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Valérie Mongrain^1,2,, Xuan Ruan^1,3,, Hugues Dardente^1,2,a, Erin E. Fortier^1,3 and Nicolas Cermakian^1,2,3,*

¹ Laboratory of Molecular Chronobiology, Douglas Mental Health University Institute, Montréal, QC, Canada, H4H 1R3
² Department of Psychiatry, and
³ Department of Neurology and Neurosurgery, McGill University, Montréal, QC, Canada, H3A 2T5

	Abstract

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Accumulating evidence indicate that molecular mechanisms generating circadian rhythms display some degree of tissue-specificity. More specifically, distinct patterns of expression for nuclear receptors of the ROR family indicate that the transcriptional control of the clock gene Bmal1 differs among tissues. This study aims to investigate the expression of Ror isoforms (Ror and Rort) and characterize the molecular mechanisms underlying their tissue-specific expression. The expression of Ror isoforms was assessed in mouse liver, muscle, thymus and testis throughout 24 h using quantitative RT-PCR. Although the expression of Ror was rhythmic in the liver and thymus, it was constitutively expressed in muscle and testis. In contrast, the expression of Rort was constitutive in all four tissues. Furthermore, rhythmic expression of Ror was impaired in Clock mutant mice whereas the mutation had no effect on Rort expression. In line with these findings, luciferase assays revealed that transcription of the Ror promoter is clock-controlled whereas that of Rort promoter is essentially clock-independent. Our results provide insights into the molecular mechanisms that lead to differential expression of Ror and Rort and are suggestive of a framework that might account for tissue-specific circadian regulation.

	Introduction

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Biological circadian rhythms are found from single-cell organisms to more complex ones such as mammals. These rhythms, with a period close to 24 h, are generated by cell-autonomous oscillators through transcriptional–translational feedback loops and associated post-translational modifications (Dardente & Cermakian 2007). Briefly, the clock proteins CLOCK and BMAL1 dimerize and activate the transcription of a number of clock genes (Cryptochromes [Crys], Periods [Pers], and Rev-Erbs, Retinoic-acid related orphan receptors [Rors]), which thereafter close two main loops. On one hand, CRYs and PERs form complexes inhibiting the activity of the CLOCK–BMAL1 heterodimer and therefore repress their own transcription (Kume et al. 1999; Sato et al. 2006). On the other hand, REV-ERBs and RORs, respectively, repress or activate the transcription of Bmal1 gene (Preitner et al. 2002; Sato et al. 2004; Akashi & Takumi 2005; Guillaumond et al. 2005).

The outcome of these feedback loops is the oscillation of messenger RNAs (mRNAs) and proteins from clock genes and clock-controlled genes. In mammals, cell-autonomous circadian oscillators are found in many brains regions and peripheral organs (Yamazaki et al. 2000; Abe et al. 2002; Schibler et al. 2003; Yoo et al. 2004). However, peripheral clocks differ in several ways from the central circadian clock, located in the suprachiasmatic nuclei of the hypothalamus (SCN) in mammals. First, molecular oscillations of individual cells within peripheral tissues desynchronize such that apparent overall tissue rhythms damp faster (Yamazaki et al. 2000; Welsh et al. 2004), likely because of lack of tight coupling between cells (Liu et al. 2007). Second, although the SCN clock is entrained to the 24-h day mostly by photic cues from the environment, peripheral clocks are rather responsive to other cues such as feeding schedule (Schibler et al. 2003). Finally, the different clocks constituting the circadian system appear to present differences in their molecular clockwork, notably in the expression of Per and Ror genes (Guillaumond et al. 2005; Feillet et al. 2008).

RORs are transcription factors of the family of orphan nuclear receptors and generally activate gene transcription through binding to retinoic acid-related orphan receptor response element (RORE) (Medvedev et al. 1996; Sato et al. 2004; Guillaumond et al. 2005). Among clock genes, Rors are undoubtedly those that show the most drastic tissue-specific expression patterns. Indeed, Ror is expressed rhythmically in the SCN and at constant levels in most peripheral tissues (e.g. liver, skeletal muscle, kidney), Rorβ is strictly expressed in the central nervous system and cycles within the SCN, retina and pineal, whereas Ror is only expressed in peripheral tissues, either in a rhythmic (liver, kidney) or constant (muscle, thymus) manner (Preitner et al. 2002; Sumi et al. 2002; Ueda et al. 2002; Guillaumond et al. 2005; Yang et al. 2006; Liu et al. 2008). Recent results support the involvement of ROR and ROR in the fine-tuning of the molecular clockwork (precision, stability) as well as in the output of the clock (Sato et al. 2004; Akashi & Takumi 2005; Kang et al. 2007; Liu et al. 2008).

