The Transcriptional Repressor STRA13 Regulates a Subset of Peripheral Circadian Outputs
2004; Elsevier BV; Volume: 279; Issue: 2 Linguagem: Inglês
10.1074/jbc.m305369200
ISSN1083-351X
AutoresAline Gréchez‐Cassiau, Satchidananda Panda, Samuel Lacoche, Michèle Teboul, Sameena Azmi, Vincent Laudet, John B. Hogenesch, Reshma Taneja, Franck Delaunay,
Tópico(s)Spaceflight effects on biology
ResumoCentral and peripheral mammalian circadian clocks regulate a variety of behavioral and physiological processes through the rhythmic transcription of hundreds of clock-controlled genes. The circadian expression of many transcriptional regulators suggests that a major part of this circadian gene network is indirectly regulated by clock genes. Here we show that the basic helix-loop-helix transcriptional repressor Stra13 is rhythmically expressed in mouse peripheral organs. The circadian transcription of Stra13 is mediated by a response element recognized by the CLOCK-BMAL1 heterodimer and located in the proximal promoter region. CLOCK-BMAL1-dependent activation of Stra13 is strongly repressed by CRY1 and also by STRA13 itself. To determine putative Stra13 output genes, we performed microarray analyses of differential gene expression in the liver between wild type and Stra13-/- mice and identified 42 target genes including a subset of 20 previously known as clock-controlled genes. Importantly, we demonstrate that circadian gene expression of the serum protein insulin-like growth factor-binding protein 1 and of the NKG2D receptor ligand retinoic acid early transcript was suppressed in Stra13-/- mice. These biochemical and genetic data establish a role for the basic helix-loop-helix repressor STRA13 as a circadian output regulator in the periphery. Central and peripheral mammalian circadian clocks regulate a variety of behavioral and physiological processes through the rhythmic transcription of hundreds of clock-controlled genes. The circadian expression of many transcriptional regulators suggests that a major part of this circadian gene network is indirectly regulated by clock genes. Here we show that the basic helix-loop-helix transcriptional repressor Stra13 is rhythmically expressed in mouse peripheral organs. The circadian transcription of Stra13 is mediated by a response element recognized by the CLOCK-BMAL1 heterodimer and located in the proximal promoter region. CLOCK-BMAL1-dependent activation of Stra13 is strongly repressed by CRY1 and also by STRA13 itself. To determine putative Stra13 output genes, we performed microarray analyses of differential gene expression in the liver between wild type and Stra13-/- mice and identified 42 target genes including a subset of 20 previously known as clock-controlled genes. Importantly, we demonstrate that circadian gene expression of the serum protein insulin-like growth factor-binding protein 1 and of the NKG2D receptor ligand retinoic acid early transcript was suppressed in Stra13-/- mice. These biochemical and genetic data establish a role for the basic helix-loop-helix repressor STRA13 as a circadian output regulator in the periphery. Circadian rhythms in physiology and behavior are observed in most organisms. They are generated by a self-sustained endogenous circadian clock that is reset by external time cues such as light and temperature (1.Panda S. Hogenesh J.B. Kay S.A. Nature. 2002; 417: 330-335Crossref Scopus (753) Google Scholar). This mechanism is believed to provide organisms with an anticipatory adaptive mechanism to the daily predictable changes in their environment. Biochemical and genetic studies in various model systems have identified a molecular oscillator generated by transcriptional/translational feedback loops. In mammals, the main loop involves the E box-mediated transcriptional activation of the Per1, Per2, Per3, Cry1, and Cry2 clock genes by the CLOCK-BMAL1 heterodimer. Then PER and CRY proteins form complexes that enter into the nucleus in a phosphorylation-dependent manner to repress the CLOCK-BMAL1-dependent transcription of their own genes, thereby generating a ∼24-h period molecular oscillator (2.Reppert S.M. Weaver D.R. Nature. 