Functional and Structural Analyses of Cryptochrome
2003; Elsevier BV; Volume: 278; Issue: 37 Linguagem: Inglês
10.1074/jbc.m305028200
ISSN1083-351X
AutoresJun Hirayama, Haruki Nakamura, Tomoko Ishikawa, Yuri Kobayashi, Takeshi Todo,
Tópico(s)Neurobiology and Insect Physiology Research
ResumoMouse mCRY1 and zebrafish zCRY1a and zCRY3 belong to the DNA photolyase/Cryptochrome family. mCRY1 and zCRY1a repress CLOCK:BMAL1-mediated transcription, whereas zCRY3 does not. Reciprocal chimeras between zCRY1a and zCRY3 were generated to determine the zCRY1a regions responsible for nuclear translocation, interaction with the CLOCK:BMAL1 heterodimer, and repression of CLOCK:BMAL1-mediated transcription. Three regions, RD-2a-(126–196), RD-1-(197–263), and RD-2b-(264–293), were identified. Proteins in this family consist of an N-terminal α/β domain and a C-terminal helical domain connected by an interdomain loop. RD-2a is within this loop, RD-1 is at the N-terminal 50 amino acids, and RD-2b at the following 31 amino acid residues of the helical domain. Either RD-2a or RD-1 is required for interaction with the CLOCK: BMAL1 heterodimer, and either RD-1 or RD-2b is required for the nuclear translocation of CRY. Both of these functions are prerequisites for the transcriptional repressor activity. The functional nuclear localizing signal in the RD-2b region also was identified. The sequence is well conserved among repressor-type CRYs, including mCRY1. Mutations in the nuclear localizing signal of mCRY1 reduce the extent of its nuclear localization. These findings show that both nuclear localization and interaction with the CLOCK:BMAL heterodimer are essential for transcriptional repression by CRY. Mouse mCRY1 and zebrafish zCRY1a and zCRY3 belong to the DNA photolyase/Cryptochrome family. mCRY1 and zCRY1a repress CLOCK:BMAL1-mediated transcription, whereas zCRY3 does not. Reciprocal chimeras between zCRY1a and zCRY3 were generated to determine the zCRY1a regions responsible for nuclear translocation, interaction with the CLOCK:BMAL1 heterodimer, and repression of CLOCK:BMAL1-mediated transcription. Three regions, RD-2a-(126–196), RD-1-(197–263), and RD-2b-(264–293), were identified. Proteins in this family consist of an N-terminal α/β domain and a C-terminal helical domain connected by an interdomain loop. RD-2a is within this loop, RD-1 is at the N-terminal 50 amino acids, and RD-2b at the following 31 amino acid residues of the helical domain. Either RD-2a or RD-1 is required for interaction with the CLOCK: BMAL1 heterodimer, and either RD-1 or RD-2b is required for the nuclear translocation of CRY. Both of these functions are prerequisites for the transcriptional repressor activity. The functional nuclear localizing signal in the RD-2b region also was identified. The sequence is well conserved among repressor-type CRYs, including mCRY1. Mutations in the nuclear localizing signal of mCRY1 reduce the extent of its nuclear localization. These findings show that both nuclear localization and interaction with the CLOCK:BMAL heterodimer are essential for transcriptional repression by CRY. Organisms ranging from bacteria to humans have daily rhythms driven by endogenous oscillators called circadian clocks that regulate various biochemical, physiological, and behavioral processes with a periodicity of approximate by 24 h (1Dunlap J.C. Cell. 1999; 96: 271-290Abstract Full Text Full Text PDF PubMed Scopus (2391) Google Scholar, 2King D.P. Takahashi J.S. Annu. Rev. Neurosci. 2000; 23: 713-742Crossref PubMed Scopus (456) Google Scholar, 3Reppert S.M. Weaver D.R. Annu. Rev. Physiol. 