Role of the CLOCK protein in the mammalian circadian mechanism

Science

Volumn 280, Issue 5369, 1998, Pages 1564-1569

  Gekakis, Nicholas ^a   Staknis, David ^a   Nguyen, Hubert B ^a   Davis, Fred C ^b   Wilsbacner, Lisa D ^c   King, David P ^c   Takahashi, Joseph S ^c   Weitz, Charles J ^a  

^a HARVARD MEDICAL SCHOOL   (United States)

^b NORTHEASTERN UNIVERSITY   (United States)

^c NORTHWESTERN UNIVERSITY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

HELIX LOOP HELIX PROTEIN; MUTANT PROTEIN; TRANSCRIPTION FACTOR;

ANIMAL TISSUE; ARTICLE; BRAIN; CIRCADIAN RHYTHM; CONTROLLED STUDY; DIMERIZATION; DROSOPHILA; GENE EXPRESSION; IN SITU HYBRIDIZATION; MAMMAL; MOUSE; NONHUMAN; OSCILLATION; PRIORITY JOURNAL; PROTEIN DNA BINDING; PROTEIN FAMILY; RETINA; TRANSCRIPTION REGULATION;

ANIMALS; BASIC HELIX-LOOP-HELIX TRANSCRIPTION FACTORS; BIOLOGICAL CLOCKS; CELL CYCLE PROTEINS; CIRCADIAN RHYTHM; CLONING, MOLECULAR; CRICETINAE; DIMERIZATION; DNA; FEEDBACK; GENE EXPRESSION; HELIX-LOOP-HELIX MOTIFS; MALE; MESOCRICETUS; MICE; MUTATION; NUCLEAR PROTEINS; PROMOTER REGIONS (GENETICS); RETINA; SUPRACHIASMATIC NUCLEUS; TRANS-ACTIVATION (GENETICS); TRANS-ACTIVATORS; TRANSCRIPTION FACTORS;

EID: 0032486330     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.280.5369.1564     Document Type: Article

