THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 40, Issue of October 3, pp. 38149–38158, 2003 © 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Constructing a Feedback Loop with Circadian Clock Molecules from the Silkmoth, Antheraea pernyi*□S Received for publication, June 30, 2003 Published, JBC Papers in Press, July 17, 2003, DOI 10.1074/jbc.M306937200 Dennis C. Chang,a,b,c,d Harriet G. McWatters,a,e Julie A. Williams,a,f Anthony L. Gotter,a,c,g Downloaded from Joel D. Levine,a,h and Steven M. Repperta,b,c,i From the aLaboratory of Developmental Chronobiology, MassGeneral Hospital for Children, Massachusetts General Hospital, Boston, Massachusetts 02114, the bProgram in Neuroscience, Harvard Medical School, Boston, Massachusetts 02115, and the cDepartment of Neurobiology, University of Massachusetts Medical Center, Worcester, Massachusetts 01605 www.jbc.org Circadian clocks are important regulators of behavior Circadian rhythms are driven by cell autonomous pacemak- and physiology. The circadian clock of Drosophila de- ers consisting of molecular feedback loops (1). In the fruit fly pends on an autoinhibitory feedback loop involving Drosophila melanogaster, circadian feedback loops require the at UNAM. DIRECCION GENERAL DE BIBLIOTECAS. DEPARTAMENTO SUSCRIPCIONES on August 8, 2006 dCLOCK, CYCLE (also called dBMAL, for Drosophila transcription factors dCLOCK (dCLK) and CYCLE (CYC, also brain and muscle ARNT-like protein), dPERIOD, and called dBMAL) (2). dCLK and CYC heterodimerize to bind dTIMELESS. Recent studies suggest that the clock E-box enhancer elements, presumably through their basic he- mechanism in other insect species may differ strikingly lix-loop-helix (bHLH)1 domains (3). In addition, dCLK and CYC from that of Drosophila. We cloned Clock, Bmal, and each possesses a PER-ARNT-SIM homology (PAS) domain (3– Timeless homologs (apClock, apBmal, and apTimeless) 5), which is thought to facilitate protein-protein interactions (6) from the silkmoth Antheraea pernyi, from which a Pe- and contains two PAS structural motifs (PAS-A and PAS-B) riod homolog (apPeriod) has already been cloned. In and a region downstream of PAS-B called PAC (7). Further Schneider 2 (S2) cell culture assays, apCLOCK:apBMAL downstream, dCLK possesses a glutamine-rich transactivation activates transcription through an E-box enhancer ele- domain (3, 4). One of the genes activated by dCLK:CYC, period -ment found in the 5؅ region of the apPeriod gene. Fur thermore, apPERIOD can robustly inhibit apCLOCK: (dper), encodes another PAS protein, dPER (2, 3). dPER inhib- apBMAL-mediated transactivation, and apTIMELESS its dCLK:CYC through a non-PAS, C-terminal domain, called can augment this inhibition. Thus, a complete feedback the dCLK:CYC inhibition domain (CCID), thus completing the loop, resembling that found in Drosophila, can be con- key circadian negative feedback loop (2, 3, 8). dCLK:CYC also structed from silkmoth CLOCK, BMAL, PERIOD, and activates transcription of the timeless (dtim) gene, whose pro- TIMELESS. Our results suggest that the circadian auto- tein product, dTIM, regulates dPER protein stability and nu- inhibitory feedback loop discovered in Drosophila is clear transport and may also contribute to the inhibition of likely to be widespread among insects. However, dCLK:CYC (2, 3, 8). dTIM also plays a role in light input to the whereas the transactivation domain in Drosophila lies clock (2). in the C terminus of dCLOCK, in A. pernyi, it lies in the The circadian feedback loops of mice depend upon mCLK: C terminus of apBMAL, which is highly conserved with mBMAL1, the elements of which are homologs of dCLK:CYC the C termini of BMALs in other insects (except Dro- (9). Cryptochromes (mCRYs) are the main inhibitors of mCLK: sophila) and in vertebrates. Our analysis sheds light on mBMAL1, whereas mouse PERs seem to regulate mCRY nu- the molecular function and evolution of clock genes in clear entry (10). Mammals lack a true ortholog of dTIM (11, 12). the animal kingdom. The presence of CLOCK, BMAL, and PERIOD (and other mol- ecules) at the heart of circadian clocks in both Drosophila and * This work was supported in part by National Institutes of Health mice suggests that both insects and vertebrates inherited their Grant R01-NS39303. The costs of publication of this article were de- clocks from a common ancestor (1). However, the differences in frayed in part by the payment of page charges. This article must fly and mouse clock mechanisms indicate that animal clocks therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. have diversified during the course of evolution. □S The on-line version of this article (available at http://www.jbc.org) Indeed, Drosophila even differs from other insects in its clock contains Figs. S1 and S2. mechanism. The temporally regulated nuclear transport of The nucleotide sequence(s) reported in this paper has been submitted dPER and dTIM (cytoplasmic during the afternoon, nuclear at to the GenBankTM/EBI Data Bank with accession number(s) AY330486 and