2001; 63: 647-676Crossref PubMed Scopus (1220) Google Scholar). Under natural conditions, rhythms are entrained to a 24-h day by environmental time cues, most commonly light. These circadian clock mechanisms have been investigated by characterizing the "clock genes" that affect the daily rhythm. The core of the clock mechanisms in Drosophila, Neurospora, mammals, and cyanobacteria is expressed by a transcription/translation-based negative feedback loop that relies on positive and negative oscillator elements. The negative feedback loop begins by activating the transcription of clock genes, the products of which then negatively regulate their own expression, setting up the rhythmic oscillations of gene expression that drive the circadian clock. Although negative feedback loop is a common mechanism in Drosophila, Neurospora, mammals, and cyanobacteria, its components differ with the species. Drosophila and mammals have common components (orthologous gene products), except for the negative elements TIM 1The abbreviations used are: TIM, TIMELESS; CRY, Cryptochrome; PER, PERIOD; GFP, green fluorescent protein; PBS, phosphate-buffered saline; NLS, nuclear localization signal; z, zebrafish; m, mouse. (TIMELESS) and CRY (Cryptochrome). PER (PERIOD), and TIM, identified as the negative elements in Drosophila, form a heterodimer that translocates to the nucleus where its components interact with the positive elements dCLOCK and CYC (4Saez L. Young M.W. Neuron. 1996; 17: 911-920Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). Formation of a complex decreases dCLOCK:CYC-mediated transcription, resulting in the repression of expression (5Gekakis N. Saez L. Delahaye-Brown A.M. Myers M.P. Sehgal A. Young M.W. Weitz C.J. Science. 1995; 270: 811-815Crossref PubMed Scopus (315) Google Scholar, 6Sehgal A. Rothenfluh-Hilfiker A. Hunter-Ensor M. Chen Y. Myers M.P. Young M.W. Science. 1995; 270: 808-810Crossref PubMed Scopus (300) Google Scholar). In mammals, CRY1 and CRY2 are partners of the PER heterodimer, rather than TIM. In mammals, the mPER and mCRY proteins form heterodimers that translocate to the nucleus where they act as negative regulators by interacting with CLOCK and BMAL1 (CYC is the Drosophila homolog of BMAL1) to inhibit transcription (7Kume 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 (1322) Google Scholar, 8Griffin Jr., E.A. Staknis D. Weitz C.J. Science. 1999; 286: 768-771Crossref PubMed Scopus (520) Google Scholar). In Drosophila a different role has been identified for dCRY (9Stanewsky R. Kaneko M. Emery P. Beretta B. Wager-Smith K. Kay S.A. Rosbash M. Hall J.C. Cell. 1998; 95: 681-692Abstract Full Text Full Text PDF PubMed Scopus (778) Google Scholar, 10Emery P. So W.V. Kaneko M. Hall J.C. Rosbash M. Cell. 1998; 95: 669-679Abstract Full Text Full Text PDF PubMed Scopus (711) Google Scholar, 11Ishikawa T. Matsumoto A. Kato Jr., T. Togashi S. Ryo H. Ikenaga M. Todo T. Ueda R. Tanimura T. Genes Cells. 1999; 4: 57-65Crossref PubMed Scopus (68) Google Scholar). It binds TIM and PER in a light-dependent manner (12Rosato E. Codd V. Mazzotta G. Piccin A. Zordan M. Costa R. Kyriacou C.P. Curr. Biol. 2001; 11: 909-917Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 13Ceriani M.F. Darlington T.K. Staknis D. Mas P. Petti A.A. Weitz C.J. Kay S.A. Science. 