Ror gene gives rise to two isoforms, ROR and RORt (Hirose et al. 1994; He et al. 1998), most probably produced by selection of alternative promoters (Villey et al. 1999; Eberl & Littman, 2003). RORt was described as a thymus-specific isoform (He et al. 1998). Previous studies addressing the circadian expression or roles of ROR did not make a distinction between the two isoforms (Preitner et al. 2002; Guillaumond et al. 2005; Kang et al. 2007; Liu et al. 2008). Likewise, some studies dealing with the role of ROR in thymocyte differentiation in the thymus studied the effects of combined isoforms (Kurebayashi et al. 2000; Sun et al. 2000). Here, we have addressed the differential regulation of Ror and Rort transcripts and their roles in tissue-specific circadian rhythmicity. More specifically, we assessed the in vivo tissue-specific expression of Ror using isoform-specific probes in wild-type mice and in mice with the Clock¹⁹ mutation. We also studied the regulation of the expression of Ror isoforms through elements present in their promoters and sought to clarify the effect of these isoforms on Bmal1 expression. Our data provide a framework to explain tissue- and isoform-specific expression of the Ror gene, and possibly the differential expression of Bmal1 and clock-controlled genes in mouse peripheral tissues.

	Results

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Tissue-specific expression of Ror isoforms in wild-type mice

The expression of Ror and Rort was assessed at Zeitgeber times (ZT) 2, 8, 14 and 20 in liver, skeletal muscle, thymus, and testis using quantitative PCR primers and probes specific for each isoform (see Fig. 1A). As it is regulated by RORs (Sato et al. 2004; Akashi & Takumi 2005; Guillaumond et al. 2005; Liu et al. 2008), the expression of the clock gene Bmal1 was also assessed. In agreement with previous findings, the expression of Bmal1 showed significant diurnal variations in liver and muscle with a peak at approximately ZT20-2 and a trough at ZT8-14 (Fig. 1B; F_3,12(11)  15.6, P  0.001). The expression of the Ror isoform also displayed significant diurnal variations in liver and thymus, peaking at ZT20 and reaching a trough at approximately ZT2-8 (Fig. 1C; F_3,12(13)  4.5, P  0.02). In contrast, Ror expression did not show time-of-day variations in muscle and testis (F_3,11(12)  0.5, P  0.7). Rort isoform expression did not exhibit rhythmicity in any of the four tissues investigated (Fig. 1D; F_3,11–13  1.9, P  0.2). A second experiment carried out using peripheral tissues from mice kept in constant darkness (DD) and sampled at six time points around the clock yielded similar results (Fig. 2). One difference is that in DD Ror RNA does not appear to be rhythmic in the thymus.

View larger version (31K): [in this window] [in a new window]   Figure 1  Expression of Ror isoforms in mouse peripheral tissues. (A) Diagram of the Ror gene showing position of Ror and Rort putative promoters and the mRNA for each isoform. The position of isoform-specific quantitative PCR primers and probe sets are indicated by dotted lines. The scheme is not drawn to scale. (B–D) Expression of Bmal1, Ror and Rort RNAs, respectively, in liver, skeletal muscle, thymus and testis of wild-type mice housed under an LD cycle, at Zeitgeber times 2, 8, 14 and 20. (E–G) Expression of Bmal1, Ror and Rort RNAs, respectively, in liver, skeletal muscle, thymus and testis of Clock mutant mice. Each data point is the mean and SEM of the individual expression of three to five animals.

View larger version (33K): [in this window] [in a new window]   Figure 2  Expression of Ror isoforms in mouse peripheral tissues. Left panels show the expression of Bmal1, Ror and Rort RNAs in liver, skeletal muscle, thymus and testis of wild-type mice housed under constant darkness, at Circadian times (CT) 2, 6, 10, 14, 18 and 22. Right panels show the expression of Bmal1, Ror and Rort RNAs in liver, skeletal muscle, thymus and testis of Clock mutant mice. Each data point is the mean and SEM of quantitative PCR triplicates from a pool of RNAs of three to five animals. CT22 data are missing for Rort in muscle of Clock mutant mice.

Ror

Rort

Expression of Ror isoforms in Clock mutant mice

In order to find out whether the molecular clockwork controls the expression of Ror isoforms, we studied the isoform-specific expression in mice having a deficient clock. The Clock mutant mouse strain was used as these animals bear a deficient clock resulting in weak or absent behavioral rhythmicity (Vitaterna et al. 1994). The Clock¹⁹ mutation affected the expression of both Bmal1 and Ror but not that of Rort (Figs 1E–G and 2, right panels). Specifically, in LD, rhythmic expression of Bmal1 in liver and muscle and of Ror in liver were severely disrupted in the Clock mutant (Group x Time interaction: F_3,24(23)  3.0, P  0.05). In contrast, the mutation had no overt impact in thymus (no significant variation with time or Group x Time interaction). However, compared to wild type, time effect of Ror expression was lost in the thymus of mutant animals (one-way ANOVA; F_3,16 = 0.4, P = 0.7). Increased expression of Bmal1 in the testis (F_1,24 = 14.1, P  0.001) and of Ror in muscle (F_1,23 = 51.9, P  0.001) were also noticed in the mutant. Similar observations were made under DD conditions (Fig. 2). Taken together, these data suggest that the transcriptional regulation of Ror is wired to the clock mechanism whereas that of Rort is independent of the clock.