2002; 418: 935-941Crossref PubMed Scopus (3334) Google Scholar). This loop controls also the rhythmic expression of the repressor REV-ERBα, which is required for rhythmic Bmal1 transcription, comprising a second loop thought to be important for the overall robustness of the oscillator (3.Preitner N. Damiola F. Lopez-Molina L. Zakany J. Duboule D. Albrecht U. Schibler U. Cell. 2002; 110: 251-260Abstract Full Text Full Text PDF PubMed Scopus (1650) Google Scholar). In mammals, the master oscillator is present in the suprachiasmatic nuclei (SCN) 1The abbreviations used are: SCN, suprachiasmatic nuclei; bHLH, basic helix-loop-helix; CCG, clock-controlled gene; CT, circadian time; DD, dark/dark; LD, light/dark; SAM, significance analysis of microarrays; ZT, Zeitgeber time; EST, expressed sequence tag; IGF, insulin-like growth factor; RPA, RNase protection assay. 1The abbreviations used are: SCN, suprachiasmatic nuclei; bHLH, basic helix-loop-helix; CCG, clock-controlled gene; CT, circadian time; DD, dark/dark; LD, light/dark; SAM, significance analysis of microarrays; ZT, Zeitgeber time; EST, expressed sequence tag; IGF, insulin-like growth factor; RPA, RNase protection assay. of the hypothalamus, which orchestrates autonomous oscillators in peripheral organs. Surprisingly, this oscillator can be observed in synchronized cultured cells ex vivo. The SCN oscillator is directly reset by light perceived and transmitted via the retinohypothalamic tract and is believed to entrain peripheral oscillators via ill-defined neurohormonal pathways. Peripheral oscillators also appear to be reset by hormonal signals and the feeding schedule (4.Balsalobre A. Brown S.A. Mercacci L. Tronche F. Kellendonk C. Reichardt H.M. Schütz G. Schibler U. Science. 2000; 289: 2344-2347Crossref PubMed Scopus (1368) Google Scholar, 5.Damiola F. LeMinh N. Preitner N. Kornmann B. Fleury-olela F. Schibler U. Genes Dev. 2000; 14: 2950-2961Crossref PubMed Scopus (1723) Google Scholar). The mechanisms by which central and peripheral oscillators regulate physiological and behavioral output pathways has remained poorly understood. To address this issue, genome-wide analyses of circadian clock-controlled gene (CCG) expression in the SCN, peripheral organs, and cultured cells have recently been performed by several groups (6.Delaunay F. Laudet V. Trends Genet. 2002; 18: 595-597Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Several hundred rhythmic transcripts were identified regulating a variety of key biological processes in a coordinated and tissue-specific manner (7.Kornmann B. Preitner N. Rifat D. Fleury-Olela F. Schibler U. Nucleic Acids Res. 2001; 29: E51Crossref PubMed Scopus (117) Google Scholar, 8.Grundschober C. Delaunay F. Pühlhofer A. Triqueneaux G. Laudet V. Bartfai T. Nef P. J. Biol. Chem. 2001; 276: 46751-46758Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 9.Akhtar R.A. Reddy A.B. Maywood E.S. Clayton J.D. King V.M. Smith A.G. Gant T.W. Hastings M.H. Kyriacou C.P. Curr. Biol. 2002; 12: 540-550Abstract Full Text Full Text PDF PubMed Scopus (647) Google Scholar, 10.Panda S. Antoch M.P. Miller B.H. Su A.I. Schook A.B. Straume M. Schultz P.G. Kay S.A. Takahashi J.S. Hogenesch J.B. Cell. 2002; 109: 307-320Abstract Full Text Full Text PDF PubMed Scopus (1857) Google Scholar, 11.Storch K.-F. Lipan O. Viswanathan N. Davis F.C. Wong W.H. Weitz C.J. Nature. 