1999; 285: 553-556Crossref PubMed Scopus (450) Google Scholar). This interaction disrupts their inhibitory effect on transactivation of the CLOCK:CYC heterodimer, leading to a phase shift in circadian rhythm. Consistent with this model, cryb mutant flies that bear a mutation within the dcry gene display free-running behavioral rhythms but lack light entrainment capability. Furthermore, these flies show rhythmicity in constant light, whereas wild-type ones are arrhythmic under such conditions (9Stanewsky R. Kaneko M. Emery P. Beretta B. Wager-Smith K. Kay S.A. Rosbash M. Hall J.C. Cell. 1998; 95: 681-692Abstract Full Text Full Text PDF PubMed Scopus (778) Google Scholar). These findings show that the role of dCRY is as a circadian photoreceptor and indicate diversity in CRY functions in different species. Mouse CRY was designated a repressor-type, Drosophila CRY a non-repressor-type. The zebrafish provides an attractive vertebrate model for biological clock analyses. Several of its clock genes have now been identified, and in vitro analyses has shown that the zebrafish negative feedback loop consists of components similar to those of mammals. zPER and zCRY act as negative regulators, and zCLOCK and zBMAL as positive elements (14Vitaterna M.H. Selby C.P. Todo T. Niwa H. Thompson C. Fruechte E.M. Hitomi K. Thresher R.J. Ishikawa T. Miyazaki J. Takahashi J.S. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12114-12119Crossref PubMed Scopus (573) Google Scholar, 15Cermakian N. Whitmore D. Foulkes N.S. Sassone-Corsi P. Proc. Natl. Acad. Sci. U. S. 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They repress the activities of mouse CLOCK and BMAL, as well as those of zebrafish CLOCK and BMAL, indicative that the basic function of repressor-type zCRYs is the same as that of mCRYs (16Kobayashi Y. Ishikawa T. Hirayama J. Daiyasu H. Kanai S. Toh H. Fukuda I. Tsujimura T. Terada N. Kamei Y. Yuba S. Iwai S. Todo T. Genes Cells. 2000; 5: 725-738Crossref PubMed Scopus (178) Google Scholar, 19Ishikawa T. Hirayama J. Kobayashi Y. Todo T. Genes Cells. 2002; 7: 1073-1086Crossref PubMed Scopus (59) Google Scholar, 20Hirayama J. Fukuda I. Ishikawa T. Kobayashi Y. Todo T. Nucleic Acids Res. 2003; 31: 935-943Crossref PubMed Scopus (34) Google Scholar). A unique feature of zebrafish CRY is the presence of extra paralogous genes. In addition to repressor-type CRYs, the zebrafish has a unique CRY, zCRY3, that despite high structural similarity to repressor-type CRYs, lacks transcriptional repression (16Kobayashi Y. Ishikawa T. Hirayama J. Daiyasu H. Kanai S. Toh H. Fukuda I. Tsujimura T. Terada N. Kamei Y. Yuba S. Iwai S. Todo T. Genes Cells. 2000; 5: 725-738Crossref PubMed Scopus (178) Google Scholar). zCRY3 therefore can be classified as a non-repressor-type CRY, but its function has yet to be identified. CRYs are members of the DNA photolyase/cryptochrome protein family (21Todo T. Ryo H. Yamamoto K. Toh H. Inui T. Ayaki H. Nomura T. Ikenaga M. Science. 1996; 272: 109-112Crossref PubMed Scopus (231) Google Scholar, 22Todo T. Mutat. Res. 1999; 434: 89-97Crossref PubMed Scopus (153) Google Scholar, 23Cashmore A.R. Jarillo J.A. Wu Y.J. Liu D. Science. 1999; 284: 760-765Crossref PubMed Scopus (810) Google Scholar) that comprise such functionally diverse members as DNA photolyase and CRY. DNA photolyase is a unique enzyme that repairs a UV-induced DNA damage in a light-dependent manner (24Todo T. Takemori H. Ryo H. Ihara M. Matsunaga T. Nikaido O. Sato K. Nomura T. Nature. 1993; 361: 371-374Crossref PubMed Scopus (260) Google Scholar, 25Sancar A. Biochemistry. 