A new gene encoding a putative transcription factor regulated by the Drosophila circadian clock affected by the different per mutations in the same way

affected by the different per mutations in the same way François Rouyer1,2, Mohammed Rachidi3, that the circadian behavior is affected, indicating that PER Claudio Pikielny4 and Michael Rosbash influences the expression of its own gene through a HHMI and Department of Biology, Brandeis University, feedback loop mechanism (Hardin et al., 1990). This Waltham MA 02254, USA and 3Dépt de Biologie Moléculaire, control appears to act mostly at the transcriptional level Institut Pasteur, Paris, France (Hardin et al., 1992). Circadian fluctuations of PER protein 1Present address: Institut Alfred Fessard, CNRS UPR 2212, abundance and phosphorylation occur in the fly head and 91198 Gif-sur-Yvette, France lag behind mRNA oscillations by ~6 h (Siwicki et al., 4Present address: UMDNJ/Robert Wood Johnson Medical School, 1988; Zerr et al., 1990; Edery et al., 1994b; Zeng et al., Piscataway, NJ, USA 1994). PER protein enters the cell in the middle of the 2Corresponding author night, a few hours before the protein reaches its peak e-mail: [email protected] level (Curtin et al., 1995). The recently isolated tim gene (Gekakis et al., 1995; Circadian rhythms of locomotor activity and eclosion Myers et al., 1995), is a major per partner in the circadian in Drosophila depend upon the reciprocal autoregulafeedback loop mechanism (Reppert and Sauman, 1995; tion of the period (per) and timeless (tim) genes. As Rosbash, 1995). tim mRNA abundance cycles with the part of this regulatory loop, per and tim mRNA levels same phase as per mRNA in fly heads, and tim as well oscillate in a circadian fashion. Other cycling tranas per mRNA oscillations depend upon both PER and tim scripts may participate in this central pacemaker protein (TIM) (Hardin et al., 1990; Sehgal et al., 1995). mechanism or represent outputs of the clock. In this Like PER levels, TIM levels fluctuate with a 6 h lag paper, we report the isolation of Crg-1, a new circabehind tim transcript cycling (Hunter-Ensor et al., 1996; dianly regulated gene. Like per and tim transcript Lee et al., 1996; Myers et al., 1996; Zeng et al., 1996). levels, Crg-1 transcript levels oscillate with a 24 h In the absence of TIM, PER levels stay very low and period in light:dark (LD) conditions, with a maximal PER does not cycle nor enter the cell nucleus (Gekakis abundance at the beginning of the night. These oscillaet al., 1995; Myers et al., 1995; Price et al., 1995). In tions persist in complete darkness and depend upon per0 flies, the TIM protein does not enter the nucleus but per and tim proteins. The putative CRG-1 proteins still cycles in LD as a result of its light-induced degradation show some sequence similarity with the DNA-binding (Hunter-Ensor et al., 1996; Myers et al., 1996; Zeng et al., domain of the HNF3/fork head family of transcription 1996). Such reciprocal effects on PER and TIM nuclear factors. In the adult head, in situ hybridization analysis entry appear to result from the formation of a PER–TIM reveals that per and Crg-1 have similar expression complex that would be responsible for the regulation of patterns in the eyes and optic lobes. per and tim transcription (Gekakis et al., 1995; Hunter