Molecular mechanism of the repressive phase of the mammalian

Xuemei Caoa, Yanyan Yanga, Christopher P. Selbya, Zhenxing Liua, and Aziz Sancara,1

aDepartment of Biochemistry and Biophysics, School of Medicine, University of North Carolina, Chapel Hill, NC 27599-7260

Contributed by Aziz Sancar, November 4, 2020 (sent for review October 12, 2020; reviewed by Ethan Buhr and Roman Kondratov) The mammalian circadian clock consists of a transcription–translation have been shown to play key roles in the clock mechanism feedback loop (TTFL) composed of CLOCK–BMAL1 transcriptional ac- (13–18), the discrepancy between our in vitro and vivo experi- tivators and CRY–PER transcriptional repressors. Previous work ments was due to lack of these kinases in the in vitro reconstituted showed that CRY inhibits CLOCK–BMAL1-activated transcription by system. In support of this view, we found that PER mutants de- a “blocking”-type mechanism and that CRY–PER inhibits CLOCK– fective in casein kinase binding failed to remove the CRY– BMAL1 by a “displacement”-type mechanism. While the mechanism CLOCK–BMAL1 repressor complex in a reporter assay. of CRY-mediated repression was explained by both in vitro and Considering the limitations of reporter gene assays, we wished to in vivo experiments, the CRY–PER-mediated repression in vivo test this model directly in vitro by using CK1δ/CK2 in the seemed in conflict with the in vitro data demonstrating PER CLOCK–BMAL1–E-box binding experiment: Here, we report removes CRY from the CLOCK–BMAL1–E-box complex. Here, we that under appropriate experimental conditions, CK1δ/CK2 phos- show that CRY–PER participates in the displacement-type repression phorylates CLOCK at multiple sites and this phosphorylation is by recruiting CK1δ to the nucleus and mediating an increased local associated with dissociation of CLOCK–BMAL1 from an E-box concentration of CK1δ at CLOCK–BMAL1-bound promoters/enhancers (17). Of special significance, the phosphorylation of CLOCK by and thus promoting the phosphorylation of CLOCK and dissocia- CK1δ/CK2 is PER dependent in vivo but not CRY or PER de- – tion of CLOCK BMAL1 along with CRY from the E-box. Our find- pendent in vitro. This is consistent with the role of CRY–PER ings bring clarity to the role of PER in the dynamic nature of the transporting CK1δ into the nucleus and mediating an increased repressive phase of the TTFL. local concentration of CK1δ at CLOCK–BMAL1-bound promoters/

enhancers in vivo and provides an explanation for our in vitro ob- BIOCHEMISTRY | | circadian clock | casein kinase | servation of CK1δ-dependent but CRY–PER-independent removal DNA binding of CLOCK–BMAL1 from an E-box.

n the canonical model for the mammalian circadian clock, Results ICLOCK and BMAL1 make a heterodimer that activates the CK1δ Is Required for Displacement of BMAL1, CLOCK, and CRY1 from transcription of Cryptochrome (CRY) and Period (PER) , the Nr1d1 E-Box. Nr1d1, which encodes the and the resulting CRY–PER complexes, after a time lag, act on NR1D1 (which is also an important component of the consoli- CLOCK–BMAL1 and inhibit their transcriptional activator dating loop of the circadian clock), is controlled almost exclu- function, thus completing the transcription–translation feedback sively from an E-box in its promoter and thus constitutes a loop (TTFL) that constitutes the core molecular clock (1–7). convenient system for investigating the working of the core clock Previous biochemical work has supported some key points of this (19). To test the suspected contributions of CK1 family kinases model but has also revealed some facts that, at face value, con- tradict the model. First, it was found that CRY (CRY1 and Significance CRY2) directly bind to the CLOCK–BMAL1–E-box complex and inhibit CLOCK–BMAL1-activated transcription even in the The circadian clock mechanism is a daily rhythmic activation of absence of PER proteins (6, 8–10). In addition, while PER circadian gene transcription by the CLOCK–BMAL1 heterodimer (PER1 and PER2) is the primary repressor in the Drosophila and repression by CRYs (CRY1 and CRY2) and PERs (PER1 and TTFL (4), in , in the absence of CRYs, it does not PER2). CRYs and PERs are highly expressed around predawn, stably bind to the CLOCK–BMAL1–E-box complex and it can- when they begin repressing transcription of cry and per and not repress CLOCK–BMAL1 transcriptional activity (6, 11). other clock genes, leading to a nadir of CRYs and PERs in the Instead, in mammals, inhibition of CLOCK–BMAL1 by PER is afternoon. CRY represses by simply binding to the CLOCK– CRY dependent, and in vivo, PER causes the removal of the BMAL1 heterodimer on DNA, and PER represses by removing entire CRY–CLOCK–BMAL1 ensemble from cognate promoters, CLOCK–BMAL1 from DNA in a CRY-dependent manner. This leading to either inhibition or activation of the relevant genes (11, work shows that removal of CLOCK–BMAL1 involves phos- 12), depending on other transcription regulatory elements in the δ ’ phorylation of CLOCK, which is accomplished by CK1 when gene s promoter and enhancer. Thus, two modes of repression in δ – “ ” CRY and PER deliver CK1 to the CLOCK BMAL1 complex in mammals were identified, blocking repression by CRY binding the nucleus. to the CLOCK–BMAL1–E-box complex, and “displacement” re- – pression in which PER removes CLOCK BMAL1 from the E-box Author contributions: X.C., C.P.S., and A.S. designed research; X.C., Y.Y., C.P.S., and Z.L. in a CRY-dependent manner. performed research; X.C., Y.Y., C.P.S., Z.L., and A.S. analyzed data; and X.C., Y.Y., C.P.S., The data explaining blocking-type and displacement-type re- and A.S. wrote the paper. pression were based on the in vitro and in vivo behavior of CRY Reviewers: E.B., University of Washington School of Medicine; and R.K., Cleveland State on CLOCK–BMAL1-controlled genes, and based on the in vivo University. effect of PER on the CRY–CLOCK–BMAL1 complex (11, 12). The authors declare no competing interest. However, the data presented a paradox: In vitro data showed that Published under the PNAS license. PER removed only CRY from the CRY–CLOCK–BMAL1–E-box 1To whom correspondence may be addressed. Email: [email protected]. complex, while in vivo it removed the entire CRY–CLOCK– This article contains supporting information online at https://www.pnas.org/lookup/suppl/ BMAL1 ensemble from E-boxes, with the consequent transcrip- doi:10.1073/pnas.2021174118/-/DCSupplemental. tional inhibition or activation (12). We reasoned that since CK1δ/e Published December 28, 2020.

