Both casein kinase 1 delta (CK1delta) and epsilon (CK1epsilon) phosphorylate core clock proteins of the mammalian circadian oscillator. To assess the roles of CK1delta and CK1epsilon in the circadian clock mechanism, we generated mice in which the genes encoding these proteins (Csnk1d and Csnk1e, respectively) could be disrupted using the Cre-loxP system. Cre-mediated excision of the floxed exon 2 from Csnk1d led to in-frame splicing and production of a deletion mutant protein (CK1delta(Delta2)). This product is nonfunctional. Mice homozygous for the allele lacking exon 2 die in the perinatal period, so we generated mice with liver-specific disruption of CK1delta. In livers from these mice, daytime levels of nuclear PER proteins, and PER-CRY-CLOCK complexes were elevated. In vitro, the half-life of PER2 was increased by approximately 20%, and the period of PER2::luciferase bioluminescence rhythms was 2 h longer than in controls. Fibroblast cultures from CK1delta-deficient embryos also had long-period rhythms. In contrast, disruption of the gene encoding CK1epsilon did not alter these circadian endpoints. These results reveal important functional differences between CK1delta and CK1epsilon: CK1delta plays an unexpectedly important role in maintaining the 24-h circadian cycle length.

The monarch butterfly (Danaus plexippus) cryptochrome 1 (DpCry1) belongs in the class of photosensitive insect cryptochromes. Here we purified DpCry1 expressed in a bacterial host and obtained the protein with a stoichiometric amount of the flavin cofactor in the two-electron oxidized, FAD(ox), form. Exposure of the purified protein to light converts the FAD(ox) to the FAD*(-) flavin anion radical by intraprotein electron transfer from a Trp residue in the apoenzyme. To test whether this novel photoreduction reaction is part of the DpCry1 physiological photocycle, we mutated the Trp residue that acts as the ultimate electron donor in flavin photoreduction. The mutation, W328F, blocked the photoreduction entirely but had no measurable effect on the light-induced degradation of DpCry1 in vivo. In light of this finding and the recently published action spectrum of this class of Crys, we conclude that DpCry1 and similar insect cryptochromes do not contain flavin in the FAD(ox) form in vivo and that, most likely, the [see text] photoreduction reaction is not part of the insect cryptochrome photoreaction that results in proteolytic degradation of the photopigment.

Every fall, Northeastern America monarch butterflies (Danaus plexippus) undergo an extraordinary migration to their overwintering site in Central Mexico. During their long migration, monarch migrants use sun compass to navigate. To maintain a southward flying direction, monarch migrants compensate for the continuously changing position of the sun by providing timing information to the compass using their circadian clock. Animal circadian clocks depend primarily on a negative transcriptional feedback loop to track time. I started my work to re-construct the monarch butterfly circadian clock negative feedback loop in cell culture, focusing on homologs of Drosophila clock genes. It turned out that in addition to a Drosophila-like cryptochrome (cry1) gene, a second mammalian-like cry2 gene exists in monarch butterflies and many other insects, except in Drosophila. The two CRYs showed distinct functions in our initial assays in cultured Drosophila Schneider 2 (S2) cells. CRY2 functions as a potent transcriptional repressor, while CRY1 is light sensitive but shows no obvious transcriptional activity. The existence of two cry genes in insects changed the Drosophila-centric view of insect circadian clock. During the course of my study, our lab obtained a monarch cell line called DpN1 cells. These cells possess a light-driven clock and contributed tremendously to the research on monarch circadian clock. Using this cell line, I provided strong evidence supporting monarch CRY2’s role as a major circadian clock repressor and identified a protein-protein protective interaction cascade underlying the CRY1-mediated resetting of the molecular oscillator in DpN1 cells. I continued my work trying to understand how insect CRY2 inhibits transcription. I provided evidence suggesting the involvement of monarch PER in promoting CRY2 nuclear entry in both S2 cells and DpN1 cells. Finally, I mapped CRY2’s transcriptional inhibitory activity onto its N-terminal domain. Collectively, my research helped to change our view of insect clocks from a Drosophila-centric standpoint to a much more diverse picture. My studies also advanced the understanding of monarch circadian clock mechanism, and provides a foundation for further studies.