Constructing a Feedback Loop with Circadian Clock Molecules from the Silkmoth, Antheraea pernyi

Abstract

Circadian clocks are important regulators of behavior and physiology. The circadian clock of Drosophila depends on an autoinhibitory feedback loop involving dCLOCK, CYCLE (also called dBMAL, for Drosophila brain and muscle ARNT-like protein), dPERIOD, and dTIMELESS. Recent studies suggest that the clock mechanism in other insect species may differ strikingly from that of Drosophila. We cloned Clock, Bmal, and Timeless homologs (apClock, apBmal, and apTimeless) from the silkmoth Antheraea pernyi, from which a Period homolog (apPeriod) has already been cloned. In Schneider 2 (S2) cell culture assays, apCLOCK:apBMAL activates transcription through an E-box enhancer element found in the 5 region of the apPeriod gene. Furthermore, apPERIOD can robustly inhibit apCLOCK: apBMAL-mediated transactivation, and apTIMELESS can augment this inhibition. Thus, a complete feedback loop, resembling that found in Drosophila, can be constructed from silkmoth CLOCK, BMAL, PERIOD, and TIMELESS. Our results suggest that the circadian autoinhibitory feedback loop discovered in Drosophila is likely to be widespread among insects. However, whereas the transactivation domain in Drosophila lies in the C terminus of dCLOCK, in A. pernyi, it lies in the C terminus of apBMAL, which is highly conserved with the C termini of BMALs in other insects (except Drosophila) and in vertebrates. Our analysis sheds light on the molecular function and evolution of clock genes in the animal kingdom.

Circadian Rhythm Gene Regulation in the Housefly Musca domestica

“…These cells possibly represent a strategic point in the neuronal network of Diptera where a physiological response to a combination of environmental variables might be amplified for entrainment (Collins et al 2005). As for the more nuclear subcellular distribution of MdTIM in the l-LNvs of Musca compared to MdPER, this could reflect a more prominent role for MdTIM as a negative regulator in the housefly, as has been suggested for A. pernyii (Chang et al 2003).…”

“…However, when transformed into per-null fliesalthough behavioral rhythmicity is markedly attenuated in the transgenic compared to control transformant fliesAntheraea pernyii PER(ApPER) does appear to locate to the nucleus of lateral neurons and photorecepeptors during the night phase (Levine et al 1995). In addition, when ApPER is used in Drosophila cell lines to reconstitute a circadian pacemaker, it does appear to act as a negative regulator of ApCLK/ApBMAL1-mediated transactivation, with the added bonus of A. pernyii TIM acting to enhance this negative regulation (Chang et al 2003). In Drosophila, TIM can shuttle in and out of the nucleus, so it may be that although TIM (or PER) cannot be seen in the nucleus at particular time points in the silkmoth, it is nevertheless present (Ashmore et al 2003;Nawathean and Rosbash 2004).…”

Molecular and phylogenetic analyses reveal mammalian-like clockwork in the honey bee (Apis mellifera) and shed new light on the molecular evolution of the circadian clock

“…amCYC protein contains highly conserved PAS-A, PAS-B, Pac, and bHLH; domains; and sequences identical (100%) to the putative N-terminal NLS, and NES signals of other CYC/BMAL proteins ( Fig. 4A; Chang et al 2003;Hirayama and Sassone-Corsi 2005). The amCYC protein is significantly longer than dCYC (675 compared with 413 amino acids) and comparable in size to BMAL proteins of other animals.…”

“…The amCYC protein is significantly longer than dCYC (675 compared with 413 amino acids) and comparable in size to BMAL proteins of other animals. Importantly, the C-terminal end of amCYC (termed "BCTR" by Chang et al 2003) is highly conserved (85% identity, 87% similarity) with those of the mouse and A. pernyi, in which this domain has potent transcriptional activity in vitro (Takahata et al 2000;Chang et al 2003). Thus, amCYC is phylogenetically related to dCYC but shares important structural similarities with BMAL proteins.…”

“…These inconsistencies may imply that not all insect clockworks are similar to Drosophila. But a detailed description of the molecular biology of the clock is only available for a few insects other than Drosophila (e.g., Sauman and Reppert 1996;Chang et al 2003;Zhu et al 2005). Thus, the degree of species-specific variation in the clockwork, as well as its functional significance, is mostly unknown.…”