Quantitative live imaging of Venus::BMAL1 in a mouse model reveals complex dynamics of the master circadian clock regulator

Autoři: Nan Yang aff001;  Nicola J. Smyllie aff003;  Honor Morris aff001;  Cátia F. Gonçalves aff001;  Michal Dudek aff001;  Dharshika Pathiranage aff001;  Johanna E. Chesham aff003;  Antony Adamson aff002;  David Spiller aff002;  Egor Zindy aff002;  James Bagnall aff002;  Neil Humphreys aff002;  Judith Hoyland aff002;  Andrew S. I. Loudon aff002;  Michael H. Hastings aff003;  Qing-Jun Meng aff001;  Dharshika R. J. Pathiranage aff001;  David G. Spiller aff002
Působiště autorů: Wellcome Centre for Cell Matrix Research, University of Manchester, Manchester, United Kingdom aff001;  Faculty of Biology, Medicine and Health, University of Manchester, Manchester, United Kingdom aff002;  Division of Neurobiology, Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, United Kingdom aff003;  NIHR Manchester Musculoskeletal Biomedical Research Centre, Manchester University NHS Foundation Trust, Manchester Academic Health Science Centre, Manchester, United Kingdom aff004
Vyšlo v časopise: Quantitative live imaging of Venus::BMAL1 in a mouse model reveals complex dynamics of the master circadian clock regulator. PLoS Genet 16(4): e32767. doi:10.1371/journal.pgen.1008729
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pgen.1008729


Evolutionarily conserved circadian clocks generate 24-hour rhythms in physiology and behaviour that adapt organisms to their daily and seasonal environments. In mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus is the principal co-ordinator of the cell-autonomous clocks distributed across all major tissues. The importance of robust daily rhythms is highlighted by experimental and epidemiological associations between circadian disruption and human diseases. BMAL1 (a bHLH-PAS domain-containing transcription factor) is the master positive regulator within the transcriptional-translational feedback loops (TTFLs) that cell-autonomously define circadian time. It drives transcription of the negative regulators Period and Cryptochrome alongside numerous clock output genes, and thereby powers circadian time-keeping. Because deletion of Bmal1 alone is sufficient to eliminate circadian rhythms in cells and the whole animal it has been widely used as a model for molecular disruption of circadian rhythms, revealing essential, tissue-specific roles of BMAL1 in, for example, the brain, liver and the musculoskeletal system. Moreover, BMAL1 has clock-independent functions that influence ageing and protein translation. Despite the essential role of BMAL1 in circadian time-keeping, direct measures of its intra-cellular behaviour are still lacking. To fill this knowledge-gap, we used CRISPR Cas9 to generate a mouse expressing a knock-in fluorescent fusion of endogenous BMAL1 protein (Venus::BMAL1) for quantitative live imaging in physiological settings. The Bmal1Venus mouse model enabled us to visualise and quantify the daily behaviour of this core clock factor in central (SCN) and peripheral clocks, with single-cell resolution that revealed its circadian expression, anti-phasic to negative regulators, nuclear-cytoplasmic mobility and molecular abundance.

Klíčová slova:

Cartilage – Circadian oscillators – Circadian rhythms – Fluorescence imaging – Fluorescence recovery after photobleaching – Chondrocytes – Chronobiology – Mice


1. Hastings M.H., Reddy A.B., Maywood E.S. (2003). A clockwork web: circadian timing in brain and periphery, in health and disease. Nat Rev Neurosci. 4, 649–661. doi: 10.1038/nrn1177 12894240

2. Partch C.L., Green C.B., Takahashi J.S. (2014). Molecular architecture of the mammalian circadian clock. Trends Cell Biol. 24, 90–99. doi: 10.1016/j.tcb.2013.07.002 23916625

3. Reppert S.M., Weaver D.R. (2002). Coordination of circadian timing in mammals. Nature 418, 935–941. doi: 10.1038/nature00965 12198538

