Systems Biology of the Mammalian Circadian Clock

Transcription

1 Systems Biology of the Mammalian Circadian Clock Hanspeter Herzel Institute for Theoretical Biology (ITB) Humboldt University Berlin together with Pal Westermark, Kasia Bozek, Angela Relogio (ITB), Samuel Bernard (Lyon), Didier Gonze (Brussels), Achim Kramer group (experimental chronobiology, Charite), Hitoshi Okamura (Kyoto), Steven Brown (Zürich), David Welsh (San Diego)

2 Chronobiology Synchronization establishes stable phase-relations between geophysical, biological and social rhythms

3 Disturbed synchronization shiftwork accidents and higher incidence of cancer traveling across time zones jet lag Exxon Valdez: 24. März 1989 extreme chronotypes social difficulties

4 Models relevant on different levels 1. The clockwork: running wheel, suprachiasmatic nucleus, molecular oscillations, feedback loops, output 2. Modeling intracellular feedbacks: clock gene degradation, mechanisms of FASPS, extreme chronotypes 3. Robust synchronization of cells in silico an ensemble of driven damped oscillators clock controlled genes: DNA-arrays, promoters, transcription factors, metabolism 5. Outlook: chronotherapy (cell cycle models, xenobiotics)

5 Light synchronizes the clock SCN-neuron nucleus The system activation Positive elements Clock genes (e.g. Period2) inhibition Regulation of physiology and behavior Negative elements Synchronization of peripheral clocks

6 Molecular Chronobiology SCN-neuron activation positive elements nucleus 3 rd ventricle clock-genes (e.g. Period2) inhibition negative elements optic chiasm Oscillation Synchronization

7 The circadian oscillator Circadian rhythm Oscillations Feedback loops Oster et al., 2002 Reppert and Weaver, 2001

8 Simplified model of the circadian core oscillator S. Becker-Weimann, J. Wolf, H. Herzel, A. Kramer: Biophys. J. 87, (2004)

9 experiments Fibroblasts as experimental model of the circadianen oscillator genetic perturbations: RNA interference pharmakological perturbations: Inhibitores Luminescence [units] control anti-cry1 Relative Amplitude solvent CKIε inhibitor time [hrs] 96 time [hrs]

10 Degradation and phosphorylation of clock protein PER2 Kramer group: experimental data on molecular phenotype Pal Westermark (ITB): models with negative feedback loop

11 β-trcp1-mediated Degradation of PERIOD2 Is Essential for Circadian Dynamics Reischl et al., 2007 Journal of Biological Rhythms β-trcp1 downregulation long period β-trcp1 binding site mutation PER2 more stable circadian rhythms disrupted

12 Kinetic model reproduces large period (red), arrythmicity (orange) and predicts downregulation of output gene Dbp

13 Molecular Chronobiology Measurements mass spec. live cell imaging Entrainment 6 days 35/39 C Rel. luminescence Constant 37 C PER2 wt FASPS biochemistry +CHX PER2 wt β-actin FASPS β-actin h cell biology Time (days) Mathematical Model Model prediction Test of prediction Normalized concentration wild-type q 1 = 0 q 12 = Time (days) Rel. luminescence solvent CKI-7 (50 µm) CKI-7 (200 µm) Time (days) Vanselow et al., Genes & Dev, 2006

14 Molecular mechanisms of extreme chronotypes HO > 60 HO < 30

15 biopsy 25,5 SAD circadian promoter luciferase skin fibroblasts lentiviral infection Human period length 25 24,5 24 cpm , HO score hours Direct measurement of human circadian period! Brown et al., PNAS 2008

16 Fibroblast Period Length and Fibroblast Transcriptional Phase Correlate Under Entrained Conditions Brown et al., PNAS 2008

17 Mathematical modeling predicts that oscillator amplitude and circadian input intensity can also affect circadian phase Amplitude Effect of Light Brown et al., PNAS 2008

18 The Amplitude of Transcription of Clock Genes Varies in Fibroblasts from Different Individuals Brown et al., PNAS 2008

19 The real challenge: How to synchronize a network of heterogeneous limit cycle oscillators within a few cycles? Suprachiasmatic nucleus Located in the hypothalamus Contains about neurons Circadian pacemaker Two regions: - Ventro-lateral (VL): VIP, light-sensitive - Dorso-medial (DM): AVP

