Mathematical Study on the Mystery of Circadian Rhythms Temperature Effect on Clock in Cultured Cells

iTHEMS-iCeMS joint workshop 2018.7.4 (Wednesday) 1600-1620@201 Maskawa Buil, Kyoto-U

Mathematical study on the mystery of circadian rhythms Temperature effect on clock in cultured cells

37 °C was 3.3 (Fig. 2). Comparing the Q10 values over the temperature range of 33–37 °C, in which reliable data were available for both circadian rhythms and the

cell cycle, the Q10 values were 0.88 for circadian rhythms Gen Kurosawa and 2.7 for the cell cycle. These results indicate that circadian rhythms are strongly temperature-compensated in cultured ﬁbroblasts and that temperature compensa- Theoretical Biology Lab, RIKEN tion is among inherent properties of the interlocked iTHEMS, RIKEN feedback loop of the core clock system. Accumulation speed of mPER proteins in vitro is dependent on ambient temperature It is assumed that the period length of the core feedback loop is regulated by various processes such as accumula- tions, phosphorylations, and the timing of nuclear trans- Figure 2 Period Tsuchiya,..Nishidaand frequency estimates of circadian(2003) rhythm and locations of core clock proteins including mPERs. To cell cycle of NIH3T3 ﬁbroblasts. The mean period and frequency examine the temperature effect on the accumulation of circadian rhythm (᭹) and cell cycle (᭡) were calculated for each speed of mPER proteins, Myc-tagged mPer1, mPer2, and treatment group. The value of each gene in the treatment group mPer3 were expressed with hCKIε in COS7 cells, and is plotted as an open circle. cells were transferred to different temperature conditions as 33 °C, 37 °C or 42 °C. CKIε is thought to be an essential component of circadian rhythms (Lowrey et al. circadian gene expressions are able to oscillate in cultured 2000) and play a role in changing the subcellular locali- cells even when the cell cycle is arrested (Balsalobre zation and stability of mPER proteins by phosphorylat- et al. 1998). We made a graph showing the relationship ing them (Vielhaber et al. 2000; Keesler et al. 2000; between ambient temperature and the period lengths of Takano et al. 2000; Akashi et al. 2002). Whole-cell lysate oscillations of gene expressions shown in Fig. 1 (Fig. 2). was extracted at each time point and immunoblotted

The temperature coefﬁcient, Q10, over the temperature with anti-Myc antibody (Fig. 3). Compared with 37 °C range of 33–42 °C was 0.88. In contrast, the period control state, the accumulation speed of mPER proteins length of the cell cycle was dependent on ambient was slower at 33 °C and rather faster at 42 °C. Although temperature; the Q10 over the temperature range of 30− the degradation speed of mPER proteins also increased

Figure 3 The effect of temperature on the accumulation speed of mPER proteins. COS7 cells were transfected with 0.7 mg of Myc-tagged mPer and 0.3 mg of CKIε per 35 mm dish (t = 0). 3 h after trans- fection, the medium was exchanged for DMEM supplemented with 10% foetal calf serum and the cells were exposed to 33 °C, 37 °C or 42 °C. Protein extracts prepared at indicated time points were subjected to immunoblotting with anti- Myc antibody (A-14). Shifted bands are phosphorylated form of mPER proteins.

Human (~24.5h period) Sleep>wake*cycle awake asleep Consecu(ve*days*in*cave

Time*of*day*(h) Palmer*(2002) --

(A movie of synchronized gene expression rhythms in brain)