4. Bass J., Takahashi J.S. (2010). Circadian integration of metabolism and energetics. Science 330, 1349–1354. doi: 10.1126/science.1195027 21127246

5. Shearman L.P., Sriram S., Weaver D.R., Maywood E.S., Chaves I., Zheng B., Kume K., Lee C.C., van der Horst G.T.J., Hastings M.H., and Reppert S.M. (2000). Interacting molecular loops in the mammalian circadian clock. Science 288, 1013–1019. doi: 10.1126/science.288.5468.1013 10807566

6. McDearmon E.L., Patel K.N., Ko C.H., Walisser J.A., Schook A.C., Chong J.L., Wilsbacher L.D., Song E.J., Hong H.K., Bradfield C.A., and Takahashi J.S. (2006). Dissecting the functions of the mammalian clock protein BMAL1 by tissue-specific rescue in mice. Science 314, 1304–1308. doi: 10.1126/science.1132430 17124323

7. Storch K.F., Paz C., Signorovitch J., Raviola E., Pawlyk B., Li T., Weitz C.J. (2007). Intrinsic circadian clock of the mammalian retina: importance for retinal processing of visual information. Cell 130, 730–741. doi: 10.1016/j.cell.2007.06.045 17719549

8. Lipton J.O., Yuan E.D., Boyle L.M., Ebrahimi-Fakhari D., Kwiatkowski E., Nathan A., Güttler T., Davis F., Asara J.M., and Sahin M. (2015). The Circadian Protein BMAL1 Regulates Translation in Response to S6K1-Mediated Phosphorylation. Cell 161, 1138–1151. doi: 10.1016/j.cell.2015.04.002 25981667

9. Michael A.K., Asimgil H., and Partch C.L. (2015). Cytosolic BMAL1 moonlights as a translation factor. Trends Biochem. Sci. 40, 489–490. doi: 10.1016/j.tibs.2015.07.006 26256246

10. Kiyohara Y.B., Tagao S., Tamanini F., Morita A., Sugisawa Y., Yasuda M., Yamanaka I., Ueda H.R., van der Horst G.T., Kondo T., and Yagita K. (2006). The BMAL1 C terminus regulates the circadian transcription feedback loop. Proc. Natl. Acad. Sci. USA. 103, 10074–10079. doi: 10.1073/pnas.0601416103 16777965

11. Xu H., Gustafson C.L., Sammons P.J., Khan S.K., Parsley N.C., Ramanathan C., Lee H.W., Liu A.C., Partch C.L. (2015). Cryptochrome 1 regulates the circadian clock through dynamic interactions with the BMAL1 C terminus. Nat. Struct. Mol. Biol. 22, 476–484. doi: 10.1038/nsmb.3018 25961797

12. Smyllie N.J., Pilorz V., Boyd J., Meng Q.J., Saer B., Chesham J.E., Maywood E.S., Krogager T.P., Spiller D.G., Boot-Handford R., et al. (2016). Visualizing and Quantifying Intracellular Behavior and Abundance of the Core Circadian Clock Protein PERIOD2. Curr. Biol. 26, 1880–1886. doi: 10.1016/j.cub.2016.05.018 27374340

13. Karatsoreos I.N., Yan L., LeSauter J., and Silver R. (2004). Phenotype matters: identification of light-responsive cells in the mouse suprachiasmatic nucleus. J. Neurosci. 24, 68–75. doi: 10.1523/JNEUROSCI.1666-03.2004 14715939

14. Dudek M., Gossan N., Yang N., Im H.J., Ruckshanthi J.P., Yoshitane H., Li X., Jin D., Wang P., Boudiffa M., et al. (2016). The chondrocyte clock gene Bmal1 controls cartilage homeostasis and integrity. J. Clin. Invest. 126, 365–376. doi: 10.1172/JCI82755 26657859

