V11 Circadian clocks in mammals and plants

1. V11 Circadian clocks in mammals and plants Most organisms (animals, plants, fungi and cyanobacteria) enhance their fitness by coordinating their development with daily environmental changes through molecular timekeepers (circadian clocks) Mammals display circadian rhythms in behavioural and physiological processes, such as - sleep - feeding - blood pressure and - metabolism Roles in plants e.g.: - opening of flowers in the morning and their closure at night Circadian rhythms are guided by external light–dark signals that are integrated through intrinsic central and peripheral molecular clocks McClung Plant Cell 18, 792 (2006) SS 2009 lecture 11 Biological Sequence Analysis 1

2. Circadian rhythms (1) Circadian rhythms are the subset of biological rhythms with period of 24 h. The term circadian combines the Latin words ‘‘circa’’ (about) and ‘‘dies’’ (day). (2) Circadian rhythms are endogenously generated and self-sustaining. They persist under constant environmental conditions, typically constant light (or dark) and constant temperature. Under these controlled conditions, the free-running period of 24 h is observed. (3) For all circadian rhythms the period remains relatively constant over a range of ambient temperatures. This is thought to be one property of a general mechanism that buffers the clock against changes in cellular metabolism. What effect does temperature usually have on chemical reactions? McClung Plant Cell 18, 792 (2006) Biological Sequence Analysis

3. Essential elements of biological clocks Our biological clocks contain 3 essential elements: (1) a central oscillator that keeps time; (2) the ability to sense time cues in the environment and to reset the clock as the seasons change; and (3) a series of outputs tied to distinct phases of the oscillator that regulate activity and physiology. Gallego et al. Nat.Rev.Mol.Cell.Biol. 8, 140 (2007) SS 2009 lecture 11 Biological Sequence Analysis 3

4. Parameters of Circadian clocks Period : time to complete one cycle. Amplitude of the rhythm : one-half the peak-to-trough distance. Phase : time of day for any given event. E.g. if the peak in a rhythm occurred at dawn, the phase of the peak would be defined as 0 h. Phase is often defined in zeitgeber time (ZT). Zeitgeber is German for „time giver“, and any stimulus that imparts time information to the clock is a zeitgeber. The onset of light is a powerful zeitgeber, and dawn is defined as ZT0. McClung Plant Cell 18, 792 (2006) Biological Sequence Analysis

5. Suprachiasmatic nucleus (SCN) In mammals, the central clock resides in the suprachiasmatic nucleus (SCN), a small region of the brain that contains ca. 20,000 neurons. The SCN produces a rhythmic output that consists of a multitude of neural and hormonal signals that influence sleep and activity. Most importantly, the SCN signals set the peripheral clocks present throughout the body. The SCN clock is reset by external light, which is sensed by the ganglion cells of the retina. Gallego et al. Nat.Rev.Mol.Cell.Biol. 8, 140 (2007) SS 2009 lecture 11 Biological Sequence Analysis 5

6. Autonomous oscillators everywhere Remarkably, autonomous circadian oscillators are also present in all tissues of the body, where they are synchronized by unidentified signals to regulate, in a tissue-specific manner, transcriptional activity throughout the day. Paolo Sassone-Corsi, UC Irvine Eckel-Mahan & Sassone-Corsi, Nat. Struct. Mol. Biol. 16, 462 (2009) SS 2009 lecture 11 Biological Sequence Analysis 6

7. Basic molecular elements of the mammalian clock (a) 2 TFs CLOCK and BMAL1 heterodimerize. (b) BMA1:CLOCK binds to the E-boxes in the promoters of the PER and CRY genes, as well as in the clock-controlled genes, activating their transcription. (c) The PER and CRY proteins dimerize, enter the nucleus and inhibit CLOCK-BMAL1–activated transcription. This is the minimal scheme for the mammalian clock. It requires several interconnecting transcriptional, translational and post-translational loops to achieve gene expression with circadian periodicity Sancar, Nat. Struct. Mol. Biol. 15, 23 (2008) SS 2009 lecture 11 Biological Sequence Analysis 7

8. Full (?) circuit of circadian rhythms in mammals PER: period CRY: cryptochrome CK1: casein kinase Rev-erb, ROR: retinoic acid- related orphan nuclear receptors Cdg: clock-controlled gene(s) Ko & Takahashi Hum Mol Genet 15, R271 (2006) Biological Sequence Analysis

9. Circadian clocks in Arabidopsis thaliana Plants were the first organisms for which the observation of a circadian rhythm was published (de Mairan, 1729). The molecular study of plant clocks began in 1985 with the observation that the mRNA abundance of the light-harvesting chlorophyll a/b-binding protein genes (LHCB) of peas oscillated with a circadian rhythm. This is still the most extensively studied clock-regulated gene in Arabidopsis. Salomé et al. J. Biol. Rhythms 19, 425 (2004) Biological Sequence Analysis

