Fbxl21 in the Circadian System Figure 4. Functional transcriptional effects of Fbxl3 and Fbxl21. A. oFbxl3 and oFbxl21 dose-dependently reverse oCRY1 inhibition of oCLK/ oBM1 induced transactivation of an oRev-erb a reporter construct. Transfection conditions per well: 100 ng of oClock, 100 ng of oBmal1, 100 ng of oCry1, 100 or 200 ng of each Fbxl or ev, 50 ng of oRev-erb alpha promoter reporter, 50 ng of b-Gal reporter and/or necessary amount of empty vector to make a final amount of 600 ng. B. mFbxl21 dose-dependently reverses mCRY1 inhibition of mCLK/mBM1 induced transactivation of an mPer1 reporter construct. Transfection conditions per well: 100 ng of mClock, 100 ng of mBmal1, 20 ng of oCry1, 100 or 200 ng of mFbxl21 or ev, 50 ng of Per1 promoter reporter, 50 ng of b-Gal reporter and/or necessary amount of empty vector to make a final amount of 600 ng. Data in A/B are mean6SEM of one triplicate representative from 3 replicate experiments. Different letters above bars indicate significantly different groups (p,0.05). doi:10.1371/journal.pone.0003530.g004 (Fig 5A). This pattern contrasts markedly with that for Fbxl3, which showed relatively weak and diffuse labelling (Fig 5A). Further experiments in mice confirmed this SCN-restricted pattern of Fbxl21 expression, and demonstrated pronounced diurnal and circadian expression rhythms, rising rapidly at the start of the day (or ‘‘subjective day’’ in animals run into constant darkness), and declining at the onset of the (subjective) night (Fig 5B). In contrast, Fbxl3 was diffusely expressed in the SCN of mice where it displayed no diurnal or circadian oscillation (data not shown and Fig 5C), in-line with previous findings [24,25]. Representative hybridization images from brains of animals sampled either in the beginning/middle of the day (ZT4) or beginning/middle of the night (ZT16) are shown in Fig 5C. These data suggest that Fbxl21 may be a clock-controlled gene that plays a specific role in SCN pacemaker function. Fbxl21 is a clock-controlled gene The circadian pattern of Fbxl21 expression in the mouse SCN is reminiscent of that seen for other genes whose expression is driven by CLK/BM1 such as Per1 or Dbp. Analysis of the mFbxl21 gene promoter revealed no canonical E-Box (CACGTG) but 11 E’ Boxes (CANNTG). Only 4 of these are conserved in the rat promoter (Fig 6A, E’1–4). Interestingly we noticed a putative D- Box element (Fig 6A), which is also conserved in the rat, human, bovine, dog and chicken Fbxl21 gene promoters in spite of the very poor sequence conservation between species (data not shown). Such a response element is known to mediate transactivation by members of the PAR-bZIP transcription factors family (DBP: albumin D-site binding protein, GenBank EU293835; HLF: Hepatic leukemia factor, Genbank EU293836 and TEF: thyro- troph embryonic factor, GenBank EU293837) that are clock- controlled genes and display strong circadian oscillation within the SCN peaking early into the light phase [12–13]. We therefore cloned a 1.3 kb fragment of the proximal mFbxl21 gene promoter and used luciferase assays to assess transactivation by CLK/BM1 and the PAR-bZIP proteins (Fig 6B). This revealed modest (,2- fold) transactivating effects of CLK/BM1 and DBP, and stronger (4–7 fold) effects of HLF and TEF. The modest (n.s., p = 0.08) effect of DBP could be due to differences in binding affinities of the various PAR-bZIP proteins towards this D-Box [35]. Alternative- ly, transcriptional co-factors required for DBP might be missing in this cell type. Taken together, these data suggest that the rhythmic expression of Fbxl21 in the mouse SCN derives from combined transactiva- tion through E’boxes and a conserved D-Box element. Conclusion Altogether, these data indicate a broad conservation of transcriptional and post-translational mechanisms of the circadian clock between a nocturnal rodent and a diurnal ungulate and identify Fbxl21 as a new clock component whose expression is driven by the core-clock. The functional characterization of new splice-variants also suggests that AS might be an important mechanism to fine-tune the clock. This idea is also supported by data for Bmal2 [34,36], Timeless [37] or the finding of a temperature-dependent splicing of the Period gene in Drosophila [38]. Since Fbxl21 directs degradation of CRY1 and displays strong and specific rhythmic circadian expression within the central pacemaker of the SCN, we suggest that Fbxl21 stands at a crucial crossroads between activation and repression in central clocking (Fig 7). Finally, the present data also raise the possibility of functional redundancy between Fbxl3 and Fbxl21 in the SCN akin to what was recently reported for Clock and Npas2 [39]. Future studies defining FBXL21 protein profiles within the SCN and targeted deletion of the gene should help delineate further the role of Fbxl21 in circadian timing. Materials and Methods Sheep tissue collection Hypothalamic blocks for in-situ hybridisation and central tissue samples were obtained from Blackface sheep killed at a local PLoS ONE | www.plosone.org 5 October 2008 | Volume 3 | Issue 10 | e3530