PNAS 2021 Vol. 118 No. 2 e2021174118 https://doi.org/10.1073/pnas.2021174118 | 1of9 Downloaded by guest on October 6, 2021 on the regulation of Nr1d1 by the core clock, we used the well- decreases PER2 and CLOCK phosphorylation levels but not characterized CK1δ/e inhibitor PF670462 (slightly selective for BMAL1 phosphorylation. CK1δ), and as a control we used PF4800567, which is known to To follow up on the CK inhibition experiments, we con- −/− −/− −/− −/− specifically inhibit CK1e activity (20, 21). We tested these in- structed Per1/2 ; Ck1δ and Per1/2 ; Ck1« cell lines us- hibitors in our “cell biochemical system” based on targeted de- ing CRISPR/Cas9 technology to knock out the Ck1δ and Ck1« livery of the desired (PER2) into the nucleus by the genes (SI Appendix, Fig. S2). Then we tested the effect of 4-OHT on CLOCK–BMAL1 binding to the Nr1d1 E-box in these cell 4-hydroxytamoxifen (4-OHT)/* system. Per1/ − − − − / – 2 / ;PER2–ER* mouse embryo cells (Per1, Per2 double-knockout lines and the control Per1/2 ; PER2 ER* cell line (Fig. 1B). Both BMAL1 and CLOCK dissociate from the Nr1d1 promoter cells expressing PER2–Estrogen Receptor fusion protein) were − − − − upon addition of 4-OHT to Per1/2 / ; PER2–ER* and Per1/2 / ; incubated with DMSO (dimethyl sulfoxide) as a solvent control, or − − Ck1« / ; PER2–ER* cell lines. However, the absence of CK1δ in with PF670462/PF4800567, and then 4-OHT was added to the − − − − the Per1/2 / ; Ck1δ / ; PER2–ER* cell line produced two in- cultures to promote PER–ER* entry into the nucleus. At 0 and 4 h teresting results. First, prior to addition of 4-OHT, there are after addition of 4-OHT, we measured the binding of BMAL1, more CLOCK–BMAL1 complexes bound to the Nr1d1 E-box, and CLOCK, and CRY1 to the Nr1d1 E-box by ChIP (chromatin im- – – second, the entry of PER2 ER* into the nucleus after addition of munoprecipitation). Fig. 1A shows that entry of PER2 ER* into 4-OHT does not significantly alter the amount of CLOCK– the nucleus strongly reduces binding of all three proteins (CLOCK, BMAL1 bound to the promoter. Interestingly, a change in overall e − − BMAL1, and CRY1) when DMSO or the control CK1 kinase CLOCK phosphorylation is not seen in the Ck1δ / versus inhibitor are in the medium. In contrast, the binding of all three Ck1δ+/+ fibroblasts following nuclear entry of PER2 (SI Appendix, proteins does not diminish when the CK1δ/e inhibitor is present in Fig. S1C), suggesting that CLOCK possesses multiple potential the medium, and for some reason the binding of CRY actually phosphorylation sites (22–24), and only a subset of these sites is increases (Fig. 1A), indicating that CK1δ kinase plays an essential targeted by CK1δ and destabilize CLOCK–BMAL1 on DNA role in PER-mediated displacement-type repression in the core when phosphorylated. clock mechanism. A control experiment (SI Appendix,Fig.S1A) shows that, under the conditions employed, the CK1δ/e inhibitor Requirement for Casein Kinase Binding Domains of PER2 in Displacement of CLOCK and BMAL1 from Promoters. Casein kinases bind to PER2 through two casein kinase binding domains (CKBDs), CKBDa (CK2 binding domain) and CKBDb (CK1e binding do- main), which are indicated in the schematic of PER2 protein in Fig. 2A (25–27). To examine the importance of a direct interaction between CK proteins and PER2 in displacement of BMAL1 and CLOCK from E-box promoters, we made constructs, shown in Fig. 2A, to express one mutant PER2–ER* protein with a deletion of the CK binding domain (CKBDb) and another protein with both the CKBDa and the CKBDb deleted (CKBDa/b). We also made a construct expressing PER2S659A, because it has been proposed that phosphorylation of PER2S659 by CK1 primes the phosphorylation of additional sites of PER2, which leads to increasing PER2 abun- dance (28, 29). We tested these constructs in our cell biochemical system based on targeted delivery of each PER2 construct into the nucleus by the OH-T/Estrogen Receptor* system (Fig. 2 B–D). As expected, entry of full-length (FL) PER2–ER* into the nucleus reduces BMAL1 binding to Nr1d1 E-boxestobackgroundlevel. Importantly, the entry of PER2–ER* with CKBDa/b double dele- tions fails to reduce BMAL1 binding to the E-box (Fig. 2B and SI Appendix,Fig.S3).SimilareffectswereobtainedwiththesePER2 mutants on CLOCK and CRY1 binding to the E-box (Fig. 2 C and D). Overall, this experiment and a prior report (11) showing efficient removalofCLOCK/BMAL1byPER2lackingaminoacids1to595 (CKBDa) reveal that deletion of CKBDa and CKBDb together but not singly eliminates CK1δ-mediated removal of CLOCK–BMAL1 from an E-box (summarized in Fig. 2A, Right).

