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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 the ES cell differentiation system to detect precise period length.(A) Schematic diagram of ES cell differentiation system. (B) Real-time monitoring of luminescence of Per2::Luc KI/KI ES cells on day 20 with photomultiplier tube; cpm, counts per min. (C) Histogram and (D) dot plot of results of autocorrelation analysis of period length using Per2::Luc KI/KI ES cells. Values in (C) are mean % of total samples of six independent experiments. Values in (D) represent individual samples in six independent experiments. GMEM, Glasgow Minimum Essential Medium; KSR, KnockOut Serum Replacement; FBS, foetal bovine serum; 2i, 2 inhibitors (CHIR99021 and PD0325901); LIF, leukaemia inhibitory factor; DMEM, Dulbecco’s Modified Eagle Medium; NEAA, Non-Essential Amino Acids; L-Glu: L-glutamine. β-ME: β-mercaptoethanol; RA, retinoic acid. Each line and dot indicates each 35-mm dish sample.
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f4: Establishment of the ES cell differentiation system to detect precise period length.(A) Schematic diagram of ES cell differentiation system. (B) Real-time monitoring of luminescence of Per2::Luc KI/KI ES cells on day 20 with photomultiplier tube; cpm, counts per min. (C) Histogram and (D) dot plot of results of autocorrelation analysis of period length using Per2::Luc KI/KI ES cells. Values in (C) are mean % of total samples of six independent experiments. Values in (D) represent individual samples in six independent experiments. GMEM, Glasgow Minimum Essential Medium; KSR, KnockOut Serum Replacement; FBS, foetal bovine serum; 2i, 2 inhibitors (CHIR99021 and PD0325901); LIF, leukaemia inhibitory factor; DMEM, Dulbecco’s Modified Eagle Medium; NEAA, Non-Essential Amino Acids; L-Glu: L-glutamine. β-ME: β-mercaptoethanol; RA, retinoic acid. Each line and dot indicates each 35-mm dish sample.

Mentions: First, we focused on the fact that ES cells do not have a circadian rhythm, but circadian oscillation appears with retinoic acid (RA)-induced differentiation40, and we can see circadian oscillation in ES cells of any genotype without generating an organism. We performed a broad-parameter search to produce sustained oscillation with high amplitude to detect precise period length. Finally, we improved the ES differentiation system40 (Fig. 4A) and succeeded in detecting 10 peaks of oscillation in Per2::luciferase knockin (KI)/KI ES cells (Fig. 4B)41. We also used this protocol in several independent experiments and observed reproducible, precise period length, that is, most of the cell culture dishes were included within approximately 1-h, although some outliers were apparent (Fig. 4C,D).


Rigid Cooperation of Per1 and Per2 proteins
Establishment of the ES cell differentiation system to detect precise period length.(A) Schematic diagram of ES cell differentiation system. (B) Real-time monitoring of luminescence of Per2::Luc KI/KI ES cells on day 20 with photomultiplier tube; cpm, counts per min. (C) Histogram and (D) dot plot of results of autocorrelation analysis of period length using Per2::Luc KI/KI ES cells. Values in (C) are mean % of total samples of six independent experiments. Values in (D) represent individual samples in six independent experiments. GMEM, Glasgow Minimum Essential Medium; KSR, KnockOut Serum Replacement; FBS, foetal bovine serum; 2i, 2 inhibitors (CHIR99021 and PD0325901); LIF, leukaemia inhibitory factor; DMEM, Dulbecco’s Modified Eagle Medium; NEAA, Non-Essential Amino Acids; L-Glu: L-glutamine. β-ME: β-mercaptoethanol; RA, retinoic acid. Each line and dot indicates each 35-mm dish sample.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Establishment of the ES cell differentiation system to detect precise period length.(A) Schematic diagram of ES cell differentiation system. (B) Real-time monitoring of luminescence of Per2::Luc KI/KI ES cells on day 20 with photomultiplier tube; cpm, counts per min. (C) Histogram and (D) dot plot of results of autocorrelation analysis of period length using Per2::Luc KI/KI ES cells. Values in (C) are mean % of total samples of six independent experiments. Values in (D) represent individual samples in six independent experiments. GMEM, Glasgow Minimum Essential Medium; KSR, KnockOut Serum Replacement; FBS, foetal bovine serum; 2i, 2 inhibitors (CHIR99021 and PD0325901); LIF, leukaemia inhibitory factor; DMEM, Dulbecco’s Modified Eagle Medium; NEAA, Non-Essential Amino Acids; L-Glu: L-glutamine. β-ME: β-mercaptoethanol; RA, retinoic acid. Each line and dot indicates each 35-mm dish sample.
Mentions: First, we focused on the fact that ES cells do not have a circadian rhythm, but circadian oscillation appears with retinoic acid (RA)-induced differentiation40, and we can see circadian oscillation in ES cells of any genotype without generating an organism. We performed a broad-parameter search to produce sustained oscillation with high amplitude to detect precise period length. Finally, we improved the ES differentiation system40 (Fig. 4A) and succeeded in detecting 10 peaks of oscillation in Per2::luciferase knockin (KI)/KI ES cells (Fig. 4B)41. We also used this protocol in several independent experiments and observed reproducible, precise period length, that is, most of the cell culture dishes were included within approximately 1-h, although some outliers were apparent (Fig. 4C,D).

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