10. Key players in Arabidopsis thaliana LHCB transcription is induced by light and shows a circadian pattern of expression with a peak in the middle of the subjective day. The red-light photoreceptors, the phytochromes (PHY), mediate the light induction of LHCB through a motif in the LHCB promoter. Comment: LHs absorb maximally at 850 nm (red light). Minimal promoter fragments necessary and sufficient for light and circadian regulation of LHCB were identified. Tobin’s group identified a protein with affinity to this promoter fragment. This TF was named CCA1 for CIRCADIAN CLOCK ASSOCIATED 1. LATE ELONGATED HYPOCOTYL (LHY) is another gene encoding a protein closely related to CCA1. Salomé et al. J. Biol. Rhythms 19, 425 (2004) Biological Sequence Analysis

11. Model of the Arabidopsis thaliana oscillator Light perceived by the PHYs and CRYs induces the expression of 2 transcription factors, CCA1and LHY. CCA1 and LHY mRNA abundance peaks shortly after dawn. CCA1 requires phosphorylation by CK2 prior to binding to DNA. PRR9, PRR7, PRR5, and PRR3 show clock-regulated mRNA abundances, peaking in that sequence at 2-h intervals throughout the day. Salomé et al. J. Biol. Rhythms 19, 425 (2004) Biological Sequence Analysis

12. Model of the Arabidopsis thaliana oscillator One known target of the repressive activity of CCA1 and LHY is TOC1 (Timing of Cab Expression 1), with the result that TOC1 (RRR1) mRNA abundance peaks around dusk, following the turnover of CCA1 and LHY proteins. TOC1 then feeds back onto CCA1 and LHY and induces their expression for the next cycle. TOC1 may require a DNA-binding protein as a cofactor, as it is not predicted to directly bind to DNA. Salomé et al. J. Biol. Rhythms 19, 425 (2004) SS 2009 lecture 11 Biological Sequence Analysis 12

13. Model of the Arabidopsis thaliana oscillator TOC1 degradation is mediated by the F-box protein ZTL (Zeitlupe = slow motion), whose activity is negatively regulated by light. CCA1 and LHY also negatively regulate their own promoters, possibly directly but possibly indirectly via TOC1. Light resetting may involve induction of CCA1 and LHY, possibly mediated through phytochrome and cryptochrome photoreceptors and PIF and PIF-like (PIL) transcription factors. Salomé et al. J. Biol. Rhythms 19, 425 (2004) SS 2009 lecture 11 Biological Sequence Analysis 13

14. Detect unknown control mechanisms:Probe gene expression by microarrays Harmer et al. used oligonucleotide-based arrays to determine steady-state mRNA levels in Arabidopsis at 4-hour intervals during the subjective day and night.  identify temporal patterns of gene expression in Arabidopsis plants under constant light conditions using GeneChip arrays representing about 8200 different genes. This is done by scoring genes with a greater than 95% probable correlation with a cosine test wave with a period between 20 and 28 hours were as circadian-regulated. How is this done? Give formula ...  453 genes (6% of the genes on the chip) were classified as cycling. Harmer et al. Science 290, 2110 (2000) Biological Sequence Analysis

15. Photosynthesis genes peak near the middle of the day • Results after normalization of peak maximum. • LHCA genes are in blue;. LHCB genes are in pink;. • (B) Photosystem I genes are in red;. Photosystem II genes are in green;. • (C) Model for function of photosynthesis gene products in photosystems II (left) and I (right). Colors of proteins match colors of corresponding gene traces. Harmer et al. Science 290, 2110 (2000) Biological Sequence Analysis

16. Energy conversion: Photosynthetic Unit Structure suggested by force field based molecular docking. http://www.ks.uiuc.edu/Research/vmd/gallery Bioinformatics III

17. Synchronized production of photoprotective pigments „Phenolic sunscreen“ Substances absorb light in the visible and UV range. Harmer et al. Science 290, 2110 (2000) Biological Sequence Analysis

18. Circadian regulation of sugar metabolism Genes encoding starch-mobilizing enzymes peak during the subjective night. Why? Plant stores starch in chloroplast for use during the night when the plant cannot do photosynthesis. (A) Cycling genes encode a putative starch kinase that is related to potato R1 protein (dark blue); a β-amylase (gold); fructose-bisphosphate aldolase, (red); a putative sugar transporter (light blue); and a sucrose-phosphate synthase homolog (green). (B) Model for the enzymatic functions of these gene products in the mobilization of starch. Colored arrows indicate the function of the corresponding gene indicated in (A). The chloroplast is bounded by a green box and the cytoplasm by a black box. Harmer et al. Science 290, 2110 (2000) Biological Sequence Analysis