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26. We used LEXA-CLOCK(1-580) as bait, which is lacking most of the COOH-terminal region predicted to be a transcriptional activation domain (7) but includes the region deleted from CLOCK-Δ19. The two-hybrid screen was carried out as described (18), except library transformants were plated onto X-Gal plates lacking tryptophan, histidine, uracil, lysine, and leucine (18) that were supplemented with 30 mM or 50 mM 3-aminotriazole. The cDNA library was generated as follows: 32 male Syrian hamsters (Charles River Laboratories) were maintained on a 14:10 light:dark cycle for ≥3 weeks, transferred to constant dim light (<1 lux) at the time of lights off, and killed by decapitation 24 to 42 hours later, eight at each of four circadian times (CT 1, 7, 13, and 19). Brains were removed and placed in phosphate-buffered saline (4°C) for 30 s, a 1.5-mm coronal slice was cut, and a pyramid of anterior hypothalamus containing the SCNs was dissected out, frozen on dry ice, and stored at -70°C. Purification of mRNA and construction of a randomly primed cDNA library in pVP16 were performed as described (18). Care of hamsters and all procedures were in full compliance with institutional guidelines for animal experimentation.
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28. 2-terminal regions of 59 and 57 amino acids, respectively. All constructs used in this work corresponded to BMAL1b, but because the existence of the different BMAL1 proteins has not been established, we refer here to the protein generically as BMAL1.
29. The 1.7-kb clone corresponded to codons 1 to 518 of mouse ARNT2 [K. Hirose et al., Mol. Cell. Biol. 16, 1706 (1996)] preceded by 6 bp of apparent 5′ untranslated sequence.
30. Preparation of coronal brain sections from mice (<1 lux; killed at CT 7 or CT 19) and in situ hybridizations were performed as described [M. E. Morris, N. Viswanathan, S. Kuhlman, F. C. Davis, C. J. Weitz, Science 279, 1544 (1998)]. Eyes were removed from perfused mice, the anterior segments were removed by dissection and discarded, and the resulting eye cups were postfixed and stored as described for brains. Parasaggital sections (12-μm) were cut on a cryostat, and in situ hybridizations were performed as for brain sections.
31. With a mouse arnt riboprobe (codons 70 to 474), we observed weak or no hybridization to SCN, but hybridization to periventricular nuclei, piriform cortex, subcortical nuclei, and cerebral cortex was consistent. With a hamster arnt2 riboprobe (6 nucleotides of 5′ untranslated plus codons 1 to 518), we observed very strong hybridization throughout the brain, especially in cerebral cortex, subcortical nuclei, and hypothalamus, likely including the SCN, although it was difficult to distinguish SCN in the context of strong signals from many hypothalamic nuclei. Although they do not show a Clock-and mper1-like pattern of expression, it is possible that arnt and arnt2 transcripts are localized in the SCN, and we cannot exclude a possible role for either in the circadian clock. Riboprobes were synthesized from polymerase chain reaction (PCR) products that incorporated T3 (for sense strand) and T7 (for antisense strand) RNA polymerase-binding sites. The mouse Clock riboprobe corresponded to codons 1 to 389, the mouse per1 riboprobe corresponded to codons 738 to 835, and the hamster bmal1 riboprobe corresponded to full-length BMAL1b. No obvious oscillation in the level of bmal1 transcripts was observed when comparing circadian times 7 and 19.
32. N. Gekakis et al., unpublished data.
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34. The indicated DNA fragments were ligated into the pBgl-lacZ reporter plasmid [J. J. Li and I. Herskowitz, Science 262, 1870 (1993)], and one-hybrid yeast strains were constructed by integration of recombinant pBgl-lacZ reporter plasmids into the ura3 locus of YPH 499 [R. S. Sikorsky and P. Hieter, Genetics 122, 19 (1989)]. Negative control strains were constructed identically with nonrecombinant pBgl-lacZ. One-hybrid strains were transformed with the indicated p424-Met25 [D. Mumberg, R. Muller, M. Funk, Nucleic Acids Res. 22, 5767 (1994)] and pVP16 (18) expression plasmids, and transformants were patched onto X-Gal plates (18) for detection of β-Gal activity. We validated the one-hybrid system by comparing results in electrophoretic mobility shift assays with one-hybrid assays using TFEB, a bHLH protein known to bind the CACGTG E-box as a homodimer [D. E. Fisher, C. S. Carr, L. A. Parent, P. A. Sharp, Genes Dev. 5, 2342 (1991)]. The Drosophila per gene 21-mer was derived from the 69-bp clock control region (16); it contained the E-box with 8 bp of 5′ and 7 bp of 3′ flanking sequences, respectively. Mutated E-boxes within the 21-mer were generated by scrambling the E-box sequence by means of a random number program: E-box mutant 1, 5′-ATTCGC; E-box mutant 2, 5′-GTAACT.
35. The indicated DNA fragments were ligated into the pBgl-lacZ reporter plasmid [J. J. Li and I. Herskowitz, Science 262, 1870 (1993)], and one-hybrid yeast strains were constructed by integration of recombinant pBgl-lacZ reporter plasmids into the ura3 locus of YPH 499 [R. S. Sikorsky and P. Hieter, Genetics 122, 19 (1989)]. Negative control strains were constructed identically with nonrecombinant pBgl-lacZ. One-hybrid strains were transformed with the indicated p424-Met25 [D. Mumberg, R. Muller, M. Funk, Nucleic Acids Res. 22, 5767 (1994)] and pVP16 (18) expression plasmids, and transformants were patched onto X-Gal plates (18) for detection of β-Gal activity. We validated the one-hybrid system by comparing results in electrophoretic mobility shift assays with one-hybrid assays using TFEB, a bHLH protein known to bind the CACGTG E-box as a homodimer [D. E. Fisher, C. S. Carr, L. A. Parent, P. A. Sharp, Genes Dev. 5, 2342 (1991)]. The Drosophila per gene 21-mer was derived from the 69-bp clock control region (16); it contained the E-box with 8 bp of 5′ and 7 bp of 3′ flanking sequences, respectively. Mutated E-boxes within the 21-mer were generated by scrambling the E-box sequence by means of a random number program: E-box mutant 1, 5′-ATTCGC; E-box mutant 2, 5′-GTAACT.