1994; 33: 2-9Crossref PubMed Scopus (570) Google Scholar, 26Hitomi K. Nakamura H. Kim S.T. Mizukoshi T. Ishikawa T. Iwai S. Todo T. J. Biol. Chem. 2001; 276: 10103-10109Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), and CRYs function in the circadian system. Despite functional diversity, the members of this protein family have a high degree of sequence similarity and flavin adenine dinucleotide (FAD) as a common cofactor. Although animal CRYs have a crucial role in the central circadian clock, because of structural complexity their functional domains have not been well characterized. CRYs require FAD as a cofactor to maintain proper conformation, and FAD binding sites are present within the N-terminal conserved regions (27Hsu D.S. Zhao X. Zhao S. Kazantsev A. Wang R.P. Todo T. Wei Y.F. Sancar A. Biochemistry. 1996; 35: 13871-13877Crossref PubMed Scopus (263) Google Scholar, 28Todo T. Tsuji H. Otoshi E. Hitomi K. Kim S.T. Ikenaga M. Mutat. Res. 1997; 384: 195-204Crossref PubMed Scopus (43) Google Scholar, 29Sancar A. Annu. Rev. Biochem. 2000; 69: 31-67Crossref PubMed Scopus (241) Google Scholar, 30Yamamoto K. Okano T. Fukada Y. Neurosci. Lett. 2001; 313: 13-16Crossref PubMed Scopus (68) Google Scholar). Any deletion in the N-terminal region therefore causes complete loss of the CRY function. This interferes with the deletion analysis commonly used to determine the functional domains of a protein. We took advantage of the similarity in nucleotide/amino acid sequences and difference in transcriptional repressor activities of the repressor-type zCRY (zCRY1a) and non-repressor-type zCRY (zCRY3) to form chimeras, which allowed us to map the region responsible for transcriptional repressor activity. We identified the sequence elements required for the interaction of CRY with other clock proteins, as well as for regulation of the subcellular distribution and transcriptional repression. The findings for these chimeras show that both the nuclear localization and interaction with the CLOCK:BMAL heterodimer are essential for transcriptional repression by CRY. Plasmid Construction—Novel restriction enzyme sites were introduced within zCRY1a and -3 to prepare the zCRY1a-3 chimeras. Site-directed mutagenesis system Mutan-Super Express KM kit (Takara) was used, which included a PmaCI site at bp 375 of the cDNA encoding zCRY1a, and PmaCI at bp 375, SacI at bp 589, SalI at bp 785, and EcoRI at bp 880 of the nucleotide sequence encoding zCRY3. Synthetic oligonucleotides were used for mutagenesis: for PmaCI in the zCRY1a sequence, 5′-GGAGGTGATCACGTGCATCTCTCA-3′ (covering bp 366–389); for PmaCI in the zCRY3 sequence, 5′-GGAAACCGTCACGTGTAACACTCAC-3′ (covering bp 366–390); for SacI in the zCRY3 sequence, 5′-CTCTCTAGAGGAGCTCGGTTTTAGG-3′ (covering bp 589–603); for SalI in the zCRY3 sequence, 5′-TGTCATGTCGACTGTTTTACTA-3′ (covering bp 789–800); and for EcoRI in the zCRY3 sequence, 5′-GGCGGGAATTCTTCTACACG-3′ (covering bp 875–894). Each of these enzyme sites is underlined above. The zCRY1a-3 chimeras shown in Figs. 2A and 3A were generated by switching each segment between zCRY1a and -3 at the newly introduced restriction enzyme sites, the Eco52I site (at bp 1254 of the cDNA encoding zCRY1a or -3), or both. The chimeras then were ligated into pcDNA3.1(+), pcDNA-GAL4, pcDNA-VP16, or pcDNA-V5, which generated each chimera construct used.Fig. 3The zCRY1a region required for interaction with CLOCK or BMAL proteins. zCRY1a, zCRY3, or an indicated chimera (A) were tested in a mammalian two-hybrid assay (B) and by co-immunoprecipitation (C) for its ability to interact with zCLOCK1 or zBMAL3. In the mammalian two-hybrid assay, each GAL-chimeric CRY was co-expressed with VP16-zCLOCK1 or VP16-zBMAL3. Effects on transactivation of the pGL-5G reporter plasmid were assayed. Results are given as -fold activation relative to the pGL-5G reporter plasmid alone. Values are mean ± S.E. of three independent experiments. In the co-immunoprecipitation, FLAG-zCLOCK1 and zBMAL3-V5 were co-expressed in the COS7 cells. The cell extract was mixed with extract containing each singly expressed VP16-zCRY or non-tagged zCRY protein, incubated on ice for 1 h, then immunoprecipitated with the anti-FLAG antibody. The immunoprecipitated materials and whole cell extracts were analyzed by immunoblotting with anti-VP16 (zCRY1a and chimera 17), anti-zCRY3 (chimeras 11, 18, and zCRY3), anti-V5 (zB-MAL3), or anti-FLAG antibodies (zCLOCK1). The abbreviations c5, 11, 12, 13, 14, 16, 17, and 18 respectively indicate chimeras 5, 11, 12, 13, 14, 16, 17, and 18.View Large Image Figure ViewerDownload Hi-res image Download (PPT) GFP fusions were generated as follows: the SalI-EcoRI fragment bearing zCRY1a NLS (amino acids 265–282) was excised from pcDNA-zCRY1a, then ligated into the corresponding site of pGFP-CI, generating pGFP-zCRY1a (amino acids 265–282). The segment encoding amino acids 265–282 of zCRY3 was amplified by the use of oligonucleotides with SalI or EcoRI then ligated into the SalI-EcoRI site of pGFP-CI, generating pGFP-zCRY3 (amino acids 265–282). The coding region of mouse Cry1 cDNA was obtained from mouse brain RNA by a reverse transcriptase-PCR then ligated into pcDNA-V5, generating pV5-mCRY1. Amino acid substitutions in mCRY1 NLS (amino acids 265–282) of mCRY1-V5 were introduced by a two-step PCR scheme that used primers encoding the mutated nucleotide. The PmaCI site was introduced at bp 375 of the nucleotide sequence encoding mCRY1 by a two-step PCR scheme, generating pV5-mCRY1 (PmaCI) and pV5-NLS-mutated mCRY1 (PmaCI). The HindIII-PmaCI fragment bearing zCRY3 (amino acids 1–125) was ligated into the corresponding sites of pV5-mCRY1 (PmaCI) and pV5-NLS-mutated mCRY1 (PmaCI), generating the zC3-mCRY1 chimeras (Fig. 7). All the constructs described were verified by sequence analysis. Other plasmids used in this study have been described elsewhere (16Kobayashi Y. Ishikawa T. Hirayama J. Daiyasu H. Kanai S. Toh H. Fukuda I. Tsujimura T. Terada N. Kamei Y. Yuba S. Iwai S. Todo T. Genes Cells. 2000; 5: 725-738Crossref PubMed Scopus (178) Google Scholar, 19Ishikawa T. Hirayama J. Kobayashi Y. Todo T. Genes Cells. 2002; 7: 1073-1086Crossref PubMed Scopus (59) Google Scholar, 20Hirayama J. Fukuda I. Ishikawa T. Kobayashi Y. Todo T. Nucleic Acids Res. 2003; 31: 935-943Crossref PubMed Scopus (34) Google Scholar). Cells, Transfection, and Luciferase Assay—NIH3T3 cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 5% calf serum. COS7 cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum. The day before transfection, both types of the cells were each plated in 12-well plates, and then were transfected the next day with 50 ng of firefly luciferase reporter plasmid, 20 ng of sea pansy luciferase reporter plasmid (pRL-SV40 (Promega)), and expression plasmids (indicated in each figure), by the use of LipofectAMINE Plus according to the manufacturer's instructions (Invitrogen). As reporter plasmids, the E-box element and its flanking sequences within the promoter/enhancer region of mouse vasopressin and five GAL4-binding sites were, respectively, cloned into the pGL-Basic vector (Promega) for the luciferase-reporter and two-hybrid assays (designated as mAVP-pGL and 5G-pGL). Total amounts of expression plasmids were adjusted by adding the pcDNA3.1 vector as the carrier. The preparation of cell lysates and the dual luciferase assays, using the dual-luciferase reporter assay system according to the manufacturer's instructions (Promega), were performed 