2002; 417: 78-83Crossref PubMed Scopus (1232) Google Scholar, 12.Ueda H.R.W.C. Adachi A. Wakamatsu H. Hayashi S. Takasugi T. Nagano M. Nakahama K.-I. Suzuki Y. Sugano S. Lino M. Shigeyoshi Y. Hashimoto S. Nature. 2002; 418: 534-539Crossref PubMed Scopus (701) Google Scholar). Interestingly, a significant proportion of transcriptional regulators have been found among these CCGs, suggesting that many physiological outputs may be indirectly or not exclusively controlled by the circadian oscillators in mammals as previously proposed in Drosophila (13.McDonald M.J. Rosbash M. Cell. 2001; 107: 567-578Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar). Accordingly, response elements for several rhythmically expressed transcription factors have been identified in the promoter region of CCGs (12.Ueda H.R.W.C. Adachi A. Wakamatsu H. Hayashi S. Takasugi T. Nagano M. Nakahama K.-I. Suzuki Y. Sugano S. Lino M. Shigeyoshi Y. Hashimoto S. Nature. 2002; 418: 534-539Crossref PubMed Scopus (701) Google Scholar). One of these clock-controlled transcription factors is Stra13 (also known as Dec1, Sharp2, or Bhlhb2), a member of the basic helix-loop-helix (bHLH) family of transcription factors, which are important regulators of cell growth, differentiation, and apoptosis. Stra13 is a transcriptional repressor that is up-regulated by multiple cell growth arrest triggers and plays a critical role in the immune system (14.Boudjelal M. Taneja R. Matsubara S. Bouillet P. Dollé P. Chambon P. Genes Dev. 1997; 11: 2052-2065Crossref PubMed Scopus (215) Google Scholar, 15.Sun H. Taneja R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4058-4063Crossref PubMed Scopus (159) Google Scholar, 16.Sun H. Lu B. Li R.-Q. Flawell R.A. Taneja R. Nat. Immunol. 2001; 2: 1040-1047Crossref PubMed Scopus (145) Google Scholar). Here we provide biochemical and genetic evidence supporting a role for Stra13 as a clock-controlled transcription factor that regulates a subset of circadian physiological outputs in peripheral organs. Animals—Eight-week-old C57BL/6J mice were purchased at IFFACredo (Lyon, France), housed in a 12 h of light and 12 h of dark cycle (LD12:12) with the lights on (defined as Zeitgeber time (ZT) 0) at 7:00 a.m. in a temperature- and humidity-controlled environment, and fed ad libitum. After 2 weeks in LD 12:12, the mice were transferred to constant darkness (DD) and sacrificed during the DD first cycle under red dim light at various circadian times (CT). The tissues were collected, rapidly frozen in liquid nitrogen, and stored at -80 °C. Stra13-/- mice in the C57BL/6J-Sv129 background have been described and were kept under the same conditions as above (16.Sun H. Lu B. Li R.-Q. Flawell R.A. Taneja R. Nat. Immunol. 2001; 2: 1040-1047Crossref PubMed Scopus (145) Google Scholar). Littermates were used as controls. The animal procedures were in accordance with Mount Sinai Institutional animal guidelines. Plasmid Constructs—cDNA fragments for mouse Stra13 (NM_011498), Per2 (NM_011066), Per3 (NM_011067), Bmal1 (NM_007489), Clock (NM_007715) Rev-erbα (NM_145434), Dbp (NM_016974), Sharp-1 (NM_024469), Alas1 (NM_020559), and Igfbp1 (NM_008341) were reverse transcription-PCR-amplified from total liver RNA using the following primers: 5′-TAAGCAAGAATCCGAAGAGCCC-3′ and 5′-GGTGGGATGAGATAGAAGGGAAGG-3′ (Stra13); 5′-AACACCATAGTTTTCTGGCG-3′ and 5′-ACTGGAGAACTCAGGGCAGC-3′ (Per2); 5′-AAGTTGCCAGCACCTGTGGA-3′ and 5′-ATACTGCTGGCACTGCTTCC-3′ (Per3); 5′-CATTGATGCCAAGACTGGACTTCC-3′ and 5′-GCCTTCCAGGACATTGGCTAAAAC-3′ (Bmal1); 5′-CCAGCTCCTTAATGAGGACA-3′ and 5′-CCTTGTCATCTTCTTCCACC-3′ (Clock); 5′-ATGCTTGCCGAGATGCA-3′ and 5′-CATAGAGAAGTCTTCCCA-3′ (Rev-erbα); 5′-TCCAGGCCATGAGACTTTTGACCC-3′ and 5′-AT-GACGTTCTTCGGGCACCTAGCT-3′ (Dbp); 5′-TATGTGTAAACCCAAAAGGAGC-3′ and 5′-ATTATCTTCTGATGCTGCTGCT-3′ (Sharp-1); 5′-GAGGTCCATGCAGTGGGGC-TCTAT-3′ and 5′-ACGGTGTCGATCAGCAAACTCGTG-3′ (Alas1); and 5′-CGTCTTCTCATCTCTCTCGTA-3′ and 5′-CTGTGTGAGACGATGAGGAAT-3′ (Igfbp1). The fragments were cloned into pKSII+ Bluescript and sequenced. The 36B4 probe has been described elsewhere (8.Grundschober C. Delaunay F. Pühlhofer A. Triqueneaux G. Laudet V. Bartfai T. Nef P. J. Biol. Chem. 2001; 276: 46751-46758Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). The Stra13 promoter deletion constructs Stra13Δ595::Luc, Stra13 Δ540::Luc, and Stra13 Δ312::Luc were generated by digesting the pGL3KN luciferase reporter plasmid (15.Sun H. Taneja R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4058-4063Crossref PubMed Scopus (159) Google Scholar) with KpnI and either ApaI or PmlI or BpU10I (partial digest), respectively, followed by fill-in with Klenow and religation. The Stra13(E3E4)3x::Luc, Stra13(E3E4)m3x::Luc, Per1(E)3x::Luc, and Per1(E)m3X::Luc reporters were made by annealing phosphorylated oligonucleotides containing wild type or mutated E box elements from the Stra13 (E3 and E4) or Per1 (proximal E box) promoter and ligating three copies of the resulting double strand fragments into a TATA box containing pGL2 reporter plasmid. The following oligonucleotides were used: 5′-GATCTGAGCGTTGTCCAACACGTGAGGCTCATGTGATG-3′ and 5′-GATCCATCACATGAGCCTCACGTGTTGGACAACGCTCA-3′ (Stra13(E3E4)3x::Luc); 5′-GATCTGAGCGTTGTCCAACATTGCAGGCTCATTGCATG-3′ and 5′-GATCCATGCAATGAGCCTGCAATGTTGGACAACGCTCA-3′ (Stra13(E3E4)m3x::Luc); 5′-GATCCAGCACCCAAGTCCACGTGCAGGGATGTGTGA-3′ and 5′-GATCTCACACATCCCTGCACGTGGACTTGGGTGCTG-3′ (Per1(E)3x::Luc); 5′-GATCCAGCACCCAAGTCCAATTGCAGGGATGTGTGA-3′ and 5′-GATCTCACACATCCCTGCAATTGGACTTGGGTGCTG-3′ (Per1(E)m3X::Luc) (E box elements are in bold, and mutated nucleotides are underlined). To construct expression vectors for CLOCK and BMAL1, the respective open reading frames were PCR amplified from pBKS-Clock (17.King D.P. Zhao Y. Sangoram A.M. Wilsbacher L.D. Tanaka M. Antoch M.P. Steeves T.D.L. Hotz Vitaterna M. Kornhauser J.M. Lowrey P. Turek F.W. Takahashi J.S. Cell. 1997; 89: 641-653Abstract Full Text Full Text PDF PubMed Scopus (1140) Google Scholar) and pCR2-Bmal1 (18.Ikeda M. Nomura M. Biochem. Biophys. Res. Commun. 1997; 233: 258-264Crossref PubMed Scopus (173) Google Scholar) using the following primers: 5′-CAAGACGGATCCATAATCCACCATGGTGTTTAC-3′ and 5′-GAGAGGAAGCTCGAGTGCTACTGTGGCTGGACC-3′ (Clock); and 5′-GATGCCGGATCCGAGCCACCATGATTAATATAG-3′ and 5′-CAAAGCAACCTCGAGTGTTTACAGCGGCCATGG-3′ (Bmal1), digested by BamH1 and XhoI and ligated into pcDNA3.1 to generate pcDNA-Clock and pcDNA-Bmal1. All of the constructs were verified by sequencing. The pSG5-Stra13 and pcDNA-Cry1 expression vectors have been described (14.Boudjelal M. Taneja R. Matsubara S. Bouillet P. Dollé P. Chambon P. Genes Dev. 1997; 11: 2052-2065Crossref PubMed Scopus (215) Google Scholar, 19.Yagita K. Yamaguchi S. Tamanini F. van Der Horst G.T. Hoeijmakers J.H. Yasui A. Loros J.J. Dunlap J.C. Okamura H. Genes Dev. 2000; 14: 1353-1363PubMed Google Scholar). RNase Protection Assay—The plasmids were linearized with the appropriate restriction enzyme, and antisense Stra13 (+1050 to +1205) Per2 (+4544 to +4852), Per3 (+2437 to +2683), Bmal1 (+657 to +829), Clock (+1761 to +1999), Rev-erbα (+1207 to +1400), Dbp (+1041 to +1457), Sharp-1 (+157 to +405), Alas1 (+1330 to +1487), Igfbp1 (+1126 to +1287), and 36B4 (+24 to +143) probes were synthesized with T7 or T3 RNA polymerase using [α-32P]UTP (3000 Ci/mmol). RNase protection assays (RPA) were performed with 30 μg of total liver RNA using the RPA III kit (Ambion) according to the manufacturer's recommendations. Hybridization was carried out at 55 °C overnight. The signals were quantified with a Storm 840 PhosphorImager and ImageQuant