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21 mper1-luc bioluminescence in single SCN cells Experimental findings: - Synchronization is achieved within a few cycles - Phase relations are re-established after transient desynchronization - Driven DM region is phase leading

22 Organotypic SCN slices: periods of synchronized and desynchronized cells unpublished data from Hitoshi Okamura (Kyoto) analyzed by Pal Westermark

23 Comparison of synchronized and desynchronized cells Desynchronized cells exhibit: sync. -variable amplitudes and phases -higher noise level -ultradian periodicities desync. red: desynchronized cells

24 Coupling through the mean field Neurotransmitter Mean field

25 Coupling two cells through the mean field Synchronization requires delicate balance of coupling and period ratio

26 Coupling of neurons D. Gonze, S. Bernard, C. Waltermann, A. Kramer, H. Herzel: Biophys. J., 89, (2005)

27 Different topologies and coupling schemes within the SCN S. Bernard, D. Gonze, B. Cajavec, H. Herzel, and A. Kramer: Synchronization-Induced Rhythmicity of Circadian Oscillators in the Suprachiasmatic Nucleus, PLoS Comp. Biol. (2007) 3:e68.

28 How circadian oscillators can be synchronized quickly: Individual oscillators are (partly) damped The oscillating part of the mean field drives the damped oscillators Self-sustained oscillations and synchronization are linked via neurotransmitter ( community effect ) Predictions: Internal periods determine the phase relations and damping allows fast synchronizability

29 Experimental verification of predictions

30 Analysis of single cell data (Pal Westermark et al.) WT Fibroblasts SCN Neurons A.C. Liu et al. (2007) Cell (data provided by David Welsh)

31 WT (-/-) Cry2 Fibroblasts SCN Neurons A.C. Liu et al. (2007) Cell

32 WT (-/-) Cry2 Fibroblasts (-/-) Cry1 SCN Neurons A.C. Liu et al. (2007) Cell

33 WT (-/-) Cry2 Fibroblasts (-/-) Cry1 (-/-) Per1 SCN Neurons A.C. Liu et al. (2007) Cell

34 Linear AND nonlinear models fit most data well P. Westermark et al., in preparation

35 Quantification of single cell data Effectively Damped Selfsustained

36 Summary and discussion of part I mathematical models can describe intracellular clock based on transcriptional/translational feedback loops open problems: parameter estimations? origin of 6 h delay? which nonlinearities essential? possible synchronization mechanism: dampening of selfsustained single cell oscillations & forcing by periodic force open problems: alternative scenarios? (specific PRCs might allow quick and robust synchronization; pacemaker cells?) coupling mechanisms? (neurotransmitters versus synapses versus gap junctions)

37 Circadian rhythms of hormones, physiology, metabolism Fu & Lee: Nat. Rev. Cancer 3, 350 (2003)

38 5 selected microarray studies promoter sequences of: CCGs all mouse genes unified list of 2065 CCGs annotated with tissue, expression phase and amplitude 167 genes appearing in at least 3 lists 4 sublists of genes expressed periodically in different tissues within 4 phase intervals...

39 CCG promoters have relatively high GC-content Background model: we sample from all mouse promoters 100 times genes with the same GC-content and count TFBS in CCGs and background sets

40 each list of CCGs sampling of GC-matched background set search for TFBSs 100x count of predicted sites of each motif count of sites mean, standard deviation z-score selection of sites: over-represented vertebrate motifs clock-related

