Implication of the F-Box Protein FBXL21 in Circadian Pacemaker Function in Mammals Hugues Dardente1*, Jorge Mendoza2, Jean-Michel Fustin1¤, Etienne Challet2, David G. Hazlerigg1 1 School of Biological Sciences, Aberdeen University, Aberdeen, Scotland, United Kingdom, 2 Institut de Neurosciences Cellulaires et Inte´gratives, De´partement de Neurobiologie des Rythmes, UMR7168, Strasbourg, France Abstract In mammals, the circadian clock relies on interlocked feedback loops involving clock genes and their protein products. Post- translational modifications control intracellular trafficking, functionality and degradation of clock proteins and are keys to the functioning of the clock as recently exemplified for the F-Box protein Fbxl3. The SCFFbxl3 complex directs degradation of CRY1/2 proteins and Fbxl3 murine mutants have a slower clock. To assess whether the role of Fbxl3 is phylogenetically conserved, we investigated its function in the sheep, a diurnal ungulate. Our data show that Fbxl3 function is conserved and further reveal that its closest homologue, the F-Box protein Fbxl21, also binds to CRY1 which impairs its repressive action towards the transcriptional activators CLOCK/BMAL1. However, while Fbxl3 appears to be ubiquitously expressed, Fbxl21 expression is tissue-specific. Furthermore, and in sharp contrast with Fbxl3, Fbxl21 is highly expressed within the suprachiasmatic nuclei, site of the master clock, where it displays marked circadian oscillations apparently driven by members of the PAR-bZIP family. Finally, for both Fbxl3 and Fbxl21 we identified and functionally characterized novel splice- variants, which might reduce CRY1 proteasomal degradation dependent on cell context. Altogether, these data establish Fbxl21 as a novel circadian clock-controlled gene that plays a specific role within the mammalian circadian pacemaker. Citation: Dardente H, Mendoza J, Fustin J-M, Challet E, Hazlerigg DG (2008) Implication of the F-Box Protein FBXL21 in Circadian Pacemaker Function in Mammals. PLoS ONE 3(10): e3530. doi:10.1371/journal.pone.0003530 Editor: Sebastian D. Fugmann, National Institute on Aging, United States of America Received July 8, 2008; Accepted October 5, 2008; Published October 27, 2008 Copyright: 2008 Dardente et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: HD and DGH are supported by the UK Biotechnology and Biological Sciences Research Council for continuing support. JM and EC are supported by grants from the Agence Nationale pour la Recherche ‘Jeunes Chercheurs/Jeunes Chercheuses’ and Centre National de la Recherche Scientifique. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: h.dardente@abdn.ac.uk ¤ Current address: Department of Systems Biology, Graduate School of Pharmaceutical Science, Kyoto University, Kyoto, Japan Introduction Endogenous circadian clocks generate rhythms of physiology and behaviour, and are synchronised to the environment through rhythmical stimuli, principally the light/dark cycle. These clocks allow organisms to anticipate and adapt to cyclical changes in environmental favourability. In mammals, the circadian clock relies on interlocked feedback loops in which a heterodimer of transcription factors, CLOCK/ BMAL1 (CLK/BM1), drives transcription of Period1-3 (Per1-3) and Cryptochrome1-2 (Cry1-2) genes through E-Box cis-elements. The PER1-3 and CRY1-2 proteins then act as transcriptional inhibitors of CLK/BM1. Apart from Per1-3 and Cry1-2, the CLK/BM1 heterodimer acts on other genes, some of which are themselves clock components, such as the orphan nuclear receptors Rev-erb a or Ror a, while others are outputs from the clock, resulting in the regulation of cell- and tissue-specific processes [1–3]. The orphan nuclear receptors of the ROR and REV-ERB families act on DNA motifs known as ROREs to control rhythmic transcription of the Bmal1 gene, with activating and repressive action, respectively [4–7]. Other ancillary inter- locked feedback loops, such as that through which REV-ERBs and RORs impinge on the transcriptional control of Rev-erb alpha [8– 10] or that closed by the clock-controlled PAR-bZIP proteins through D-Boxes [11–13] add to the robustness of the clock mechanism [14]. Patterns of rhythmical transcription depend heavily on post- translational mechanisms operating within the circadian clock [3,15–17]. Post-translational modifications of clock proteins include phosphorylation [16], SUMOylation [18] or acetylation [19], most of which are induced upon formation of heterodimers or larger protein complexes. These processes control intracellular trafficking of clock proteins, their functionality and ultimately their degradation. A timely orchestrated degradation of clock proteins is indispensable to the proper functioning of the clock as illustrated by the recently uncovered role for the F-Box protein Fbxl3. In mammals, the F-Box protein family comprises about 40 members, which direct ubiquitination and proteasome-mediated degradation of specific proteins through SCF (Spk/Cullin/F-Box protein) E3 ubiquitin ligase complex [20–22]. Accordingly, the SCFFbxl3 complex was recently shown to direct degradation of CRY1 and CRY2 proteins, with mutations in Fbxl13 leading to a slowed circadian clock in mice [23–25]. We sought to test whether the regulating effect of Fbxl3 was conserved in the diurnal sheep and investigate whether its closest homologue, Fbxl21 [26], is also involved in circadian function. Results and Discussion Ovine orthologues of Fbxl3 and Fbxl21 Cloning of ovine Fbxl3 generated a full-length (oFbxl3fl, GenBank EF643523) coding sequence (cds) of 1290 nucleotides PLoS ONE | www.plosone.org 1 October 2008 | Volume 3 | Issue 10 | e3530