software (Molecular Dynamics). Real Time PCR—Quantitative PCR was performed with a Lightcycler (Roche Applied Science) using SYBR green I dye detection according to the manufacturer's recommendations. The following primers were used: 5′-GCTGATGGGCAAGAACACCA-3′ and 5′-CCCAAAGCCTGGAAGAAGGA-3′ (36B4); 5′-ATGGCATAGCCTTCAGCAGC-3′ and 5′-GTGGGGTCAATGAAAGCACC-3′ (Cyp2a4); and 5′-AAGGCAGCAGTGACCAAGCG-3′ and 5′-TGGGGTAGGAGCCTTGATGG-3′ (Raet1c). Liver cDNA from wild type and Stra13-/- mice or calibrator (dilution 1:100) was added to a reaction mixture (Faststart DNA SYBR Green I; Roche Applied Science) with appropriate primers at 0.5 μm each and amplified using the following PCR conditions: 10 min at 95 °C; 30 s at 95 °C, 10 s at 58 °C, and 10 s 72 °C for 45 cycles; and then melting curve analysis. The expression levels were normalized to the levels of 36B4. Relative mRNA abundance was calculated using a standard curve method. Western Blotting—Western blot analysis was performed according to standard procedures using 50 μg of liver nuclear extract from mice kept in DD conditions. STRA13 was detected using a specific affinity-purified rabbit polyclonal antibody (Sigma GenoSys) that was raised against a C-terminal peptide 2S. Azmi and R. Taneja, unpublished data. and used at a dilution of 1:100 followed by chemiluminescence detection according to the manufacturer's recommendations (Amersham Biosciences). Equal loading was checked by reprobing the membrane with an EF-1α polyclonal antibody (Upstate Biotechnology Inc.). The signals were quantified with ImageQuant software (Molecular Dynamics). Cell Culture and Transient Transfections—NIH 3T3 cells and COS-7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 IU/ml penicillin, and 100 mg/ml streptomycin (Invitrogen) at 37 °C in 5% CO2. NIH 3T3 cells were serum-shocked as described (20.Balsalobre A. Damiola F. Schibler U. Cell. 1998; 93: 929-937Abstract Full Text Full Text PDF PubMed Scopus (1550) Google Scholar). COS-7 cells were seeded at a density of 105 cells/well in 12-well multidishes. On the following day, the cells were cotransfected with reporter vector (40 ng) and expression vector (200 ng) for 6 h using LipofectAMINE (Invitrogen). The cells were then incubated for 48 h in fresh medium and lysed in 50 mm Tris, pH 7.8, 1 mm dithiothreitol, 0.1% Triton X-100 lysis buffer, and luciferase assays were performed using a Trilux luminometer (Wallac). The activities were normalized to the total protein concentrations determined with the Bradford method. DNA Microarrays Experiments—For each genotype, the livers from two (CT4) or three (CT12) animals were dissected, and total RNA was prepared. cDNA synthesis, biotin labeling of cRNA, and hybridization to murine U74Av2 Genechips (one chip/sample) were performed according the recommendations of the manufacturer (Affymetrix). The .cel files were condensed using the robust multiarray average algorithm (21.Irizarry R.A. Hobbs B. Collin F. Beazer-Barclay Y.D. Antonellis K.J. Scherf U. Speed T.P. Biostatistics. 2003; 4: 249-264Crossref PubMed Scopus (8407) Google Scholar). To identify differentially expressed genes, we used the significance analysis of microarrays (SAM) method with a maximal false discovery rate of 20% and a fold change of at least 1.5 for genes called significant by SAM (22.Tusher V.G. Tibshirani R. Chu G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5116-5121Crossref PubMed Scopus (9715) Google Scholar). Fold change was calculated as the mean from two (CT4) or three (CT12) knockout animals/mean from two (CT4) or three (CT12) wild type animals. Functional annotation was done using NetAffx (www.affymetrix.com/analysis/index.affx; released August 2003). The resulting data sets were compared with publicly available liver data