CLOCK deubiquitylation by USP8 inhibits CLK/CYC transcription in drosophila

Howard Hughes Medical Institute.
Genes & development (Impact Factor: 10.8). 11/2012; 26(22):2536-49. DOI: 10.1101/gad.200584.112
Source: PubMed


A conserved transcriptional feedback loop underlies animal circadian rhythms. In Drosophila, the transcription factors CLOCK (CLK) and CYCLE (CYC) activate the transcription of direct target genes like period (per) and timeless (tim). They encode the proteins PER and TIM, respectively, which repress CLK/CYC activity. Previous work indicates that repression is due to a direct PER-CLK/CYC interaction as well as CLK/CYC phosphorylation. We describe here the role of ubiquitin-specific protease 8 (USP8) in circadian transcriptional repression as well as the importance of CLK ubiquitylation in CLK/CYC transcription activity. usp8 loss of function (RNAi) or expression of a dominant-negative form of the protein (USP8-DN) enhances CLK/CYC transcriptional activity and alters fly locomotor activity rhythms. Clock protein and mRNA molecular oscillations are virtually absent within circadian neurons of USP8-DN flies. Furthermore, CLK ubiquitylation cycles robustly in wild-type flies and peaks coincident with maximal CLK/CYC transcription. As USP8 interacts with CLK and expression of USP8-DN increases CLK ubiquitylation, the data indicate that USP8 deubiquitylates CLK, which down-regulates CLK/CYC transcriptional activity. Taken together with the facts that usp8 mRNA cycles and that its transcription is activated directly by CLK/CYC, USP8, like PER and TIM, contributes to the transcriptional feedback loop cycle that underlies circadian rhythms.

Download full-text


Available from: Yue Li
  • Source
    • "We confirmed that CG14619, which is a CCG[37,38], is the orthologue of mUsp2 by protein sequence alignments (S1 Fig) andthat dUsp2-kd suffer from sub viability ([33]; Fig 2C–2F,S1 and S2 Files). Interestingly, dUsp2 and dUsp8, which is a core clock cogwheel in Drosophila are paralogues and possibly differentially evolved towards output effector and core cogwheel status, respectively[36,48]. We found no significant alteration of the circadian free-running period in both clock-neuron specific knockdown of dUsp2 in Drosophila and in our Usp2-KO mouse (Fig 1, Fig 2A and 2B, Table 1). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The mammalian circadian clock influences most aspects of physiology and behavior through the transcriptional control of a wide variety of genes, mostly in a tissue-specific manner. About 20 clock-controlled genes (CCGs) oscillate in virtually all mammalian tissues and are generally considered as core clock components. One of them is Ubiquitin-Specific Protease 2 (Usp2), whose status remains controversial, as it may be a cogwheel regulating the stability or activity of core cogwheels or an output effector. We report here that Usp2 is a clock output effector related to bodily Ca2+ homeostasis, a feature that is conserved across evolution. Drosophila with a whole-body knockdown of the orthologue of Usp2, CG14619 (dUsp2-kd), predominantly die during pupation but are rescued by dietary Ca2+ supplementation. Usp2-KO mice show hyperabsorption of dietary Ca2+ in small intestine, likely due to strong overexpression of the membrane scaffold protein NHERF4, a regulator of the Ca2+ channel TRPV6 mediating dietary Ca2+ uptake. In this tissue, USP2-45 is found in membrane fractions and negatively regulates NHERF4 protein abundance in a rhythmic manner at the protein level. In clock mutant animals (Cry1/Cry2-dKO), rhythmic USP2-45 expression is lost, as well as the one of NHERF4, confirming the inverse relationship between USP2-45 and NHERF4 protein levels. Finally, USP2-45 interacts in vitro with NHERF4 and endogenous Clathrin Heavy Chain. Taken together these data prompt us to define USP2-45 as the first clock output effector acting at the post-translational level at cell membranes and possibly regulating membrane permeability of Ca2+.
    Full-text · Article · Jan 2016 · PLoS ONE
  • Source
    • "In addition to DBT, kinases known to be involved in CLK phosphorylation include NEMO (NMO), which destabilizes CLK (Yu et al., 2011), and CK2a that stabilizes CLK and inhibits its transcriptional activity (Szabó et al., 2013). CLK is also regulated by ubiquitylation with the HECT-domain ubiquitin ligase CTRIP destabilizing CLK (Lamaze et al., 2011), and the USP8 ubiquitin protease decreasing its transcriptional activity (Luo et al., 2012). Finally, CLK activity is controlled by repressors/activators such as CLOCKWORK ORANGE (CWO) (Matsumoto et al., 2007; Kadener et al., 2007; Lim et al., 2007; Richier et al., 2008) and the FOS ortholog KAYAK (KAY) (Ling et al., 2012). "
    [Show abstract] [Hide abstract]
    ABSTRACT: In the Drosophila circadian oscillator, the CLOCK/CYCLE complex activates transcription of period (per) and timeless (tim) in the evening. PER and TIM proteins then repress CLOCK (CLK) activity during the night. The pace of the oscillator depends upon post-translational regulation that affects both positive and negative components of the transcriptional loop. CLK protein is highly phosphorylated and inactive in the morning, whereas hypophosphorylated active forms are present in the evening. How this critical dephosphorylation step is mediated is unclear. We show here that two components of the STRIPAK complex, the CKA regulatory subunit of the PP2A phosphatase and its interacting protein STRIP, promote CLK dephosphorylation during the daytime. In contrast, the WDB regulatory PP2A subunit stabilizes CLK without affecting its phosphorylation state. Inhibition of the PP2A catalytic subunit and CKA downregulation affect daytime CLK similarly, suggesting that STRIPAK complexes are the main PP2A players in producing transcriptionally active hypophosphorylated CLK. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Full-text · Article · May 2015 · Cell Reports
  • Source
    • "Instead, USP2 appears to regulate PER1 intracellular localization (Yang et al., 2014). Interestingly, the only other DUB that to our knowledge has been implicated in clock mechanisms, Drosophila USP8, also seems to act in a non-degradative manner: it deubiquitinates CLOCK, thereby inhibiting transcriptional activity of CLOCK/CYCLE (CYCLE is the Drosophila homolog of BMAL1; Luo et al., 2012). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Circadian rhythms, endogenous cycles of about 24 h in physiology, are generated by a master clock located in the suprachiasmatic nucleus of the hypothalamus and other clocks located in the brain and peripheral tissues. Circadian disruption is known to increase the incidence of various illnesses, such as mental disorders, metabolic syndrome, and cancer. At the molecular level, periodicity is established by a set of clock genes via autoregulatory translation-transcription feedback loops. This clock mechanism is regulated by post-translational modifications such as phosphorylation and ubiquitination, which set the pace of the clock. Ubiquitination in particular has been found to regulate the stability of core clock components but also other clock protein functions. Mutation of genes encoding ubiquitin ligases can cause either elongation or shortening of the endogenous circadian period. Recent research has also started to uncover roles for deubiquitination in the molecular clockwork. Here, we review the role of the ubiquitin pathway in regulating the circadian clock and we propose that ubiquitination is a key element in a clock protein modification code that orchestrates clock mechanisms and circadian behavior over the daily cycle.
    Full-text · Article · Aug 2014 · Frontiers in Molecular Neuroscience
Show more