Quantification of nuclear protein dynamics reveals chromatin remodeling during acute protein degradation
Year: 2018
Type: preprint
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.
DOI: 10.1101/345686
Authors: 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
Language: English
Subfield: Spectroscopy
Field: Chemistry
Domain: Physical Sciences
Open Access status: green
OpenAlex ID: W2807897000