Cell-type specificity in the expression of Ror isoforms

In order to clarify the possible relationships between the expression of Bmal1 and Ror isoforms, we looked at their expression in different cell types in the thymus. Thymi were collected at ZT20, time of peak expression of Ror, and thymocytes were sorted by FACS according to surface markers CD4 and CD8. Figure 3 shows that among thymocyte populations, Ror, Rort and Bmal1 were all mostly if not exclusively expressed in double positive (DP) thymocytes that represent a pool of cells undergoing maturation (double negative [DN] thymocytes are immature cells whereas cells expressing exclusively CD4 or CD8 [SP4 or SP8] are fully differentiated thymocytes). In fact, no expression of Ror isoform could be consistently detected in either immature or mature thymocytes. Furthermore, Ror, Rort and Bmal1 were also co-expressed in thymic epithelial cells. The concomitant expression of Ror and Rort within the same cell types supports a role for both isoforms in the transcriptional activation of Bmal1.

View larger version (11K): [in this window] [in a new window]   Figure 3  Expression of Bmal1, Ror and Rort in thymus cell populations at Zeitgeber time 20. Mouse thymic cells were dissociated, stained with fluorescent antibodies and sorted. Quantitative PCR was carried out on RNA from each of the five following populations: DN, double negative immature thymocytes; DP, double positive CD4–CD8 differentiating thymocytes; SP4 and SP8, single positive mature thymocytes; EPI, thymic epithelial cells. Each data point is the mean and SEM of quantitative PCR triplicates from a pool of RNAs of two animals.

Transcriptional elements in Ror and Rort regulatory regions

We sought to understand the differential patterns of expression of Ror isoforms by looking at regulatory elements present in their respective promoters. In the murine Ror gene, we identified three consensus E-boxes (CACGTG) in the 5 kb upstream of Ror and one in the region upstream of Rort-specific exon (Fig. 4A). In addition, we found a putative RORE regulatory sequence in the Ror promoter. Importantly, most of these elements were also found in the rat and human genomes, suggesting a conserved role in the transcriptional regulation of Ror. These observations suggest that the transcription of Ror is regulated by both CLOCK/BMAL1 and orphan nuclear receptors. In contrast, transcription of Rort could potentially be regulated only by the CLOCK/BMAL1 heterodimer.

View larger version (31K): [in this window] [in a new window]   Figure 4  Transcriptional elements in Ror gene promoters and their regulation by CLOCK and BMAL1. (A) Schematic view of the position of E-boxes and RORE in putative promoters of Ror and Rort in the human, rat and mouse genomes and diagrams showing the genomic sequences cloned in the luciferase expression vector for luciferase assays. E1, E2 and E3 are the Ror-specific E-boxes and Et is the Rort-specific E-box. Numbers refer to position relative to the respective transcription start sites (+1). The scheme is not drawn to scale. (B) Transcriptional activation by CLOCK/BMAL1 via Ror or Rort E-box-containing genomic regions using luciferase reporters described in (A). Data represent the mean and SEM of at least four different experiments performed in triplicates (n  12). *P < 0.01 compared to control without CLOCK/BMAL1. (C) Transcriptional activation by CLOCK/BMAL1 via Ror putative promoter containing wild type (CACGTG) or mutated E-boxes (CTCGAG). Data are mean and SEM of four different experiments performed in triplicates (n = 12). *P < 0.001 compared to wild-type E-boxes with CLOCK/BMAL1; #P < 0.001 compared to mutant E2 or mutant E1. (D) Transcriptional activation by CLOCK/BMAL1 via Rort putative promoter containing wild type (CACGTG) or mutated E-box (CTCGAG). Data are mean and SEM of two different experiments performed in triplicates (n = 6). *P < 0.01 compared to controls without CLOCK/BMAL1. No difference was found between wild type and mutated E-box with CLOCK/BMAL1 (P = 0.2).

Transcriptional activation of Ror isoforms by CLOCK/BMAL1

To assess the role of CLOCK/BMAL1 in the transcriptional regulation of Ror isoforms, we generated reporter constructs in which the expression of the luciferase gene is under the control of the two most proximal E-boxes of Ror (E1 and E2, –2526 to –751, see Fig. 4A) or the single E-box of Rort (–568 to +190, see Fig. 4A). These constructs were co-transfected in COS-7 cells with CLOCK/BMAL1 expression vectors or empty controls. As shown in Fig. 4B, CLOCK/BMAL1 potently induced the transcription via Ror E-boxes (approximately 15-fold, F_3,44 = 59.5, P  0.001) but had only minor effect upon the Rort E-box (less than twofold, F_3,50 = 5.8, P  0.01). To further delineate the specific contribution of Ror and Rort E-boxes to transcriptional activation, we generated constructs with mutant E-boxes (CACGTG CTCGAG). As seen in Fig. 4C, transcriptional activation was reduced by 16% when the most proximal E-box (E1) was mutated. Although mutation of E2 did not have any effect, transcriptional activation of the double mutant construct (E1 + E2) was blunted by 62% (one-way ANOVA including conditions with CLOCK/BMAL1; F_3,44 = 32.2, P  0.001). These data show that both Ror E-boxes are functional and that either one or the other is sufficient to mediate activation by CLOCK and BMAL1. In contrast, mutation of Rort E-box had no significant impact (Fig. 4D; unpaired t-test including conditions with CLOCK/BMAL1; t₁₀ = 1.4, P = 0.2), implying that CLOCK/BMAL1 cannot activate transcription from this element.

