, Morphology changes seen in iAs-transformed BEAS-2B cells. BEAS-2B cells become elongated and spindle-shaped with 0.5 μm iAs treatment. B, Western blot analyses of EMT markers showing changes in the EMT markers. For instance, β-Catenin levels decrease and Slug levels increase in iAs transformed cells. C, qRT-PCR confirmation of iAs-induced EMT markers in HeLa (top) and BEAS-2B (bottom). qRT-PCR confirms the changes in expression of EMT markers in iAs-transformed cells. Error bars represent standard deviation (S.D.) from three biological replicates, each containing three technical replicates. Asterisks denote p values < 0.05 as determined by Student's t test.

Fig. 2.

Electron Capture Dissociation top-down MS/MS of H2B isoforms. A, Off-line reversed-phase liquid chromatogram for histones. B, Representative mass spectrum of histone H2B sequence variant isotopic distributions for charge state, Z = 19. Precursor m/z 726.75 ions were isolated by SWIFT and fragmented with ECD.

Fig. 3.

Upper left. ECD product ion spectrum for precursor (H2B1N/2E/2F) m/z 726. 75 ions. Upper right and bottom. ECD fragmentation maps generated by our custom software. When peptides are dissociated, c ions contain the charge at the N terminus, whereas z ions contain the charge at the C terminus.

Fig. 4.

Mass spectrometry profiling of H2B variants in NT and iAs-T cells. A, Profiles of H2B variants in HeLa cells show differential abundance of 10 histone H2B variants in iAs-transformed cells compared with time-matched NT cells, after 16 days of treatment. Additionally, there were no major changes in the expression levels in cells transformed with 0.5 μm and 1 μm iAs. B, Changes in the abundance of these variants were confirmed in BEAS-2B cells. Although most of the variants show patterns similar to iAs-T HeLa cells, H2B1J, H2B1O, and H2B2E, show different patterns of expression in the iAs-T BEAS-2B cells. C, Western blots of total histones H2AB and H3 show equal expression in treated and nontreated cells. Equal amounts of nuclear protein (25 ng) were loaded onto a 15% SDS-PAGE gel and blotted for H2B or H3. Error bars represent standard deviation from three technical replicates. Biological replicates in HeLa and BEAS-2B cells show a similar trend (supplemental Fig. 7). Asterisks denote p values < 0.05 as determined by Student's t test.

Fig. 5.

Time-dependent changes in H2B variants as cells undergo iAs-mediated EMT. The abundance of histone H2B variants was monitored at different times during the iAs-transformation process. Changes by day (Top-4 days, Middle-8 days, Bottom-16 days) of the H2B variants showed that the most overall up-regulated histone H2B variants were H2B1K and H2B1C, whereas the most downregulated variants were H2B1D and H2B1B. Shown next to the graphs are the obtained ECD spectra for these four variants. The full extent of the changes in the relative abundance of the 10 observed H2B variants changes and a consequence of exposure time (days of exposure) are shown in supplemental Fig. 6 and ECD fragmentation maps in supplemental Fig. 5. Untreated cells are from the same days as the treated groups. Error bars represent S.D. from three technical replicate experiments. *, p value < 0.05 as determined by Student's t test.

Fig. 6.

Validation of H2B changes with qRT-PCR. A, qRT-PCR in HeLa cells validated the expression patterns of 7 of the 10 H2B variants investigated through MS. B, qRT-PCR in BEAS-2B cells validates the expression patterns of all 10 H2B variants. Error bars represent S.D. from three biological replicates, each containing three technical replicates. *, p value < 0.05, determined by Student's t test.

Fig. 7.

Effects with the removal of iAs-treatment after transformation (iAs-rev cells). A, Western blot analyses of EMT markers. Claudin-3 and Slug in iAs-rev cells show an intermediate level of expression, whereas others such as β-Catenin and Vimentin revert to near NT levels. B, qRT-PCR for EMT markers. qRT-PCR for EMT markers in iAs-Rev cells mostly show changes in expression opposite of the iAs-T cells. C, qRT-PCR of the H2B variants. qRT-PCR for H2B variants in iAs-Rev cells mostly show changes in expression reverting to near NT cells. Error bars represent S.D. from three biological replicates, each containing three technical replicates. *, p < 0.05 compared with NT; †, p < 0.05 between iAs-T and iAs-Rev.