sets of rhythmic transcripts (7.Kornmann B. Preitner N. Rifat D. Fleury-Olela F. Schibler U. Nucleic Acids Res. 2001; 29: E51Crossref PubMed Scopus (117) Google Scholar, 9.Akhtar R.A. Reddy A.B. Maywood E.S. Clayton J.D. King V.M. Smith A.G. Gant T.W. Hastings M.H. Kyriacou C.P. Curr. Biol. 2002; 12: 540-550Abstract Full Text Full Text PDF PubMed Scopus (647) Google Scholar, 10.Panda S. Antoch M.P. Miller B.H. Su A.I. Schook A.B. Straume M. Schultz P.G. Kay S.A. Takahashi J.S. Hogenesch J.B. Cell. 2002; 109: 307-320Abstract Full Text Full Text PDF PubMed Scopus (1857) Google Scholar, 11.Storch K.-F. Lipan O. Viswanathan N. Davis F.C. Wong W.H. Weitz C.J. Nature. 2002; 417: 78-83Crossref PubMed Scopus (1232) Google Scholar, 12.Ueda H.R.W.C. Adachi A. Wakamatsu H. Hayashi S. Takasugi T. Nagano M. Nakahama K.-I. Suzuki Y. Sugano S. Lino M. Shigeyoshi Y. Hashimoto S. Nature. 2002; 418: 534-539Crossref PubMed Scopus (701) Google Scholar). Circadian Expression of Stra13 in Peripheral Tissues—The Stra13 gene is expressed in many embryonic and adult tissues with high levels in the liver, kidney, and lung (14.Boudjelal M. Taneja R. Matsubara S. Bouillet P. Dollé P. Chambon P. Genes Dev. 1997; 11: 2052-2065Crossref PubMed Scopus (215) Google Scholar). Here we show using a quantitative RNase protection assay that Stra13 mRNA exhibits a robust rhythmic expression pattern in the liver of mice kept in a LD12:12 cycle, with a ∼5-fold amplitude and trough and peak levels at ZT4 and ZT8-ZT12, respectively (Fig. 1A). Interestingly, this rhythmic expression pattern was also observed in zebrafish liver, suggesting that Stra13 oscillating expression plays a physiologically important role in the vertebrate circadian system (data not shown). Under constant darkness conditions a similar circadian expression pattern was observed in liver as well as in several other peripheral tissues including the heart, kidney, and lung, indicating that Stra13 cyclic expression is driven by endogenous circadian oscillators in these organs (Fig. 1B). A 2-h serum shock of cultured cells followed by serum starvation has previously been shown to trigger circadian gene expression in vitro (20.Balsalobre A. Damiola F. Schibler U. Cell. 1998; 93: 929-937Abstract Full Text Full Text PDF PubMed Scopus (1550) Google Scholar). This treatment was also able to induce Stra13 oscillating expression in mouse NIH 3T3 fibroblasts (Fig. 1C). Western blot analysis of STRA13 protein expression in mouse liver nuclear extracts showed a circadian pattern consistent with the mRNA expression profile with a peak at CT12 (Fig. 1D). Together these expression data indicate that Stra13 is under a circadian regulation in mouse peripheral tissues, in accordance with two recent microarray analyses of the liver gene expression (10.Panda S. Antoch M.P. Miller B.H. Su A.I. Schook A.B. Straume M. Schultz P.G. Kay S.A. Takahashi J.S. Hogenesch J.B. Cell. 2002; 109: 307-320Abstract Full Text Full Text PDF PubMed Scopus (1857) Google Scholar, 12.Ueda H.R.W.C. Adachi A. Wakamatsu H. Hayashi S. Takasugi T. Nagano M. Nakahama K.-I. Suzuki Y. Sugano S. Lino M. Shigeyoshi Y. Hashimoto S. Nature. 2002; 418: 534-539Crossref PubMed Scopus (701) Google Scholar). Notably the peak of Stra13 mRNA expression corresponds to the known maximal activity of the CLOCK-BMAL1 heterodimer in liver, and Stra13 mRNA levels are depressed in the livers of Clock/Clock mice (10.Panda S. Antoch M.P. Miller B.H. Su A.I. Schook A.B. Straume M. Schultz P.G. Kay S.A. Takahashi J.S. Hogenesch J.B. Cell. 2002; 109: 307-320Abstract Full Text Full Text PDF PubMed Scopus (1857) Google Scholar). The CLOCK-BMAL1 Heterodimer Activates the Stra13 Promoter—The components of the positive limb of the main feedback loop of the circadian oscillator, CLOCK and BMAL1, have been shown to directly regulate the expression of several clock genes and CCGs through E box elements generally localized in the proximal promoter region or the first intron (23.Gekakis N. Staknis D. Nguyen H.B. Davis F.C. Wilsbacher L.D. King D.P. Takahashi J.S. Weitz C.J. Science. 