In silico APC/C substrate discovery reveals cell-cycle-dependent degradation of UHRF1 and other chromatin regulators

E3 ubiquitin ligases are the final regulatory enzymes in the ubiquitin cascade, responsible for choosing substrates for ubiquitylation. Despite their central role in governing ubiquitin pathways, technical challenges have precluded development of simple and robust methods to systematically map their substrates. The Anaphase Promoting Complex/Cyclosome (APC/C) is an E3 ligase and master regulator of cell cycle progression and genome maintenance. Here, we develop a sensitive and specific computational strategy to predict APC/C substrates, leveraging orthogonal features among known targets. Interestingly, chromatin regulatory proteins are enriched among putative substrates and we validate several here. We reveal detailed ubiquitylation mechanisms of a key reader and writer of histone modifications, UHRF1, and show that its degradation regulates cell cycle and D methylation maintenance. Our results uncover mechanisms underlying the coordination between cell cycle and the chromatin landscape, and suggest an extensive, post-translational crosstalk with far-reaching consequences in normal cell physiology and disease. Genomic D was isolated from Parental U2OS cells and U2OS cells overexpressing either GFP-UHRF1WT or GFP-UHRF1KEN/AAA. All samples groups (total of 9 samples all together) were processed in biological triplicate, and the Parental U2OS cells served as the control.