SnapShot: Circadian Clock Proteins Elizabeth E. Hamilton and Steve A. Kay Division of Biological Sciences, University of California San Diego, La Jolla, CA 92093, USA Peak Expression Post- Protein Clock Function translational CK1δ: Kinase Regulates PER Protein Clock Function RNA Protein Regulation CK1ε: Kinase Regulates PER, CRY, BMAL1 CLOCK: bHLH-PAS Dimerizes with BMAL1 to bind E box Constant Constant Rhythmic transcription factor, (CACGTG), activating transcription of per, phosphorylation GSK3β: Kinase Regulates PER, CRY, REV- HAT cry, rev-erb ERBα BMAL1: bHLH-PAS Dimerizes with CLOCK to bind E box 15–21 0–8 Phosphorylation PP1: Phosphatase Regulates PER transcription factor (CACGTG), activating transcription; PP2A: Phosphatase Regulates PER ortholog of fl y CYC Mammal PER1,2: PAS domain Dimerizes with CRY to inhibit CLOCK- 4–6, 10–14 Progressive PP5: Phosphatase Regulates CK1ε BMAL1 transcriptional activity 6–12 phosphorylation Core Loop (CL) Core βTrCP: F box protein Targets PER for degradation CRY1,2: Flavoprotein; Dimerizes with PER to inhibit CLOCK- 8–14 12–18 No FBXL3: F box protein Targets CRY for degradation repressor domain BMAL1 DBT: Kinase Regulates PER and CLK RORα,β: Orphan Activates Bmal1 transcription 8–12 — — nuclear receptor CK2: Kinase Regulates PER Mammal REV-ERBα,β: Orphan Represses Bmal1 transcription 2–6 — — SGG: Kinase Regulates TIM nuclear receptor PP2A: Phosphatase Regulates PER and CLK DEC1,2: bHLH- Represses transcription from E box 2, 6 ~2, ~6 — ORANGE transcription (CACGTG) driven promoters; ortholog of Drosophila SLMB: F box protein Targets PER for degradation factor fl y CWO Regulators JET: F box protein Targets TIM for degradation DBP: bZIP-PAR Activates per transcription 4–6 6 — transcription factor CK1α: Kinase Regulates FRQ Interlocking Loops (IL) E4BP4: bZIP Represses per transcription; ortholog of 16 16–20 — CK2: Kinase Regulates FRQ transcription factor fl y VRI PP1: Phosphatase Regulates FRQ CLK: bHLH-PAS Dimerizes with CYC to bind E box ~0 Constant Rhythmic PP2A: Phosphatase Regulates FRQ and WC-1/ transcription factor (CACGTG), activates per, tim, vri, Pdp1ε phosphorylation WC-2 dimer transcription Neurospora FWD1: F box protein Targets FRQ for degradation CYC: bHLH-PAS Dimerizes with CLK to bind E box Constant Constant No transcription factor; (CACGTG), activates per, tim, vri, Pdp1ε CK2: Kinase Phosphorylates CCA1, LHY HAT transcription; ortholog of mammal BMAL1 CL PER: PAS domain Dimerizes with TIM to inhibit CLK-CYC ~15 17–21 Progressive ZTL: F box protein Targets TOC1 and PRR5 for transcription activity phosphorylation and PAS/LOV degradation domain TIM: Putative Armadillo Dimerizes with PER ~12 ~20 Progressive Arabidopsis and HEAT domains phosphorylation Drosophila PDP1ε: bZIP Activates Clk transcription ~18 ~21 No transcription factor VRI: bZIP transcription Represses Clk transcription; ortholog of ~14 ~14 Phosphorylation IL factor mammal E4BP4 CWO: bHLH-ORANGE Binds to E box (CACGTG) to repress 12–15 — — transcription factor CLK-mediated transcription; ortholog of mammal DEC1/2 WC-1: PAS/LOV Dimerizes with WC-2 to activate frq Constant 18–20 Phosphorylation transcription factor; HAT transcription; blue-light photoreceptor WC-2: PAS Dimerizes with WC-1 to activate frq Constant Constant No CL transcription factor transcription FRQ: Coiled-coil, Homodimerizes to regulate WC-1/WC-2 0–6 ~8 Progressive PEST domain dimer activity and abundance phosphorylation Neurospora VVD: PAS/LOV domain Negative regulator of light responses to 0 1–2 — IL core loop; blue-light photoreceptor TOC1: Pseudo- Activates transcription of LHY and CCA1 10–12 12 Progressive receiver and CCT phosphorylation domains LUX: MYB Activates transcription of LHY and CCA1 12 12 — transcription factor CL ELF4: Unknown Activates transcription of LHY and CCA1 ~12 — — CCA1/LHY: MYB Binds to evening element (AAAATATCT) 0 1–3 Phosphorylation transcription factor inhibiting transcription of TOC1, LUX, ELF4, GI, ELF3; activates PRR5,7,9 transcription GI: Unknown Stabilizes ZTL promoting TOC1 degradation 8–10 11–13 — PRR3: Pseudo-receiver Stabilizes TOC1 inhibiting ZTL binding 10 ~13 Progressive Arabidopsis and CCT domains to TOC1 phosphorylation PRR5: Pseudo-receiver Inhibits CCA1/LHY transcription 8–9 11 Phosphorylation and CCT domains IL PRR7: Pseudo-receiver Inhibits CCA1/LHY transcription 6–8 10 Progressive and CCT domains phosphorylation PRR9: Pseudo-receiver Inhibits CCA1/LHY transcription 2–4 8–10 — and CCT domains ELF3: Unknown Negative regulator of light responses to CL 16 16 — 368 Cell 135, October 17, 2008 ©2008 Elsevier Inc. DOI 10.1016/j.cell.2008.09.042 See online