A quantitative atlas of histone modification signatures from human cancer cells

Abstract

BackgroundAn integral component of cancer biology is the understanding of molecular properties uniquely distinguishing one cancer type from another. One class of such properties is histone post-translational modifications (PTMs). Many histone PTMs are linked to the same diverse nuclear functions implicated in cancer development, including transcriptional activation and epigenetic regulation, which are often indirectly assayed with standard genomic technologies. Thus, there is a need for a comprehensive and quantitative profiling of cancer lines focused on their chromatin modification states.ResultsTo complement genomic expression profiles of cancer lines, we report the proteomic classification of 24 different lines, the majority of which are cancer cells, by quantifying the abundances of a large panel of single and combinatorial histone H3 and H4 PTMs, and histone variants. Concurrent to the proteomic analysis, we performed transcriptomic analysis on histone modifying enzyme abundances as a proxy for quantifying their activity levels. While the transcriptomic and proteomic results were generally consistent in terms of predicting histone PTM abundance from enzyme abundances, several PTMs were regulated independently of the modifying enzyme expression. In addition, combinatorial PTMs containing H3K27 methylation were especially enriched in breast cell lines. Knockdown of the predominant H3K27 methyltransferase, enhancer of zeste 2 (EZH2), in a mouse mammary xenograft model significantly reduced tumor burden in these animals and demonstrated the predictive utility of proteomic techniques.ConclusionsOur proteomic and genomic characterizations of the histone modification states provide a resource for future investigations of the epigenetic and non-epigenetic determinants for classifying and analyzing cancer cells.

Histone H4 Lysine 20 (H4K20) Methylation, Expanding the Signaling Potential of the Proteome One Methyl Moiety at a Time

“…Depending on the cell type, up to ϳ80% of H4 molecules can be dimethylated, whereas H4K20me1 and H4K20me3 are generally less abundant, for example being present on 10 and 5% of nucleosomes in asynchronous HeLa cells, respectively, with similar ratios observed in mouse embryonic fibroblasts (MEFs) and other cell types (13)(14)(15)(16). Three distinct SET domains containing lysine (K) methyltransferase (KMT) enzymes, SETD8 (SET8, PR-SET7), SUV4-20H1, and SUV4-20H2, are responsible for the generation of the three different methyl states of H4K20 (17)(18)(19)(20).…”

Quantitative Proteomic Discovery of Dynamic Epigenome Changes that Control Human Cytomegalovirus (HCMV) Infection

“…The relative abundance of a histone PTM isoform is computed as the peak area for that isoform normalized by the sum of the peak areas for all PTM isoforms on the same histone peptide. We have previously shown this to be an accurate method for examining relative quantitative changes in PTMs between samples (19,20), and in this study we will be investigating the fold-change of histone PTMs in HCMV-infected samples relative to mock-infected samples at different time points. Fixed modifications considered were propionylation of all unmodified and monomethylated lysine residues, and variable modifications consisted of acetylation and methylation (mono-, di-, and tri-) of lysine residues.…”

Streamlined discovery of cross-linked chromatin complexes and associated histone modifications by mass spectrometry

“…In contrast to the analysis of most proteins associated with BioTAP-tagged baits, analysis of the extensively modified histones presents a unique challenge for standard proteomics. Derivatization with propionic anhydride is one approach to facilitate histone analysis that has been used by various groups (17)(18)(19)(20)(21)(22). The anhydride reaction protects unmodified and monomethylated lysines from trypsin cleavage and increases overall hydrophobicity.…”

“…Propionylated peptides were desalted with C 18 -STAGE tips for LC-MS. Genomic histones were prepared by lysing cells with RIPA buffer followed by high salt and sulfuric acid extraction. Histones were precipitated with trichloroacetic acid, washed with acetone, and prepared by propionic anhydride as described (17). To induce histone acetylation, 5 mM sodium butyrate was added to S2 tissue culture medium for overnight incubation.…”

“…Washed beads were incubated with trypsin in AMBIC overnight at 37°C. The supernatant was then derivatized with propionic anhydride as described (17). Propionylated peptides were desalted with C 18 -STAGE tips for LC-MS. Genomic histones were prepared by lysing cells with RIPA buffer followed by high salt and sulfuric acid extraction.…”