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Quantitative Mass Spectrometry Reveals Changes in Histone H2B University of Kentucky UKnowledge Molecular and Cellular Biochemistry Faculty Molecular and Cellular Biochemistry Publications 5-11-2016 Quantitative Mass Spectrometry Reveals Changes in Histone H2B Variants as Cells Undergo Inorganic Arsenic-Mediated Cellular Transformation Matthew Rea University of Kentucky, [email protected] Tingting Jiang Florida State University Rebekah Eleazer University of Kentucky, [email protected] Meredith Eckstein University of Kentucky, [email protected] Alan G. Marshall Florida State University FSeoe nelloxtw pa thige fors aaddndition addal aitutionhorsal works at: https://uknowledge.uky.edu/biochem_facpub Part of the Biochemistry, Biophysics, and Structural Biology Commons, Cellular and Molecular Right click to open a feedback form in a new tab to let us know how this document benefits oy u. Physiology Commons, and the Inorganic Chemicals Commons Repository Citation 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 Publications. 110. https://uknowledge.uky.edu/biochem_facpub/110 This Article is brought to you for free and open access by the Molecular and Cellular Biochemistry at UKnowledge. It has been accepted for inclusion in Molecular and Cellular Biochemistry Faculty Publications by an authorized administrator of UKnowledge. For more information, please contact [email protected]. Authors Matthew Rea, Tingting Jiang, Rebekah Eleazer, Meredith Eckstein, Alan G. Marshall, and Yvonne N. Fondufe- Mittendorf Quantitative Mass Spectrometry Reveals Changes in Histone H2B Variants as Cells Undergo Inorganic Arsenic- Mediated Cellular Transformation Notes/Citation Information Published in Molecular & Cellular Proteomics, v. 15, no. 7, p. 2411-2422. This research was originally published in Molecular & Cellular Proteomics. Rea, M., Jiang, T., Eleazer, R., Eckstein, M., Marshall, A., and Fundufe-Mittendorf, Y. Quantitative Mass Spectrometry Reveals Changes in Histone H2B Variants as Cells Undergo Inorganic Arsenic-Mediated Cellular Transformation. Molecular & Cellular Proteomics. 2016; 57: 2411-2422. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc. The opc yright holder has granted the permission for posting the article here. Digital Object Identifier (DOI) https://doi.org/10.1074/mcp.M116.058412 This article is available at UKnowledge: https://uknowledge.uky.edu/biochem_facpub/110 crossmark Research © 2016 by The American Society for Biochemistry and Molecular Biology, Inc. This paper is available on line at http://www.mcponline.org Quantitative Mass Spectrometry Reveals Changes in Histone H2B Variants as Cells Undergo Inorganic Arsenic-Mediated Cellular Transformation*□S Matthew Rea‡**, Tingting Jiang§**, Rebekah Eleazer‡, Meredith Eckstein‡, Alan G. Marshall§¶, and Yvonne N. Fondufe-Mittendorf‡ʈ Exposure to inorganic arsenic, a ubiquitous environmen- ants provides a first step toward an understanding of the tal toxic metalloid, leads to carcinogenesis. However, the functional significance of the diversity of histone struc- mechanism is unknown. Several studies have shown that tures, especially in inorganic arsenic-mediated gene ex- inorganic arsenic exposure alters specific gene expres- pression and carcinogenesis. Molecular & Cellular Pro- sion patterns, possibly through alterations in chromatin teomics 15: 10.1074/mcp.M116.058412, 2411–2422, 2016. structure. While most studies on understanding the mech- anism of chromatin-mediated gene regulation have fo- cused on histone post-translational modifications, the Millions of people worldwide are chronically exposed to role of histone variants remains largely unknown. Incor- inorganic arsenic (iAs)1, a ubiquitous environmental carcino- poration of histone variants alters the functional proper- ties of chromatin. To understand the global dynamics of gen, through drinking water and food, with consequences chromatin structure and function in arsenic-mediated ranging from acute toxicities to malignant transformations (1, carcinogenesis, analysis of the histone variants incorpo- 2). Despite well-known deleterious health effects, the molec- rated into the nucleosome and their covalent modifica- ular mechanisms in iAs-mediated toxicity and disease are not tions is required. Here we report the first global mass fully understood. Several possible mechanisms have been spectrometric analysis of histone H2B variants as cells proposed, including oxidative stress and genotoxic DNA undergo arsenic-mediated epithelial to mesenchymal damage (2–5). Recent evidence suggests that an even less transition. We used electron capture dissociation-based understood—but