Quantification of nuclear protein dynamics reveals chromatin remodeling during acute protein degradation

Federation, A. J.; Nandakumar, V.; Wang, H.; Searle, B.; Pino, L.; Merrihew, G.; Ting, Y. S.; Howard, N.; Kutyavin, T.; MacCoss, M. J.; Stamatoyannopoulos, J. A.

doi:10.1101/345686

bioRxiv · 10.1101/345686

Quantification of nuclear protein dynamics reveals chromatin remodeling during acute protein degradation

Federation, A. J.·Nandakumar, V.·Wang, H.·Searle, B.·Pino, L.·Merrihew, G.·Ting, Y. S.·Howard, N.·Kutyavin, T.·MacCoss, M. J.·Stamatoyannopoulos, J. A.

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Abstract

Sequencing-based technologies cannot measure post-transcriptional dynamics of the nuclear proteome, but unbiased mass-spectrometry measurements of chromatin-associated proteins remain difficult. In this work, we have combined facile nuclear sub-fractionation approaches with data-independent acquisition mass spectrometry to improve detection and quantification of nuclear proteins in human cells and tissues. Nuclei are isolated and subjected to a series of extraction conditions that enrich for nucleoplasm, euchromatin, heterochromatin and nuclear-membrane associated proteins. Using this approach, we can measure peptides from over 70% of the expressed nuclear proteome. As we are physically separating chromatin compartments prior to analysis, proteins can be assigned into functional chromatin environments to illuminate systems-wide nuclear protein dynamics. The integrity of nuclear sub-compartments were validated with immunofluorescence, which confirms the presence of key markers during chromatin extraction. We then apply this method to study the nuclear proteome-wide response to pharmacological degradation of the BET bromodomain proteins. BET degradation leads to widespread changes in chromatin composition, and we discover global HDAC1/2-mediated remodeling of chromatin previously bound by BET bromodomains. In summary, we have developed a technology for reproducible, comprehensive characterization of the nuclear proteome to observe the systems-wide nuclear protein dynamics.

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Federation, A. J., Nandakumar, V., Wang, H., Searle, B., Pino, L., Merrihew, G., Ting, Y. S., Howard, N., Kutyavin, T., MacCoss, M. J., Stamatoyannopoulos, J. A.. 2018-06-14. Quantification of nuclear protein dynamics reveals chromatin remodeling during acute protein degradation. https://doi.org/10.1101/345686

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