Transcriptional activation of Ror by orphan nuclear receptors

To assess the role of orphan nuclear receptors in the transcriptional regulation of Ror, we generated a reporter construct in which the expression of the luciferase gene is under the control of the Ror RORE (–6622 to –6247; see Fig. 4A). This construct was co-transfected in COS-7 cells with vectors expressing REV-ERB, β, ROR, β, , t, or empty vectors. As shown in Fig. 5A, the transcriptional levels were increased by ROR, and t (F_4(2),55(32)  8.9, P  0.01) whereas RORβ and REV-ERB, β had no apparent effect. However, electrophoretic mobility shift assay indicated that all REV-ERBs and RORs are able to bind to Ror RORE (Fig. 5B). The apparent absence of effect of RORβ could be because of lack of appropriate co-factors in the cell line used (Greiner et al. 1996) or to low expression of the protein in this cellular system. As the apparent absence of repression by REV-ERBs might arise from the low basal level of expression of the reporter, we performed a competition assay in which constant amounts of ROR or RORt were transfected in combination with increasing amounts of the transcriptional repressors REV-ERB or REV-ERBβ. As shown in Fig. 5C, the induction by ROR or RORt was efficiently repressed in a dose-dependent manner by both REV-ERB (F_2,24  7.2, P  0.01) and REV-ERBβ (F_2,14 = 16.4, P  0.001). These results indicate that ROR, ROR, and RORt can act as transcriptional activators of Ror gene whereas REV-ERB and REV-ERBβ can act as transcriptional repressors.

View larger version (28K): [in this window] [in a new window]   Figure 5  Transcriptional regulation of the Ror isoform by orphan nuclear receptors. (A) Luciferase assays to study the effect of REV-ERBs or RORs on Ror upstream genomic region containing a RORE (luciferase construct shown in Fig. 4A). Data are mean and SEM of four different experiments performed in triplicates (n = 12). *P < 0.01 compared to controls without orphan nuclear receptors (pCMX for REV-ERB, β, ROR, β [dark grey control], and pSG5 for ROR and t [light grey control]). (B) Binding of REV-ERB and ROR proteins to the RORE (retinoic acid-related orphan receptor response element) site within Ror gene promoter. Electrophoretic mobility shift assays with a labeled RORE double-stranded oligonucleotide and nuclear proteins from COS-7 cells transfected with either the empty pCMX vector (control) or one of the five orphan nuclear receptor expression vector (REV-ERB, REV-ERBβ, ROR1, RORβ and ROR). Specificity of protein–DNA complexes was demonstrated by the addition of a 100-fold excess of unlabeled wild type (100x wt) or mutated (100x mutant) RORE oligonucleotide. (C) Competition between ROR isoforms and REV-ERB or β for Ror RORE. Luciferase assays were carried out as in (A), with either ROR or RORt, and increasing concentrations of REV-ERB or β. Data are mean and SEM of two or three different experiments performed in triplicates (n = 6 or 9). +P = 0.1 compared to full activation by ROR; *P < 0.05 compared to full activation by ROR or RORt alone.

Transcriptional activation of Bmal1 by ROR isoforms

As Bmal1, Ror and Rort are expressed in all four tissues examined, we wished to evaluate whether both ROR isoforms could contribute to Bmal1 gene regulation. We thus generated a reporter construct in which the expression of the luciferase gene is under the control of Bmal1 promoter region encompassing the two ROREs (–965 to +65; Guillaumond et al. 2005). As shown in Fig. 6, Bmal1 transcription was similarly induced by both ROR and RORt (F_2,18 = 9.4, P  0.01). We conclude that both ROR isoforms can act as transcriptional activators on the Bmal1 promoter.

View larger version (16K): [in this window] [in a new window]   Figure 6  Transcriptional activation of Bmal1 promoter by ROR isoforms. The graph shows the activation of the luciferase gene by ROR isoforms via Bmal1 upstream genomic region including two ROREs. Data are mean and SEM of three different experiments performed in triplicates (n = 9). *P < 0.05 compared to negative control without ROR or RORt.