24 h after transfection. Firefly and sea pansy luciferase activities were quantified by means of a luminometer, with the firefly luciferase activity normalized for transfection efficiency based on the sea pansy luciferase activity. All experiments were done three times. Antibodies—Polyclonal antisera against the glutathione S-transferase-fused zCRY3 C-terminal polypeptide (amino acids 506–598) were raised in rabbits. Anti-VP16 antibody was purchased from Clontech, anti-FLAG from Sigma, anti-V5 from Invitrogen, and anti-GFP from Roche Diagnostics Corp. Co-immunoprecipitation—Co-immunoprecipitation was done as previously described (7Kume 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 (1322) Google Scholar), with some modifications. COS7 cells were seeded in 6-cm dishes, and were transfected the following day with the expression plasmids described in each figure. The cells were washed twice with phosphate-buffered saline (PBS) 24 h after transfection, homogenized in binding buffer (150 mm NaCl, 5 mm EDTA, 0.5% Nonidet P-40, and 50 mm Tris-HCl, pH 7.5) containing protease inhibitor mixture tablets, and then clarified by centrifugation for 10 min at 10,000 × g. Total protein (100 μg) from the supernatant was incubated with 15 μl of protein A/G-agarose beads (Santa Cruz) for 1 h at 4 °C, after which the material was centrifuged. The supernatant was incubated for 12 h at 4 °C with either the anti-V5 mouse antibodies (Invitrogen) or the anti-VP16 rabbit antibody, and 15 μl of protein A/G-agarose beads. The beads were then washed three times with binding buffer, boiled in SDS sample buffer, and centrifuged. The supernatant was separated by SDS-PAGE and analyzed by Western blotting, as described below. Western Blot Analysis—Total protein (10 μg), extracted from the cells as described previously, was separated by SDS-PAGE in a 6.5% gel and transferred electrophoretically onto a polyvinylidene difluoride membrane. The membrane was blocked with 7% nonfat milk and incubated with the mouse anti-FLAG antibody (Sigma), the mouse anti-V5 antibody (Invitrogen), the rabbit anti-zCRY3 antibody, or the rabbit anti-VP16 antibody for 1 h at room temperature. The blots were incubated with the appropriate secondary antibody, peroxidase-conjugated anti-mouse or anti-rabbit IgG antibody (Santa Cruz), and blots were developed with the ECL Western blotting detection system (Amersham Biosciences). Immunofluorescence—NIH cells (3 × 105) were seeded on glass coverslips in 6-well dishes and transfected the following day (as described above) with 1 μg of total DNA per well. Thirty hours after transfection, the cells were washed twice with PBS, fixed with 4% paraformaldehyde in PBS, washed, and blocked for 30 min at 37 °C in 1% bovine serum albumin, 0.1% Triton X-100 in PBS. The anti-V5, anti-zCRY3, or anti-VP16 antibodies were diluted in 0.5% bovine serum albumin in PBS, and then incubated with the cells for 1 h at 37 °C. The cells were washed three times with 0.1% Triton X-100 in 10% PBS, and then the cells were incubated with the fluorescein isothiocyanate- (Santa Cruz) and/or the Cy3-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, Inc.) for1hat37 °C. After several washes, the cell nuclei were stained with 4′,6′-diamidino-2-phenylindole, and the cells were mounted for fluorescence microscopy. Protein Modeling—The amino acid sequences of Escherichia coli CPD photolyase, mCRY1, zCRY1a, and zCRY3 were aligned by the use of commercially available software, GENENTYX-MAC version 8.0 (Software Development Co., Ltd.). Tertiary models of mCRY1 and zCRY1a were constructed by