Fig. 1. PER2-mediated displacement of CLOCK, BMAL1, and CRY1 from an δ − − CLOCK Hyperphosphorylation and CK1 Nuclear Localization Are PER E-box in vivo is CK1δ dependent. (A) After treatment of Per1/2 / ; PER2–ER* μ δ e μ and CRY Dependent in Vivo. To determine the mechanism by which cells for 24 h with 10 M PF670462 (CK1 / inhibitor) or 10 M PF4800567 δ (CK1e-selective inhibitor), 1 μM 4-OHT was added for 0 or 4 h to induce CK1 promotes CLOCK removal from E-boxes in vivo, we first nuclear entry of PER2–ER*. CLOCK, BMAL1, and CRY1 binding to the Nr1d1 tested the phosphorylation of CLOCK, BMAL1, and PER2 E-box was then measured by ChIP. (B) Experiments were then repeated in vitro by the main members of the casein kinase family. Fig. 3 A (without inhibitors) to test cells with knocked-out CK1δ and CK1« genes and B utilized the constitutively active form of CK1δ, CK1δΔC, (Per1/2−/−; Ck1δ−/−; PER2–ER* and Per1/2−/−; Ck1«−/−; PER2–ER* cells), using purchased from a commercial source, which lacks its auto- − − Per1/2 / ; PER2–ER* cells as a control. Results indicate a role of CK1δ but not inhibitory C terminus and retains 97% identity with CK1δ in the CK1e in removal of the CRY1–CLOCK–BMAL1 complex in vivo. Data for each kinase domain. The autoradiograms in Fig. 3 A and B show that panel were normalized to a value of 1 given to a control signal obtained δ e – CLOCK, BMAL1, and PER2 are all substrates of CK1 , CK1 , with 0-h 4-OHT treatment (DMSO for A; PER2 ER* in B). Three biological and CK2 for labeling by [γ-32P]ATP. We next performed in vivo repeats were used for quantification. Data are represented as dots for in- – dividual experiments and as columns for means. Error bars represent SDs. n.s, experiments to examine CLOCK BMAL1 phosphorylation lev- not significant; *P < 0.05, **P < 0.01, ***P < 0.001, as determined by t test els as a function of CT (circadian time) in wild-type (WT) as well between 0 and 4 h in same cell line and two-way ANOVA between different as CRY and PER knockout mice. Phosphorylation levels were cell lines. determined by examining bandshifts in immunoblots. The blots

2of9 | PNAS Cao et al. https://doi.org/10.1073/pnas.2021174118 Molecular mechanism of the repressive phase of the mammalian circadian clock Downloaded by guest on October 6, 2021 CK1δ oscillate with a pattern similar to the patterns of CRY1 and PER2. Of course, we cannot rule out the possibility that PER1/2 and CRY1/2 regulate CK1δ nuclear retention/nuclear export. Consequently, nuclear levels of CK1δ become elevated during the repressive phase of the clock when the substrate of CK1δ, CLOCK, becomes hyperphosphorylated and exhibits re- duced activity. Taken together, these data are consistent with the idea that PER1/2 plays a role in the localization of CK1δ into the nucleus, which in turn phosphorylates CLOCK and causes the dissociation of the CLOCK–BMAL1 complex from the cognate promoter and ultimately ubiquitination and degradation by the proteasome (24, 32).

Displacement of CLOCK–BMAL1 from an E-Box by CK1δ in Vitro: Electrophoretic Mobility Shift Assay. To further test the model of CRY1 and PER2-mediated recruitment of CK1δ to repress

Fig. 2. Mapping PER2 domains required for removing CRY1–CLOCK–BMAL1 from an E-box in vivo. (A) Illustration of the PER2–ER* constructs expressed − − in the Per1/2 / cell line. The numbers indicate amino acid residues bordering deletions made in the full-length, 1,257-amino acid-long PER2. CBD, CRY- binding domain; CKBDa, casein kinase 2 binding domain; CKBDb, casein kinase 1e binding domain; ER*, estrogen receptor “tag”; PAS, PER–ARNT– SIM domain. The dashed lines indicate regions deleted (Δ) in the constructs. Locations of the CK binding domains a and b (CKBDa and CKBDb) are shown. (B−D) ChIP analyses of BMAL1, CLOCK, and CRY1 binding to an E-box in the

− − BIOCHEMISTRY Nr1d1 promoter in Per1/2 / cell lines expressing different PER2–ER* pro- teins. Full-length PER2 (1–1,257), S659A PER2, and ΔCKBDb PER2 disrupt CRY1–CLOCK–BMAL1 binding to chromatin. Only the PER2 construct lacking both CKBDs (ΔCKBDa/b PER2) is unable to remove CRY1–CLOCK–BMAL1 from chromatin. S659A PER2, and ΔCKBDa PER2 appear to have only partial ability to remove CRY1 from the CLOCK–BMAL1–E-box complex. All data were normalized to a value of 1 for full-length PER2 (1–1,257) at 0-h 4-OHT. Three biological repeats were used for quantification. Data are represented as dots for individual experiments and as columns for means. Error bars represent SDs. n.s, not significant; *P < 0.05, **P < 0.01, ***P < 0.001, as determined by t test.