	Discussion

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Ror

Rort

Ror

Ror

Rort

Ror

Rort,

Our data show that the expression of Ror is rhythmic in the liver but constant in skeletal muscle, which is consistent with previous findings (Preitner et al. 2002; Ueda et al. 2002; Guillaumond et al. 2005; Yang et al. 2006; Gréchez-Cassiau et al. 2008; Liu et al. 2008). As Ror represents the bulk of the two isoforms in these tissues (i.e. Rort is only expressed at very low levels), the use of primers that do not discriminate between the two isoforms in previous work gives an accurate picture in these tissues. In contrast, assessing the expression of each isoform independently in the thymus, where levels of Rort are high, uncovers the rhythmic expression of Ror. Moreover, in this tissue, we observe that both isoforms are expressed in the same cell types, CD4/CD8 double-positive thymocytes, in which the requirement of Rort for thymocytes differentiation has been reported (He et al. 1998; Villey et al. 1999; Eberl et al. 2004), as well as in epithelial cells. Our results contrast with previous reports showing that Rort was only expressed in the thymus (He et al. 1998; Villey et al. 1999), which might be because of difference in sensitivity of the methods used. Further studies will need to address the functional role of this isoform in non-thymic tissues.

The Clock¹⁹ mutation affects the expression of clock genes, such as Bmal1, Per2 and Dec1/2, in a tissue-specific manner (Oishi et al. 2000; Noshiro et al. 2005). Here, the Clock¹⁹ mutation affected solely the expression of the Ror isoform, and not the Rort isoform. In Clock mutant mice, the Ror isoform is no longer rhythmic in liver and thymus and is increased in muscle. This is reminiscent of the absence of rhythmic Ror expression in the liver of Bmal1^–/– mice (Liu et al. 2008). In the liver of wild-type mice, the low expression of Ror at ZT2-8 could permit the low Bmal1 expression at ZT8-14. As Ror is no longer rhythmic but expressed at intermediate levels in the Clock mutant, this would explain why trough levels of Bmal1 are higher (see Fig. 1). Likewise, in muscle, the increase in Ror expression is paralleled by higher trough levels of Bmal1 expression in the mutant resulting in a rhythm of decreased amplitude. These results support a role for ROR in the transcriptional activation of Bmal1 in liver and muscle. However, these data need to be interpreted with caution. For example, the absence of rhythmicity of the Ror isoform in muscle whereas Bmal1 cycles in the same tissue implies that other factors contribute to Bmal1 rhythmic expression, for example, REV-ERB factors.

In contrast, we did not observe any oscillation in the expression of Bmal1 in the thymus of wild-type mice. This is consistent with the reduced amplitude or absence of rhythm reported in previous studies (Alvarez & Sehgal 2005; Guillaumond et al. 2005). We observed a damped rhythm of expression of Ror in the thymus of Clock mutant mice. However, the expression of Rort was not affected by the mutation and remained at high levels. As both ROR isoforms can activate transcription of Bmal1 (Fig. 6) and are expressed in the same thymic cell subsets (Fig. 3), Bmal1 expression profile is probably imparted by the most abundant isoform in this tissue (i.e. RORt). Consistent with this Bmal1 RNA levels are constant in the thymus of WT mice, no matter whether the weakly expressed isoform Ror is expressed rhythmically (in LD; Fig. 1) or in a constant manner (in DD; Fig. 2).

The presence of a molecular clock in the testis has been questioned given the observation that the expression of clock genes is not rhythmic therein (Morse et al. 2003; Alvarez & Sehgal 2005). However, other studies noticed weak oscillations in clock genes expression in this tissue (Yamamoto et al. 2004) and more specifically in Leydig cells (Alvarez et al. 2008). Here, we found no circadian oscillation in Bmal1, Ror and Rort expression in the testis, suggesting a role for these genes in non-circadian physiology in this tissue. Nevertheless, we observed a higher expression of Bmal1 in the testis of Clock mutant animals, suggesting that Bmal1 gene is somehow regulated by CLOCK in this tissue.

Overall, our in vivo gene expression results strongly suggest that the expression of the Ror isoform is under the control of CLOCK/BMAL1 and that this isoform can regulate the expression of the core-clock component BMAL1. In contrast, the constant pattern of expression of Rort and the absence of effects of the Clock mutation on its expression support a clock-independent transcriptional regulation of this isoform. By analyzing the genomic sequences of Ror- and Rort-specific putative promoters, we found putative elements of transcriptional regulation, which are conserved across species: three E-boxes (CACGTG) and an RORE (AAAGTGGGTCA) in Ror upstream region, which extends previous observations (Ueda et al. 2005), and an E-box in Rort-specific regulatory region. Most importantly, luciferase assays showed that these Ror regulatory elements are functional whereas the one in the putative Rort promoter is unresponsive to CLOCK/BMAL1. Consistent with this, Ueda et al. (2005) showed that one of Ror E-boxes or Ror RORE can mediate transcriptional oscillation in cultured cells. Information about the orphan nuclear receptors involved in Ror gene regulation in vivo will require additional studies in which the expression of these transcription factors is down-regulated. Also, E-box-like elements could be involved in the transcriptional regulation of Ror, as shown for other genes (Yoo et al. 2005; Nakahata et al. 2008). Indeed, we observed several potential E-box-like sequences in Ror upstream region, which could also explain the activation of the promoter remaining in our assays when both E1 and E2 are mutated. Therefore, our luciferase assay data converge with our in vivo expression assays and support a clock-dependent transcriptional regulation of Ror and a clock-independent mechanism for Rort.

