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

View ORCID ProfileAlexander J. Federation, Vivek Nandakumar, Hao Wang, Brian C. Searle, Lindsay K. Pino, Gennifer Merrihew, Ying S. Ting, Nicholas Howard, Tanya Kutyavin, Michael J. MacCoss, John A. Stamatoyannopoulos
doi: https://doi.org/10.1101/345686
Alexander J. Federation
1Altius Institute for Biomedical Sciences; Seattle, WA 98121
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Vivek Nandakumar
1Altius Institute for Biomedical Sciences; Seattle, WA 98121
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Hao Wang
1Altius Institute for Biomedical Sciences; Seattle, WA 98121
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Brian C. Searle
2University of Washington, Department of Genome Sciences; Seattle, WA 98195
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Lindsay K. Pino
2University of Washington, Department of Genome Sciences; Seattle, WA 98195
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Gennifer Merrihew
2University of Washington, Department of Genome Sciences; Seattle, WA 98195
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Ying S. Ting
2University of Washington, Department of Genome Sciences; Seattle, WA 98195
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Nicholas Howard
1Altius Institute for Biomedical Sciences; Seattle, WA 98121
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Tanya Kutyavin
1Altius Institute for Biomedical Sciences; Seattle, WA 98121
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Michael J. MacCoss
2University of Washington, Department of Genome Sciences; Seattle, WA 98195
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John A. Stamatoyannopoulos
1Altius Institute for Biomedical Sciences; Seattle, WA 98121
2University of Washington, Department of Genome Sciences; Seattle, WA 98195
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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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Posted June 30, 2018.
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Quantification of nuclear protein dynamics reveals chromatin remodeling during acute protein degradation
Alexander J. Federation, Vivek Nandakumar, Hao Wang, Brian C. Searle, Lindsay K. Pino, Gennifer Merrihew, Ying S. Ting, Nicholas Howard, Tanya Kutyavin, Michael J. MacCoss, John A. Stamatoyannopoulos
bioRxiv 345686; doi: https://doi.org/10.1101/345686
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Quantification of nuclear protein dynamics reveals chromatin remodeling during acute protein degradation
Alexander J. Federation, Vivek Nandakumar, Hao Wang, Brian C. Searle, Lindsay K. Pino, Gennifer Merrihew, Ying S. Ting, Nicholas Howard, Tanya Kutyavin, Michael J. MacCoss, John A. Stamatoyannopoulos
bioRxiv 345686; doi: https://doi.org/10.1101/345686

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