Yamaguchi,.., Okamura (2003) Science 302, 1408-12 sponding stochastic version (28). All of these models produce PER CRY sustained rhythms with 24-hour periodicity but are deficient in A several important respects. First, it is now known that circadian phenotypes in the SCN and CLK BMAL1 at the behavioral levels are not always cell-autonomous. The SCN E-BOX Per1, Per2, Rev-erbα in vivo is a network of neurons that are synchronized through cell–cell communication mediating intercellular coupling, and con- sequently, the SCN ensemble is robustly rhythmic (13, 14). How- REV-ERBα ever, dispersed SCN neurons or peripheral tissues/cells lack func- RORc tional intercellular coupling and thus display independently phased, RORE Clk, Bmal1 cell-autonomous rhythms (14, 29, 30). Compared with coupled oscillators in the SCN, there is considerably more dispersion in the PER CRY period lengths of cell-autonomous oscillators and an increase in REV-ERBα cycle-to-cycle variability. Even more significantly, in mutants, oscillations of the SCN clock and its regulated behavior are not RORc CLK BMAL1 necessarily cell-autonomous. The effect of knockout mutations on RORE E-BOX Cry1, Cry2, Rorc A mechanism for robust circadian timekeeping phenotype can be radically different depending on whether one 21 variable-model JK Kim and DB Forger assesses at the cellular level or at the SCN tissue or organismal 180 variable model levels. For example, SCN explants of both Per1 and Cry1 knockouts B A retain rhythmicity, whereas dispersed SCN neurons of both knockout types (cell-autonomous) are largely arrhythmic (29). Thus, Per1 Per2 Cry1 Cry2 intercellular coupling confers robustness to the SCN clock network 2 2 Per1 Per2 Cry1 Cry2 2 2 2 2 but can mask cell-autonomous circadian phenotypes. In this con- CLK/BMAL1 mRNA mRNA mRNA mRNA 2 2 text, previous models were developed in part by fitting to the SCN 2 tissue-level and/or behavioral knockout phenotypes and do a good 2 2 CLK BMAL1 PER1 PER2 CRY1 CRY2 REV-ERB 1 2 2 job at matching these phenotypes. However, because cell- α PER2/CRY1 autonomous and behavioral phenotypes may differ, previous mod- PER2/CRY2 PER1/CRY1 els are sometimes inaccurate as cell-level models. Equally impor- PER1/CRY2 tant, while intercellular coupling comprises a special attribute of the 2 2 2 2 Bmal1 Bmal1 Rorc Rev-erbα Rev-erbα 2 2 2 SCN, the SCN clockwork is similar to oscillators in peripheral mRNA mRNA CLOCK/ Clk Transcription inhibition PER GSK3! tissues at the molecular level. The model developed here makes Clk Rorc NPAS2 mRNA Transcription promotion CRY REV-ERBs exclusive use of cell-level data for both parametric fitting and mRNA Translation Phosphorylation BMAL CKI validation. RORc Second, previous models do not include all essential mo- Simplification lecular components, whereas we developed our model with 8 Fig. 1. Model structure. (A) Gene regulation scheme for the mouse circadian 2012 Kim and Forger genes (Per1, Per2, Cry1, Cry2, Clk, Bmal1, Rev-erb␣,andRorc) clock. Per1, Per2Mirsky,..Kay, and Rev-erb␣ are, Doyle activated at(2009) E-Boxes byPNAS CLK/BMAL1 and and thereby provide a more complete network and offer deactivated when PER/CRY also binds. Clk and Bmal1 are activated by RORc B Molecular Systems Biology greater opportunities for validation. The 73-state model (27) and deactivated by REV-ERB␣ at RORE. Cry1, Cry2, and Rorc have both an E-Box Bmal1/Npas2 and a RORE and are regulated accordingly. (B) Schematic of the mouse Per1 Per2 Cry1 Cry2 Rev erb␣ REV"/! includes , , , ,and - ,butwithCLK circadian clock model incorporating genetic regulation at Per1, Per2, Cry1, BMAL CRY and BMAL1 present only implicitly at constitutively high levels BMAL PERCRY Cry2, Rev-erb␣, Clk, Bmal1, and Rorc, the degradation of mRNAs, the trans- BMAL + BMAL BMAL BMAL CRY CLK CLK and REV-ERB␣ present only at extremely low levels. The lation of mRNAs to protein, the formationNegative and dissociation offeedback PER/CRY and of gene expression CLK CLKPER PER 16-state model (26) contains only Bmal1, Per,andCry (plus CLK/BMAL1, and the degradation of PER1, PER2, CRY1, CRY2, REV-ERB␣, CLK, 2nd NF Core NF (Inactive) Rev-erb␣ in its 19-state form). This model incorporates Rev- BMAL1, and RORc. The loops involving Rev-erb␣ and Rorc are shown in red. is necessary for circadian rhythmsBMAL erb␣ Rorc Clk BMAL but includes neither nor , and it does not Rev-Erb"/! CCLKLK Per1/2, Cry1/2 distinguish between the several types of Per and Cry.Most Figure 1 Schematic of the detailed mammalian circadian clock model. (A) Only some of the relevant species are shown. Circles refer to transcripts and squares are importantly, our model addresses the overlapping but differ- define gene function. The new model must match phase subtletiesproteins, possibly in complex. Small circles refer to phosphorylation states that are color coded by the kinases that perform the phosphorylation. See section ‘Description of the detailed model’ in Supplementary information for details. (B) The detailed model consists of a core negative feedback loop and an additional negative feedback ential functions of CRY1 and CRY2 in the clock mechanism: and predict cell-autonomous knockout phenotypes at the cellularloop (the NNF structure). The repressors (PER1–2 and CRY1–2) inactivate the activators (BMALs and CLOCK/NPAS2) of their own transcription expression through the They antagonistically regulate period length and differentially level. Although a network diagram can be constructed fromcore negative feedback loop. The activators inactivate their own transcription expression by inducing the Rev-erbs through the secondary negative feedback loop. biological data, the kinetics of the individual reactions is insuffi- control rhythm persistence and amplitude (29, 31, 32). of activators (all forms of BMAL–CLOCK/NPAS2 in the a 1–1 stoichiometry, the model shows sustained oscillations Third, previous models have not captured the precise phase ciently characterized to parameterize a model. However, given thenucleus) over a period. Moreover, we specifically refer to with high amplitude (Figure 3B). However, if the stoichiometry phase relationships in the system and an awareness of their funda-repressors and activators of E/E’-boxes when discussing was too high or too low, rhythms are dampened or completely relationships among molecular components of the circadian clock- stoichiometry. We found that mutations that caused the absent (Figure 3B). This matches a recent experimental study work revealed in recent experimental work at the intracellular level, mental importance to the correct operation of the clock, it isstoichiometry to be too high or too low, yielded arrhythmic showing that the amplitude and sustainability of population possible to use iterative computation, with an evolutionary strategy,phenotypes (Figure 3A). So long as the mutations allowed the rhythms increase when the level of PER–CRY is increased which reflect the complex and often combinatorial regulation of the stoichiometry to be around a 1–1 ratio, relatively high closer to that of BMAL1–CLOCK in mouse fibroblasts (Lee circadian genes (16, 20). Specifically, Rev-erb␣ mRNA leads Per1 to develop a parameter set (33). This approach was used previouslyamplitude oscillations were seen. Thus, we predict that et al, 2011). stoichiometry provides a unifying principle to determine the We defined the stoichiometry as the average ratio between and Per2 mRNAs by 4 hours; Per1 and Per2 mRNAs lead Cry1, to good effect in the development of a model for the circadian clockrhythmicity of mutations of the mammalian circadian the total concentrations of repressors to that of activators Cry2,andRorc mRNAs by 4 hours; Cry1, Cry2,andRorc mRNAs in Arabidopsis (22). The model can then be tested by using it toclock. Tofurther test this principle, we constitutively expressed over a period. However, recent work has shown that CRY1 either the Per2 gene (the dominant repressor gene) or the has stronger repressor activity than CRY2. The underlying lead Clk and Bmal1 mRNAs by 8 hours; and Clk and Bmal1 predict a host of additional behaviors for which experimental dataBmal and Clock genes (the dominant activator genes) biochemical mechanisms for this result have not been fully mRNAs lead Rev-erb␣ mRNA by 8 hours. Correct operation of the exist, including the ability of the system to retain rhythmicity whenat different levels. Interestingly, within a range centered near identified (Khan et al, 2012). If the difference is due to a circadian system is in many respects a matter of correct phasing one or more genes are removed, as well as the resulting changes in& 2012 EMBO and Macmillan Publishers Limited Molecular Systems Biology 2012 3 (10). Although the constituents of the prior models are correctly expression levels of clock components. phased in a general sense, they do not incorporate the recently revealed subtleties because of combinatorial gene regulation. The Results 73-state model (27) places all components approximately in-phase, Model Development. The gene regulatory scheme for the mouse whereas the 16- and 19-state models (26) have Per and Cry in-phase circadian clock, depicted in Fig. 1A,wasusedtoconstructthe and Bmal1 in anti-phase. network diagram shown in Fig. 1B.Thisnetworkwasthentrans- Thus, the need arises for a new mammalian circadian model that lated into a system of 21 ordinary differential equations [supporting takes into consideration the complex transcriptional regulatory information (SI) Appendix]containing132(unknown)parameters. network and cell-autonomous clock phenotypes that more precisely Michaelis–Menten kinetics was assumed for transcriptional rates,

11108 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0904837106 Mirsky et al.

Takahashi (2017) Nat Rev Genet

Based on experimental knowledge, we theoretically study fundamental questions in circadian rhythms. We make testable predictions from model.

Mechanism of temperature compensation in circadian rhythms Kurosawa, Fujioka, Koinuma, Mochizuki, Shigeyoshi (2017) PLOS Computational Biology

Regulation of circadian rhythms by RNA methylation Fustin,.., Gibo, Kurosawa,.., Okamura (2018) PNAS

Temperature effect on clock in cultured cells (Ex) Mammalian NIH3T3 cells

37 °C was 3.3 (Fig. 2). Comparing the Q10 values over the temperature range of 33–37 °C, in which reliable data were available for both circadian rhythms and the

cell cycle, the Q10 values were 0.88 for circadian rhythms and 2.7 for the cell cycle. These results indicate that circadian rhythms are strongly temperature-compensated in cultured ﬁbroblasts and that temperature compensation is among inherent properties of the interlocked Circadian feedback loop of the core clock system.

Cell cycle Accumulation speed of mPER proteins in vitro is dependent on ambient temperature It is assumed that the period length of the core feedback loop is regulated by various processes such as accumula- tions, phosphorylations, and the timing of nuclear trans- Akin (2011) Figure 2 Period and frequencyTsuchiya,..Nishida estimates of circadian(2003) rhythm and locations of core clock proteins including mPERs. To cell cycle of NIH3T3 ﬁbroblasts. The mean period and frequency examine the temperature effect on the accumulation ..of circadian rhythm (᭹) and cell cycle (᭡) were calculated for each speed of mPER proteins, Myc-tagged mPer1, mPer2, and ...treatment group. The value of each gene in the treatment group mPer3 were expressed with hCKIε in COS7 cells, and is plotted as an open circle. cells were transferred to different temperature conditions as 33 °C, 37 °C or 42 °C. CKIε is thought to be an essential component of circadian rhythms (Lowrey et al. circadian gene expressions are able to oscillate in cultured 2000) and play a role in changing the subcellular locali- cells even when the cell cycle is arrested (Balsalobre zation and stability of mPER proteins by phosphorylat- et al. 1998). We made a graph showing the relationship ing them (Vielhaber et al. 2000; Keesler et al. 2000; between ambient temperature and the period lengths of Takano et al. 2000; Akashi et al. 2002). Whole-cell lysate oscillations of gene expressions shown in Fig. 1 (Fig. 2). was extracted at each time point and immunoblotted

