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Rigid Cooperation of Per1 and Per2 proteins

View Article: PubMed Central - PubMed

ABSTRACT

Period circadian clock (Per) genes Per1 and Per2 have essential roles in circadian oscillation. In this study, we identified a new role of Per1-Per2 cooperation, and its mechanism, using our new experimental methods. Under constant light conditions, the period length of Per1 and Per2 knockout mice depended on the copy number ratio of Per1:Per2. We then established a light-emitting diode-based lighting system that can generate any pattern of light intensity. Under gradually changing light in the absence of phase shift with different periods, both Per1(−/−) and Per2(−/−) mice were entrained to a broader range of period length than wild-type mice. To analyse Per1-Per2 cooperative roles at the cell culture level, we established a Per2 knockout-rescue system, which can detect period shortening in a familial advanced sleep phase syndrome (FASPS) mutant. Upon introduction of the Per1 coding region in this system, we saw period shortening. In conclusion, short period-associated protein Per1 and long period-associated Per2 cooperated to rigidly confine the circadian period to “circa” 24-h. These results suggest that the rigid circadian rhythm maintained through the cooperation of Per1-Per2 could negatively impact modern society, in which the use of artificial lighting is ubiquitous, and result in circadian disorders, including delirium.

No MeSH data available.


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Establishment of Per2 knockout-rescue system to detect period shortening in FASPS.(A) Schematic diagram of Per2 knockout-rescue targeting. We introduced Per2 into the Rosa26 locus of Per2(−/−) ES cells using TALEN, picked colonies, and checked genome structure with Arm PCR and quantitative PCR. ES clones containing only one copy of the rescue construct were differentiated. (B) Schematic diagram of homologous recombination between targeting vector and Rosa26 locus. (C) Detrend/baseline oscillation of two independent lines each of WT and FASPS mutant ES cells. (D) Period length calculated by autocorrelation analysis in individual (a) lines and (b) genotypes. FASPS, familial advanced sleep phase syndrome; FRT, flippase recognition target; P(PGK), mouse phosphoglycerate kinase 1 promoter; DT-A, diphtheria toxin fragment A.
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f5: Establishment of Per2 knockout-rescue system to detect period shortening in FASPS.(A) Schematic diagram of Per2 knockout-rescue targeting. We introduced Per2 into the Rosa26 locus of Per2(−/−) ES cells using TALEN, picked colonies, and checked genome structure with Arm PCR and quantitative PCR. ES clones containing only one copy of the rescue construct were differentiated. (B) Schematic diagram of homologous recombination between targeting vector and Rosa26 locus. (C) Detrend/baseline oscillation of two independent lines each of WT and FASPS mutant ES cells. (D) Period length calculated by autocorrelation analysis in individual (a) lines and (b) genotypes. FASPS, familial advanced sleep phase syndrome; FRT, flippase recognition target; P(PGK), mouse phosphoglycerate kinase 1 promoter; DT-A, diphtheria toxin fragment A.

Mentions: We then introduced Per2 into the Rosa26 locus of Per2(−/−) ES cells using TAL effector nuclease (TALEN) (Fig. 5A). Specifically, we constructed a vector containing the Per2 promoter (3.5 kb, P(Per2L)), Per2 coding region, and luciferase gene between the Rosa26 long arm (4 kb, modified from 8 kb) and short arm (4 kb) (Fig. 5B). We transiently cotransfected Per2(−/−) ES cells with this vector and TALEN vectors42, which create a double-strand break in the Rosa26 region, to improve recombination efficiency. Then we selected ES cells with puromycin and picked colonies under a phase contrast microscope. We checked the structure of the Rosa26 locus by polymerase chain reaction (PCR) (targeted arm 5′, 3′, WT arm 5′, 3′) and quantitative PCR of the puromycin resistance gene (Supplemental Figure S4). We obtained several rescued Per2 knockout ES cell lines that contained only one copy of the Per2 gene cassette correctly introduced into Rosa26. By introducing the WT Per2 gene, we were able to detect the 24-h oscillation from two independent ES clones (Fig. 5C,D, Supplemental Figure S5A).