15. Dudek M., Yang N., Ruckshanthi J.P., Williams J., Borysiewicz E., Wang P., Adamson A., Li J., Bateman J.F., White M.R., et al. (2017). The intervertebral disc contains intrinsic circadian clocks that are regulated by age and cytokines and linked to degeneration. Ann. Rheum. Dis. 76, 576–584. doi: 10.1136/annrheumdis-2016-209428 27489225

16. Aryal R.P., Kwak P.B., Tamayo A.G., Gebert M., Chiu P.L., Walz T., and Weitz C.J. (2017). Macromolecular Assemblies of the Mammalian Circadian Clock. Mol. Cell. 67, 770–782.e6. doi: 10.1016/j.molcel.2017.07.017 28886335

17. von Gall C., Noton E., Lee C., and Weaver D.R. (2003). Light does not degrade the constitutively expressed BMAL1 protein in the mouse suprachiasmatic nucleus. Eur. J. Neurosci. 18, 125–133. doi: 10.1046/j.1460-9568.2003.02735.x 12859345

18. Liu A.C., Tran H.G., Zhang E.E., Priest A.A., Welsh D.K., and Kay S.A. (2008) Redundant function of REV-ERBalpha and beta and non-essential role for Bmal1 cycling in transcriptional regulation of intracellular circadian rhythms. PLoS. Genet. 4, e1000023. doi: 10.1371/journal.pgen.1000023 18454201

19. Koike N., Yoo S.H., Huang H.C., Kumar V., Lee C., Kim T.K., and Takahashi J.S. (2012). Transcriptional architecture and chromatin landscape of the core circadian clock in mammals. Science 338, 349–354. doi: 10.1126/science.1226339 22936566

20. Carmo-Fonseca M. (2002). The contribution of nuclear compartmentalization to gene regulation. Cell 108, 513–521. doi: 10.1016/s0092-8674(02)00650-5 11909522

21. Raccaud M., and Suter D.M. (2018). Transcription factor retention on mitotic chromosomes: regulatory mechanisms and impact on cell fate decisions. FEBS Lett. 592, 878–887. doi: 10.1002/1873-3468.12828 28862742

22. Gaucher J., Montellier E., and Sassone-Corsi P. (2018). Molecular Cogs: Interplay between Circadian Clock and Cell Cycle. Trends Cell Biol. 28, 368–379. doi: 10.1016/j.tcb.2018.01.006 29471986

23. Nagoshi E., Saini C., Bauer C., Laroche T., Naef F., and Schibler U. (2004). Circadian gene expression in individual fibroblasts: cell-autonomous and self-sustained oscillators pass time to daughter cells. Cell 119, 693–705. doi: 10.1016/j.cell.2004.11.015 15550250

24. Kwon I., Lee J., Chang S.H., Jung N.C., Lee B.J., Son G.H., Kim K., and Lee K.H. (2006). BMAL1 shuttling controls transactivation and degradation of the CLOCK/BMAL1 heterodimer. Mol. Cell. Biol. 26, 7318–7330. doi: 10.1128/MCB.00337-06 16980631

25. Kondratov R.V., Chernov M.V., Kondratova A.A., Gorbacheva V.Y., Gudkov A.V., and Antoch M.P. (2003). BMAL1-dependent circadian oscillation of nuclear CLOCK: posttranslational events induced by dimerization of transcriptional activators of the mammalian clock system. Genes Dev. 17, 1921–1932. doi: 10.1101/gad.1099503 12897057

26. Gossan N., Zeef L., Hensman J., Hughes A., Bateman J.F., Rowley L., Little C.B., Piggins H.D., Rattray M., Boot-Handford R.P., and Meng Q.J. (2013). The circadian clock in murine chondrocytes regulates genes controlling key aspects of cartilage homeostasis. Arthritis Rheum. 65, 2334–2345. doi: 10.1002/art.38035 23896777