1998; 280: 1564-1569Crossref PubMed Scopus (1544) Google Scholar, 24.Jin X. Shearman L.P. Weaver D.R. Zylka M.J. DeVries G.J. Reppert S.M. Cell. 1999; 96: 57-68Abstract Full Text Full Text PDF PubMed Scopus (786) Google Scholar, 25.Ripperger J.A. Shearman L.P. Reppert S.M. Schibler U. Genes Dev. 2000; 14: 679-689PubMed Google Scholar). Four such E box sequences were identified within the proximal 5′-flanking region of the mouse Stra13 gene, two of them (E3 and E4) being also conserved in the puffer fish (fugu) and human promoters (Fig. 2A). When a luciferase reporter construct containing the mouse Stra13 proximal promoter region (Stra13Δ595::Luc construct) and expression vectors for CLOCK and BMAL1 were cotransfected in COS-7 cells, a significant (∼3-fold) induction over basal activity level was observed (Fig. 2B). Deletion constructs lacking either part of the E box element E4 (Stra13Δ540::Luc construct) or both E box elements E3 and E4 (Stra13Δ312::Luc construct) were unresponsive to CLOCK-BMAL1 (Fig. 2B), demonstrating that element E4 is critical for CLOCK-BMAL1 responsiveness. Interestingly the region including elements E3 and E4 had a configuration very close to that recently defined as being optimal for mediating rhythmic transcription, a perfect class B E box followed by a divergent E box (Fig. 2A) (26.Munoz E. Brewer M. Baler R. J. Biol. Chem. 2002; 277: 36009-36017Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). To test whether this composite element was sufficient to confer responsiveness to the CLOCK-BMAL1 heterodimer, we cloned this sequence in triplicate in front of minimal promoter driving the luciferase reporter gene. This construct was strongly stimulated by the CLOCK-BMAL1 heterodimer, whereas a mutated version was inactive (Fig. 2C). As expected this activation could be totally inhibited by coexpressing CRY1, a strong repressor of the negative limb of the circadian oscillator (27.Kume K. Zylka M.J. Sriram S. Shearman L.P. Weaver D.R. Jin X. Maywood E.S. Hastings M.H. Reppert S.M. Cell. 1999; 98: 193-205Abstract Full Text Full Text PDF PubMed Scopus (1296) Google Scholar). Interestingly, cotransfection of a STRA13 expression vector also resulted in a potent repression of CLOCK-BMAL1-dependent activation as previously described for the Per1 promoter (28.Honma S. Kawamoto T. Takagi Y. Fujimoto K. Sato F. Noshiro M. Kato Y. Honma K.-I. Nature. 2002; 419: 841-844Crossref PubMed Scopus (512) Google Scholar). These functional data provide a direct and simple mechanism for the rhythmic transcriptional regulation of Stra13 by circadian oscillators and furthermore suggest a role for Stra13 in negative autoregulation of this mechanism. Stra13 Is Not Required for Normal Circadian Oscillator Activity—The transcriptional activators CLOCK and BMAL1 contain bHLH DNA-binding domains also present in the STRA13 repressor protein. Consequently, the role of STRA13 in the circadian system could be to negatively regulate the circadian oscillator through DNA binding interference with the CLOCK-BMAL1 heterodimer as previously suggested from in vitro studies (28.Honma S. Kawamoto T. Takagi Y. Fujimoto K. Sato F. Noshiro M. Kato Y. Honma K.-I. Nature. 