in Fig. 3 C and D clearly show circadian oscillations of CLOCK– BMAL1 phosphorylation levels in WT mouse liver nuclei. Notably, in the repressive phase when CLOCK–BMAL1 binding to the E-box becomes weak (CT12–22) (30, 31), CLOCK is hyper- phosphorylated. In the active phase when CLOCK/BMAL1 bind- ing to the E-box is strong (CT4–8) (30, 31), CLOCK is Fig. 3. CLOCK hyperphosphorylation and CK1δ nuclear translocation are hypophosphorylated. CLOCK is also hypophosphorylated at all PER and CRY-dependent in vivo. Preliminary experiments were done in vitro time points in the knockout mice lacking CRY and PER repressors. to assess phosphorylation of core clock proteins by CKs. Purified proteins were incubated with [γ-32P]ATP and separated by SDS-PAGE (sodium dodecyl Thus, by analyzing both circadian expression and knockout mice, we see sulfate–polyacrylamide gel electrophoresis), and reaction products were vi- CLOCK–BMAL1 repression associated with hyperphosphorylation of sualized by autoradiography. Autoradiograms show that PER2 (A)and CLOCK, and activation associated with hypophosphorylation. In CLOCK–BMAL (B) can be directly phosphorylated by CK1δ and CK1e,and contrast, in WT liver BMAL1 is hypophosphorylated between CT12 marginally phosphorylated by CK2. (C and D) CLOCK–BMAL1 phosphoryla- − − − − and CT24, and hyperphosphorylated between CT8 and CT12, and tion levels in WT, Per1/2 / , and Cry1/2 / mice as detected by bandshifts in in the knockout mice, BMAL1 was hyperphosphorylated at all time immunoblots. Nuclear protein extracts from mouse livers were prepared at – points due to an unknown mechanism (Fig. 3 C and D). six CT points for analysis. In WT mice, when CLOCK BMAL1 binding to δ E-boxes is high at CT4–8, CLOCK is hypophosphorylated. However, when We next examined the apparent link between CK1 and CLOCK–BMAL1 binding to E-boxes is low at CT12–22, CLOCK is hyper- CLOCK phosphorylation by comparing the subcellular localiza- phosphorylated. BMAL1 is hyperphosphorylated between CT8 and CT12, and tion of CK1δ and the negative effectors CRY1 and PER2 in WT BMAL1 is hypophosphorylated between CT16 and CT24. In Per1/2−/− and and knockout mouse liver as a function of CT. The immunoblot Cry1/2−/− mice, CLOCK is hypophosphorylated but BMAL1 is hyper- results in Fig. 3 E and F show that as expected, CRY1 and PER2 phosphorylated at all time points. (E) Nuclear (“Nuc”) and cytoplasmic − − both oscillate in cytosol and nucleus of WT mice with peaks (“Cyto”) temporal expression of core clock proteins in WT and Per1/2 / mouse liver. (F) Nuclear (Nuc) and cytoplasmic (Cyto) temporal expression of during the repressive phase at approximately CT20. The results −/− from knockout mice show that CRY1 enters the nucleus in the core clock proteins in WT and Cry1/2 mouse liver. Equal amounts of cy- toplasmic protein (120 μg) and nuclear protein (6 μg) were loaded in each absence of PER1/2, but PER2 requires CRY1/2 for nuclear entry α δ protein lane. -Tubulin (cytoplasmic) and Lamin B1 (nuclear) were probed to (13). Importantly, nuclear entry of CK1 directly requires PER1/ provide loading controls. All the immunoblots were performed with at least 2 and indirectly requires CRY1/2 since both CRY1/2 are nec- two biological repeats and some had three technical repeats. All data essary for PER2 entry in the nucleus. Also, nuclear levels of yielded similar results. Representative images are shown.

Cao et al. PNAS | 3of9 Molecular mechanism of the repressive phase of the mammalian circadian clock https://doi.org/10.1073/pnas.2021174118 Downloaded by guest on October 6, 2021 CLOCK–BMAL1–E-box complexes, we performed in vitro independently of PER2 and CRY1 (SI Appendix, Fig. S7). We studies with purified CLOCK/BMAL1, PER2, CRY1, CK1δ, and wished to know whether the observed removal was due to non- CK1e proteins (SI Appendix, Fig. S4). Since PER2 appears to be specific phosphorylation by kinases and thus tested CK1e more important than PER1 for normal clock function in mice and CK2 kinases. At concentrations similar to those at which (33, 34), and CRY1 is a stronger repressor than CRY2 because CK1δ was effective, CK2 has only a minor effect on a of higher affinity to CLOCK/BMAL1 (35, 36), we used PER2 CLOCK–BMAL1–E-box complex and CK1e has no effect and CRY1 in our in vitro experiments. In electrophoretic mo- (Fig. 4 C and I). bility shift assays (EMSAs), the purified CLOCK/BMAL1 com- A more comprehensive analysis of CLOCK–BMAL1–E-box plex binds specifically to an E-box sequence in a 30-bp duplex, stability in the presence of kinases, CRY1, and PER2 is shown and binding is concentration dependent (SI Appendix, Fig. S5 A in Fig. 4D. Here, addition of CK1δ directly dissociates nearly the and B). Fig. 4A recapitulates our previous finding that CRY1 entire CLOCK–BMAL1–E-box complex, and the inclusion of makes a CRY1–CLOCK–BMAL1–E-box complex (lane 4) and CK2 in the reaction makes only a modest difference (Fig. 4D, that addition of PER2 to this complex results in removal of lanes 1 to 5, and Fig. 4I). Binding results obtained under con- CRY1 without affecting the level of CLOCK–BMAL1 at the ditions that mimic the in vivo conditions necessary for dis- E-box (lane 5), which, in vivo, depending on the types of regu- placement of CLOCK–BMAL1 from the E-box, shown in latory elements in the promoter/enhancer may result in tran- Fig. 4D (lanes 6 to 12) and Fig. 4I, confirm earlier observations scriptional repression or activation. Interestingly, Fig. 4B and SI of PER2 displacing CRY1 from a CRY–CLOCK–BMAL1–E- Appendix, Fig. S6 show that above 40 nM, and in the presence of box complex (lanes 8 and lane 9). Lane 10 shows strong dis- ATP, CK1δΔC reduces the amount of CLOCK–BMAL1 bound placement of the entire CLOCK/BMAL1 complex by CK1δ even to the E-box independently of PER and CRY. Both full-length when PER2 and CRY are present in the binding reaction. CK2 mCK1δ purified by us and hCK1δ purchased from commer- has only a minor effect on its own (lane 11), but it seems to cial resource also remove CLOCK/BMAL1 from the E-box enhance the displacement effect of CK1δ (lane 12), suggesting

Fig. 4. CK1δ and CK2 reduce CLOCK/BMAL1 binding to an E-box in vitro. (A) Effect of CRY1 and PER2 on the mobility of the CLOCK–BMAL1–E-box complex. EMSA was performed with a 32P-labeled 30-bp duplex containing an E-box (1 nM) and CLOCK–BMAL1 (2 nM). A supershift was caused by CRY1 (15 nM) but not PER2 (15 nM) (lanes 3 and 4). PER2 removes only CRY1 from the CLOCK–BMAL1–CRY1–E-box complex; the CLOCK–BMAL1–E-box remains (lane 5). (B) Effect of CK1δΔC, CRY1, and PER2 on the mobility of the CLOCK–BMAL1–E-box complex. The E-box duplex (1 nM) was incubated with CLOCK–BMAL1 complex at 2 nM and increasing amounts of CK1δΔC (40, 100, and 200 nM). CRY1 and PER2 were added as in A.(C) Effect of CK2 and CK1e on the amount of

