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Decoding the protein composition of whole nucleosomes with Nuc-MS

Abstract

Current proteomic approaches disassemble and digest nucleosome particles, blurring readouts of the ‘histone code’. To preserve nucleosome-level information, we developed Nuc-MS, which displays the landscape of histone variants and their post-translational modifications (PTMs) in a single mass spectrum. Combined with immunoprecipitation, Nuc-MS quantified nucleosome co-occupancy of histone H3.3 with variant H2A.Z (sixfold over bulk) and the co-occurrence of oncogenic H3.3K27M with euchromatic marks (for example, a >15-fold enrichment of dimethylated H3K79me2). Nuc-MS is highly concordant with chromatin immunoprecipitation-sequencing (ChIP-seq) and offers a new readout of nucleosome-level biology.

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Fig. 1: Three strategies for histone analysis, including Nuc-MS for the direct interrogation of intact nucleosomes.
Fig. 2: Nuc-MS analysis of endogenous mononucleosomes from HEK293T cells.
Fig. 3: Nuc-MS of endogenous nucleosomes prepared from cells with H3.3-FLAG-HA WT or K27M.

Data availability

MS1, MS2 and MS3 spectra presented in the paper are available online in the MassIVE database under accession code MSV000085238 and can be visualized with Thermo Qual Browser. Source data are provided with this paper.

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Acknowledgements

This work was supported by the National Institute of General Medical Sciences (P41 GM108569) for the National Resource for Translational and Developmental Proteomics at Northwestern University and NIH grants S10OD025194 and RF1AG063903 (Kelleher laboratory) and R44GM116584, R44CA212733 and R44CA214076 (EpiCypher). L.F.S. is a Gilliam Fellow of the Howard Hughes Medical Institute. Research in this publication is also supported by Thermo Fisher Scientific and a fellowship associated with the Chemistry of Life Processes Predoctoral Training grant T32GM105538 at Northwestern University. A.P. is supported by the Transition to Independence grant K99CA234434-01. We also thank M. Senko, P. Compton, C. Koo, L. Szymczak and M. McAnnally for technical assistance and S. Judge and A. Rosenzweig for providing thoughtful suggestions to improve the manuscript.

Author information

Affiliations

Authors

Contributions

L.F.S. performed Nuc-MS data acquisition and analysis. K.J. assisted with proteoform quantitation and MS analysis. A.P., M.A.M., A.S.L. and A.S. prepared and made available H3.3K27M-FLAG and H3.3WT-FLAG mononucleosomes. M.A.M. and M.I. conducted and analyzed ChIP-seq experiments. J.O.K. assisted with acquisition of multiplexed I2MS data on endogenous nucleosomes. M.J.M., M.A.C., J.M.B. and S.A.H. assisted with synthesis, purification and verification of modified nucleosomes. M.-C.K. coordinated modified nucleosome synthesis and provided insightful feedback on the manuscript. L.F.S. and N.L.K. conceived the project and wrote the manuscript.

Corresponding author

Correspondence to Neil L. Kelleher.

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Competing interests

N.L.K. serves as a consultant to Thermo Fisher Scientific. EpiCypher is a commercial developer and supplier of reagents, including the recombinant semi-synthetic modified nucleosomes (dNucs) used in this study. The other authors declare no competing interests.

Additional information

Peer review information Arunima Singh was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figs. 1–19, Tables 1–3 and Protocol.

Reporting Summary

Supplementary Data 1

Statistical source data and analysis for quantitative comparison of two equimolar synthetic Nucs presented in Supplementary Fig. 4b.

Supplementary Data 2

Statistical source data and analysis for quantitative comparison of H3.3 and HEK293T bulk Nucs presented in Supplementary Fig. 9.

Supplementary Data 3

Statistical source data and analysis for quantitative comparison of H3.3K27M fragments that localize to K79me2 in Supplementary Fig. 14.

Supplementary Data 4

Uncropped western blots for data presented in Supplementary Fig. 17.

Source data

Source Data Fig. 3

Statistical source data and analysis for quantitative comparison of H3.3K27M and WT Nucs presented in Fig. 3c–e.

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Schachner, L.F., Jooß, K., Morgan, M.A. et al. Decoding the protein composition of whole nucleosomes with Nuc-MS. Nat Methods 18, 303–308 (2021). https://doi.org/10.1038/s41592-020-01052-9

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