equally important—mechanism is toxicity top-down tandem mass spectrometry analysis validated produced via changes in epigenetic-regulated gene expres- with quantitative reverse transcription real-time polymer- sion. Proper gene expression requires regulatory proteins to ase chain reaction to identify changes in the expression bind to their target sites on DNA, found in eukaryotic cells as levels of H2B variants in inorganic arsenic-mediated epi- thelial-mesenchymal transition. We identified changes in chromatin, many of which are directed by epigenetic marks. the expression levels of specific histone H2B variants in In eukaryotes, genomic DNA is organized into nucleo- two cell types, which are dependent on dose and length of somes, the fundamental repeating unit of chromatin (6). Each exposure of inorganic arsenic. In particular, we found nucleosome consists of 147 base pairs of DNA wrapping 1.7 increases in H2B variants H2B1H/1K/1C/1J/1O and turns around a histone octamer comprised of two copies each H2B2E/2F, and significant decreases in H2B1N/1D/1B as of H2A, H2B, H3, and H4 (7–9). The packaging of eukaryotic cells undergo inorganic arsenic-mediated epithelial-mes- genomes with histones to form chromatin is essential for the enchymal transition. The analysis of these histone vari- necessary condensation and protection of DNA. Changes in the structure of chromatin are essential for the proper regu- From the ‡Department of Molecular and Cellular Biochemistry, lation of cellular processes, including gene activation and University of Kentucky, Lexington, Kentucky 40536; §Department of silencing, DNA repair, replication and recombination. Altera- Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32306; ¶Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory, Florida State University, Tallahassee, Flor- 1 The abbreviations used are: iAs, inorganic arsenic; ECD, electron ida 32310 capture dissociation; EMT, epithelial-to-mesenchymal transition; FT- Received January 22, 2016, and in revised form, May 9, 2016 ICR, Fourier transform ion cyclotron resonance; HPLC: high-perform- Published, MCP Papers in Press, May 11, 2016, DOI 10.1074/ ance liquid chromatography; iAs-Rev, inorganic arsenic reverse cells; mcp.M116.058412 iAs-T, inorganic arsenic transformed cells; MS/MS, tandem mass Author contributions: Conceived and designed the experiments: spectrometry; NP-40, nonyl phenoxypolyethoxylethanol; PBS, phos- YNF-M. Performed the experiments: MR, TJ, RE, ME, YFN-M. Ana- phate buffered saline; PBST, phosphate buffered saline with Tween lyzed the data: MR, TJ, AGM, YNF-M. Contributed reagents/materi- 20; qRT-PCR, quantitative reverse transcription real-time polymerase als: AGM, YNF-M. Wrote the paper: MR, TJ, RE, ME, AGM, YNF-M. chain reaction; SWIFT, Stored Waveform Inverse Fourier Transform. Molecular & Cellular Proteomics 15.7 2411 H2B Variants Expression During iAs-mediated EMT tions of the chromatin template during these processes can expression and incorporation of these variants depends on occur through at least 3 interrelated mechanisms: post-trans- developmental and environmental cues. lational modifications of histones, ATP-dependent chromatin How do Histone Variants Get Incorporated?—Due to the remodeling, and the incorporation of specialized histone vari- weaker contacts between H2A/H2B dimers and DNA, the ants into chromatin. Of these mechanisms, the exchange of DNA at the entry/exit sites of the nucleosome is more prone to variants into and out of chromatin is the least understood or transiently unwrap (39). This allows for higher turnover rates studied. It is now known that the exchange of conventional for histones H2A and H2B than for H3 and H4 (40, 41). In this histones for variant histones has distinct and profound con- study, we focused on the variants of histone H2B, in partic- sequences within the cell (10, 11). ular, as their expression changes during iAs-mediated epithe- The intricate mechanisms of regulation that involve chro- lial-to-mesenchymal transition (EMT) (42). To date, no func- matin are largely based on the enormous complexity built into tional role for the H2B variants has been elucidated (35, 43). its structure. A number of studies have demonstrated that iAs However, the clinical importance of histone variants is high- exposure induces global and gene-specific post-translational lighted by recent reports that identified alterations in specific histone modifications such as reduction of acetylation in his- histone H2B variants in specific cancer cell types, suggesting tone H3 and H4: loss of H4Lys16ac, increases in H3Lys14ac, cancer or tissue-specific
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