CLOCK–BMAL1–E-box complex. (D) Effect of CK1δ and CK2 on the entire circadian protein assembly binding to the E-box. MgCl2 and ATP were present in all reactions in Fig. 4 A–D. EMSA showing the effect of CK1δ (E), CK2 (F), and CK1δ and CK2 together (G) on the amount of CLOCK–BMAL1–E-box complexes in the presence of ADP, ATP, or no nucleotide. (H) Effect of both CK1δ and CK2 on the entire circadian protein assembly binding to the E-box with/without ATP. The EMSAs data are representative of three independent experiments. (I) Quantitative analysis of the amount of CLOCK–BMAL1–E-box complex when adding core clock proteins with/without ATP. Three biological repeats were used for quantification. Data are represented as dots for individual experiments and as columns for means. Error bars are SDs of triplicate data. n.s, not significant; *P < 0.05, **P < 0.01, ***P < 0.001 were determined by two-way ANOVA.

4of9 | PNAS Cao et al. https://doi.org/10.1073/pnas.2021174118 Molecular mechanism of the repressive phase of the mammalian circadian clock Downloaded by guest on October 6, 2021 that in addition to CK1δ, CK2 may also contribute toward BMAL1 is nonphosphorylated; in fact, there appears to be a phosphorylation and displacement of CLOCK/BMAL1 from the slight enrichment in nonphosphorylated BMAL1 in the released E-box. We further examined whether PER2 might assist CK1δ in fractions compared to bound. These results support and extend displacing CLOCK–BMAL1, and found that it did not, even our findings above in showing the CK1δ-dependent phosphory- following prolonged reaction times or using high concentrations lation of CLOCK and consequent dissociation of the CLOCK– of PER2 in combination with a low concentration of CK1δ (SI BMAL complex from an E-box. Appendix, Fig. S8 A and B). To test whether the CK1δ effect The pull-down experiment in Fig. 5F, which included CRY1 observed was due to the phosphorylation of target proteins and and PER2 proteins, confirms earlier data in demonstrating the not an artifact of the presence of highly negatively charged nu- binding of CRY1 to the CLOCK–BMAL1–E-box complex (Re- cleotides in the binding reaction, we performed experiments with action 4, “Bound”), and shows removal of the entire CRY1– ATP or ADP in the binding reactions. Fig. 4 E and I shows that CLOCK–BMAL1 complex from the E-box by CK1δΔC (Reac- in a reaction containing only CLOCK–BMAL1 and CK1δ, ADP tion 6, “Released”). Unexpectedly, removal by CK1δΔCisless does not affect the amount of CLOCK/BMAL1–E-box (lane 5), efficient in the presence of PER2 (Reactions 6 versus 2, Re- but ATP significantly reduces the amount of bound E-box (lane leased); this result may be unique to the experimental condition in 6). Fig. 4F shows that the marginal effect of CK2 on the complex which PER2 may compete with CLOCK for phosphorylation by is also ATP dependent. Importantly, Fig. 4G shows that the CK1δΔC. More importantly, with respect to PER2, our results combination of CK1δ and CK2 in the presence of ATP dissoci- overall indicate an important role of PER2 in transporting CK1δ ates virtually the entire complex (lane 7). Finally, when the effect into the nucleus to promote hyperphosphorylation of CLOCK leading of CK1δ and CK2 is tested in the presence of the entire circadian to dissociation of CLOCK–BMAL1 from E-boxes. Similar models protein assembly, results similar to those observed with have been proposed for PER-dependent phosphorylation of CLOCK CLOCK–BMAL1–E-box alone are observed (compare Fig. 4G, leading to its displacement from cognate promoters in Drosophila lane 7, and Fig. 4H, lane 10), showing that CK1δ/CK2 dissocia- (38–40), and FRQ–FRH-dependent hyperphosphorylation of WCC tion of CLOCK–BMAL1 from an E-box is ATP-dependent and and its dissociation from target promoters in Neurospora (41, 42). independent of the CRY1 and PER2 negative effectors in vitro. We note that only 70% of 2 nM CLOCK/BMAL1 was removed Nuclear Clock Protein Complexes Present during the Repressive from the E-box even with the high concentration of CK1δ/CK2 Phase. The dependence of CK1δ nuclear entry on CRYs and (200 nM) in vitro (Fig. 4I). However, it is also noted that only a PERs described above suggests that these proteins are trans- small fraction of CK1δ/CK2 is active and the reported activity of located into the nucleus as a complex. To examine possible clock CK1δ/CK2 from the commercial source was established under protein–protein interactions in vivo during the repressive phase BIOCHEMISTRY high substrate concentration. Furthermore, the presence of a of the circadian cycle, we analyzed proteins in nuclear extract relatively low amount of substrate (CLOCK–BMAL1–E-box) from WT mouse liver obtained at ZT19 (zeitgeber time) by relative to enzyme may recapitulate the in vivo condition (37). glycerol gradient centrifugation. Fig. 6A and SI Appendix, Fig. Taken as whole, the data in Fig. 4 show that CK1δ and CK2 are S9A show that circadian proteins sedimented principally in two capable of disrupting the CLOCK–BMAL1–E-box complex un- complexes; CRY, PER, and CK1δ comigrated at a position assisted, provided they are transported to the nucleus. This un- corresponding to ∼550 kDa (which is consistent with multimers expected finding led us to investigate in more detail the role of of PER1, PER2, CRY1, CRY2, and CK1δ), and CLOCK and phosphorylation in clock repression. BMAL1 comigrated at a position corresponding to about 200 kDa, which is approximately the size of a CLOCK–BMAL1 Release of the CLOCK–BMAL1 Complex from an E-Box by CK1δ in heterodimer. Most of CRY1 is associated with PER in 550-kDa Vitro: Pull-Down Assay. We complemented the above studies by complexes in WT at ZT19; a small fraction of CRY1 is monomer using a pull-down assay to more directly monitor release of (Fig. 6A and SI Appendix, Fig. S9A). Naturally, there is no − − − − CLOCK–BMAL1 from an E-box in response to CKs, PER, and high–molecular-weight PER complex in Per1/2 / and Cry1/2 / CRY. To do this, we first isolated CLOCK–BMAL1–E-box com- mice; CLOCK–BMAL1 migrate as a heterodimer. Most of − − plexes using streptavidin beads bound to a biotin-tagged, E-box CRY1 is monomer in Per1/2 / mice (Fig. 6 and SI Appendix, containing DNA duplex, as illustrated in the schematic in Fig. Fig. S9 B and C). These results are consistent with the idea that a 5 A, Left. The immunoblot in Fig. 5B shows that the CLOCK– CRY–PER–CK1δ complex translocates into the nucleus and BMAL1 complex is pelleted (IP) with the streptavidin beads when interacts transiently to phosphorylate CLOCK and inactivate the the DNA duplex contains an E-box (lane 5), but not when the CLOCK–BMAL1 complex. These results also explain why PER duplex has a mutant E-box (Mut) or when there is no DNA, and CRY are not necessary for removal of CLOCK–BMAL1 demonstrating the binding specificity. For the pull-down assays (as from E-boxes in vitro, where nuclear entry is not required. We outlined in the schematic, Fig. 5 A, Right)showninFig.5C–E, should note, however, that our findings of a CLOCK–BMAL1 CLOCK–BMAL1–E-box complexes specifically bound to the complex in the 200-kDa range separated from the CRY–PER– E-box duplex and pelleted in this manner (Fig. 5 B,lane5)were CK1δ complex in the 550-kDa range differs from reports of so- resuspended and used as input. (In Fig. 5F, the input for pull-down called PER megadalton complexes with 1.1- and 1.9-MDa size in assays consisted of CLOCK–BMAL1 plus CRY1 bound to the the cytoplasm and nucleus, respectively (17). Moreover, these E-box.) CLOCK–BMAL1–E-box complexes were incubated for megadalton complexes reportedly contained the core clock 45 min at room temperature in the presence or absence of added proteins (CLOCK, BMAL1, CRY, PER, CK1δ) along with ad- ATP and clock proteins, and then pulled down to separate Bound ditional proteins in the nucleus (17). It must be noted, however, and Released fractions. that these supercomplexes were detected by gel filtration chro- Experimental results (Fig. 5 C, E, and F) consistently show matography or blue native-agarose polyacrylamide gel electro- CK1δΔC-dependent release of CLOCK–BMAL1 from the phoresis. We believe these isolation methods have some limitations E-box. Most of the CLOCK and a portion of the BMAL1 are in and need to be supported by complementary biochemical ap- phosphorylated form after incubation with CK1δΔC, as indi- proaches such as ultracentrifugation. cated by bandshifts in the immunoblots (Fig. 5 C, E, and F), and by 32P labeling when [γ-32P]ATP was included in binding reac- Discussion tions (Fig. 5D). Release is in fact ATP dependent (Fig. 5E). In our previous studies, we demonstrated that: 1) CRY (CRY1 While both the bound and released CLOCK appear to be in or CRY2) bound to a CLOCK–BMAL1–E-box complex phosphorylated form, a significant proportion of the released in vitro, 2) but PER (PER1 or PER2) did not bind to this