Rigid Cooperation of Per1 and Per2 proteins
Establishment of Per2 knockout-rescue system to detect period shortening in FASPS.(A) Schematic diagram of Per2 knockout-rescue targeting. We introduced Per2 into the Rosa26 locus of Per2(−/−) ES cells using TALEN, picked colonies, and checked genome structure with Arm PCR and quantitative PCR. ES clones containing only one copy of the rescue construct were differentiated. (B) Schematic diagram of homologous recombination between targeting vector and Rosa26 locus. (C) Detrend/baseline oscillation of two independent lines each of WT and FASPS mutant ES cells. (D) Period length calculated by autocorrelation analysis in individual (a) lines and (b) genotypes. FASPS, familial advanced sleep phase syndrome; FRT, flippase recognition target; P(PGK), mouse phosphoglycerate kinase 1 promoter; DT-A, diphtheria toxin fragment A.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5016722&req=5

f5: Establishment of Per2 knockout-rescue system to detect period shortening in FASPS.(A) Schematic diagram of Per2 knockout-rescue targeting. We introduced Per2 into the Rosa26 locus of Per2(−/−) ES cells using TALEN, picked colonies, and checked genome structure with Arm PCR and quantitative PCR. ES clones containing only one copy of the rescue construct were differentiated. (B) Schematic diagram of homologous recombination between targeting vector and Rosa26 locus. (C) Detrend/baseline oscillation of two independent lines each of WT and FASPS mutant ES cells. (D) Period length calculated by autocorrelation analysis in individual (a) lines and (b) genotypes. FASPS, familial advanced sleep phase syndrome; FRT, flippase recognition target; P(PGK), mouse phosphoglycerate kinase 1 promoter; DT-A, diphtheria toxin fragment A.
Mentions: We then introduced Per2 into the Rosa26 locus of Per2(−/−) ES cells using TAL effector nuclease (TALEN) (Fig. 5A). Specifically, we constructed a vector containing the Per2 promoter (3.5 kb, P(Per2L)), Per2 coding region, and luciferase gene between the Rosa26 long arm (4 kb, modified from 8 kb) and short arm (4 kb) (Fig. 5B). We transiently cotransfected Per2(−/−) ES cells with this vector and TALEN vectors42, which create a double-strand break in the Rosa26 region, to improve recombination efficiency. Then we selected ES cells with puromycin and picked colonies under a phase contrast microscope. We checked the structure of the Rosa26 locus by polymerase chain reaction (PCR) (targeted arm 5′, 3′, WT arm 5′, 3′) and quantitative PCR of the puromycin resistance gene (Supplemental Figure S4). We obtained several rescued Per2 knockout ES cell lines that contained only one copy of the Per2 gene cassette correctly introduced into Rosa26. By introducing the WT Per2 gene, we were able to detect the 24-h oscillation from two independent ES clones (Fig. 5C,D, Supplemental Figure S5A).

View Article: PubMed Central - PubMed

ABSTRACT

Period circadian clock (Per) genes Per1 and Per2 have essential roles in circadian oscillation. In this study, we identified a new role of Per1-Per2 cooperation, and its mechanism, using our new experimental methods. Under constant light conditions, the period length of Per1 and Per2 knockout mice depended on the copy number ratio of Per1:Per2. We then established a light-emitting diode-based lighting system that can generate any pattern of light intensity. Under gradually changing light in the absence of phase shift with different periods, both Per1(−/−) and Per2(−/−) mice were entrained to a broader range of period length than wild-type mice. To analyse Per1-Per2 cooperative roles at the cell culture level, we established a Per2 knockout-rescue system, which can detect period shortening in a familial advanced sleep phase syndrome (FASPS) mutant. Upon introduction of the Per1 coding region in this system, we saw period shortening. In conclusion, short period-associated protein Per1 and long period-associated Per2 cooperated to rigidly confine the circadian period to “circa” 24-h. These results suggest that the rigid circadian rhythm maintained through the cooperation of Per1-Per2 could negatively impact modern society, in which the use of artificial lighting is ubiquitous, and result in circadian disorders, including delirium.

No MeSH data available.


Related in: MedlinePlus