© Blackwell Publishing Limited Genes to Cells (2003) 8, 713–720 715 sponding stochastic version (28). All of these models produce PER CRY sustained rhythms with 24-hour periodicity but are deficient in A several important respects. First, it is now known that circadian phenotypes in the SCN and CLK BMAL1 at the behavioral levels are not always cell-autonomous. The SCN E-BOX Per1, Per2, Rev-erbα in vivo is a network of neurons that are synchronized through cell–cell communication mediating intercellular coupling, and con- sequently, the SCN ensemble is robustly rhythmic (13, 14). How- REV-ERBα ever, dispersed SCN neurons or peripheral tissues/cells lack func- RORc tional intercellular coupling and thus display independently phased, RORE Clk, Bmal1 cell-autonomous rhythms (14, 29, 30). Compared with coupled oscillators in the SCN, there is considerably more dispersion in the PER CRY period lengths of cell-autonomous oscillators and an increase in REV-ERBα cycle-to-cycle variability. Even more significantly, in mutants, oscillations of the SCN clock and its regulated behavior are not RORc CLK BMAL1 necessarily cell-autonomous. The effect of knockout mutations on RORE E-BOX Cry1, Cry2, Rorc A mechanism for robust circadian timekeeping phenotype can be radically different depending on whether one 21 variable-model JK Kim and DB Forger assesses at the cellular level or at the SCN tissue or organismal 180 variable model levels. For example, SCN explants of both Per1 and Cry1 knockouts B A retain rhythmicity, whereas dispersed SCN neurons of both knockout types (cell-autonomous) are largely arrhythmic (29). Thus, Per1 Per2 Cry1 Cry2 intercellular coupling confers robustness to the SCN clock network 2 2 Per1 Per2 Cry1 Cry2 2 2 2 2 but can mask cell-autonomous circadian phenotypes. In this con- CLK/BMAL1 mRNA mRNA mRNA mRNA 2 2 text, previous models were developed in part by fitting to the SCN 2 tissue-level and/or behavioral knockout phenotypes and do a good 2 2 CLK BMAL1 PER1 PER2 CRY1 CRY2 REV-ERB 1 2 2 job at matching these phenotypes. However, because cell- α PER2/CRY1 autonomous and behavioral phenotypes may differ, previous mod- PER2/CRY2 PER1/CRY1 els are sometimes inaccurate as cell-level models. Equally impor- PER1/CRY2 tant, while intercellular coupling comprises a special attribute of the 2 2 2 2 Bmal1 Bmal1 Rorc Rev-erbα Rev-erbα 2 2 2 SCN, the SCN clockwork is similar to oscillators in peripheral mRNA mRNA CLOCK/ Clk Transcription inhibition PER GSK3! tissues at the molecular level. The model developed here makes Clk Rorc NPAS2 mRNA Transcription promotion CRY REV-ERBs exclusive use of cell-level data for both parametric fitting and mRNA Translation Phosphorylation BMAL CKI validation. RORc Second, previous models do not include all essential mo- Simplification lecular components, whereas we developed our model with 8 Fig. 1. Model structure. (A) Gene regulation scheme for the mouse circadian 2012 Kim and Forger genes (Per1, Per2, Cry1, Cry2, Clk, Bmal1, Rev-erb␣,andRorc) clock. Per1, Per2Mirsky,..Kay, and Rev-erb␣ are, Doyle activated at(2009) E-Boxes byPNAS CLK/BMAL1 and and thereby provide a more complete network and offer deactivated when PER/CRY also binds. Clk and Bmal1 are activated by RORc B Molecular Systems Biology greater opportunities for validation. The 73-state model (27) and deactivated by REV-ERB␣ at RORE. Cry1, Cry2, and Rorc have both an E-Box Bmal1/Npas2 and a RORE and are regulated accordingly. (B) Schematic of the mouse Per1 Per2 Cry1 Cry2 Rev erb␣ REV"/! includes , , , ,and - ,butwithCLK circadian clock model incorporating genetic regulation at Per1, Per2, Cry1, BMAL CRY and BMAL1 present only implicitly at constitutively high levels BMAL PERCRY Cry2, Rev-erb␣, Clk, Bmal1, and Rorc, the degradation of mRNAs, the trans- BMAL + BMAL BMAL BMAL CRY CLK CLK and REV-ERB␣ present only at extremely low levels. The lation of mRNAs to protein, the formationNegative and dissociation offeedback PER/CRY and of gene expression CLK CLKPER PER 16-state model (26) contains only Bmal1, Per,andCry (plus CLK/BMAL1, and the degradation of PER1, PER2, CRY1, CRY2, REV-ERB␣, CLK, 2nd NF Core NF (Inactive) Rev-erb␣ in its 19-state form). This model incorporates Rev- BMAL1, and RORc. The loops involving Rev-erb␣ and Rorc are shown in red. is necessary for circadian rhythmsBMAL erb␣ Rorc Clk BMAL but includes neither nor , and it does not Rev-Erb"/! CCLKLK Per1/2, Cry1/2 distinguish between the several types of Per and Cry.Most Figure 1 Schematic of the detailed mammalian circadian clock model. (A) Only some of the relevant species are shown. Circles refer to transcripts and squares are importantly, our model addresses the overlapping but differ- define gene function. The new model must match phase subtletiesproteins, possibly in complex. Small circles refer to phosphorylation states that are color coded by the kinases that perform the phosphorylation. See section ‘Description of the detailed model’ in Supplementary information for details. (B) The detailed model consists of a core negative feedback loop and an additional negative feedback ential functions of CRY1 and CRY2 in the clock mechanism: and predict cell-autonomous knockout phenotypes at the cellularloop (the NNF structure). The repressors (PER1–2 and CRY1–2) inactivate the activators (BMALs and CLOCK/NPAS2) of their own transcription expression through the They antagonistically regulate period length and differentially level. Although a network diagram can be constructed fromcore negative feedback loop. The activators inactivate their own transcription expression by inducing the Rev-erbs through the secondary negative feedback loop. biological data, the kinetics of the individual reactions is insuffi- control rhythm persistence and amplitude (29, 31, 32). of activators (all forms of BMAL–CLOCK/NPAS2 in the a 1–1 stoichiometry, the model shows sustained oscillations Third, previous models have not captured the precise phase ciently characterized to parameterize a model. However, given thenucleus) over a period. Moreover, we specifically refer to with high amplitude (Figure 3B). However, if the stoichiometry phase relationships in the system and an awareness of their funda-repressors and activators of E/E’-boxes when discussing was too high or too low, rhythms are dampened or completely relationships among molecular components of the circadian clock- stoichiometry. We found that mutations that caused the absent (Figure 3B). This matches a recent experimental study work revealed in recent experimental work at the intracellular level, mental importance to the correct operation of the clock, it isstoichiometry to be too high or too low, yielded arrhythmic showing that the amplitude and sustainability of population possible to use iterative computation, with an evolutionary strategy,phenotypes (Figure 3A). So long as the mutations allowed the rhythms increase when the level of PER–CRY is increased which reflect the complex and often combinatorial regulation of the stoichiometry to be around a 1–1 ratio, relatively high closer to that of BMAL1–CLOCK in mouse fibroblasts (Lee circadian genes (16, 20). Specifically, Rev-erb␣ mRNA leads Per1 to develop a parameter set (33). This approach was used previouslyamplitude oscillations were seen. Thus, we predict that et al, 2011). stoichiometry provides a unifying principle to determine the We defined the stoichiometry as the average ratio between and Per2 mRNAs by 4 hours; Per1 and Per2 mRNAs lead Cry1, to good effect in the development of a model for the circadian clockrhythmicity of mutations of the mammalian circadian the total concentrations of repressors to that of activators Cry2,andRorc mRNAs by 4 hours; Cry1, Cry2,andRorc mRNAs in Arabidopsis (22). The model can then be tested by using it toclock. Tofurther test this principle, we constitutively expressed over a period. However, recent work has shown that CRY1 either the Per2 gene (the dominant repressor gene) or the has stronger repressor activity than CRY2. The underlying lead Clk and Bmal1 mRNAs by 8 hours; and Clk and Bmal1 predict a host of additional behaviors for which experimental dataBmal and Clock genes (the dominant activator genes) biochemical mechanisms for this result have not been fully mRNAs lead Rev-erb␣ mRNA by 8 hours. Correct operation of the exist, including the ability of the system to retain rhythmicity whenat different levels. Interestingly, within a range centered near identified (Khan et al, 2012). If the difference is due to a circadian system is in many respects a matter of correct phasing one or more genes are removed, as well as the resulting changes in& 2012 EMBO and Macmillan Publishers Limited Molecular Systems Biology 2012 3 (10). Although the constituents of the prior models are correctly expression levels of clock components. phased in a general sense, they do not incorporate the recently revealed subtleties because of combinatorial gene regulation. The Results 73-state model (27) places all components approximately in-phase, Model Development. The gene regulatory scheme for the mouse whereas the 16- and 19-state models (26) have Per and Cry in-phase circadian clock, depicted in Fig. 1A,wasusedtoconstructthe and Bmal1 in anti-phase. network diagram shown in Fig. 1B.Thisnetworkwasthentrans- Thus, the need arises for a new mammalian circadian model that lated into a system of 21 ordinary differential equations [supporting takes into consideration the complex transcriptional regulatory information (SI) Appendix]containing132(unknown)parameters. network and cell-autonomous clock phenotypes that more precisely Michaelis–Menten kinetics was assumed for transcriptional rates,