Although we demonstrated that CLOCK/BMAL1 can activate Ror E-boxes, it is possible that the observed in vivo effects were due to other transcriptional repressors such as DEC1/2, which also bind E-boxes and whose transcription is regulated by CLOCK/BMAL1 in a circadian manner (Gréchez-Cassiau et al. 2004; Hamaguchi et al. 2004; Sato et al. 2004). The absence of CLOCK/BMAL1 activation in Clock mutant mice could decrease levels of DEC1/2, which could increase Ror transcription and therefore contribute to the constant intermediate levels observed in liver and increased levels of Ror mRNA in muscle of mutant mice. Moreover, we found one putative cAMP response element (CRE, TGACGTCA; –6190 to –6183) in Ror promoter. This element may mediate control by CREB or CREM, as was proposed for the CRE in the promoter of Per1 (Travnickova-Bendova et al. 2002; Morse et al. 2003). In the case of the Rort-specific promoter, different E-box-binding transcription factors could be involved, such as E proteins (E12, E47, HEB) (Hu et al. 1992), which are expressed in the thymus and were shown to bind Rort promoter E-box-like sequences and activate transcription from this promoter (Xi et al. 2006).

Microarray studies have shown extensive tissue-specificity in the expression of clock-controlled genes (Panda et al. 2002; Storch et al. 2002; Ueda et al. 2002; Duffield, 2003). It was also suggested that a small number of these rhythmic transcripts are under the direct control of the core-clock mechanisms (Delaunay & Laudet 2002; Schibler et al. 2003). Our results provide a possible mechanism to explain tissue-specific circadian rhythmicity in gene expression and clock outputs. The ROR isoforms, which originate from alternative promoters and differ at their amino-terminus, are either rhythmically or constantly regulated, and their targets are likely regulated in a rhythmic or constant fashion, respectively. For example, in the liver, Ror is a rhythmically expressed gene that is an important link between circadian function and metabolism (Yang et al. 2006; Kang et al. 2007; Gréchez-Cassiau et al. 2008) whereas in the thymus, Rort RNA is constantly high in a specific cell population where it is essential for thymocyte differentiation (Kurebayashi et al. 2000; Eberl et al. 2004). The patterns of expression and the function of Ror isoforms can therefore be explained by regulatory sequences present in their specific promoters.

Conclusion

Figure 7 presents a model for the involvement of Ror in tissue-specific circadian rhythmicity. According to our observations, CLOCK/BMAL1 activates Ror isoform transcription in most peripheral tissues, which results, also depending on the presence of other transcription factors, in either a rhythmic (liver and thymus) or a constitutive (muscle and testis) pattern of expression. ROR is then proposed to activate the transcription of the core-clock component Bmal1 and increases the amplitude of its expression. ROR is also likely to contribute to the high amplitude oscillations of other core-clock components such as Cry1 (Liu et al. 2008). Rort-specific promoter is apparently not regulated by CLOCK/BMAL1 but could possibly be regulated by other transcription factors binding to E-boxes. Lastly, our results indicate that in some tissues (e.g. thymus) the combined expression of Ror and Rort governs the regulation of the Bmal1 gene. Overall, we demonstrated how a transcription factor can be regulated in an isoform- and tissue-specific manner and contribute to the circadian transcriptome in some tissues or to constitutive expression in others.

View larger version (19K): [in this window] [in a new window]   Figure 7  Model for the tissue-specific expression of Ror isoforms. The transcription of the Ror isoform is activated by CLOCK/BMAL1 heterodimer and could also be activated or repressed by other transcription factors of the basic-Helix-Loop-Helix family such as DEC1/2, by orphan nuclear receptors including ROR itself, and by CREB and CREM via a putative CRE site. Depending on the combination of transcription factors and on post-transcriptional events, Ror expression can be either rhythmic or constant, and activate rhythmically or constantly the expression of Bmal1 gene and other target genes. The expression of the Rort isoform is likely to involve clock-independent components such as E proteins. Nevertheless, this isoform has the capacity to activate the transcription of Bmal1 gene in a constant manner.

	Experimental procedures

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Clock¹⁹ heterozygous mutant mice (Vitaterna et al. 1994) were obtained from Jackson Laboratory (Bar Harbor, ME), and used to start a colony. To increase breeding efficiency, animals of the inbred strain C57BL/6J carrying the mutation were backcrossed with BALB/c mice (Charles River Laboratories, Wilmington, DE) in order to maintain the Clock¹⁹ mutation on a 50–50 background. Only Clock homozygous mice (Clock^19/19) and wild-type littermates (Clock^+/+) from F2 generation were used in the experiments. Mice were entrained to a 12 h light : 12 h dark cycle (LD 12 : 12) for 2 weeks (with Zeitgeber time 0 [ZT0] = lights ON). For the first experiment, mice were killed by decapitation every 6 h over 24 h under LD 12 : 12 conditions (ZT2, 8, 14, 20), and liver, skeletal muscle, thymus and testis were sampled, immediately frozen and stored at –80 °C until RNA extraction. For the second experiment, mice were transferred to constant darkness (DD) and killed by decapitation every 4 h over 24 h (circadian time [CT] 2, 6, 10, 14, 18, 22), and liver, skeletal muscle, thymus and testis were sampled. Procedures involving animals were carried out in accordance with guidelines of the Canadian Council on Animal Care.