Cao et al. PNAS | 5of9 Molecular mechanism of the repressive phase of the mammalian circadian clock https://doi.org/10.1073/pnas.2021174118 Downloaded by guest on October 6, 2021 Fig. 5. Release of CLOCK–BMAL1 from an E-box by CK1δ in vitro. (A) Schematic showing the DNA pull-down assay to test the roles of PER2, CRY1, and CK1δ ΔC on CLOCK–BMAL1 release from an E-box. The E-box–containing duplex is tagged with biotin (“B”) and binds to and pellets with streptavidin (“S”) beads. CLOCK–BMAL1, bound to the E-box, also pellets with the beads. (In F, beads are prepared by adding CRY1 together with CLOCK–BMAL1 at this step, and in this case, the CLOCK–BMAL1–CRY1 complex pellets with the beads.) For pull-down assay, beads with CLOCK–BMAL1–E-box (or CLOCK–BMAL1–CRY1–E-box complexes) are then resuspended and incubated with PER2, CRY1, or CK1 δ, then pulled down to assess E-box bound and released proteins. (B) Preparation of CLOCK–BMAL1–E-box complexes (A, Left). CLOCK–BMAL1 (20 nM) binds to the DNA duplex (10 pmol) containing the E-box sequence and is pulled down (“IP”) by the streptavidin beads as shown in lane 5 of the immunoblot. When the duplex has a mutated E-box sequence, or in the absence of DNA, CLOCK–BMAL1 remain entirely in the “Free” fraction following pull-down, demonstrating specificity of binding. (C) For pull-down assay (A, Right), CLOCK–BMAL1–E-box complexes immunoprecipitated as in B, lane 5, were resuspended, incubated with CK1δΔC, and then pulled down again to separate Bound and Released fractions. The immunoblot analysis shows that CLOCK–BMAL1 is released from the E-box after adding 200 nM CK1δΔC. (D) SDS-PAGE autoradiogram showing that the bound and released CLOCK is hyperphosphorylated, but only bound BMAL1 is hyperphosphorylated after addition of 200 nM CK1δΔC. Proteins were radiolabeled by adding [γ-32P]ATP to the binding reactions. (E) Release of CLOCK–BMAL1 from the E-box by 200 nM CK1δΔC is ATP dependent in the pull-down assay. (F) Effect of 15 nM PER2, and 200 nM CK1δΔC on the CLOCK–BMAL1–CRY1–E-box complex. Note that the CRY1 amount associated with CLOCK–BMAL1 in the bound fraction (lanes 4 to 6) is lower than that observed in the EMSAs (Fig. 4 A, B, and D) because the multiple washes in the pull-downs causes dissociation of CRY from the CLOCK–BMAL1 complex. The DNA pull-down data are representative of three independent experiments.