11108 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0904837106 Mirsky et al. AAC3CA833?DC

ModelModel acZvator( dX ! vX γ $γ 1 ac$vator! dX k vX change( ε = #k + & − aX degradaCon degradation change! ε dt = # K γ X γ & h Y− aX ex.(mRNA " vn+ % γ+ gs ex.!mRNA dt h +Ygene0(v'+ Xgene0( a d acZvaZon! inacZvaZon! v X Y inhibitor( dY sX α change( = α − dY inhibitor! α α ex.proteindYdt KgX+ X k change! s dY = α − ex.proteindt gtranslaZon'+ X degradaZon KurosawaKurosawa,.. et al (2017)Shigeyoshi! PLOSubmi%ed Comp Biol Question:QuesZon:(Suppose(that(reacZon(becomes(faster(with(temperature.( Suppose that reaction becomes faster with temperature. (( " dk da dv ds dd % " dk$i.e. ≡dsa, ≡ s d, v ≡ s , ds≡ s , ≡dsd and s ,s ,s ,s ,s > 0' % dT k dT a dT v dT s dT d k a v s d ((((((((((((((((((($i.e. # ≡ sk, ≡ sa, ≡ sv, ≡ ss, ≡ sd and sk, sa, s&v, ss, sd > 0' # dT dT dT dT dT & When(period(can(be(maintained(as(temperature(increases?( When period can be maintained as temperature increases? -- --- -- --- When#period#is#stable,#amplitude#increases#as#temperature#increases. For#many#cases,#period#tends#to#decrease#as#reac.on#speed#increases. mRNA''[nM] mRNA''[nM] 10 10 0 0 0 0 20 20 - 5 5 Time'[days] Time'[days] 25 25 - 10 10 30 30 15 15

Model γ γ γ k+v -h ε dX dt = k + vX K + X h +Y aKS Y nullcline ( ( V )) ( ) ) Y A D −aX 8 α α α dY dt = sX (K S + X ) − dY “slow”

4 X nullcline C k -h aKS B

Abundance of protein ( “fast”

0 KSk 1 2 KS(k+v) k+v k Abundance of mRNA (X) By assuming that time scale of mRNA dynamics and protein dynamics is different (i.e. e very small), we can derive approximated form of period: ! $ ! ! $$ Approximated 1 log"(s + dh − dk (aKS )) (s + dh − d (k + v) (aKS ))% # & = # & # period & d # ! $ & " % "+log"((k + v) (aKS ) − h) (k (aKS ) − h)% %

FigureFigure 4 4 AA BB k+vk+v-h h aKaKS S - ) ) A’ D’ D’(A’)D’(A’) CycleCycle at at high high T T

A’ D’ ) ) Y Y Y 88AA DD Y 88 D D CycleCycle at at high high T T (A)(A) (τ (~24h)τ~24h) CycleCycle at at CycleCycle at at low low T T lowlow T T (τ (~24h)τ~24h) B B (C)(C) 44 CC 44 BB k k-h h C’C’ aKaKS S - B’B’ B’(C’)B’(C’) Abundance of protein ( Abundance of protein ( Abundance of protein ( Abundance of protein ( KSKkSk KSK(k+v)S(k+v) 00 20 40 60 80 00k+vk+v 11 22 k k 20 40 60 80 AbundanceAbundance of of mRNA mRNA ( X(X) ) TimeTime (h) (h) CC for biologically plausible DparameterD condi7on (ds/dT>dd/dT) 1616We prove ( ): ) 8) 8 Y Y “it is impossible to maintain4040 stable period if Y Y8 8 4 4 4848 protein ( maximums of variables are smaller at higher temperatureprotein ( and, Abundance of Abundance of 0 0 0 0 2 2 4 4 0 0 minimumsv v of variables are larger at higher temperature”.2020 30 30 Period (h) Period (h) TemperatureTemperature

20 Period (h) 20 Period (h) 2424

00 00 00 22 44 1010 2020 3030 40 40 PositivePositive feedback feedback strength strength ( v()v) TemperatureTemperature (°C) (°C) Figure 4 A B k+v h aKS - ) A’ D’ D’(A’) Cycle at high T ) Y 8 A D Y 8 D Cycle at high T (A) (τ~24h) Cycle at Cycle at low T low T (τ~24h) B (C) 4 C 4 B k h C’ aKS - B’ B’(C’) Abundance of protein ( Abundance of protein ( KSk KS(k+v) 0 0 k+v 1 2 k 20 40 60 80 Abundance of mRNA (X) Time (h) C D 16

) 8 Y 40 Y 8 4 48 protein ( Abundance of 0 0 2 4 0 v 20 30 Period (h) Temperature

20 Period (h) 24

0 0 0 2 4 10 20 30 40 Positive feedback strength (v) Temperature (°C) High temperature dependency of amplitude should be observed at daily rhythms. (We call it as “temperature-amplitude coupling”.) --(-1 -)(1- 1)---()( By courtesyFigure of 1 Prof. Yasufumi Shigeyoshi, Kindai University (Kurosawa et al (2017) PLOS Comp Biol ) A rBmal1 rCry1 rDbp rE4bp4 600 200 200 200 38℃ 400 100 100 200 100 35℃ 0 0 0 0 32 48 64 80 96 32 48 64 80 96 32 48 64 80 96 32 48 64 80 96 rPer2 rPer3 rRev-erb B 400 200 200 1000 mRNA 200

mRNA abundance (% to time 0) 100 100 500 Bmal1 r 0 0 0 32 48 64 80 96 32 48 64 80 96 32 48 64 80 96 0 100 200 Time (h) rCry1 mRNA C rBmal1 rCry1 rDbp rE4bp4 rPer2 rPer3 rRev-erb

10 10 10 10 10 10 10 Q = 1.35 Q = 1.18 Q = 0.99 40 Q = 1.20 Q = 1.15 Q = 1.25 Q = 1.07 Temperature40 40 -amplitude40 coupling was40 verified40 experimentally.40 20 20 20 20 20 20 Period (h) 20

0 0 0 0 0 0 0 35 38 35 38 35 38 35 38 35 38 35 38 35 38 Temperature (°C)

D rBmal1 rCry1 rDbp rE4bp4 rPer2 rPer3 rRev-erb Q10 = 2.20 Q10 = 1.30 Q10 = 4.07 Q10 = 3.34 Q10 = 1.67 2 2 2 2 2 2 2

1 1 1 1 1 1 1 Amplitude

Q10 = 10.57 Q10 = 7.92 0 0 0 0 0 0 0 35 38 35 38 35 38 35 38 35 38 35 38 35 38 Temperature (°C)

We theoretically showed that higher amplitude at higher temperature can stabilize the period to temperature (temperature-amplitude coupling).

Our collaborators verified it experimentally.

