Skip to main content

Advertisement

Log in

SILAC-based proteomic analysis to dissect the “histone modification signature” of human breast cancer cells

  • Original Article
  • Published:
Amino Acids Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

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

References

  • Beck HC (2010) Mass spectrometry in epigenetic research. Methods Mol Biol 593:263–282

    Article  PubMed  CAS  Google Scholar 

  • Bracken AP, Pasini D, Capra M, Prosperini E, Colli E, Helin K (2003) EZH2 is downstream of the pRB-E2F pathway, essential for proliferation and amplified in cancer. EMBO J 22(20):5323–5335

    Article  PubMed  CAS  Google Scholar 

  • Burlingame AL, Zhang X, Chalkley RJ (2005) Mass spectrometric analysis of histone posttranslational modifications. Methods 36(4):383–394

    Article  PubMed  CAS  Google Scholar 

  • Cedar H, Bergman Y (2009) Linking DNA methylation and histone modification: patterns and paradigms. Nat Rev Genet 10(5):295–304

    Article  PubMed  CAS  Google Scholar 

  • Cosgrove MS (2007) Histone proteomics and the epigenetic regulation of nucleosome mobility. Exp Rev Proteomics 4(4):465–478

    Article  CAS  Google Scholar 

  • Cox J, Mann M (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26(12):1367–1372

    Article  PubMed  CAS  Google Scholar 

  • Dobosy JR, Selker EU (2001) Emerging connections between DNA methylation and histone acetylation. Cell Mol Life Sci 58(5–6):721–727

    Article  PubMed  CAS  Google Scholar 

  • Elsheikh SE, Green AR, Rakha EA, Powe DG, Ahmed RA, Collins HM, Soria D, Garibaldi JM, Paish CE, Ammar AA, Grainge MJ, Ball GR, Abdelghany MK, Martinez-Pomares L, Heery DM, Ellis IO (2009) Global histone modifications in breast cancer correlate with tumor phenotypes, prognostic factors, and patient outcome. Cancer Res 69(9):3802–3809

    Article  PubMed  CAS  Google Scholar 

  • Esteller M (2002) CpG island hypermethylation and tumor suppressor genes: a booming present, a brighter future. Oncogene 21(35):5427–5440

    Article  PubMed  CAS  Google Scholar 

  • Feinberg AP, Vogelstein B (1983) Hypomethylation distinguishes genes of some human cancers from their normal counterparts. Nature 301(5895):89–92

    Article  PubMed  CAS  Google Scholar 

  • Feng W, Lu Z, Luo RZ, Zhang X, Seto E, Liao WS, Yu Y (2007) Multiple histone deacetylases repress tumor suppressor gene ARHI in breast cancer. Int J Cancer 120(8):1664–1668

    Article  PubMed  CAS  Google Scholar 

  • Fraga MF, Ballestar E, Villar-Garea A, Boix-Chornet M, Espada J, Schotta G, Bonaldi T, Haydon C, Ropero S, Petrie K, Iyer NG, Perez-Rosado A, Calvo E, Lopez JA, Cano A, Calasanz MJ, Colomer D, Piris MA, Ahn N, Imhof A, Caldas C, Jenuwein T, Esteller M (2005) Loss of acetylation at Lys16 and trimethylation at Lys20 of histone H4 is a common hallmark of human cancer. Nat Genet 37(4):391–400

    Article  PubMed  CAS  Google Scholar 

  • Freitas MA, Sklenar AR, Parthun MR (2004) Application of mass spectrometry to the identification and quantification of histone post-translational modifications. J Cell Biochem 92(4):691–700

    Article  PubMed  CAS  Google Scholar 

  • Garcia BA, Shabanowitz J, Hunt DF (2007) Characterization of histones and their post-translational modifications by mass spectrometry. Curr Opin Chem Biol 11(1):66–73

    Article  PubMed  CAS  Google Scholar 

  • Herman JG, Baylin SB (2003) Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med 349(21):2042–2054

    Article  PubMed  CAS  Google Scholar 

  • Ikegami K, Iwatani M, Suzuki M, Tachibana M, Shinkai Y, Tanaka S, Greally JM, Yagi S, Hattori N, Shiota K (2007) Genome-wide and locus-specific DNA hypomethylation in G9a deficient mouse embryonic stem cells. Genes Cells 12(1):1–11