27. Godinho S.I., Maywood E.S., Shaw L., Tucci V., Barnard A.R., Busino L., Pagano M., Kendall R., Quwailid M.M., Romero M.R., et al. (2007). The after-hours mutant reveals a role for Fbxl3 in determining mammalian circadian period. Science 316, 897–900. doi: 10.1126/science.1141138 17463252

28. Lee C., Etchegaray J.P., Cagampang F.R., Loudon A.S., and Reppert S.M. (2001). Posttranslational mechanisms regulate the mammalian circadian clock. Cell 107, 855–867. doi: 10.1016/s0092-8674(01)00610-9 11779462

29. Maywood E.S., Chesham J.E., Meng Q.J., Nolan P.M., Loudon A.S.I., and Hastings M.H. (2011). Tuning the Period of the Mammalian Circadian Clock: Additive and Independent Effects of CK1 epsilon(Tau) and Fbxl3(Afh) Mutations on Mouse Circadian Behavior and Molecular Pacemaking. J. Neurosci. 31, 1539–1544. doi: 10.1523/JNEUROSCI.4107-10.2011 21273438

30. Meng Q.J., Logunova L., Maywood E.S., Gallego M., Lebiecki J., Brown T.M., Sládek M., Semikhodskii A.S., Glossop N.R.J., Piggins H.D., et al. (2008). Setting clock speed in mammals: the CK1 epsilon tau mutation in mice accelerates circadian pacemakers by selectively desta- bilizing PERIOD proteins. Neuron 58, 78–88. doi: 10.1016/j.neuron.2008.01.019 18400165

31. Siepka S.M., Yoo S.H., Park J., Lee C., and Takahashi J.S. (2007). Genetics and neurobiology of circadian clocks in mammals. Cold Spring Harb. Symp. Quant. Biol. 72, 251–259. doi: 10.1101/sqb.2007.72.052 18419282

32. Etchegaray J.P., Lee C., Wade P.A., and Reppert S.M. (2003). Rhythmic histone acetylation underlies transcription in the mammalian circadian clock. Nature 421, 177–182. doi: 10.1038/nature01314 12483227

33. Griffin E.A. Jr., Staknis D., and Weitz C.J. (1999). Light-independent role of CRY1 and CRY2 in the mammalian circadian clock. Science. 286, 768–771. doi: 10.1126/science.286.5440.768 10531061

34. Kume K., Zylka M.J., Sriram S., Shearman L.P., Weaver D.R., Jin X., Maywood E.S., Hastings M.H., and Reppert S.M. (1999). mCRY1 and mCRY2 are essential components of the negative limb of the circadian clock feedback loop. Cell 98, 193–205. doi: 10.1016/s0092-8674(00)81014-4 10428031

35. Pett J.P., Korenčič A., Wesener F., Kramer A., and Herzel H. (2016). Feedback Loops of the Mammalian Circadian Clock Constitute Repressilator. PLoS Comput. Biol. 12, e1005266. doi: 10.1371/journal.pcbi.1005266 27942033

36. Sekiya T., Muthurajan U.M., Luger K., Tulin A.V., and Zaret K.S. (2009). Nucleosome-binding affinity as a primary determinant of the nuclear mobility of the pioneer transcription factor FoxA. Genes Dev. 23, 804–809. doi: 10.1101/gad.1775509 19339686

37. Narumi R., Shimizu Y., Ukai-Tadenuma M., Ode K.L., Kanda G.N., Shinohara Y., Sato A., Matsumoto K., Ueda H.R. (2016). Mass spectrometry-based absolute quantification reveals rhythmic variation of mouse circadian clock proteins. Proc. Natl. Acad. Sci. USA. 113, E3461–3467. doi: 10.1073/pnas.1603799113 27247408

38. Stratmann M., Suter D.M., Molina N., Naef F., and Schibler U. (2012). Circadian Dbp transcription relies on highly dynamic BMAL1-CLOCK interaction with E boxes and requires the proteasome. Mol. Cell. 48, 277–287. doi: 10.1016/j.molcel.2012.08.012 22981862