Mechanism of temperature compensation in circadian rhythms Kurosawa, Fujioka, Koinuma, Mochizuki, Shigeyoshi (2017) PLOS Computational Biology

Regulation of circadian rhythms by RNA methylation Fus>n,.., Gibo, Kurosawa,.., Okamura (2018) under review 1 Per1 mRNA level daytime feeding nighttime feeding Time [hours] (Damiola et al., Genes & Dev., 2000)

How does food and/or metabolic state regulate the clock?

19 REVIEWS a conserved GAC[U>A] motif; thus, the occupancy of S. cerevisiae Mrb1 YTH domain YTHDF2 resembles the distribution pattern of m6A on mRNA. Notably, the knockdown of YTHDF2 led to S. pombe Mmi1 YTH domain decreased half-lives of these RNA targets but had minor Human YTHDF1 P/Q/N-rich YTH domain effects on the mRNA levels in the actively translating pool. Ribosome profiling further suggests that YTHDF2 Human YTHDF2RNA CH3P/Q/N-rich YTH domain alters ribosome occupancy of its mRNA targets. These Human YTHDF3 P/Q/N-rich YTH domain results suggest that YTHDF2 has a role in RNA decay. (destabilized) Fluorescence immunostaining of YTHDF2 and fluorescence in situ hybridization of its cognate mRNA revealed b 600 that YTHDF2 binds to m6A through the C-terminal RNA methylated Non-m6A YTH domain and localizes the cognate mRNA to 6 500 m A processing bodies (P-bodies) for accelerated degradation through its N-terminal Pro/Gln/Asn-rich domain (FIG. 4). 400 Fu et al. (2014) Nat Rev Genet The exact RNA degradation mechanism needs to be further elucidated; however, YTHDF2 binds to mRNA 300 with shorter poly(A) tails and does not seem to affect the deadenylation process76. 200 Several cytoplasmic mRNA decay pathways are Non-RNA methylated known77–86. The YTHDF2-mediated mRNA degrada- eieeAs 100 tion, which affects thousands of mRNA molecules, is a unique process that is dependent on the methylation Number of different mRNAs different of Number 0 of the target mRNA and could therefore be reversibly 0 3 6 9 12 15 18 tuned through m6A methylation and demethylation. mRNA lifetime (hours) mRNA lifetime (h) This discovery, together with the negative correlation of m6A with mRNA stability in general as revealed by “RNA methylation may control gene-protein dynamics by knockdownRNA destabilization.” of methyltransferases40, suggests one main c Translation function of m6A on RNA: the regulated degradation of methylated RNA. This process is mediated through ? P/Q/N- selective m6A recognition and subsequent relocaliza- rich Me tion by a reader or effector protein. The control of the YTHDF2 YTH stability of the non-translating pool of mRNA (or other m7Gppp A n Me RNA species) through the YTHDF2-dependent mecha- RNA 7 Transcription m Gppp An nism could be important under various circumstances 77 ? for the selective elimination of a group of RNAs . Interestingly, Mmi1 — the homologue of YTHDF pro- DNA teins in Schizosaccharomyces pombe (FIG. 4a) — is essen- Localization Degradation tial for the elimination of meiosis-specific transcripts (strorage or transport) during meiosis87. However, the presence of m6A has not been reported in S. pombe, which lacks homologues of Figure 4 | Functions of the reader (that is, effector) proteins of m6A. a | The METTL3 and METTL14. The potential presence of m6A characterized YTHDF proteins serve as N6-methyladenosine (mNature6A) ‘readers’. Reviews Human | Genetics in mRNA and its functional roles in S. pombe should be YTHDF1–3 proteins contain a carboxy-terminal YTH RNA-binding domain and an further investigated. i-ei-iceieipeiiscseeieissi yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae. hnRNPs could be potential nuclear m6 A readers. Besides b | The m6A modification is enriched in mRNAs with shorter half-lives in general, which supports the proposed main role of m6A in regulating mRNA stability. c | The the YTH domain family of proteins and other cytoplas- m6A-specific RNA-binding proteins are engaged in post-transcriptional regulation mic mRNA-binding proteins, pulldown experiments of gene expression. YTHDF2 regulates the methylation (me)-dependent RNA have also identified proteins of the heterogeneous degradation. Other reader proteins may exist and affect RNA splicing, storage, nuclear ribonucleoprotein (hnRNP) type as potential trafficking and translation. Data in part bces m6A-selective binding proteins17. Known to form ribonucleoprotein granules that could affect mRNA localization and transport, hnRNPs could also block binding of contain adenine, hm6A or f 6A, or that have m6A in non- splicing factors and affect alternative splicing. Additional Ribosome profiling Qualitative and quantitative consensus sequences have decreased binding affinity. experiments are required to investigate connections 6 6 sequencing of the RNA attached The enrichment of m A in RNA immunoprecipitated between hnRNPs and m A. to ribosomes as a signature of with YTHDF1–3 further supports the role of YTHDF genes that are expressed. proteins as m6A-specific RNA-binding proteins. Anti-readers of m6 A and m6 A-derived modifications. RNA immunoprecipitation and PAR–CLIP experi- The presence of the methyl group can also disfavour Processing bodies (P-bodies). Distinct foci in the ments revealed mostly mRNA as targets of YTHDF2, binding of an RNA-binding protein to the modified cytoplasm that are enriched in addition to some lncRNA targets. The binding sites RNA. This mechanism of anti-reading has yet to be with RNA degradation factors. localize around stop codons and at 3ʹUTRs with a observed for m6A. The m6A modification is widely

300 | MAY 2014 | VOLUME 15 www.nature.com/reviews/genetics

SAH CH3

SAM

....

RNA CH3 RNA methylation (destabilized) WT inhibition

Fustin,.., Okamura, Cell, 2013 “How is the period elongated? “

methylated CT4

CT16

(thick segment: exon, thin line: intron) We focused on Ck1d. © 1999© 1999 Nature Nature America America© 1999 Inc. NatureInc. • http://medicine.nature.com • http://medicine.nature.com America Inc. • http://medicine.nature.com ©© 1999 1999 Nature Nature America America Inc. Inc. • http://medicine.nature.com• http://medicine.nature.com ARTICLESARTICLESARTICLES ARTICLESARTICLES

Fig.Fig. 1 1PedigreesPedigrees of three Fig.of three 1FASPS PedigreesFASPS kindreds. kindreds. of threeCircles, Circles, FASPS kindreds. Circles, males;Fig.Fig.males; 1 1squares,Pedigrees squares,Pedigrees females. females.of of males;three three Filled FASPSFilled squares,FASPS symbols, symbols, kindreds. kindreds. females. affected affected Circles, Circles,Filled in- in-symbols, affected individuals;males;males;dividuals; squares, squares,open open symbols,females. females. symbols,dividuals; Filledunaffected Filled unaffected symbols, opensymbols, subjects; symbols, subjects;affected affected sym- unaffected in-sym- in- subjects; sym- bolsdividuals;dividuals;bols with with open central open central symbols, symbols, dots, bols dots, individualsunaffected withunaffected individuals central subjects; of subjects; dots,unknownof unknown sym- individualssym- of unknown phenotype;bolsbolsphenotype; with with diamonds, central central diamonds, dots,phenotype; dots, sibships sibships individuals individuals of diamonds,children of children of of unknownwith unknown sibships with un- un- of children with un- knownphenotype;phenotype;known phenotype phenotype diamonds, diamonds, (numberknown (numbersibships sibships phenotype in of diamond, inof children children diamond, (number with sibship with sibshipun- un- in diamond, sibship size).knownknownsize). Number phenotype Number phenotype at upper at upper (number size). (number left leftof Number symbol, of in in symbol, diamond, diamond, at inpatient upper inpatient sibshipleft sibshippar- of par- symbol, inpatient par- ticipantsize).size).ticipant Number Numberidentifier identifier at at upper(age upper (ageticipant in left years).left in of years).of symbol,identifier symbol, Arrows, Arrows, inpatient (ageinpatient probands. probands.in years).par- par- Arrows, probands. ticipantticipant identifier identifier (age (age in in years). years). Arrows, Arrows, probands. probands.

