Multi-faceted quantitative proteomics analysis of histone H2B isoforms and their modifications

Abstract

BackgroundHistone isoforms and their post-translational modifications (PTMs) play an important role in the control of many chromatin-related processes including transcription and DNA damage. Variants of histones H2A and H3 have been studied in depth and have been found to have distinct functions. Although 13 somatic histone H2B isoforms have been identified by various biochemical and mass spectrometric (MS) approaches, the distinct roles of these isoforms within human cells are as yet unknown. Here, we have developed quantitative MS techniques to characterize isoform-specific H2B expression across the cell cycle, in differentiated myogenic cells, and in different cancer cell lines to illuminate potential functional roles.ResultsUsing the MS strategies that we developed, we identified differences in H2B isoform levels between different cancer cell types, suggesting cancer or tissue-specific H2B isoform regulation. In particular, we found large variations in the levels of isoforms H2B1B and H2B1M across the panel of cell lines. We also found that, while individual H2B isoforms do not differ in their acetylation levels, trends in the acetylation on all H2B isoforms correlated with acetylation on other histone family members in the cancer cell line panel. We also used the MS strategies to study H2B protein expression across the cell cycle and determined that H2B isoforms that are alternatively spliced to carry a polyadenylation signal rather than the standard histone downstream element are expressed independently of the cell cycle. However, the level of protein produced from the polyadenylated transcripts does not contribute significantly to the total pool of H2B isoforms translated across the cell cycle or in non-cycling myogenic cells.ConclusionsOur results show that H2B isoforms are expressed at varying levels in different cells, suggesting isoform-specific, and possibly cell-type-specific, H2B gene regulation. The bottom-up mass spectrometry technique we developed for H2B quantification is compatible with the current standard histone H3 and H4 bottom-up ‘one-pot’ analysis platform so that H2B isoforms and their modifications can be studied in future experiments at the same time as histone H3 and H4 modifications. Therefore, we have expanded the histone landscape that can be interrogated in future experiments.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-015-0006-8) contains supplementary material, which is available to authorized users.

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

“…Underscoring that importance, the expression of H2B histone variants is cell-type specific, differentiation stage-dependent as well as cancer-stage and type dependent (38). However, the study of these variants has remained difficult largely because they differ by very few amino acids (Table I; supplemental Fig.…”

“…S1), making variant-specific antibodies virtually impossible to develop. However, the use of top-down MS/MS has proven to be an invaluable tool to study such closely related histone variants (38). This type of mass spectrometry characterizes the histone proteome in an unbiased approach, providing a sequence that does not rely on the existence of modification-specific antibodies.…”

“…To date, no functional role for the H2B variants has been elucidated (35,43). However, the clinical importance of histone variants is highlighted by recent reports that identified alterations in specific histone H2B variants in specific cancer cell types, suggesting cancer or tissue-specific H2B variant regulation (38). In fact, there are 22 genes encoding the different H2B variants within the human genome spread across the three major histone gene clusters.…”

“…This high degree of sequence identity makes the H2B variants challenging to study with standard protein techniques that would require the use of variant-specific antibodies. However, MS can readily resolve them because of the changes in mass from the differences in amino acids found in the variants (35,38,43). Thus, in this study, we used top-down ultrahigh resolution Fourier transform ion cyclotron resonance (FT-ICR) tandem mass spectrometry (MS/MS) to quantify the relative abundance of histone H2B variants in cells undergoing iAsmediated EMT.…”

Epigenomic reprogramming in inorganic arsenic-mediated gene expression patterns during carcinogenesis

“…Specifically, we showed that histone H2B variants were abnormally expressed following iAs exposure, and during the subsequent iAs-mediated epithelial-mesenchymal transition (EMT) (73). We identified a total of 16 H2B variants, 13 somatic and three testis-specific; of the somatic variants, seven were upregulated while three were downregulated (73)(74)(75)(76)(77). The most significant changes observed in our study were the upregulation of histones H2B1K and H2B1C, and downregulation of histones H2B1D and H2B1B.…”