title: Ubiquitin ligase Siah2 regulates RevErbα degradation and the mammalian circadian clock.
abstract: Regulated degradation of proteins by the proteasome is often critical to their function in dynamic cellular pathways. The molecular clock underlying mammalian circadian rhythms relies on the rhythmic expression and degradation of its core components. However, because the tools available for identifying the mechanisms underlying the degradation of a specific protein are limited, the mechanisms regulating clock protein degradation are only beginning to be elucidated. Here we describe a cell-based functional screening approach designed to quickly identify the ubiquitin E3 ligases that induce the degradation of potentially any protein of interest. We screened the nuclear hormone receptor RevErbα (Nr1d1), a key constituent of the mammalian circadian clock, for E3 ligases that regulate its stability and found Seven in absentia2 (Siah2) to be a key regulator of RevErbα stability. Previously implicated in hypoxia signaling, Siah2 overexpression destabilizes RevErbα/β, and siRNA depletion of Siah2 stabilizes endogenous RevErbα. Moreover, Siah2 depletion delays circadian degradation of RevErbα and lengthens period length. These results demonstrate the utility of functional screening approaches for identifying regulators of protein stability and reveal Siah2 as a previously unidentified circadian clockwork regulator that mediates circadian RevErbα turnover.
authors.affiliation: Neuroscience Institute, Department of Pharmacology and Toxicology, Morehouse School of Medicine, Atlanta, GA 30310; jdebruyne@msm.edu hogenesc@mail.med.upenn.edu.
authors.forename: Jason P
authors.initials: JP
authors.lastname: DeBruyne
authors.affiliation: Neuroscience Institute, Department of Pharmacology and Toxicology, Morehouse School of Medicine, Atlanta, GA 30310;
authors.forename: Julie E
authors.initials: JE
authors.lastname: Baggs
authors.affiliation: Department of Pharmacology, Penn Genome Frontiers Institute, Institute for Translational Medicine and Therapeutics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia PA, 19104.
authors.forename: Trey K
authors.initials: TK
authors.lastname: Sato
authors.affiliation: Department of Pharmacology, Penn Genome Frontiers Institute, Institute for Translational Medicine and Therapeutics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia PA, 19104 jdebruyne@msm.edu hogenesc@mail.med.upenn.edu.
authors.forename: John B
authors.initials: JB
authors.lastname: Hogenesch
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D057906:Circadian Clocks / Q000235:genetics*; D004622:Embryo, Mammalian / Q000166:cytology; D005347:Fibroblasts / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D056953:Nuclear Receptor Subfamily 1, Group D, Member 1 / Q000235:genetics* / Q000378:metabolism; D059748:Proteolysis; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D044767:Ubiquitin-Protein Ligases / Q000235:genetics* / Q000378:metabolism; D000098563:Seven in Absentia Proteins
chemical_list: C064353:NR1D1 protein, human; D009687:Nuclear Proteins; D056953:Nuclear Receptor Subfamily 1, Group D, Member 1; D044767:Ubiquitin-Protein Ligases; D000098563:Seven in Absentia Proteins
keywords: RevErbα/Nr1d1; Siah2; circadian clock; ubiquitin ligase screen