41 Counts of TFBS in background sets (black) and in 167 CCGs z=6.24 z=3.21 z=2.79 z=2.22 z=2.80 z=2.55 z=2.07 z=3.96

42 Over-represented TFBS E-box motifs E-box binding TFs D-box binding TFs

43 Over-represented TFBS D-box motifs E-box binding TFs D-box binding TFs

44 Over-represented TFBS (all known & novel TFs) Z-score in Overrepresentation in: Motif Consensus Sequence all CCGs Sel. genes Heart Liver SCN Muscle Sp1 DGGGYGGGVN 9.04 x x x x EGR E-box GYGGGSGSRRV binding TFs 8.58 x x x x x Pax-4 RNWAAWWRNNNNNNHN NNNNNNHHSAYHSB 7.06 x x KROX BBCGCCCMCDYNNM 6.65 x x ZF5 GSGCGCNR 6.6 x x x x AP-2 VDCCCSSVGRMS 6.35 x x x x C/EBP NNNHKNDGNAAN 5.86 x x x x x CRE-BP1 TTACGTAA 5.65 x x x x MEF-2 BTCTAAAAATAACYCY 5.57 x x x x x HMGIY HNDKNAWWTTNYYND 5.33 x x x Evi-1 DGATADGAHWRGATA 5.04 x x x x AHRHIF NRCGTGNNN 4.92 x x c-myc:max VSCAYGYGSN 4.91 x HLF RTTACRYMAY 4.74 x x x x x VBP RTTACRTMAK 4.27 x x x x E4BP4 NRTTAYGTAAYN 4.19 x x x x x TATA NCTATAAAAN 4.1 x x x Oct-1 D-box WNTATGBTAATT binding TFs 3.82 x HNF-1 RGTTAATNWTTRNMN 3.67 x x WT1 SVCHCCBVC 3.35 STAT5A NNNTTCYN 3.31 IRF BNNNSTTTCWNTTYY 3.3 x MEIS1A:HOXA9 TGACAGKTTWAYGA 3.29 Nrf-1 CGCRTGCGCR 3.28 x x AhR:Arnt GDBNATYGCGTGMSWD BCC 3.2 x x E2F TTTSGCGC 3.13 x x x

45 endocrine regulation LXR, AR, PR(GR), T3R, ERRα, XBP1, Pax-4, AP-2, Fox metabolism C/EBP, HNF4, SREBP-1, PPARγ, Evi-1, Nrf-1, IPF1, NF-Y, HIF-1 pharmacokinetics AhR, DBP, HLF, TEF, HTF, HSF1, Nrf1, Myc core clock CLOCK:BMAL1, RORα, CREB, DEC1, DBP, HLF, TEF, E4BP4 liver specific HNF1, HNF3, HNF4, C/EBP, HTF immune system STATs, IRF, NF-κB, GATA3, STAT4, HMGIY, AP1, NFAT muscle/heart specific MEF2, MyoD, Myogenin, SP1, SRF, TEF, Nkx2-5, GATA-4

46 Erythropoietin promoter with predicted TFBS False discovery rate fixed at 0.05 (Rahmann, Moeller, Vingron 2003) False discovery rate fixed at 0.05 (Rahmann, Moeller, Vingron 2003)

47 Another CCG: rhythmic transcription of Epo independent of Bmal1:Clock B 15 A relative mrna levels time after release in constant darkness [hrs] Quantitative PCR in murine kidney. filled: Epo. open: Per2 control relative transactivation activity Epo reporter Clock Bmal1 Hif1-α relative transactivation activity E-box reporter Clock Bmal1 0

48 Chronotherapy: clock + cell cycle + drug + metabolism Claudel et al (2007). FEBS Letters

49 It s a question of timing Chronotherapy aims to adapt the timing of drug administration according to the circadian rhythms of cancer and normal cells. Administration of drug at a circadian time when it is best tolerated can achieve best antitumour activity. Mormont and Lévi, 2003

50 Simulating chronotherapy Time course for two strengths of the clock Effectiveness of the treatment vs strength of the clock S. Bernard et al., in preparation

51 Current projects Details of PER2 phosphorylation, decay via ubiquitination and nuclear export/import (with A. Kramer group, A. Relogio) Diverse topologies and coupling mechanisms within the SCN (collaborations with H. Okamura, A. Herzog, D. Welsh) Optimization of kinetic parameters & jet lag simulation (J. Locke, P. Westermark, A. Relogio et al.) Phase response curves and synchronization (A. Granada et al.) Regulation of clock controlled genes (array data & promoter analysis K. Bozek et al.) Cyanobacterial (in vitro) clock phosphorylation of KaiC hexamers (Mol. Sys. Biol., 2007; 3:90)

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53 Synchronization of circadian clocks to light input Entrainment zone for different periods and coupling Phase-locking of internal variables (mrna peak) to sunset for night-active animals Problem: How can the internal clock follow changes of the photoperiod? Simulation & PRC: Small free running period & gating allows to track light offset F. Geier, S. Becker-Weimann, A. Kramer, H.Herzel: J. Biol. Rhythms, 20, (2005)