icalical assessment assessment thatical that they assessment they were were not that not sleep they sleep were not sleep phase-delayed.icalicalphase-delayed. assessment assessment that phase-delayed. that they they were were not not sleep sleep phase-delayed.phase-delayed.ThereThere was was also also a profound aThere profound was qualitative alsoqualitative a profound dif- dif- qualitative dif- ferenceThereferenceThere between was was between also also groups.a ference aprofound groups.profound People between Peoplequalitative qualitative with groups. with con- dif- dif- con- People with con- ventionalferenceferenceventional between between sleep sleep schedules groups. ventional groups. schedules People tend People sleep tend to with schedules with tostay stay con- up con- up tend to stay up laterventionalventionallater and and wake sleep sleepwake up schedules scheduleslateruplater later andwhen when tend wake tend on to on vacation.up to stay vacation.later stay up upwhen on vacation. Inlaterlater In contrast, and contrast,and wake wake FASPS up FASPSupIn later later subjects contrast, subjectswhen when tend on FASPSon tend vacation. vacation. to to subjects fall fall tend to fall asleepInIn asleep contrast, contrast, even even earlier FASPSearlier FASPS asleepand subjectsand subjectsalso even also to wakeearlierto tend tend wake up toand to upear- fall also fallear- to wake up ear- lierasleepasleeplier during duringeven even vacation, earlier earliervacation,lier and and consistentduring also consistentalso to tovacation, wake wakewith with up uptheir ear-consistent theirear- with their substantiallierliersubstantial during during tendencyvacation, vacation, tendencysubstantial consistent towards consistent towards tendency sleep-phase with sleep-phasewith their their towards sleep-phase advancesubstantialsubstantialadvance (data (data tendency tendencynot not shown).advance shown). towards towards (data sleep-phase not sleep-phase shown). 83.48;83.48; % stage % stage 1 sleep, 183.48; sleep, 11.72 11.72% stage ± 3.79 ± 3.791 andsleep, and 12.83 11.72 12.83 ± 4.37;± ± 3.79 4.37; % and rapid % rapid12.83 eye eye± 4.37;We %We studied rapid studied eye one one 69-year-oldWe advance69-year-oldadvance studied (datasubject (data one subject not 69-year-oldnot in shown). ainshown). time a time isolation subject isolation infacility a facility time isolation facility movement83.48;83.48;movement % % stage stage sleep, sleep,1 1sleep, sleep, 20.08movement 20.08 11.72 11.72 ± 3.72 ± ± 3.72± sleep, 3.79 3.79 and andand and 21.0020.08 12.83 21.00 12.83 ± 7.58; 3.72± ±± 4.37; 7.58;4.37; and and % % and rapid 21.00 %rapid slow % eye ±sloweye 7.58;to determineWeto andWe determine studied studied % slow the one onethe endogenous to69-year-old 69-year-oldendogenous determine periodsubject thesubject period endogenous of in inherof a atimeher circadiantime circadian isolationperiod isolation clock. of clock.facility herfacility We circadian We clock. We wavemovementmovementwave sleep, sleep, 10.30 sleep, sleep, 10.30 ±wave 20.08 20.08 7.02 ± 7.02 sleep, ± and ± 3.72 3.72and 10.4410.30 and 10.44 and ±± 21.00 21.00 3.59. 7.02± 3.59. ± andOne ± 7.58; 7.58;One 10.44FASPS and FASPS and ± subject % 3.59. % subject slow slow Onedeterminedto FASPSto determineddetermine determine subject sleep–wake the sleep–wakethe endogenousdetermined endogenous and and temperature sleep–wakeperiod temperatureperiod of of her her data and circadian data circadian during temperature during clock. the clock. the time dataWe timeWe during the time hadwavewavehad evidence sleep, evidencesleep, 10.30 of10.30 moderateof had ±moderate ± 7.02 7.02evidence obstructiveand and obstructive 10.44 10.44of moderate ± sleep± 3.59. 3.59.sleep apnea obstructiveOne Oneapnea andFASPS FASPS and one sleepsubject onesubject con- con-apneaisolationdetermined determinedandisolation one study con- study sleep–wake sleep–wake (Fig. (Fig.isolation 2). 2).Periodograms and Periodograms and study temperature temperature (Fig. showed 2). showed Periodograms data data a very duringa duringvery short short showed the theτ (23.3 time τ time(23.3 a very short τ (23.3 trolhadhadtrol subject evidence evidence subject had ofhad of periodicmoderate moderatetrol periodic subject limb obstructive obstructivelimb hadmovements movements periodic sleep sleep in limbapnea apneasleepin sleepmovements and withand with one onemicro- con-micro- con- in sleephours)isolationisolationhours) with for micro- study forboth study both (Fig.rhythms (Fig. hours)rhythms 2). 2). Periodograms compared Periodogramsfor compared both rhythms with showed with showed those comparedthose a of avery very aof sex- ashort shortwithsex- and τand those τ(23.3age-(23.3 age- of a sex- and age- arousals.troltrolarousals. subject subject None Nonehad had of periodic periodicarousals. theof the multiple limb multiple limb None movements sleepmovements of sleep the latency latency multiple in in sleep test sleep test sleepresults with with results latencymicro- frommicro- from testmatchedhours)hours) matched results for forcontrol fromboth controlboth rhythms subjectrhythmsmatched subject (24.2 compared compared (24.2control hours) hours) subjectwith withand and those with those (24.2 with estimatesof ofhours) aestimates asex- sex- and ofand 24.0 ofwithage- age- 24.0 estimates of 24.0 controlarousals.arousals.control or None FASPSor None FASPS of subjects of control the subjects the multiple multiplewere or wereFASPS indicative sleep indicative sleep subjects latency latency of narcolepsywereof narcolepsy test test indicative results results or other or from of fromother narcolepsytomatchedmatched 24.5to 24.5 hoursor control otherhours control in otherin subject to subjectother 24.5 studies studies(24.2 hours (24.214. hours) 14inhours). other and and studies with with 14estimates .estimates of of 24.0 24.0 14 causecontrolcontrolcause of excessiveorof or FASPSexcessive FASPS subjectsdaytime causesubjects daytime of were sleepiness. excessivewere sleepiness. indicative indicative daytime of of narcolepsy sleepiness. narcolepsy or or other other totoOur 24.5 24.5Our results hours hoursresults define in in defineother other aOur hereditary studiesa studies hereditaryresults14. define.circadian circadian a hereditary rhythm rhythm variant circadian variant in hu-rhythmin hu- variant in hu- causecauseWeWe determinedof of excessivedetermined excessive circadiandaytime daytime Wecircadian determined sleepiness. phasesleepiness. phase using circadianusing plasma plasma phase melatonin melatonin using plasmaand andmans melatoninOurmansOur associated results results associated and define define withmans witha ahereditary a hereditary shortassociated a short endogenous circadian endogenouscircadian with a rhythm short period.rhythm period. endogenous variant Thevariant The clinical in clinicalin hu- hu- period. The clinical http://medicine.nature.com http://medicine.nature.com http://medicine.nature.com • • 13 13 • bodyWebodyWe core determined determinedcore temperature temperature bodycircadian circadian measurements core measurements temperaturephase phase using using13. The measurementsplasma. Theplasma melatonin melatonin melatonin melatonin and. and The tem- and and tem-melatoninhistories,mansmanshistories, and associated associated sleep tem- sleep logs withlogs histories, withand and aambulatory a short ambulatory short sleep endogenous endogenous logs actigraphy and actigraphy ambulatory period. period. patterns patterns The Theactigraphy of clinical FASPS of clinical FASPS patterns of FASPS http://medicine.nature.com http://medicine.nature.com