36. The indicated DNA fragments were ligated into the pBgl-lacZ reporter plasmid [J. J. Li and I. Herskowitz, Science 262, 1870 (1993)], and one-hybrid yeast strains were constructed by integration of recombinant pBgl-lacZ reporter plasmids into the ura3 locus of YPH 499 [R. S. Sikorsky and P. Hieter, Genetics 122, 19 (1989)]. Negative control strains were constructed identically with nonrecombinant pBgl-lacZ. One-hybrid strains were transformed with the indicated p424-Met25 [D. Mumberg, R. Muller, M. Funk, Nucleic Acids Res. 22, 5767 (1994)] and pVP16 (18) expression plasmids, and transformants were patched onto X-Gal plates (18) for detection of β-Gal activity. We validated the one-hybrid system by comparing results in electrophoretic mobility shift assays with one-hybrid assays using TFEB, a bHLH protein known to bind the CACGTG E-box as a homodimer [D. E. Fisher, C. S. Carr, L. A. Parent, P. A. Sharp, Genes Dev. 5, 2342 (1991)]. The Drosophila per gene 21-mer was derived from the 69-bp clock control region (16); it contained the E-box with 8 bp of 5′ and 7 bp of 3′ flanking sequences, respectively. Mutated E-boxes within the 21-mer were generated by scrambling the E-box sequence by means of a random number program: E-box mutant 1, 5′-ATTCGC; E-box mutant 2, 5′-GTAACT.
37. The indicated DNA fragments were ligated into the pBgl-lacZ reporter plasmid [J. J. Li and I. Herskowitz, Science 262, 1870 (1993)], and one-hybrid yeast strains were constructed by integration of recombinant pBgl-lacZ reporter plasmids into the ura3 locus of YPH 499 [R. S. Sikorsky and P. Hieter, Genetics 122, 19 (1989)]. Negative control strains were constructed identically with nonrecombinant pBgl-lacZ. One-hybrid strains were transformed with the indicated p424-Met25 [D. Mumberg, R. Muller, M. Funk, Nucleic Acids Res. 22, 5767 (1994)] and pVP16 (18) expression plasmids, and transformants were patched onto X-Gal plates (18) for detection of β-Gal activity. We validated the one-hybrid system by comparing results in electrophoretic mobility shift assays with one-hybrid assays using TFEB, a bHLH protein known to bind the CACGTG E-box as a homodimer [D. E. Fisher, C. S. Carr, L. A. Parent, P. A. Sharp, Genes Dev. 5, 2342 (1991)]. The Drosophila per gene 21-mer was derived from the 69-bp clock control region (16); it contained the E-box with 8 bp of 5′ and 7 bp of 3′ flanking sequences, respectively. Mutated E-boxes within the 21-mer were generated by scrambling the E-box sequence by means of a random number program: E-box mutant 1, 5′-ATTCGC; E-box mutant 2, 5′-GTAACT.
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40. The 2.0-kb mper1 upstream fragment (-2122 bp to -129 bp with respect to the putative transcription start site) was generated by PCR using pfu and tag polymerases together on a cloned lambda phage template (primers: 5′-CATCCGCTCGAGCTCTTTGGTACCTGGCCAGCAACC and 5′-CATCCGCTCGAGACTGAGGTCAGGGCTGTGTCACAC; both primers included Xho I sites added at 5′ ends). Transfections were performed with a pool of 18 independent recombinants derived from different PCRs. The 54-mer derived from the mouse per) gene 5′ flanking region consisted of the three 18-bp sequences shown in Fig. 4A; they were linked together in the order shown from 5′ to 3′. The E-box mutant 54-mer was the same except that it had each E-box site independently scrambled by means of a random number program: 5′-GCTAGT (distal), 5′-TTTAGT (middle), and 5′-TCGCTC (proximal). Individual E-box constructs consisted of the 18-bp sequences shown in Fig. 4A.
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42. Mouse NIH-3T3 cells were transfected with Lipofectamine-Plus (GibcoBRL). Cells were grown in Dulbecco's minimum essential medium with 10% fetal bovine serum (GibcoBRL) in six-well plates, and cells in each well were transfected with 1 μg (total) of the pcDNA3 expression plasmids (Invitrogen) with the indicated inserts, 10 ng of the pGL3 firefly luciferase reporter plasmid containing the minimal SV40 promoter (Promega) and the indicated inserts, and 0.5 ng of pRL-CMV (Renilla) luciferase internal control plasmid (Promega). After 48 hours, cells in each well were extracted with 200 μl of Promega passive lysis buffer, and separate portions of extract were taken for assays of firefly luciferase [A. R. Brasier, J. E. Tate, J. F. Habener, Biotechniques 7, 1117 (1988)] and Renilla luciferase [S. Inouye and O. Shinomura, Biochem. Biophys. Res. Commun. 233, 349 (1997)] by luminometry. For each sample, luciferase activity was corrected for transfection efficiency by dividing the measured firefly luciferase activity (from the reporter construct) by the measured Renilla luciferase activity (from the constitutively driven construct). A full-length Clock cDNA was constructed by fusion at a Pst I site of clones YZ50 and D1 (7). Clock-Δ19 cDNA was generated by subcloning of full-length Clock cDNA into pGEX-4-T3 (Pharmacia Biotech) for oligonucleotide-directed deletion of exon 19 with the U.S.E. Mutagenesis kit (Pharmacia Biotech).
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48. We thank J. Li, F. Winston, and K. Buckley for plasmids, E. Raviola and H. Regan for generous help with retinal anatomy and histology, L. Buck for the use of her microscope, M. Greenberg for helpful comments on the manuscript, and J. Lee and Q. Shen for technical assistance. Supported by the NSF (C.J.W.), a McKnight Scholars Award (C.J.W.), the NIH (F.C.D. and J.S.T.), and the NSF Center for Biological Timing (J.S.T.). J.S.T is an Investigator of the Howard Hughes Medical Institute.