In silico APC/C substrate discovery reveals cell cycle-dependent degradation of UHRF1 and other chromatin regulators
Fig 2
Putative APC/C substrates are enriched for roles in chromatin regulation.
(A) The set of 145 known and putative APC/C substrates is enriched for proteins involved in various chromatin-related process. This includes chromatin readers and writers, chaperones, RNA regulation and processing, DNA damage repair, and others. (Note that AURORA B, a mitotic kinase that phosphorylates histone H3, is listed here and in Fig 1E) (B) GO analysis of the overlapping KEN-box containing cell cycle–regulated transcripts. This set is enriched for the indicated biological process, including DNA metabolism, protein-DNA complex assembly, DNA packaging, and DNA conformation. (S1 Data) (C) APC/C activation assay to monitor substrate degradation. Following synchronization in mitosis, cells were washed 1 time and treated with CDK inhibitors to remove inhibitory phosphorylation marks that hinder the formation of APC/CCdh1 needed for the M/G1 phase transition. Protein degradation was monitored by immunoblot. CHAF1B and PCAF are putative APC/C substrates, and FoxM1 and Cyclin B are known targets. Data representative of n = 3 experiments. (D) coIP of HA-Cdh1 with Myc-CHAF1B in transiently transfected HEK-293T cells treated with proteasome inhibitors prior to harvesting. The underline indicates which protein or tag was blotted for in a particular panel (here and below). Input equal to 1% of IP, here and below. Data representative of n = 2 experiments. (E) coIP of HA-Cdh1 with FLAG-PCAF in transiently transfected 293T cells treated with proteasome inhibitors prior to harvesting. Data representative of n = 2 experiments. (F) coIP of HA-Cdh1 with Myc-NCOA3 in transiently transfected 293T cells treated with proteasome inhibitors prior to harvesting. Data representative of n = 3 experiments. (G) coIP of HA-Cdh1 with Myc-TTF2 in transiently transfected 293T cells treated with proteasome inhibitors prior to harvesting. Data representative of n = 4 experiments. (H) Mitotic shake-off of synchronized U2OS cells collected after release at the indicated timepoints. Immunoblotting for select endogenous proteins that are putative APC/C substrates or the positive control cyclin B. Data representative of n = 3 experiments. APC/C, anaphase-promoting complex/cyclosome; coIP, co-immunoprecipitation; GO, gene ontology.