• 13 • peraturebodybodyperature core core rhythms temperature temperature rhythms wereperature were measurements bothmeasurements both rhythms phase-advanced phase-advanced were13. The. The both melatonin melatonin by phase-advanced by 3–4 3–4 hoursand and hours tem- tem- in in bysubjectshistories, histories, 3–4subjects hours demonstrated sleep sleep demonstrated in logs logssubjects and and ambulatory a ambulatory significant a demonstrated significant actigraphy actigraphy phase phase a significant advancepatterns patterns advance of ofof phaseFASPS FASPS of the the advance of the FASPSperatureperatureFASPS subjects subjects rhythms rhythms compared compared FASPS were were bothsubjects with both with control phase-advanced phase-advanced controlcompared subjects subjects with (Table by bycontrol (Table 3–4 3–4 1). hours 1). hourssubjects in in (Tablesleep–wakesubjectssubjectssleep–wake 1). demonstrated demonstratedrhythm rhythmsleep–wake in normalin anormal a significant significantrhythm life life settings settings in phase phasenormal relative relative advance advancelife not settings not only of ofonly the torelative the to not only to FASPSFASPSTo To eliminate subjects subjects eliminate compared compared the the To possibility possibility eliminate with with control control of the of sleep subjects sleepsubjects possibility deprivation deprivation (Table (Table of 1). 1). sleep or or self- deprivation self-oursleep–wakesleep–wakeour control control or self- rhythm subjects, rhythm subjects,our in butin control normal butnormal also also subjects, tolife life tosleep–wake settings settings sleep–wake but relative also relative schedules schedules to not sleep–wake not only widely only widely to to schedules widely imposedToimposedTo eliminate eliminate unconventional unconventional theimposed the possibility possibility sleep–wake unconventional sleep–wake of of sleep sleep schedules, schedules, deprivation deprivation sleep–wake subjects subjects or schedules, or kept self- self- keptheldour our subjectsheld controlto control beto conventionalbe kept subjects, conventional subjects,held but to butand be also and also toconventional published to to published sleep–wake sleep–wake values and values schedules to schedulesfor published forsleep–wake sleep–wake widely widely values for sleep–wake ‘sleepimposedimposed‘sleep logs’ logs’ unconventional unconventionalat homeat home‘sleep for for 1logs’ week sleep–wake 1 sleep–wake weekat beforehome before schedules, foradmission schedules, admission1 week subjectsto before subjects andto and admissionfor kept kept for2 2schedulesheldheld schedulesto toand to be 8be . for conventionalInpatient 8conventional. 2Inpatientschedules recordings recordings and and8. toInpatient to confirmedpublished published confirmed recordings valuesthe values the early forearly confirmedfor sleep sleep–wake sleep–wake sleep phase phase the early sleep phase 8 weeks‘sleep‘sleepweeks afterlogs’ logs’ after leavingat at leavinghome homeweeks the for thefor Clinical after1 1Clinicalweek week leaving Research before before Research the admission admissionCenter.Clinical Center. There Researchto toThere and andwas forwas forCenter.no 2 no2 andschedules schedulesThereand showed showed was. 8 Inpatient. that noInpatient that theand the melatoninrecordings showedrecordings melatonin that andconfirmed confirmed andthe temperature melatonintemperature the the early early rhythms and rhythmssleep sleep temperature phasewere phase were rhythms were consistentweeksweeksconsistent after after seasonal leaving leaving seasonalconsistent the bias the biasClinical forClinical for date seasonal dateResearch Research of inpatientof bias inpatient Center. Center. for study date There study There of in inpatient was inFASPS was FASPS no no also studyandandalso advanced.showed showed in advanced. FASPS that thatFASPS theFASPSalso the subjectsmelatonin advanced.melatonin subjects tended andtended FASPSand temperatureto temperature subjects fallto fall asleep asleep tended rhythms duringrhythms during to solar fallwere were solar asleep during solar comparedconsistentconsistentcompared with seasonal seasonalwith control controlcompared bias bias subjects. for subjects. for with date date control of of inpatient inpatient subjects. study study in in FASPS FASPS clockalsoalsoclock advanced.times advanced. times that that FASPScorrespond FASPSclock correspond subjects subjectstimes to that thetendedto tended the ‘maintenancecorrespond ‘maintenance to to fall fall asleep toasleep ofthe wakefulnessofduring ‘maintenanceduring wakefulness solar solar of wakefulness 1999 Nature America Inc. 1999 Nature America Inc. 1999 Nature America Inc. 15,16 15,16

15,16 comparedcomparedActivityActivity levelswith with levels control (actigraphy) control (actigraphy)Activity subjects. subjects. levelswere were also(actigraphy) also recorded recorded were during during also the recorded the in- in-zone’clock duringclockzone’ times in times the in conventional thatin- conventionalthat correspond correspondzone’ sleepers in sleepers conventionalto to the the ‘maintenance. ‘maintenance Similarly,. Similarly, sleepers FASPS of of FASPS wakefulness wakefulness. subjects Similarly, subjects FASPS subjects © © © 1999 Nature America Inc.

1999 Nature America Inc. 15,16 15,16 ActivitypatientActivity staylevels levels and (actigraphy) patient(actigraphy) for 3 stay weeks were were and after also foralso subjectsrecorded 3 recorded weeks wentduring after during home. subjects the the in- in- The wentzone’zone’tended home. in in to conventional The conventional waketended up during sleepersto sleepers wake solar up clock. . duringSimilarly, Similarly, times solar that FASPS FASPSclock correspond subjectstimes subjects that to correspond to