comparative modeling based on the structure of E. coli CPD photolyase (Protein Data Bank code 1dnp), using our original programs: a loop search method for the backbone structure (31Nakamura H. Katayanagi K. Morikawa K. Ikehara M. Nucleic Acids Res. 1991; 19: 1817-1823Crossref PubMed Scopus (32) Google Scholar), a dead-end elimination method for the side chain structure (32Tanimura R. Kidera A. Nakamura H. Protein Sci. 1994; 3: 2358-2365Crossref PubMed Scopus (61) Google Scholar), and a conformation energy minimization method for structure refinement (33Morikami K. Nakai T. Kidera A. Saito M. Nakamura H. Comput. Chem. 1992; 16: 243-248Crossref Scopus (189) Google Scholar), using the AMBER force field (34Weiner S.J. Kollman P.A. Nguyen D.T. Case D. J. Comput. Chem. 1986; 7: 230-252Crossref PubMed Scopus (3604) Google Scholar). The quality of the model structure was examined by the program PROCHECK (35Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar), and it was shown to have the quality corresponding to a crystal structure with 2.0 to 2.5-Å resolution. The atom coordinates of the model structure are available from the corresponding author when requested. Construction of Chimeric zCRYs with Exchanged zCRY1a and zCRY3 Regions—A comparison of the predicted amino acid sequences of mCRY1 and zCRY1a and -3 shows that the zebrafish proteins are close structural homologs of mCRY1 (Fig. 1A). CRY protein consists of the N-terminal chromophore-binding and C-terminal extension domains, the former well conserved among all the CRY proteins, the latter having no known homology (22Todo T. Mutat. Res. 1999; 434: 89-97Crossref PubMed Scopus (153) Google Scholar, 23Cashmore A.R. Jarillo J.A. Wu Y.J. Liu D. Science. 1999; 284: 760-765Crossref PubMed Scopus (810) Google Scholar, 29Sancar A. Annu. Rev. Biochem. 2000; 69: 31-67Crossref PubMed Scopus (241) Google Scholar). In fact, the sequence of the former domain is highly conserved between mCRY and zCRY1a and -3 with 77–81% identity (Fig. 1A). Despite sharing a well conserved primary structure, the three CRYs differ markedly in their functional activities. zCRY1a, as well as mCRY1, represses CLOCK:BMAL-mediated transcription, whereas zCRY3 does not (16Kobayashi Y. Ishikawa T. Hirayama J. Daiyasu H. Kanai S. Toh H. Fukuda I. Tsujimura T. Terada N. Kamei Y. Yuba S. Iwai S. Todo T. Genes Cells. 2000; 5: 725-738Crossref PubMed Scopus (178) Google Scholar, 20Hirayama J. Fukuda I. Ishikawa T. Kobayashi Y. Todo T. Nucleic Acids Res. 2003; 31: 935-943Crossref PubMed Scopus (34) Google Scholar). We took advantage of the similarities between the nucleotide/amino acid sequences and the differences in transcriptional repressor activities of zCRY1a and -3 to produce zCRY1a-3 chimeras, which permitted mapping of the region responsible for transcriptional repressor activity. Alignment of the amino acid sequences of zCRY1a and -3 showed no insertion or deletion of the amino acid sequences between the N-terminal chromophore-binding domains of the two proteins (Fig. 1A), indicative of minimized disruption of the native conformation by chimera construction. To generate the chimeric constructs, restriction recognition sites were created in each cDNA clone by site-directed mutagenesis. In each mutagenesis, alterations in the nucleotide sequences were designed to minimize changes in the encoded amino acids. To determine which domain to exchange reciprocally, we constructed a structural model of zCRY1a, using the crystal structure of E. coli photolyase, a member of the DNA photolyase/Cryptochrome family, as the starting point (Fig. 1B). With