    Article  PubMed  CAS  Google Scholar 

  • Jenuwein T, Allis CD (2001) Translating the histone code. Science 293(5532):1074–1080

    Article  PubMed  CAS  Google Scholar 

  • Jung HR, Pasini D, Helin K, Jensen ON (2010) Quantitative mass spectrometry of histones H3.2 and H3.3 in Suz12-deficient mouse embryonic stem cells reveals distinct, dynamic post-translational modifications at Lys-27 and Lys-36. Mol Cell Proteomics 9(5):838–850

    Article  PubMed  CAS  Google Scholar 

  • Kall L, Storey JD, MacCoss MJ, Noble WS (2008) Assigning significance to peptides identified by tandem mass spectrometry using decoy databases. J Proteome Res 7(1):29–34

    Article  PubMed  Google Scholar 

  • Kelleher NL, Zubarev RA, Bush K, Furie B, Furie BC, McLafferty FW, Walsh CT (1999) Localization of labile posttranslational modifications by electron capture dissociation: the case of gamma-carboxyglutamic acid. Anal Chem 71(19):4250–4253

    Article  PubMed  CAS  Google Scholar 

  • Kouzarides T (2007) Chromatin modifications and their function. Cell 128(4):693–705

    Article  PubMed  CAS  Google Scholar 

  • Kovalchuk O, Tryndyak VP, Montgomery B, Boyko A, Kutanzi K, Zemp F, Warbritton AR, Latendresse JR, Kovalchuk I, Beland FA, Pogribny IP (2007) Estrogen-induced rat breast carcinogenesis is characterized by alterations in DNA methylation, histone modifications and aberrant microRNA expression. Cell Cycle 6(16):2010–2018

    Article  PubMed  CAS  Google Scholar 

  • Loyola A, Bonaldi T, Roche D, Imhof A, Almouzni G (2006) PTMs on H3 variants before chromatin assembly potentiate their final epigenetic state. Mol Cell 24(2):309–316

    Article  PubMed  CAS  Google Scholar 

  • Mann M (2006) Functional and quantitative proteomics using SILAC. Nat Rev Mol Cell Biol 7(12):952–958

    Article  PubMed  CAS  Google Scholar 

  • Medzihradszky KF, Zhang X, Chalkley RJ, Guan S, McFarland MA, Chalmers MJ, Marshall AG, Diaz RL, Allis CD, Burlingame AL (2004) Characterization of Tetrahymena histone H2B variants and posttranslational populations by electron capture dissociation (ECD) Fourier transform ion cyclotron mass spectrometry (FT-ICR MS). Mol Cell Proteomics 3(9):872–886

    Article  PubMed  CAS  Google Scholar 

  • Olsen JV, de Godoy LM, Li G, Macek B, Mortensen P, Pesch R, Makarov A, Lange O, Horning S, Mann M (2005) Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap. Mol Cell Proteomics 4(12):2010–2021

    Article  PubMed  CAS  Google Scholar 

  • Ong SE, Mittler G, Mann M (2004) Identifying and quantifying in vivo methylation sites by heavy methyl SILAC. Nat Methods 1(2):119–126

    Article  PubMed  CAS  Google Scholar 

  • Pesavento JJ, Kim YB, Taylor GK, Kelleher NL (2004) Shotgun annotation of histone modifications: a new approach for streamlined characterization of proteins by top down mass spectrometry. J Am Chem Soc 126(11):3386–3387

    Article  PubMed  CAS  Google Scholar 

  • Pfister S, Rea S, Taipale M, Mendrzyk F, Straub B, Ittrich C, Thuerigen O, Sinn HP, Akhtar A, Lichter P (2008) The histone acetyltransferase hMOF is frequently downregulated in primary breast carcinoma and medulloblastoma and constitutes a biomarker for clinical outcome in medulloblastoma. Int J Cancer 122(6):1207–1213

    Article  PubMed  CAS  Google Scholar 

  • Pokholok DK, Harbison CT, Levine S, Cole M, Hannett NM, Lee TI, Bell GW, Walker K, Rolfe PA, Herbolsheimer E, Zeitlinger J, Lewitter F, Gifford DK, Young RA (2005) Genome-wide map of nucleosome acetylation and methylation in yeast. Cell 122(4):517–527