Quantitative PCR

Total RNAs from mouse peripheral tissues (liver, muscle, thymus and testis) were extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) according to manufacturer's instructions. Briefly, for each tissue, 2–3 mL of TRIzol reagent was added, the tissue was homogenized, 200 µL of chloroform/mL of TRIzol were added, and RNA was precipitated from the aqueous phase with 0.5 mL of isopropanol/mL of TRIzol. RNA pellets were dissolved in RNase-free water. All individual RNA extracts were verified on agarose gel, quantified by spectrophotometry, and stored at –80 °C. For reverse transcription, 500 ng of RNA was transcribed to cDNA using high capacity cDNA reverse transcription kit (Applied Biosystems, Foster city, CA).

Quantitative PCR was performed according to Applied Biosystems protocol using a real-time cycler ABI Prism 7500 (Applied Biosystems). Briefly, 10 ng of cDNA was used for quantitative PCR with Master Mix reagent (Applied Biosystems), under the following cycling conditions: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Mouse β-Actin, Bmal1, Ror and Rort primers and probes were purchased as individual kits from Applied Biosystems (Product #: β-Actin = 4352341E; Taqman gene expression assay ID: Bmal1 = Mm00500226_m1; Ror = Mm00441139_m1). The assay for Rort PCR was designed at the Exon t–Exon 3 junction by the authors (see Fig. 1A), and the sequence of primers and probe are reported in Table 1. Each PCR reaction was carried out in triplicate. β-Actin was used as an endogenous control and relative quantification of mRNA levels was evaluated using the Ct method (Livak & Schmittgen 2001).

View this table: [in this window] [in a new window]   Table 1  Primers and probes (5' 3') used in this study

Thymi from three mice sacrificed at ZT20 were immediately dissociated between two sterile frosted microscope slides and put in RPMI-1640 completed with 10% FBS, 1% L-glutamine, HEPES, non-essential amino acids, sodium pyruvate, penicillin–streptomycin and 0.1% 50 mM β-mercaptoethanol (Gibco, Grand Island, NY). Twenty million cells were stained for CD4 and CD8 surface markers. Briefly, thymus cells were incubated on ice for 30 min in the fluorescent antibodies anti-CD4PE and anti-CD8APC (Cederlane Laboratories Ltd, Burlington, Canada) diluted in PBS 1x. Next, cells were washed twice with PBS, filtered with a 70-µm mesh filter and sorted with a Becton Dickson FacsVantage (Franklin Lakes, NJ). All voltages were kept constant during sort. Double negative (DN), double positive (DP), single CD4 positive (SP4) and single CD8 positive (SP8) thymocyte subset were sorted in TRIzol at 20 000 cells/mL. Purification of epithelial cells from thymic capsules was performed as described (Gray et al. 2002; Gavanescu et al. 2007). Cells were stained with antibodies against CD45.2 APC to exclude haematopoietic cells and MHCII IA–IE biotin/Streptavidin PE to select for epithelial cells. Cells were then sorted on a Becton Dickson FacsVantage. All voltages were kept constant during analysis. MHCII + CD45.2– cells were sorted at 20 000 cells/mL TRIzol. RNA was extracted as detailed above with the addition of 40 µg glycogen (Amersham, Piscataway, NJ) prior to extraction. Quantitative PCR was performed as described above.

Molecular cloning

Genomic regions including Ror or Rort upstream sequences were PCR-amplified from mouse tail genomic DNA (100–400 ng genomic DNA in 25 µL PCR mixture). Whole open reading frame (ORF) of Ror was amplified by PCR from pCMX-Ror (Guillaumond et al. 2005) whereas Rort ORF was amplified by PCR from thymus cDNA. Ror and Rort E-box mutations were generated by overlapping PCR mutagenesis as previously described (Guillaumond et al. 2005). Primers used in PCR reactions are listed in Table 1.

Wild type and mutant Ror and Rort genomic sequences were cloned in the pGL2-Basic-TATA reporter vector, composed of a TATA box cassette, made up of two annealed oligos (see Table 1) inserted into pGL2-Basic plasmid (Promega, Madison, WI) between BglII and HindIII sites. The use of this TATA-box reporter construct was validated by cloning Per1 E-box tandem repeats in this plasmid and checking that CLOCK/BMAL1 activated luciferase from the resulting reporter (data not shown). Bmal1 gene upstream fragment including two ROREs (Guillaumond et al. 2005) was sub-cloned in pGL2-Basic. The coding sequences of Ror and Rort were cloned in pSG5 (Stratagene, La Jolla, CA) in which sequence for V5 and His tags was inserted. Plasmids were sequenced at McGill University and Genome Quebec Innovation Centre (Montreal, Canada).