this structural model as a guide and considering restrictions within the nucleotide sequence for creating new restriction enzyme sites, six zCRY1a regions were selected for reciprocal domain swaps: residues 1–125, 126–196, 197–263, 264–293, 294–419, and 420–557 (Fig. 1, A–C). As seen in Fig. 1C, zCRY1a is folded into two domains, an N-terminal α/β domain (residues 1–127) and a C-terminal helical one (residues 214–557), which are connected by a long interdomain loop (residues 128–213). The first (residues 1–125) and second (residues 126–196) regions were designed, respectively, to carry the N-terminal α/β domain and subsequent interdomain loop. The remaining 197–557 region has a helical domain that is the FAD binding site, and its primary structure is well conserved within this protein family. Initially, we planned to divide this region into three parts: residues 197–293, 294–419, and 420–557, because the amino acid sequence in 294–419 is the best conserved, whereas those in 197–293 and 420–557 have diverged to some extent (Fig. 1A). The region was further divided into two subregions, 197–263 and 264–293, to analyze the functionally important 197–293 region more precisely. As shown schematically in Fig. 2A, 19 chimeras composed of reciprocal domain swaps were generated between zCRY1a and -3. Determination of Those Sequence Elements of zCRY1a Sufficient for Transcriptional Repression—Effects of the chimeric zCRYs on zCLOCK:zBMAL-mediated transcription were examined in a luciferase reporter gene assay. As reported elsewhere (16Kobayashi Y. Ishikawa T. Hirayama J. Daiyasu H. Kanai S. Toh H. Fukuda I. Tsujimura T. Terada N. Kamei Y. Yuba S. Iwai S. Todo T. Genes Cells. 2000; 5: 725-738Crossref PubMed Scopus (178) Google Scholar, 20Hirayama J. Fukuda I. Ishikawa T. Kobayashi Y. Todo T. Nucleic Acids Res. 2003; 31: 935-943Crossref PubMed Scopus (34) Google Scholar), co-expression of zCRY1a efficiently inhibits zCLOCK1:zBMAL3-mediated transcription (Fig. 2B, lane 3), whereas that of zCRY3 does not (lane 4). First, two chimera series (chimeras 1–8), in which the N- or C-terminal regions of zCRY1a were replaced sequentially by the corresponding regions of zCRY3, were tested. Two chimeras, 3 with amino acids 197–557 and 6 with 1–293 of zCRY1a, maintained transcriptional repression activity (lanes 7 and 10), whereas their reciprocals, chimeras 4 and 5, lacked that activity (lanes 8 and 9). This showed that the region between amino acids 197 and 293 of zCRY1a is necessary for transcriptional repression. In fact, chimera 9, which had amino acids 197–293 of zCRY1a, had repressor activity (lane 13), whereas its reciprocal, chimera 10, did not (lane 14). To identify precisely the critical domain, the 197–293 region was divided into two subregions, 197–263 and 264–293. Four chimeras, 11, 12, 13, and 14, carrying each region, were tested for repressor activity. Chimera 11 with the 197–263 residues of zCRY1a had activity (lane 15), whereas chimera 13 with the rest of the region (264–293) lacked it (lane 17), evidence that the 197–263 region of zCRY1a is sufficient to repress zCLOCK1:zBMAL3-mediated transcription. Consistent with this conclusion, chimera 14, with all zCRY1a sequences except the 264–293 region, which was replaced by zCRY3, had repressor activity (lane 18). Unexpectedly, chimera 12, in which the 197–263 region of zCRY1a was replaced with that of zCRY3, also had repressor activity (lane 16). This suggests that besides the 197–263 region of zCRY1a sufficient for repressor activity, a second region als
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