19. Chilling resistance Chilling resistance is an important trait in plants. A number of enzymes involved in lipid modification, including two desaturases, were found to be under clock regulation and peaked near subjective dusk. This is consistent with previously observed rhythms in membrane lipid desaturation levels that correlate with increased resistance to cold treatments during the subjective night. Speculate about mechanism ... Gallego et al. Nat.Rev.Mol.Cell.Biol. 8, 140 (2007) Biological Sequence Analysis

20. Genes implicated in cell elongation are circadian-regulated (B) Proposed mode of action of the products of these clock-controlled genes in cell wall remodeling. The rigid plant cell wall normally prevents cell expansion, but a simultaneous loosening of cell wall components, uptake of water, and synthesis of cell wall components seems allowed. (A) Genes encoding the auxin efflux carriers PIN3 and PIN7 (red), a putative expansin (green), a putative polygalacturonase (light blue), and aquaporin d-TIP (dark blue) all peak toward the end of the subjective day. Auxins are phytohormones – they regulate cell extension. 3 enzymes implicated in cell wall synthesis (all in gold) peak toward the end of the subjective night. Harmer et al. Science 290, 2110 (2000) Biological Sequence Analysis

21. Master regulator sequence of circadian-regulated genes? Check genomic DNA regions upstream of cycling genes for overrepresented promoter elements  absolutely conserved motif, AAAATATCT “evening element,” that occurs 46 times in the promoters of 31 cycling genes. All genes demonstrated impressive coregulation. All but one peak toward the end of the subjective day. Mutation of the conserved AAAATATCT, but not a closely related motif, greatly reduced the ability of a promoter to confer circadian rhythmicity on a luciferase reporter gene in plants. Harmer et al. Science 290, 2110 (2000) Biological Sequence Analysis

22. Review: Feedback loops control the mammalian circadian core clock Gallego et al. Nat.Rev.Mol.Cell.Biol. 8, 140 (2007) The mammalian circadian rhythms core clock is a transcription–translation negative-feedback loop with a delay between transcription and the negative feedback. It is initiated by a heterodimeric transcription factor that consists of CLOCK and BMAL1. CLOCK and BMAL1 drive expression of their own negative regulators, the period proteins PER1 and PER2 and the cryptochromes CRY1 and CRY2. Over the course of the day, the PER and CRY proteins accumulate and multimerize in the cytoplasm, where they are phosphorylated by casein kinase Iε (CKIε) and glycogen synthase kinase-3 (GSK3). They then translocate to the nucleus in a phosphorylation-regulated manner where they interact with the CLOCK–BMAL1 complex to repress their own activator. At the end of the circadian cycle, the PER and CRY proteins are degraded in a CKI-dependent manner, which releases the repression of the transcription and allows the next cycle to start. An additional stabilizing feedback loop, which involves the activator Rora and the inhibitor Rev-Erbα, controls BMAL1 expression and reinforces the oscillations. RRE, R-response element. Biological Sequence Analysis

23. Circadian clock in D. melanogaster (1) Clock (CLK) and cycle (CYC) activate the transcription of the circadian genes in D. melanogaster. (2) Period (PER) and timeless (TIM) form heterodimers in the cytoplasm where they are phosphorylated by double-time (DBT) and shaggy (SGG). (3) PER and TIM then translocate to the nucleus where PER inhibits the transcriptional activity of the CLK–CYC complex. (4) Similarly to the mammalian clock, a number of kinases regulate PER and TIM. (5) In the stabilizing loop, the protein vrille (VRI) inhibits, whereas PAR-domain protein-1 (PDP1) activates the transcription of Clk. Gallego et al. Nat.Rev.Mol.Cell.Biol. 8, 140 (2007) Biological Sequence Analysis

24. Why add phosphorylation to the clock? Why are post-transcriptional modifications of crucial importance? Transcription–translation feedback cycles generally operate on a timescale of up to a few hours. If, following synthesis, the repressor proteins PER and CRY translocated to the nucleus to repress CLOCK and BMAL1, the whole cycle would take just a few hours rather than one day. To maintain the daily oscillations of clock proteins, a significant delay between the activation and repression of transcription is required. This is ensured by regulation through post-translational modifications. Reversible phosphorylation regulates important processes such as nuclear entry, formation of protein complexes and protein degradation. Each of these can individually contribute to introduce the delay that keeps the period at ~24 hours. Gallego et al. Nat.Rev.Mol.Cell.Biol. 8, 140 (2007) SS 2009 lecture 11 Biological Sequence Analysis 24