AY330487. night) in pacemaker neurons (13, 14) is thought to be critical d Supported by a Howard Hughes Medical Institute Predoctoral for Drosophila clock function (2). In contrast, PER and TIM in Fellowship. the Chinese oak silkmoth, Antheraea pernyi (apPER and ap- e Current address: Dept. of Plant Sciences, Oxford University, South TIM), are primarily cytoplasmic at all times of day in brain Parks Rd., Oxford OX1 3RB, United Kingdom. f Current address: Dept. of Neuroscience, Howard Hughes Medical neurons (15). PER in a damselfly (order Odonata) is also cyto- Institute, University of Pennsylvania, 232 Stemmler Hall/6074, Phila- plasmic throughout the day (16). In fact, in the early morning, delphia, PA 19104. when dPER is nuclear in Drosophila (14), PER is cytoplasmic g Current address: Division of Human Genetics and Molecular Biol- ogy, Children’s Hospital of Philadelphia, 3516 Civic Center Blvd., ARC 1002, Philadelphia, PA 19104. h Current address: Dept. of Biology, MS-008, Brandeis University, 1 The abbreviations used are: bHLH, basic helix-loop-helix; PAS, Waltham, MA 02454. PER-ARNT-SIM homology; CCID, dCLK:CYC inhibition domain; i To whom correspondence should be addressed: Dept. of Neurobiol- mCRY, cryptochrome; S2, Schneider 2; NLS, nuclear localization se- ogy, University of Massachusetts Medical School, 364 Plantation St., quence; SFM, serum-free medium; PBS, phosphate-buffered saline; Worcester, MA 01605. Tel.: 508-856-6148; Fax: 508-856-6233; E-mail: CLD, cytoplasmic localization domain; aa, amino acid(s); BCTR, BMAL [email protected]. C-terminal region; DAPI, 4Ј,6-diamidino-2-phenylindole. Supplemental Material can be found at: This paper is available on line at http://www.jbc.org 38149 http://www.jbc.org/cgi/content/full/M306937200/DC1 38150 A Silkmoth Circadian Feedback Loop in representatives of many insect orders: Thysanura, Ephemer- 25 °C in Schneider’s Drosophila medium (Invitrogen) with 9% heat- optera, Orthoptera, Plecoptera, Hemiptera, Coleoptera, Hy- inactivated fetal bovine serum (Invitrogen), and Sf9 cells were main- menoptera, Trichoptera, and even Diptera (16). Thus the pace- tained at 27 °C in Sf-900 II serum-free medium (SFM, Invitrogen). Transient transfection of plasmids involved mixing DNA with 5–10 ␮l maker mechanism in A. pernyi (Lepidoptera) may be more of CellFECTIN reagent (Invitrogen), and 15 min later, applying the representative of insect clocks than the mechanism in mixture to S2 cells in Drosophila SFM (Invitrogen) supplemented with Drosophila. 2mML-glutamine, or to Sf9 cells in Sf-900 II SFM. Approximately 4 h To further our understanding of insect clock function and later, S2 cells were fed with an equal volume of 9% fetal bovine serum evolution, we cloned Clock, Bmal, and Timeless homologs from in Schneider’s medium, and Sf9 cells were given fresh SFM. Cells were Downloaded from ϳ A. pernyi (apClk, apBmal, and apTim). We characterized their incubated for 48 h before use. Transcription Assays—To test the ability of apCLK:apBMAL to ac- functions and interactions with apPer using Drosophila tivate transcription in S2 cells, we used a modified version of the Schneider 2 (S2) cells, because many aspects of both fly and transcription assay of Darlington and coworkers (3). In brief, 1 ng of Ϯ mammalian circadian biochemistry can be simulated in S2 apClk 1ngofapBmal was co-transfected with 10 ng of apPer 4Ep- cells. These include dCLK:CYC-mediated transactivation via hs-luc and 30 ng of ␤gal pAc5.1V5/HisA (␤gal-V5). For each assay, a E-boxes, dPER inhibition of dCLK:CYC (3, 8), mCLK:mBMAL1 control transfection, including only the reporters apPer4Ep-hs-luc and www.jbc.org ␤ transactivation, and mCRY inhibition of that activation (17, gal-V5, was used to establish baseline reporter activity. In the apPer promoter assays, we substituted apPer genomic DNA constructs for 18). In S2 cells, apCLK:apBMAL can activate transcription apPer4Ep-hs-luc. To measure the inhibitory activity of apPer/apTim,we through an E-box in the apPer promoter, apPER inhibits ap- co-transfected 2–250 ng of apPer, apTim,orapPer mutant constructs CLK:apBMAL-mediated transcription, and apTIM can aug- with apClk, apBmal, and the reporters. The total amount (nanograms) at UNAM. DIRECCION GENERAL DE BIBLIOTECAS. DEPARTAMENTO SUSCRIPCIONES on August 8, 2006 ment the inhibitory activity of apPER. Like dPER, apPER of DNA in each transfection was normalized using empty vector inhibits apCLK:apBMAL through a C-terminal domain that (pAc5.1V5). After transfection and a 48-h incubation at 25 °C, cells were also contains the primary nuclear localization sequence (NLS) harvested, washed in phosphate-buffered saline (Dulbecco’s PBS, In- vitrogen), and lysed in lysis buffer (Promega). Cell extracts were as- of apPER. Thus, despite initial appearances, a Drosophila-like sayed for ␤-galactosidase and luciferase activities using commercial negative feedback loop involving CLOCK, BMAL, PERIOD, assay kits (Tropix and Promega, respectively) and an MLX microtiter and TIMELESS may exist in the silkmoth A.