version for legend and references SnapShot: Circadian Clock Proteins Elizabeth E. Hamilton and Steve A. Kay Division of Biological Sciences, University of California San Diego, La Jolla, CA 92093, USA Table The earth’s rotation about its axis generates predictable changes in light and temperature, which define the 24 hr day-night cycle. Organisms from bacteria to humans have evolved an internal timekeeper to anticipate these environmental changes. This timekeeper—the circadian clock—allows organisms to parse biological processes to the appropriate time of day or night. Circadian clocks regulate behavioral, physiological, and metabolic rhythms via transcriptional feedback loops. Although clocks in different species use distinct protein constituents, the general architecture is conserved: A network of interconnected transcriptional feedback loops regulates rhythmic transcription of loop components as well as clock output genes. These interconnected loops consist of a core loop (CL) with a number of interlocking loops (IL) intersecting it. The primary difference between these loops is the effect of mutations on clock behavior. Mutating components of the core loop severely affects the regulation of the clock, whereas the effects are much milder for components in the interlocking loops. Posttranslational regulation such as phosphorylation introduces small delays ensuring that feedback regulators function at the correct time and cellular location to maintain ~24 hr rhythms in transcription. The complexity of these transcriptional feedback loops for simply maintaining 24 hr timekeeping may not be a universal requirement. Recent work in cyanobacteria, a prokaryote, shows that a 24 hr oscillator can be reconstituted in vitro by three purified proteins (see Nakajima et al., 2005). Peak phases for RNA and protein expression outlined in the table are in Zeitgeber time (ZT), where dawn (lights on) is ZT0 and dusk (lights off) is ZT12. A dash (“–”) indicates data that have not been reported. Mammalian phase data were determined for the suprachiasmatic nucleus (SCN). Figure At the heart of the eukaryotic clock are interlocking, autoregulatory feedback loops. In the core loop of the mammalian clock, positive elements CLOCK and BMAL1 (circles) heterodimerize and initiate transcription of their negative regulators PER and CRY (rounded squares). As the concentration of PER and CRY accumulates, these proteins form multimers, are phosphorylated, and translocate to the nucleus where they repress their own transcription by interacting with CLOCK-BMAL1 heterodimers. Phosphorylation- induced degradation of PER and CRY decreases their concentrations, which reactivates the positive elements, allowing the cycle to start again. Core loop components also activate multiple ccgs (clock-controlled genes) to form interlocking positive- and negative-feedback loops that are important for maintaining robustness of the clock. In mam- mals, CLOCK-BMAL1 heterodimers activate transcription of RORα and REV-ERBα (green square), which provide additional positive and negative feedback, respectively, to transcription of BMAL1. Abbreviations βTrCP, β-transduction repeat-containing protein; bHLH, basic helix-loop-helix; BMAL1, brain and muscle ARNT-like protein 1; bZIP, basic leucine zipper motif; CCA1, cir- cadian clock associated 1; ccgs, clock-controlled genes; CCT, constans, constans-like, TOC1; CK, casein kinase; CLK/CLOCK, circadian locomotor output cycle kaput (CLOCK); CRY, cryptochrome; CWO, clockwork orange; CYC, cycle; DBP, D binding protein; DBT, doubletime; DEC, differentially expressed in chondrocytes; E4BP4, E4 promoter-binding protein; ELF, early flowering; FBXL3, F box and leucine-rich repeat protein 3; FRQ, frequency; FWD-1, F box and WD40-repeat-contiaining protein-1; GI, gigantea; GSK3β, glycogen synthase kinase-3β; HAT, histone acetyltransferase; HEAT, Huntington elongation factor 3, A subunit of protein phosphotase 2A and TOR1; JET, jetlag; LHY, late elongated hypocotyl; LOV, light, oxygen, voltage domain; LUX, lux arrhythmo; PAR, proline and acidic amino acid-rich; PAS, period-ARNT-single-minded; PDP1ε, PAR domain protein1ε; PER, period; PEST, proline, glutamic acid, serine, and threonine; PP, protein phosphotase; PRR, pseudo-response regulator; ROR, retinoic acid-related orphan receptor; SGG, shaggy; SLMB, supernumerary limbs (SLIMB); TIM, timeless; TOC1, timing of CAB expression 1; VRI, vrille; VVD, vivid; WC, white collar; ZTL, zeitlupe REFERENCES Bell-Pedersen, D., Cassone, V.M., Earnest, D.J., Golden, S.S., Hardin, P.E., Thomas, T.L., and Zoran, M.J. 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