Figure 3. Fbxl21 and Fbxl3 promote degradation of ovine CRY1 and BM1. A. COS7 cells were transfected with oCry1, oBm1 and oClk, and an oFbxl expression vector as indicated above each lane. Extracts were made at the start of cycloheximide (CHX) treatment (t = 0) Fbxl21 in the Circadian System or 2 or 8 h later as indicated and protein levels assessed by WB. Transfection conditions per well: 1 mg of oClock, 600 ng of oBmal1, 600 ng of each oFbxl/ev and 400 ng of oCry1. B. Quantitation of baseline levels (t = 0) of oCRY1, oBM1 or oCLK proteins. Data are normalised to values observed in cells not transfected with any oFbxl (ev). **: significantly lower density than for ev, p,0.01, One-way ANOVA followed by Student-Newman-Keuls post-hoc test. C. Quantitation of temporal decline in protein levels following the start of CHX treatment. Data are expressed relative to t = 0 values for each vector combination. p values for the interaction (treatment6time, two-way ANOVA) are given on the right. Data in B/C are mean6SEM of observations from 3 replicate experiments. doi:10.1371/journal.pone.0003530.g003 levels to less than 25% of t = 0 values after 8-h of protein synthesis blockade. A qualitatively similar but quantitatively more modest effect (non-significant, post-hoc test p,0.1) was seen for oFbxl21fl. By contrast, degradation rates for oCRY1 (but not oCLK or oBM1) in cells transfected with oFbxl3sv showed a trend towards reduction compared to controls (non-significant, post-hoc test p,0.1), confirming predictions that the truncated F-box motif in this protein leads to a protein stabilising effect. Collectively these data indicate that Fbxl21fl and Fbxl3sv, in addition to Fbxl3fl, govern the kinetics of degradation of CRY1. Fbxl3 and Fbxl21 modulate CRY1 suppression of CLK/ BM1 induced E-box transactivation To investigate whether these differential effects on protein stability result in different clock gene function in vitro, we constructed a luciferase reporter with the promoter region of the sheep Rev-erb a gene (GenBank EF494248), a gene whose transcription is regulated through circadian E-boxes, responsive to CLK/BM1 [1–3]. We then performed further co-transfection experiments in which the impact of the various Fbxl on oCRY1-dependent transcriptional repression of oRev-erb a expression could be assessed. Similarly to results obtained in the mouse [25], this approach revealed that oCRY1 is a potent inhibitor of oCLK/oBM1- induced oRev-erb a transactivation, reducing expression by approximately 5-fold (Fig 4A). This repressive effect was attenuated by approximately 75% upon co-transfection with oFbxl3fl or oFbxl21fl, consistent with the oCRY1 degradation promoting effects of these proteins. According to predictions, oFbxl3sv and oFbxl21sv proteins failed to reduce the repressive effects of oCRY1, likely due to the truncated F-box motif and lack of the CBD, respectively. A similar set of experiments was performed using previously validated [34] murine clock components (mClk, mBm1, mCry1 and an mPer1-luc reporter gene construct). Akin to what is observed for oFbxl21fl, mFbxl21fl is able to reverse mCRY1-induced repression of mCLK/mBM1 transactivation (Fig 4B). Rhythmical Fbxl21 expression is confined to the SCN within the hypothalamus Our RT-PCR screen showed that Fbxl3 is ubiquitously expressed while Fbxl21 has a highly tissue-specific expression pattern. We therefore wondered if Fbxl21 also shows localised expression within the hypothalamus. To address this, we performed radioactive in situ hybridisation in brain tissue from sheep sampled at ZT 4-6 and mice sampled around the clock using homologous probes. Remarkably, in the sheep hypothalamus we observed strong labelling within the SCN, which harbours the mammalian master clock, in a region overlapping with sites of arginine vasopressin (Avp, GenBank EU045357) and vaso-active intestinal polypeptide (Vip, GenBank EU016225) expression PLoS ONE | www.plosone.org 4 October 2008 | Volume 3 | Issue 10 | e3530