© patient stay and for 3 weeks after subjects went home. The tended to wake up during solar clock times that correspond to © phasepatientpatientphase advance stay advance stay and of and self-reportedofphase for for self-reported 3 3 advance weeks weeks sleep after after ofsleep self-reportedtimes subjects subjectstimes in FASPSin went wentFASPS sleep and home. home. andtimes control control The in The FASPSthetendedtendedthe circadianand circadian tocontrol to wake wake peak peakup uptheof during sleepinessofduring circadian sleepiness solar solar inpeak clock conventionalinclock ofconventional timessleepiness times that that sleepers incorrespond sleeperscorrespond conventional15,1615,16. .to to sleepers15,16. 15,16 subjectsphasephasesubjects advance advancewas was consistent ofconsistent of self-reportedsubjects self-reported with withwas ambulatory sleepconsistent sleepambulatory times times actigraphy within in actigraphyFASPS FASPS ambulatory and andand controland controlsleep actigraphysleep thetheTo circadian Toourcircadianand our knowledge, sleep knowledge, peak peak of of Tono sleepiness sleepiness ournowell-characterized well-characterizedknowledge, in in conventional conventional no well-characterizedmonogenic monogenic sleepers sleepers circadian 15,16circadian. . monogenic circadian logsubjectssubjectslog data. data. Bywas was Byall consistent threeallconsistent threelog measures, data. measures, with with By ambulatory all FASPSambulatory threeFASPS patients measures, patients actigraphy actigraphy were wereFASPS sleep andsleep and patients phase- sleep phase-sleep wererhythmTorhythm sleepTo our our phase- variantknowledge, knowledge, variant hasrhythm has no previouslyno well-characterized previously well-characterized variant been has been reported previously monogenic reportedmonogenic in been in humans.circadian circadian humans. reported in humans. advancedloglogadvanced data. data. Byby By all by 3–4all three 3–4three hoursadvanced hoursmeasures, measures, compared compared by FASPS3–4 FASPS with hours patientswith patients control comparedcontrol were were subjects sleepsubjects sleep with phase- (dataphase- control (dataFurthermore,rhythm subjectsrhythmFurthermore, variant(data variant a profoundly a Furthermore, has profoundly has previously previously advanced advanced a profoundly been been sleep–wake reported sleep–wake reported advanced rhythm in in rhythm humans. humans. sleep–wake has has rhythm has notadvancedadvancednot shown). shown). by by The3–4 3–4 The largenothours hours large shown). difference compared compared difference The in with large with Horne-Östbergin Horne-Östberg control differencecontrol subjects subjects in scores Horne-Östberg scores (data (data for forbeenFurthermore,Furthermore,been thought scores thought for toa a profoundly tobe profoundlybeen be exceedingly exceedinglythought advanced advanced to rare be rare in exceedingly sleep–wake sleep–wake inhealthy, healthy, young, rhythm rare rhythm young, in non- healthy, has non- has young, non- FASPSnotnotFASPS shown). shown).patients“Why patients The (77 The (77 FASPS largeCk1d± large 6.7; ± 6.7; difference npatients difference =5) n(Casein =5) and (77and incontrol in ± Horne-Östbergcontrol 6.7; Horne-Östberg Kinase subjectsn =5) subjects and (48.2 scorescontrol 1d)?”(48.2 scores ±4.6; ±4.6; for subjects for depressedbeenbeendepressed (48.2 thought thought adults±4.6; adults to 5. to However, bedepressed 5. be However, exceedingly exceedingly in adults thein the families5 rare . rareHowever, families in in reported healthy, healthy, reported in the here, young, families young, here, there non-there reported non- is is here, there is 5 nFASPS =6)FASPSn =6) ( Ppatients patients=0.006) (P =0.006) (77 (77is n consistent± is =6)± 6.7; consistent 6.7; ( Pn n=0.006)=5) =5) with and withand a is controlphase controlconsistenta phase advance subjects subjectsadvance with of (48.2 a (48.2 thisof phase this ±4.6;large ±4.6; advancelargeadepressed depressed cleara of clear this autosomal adultslarge autosomal adults. 5However,a . dominantHowever, clear dominant autosomal in in the transmission the transmission families families dominant reported reported of of profound transmission profound here, here, there there sleep sleep is of is profound sleep magnitude.n n=6)magnitude. =6) ( P( P=0.006) =0.006) TheFamilial The averageis magnitude.is consistent average consistent Advanced Horne-Östberg Horne-Östberg with The with a average aphase Sleepphase score advance Horne-Östberg scoreadvance Phase of of48.2 of of 48.2Syndrome this this for large for the scorelarge the phase ofa a clearphase 48.2 clear advance, for autosomal advance, autosomal the indicating phaseindicating dominant dominant advance, that that ASPS transmission transmission indicatingASPS in thein the young that of young of profound ASPS profoundis more is inmore the com- sleep sleep com-young is more com- controlmagnitude.magnitude.control subjects subjects The originates The also average control averagealso supports supports Horne-Östbergsubjects from Horne-Östberg our our sleep aalso sleepmutated log,supports log, score actigraphy score actigraphy our ofCk1d of 48.2 sleep 48.2 and. for andlog, for clin- the clin-actigraphy the monphasephasemon than andadvance, thanadvance, previously clin- previously indicating indicatingmon thought. than thought. that thatpreviously ASPS ASPS in in thought.the the young young is is more more com- com- controlcontrol subjects subjects also also supports supports our our sleep sleep log, log, actigraphy actigraphy and and clin- clin- monmon than than previously previouslySeveral thought. thought.Several observations observationsSeveral support support observations the the assertion assertion support the assertion TableTable 1 1PhasePhase markers markersTable of 1 overt ofPhase overt rhythms markersrhythms of overt rhythms (Jones et al (1999) Nat Med, Toh et al. (2001) Science; Xu et al. (2005) Nature) SeveralthatSeveral FASPS observations observations is a geneticthat FASPS support supporttrait is and athe geneticthe not assertion assertion a learned trait and not a learned TableTable 1 1 PhasePhase markers markers of of overt overt rhythms rhythms that FASPS is a genetic trait and not a learned ControlControl FASPS( FASPS(nControl =6)n =6) Difference FASPS( Differencen =6)P value DifferenceP value habit.thatthathabit. FASPS FASPSSiblingsP valueSiblings is is a aingenetic genetic theseinhabit. these trait kindredstraitSiblings kindredsand and not notinwere athesewere alearned learnedoften oftenkindreds were often (ControlnControl =6)(n =6) FASPS(Mean FASPS( Mean ±s.dn(n n=6) =6)±s.d=6) (hours:minutes) Difference(hours:minutes) Difference Mean ±s.d (hours:minutes)P Pvaluevalue widelyhabit.habit.widely Siblings divergentSiblings divergent in in widely forthese these for morning, kindreds morning,kindreds divergent evening were were evening for often morning, often or or evening or Mean(Meann( n =6)± =6) s.d. ± s.d. Mean MeanMean ±s.d ±s.d ± s.d. (hours:minutes) (hours:minutes) conventionalwidelywidelyconventional divergent divergent sleep–wake sleep–wake conventional for for morning, morning, preference; preference; sleep–wake evening evening more- more- or or preference; more- MeanMean ± ± s.d. s.d. conventional sleep–wake preference; more- SleepSleep Onset OnsetSleep Onset 23:10 23:10 ± 0:40 ± 0:40 19:25 19:25 23:10 ± 1:44 ±± 1:44 0:40 3:45 19:25 3:45 ± 1:44 < 0.0005 < 0.0005 3:45over,conventionalover, the < 0.0005 the onset onset sleep–wake ofover, of the the thephenotype phenotype preference; onset of is the often is more- often phenotype is often SleepSleepSleepSleep Offset Onset Onset Offseta a Sleep Offset07:44 23:10a 23:1007:44 ± ± 1:13± 0:40 ± 0:40 1:13 04:18 19:25 19:25 04:1807:44 ± ± 2:00± 1:44 ±± 1:44 2:00 1:13 3:26 04:18 3:45 3:45 3:26 ± 2:00 < < 0.0005 <0.0005 <0.0005 0.0005 3:26afterover,over,after young

transcripZon Ck1dMk(RNA WT

splicing ( stable stablestable Ck1d1Mk1 Ck1d2Mk2 Ck1d1 ox

Me Me unstable unstableunstable Ck1d1Mk1 Ck1d2Mk2 (FusZn,..,Kurosawa,..(submi8ed) translaZon Ck1d2 ox

protein Ck1d1MK1 Ck1d2MK2 Effects of the two splice variants on period are opposite. (,,22,1, 2,2122,) cf. Zhou et al. (2015) Mol Cell

Per2 P h1 h2 h3

b h4 CK1D1,2

RNA methylation

Casein Kinase 1D (CK1D) is known to phosphorylate circadian proteins. CK1D possibly activates six phosphorylation processes, above. We didn’t know which processes are actually acHvated by CK1D1,2.

26 190 variable model

mRNA PER2 p h1 h2 h3 b ? ? h4 ? ? ? CK1D1,2 ? RNA methylation

If CK1D2 ac;vates p.. If CK1D2 activates h3..

consistent inconsistent

protein CTL 0 Time (h) 100 0 Time (h) 100

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shorter -0.1 Fustin,..,Gibo, Kurosawa,..,Okamura 2018 PNAS

Mechanism of temperature compensation in circadian rhythms We predict that larger amplitude at higher temperature. Kurosawa, Fujioka, Koinuma, Mochizuki, Shigeyoshi (2017) PLOS Computational Biology

Regulation of circadian rhythms by RNA methylation We predict the PER2 phosphorylation process (p) which is regulated by RNA methylation via Ck1d2. Fustin,.., Gibo, Kurosawa,.., Okamura (2018) PNAS

Atsushi Mochizuki (RIKEN) Shingo Gibo (RIKEN)

Yasufumi Shigeyoshi Kindai Univ Atsuko Fujioka (Kindai Univ) Satoshi Koinuma (Kindai Univ)

Hitoshi Okamura (Kyoto Univ) Jean-Michel Fustin (Kyoto Univ)

Funding: CREST “Chrono-metabolism” project (PI: Hitoshi Okamura, Kyoto Univ)

Under constant darkness daytime feeding (6:00-18:00) Assuming.. nighttime feeding (18:00-6:00) 20 night day night …

Methionine 10

RNA methylation Per1mRNA level

0 Circadian kinase 4800 490 500 24 (CK1D2) Time [hours]Time (after[Hours] 20 days)