39. Mirsky H.P., Liu A.C., Welsh D.K., Kay S.A., and Doyle F.J. 3rd. (2009). A model of the cell-autonomous mammalian circadian clock. Proc. Natl. Acad. Sci. USA. 106, 11107–11112. doi: 10.1073/pnas.0904837106 19549830

40. Yoo S.H., Yamazaki S., Lowrey P.L., Shimomura K., Ko C.H., Buhr E.D., Siepka S.M., Hong H.K., Oh W.J., Yoo O.J., et al. (2004). PERIOD2:LUCIFERASE real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues. Proc. Natl. Acad. Sci. USA 101, 5339–5346. doi: 10.1073/pnas.0308709101 14963227

41. Shen B., Zhang W., Zhang J., Zhou J., Wang J., Chen L., Wang L., Hodgkins A., Iyer V., Huang X., Skarnes W.C.(2014). Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Nat Methods. 11(4), 399–402. doi: 10.1038/nmeth.2857 24584192

42. Waldo G., Standish B., Berendzen J., Terwilliger T. (1999). Rapid protein-folding assay using green fluorescent protein, Nat. Biotechnol. 17, 691–695. doi: 10.1038/10904 10404163

43. Reddy A.B., Karp N.A., Maywood E.S., Sage E.A., Deery M., O'Neill J.S., Wong G.K., Chesham J, Odell M., Lilley K.S., Kyriacou C.P., Hastings M.H. (2006). Circadian orchestration of the hepatic proteome. Curr Biol. 16(11),1107–1115. doi: 10.1016/j.cub.2006.04.026 16753565

44. Yang N., Williams J., Pekovic-Vaughan V., Wang P., Olabi S., McConnell J., Gossan N., Hughes A., Cheung J., Streuli C. H. et al. (2017). Cellular mechano-environment regulates the mammary circadian clock. Nat. Commun. 8, 14287. doi: 10.1038/ncomms14287 28134247

45. Zielinski T., Moore A.M., Troup E., Halliday K.J., Millar A.J. (2014). Strengths and Limitations of Period Estimation Methods for Circadian Data. PLoS ONE 9(5), e96462. doi: 10.1371/journal.pone.0096462 24809473

46. Thirion S. and Berenbaum F (2004). Culture and phenotyping of chondrocytes in primary culture. Methods Mol Med. 100, 1–14. doi: 10.1385/1-59259-810-2:001 15280583

47. Xu J (2005). Preparation, culture, and immortalization of mouse embryonic fibroblasts. Current Protocols in Molecular Biology, Chapter 28: Unit 28.1. doi: 10.1002/0471142727.mb2801s70.

48. Dorostkar M.M., Dreosti E., Odermatt B., and Lagnado L. (2010). Computational processing of optical measurements of neuronal and synaptic activity in networks. J Neurosci Meth 188, 141–150.

Článek vyšel v časopise

PLOS Genetics

2020 Číslo 4
Nejčtenější tento týden
Nejčtenější v tomto čísle

Zvyšte si kvalifikaci online z pohodlí domova

Úloha kombinovaných preparátů v léčbě arteriální hypertenze
nový kurz
Autoři: prof. MUDr. Martin Haluzík, DrSc.

Třikrát z interní medicíny
Autoři: Mgr. Jana Kubátová, Ph.D.

Pokročilá Parkinsonova nemoc − úskalí a možnosti léčby
Autoři: doc. MUDr. Marek Baláž, Ph.D.

Léčba diabetes mellitus 2. typu pomocí GLP- 1 RA

Depresivní porucha a zánětlivé procesy
Autoři: MUDr. Juraj Tkáč

Všechny kurzy
Zapomenuté heslo

Zadejte e-mailovou adresu, se kterou jste vytvářel(a) účet, budou Vám na ni zaslány informace k nastavení nového hesla.


Nemáte účet?  Registrujte se