title: Knockout-Rescue Embryonic Stem Cell-Derived Mouse Reveals Circadian-Period Control by Quality and Quantity of CRY1.
abstract: To conduct comprehensive characterization of molecular properties in organisms, we established an efficient method to produce knockout (KO)-rescue mice within a single generation. We applied this method to produce 20 strains of almost completely embryonic stem cell (ESC)-derived mice ("ES mice") rescued with wild-type and mutant Cry1 gene under a Cry1-/-:Cry2-/-background. A series of both phosphorylation-mimetic and non-phosphorylation-mimetic CRY1 mutants revealed that multisite phosphorylation of CRY1 can serve as a cumulative timer in the mammalian circadian clock. KO-rescue ES mice also revealed that CRY1-PER2 interaction confers a robust circadian rhythmicity in mice. Surprisingly, in contrast to theoretical predictions from canonical transcription/translation feedback loops, the residues surrounding the flexible P loop and C-lid domains of CRY1 determine circadian period without changing the degradation rate of CRY1. These results suggest that CRY1 determines circadian period through both its degradation-dependent and -independent pathways.
authors.affiliation: Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; Laboratory for Synthetic Biology, RIKEN Quantitative Biology Center, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Koji L
authors.initials: KL
authors.lastname: Ode
authors.affiliation: Laboratory for Synthetic Biology, RIKEN Quantitative Biology Center, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Ukai
authors.affiliation: Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; Laboratory for Synthetic Biology, RIKEN Quantitative Biology Center, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan; PRESTO, Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama, 332-0012, Japan.
authors.forename: Etsuo A
authors.initials: EA
authors.lastname: Susaki
authors.affiliation: Laboratory for Synthetic Biology, RIKEN Quantitative Biology Center, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Ryohei
authors.initials: R
authors.lastname: Narumi
authors.affiliation: Laboratory for Synthetic Biology, RIKEN Quantitative Biology Center, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Matsumoto
authors.affiliation: Laboratory for Synthetic Biology, RIKEN Quantitative Biology Center, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Junko
authors.initials: J
authors.lastname: Hara
authors.affiliation: Laboratory for Synthetic Biology, RIKEN Quantitative Biology Center, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Naoshi
authors.initials: N
authors.lastname: Koide
authors.affiliation: Genetic Engineering Team, RIKEN Center for Life Science Technologies, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan.
authors.forename: Takaya
authors.initials: T
authors.lastname: Abe
authors.affiliation: PRESTO, Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama, 332-0012, Japan; Division of Molecular Cell Engineering, National Institute of Genetics, Research Organization of Information and Systems, Yata 1111, Mishima, Shizuoka 411-8540, Japan; Department of Genetics, School of Life Science, SOKENDAI, Mishima, Shizuoka 411-8540, Japan.
authors.forename: Masato T
authors.initials: MT
authors.lastname: Kanemaki
authors.affiliation: Genetic Engineering Team, RIKEN Center for Life Science Technologies, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan; Animal Resource Development Unit, RIKEN Center for Life Science Technologies, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kiyonari
authors.affiliation: Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; Laboratory for Synthetic Biology, RIKEN Quantitative Biology Center, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address: uedah-tky@umin.ac.jp.
authors.forename: Hiroki R
authors.initials: HR
authors.lastname: Ueda
mesh_terms: D000818:Animals; D001522:Behavior, Animal; D057906:Circadian Clocks; D002940:Circadian Rhythm; D056931:Cryptochromes / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D053595:Embryonic Stem Cells / Q000378:metabolism*; D005838:Genotype; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008958:Models, Molecular; D009043:Motor Activity; D009154:Mutation; D041681:NIH 3T3 Cells; D056950:Period Circadian Proteins / Q000235:genetics / Q000378:metabolism; D010641:Phenotype; D010766:Phosphorylation; D011487:Protein Conformation; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D013997:Time Factors; D014162:Transfection
chemical_list: C538963:CRY1 protein, human; C538964:Cry1 protein, mouse; C538967:Cry2 protein, mouse; D056931:Cryptochromes; C513312:Per2 protein, mouse; D056950:Period Circadian Proteins
keywords: Cry1; ES mouse; circadian clock; cryptochrome; knockin mouse; phosphorylation