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. 2004 Dec;33(3):585-608.
doi: 10.1677/jme.1.01554.

The orphan receptor Rev-erbalpha gene is a target of the circadian clock pacemaker

Gérard Triqueneaux  1 , Sandrine Thenot, Tomoko Kakizawa, Marina P Antoch, Rachid Safi, Joseph S Takahashi, Franck Delaunay, Vincent Laudet
Affiliations

The orphan receptor Rev-erbalpha gene is a target of the circadian clock pacemaker

Gérard Triqueneaux et al. J Mol Endocrinol. 2004 Dec.

Abstract

Rev-erbalpha is a ubiquitously expressed orphan nuclear receptor which functions as a constitutive transcriptional repressor and is expressed in vertebrates according to a robust circadian rhythm. We report here that two Rev-erbalpha mRNA isoforms, namely Rev-erbalpha1 and Rev-erbalpha 2, are generated through alternative promoter usage and that both show a circadian expression pattern in an in vitro system using serum-shocked fibroblasts. Both promoter regions P1 (Rev-erbalpha1) and P2 (Rev-erbalpha2) contain several E-box DNA sequences which function as response elements for the core circadian-clock components: CLOCK and BMAL1. The CLOCK-BMAL1 heterodimer stimulates the activity of both P1 and P2 promoters in transient transfection assay by 3-6-fold. This activation was inhibited by the overexpression of CRY1, a component of the negative limb of the circadian transcriptional loop. Critical E-box elements were mapped within both promoters. This regulation is conserved in vertebrates since we found that the CLOCK-BMAL1 heterodimer also regulates the zebrafish Rev-erbalpha gene. In line with these data Rev-erbalpha circadian expression was strongly impaired in the livers of Clock mutant mice and in the pineal glands of zebrafish embryos treated with Clock and Bmal1 antisense oligonucleotides. Together these data demonstrate that CLOCK is a critical regulator of Rev-erbalpha circadian gene expression in evolutionarily distant vertebrates and suggest a role for Rev-erbalpha in the circadian clock output.

