PhD degree in Molecular Medicine European School of Molecular Medicine (SEMM), University of Milan and University of Naples “Federico II” Faculty of Medicine Settore disciplinare: BIO/10 Establishment and optimization of the ChroP approach, combining ChIP and MS-based proteomics, for the characterization of the chromatome at distinct functional domains Monica Soldi IFOM-IEO Campus, Milan Matricola n. R08407 Supervisor: Dr. Tiziana Bonaldi IFOM-IEO Campus, Milan Added co-Supervisor: Dr. Gioacchino Natoli IFOM-IEO Campus, Milan Anno accademico 2011-2012 To my family: Andrea and Paolo! 1 TABLE OF CONTENT FIGURE AND TABLE INDEX ....................................................................................... 5 LIST OF ABBREVIATIONS ....................................................................................... 8 1. ABSTRACT .............................................................................................................. 10 2. INTRODUCTION .................................................................................................... 13 2.1. Chromatin, epigenetics and histone post-translational modifications ............... 13 2.2. Mass Spectrometry analysis and MS-based proteomics ...................................... 17 2.2.1. Basic concepts of Mass Spectrometry........................................................... 19 2.2.2. Different MS approaches for hPTMs analysis: from “Bottom Up” to “Top Down”, via “Middle Down” .................................................................................. 25 2.2.3. Data analysis and bioinformatics tools for hPTM analysis by MS .............. 28 2.3. Quantitation strategies in MS-based proteomics ............................................... 32 2.3.1. Stable isotope labeling with amino acids in cell culture (SILAC) ............... 34 2.4. Quantitative MS-based approaches in epigenetics research ............................... 36 2.5. Mass Spectrometry analysis of histone variants and their modifications ........... 40 2.6. Interaction proteomics to study chromatin architecture ..................................... 45 3. AIM OF THE PROJECT ........................................................................................ 49 4. MATERIALS AND METHODS ............................................................................ 51 4.1. General biochemistry buffers .............................................................................. 51 4.2. Cell culture and SILAC labeling.......................................................................... 53 4.3. Native chromatin immunoprecipitation (N-ChIP) ............................................... 54 4.3.1. Buffers for N-ChIP ........................................................................................ 55 4.4. Cross-linking chromatin immunoprecipitation (X-ChIP) .................................... 55 4.4.1. Buffers for X-ChIP ........................................................................................ 56 4.5. In-gel digestion of histones for MS analysis ........................................................ 57 2 4.6. In-gel digestion of immunopurified proteins ....................................................... 57 4.7. Liquid Chromatography and tandem Mass Spectrometry (LC-MS/MS) ............. 58 4.8. Quantitative MS analysis of hPTMs co-enriched in precipitated chromatin ...... 59 4.8.1. Masses (in Da) of site-specific identification of PTMs on Histone H3 ........ 60 4.8.2. Masses (in Da) of site-specific identification of PTMs on Histone H2A ...... 60 4.8.3. Masses (in Da) of site-specific identification of PTMs on Histone H4 ........ 61 4.9. Proteomics analysis of proteins co-associated within precipitated chromatin fractions ...................................................................................................................... 61 4.10. Quantitative RT-PCR of immunopurified DNA ................................................. 62 4.10.1. Primers for quantitative PCR upon conventional ChIP ............................ 62 4.11. Immunoblot analysis .......................................................................................... 63 4.12. Immunofluorescence analysis ............................................................................ 63 4.13. ChIP-Sequencing: preparation of ChIP DNA libraries, sequencing and data analysis ....................................................................................................................... 64 5. RESULTS ................................................................................................................. 66 5.1. Characterization of hPTM patterns co-enriched at specific chromatin regions by N-ChIP combined with high resolution MS analysis ................................................. 66 5.2. Label free quantification of hPTMs enriched in repressed and active chromatin domains ....................................................................................................................... 71 5.3. Large-scale study of chromatin-associated proteins by X-ChIP combined with high resolution MS analysis ....................................................................................... 81 5.4. X-ChroP characterizes novel players in H3K9me3 and H3K4me3 chromatomes ............................................................................................................... 88 5.5. Accumulation of histone variants at specific chromatin domains ....................... 91 5.6. Heterochromatic enrichment of WICH complex, recriuted to H2A.X ................. 97 6. DISCUSSION ......................................................................................................... 102 3 7. CONCLUSIONS AND PRESPECTIVES ........................................................... 106 8. REFERENCES ....................................................................................................... 108 9. APPENDIX ............................................................................................................. 121 4 FIGURES and TABLE INDEX Figure 1. Histone post-translational modifications ...................................................... 14 Figure 2. Domains binding modified histones ............................................................... 15 Figure 3. The distinct components contributing to define the functional state of chromatin domain .......................................................................................................... 16 Figure 4. Proteomics experiment overview ................................................................... 18 Figure 5. Schematic representation of the basic components of a mass spectrometer . 19 Figure 6. The process of electrospray ionization (ESI) ................................................. 21 Figure 7. Liquid chromatography directly coupled to the mass spectrometer (nanoLC-MS) ................................................................................................................. 21 Figure 8. Example of collision-induced dissociation (CID) MS/MS spectra ................ 23 Figure 9. Schematic representation of “peptide-centric” versus “protein-centric” MS analytical strategies. ............................................................................................... 28 Figure 10. Peptide identification by MS/MS database searching ................................. 29 Table 1. Software and search algorithms used to study hPTMs ................................... 30 Figure 11. Metabolic and chemical labeling methods .................................................. 33 Figure 12. Overview of standard SILAC experiment .................................................... 35 Figure 13. Comparison between histone variant sequences ......................................... 41 Figure 14. Binding of linker histone H1 to the nucleosomal string induces chromatin compaction ..................................................................................................................... 44 Figure 15. Different biochemical approaches for the proteomic characterization of chromatin composition and architecture ....................................................................... 48 5 Figure 16. Scheme of the N-ChroP strategy, combining N-ChIP and MS analysis ...... 67 Figure 17. Representative scheme of hPTMs analysis .................................................. 68 Figure 18. Validation of the N-ChroP approach........................................................... 69 Figure 19. Evaluation of H3K9me3 antibody specificity and efficiency ....................... 70 Figure 20. Relative enrichment of modifications in H3K9me3 and H3K4me3- mononucleosome ............................................................................................................ 71 Figure 21. Relative enrichment of modifications in (3-8) and (9-17) peptides of H3 in H3K9me3 and H3K4me3 ChIPs ................................................................................ 72 Figure 22. Identification of R26me2 in H3K4me3 regions, enriched by N-ChIP ........ 73 Figure 23. Relative enrichment of modifications on (18-23) peptides of H3 in H3K9me3 and H3K4me3 ChIPs ................................................................................ 74 Figure 24. Elution profile of H3 (27-40) modified peptide ........................................... 76 Figure 25. Relative enrichment of modifications on (27-40) and (73-83) peptides of H3 in H3K9me3 and H3K4me3 ChIPs .........................................................................