complex. 3) Rather surprisingly, when PER2 was added to the Taken in its entirety, the data presented above suggest the CRY1–CLOCK–BMAL1–E-box complex, it caused displace- following displacement-type repression model: PER is bound to ment of CRY1 from the complex. 4) In an in vivo system where CK1δ through its CKBD and carries CK1δ to the nucleus with − − CRY1 or PER2 could be delivered to the nuclei of Cry1/2 / ; CRY and in the nucleus in a CRY-dependent manner increases − − Per1/2 / cells it was found that entry of CRY1 into the nucleus local concentration of CK1δ at CLOCK–BMAL1 promoters/ inhibited the expression of genes controlled solely by CLOCK– enhancers, phosphorylates (hyperphosphorylates) CLOCK and BMAL1 without removal of CLOCK–BMAL1 from the cognate to a lesser degree BMAL1, and thereby causes the disruption E-box (blocking-type repression). 5) In the same quadruple mu- and dissociation of the entire CRY–CLOCK–BMAL1 complex tant cell line, controlled entry of PER into the nucleus did not from the E-box. This interpretation of our data confirms and affect either the binding of CLOCK–BMAL1 to an E-box, or gene extends the models proposed by Kondratov et al. (24) on the role expression controlled by CLOCK–BMAL1 binding to an E-box. of CLOCK phosphorylation in the circadian clock and by Most surprisingly, the controlled entry of PER into the nuclei Etchegaray et al. (15) on the role of CK1δ but not CK1e on − − of Cry1/2+/+; Per1/2 / cells resulted in removal of the entire circadian period length. Furthermore, in this paper by intro- CRY–CLOCK–BMAL1 complex from the E-box (in contrast to ducing CK1δ into our in vitro system, we have tested the plau- the vitro experiments in which PER2 removes CRY1 but not sibility of the proposed model while at the same time taking into CLOCK–BMAL1 from the E-box) resulting in inhibition of E-box account the extensive studies that have addressed the role of controlled (displacement-type repression) (11). CK1δ/e in the mammalian circadian clock and those studies that Furthermore, it was found that, in this in vivo system, removal of have defined the roles of the functional orthologs of CK1δ/e in CLOCK–BMAL1 from the cognate promoter was dependent on the two other well-studied systems, Neurospora and Drosophila. intact CKBD (casein kinase binding domain) and CBD (CRY Similar displacement-type repression models have been pro- binding domain) regions of PER (11). posed for PER-dependent phosphorylation of CLOCK and its

6of9 | PNAS Cao et al. https://doi.org/10.1073/pnas.2021174118 Molecular mechanism of the repressive phase of the mammalian circadian clock Downloaded by guest on October 6, 2021 CLOCK and BMAL1 are substrates for phosphorylation at multiple sites by multiple kinases including PKG, PKC, and ca- sein kinase family members (44–46). A prior study reported that phosphorylation specifically of Ser-38 and Ser-42 residues, lo- cated in the bHLH of CLOCK, reduces the transactivation ac- tivity of CLOCK (22, 23). In the present study, we find that CK1δ is the main kinase responsible for release of CLOCK– BMAL1 from an E-box; however, our data show that there is considerable “background” phosphorylation of CLOCK in vivo in addition to that produced by CK1δ and CK2 (Fig. 3). Thus, it is likely that phosphorylation of CLOCK by CK1δ/CK2 specifi- cally at a defined subset of the potential CLOCK phosphoryla- tion sites is responsible for reduced E-box binding activity of CLOCK–BMAL1. Further investigation is needed to identify amino acid residues important in mediating this inhibitory effect of CK1δ/CK2 and to further examine the roles of CLOCK res- idues Ser-38 and Ser-42. In Fig. 7, we present a model of the mammalian circadian clock that incorporates both blocking-type and displacement- type mechanisms. At CT4–8(“active phase”), the CLOCK– BMAL1 heterodimer binds to an E-box and activates transcrip- tion and expression of PER and CRY. In the cytoplasm, PER, CRY, and CK1δ form a stable complex that enters the nucleus. In the nucleus, starting at about CT12, CRY bridges the inter- action of PER with CLOCK–BMAL1–E-box. From CT12 to CT22, when the PER–CRY–CK1δ complex concentration in- Fig. 6. Analysis of circadian complexes by glycerol gradient sedimentation. creases in the nucleus, PER is hyperphosphorylated. At the same Mouse nuclear extract prepared from a mouse harvested at ZT19 and ref- erence proteins were mixed together, and the extract and reference pro- time, PER acts as scaffold to increase the local concentration of δ – BIOCHEMISTRY teins were sedimented together through a 10 to 30% (wt/wt) glycerol CK1 /CK2 at CLOCK BMAL1-bound promoters/enhancers gradient. Reference proteins included bovine thyroglobulin (669 kDa, 19 S), and allow CK1δ/CK2 kinases to phosphorylate CLOCK, and sweet potato β-amylase (222 kDa, 8.9 S), and chicken ovalbumin (43 kDa, then hyperphosphorylation of CLOCK leads to the release of the 3.55 S). Fractions were collected starting at the bottom of the gradient and complexes from the E-box (CT12–22 displacement-type repres- analyzed by loading samples to two SDS-PAGE gels. Gels showing gradient sion). Later, hyperphosphorylated PER is degraded by a ubiquitin- − − − − profiles for extracts from WT (A), Per1/2 / (B), and Cry1/2 / mice (C)atZT19 mediated proteasome pathway, so by CT0–4, CRY1, with the low were developed by immunoblot with PER1, PER2, CRY1, CRY2, CK1δ, CLOCK, concentration of PER, blocks CLOCK–BMAL1 in a poised state “ ” and BMAL1 antibodies. P stands for pellet and indicates the sample until the next cycle begins (CT0–4 blocking-type repression). This obtained by washing the empty gradient tube with a small volume (240 μL) after collecting all fractions. The purpose of the P sample was to discover model is consistent with published ChIP data, which measured whether an insoluble pellet existed following centrifugation and to examine its composition. Three percent of the original sample was loaded directly to the SDS-PAGE gel as “Input.” The arrows indicate positions of the peak fraction of each reference protein as determined from the Coomassie Blue- stained SDS-PAGE gel (see SI Appendix, Fig. S9 for additional information related to this figure). Quantification of relative intensity is shown beside the immunoblot. Data for each protein were normalized to a value of 1 given to the peak fraction. Glycerol gradient experiments were performed − − with three biological repeats for WT and Cry1/2 / , and two biological re- − − peats for Per1/2 / mice, and all data yielded essentially identical results. Representative images are shown in the figure.