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Figures

Figure 1
Figure 1
The Rev-erbα gene generates two N-terminal distinct isoforms, Rev-erbα1 and Rev-erbα2, from two different promoters, P1 and P2. Transcription start sites are indicated by arrows. Untranslated sequences are shown as dotted lines whereas translated ones are represented as thick lines. The start codons are indicated by circles showing that the Rev-erbα1 mRNA can generate both the Rev-erbα1 and Rev-erbα2 protein isoforms by alternative initiator codon usage. The A/B, C and E domains are indicated. The 58 and 38 primers used to detect specifically Rev-erbα1 and Rev-erbα2 transcripts are indicated by arrowheads on E1A, E1B and E2 exons respectively.
Figure 2
Figure 2
Circadian expression of Rev-erbα transcripts in fibroblasts after 2 h horse serum-shock treatment. T0 corresponds to the serum-shock start. Expression was observed on RNA samples collected every 4 h with an additional point at 2 h during 48 h (A) or 24 h (B and C). (A) Expressions of Rev-erbα, Per1, Per3, Cry1 and Clock as a non-cyclic gene control in Rat-1 serum-shocked fibroblasts studied by semi-quantitative PCR. Each histogram is the average result of two experiments normalized on 28S analysis (not shown). For Cry1 two independent Q-PCRs were done on triplicates to refine the quantitation. (B, C) RT-PCR analysis of Rev-erbα1 (E1A) and Rev-erbα2 (E1B) transcripts in serum-shocked 3Y1 (B) and Rat-1 (C) fibroblasts. In each case the upper panel shows the result of a representative semi-quantitative RT-PCR assay whereas the lower curves are the averages from three Q-PCR experiments done on a 5700 GeneAmp Applied Biosystems apparatus. In Q-PCR experiments, a sharp analysis was done over the first 3 h. Note the difference of scales in the histograms (lower panels) and the number of PCR cycles (upper panels). All PCR products were obtained using an equal amount of reverse-transcribed mRNA amplified with primers located on two different exons hybridized with an internal specific probe. The signal obtained with 28S is a non-cycling control (upper B, C) and Q-PCR experiments were normalized on 28S values (lower B, C).
Figure 3
Figure 3
(A) Genomic structure of Rev-erbα promoters in rat (Rattus norvegicus) and human. P1 and P2 are the two promoters. E1A and E1B are the first alternative exons spliced to exon 2. The numbered E-boxes, g1–g5 (black boxes), correspond to classical E-boxes (CACGTG) and a1–a4 (grey boxes) to the divergent ones (CACATG). Transcription start sites are indicated by arrows. (B) Alignment of the E-boxes found in P1 and P2 rat and human promoters with E-boxes found in various circadian promoters. Numbers on the right indicates the E-boxes position in each promoter. The alignment was obtained by Seqvu software analysis (The Garvan Institute of Medical Research, Sydney, Australia). Boxed bases show conserved positions and the arrow indicates the base similarity at position −1. At −3 and +10 positions were found with a higher frequency of G and C respectively. A consensus sequence derived from this comparison is boxed on the bottom of the Figure.
Figure 4
Figure 4
CLOCK–BMAL1 heterodimers activate both the P1 and P2 promoters. (A) Transactivation assays were performed on Rev-erbα promoters in Cos-1, Ros17·2/8 and 3Y1 growing cells. Results are shown in fold activation and are the means±SEM from at least three independent experiments. 40 ng pGL2-luciferase reporter plasmid were used and mixed with 100 ng pCDNA3-Clock and 100 ng pCDNA3-Bmal1 or 200 ng pCDNA3-gal as a neutral vector. 48 h later cells were harvested and luciferase activity was analyzed. (Per1E)3-Luc and (Per1Em)3-Luc are positive and negative controls, containing wild-type or mutated E-boxes respectively in front of a minimal promoter. (B) Cry1 inhibits CLOCK–BMAL1-induced activation of the Rev-erbα promoters. The left panel shows the effect of Cry1 on the positive (Per1E)3-Luc and the negative (Per1Em)3-Luc reporter vectors. The right-hand panel shows the effect of Cry1 on P1+P2, P1 and P2 promoters. This experiment was performed on Ros17·2/8 cells but similar results were obtained on Cos-1 cells. 20 ng of pCDNA3-Cry1 were used and the total amount of DNA was adjusted to 300 ng; CB, CLOCK–BMAL1.
Figure 5
Figure 5