2002; 419: 841-844Crossref PubMed Scopus (512) Google Scholar). To genetically test this assumption, we analyzed wild type and STRA13-deficient mice kept in constant darkness for the expression of Per2, Per3, Rev-erbα, and Dbp whose transcription is known to be under the control of the CLOCK-BMAL1 heterodimer (3.Preitner N. Damiola F. Lopez-Molina L. Zakany J. Duboule D. Albrecht U. Schibler U. Cell. 2002; 110: 251-260Abstract Full Text Full Text PDF PubMed Scopus (1650) Google Scholar, 25.Ripperger J.A. Shearman L.P. Reppert S.M. Schibler U. Genes Dev. 2000; 14: 679-689PubMed Google Scholar, 29.Bunger M.K. Wilsbacher L.D. Moran S.M. Clendenin C. Radcliffe L.A. Hogenesch J.B. Simon M.C. Takahashi J.S. Bradfield C.A. Cell. 2000; 103: 1009-1017Abstract Full Text Full Text PDF PubMed Scopus (1165) Google Scholar). The liver circadian expression profiles of these four genes were nearly identical in mice from both genotypes (Fig. 3A). Consistently, expression patterns of Bmal1 and Clock, which are targets of Rev-erbα, were not altered in Stra13-/- mutant mice. These data suggest that Stra13 is not a critical regulator of peripheral circadian oscillators. Alternatively the disruption of Stra13 in mutant mice may be compensated by its paralog Sharp-1 (also known as Dec2), which exhibits a robust circadian expression pattern in the rat SCN and whose promoter is down-regulated in vitro by STRA13 in human cells (28.Honma S. Kawamoto T. Takagi Y. Fujimoto K. Sato F. Noshiro M. Kato Y. Honma K.-I. Nature. 2002; 419: 841-844Crossref PubMed Scopus (512) Google Scholar, 30.Li Y. Xie M. Song X. Gragen S. Sachdeva K. Wan Y. Yan B. J. Biol. Chem. 2003; 278: 16899-16907Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Analysis of Sharp-1 mRNA expression in the liver from wild type mice kept in DD showed weaker expression, oscillating with a peak at CT8-CT12 (Fig. 3, B and C). Expression levels of Sharp-1 were increased by ∼2-fold at all time points in Stra13-/- mutant animals, consistent with a role for Stra13 in Sharp-1 expression (30.Li Y. Xie M. Song X. Gragen S. Sachdeva K. Wan Y. Yan B. J. Biol. Chem. 2003; 278: 16899-16907Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). We conclude from these observations that Sharp-1 is a Stra13 target gene in vivo and that Stra13 is not required for circadian oscillator activity in the presence of Sharp-1. STRA13 Regulates a Subset of CCGs in Liver—To further investigate the physiological role of Stra13 in the mammalian circadian clock, we sought to identify circadianly expressed STRA13 target genes. To this end, we performed a microarray analysis of liver gene expression at CT4 and CT12 in wild type and Stra13-/- mice using DNA GeneChips (Affymetrix) and compared the resulting data sets with previously established lists of rhythmic transcripts in liver. The CT4 time point was selected to minimize the possible redundant activity of Sharp-1 as Sharp-1 expression level remained low while the STRA13 protein was present at CT4 (Figs. 1D and 3C). CT12 was analyzed as the time point of maximum STRA13 protein accumulation (Fig. 1D). Using the recently developed robust multiarray average algorithm for normalization followed by a SAM statistical procedure for the detection of differentially expressed genes in mutant versus wild type mice, we identified 42 STRA13 target genes showing at least a 1.5-fold change in expression, of which 20 were known as CCGs in liver (Table I) (7.Kornmann B. Preitner N. Rifat D. Fleury-Olela F. Schibler U. Nucleic Acids Res. 2001; 29: E51Crossref PubMed Scopus (117) Google Scholar, 9.Akhtar R.A. Reddy A.B. Maywood E.S. Clayton J.D. King V.M. Smith A.G. Gant T.W. Hastings M.H. Kyriacou C.P. Curr. Biol. 2002; 12: 540-550Abstract Full Text Full Text PDF PubMed Scopus (647) Google Scholar, 10.Panda S. Antoch M.P. Miller B.H. Su A.I. Schook A.B. Straume M. Schultz P.G. Kay S.A. Takahashi J.S. Hogenesch J.B. Cell. 2002; 109: 307-320Abstract Full Text Full Text PDF PubMed Scopus (1857) Goo
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