displacement from cognate promoters in Drosophila, as well as FRQ-dependent hyperphosphorylation of WCC and its dissoci- ation from target promoters. However, there is no blocking-type repression reported in Neurospora.InDrosophila, PER has subsumed the role of the mammalian CRY as the primary re- pressor in on-DNA repression (43), indicating that increasingly more sophisticated regulation of repression evolved from Neu- Fig. 7. Model showing the role of blocking-type and displacement-type rospora,toDrosophila, to mammals. repression in the mammalian circadian clock. At around CT4–8, CLOCK– Cellular assays show that treatment with CK1δ/e inhibitor and BMAL1 binds to E-boxes to drive clock-controlled gene transcription. After knockout of CK1δ can inhibit about 70% of CLOCK–BMAL1 protein synthesis in the cytoplasm, CRY recruits PER and PER recruits CK1δ/ displacement by PER2 (Fig. 1). However, CK1δ-mediated re- CK2 through its CKBD and then enters the nucleus and phosphorylates moval of CLOCK–BMAL1 from an E-box requires deletion of CLOCK, leading to CLOCK–BMAL1 dissociation from the E-box (CT12–22 both CKBDa (CK2 binding domain) (26) and CKBDb (CK1e displacement-type repression). At around CT0–4, PER levels are too low to be δ detected and only CRY1 binds to the CLOCK–BMAL1–E-box to block binding domain) in PER2 (Fig. 2) (27). It is possible that CK1 – plays a primary role, and CK1δ/CK2 act synergistically in CLOCK BMAL1 activity (blocking-type repression), which maintains CLOCK–BMAL1 in a repressed state until the next TTFL cycle begins. Note the CLOCK–BMAL1 displacement from an E-box (Fig. 4). We δ CK2 is shown a circle with discontinuous circumference to indicate its partial cannot rule out the possibility that CK1 binds both CKBDa contribution relative to CK1δ. In addition, the entire “repressor–activator (CK2 binding domain) and CKBDb (CK1e binding domain) complex” is shown in brackets to indicate that it must exist as a kinetic in- in PER2. termediate and not a stable megadalton complex.

Cao et al. PNAS | 7of9 Molecular mechanism of the repressive phase of the mammalian circadian clock https://doi.org/10.1073/pnas.2021174118 Downloaded by guest on October 6, 2021 binding of clock proteins to E-box regions as a function of CT, and travelers rather than conjugal partners” (50). However, it must showed that at CT0–4 (when PER is degraded to very low levels) also be noted that PER binding to E-boxes was detected by ChIP there is maximal mutual binding of CLOCK, BMAL1, and using a sensitive dual–cross-linking approach (31), and clearly CRY1 (31). the activator–repressor complexes do interact, albeit transiently, The model in Fig. 7 uses brackets to denote the transience of to generate an ephemeral activator–repressor complex, thus the interaction between activator (CLOCK–BMAL1–E-box) and reconciling our findings with the so-called nuclear megadalton repressor (CRY–PER–CK1δ) complexes. The contrary notion of PER complex. Phosphorylation of CLOCK by CK1δ recruited by a stable activator–repressor interaction is implicit in reports that the CRY–PER complex reduces activator–repressor affinity, par- have described megadalton-sized, stable complexes composed of alleling the situation in Neurospora, in which hyperphosphorylation activator and repressor clock proteins together with varied of FRQ in the repressive phase induces the rapid loss of interaction combinations of the numerous clock protein-associated proteins between FRQ and WCC (51, 52). Further work is needed to refine and RNA that have been discovered since 2005 (17, 47–49). We the mammalian clock model and better define the unique roles and also found that the entire clock protein ensemble comigrated in dynamics of proteins in both the positive and negative arms of the the megadalton range by gel filtration chromatography, as molecular circadian clock. reported previously (17, 47–49). However, our sedimentation analysis found no PER supercomplexes at ZT19, as commonly Materials and Methods invoked, but instead revealed apparent separate activator and For details on chemicals, animals, plasmids and cell lines, protein expression repressor complexes of 200 kDa (containing CLOCK–BMAL1) and purification, ChIP-qPCR, nuclear and cytoplasmic protein extraction, and 550 kDa (containing CRY1/2, PER1/2, and CK1δ) (Fig. 6). radioactive kinase assays, EMSAs, DNA pull-down assays, glycerol gradient In addition, we have been unable to detect PER binding to centrifugation, and immunoblot analysis with antibodies, see SI Appendix, CLOCK–BMAL1–E-boxes in vitro (by EMSA or pull-down as- Materials and Methods. says) (Figs. 4 and 5) or PER2 binding to E-boxes in vivo by Data Availability. All study data are included in the article and SI Appendix. standard ChIP assays (6, 11). Multidomain mosaic proteins, such – as PERs are notorious for engaging in multiple protein protein ACKNOWLEDGMENTS. We thank Khagani Eynullazada for technical support interactions (48). While some of these interactions are of func- and Dr. Laura A. Lindsey-Boltz for critical comments on the manuscript. This tional significance, other interacting partners often are “fellow work was supported by NIH Grant GM118102 to A.S.

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Cao et al. PNAS | 9of9 Molecular mechanism of the repressive phase of the mammalian circadian clock https://doi.org/10.1073/pnas.2021174118 Downloaded by guest on October 6, 2021