(A) Mapping of the E-box mediating CLOCK–BMAL1 activation on the Rev-erbα promoters. Trans-activation assays were performed on mutated Rev-erbα promoter fragments in Cos-1 cells. Results are shown in fold activation and are the means±SEM from at least three independent experiments. 40 ng pGL2-luciferase reporter plasmid were used and mixed with 100 ng pCDNA3-Clock and 100 ng pCDNA3-Bmal1 or 200 ng pCDNA3-gal as a neutral vector. (Per1E)3-Luc and (Per1Em)3-Luc are positive and negative controls containing wild-type or mutated E-boxes respectively in front of a minimal promoter. Cells were harvested 48 h after transfection and luciferase activity was analyzed with a luminometer. The E-boxes discussed in the text are shown as black boxes (classical E-box CACGTG) or green boxes (divergent E-box CACATG). An asterisk indicates a mutated E-box. (B) Absolute activities of P1-Luc and a2*P1-Luc (mutated a2) promoters (grey bars) and their CLOCK–BMAL1 activation responses (blue bars) indicated the specificity of the a2 E-box element.
Figure 6
Figure 6
CLOCK–BMAL1 heterodimers bind to the E-boxes of the Rev-erbα promoters. EMSAs were performed on E-box from the mouse Per1 promoter (A) or E-boxes from the Rev-erbα promoter (B and C) as probes. For A and B, the CLOCK and BMAL1 proteins were produced independently in reticulocytes lysates whereas for C the EMSA was performed using nuclear extracts from mouse STO fibroblasts in which Clock and Bmal1 expression vectors were transiently transfected. In each case, specific complexes are indicated by black arrows whereas non-specific ones are shown by empty circles. (A) The classical E-boxes of the Rev-erbα promoters compete with the binding of CLOCK–BMAL1 to the mPer1 sequence. Lane 1, C, CLOCK protein alone; lane 2, B, BMAL1 protein alone; lanes 3–19, CB, CLOCK–BMAL1 heterodimer. Competition experiments were done with a molar excess of 10-and 100-fold of the relevant E-box as indicated by the triangles. g3+4 indicates the competition by an oligonucleotide containing both the g3 and g4 E-boxes that are adjacent in the P2 promoter. (B) Direct binding of CLOCK–BMAL1 to the divergent E-boxes. The a1, a2 and a3 E-boxes were used as probes in these EMSA. Lane 1, U, untranslated reticulocyte lysates; lanes 3 and 4, CB, CLOCK–BMAL1 heterodimer; the triangle indicates increasing amount of competitor (10-and 100-fold) which in each case is the mPer1 E-box. (C) EMSA performed with nuclear-extracts of STO fibroblasts producing BMAL1 (B, lane 2), CLOCK (CLK, lane 3) or CLOCK–BMAL1 (lanes 4–8). Nuclear extracts of cells producing the -galactosidase protein were used as controls (crude, lane 1). The probe used was the a2 element; the competitors added with a molar excess (10-or 100-fold, indicated by triangles) are indicated above each lane. Bottom panel, quantitation of the specific binding of the a2 probe.
Figure 7
Figure 7
Influence of the E-box core sequence for CLOCK–BMAL1 binding. EMSAs were performed on the divergent a2 E-box from the rat P1 promoter (A) or the canonical E-box from the Per1 promoter (B) as probes with crude nuclear extracts from STO fibroblasts. In each case, competition experiments were performed with two different molar excess (10-or 100-fold, indicated by triangles) of the Per1 E-box sequence (Per1), the Per1 E-box in which the core sequence has been changed to CACATG (Per1a), the mutated Per1 E-box (CAATTG; Per1*), the rat a2 E-box (CACATG; a2), the a2 element that has been changed toward CACGTG (a2 g), the mutated a2 E-box (CCATAG; a2*) or an unrelated oligonucleotide (unr).
Figure 8
Figure 8
(A) Northern blot analysis of Rev-erbα expression in Clock mutant mice. (A, B) The scale above the Figure indicates subjective day (hatched) or night (black), respectively. Time is indicated either as the number of hours after constant darkness (above the scale), or circadian time (CT below the scale): CT0 is the time when light would normally be switched on. Each lane was loaded with 10 µg total liver RNA. Animals were maintained in dark/dark conditions and three animals were killed at each indicated time. (A) Membranes were first probed with a Rev-erbα probe (detecting the two isoforms), exposed, washed and rehybridized with 28S probe to normalize the signal. Normalized Rev-erbα expression levels are shown as histograms at the bottom of the Figure. These histograms are the results of two independent Northern blot experiments. (B) Differential analysis of transcripts emanating from transcripts starting from P1 (E1A, upper panel) or P2 (E1B, lower panel) in wild-type or Clock mutant mice by Q-PCR experiments. Note the different scales of the histograms.