25. Multiple roles of CK1 in the mammalian circadian clock Casein kinase I (CKI) has many roles in the circadian clock. a | It has a confusing role in regulating the nuclear localization of the circadian repression protein period (here PER1). In some cell types, CKI activity promotes the cytoplasmic accumulation of PER1, whereas in others it mediates the nuclear translocation of PER1. b | Phosphorylation of PER proteins increases over the course of the circadian day, peaking when the repression of the positive transcription factors CLOCK and BMAL1 is maximal. There are many CKI sites on PER proteins, but the function of only a subset of these sites is known. c | One clear function of the phosphorylation of PER proteins is the regulation of protein stability. Phosphorylation of one or two distinct sites on PER1 and PER2 target these proteins for ubiquitin-mediated degradation by the 26S proteasome. Degradation of PER proteins can reset the clock, allowing the CLOCK–BMAL1 complex to become active. d | PER and CRY proteins are not the only substrates of CKI in the clock. CKIε-mediated phosphorylation of the circadian regulator BMAL1 increases its transcriptional activity. Gallego et al. Nat.Rev.Mol.Cell.Biol. 8, 140 (2007) Biological Sequence Analysis

26. New: Dual roles of CLOCK acetyltransferase activity CLOCK acetylates (Ac) histones H3 and H4 in nucleosomes (green) to confer ‘open’ chromatin structure and enable CLOCK-BMAL1 to bind to the E-boxes in cognate promoters and turn on transcription. CLOCK also acetylates BMAL1, making it a target for binding of the CRY repressor, concomitant with deacetylation of histones by histone deacetylases (HDAC). These dual effects of acetylation by CLOCK contribute to circadian periodicity of gene expression. Sancar, Nat. Struct. Mol. Biol. 15, 23 (2008) SS 2009 lecture 11 Biological Sequence Analysis 26

27. Summary Most organisms enhance fitness by coordinating their development with daily environmental changes through molecular timekeepers known as circadian clocks. Clocks are generated by a transcription-translation negative feedback loop with a crucial delay between stimulus and response. This system of multiple connected loops increases the clock’s robustness and provides numerous points of input and output to the clock. Many metabolic pathways are regulated by circadian clocks in plants and animals. V12: Coupling of circadian clocks and metabolism! Kay & Schroeder Science 318, 1730 (2007) Biological Sequence Analysis

28. additional slides (not used) SS 2009 lecture 11 Biological Sequence Analysis 28

29. Circadian rhythm disorders Delayed sleep-phase syndrome (DSPS) Opposite to FASPS, DSPS causes late sleep-onset and the inability to wake up at a conventional time. A polymorphism in the human PER3 gene (V647G) has been linked to the pathogenesis of DSPS111. Residue 647 locates in a region similar to the CKIε-binding region of PER1 and PER2, close to the serine residue in PER2 that is disrupted by the FASPS mutation. Therefore, this polymorphism might alter the CKIε-dependent phosphorylation of human PER3. Familial advanced sleep-phase syndrome (FASPS) FASPS is a autosomal dominant human behavioural disorder that causes early sleep times, early morning awakening and a short circadian period. Genetic analysis in one family affected by FASPS identified a single amino-acid missense mutation in the human period-2 (PER2) gene as the cause of that sleep disorder variation. The mutation, an S662G change, is in the casein kinase Iε (CKIε)-binding domain of PER2 and decreases PER2 phosphorylation in vitro. Another Thr to Ala mutation in the human CKIδ gene was found in a family with FASPS. The mutation decreases the enzymatic activity of the kinase in vitro. Gallego et al. Nat.Rev.Mol.Cell.Biol. 8, 140 (2007) Biological Sequence Analysis

30. The clock mechanism in Neurospora crassa The white collar complex (WCC) activates the transcription of the frequency (frq) gene. The FRQ protein positively and negatively regulates the WCC. Homologous genes regulate the Drosophila melanogaster and vertebrate (Neurospora crassa ) clocks, although some details might differ. In the morning, FRQ interacting RNA helicase (FRH) and casein kinases I (CKI) and CKII promote the FRQ dependent phosphorylation and inactivation of the WCC, which results in the inhibition of frq transcription. In the evening, high amounts of hyperphosphorylated FRQ in the cytoplasm support the accumulation of WCC. At night, hyperphosphorylated FRQ is degraded, the repression on WCC is relieved and transcription of frq is activated. Gallego et al. Nat.Rev.Mol.Cell.Biol. 8, 140 (2007) Biological Sequence Analysis