Transfection and luciferase assay

COS-7 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 2 mM L-glutamine (all Gibco products), in 95% humidity and 5% CO₂ at 37 °X.

COS-7 cells were transfected in 24-well plates (seeded at 1 x 10⁵ cells/well) using 2 µL Lipofectamine 2000 (Invitrogen) with a total of 600–1050 ng of expression plasmids. For the experiments testing whether E-boxes of Ror or Rort are functional, 700 ng plasmid mix included 25 ng of luciferase reporter plasmid (pGL2-Basic-TATA) containing the E-box(es) of Ror or t, 200 ng of pSG5-CLOCK (Travnickova-Bendova et al. 2002) or pSG5, 200 ng of pCS2-MTK-BMAL1 (Travnickova-Bendova et al. 2002) or pCS2-MTK, 25 ng of pCR3-LacZ and 250 ng of pBluescript vector as a carrier. For the experiments aimed to examine whether REV-ERBs or RORs can activate Ror RORE, 600 or 950 ng plasmid mix included 25 ng of luciferase reporter plasmid containing Ror RORE, 200 ng of pCMX-REV-ERBs or pCMX-RORs or pCMX (Guillaumond et al. 2005) or 900 ng pSG5-ROR or pSG5-RORt or pSG5, 25 ng of pCR3-LacZ and 0–350 ng of pBluescript vector as a carrier. For the experiments aimed to examine whether REV-ERB factors could compete with RORs for activation via Ror RORE, 1050 ng plasmid mix included 25 ng of luciferase reporter plasmid containing Ror RORE, 500 ng pSG5-ROR or pSG5-RORt or pSG5, and either 0, 200 or 400 ng of pCMX-REV-ERB or β and pCMX to 500 ng total, and 25 ng of pCR3-LacZ. For the experiments aimed to examine whether both ROR and RORt can activate Bmal1 ROREs, 600 ng plasmid mix included 25 ng of luciferase reporter plasmid containing Bmal1 ROREs, 200 ng of pSG5-ROR or pSG5-RORt or pSG5, 25 ng of pCR3-LacZ and 350 ng of pBluescript vector as a carrier.

Five to six hours following transfection, 500 µL COS-7 medium was added to each well, and cells were grown overnight. Luciferase assays were performed as described previously (Dardente et al. 2007). Briefly, cells were rinsed with PBS and lysed by shaking in 150 µL lysis buffer. Ten micro liters of lysate were transferred in a 96-well luminometer microplate and lucifease buffer was injected automatically using the Orion II Microplate Luminometer (Berthold Detection Systems, Oak Ridge, TN). The luciferase activity was measured and analyzed using the SIMPLICITY 4.0 program. All relative light unit values were normalized to β-galactosidase activity and protein levels (DC protein assay; Bio-Rad, Hercules, CA). Data represent fold induction over the negative control (empty expression vectors). Experiments were carried out in triplicate and repeated at least two times.

Electrophoretic mobility shift assay (EMSA)

EMSA was performed as described (Guillaumond et al. 2005). In brief, nuclear proteins of COS-7 cells transfected with 1.5 µg of expression plasmids were incubated with radioactive probe (double-stranded RORE oligonucleotides end-labeled with [-³²P]ATP, see Table 1). For competition experiments, a 100-fold excess of unlabeled wild type or mutant RORE oligonucleotides (Table 1) was added simultaneously with the labeled probe. Protein–DNA complexes, appearing on the gel as shifted bands, were resolved on a 7% polyacrylamide gel. After fixing and drying, gels were exposed to an imaging screen and visualized using Bio-Rad Personal Molecular Imager FX and QUANTITY ONE software (Bio-Rad Laboratories).

Statistical analysis

Statistical analyses were performed with STATISTICA (Statsoft, Tulsa, OK) or Prism (GRAPHPAD Software Inc., San Diego, CA). To analyze gene expression, one-way analyses of variance (ANOVAs) were used to look at diurnal changes in gene expression, and Group x Time ANOVAs were used with wild type and Clock mutant as groups and ZT2, 8, 14 and 20 as time points to compare expression between genotypes. One-way ANOVA was used to analyze multiple groups in luciferase assays, and the subsequent comparison was conducted using Tukey's honestly significant difference (HSD) test or Student's unpaired t-test. Data are shown as means ± SEM and statistical significance was set to P  0.05.

	Acknowledgements

 
The authors wish to thank Aude Villemain for technical assistance, Nathalie Labrecque, Miriam Marquis and Nathalie Henley for help with thymus cell-sorting, and members of the Laboratory of Molecular Chronobiology for continuous support and discussions. Research was supported by FRSQ and NSERC fellowships (VM), an FRSQ salary award (NC) and an NSERC grant (NC).

	Footnotes

 
Communicated by: Paolo Sassone-Corsi

These authors contributed equally to this work.

^aPresent address: School of Biological Sciences, University of Aberdeen, Aberdeen, Scotland, UK.

^* Correspondence: nicolas.cermakian{at}mcgill.ca

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Received: 16 June 2008
Accepted: 25 August 2008

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