Figure 9
Figure 9
(A) Genomic structure of Rev-erbα promoter in zebrafish. The two first coding exons E1 and E2 are indicated. The numbered E-boxes ZFg1–ZFg4 (black boxes) correspond to classical E-boxes (CACGTG) and ZFa (grey boxes) to the unique divergent one (CACATG) which is homologous to the rat and human a2 element. The Rev-DR2 element is indicated by an oval. The sequence of the zebrafish proximal promoter region compared with the rat proximal P1 sequence is shown under the genomic structure. E-boxes are boxed whereas the Rev-DR2 element is underlined. The start of the rat exon 1A is emboldened. The various constructs A, B and C used in transient transfections (see panel B) are indicated. (B) CLOCK–BMAL1 heterodimers activates the zebrafish Rev-erbα promoter in COS-1 cells. The construct used, A, B, C or C mutated in the two E-boxes ZFg1 and ZFa (C2 m) are indicated below the histogram. Results are the means±SEM from at least three independent experiments. 40 ng pGL2-luciferase reporter plasmid were used and mixed with 100 ng pCDNA3-Clock and 100 ng pCDNA3-Bmal1 or 200 ng pCDNA3 as a neutral vector. Cells were harvested after 48 h and luciferase activity was analyzed. (C) CLOCK–BMAL1 binds to the zebrafish E-boxes. EMSAs were performed on the a2 E-box from the rat P1 promoter as probe with crude nuclear extract from STO fibroblasts. In each case, competition experiments were performed with two different molar excess (10-or 100-fold, indicated by triangles) of either the a2 E-box (lanes 3 and 4), the ZFg1 E-box (lanes 5 and 6), the mutated version of the ZFg1 E-box (CAATTG; ZFg1*; lanes 7 and 8), the divergent ZFa E-box (lanes 9 and 10), the mutated version of the ZFa E-box Per1 E-box sequence (CCATAG; ZFa*; lanes 11 and 12) or an unrelated oligonucleotide (unr; lanes 13 and 14). Specific complexes are indicated by an arrowhead whereas non-specific ones are shown by an empty circle. (D) Whole-mount in situ hybridization analysis of Rev-erbα expression in the zebrafish epiphysis at 48 h post-fertilization (hpf). All analyses were performed on at least 25 embryos. Wild-type and injected embryos were shown as follows: random morpholino (Mo; 0·5 mg/ml), CLOCK1+CLOCK2 morpholinos (0·25 mg/ml+0·25 mg/ml) and BMAL1+BMAL2 morpholinos (0·25 mg/ml+ 0·25 mg/ml). Rev-erbα probe was labeled with UTP-digoxigenin for overnight at 70 °C. Higher magnification at the pineal gland level is depicted below each stage in the left-hand inset. In each case the right-hand inset shows the expression of Otx5, a non-circadian gene expressed in the pineal gland, the parapineal and the retina (results not shown). For both probes, all the staining was performed under precisely identical conditions.
Figure 10
Figure 10
Hypothetical model for the transcriptional loops regulating the central oscillator. Two transcription factor families, the PAR and the nuclear orphan receptors, contain activators and repressors that might form two loops regulating the circadian clock. Transcriptional activators are in green and repressors in red. Activation is shown by +, whereas repression is depicted by -. Cycling regulation is shown by | and curved arrows. CLOCK and BMAL1 have been shown to regulate some PAR proteins such as DBP (Ripperger et al. 2000, Yamaguchi et al. 2000) and Rev-erbα (this study). Whether other PAR proteins and Rev-erb are also controlled by the central clock remains to be shown. DBP regulates in turn the central clock by activating Per1 expression (Yamaguchi et al. 2000) whereas Rev-erbα represses BMAL1 expression (Preitner et al. 2002). The expression of E4 BP4 is cycling (Mitsui et al. 2001) as is the expression of at least two of the ROR genes; (Andre et al. 1998) and y (Panda et al. 2002, Preitner et al. 2002, Storch et al. 2002). Nevertheless, it is still unknown if these genes are directly controlled by the central oscillator (? in the scheme). It is likely that E4 BP4 and RORs also regulate the oscillator, even though this remains to be shown. Not depicted in the scheme is the attractive possibility that the PAR and nuclear receptor loops are interconnected in some way.
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