Abstract
In living cells, the N-terminal tails of core histones, the proteinaceous component of nucleosomes, are subjected to a range of covalent post-translational modifications (PTMs), which have specific roles in modulating chromatin structure and function. A growing body of evidence suggests that deregulation of histone modification patterns, upstream or downstream of DNA methylation, is a critical event in cancer initiation and progression. However, a comprehensive description of how histone modifications, singly or in combination, is disrupted in transformed cells is missing; consequently the issue whether and how specific changes in histone PTMs patterns correlate to particular tumor features is still elusive. In the present study, we focused on human breast cancer and comprehensively analyzed PTMs on histone H3 and H4 from four cancer cell lines (MCF7, MDA-MB231, MDA-MB453 and T-47D), in comparison with normal epithelial breast cells. We performed high-resolution mass spectrometry analysis of histones, in combination with stable isotope labeling with amino acids in cell culture (SILAC), to quantitatively track the modification changes in cancer cells, as compared to their normal counterpart. Our investigation focuses on lysine acetylation and methylation on fourteen distinct sites in H3 and H4. We observed significant changes for several modifications in cancer cells: while in a few cases those modifications had been previously described as a hallmark of human tumors, we could identify novel modifications, whose abundance is significantly altered in breast cancer cells. Overall, these modifications may represent part of a “breast cancer-specific epigenetic signature”, with implications in the characterization of histone-related biomarkers. This work demonstrates that SILAC-based proteomics is a powerful tool to study qualitatively and quantitatively histone PTMs patterns, contributing significantly to the comprehension of epigenetic phenomena in cancer biology.
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Abbreviations
- MS:
-
Mass spectrometry
- LC-MS/MS:
-
Liquid chromatography coupled to tandem mass spectrometry
- SILAC:
-
Stable isotope labeling with amino acid in cell culture
- hPTMs:
-
Histone post-translational modifications
- HDACs:
-
Histone deacetylases
- HMTs:
-
Histone methyl-transferases
- FA:
-
Formic acid
- ACN:
-
Acetonitrile
- Ac:
-
Acetylation
- me1:
-
Mono-methylation
- me2:
-
Di-methylation
- me3:
-
Tri-methylation
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Acknowledgments
TB work is supported by grants from the Giovanni Armenise-Harvard Foundation Career Development Program, the Association of International Cancer Research (AICR), the Associazione Italiana Ricerca sul Cancro (AIRC) and the Fondazione Cariplo. Work in SMi lab is supported by EEC (Epitron) and AIRC. We would like to thank Pietro Spinelli for technical support in cell culture and David Cairns for critical reading of the manuscript.
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A. Cuomo and S. Moretti contributed equally to this work.
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Fig. S1 Relative quantification of histone modifications in breast cancer cells
versusnon-tumoralbreast cells. SILAC ratios in log2 scale for: histone H3 (9-17) acetylated on K14 or mono-, di-, and trimethylatedon K9 co-exiting with K14 acetylated (left panel); H3 (18-26) unmodified, mono-, and diacetylatedon K23 and K18/K23 respectively. The value indicates by red dotted lines is the standarddeviation calculated for the ratios of non modified peptides within twelve LC runs as described in section2.4 of Materials and Methods. (EPS 981 kb)
Fig. S2 MS/MS spectra of histone H3 and H4 peptides analyzed
. Fragmentation spectra were used forthe site-specific assignment of modifications within the peptides; MASCOT search with the most intenseions identified in the MSMS spectra with the calculated score; theoretical and experimental spectra aredisplayed. (PDF 1878 kb)
Fig. S3 Identification and Relative Quantification of peptide (27-40) modified species from histoneH3.3
(A) Zoomed survey MS scan relative to elution time range (27.07-27.40 min) of LC/MSMS analysisfor MCF7/MCF10 (H/L) sample. Peptides (27-40) modified ions from the three H3 variants co-elutes,resulting in a highly complex peak pattern where H3.3 peptides are not detectable and peaks form H3.1/2are predominant. (B) The tetra-methylated (me4) specie of (27-40) from H3.3 variant becomes detectable,because of the delayed elution (27.48min), that allows its resolution in MS (C) SILAC ratios in log2 scalefor H3.3 (27-40) tetra-methylated, whose MS/MS analysis reveal the following site-attribution: K27me2K36me2. Red dotted lines represent the standard deviation calculated for the ratios of unmodified species. (PDF 380 kb)
Table S1 Ratios measured for the monoisotopic peaks from three different unmodified peptides forH3, H4 and H2A
. These ratios were used for the calculation of correction factor and the correspondingstandard deviation for each CF for each sample. (PDF 204 kb)
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Cuomo, A., Moretti, S., Minucci, S. et al. SILAC-based proteomic analysis to dissect the “histone modification signature” of human breast cancer cells. Amino Acids 41, 387–399 (2011). https://doi.org/10.1007/s00726-010-0668-2
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DOI: https://doi.org/10.1007/s00726-010-0668-2