    Article  PubMed  CAS  Google Scholar 

  • Rappsilber J, Mann M, Ishihama Y (2007) Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc 2(8):1896–1906

    Article  PubMed  CAS  Google Scholar 

  • Roth MJ, Forbes AJ, Boyne MT II, Kim YB, Robinson DE, Kelleher NL (2005) Precise and parallel characterization of coding polymorphisms, alternative splicing, and modifications in human proteins by mass spectrometry. Mol Cell Proteomics 4(7):1002–1008

    Article  PubMed  CAS  Google Scholar 

  • Seligson DB, Horvath S, Shi T, Yu H, Tze S, Grunstein M, Kurdistani SK (2005) Global histone modification patterns predict risk of prostate cancer recurrence. Nature 435(7046):1262–1266

    Article  PubMed  CAS  Google Scholar 

  • Siuti N, Roth MJ, Mizzen CA, Kelleher NL, Pesavento JJ (2006) Gene-specific characterization of human histone H2B by electron capture dissociation. J Proteome Res 5(2):233–239

    Article  PubMed  CAS  Google Scholar 

  • Su X, Ren C, Freitas MA (2007) Mass spectrometry-based strategies for characterization of histones and their post-translational modifications. Exp Rev Proteomics 4(2):211–225

    Article  CAS  Google Scholar 

  • Suzuki J, Chen YY, Scott GK, Devries S, Chin K, Benz CC, Waldman FM, Hwang ES (2009) Protein acetylation and histone deacetylase expression associated with malignant breast cancer progression. Clin Cancer Res 15(9):3163–3171

    Article  PubMed  CAS  Google Scholar 

  • Tachibana M, Sugimoto K, Fukushima T, Shinkai Y (2001) Set domain-containing protein, G9a, is a novel lysine-preferring mammalian histone methyltransferase with hyperactivity and specific selectivity to lysines 9 and 27 of histone H3. J Biol Chem 276(27):25309–25317

    Article  PubMed  CAS  Google Scholar 

  • Taverna SD, Ueberheide BM, Liu Y, Tackett AJ, Diaz RL, Shabanowitz J, Chait BT, Hunt DF, Allis CD (2007) Long-distance combinatorial linkage between methylation and acetylation on histone H3 N termini. Proc Natl Acad Sci USA 104(7):2086–2091

    Article  PubMed  CAS  Google Scholar 

  • Thomas CE, Kelleher NL, Mizzen CA (2006) Mass spectrometric characterization of human histone H3: a bird’s eye view. J Proteome Res 5(2):240–247

    Article  PubMed  CAS  Google Scholar 

  • Thorne AW, Kmiciek D, Mitchelson K, Sautiere P, Crane-Robinson C (1990) Patterns of histone acetylation. Eur J Biochem 193(3):701–713

    Article  PubMed  CAS  Google Scholar 

  • Tryndyak VP, Kovalchuk O, Pogribny IP (2006) Loss of DNA methylation and histone H4 lysine 20 trimethylation in human breast cancer cells is associated with aberrant expression of DNA methyltransferase 1, Suv4–20h2 histone methyltransferase and methyl-binding proteins. Cancer Biol Ther 5(1):65–70

    Article  PubMed  CAS  Google Scholar 

  • Wang Z, Zang C, Rosenfeld JA, Schones DE, Barski A, Cuddapah S, Cui K, Roh TY, Peng W, Zhang MQ, Zhao K (2008) Combinatorial patterns of histone acetylations and methylations in the human genome. Nat Genet 40(7):897–903

    Article  PubMed  CAS  Google Scholar 

  • Zhang K, Williams KE, Huang L, Yau P, Siino JS, Bradbury EM, Jones PR, Minch MJ, Burlingame AL (2002) Histone acetylation and deacetylation: identification of acetylation and methylation sites of HeLa histone H4 by mass spectrometry. Mol Cell Proteomics 1(7):500–508

    Article  PubMed  CAS  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Tiziana Bonaldi.

Additional information

A. Cuomo and S. Moretti contributed equally to this work.

Electronic supplementary material

Below is the link to the electronic supplementary material.

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)

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00726-010-0668-2

Keywords

Navigation