Cryptochrome 1 regulates the circadian clock through dynamic interactions with the BMAL1 C terminus

Abstract

The molecular circadian clock in mammals is generated from transcriptional activation by the bHLH-PAS transcription factor CLOCK–BMAL1 and subsequent repression by PERIOD and CRYPTOCHROME (CRY). The mechanism by which CRYs repress CLOCK–BMAL1 to close the negative feedback loop and generate 24-h timing is not known. Here we show that, in mouse fibroblasts, CRY1 competes for binding with coactivators to the intrinsically unstructured C-terminal transactivation domain (TAD) of BMAL1 to establish a functional switch between activation and repression of CLOCK–BMAL1. TAD mutations that alter affinities for co-regulators affect the balance of repression and activation to consequently change the intrinsic circadian period or eliminate cycling altogether. Our results suggest that CRY1 fulfills its role as an essential circadian repressor by sequestering the TAD from coactivators, and they highlight regulation of the BMAL1 TAD as a critical mechanism for establishing circadian timing.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Only BMAL1 can restore circadian rhythms in Bmal1−/− Per2Luc fibroblasts.
Figure 2: The BMAL1 C terminus is needed for circadian function.
Figure 3: A helical motif within the BMAL1 H domain controls circadian period length.
Figure 4: CBP (p300) and CRY1 interact with BMAL1 TAD.
Figure 5: BMAL1 TAD mutations decrease affinity for CBP (p300) and CRY1 and influence circadian period.
Figure 6: Distinct sites on CLOCK and BMAL1 facilitate repression by CRY1.
Figure 7: Regulation of the BMAL1 TAD by CRY1 contributes to determination of circadian period.

Accession codes

Primary accessions

Biological Magnetic Resonance Data Bank

Referenced accessions

Protein Data Bank

References

  1. 1

    Bass, J. Circadian topology of metabolism. Nature 491, 348–356 (2012).

    CAS  Article  Google Scholar 

  2. 2

    Partch, C.L., Green, C.B. & Takahashi, J.S. Molecular architecture of the mammalian circadian clock. Trends Cell Biol. 24, 90–99 (2014).

    CAS  Article  Google Scholar 

  3. 3

    Brown, S.A. et al. PERIOD1-associated proteins modulate the negative limb of the mammalian circadian oscillator. Science 308, 693–696 (2005).

    CAS  Article  Google Scholar 

  4. 4

    Duong, H.A., Robles, M.S., Knutti, D. & Weitz, C.J. A molecular mechanism for circadian clock negative feedback. Science 332, 1436–1439 (2011).

    CAS  Article  Google Scholar 

  5. 5

    Lee, C., Etchegaray, J., Cagampang, F., Loudon, A. & Reppert, S. Posttranslational mechanisms regulate the mammalian circadian clock. Cell 107, 855–867 (2001).

    CAS  Article  Google Scholar 

  6. 6

    Ye, R., Selby, C., Ozturk, N., Annayev, Y. & Sancar, A. Biochemical analysis of the canonical model for the mammalian circadian clock. J. Biol. Chem. 286, 25891–25902 (2011).

    CAS  Article  Google Scholar 

  7. 7

    Koike, N. et al. Transcriptional architecture and chromatin landscape of the core circadian clock in mammals. Science 338, 349–354 (2012).

    CAS  Article  Google Scholar 

  8. 8

    Stratmann, M., Stadler, F., Tamanini, F., van der Horst, G. & Ripperger, J. Flexible phase adjustment of circadian albumin D site-binding protein (DBP) gene expression by CRYPTOCHROME1. Genes Dev. 24, 1317–1328 (2010).

    CAS  Article  Google Scholar 

  9. 9

    Shearman, L.P. et al. Interacting molecular loops in the mammalian circadian clock. Science 288, 1013–1019 (2000).

    CAS  Article  Google Scholar 

  10. 10

    Ye, R. et al. Dual modes of CLOCK:BMAL1 inhibition mediated by Cryptochrome and Period proteins in the mammalian circadian clock. Genes Dev. 28, 1989–1998 (2014).

    CAS  Article  Google Scholar 

  11. 11

    Liu, A.C. et al. Intercellular coupling confers robustness against mutations in the SCN circadian clock network. Cell 129, 605–616 (2007).

    CAS  Article  Google Scholar 

  12. 12

    Evans, J.A., Pan, H., Liu, A.C. & Welsh, D.K. Cry1−/− circadian rhythmicity depends on SCN intercellular coupling. J. Biol. Rhythms 27, 443–452 (2012).

    Article  Google Scholar 

  13. 13

    Khan, S.K. et al. Identification of a novel cryptochrome differentiating domain required for feedback repression in circadian clock function. J. Biol. Chem. 287, 25917–25926 (2012).

    CAS  Article  Google Scholar 

  14. 14

    Huang, N. et al. Crystal structure of the heterodimeric CLOCK:BMAL1 transcriptional activator complex. Science 337, 189–194 (2012).

    CAS  Article  Google Scholar 

  15. 15

    Bunger, M.K. et al. Mop3 is an essential component of the master circadian pacemaker in mammals. Cell 103, 1009–1017 (2000).

    CAS  Article  Google Scholar 

  16. 16

    Shi, S. et al. Circadian clock gene Bmal1 is not essential; functional replacement with its paralog, Bmal2. Curr. Biol. 20, 316–321 (2010).

    CAS  Article  Google Scholar 

  17. 17

    Liu, A.C. et al. 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 (2008).

    Article  Google Scholar 

  18. 18

    Baggs, J.E. et al. Network features of the mammalian circadian clock. PLoS Biol. 7, e52 (2009).

    Article  Google Scholar 

  19. 19

    Hogenesch, J.B. et al. The basic helix-loop-helix-PAS protein MOP9 is a brain-specific heterodimeric partner of circadian and hypoxia factors. J. Neurosci. 20, RC83 (2000).

    CAS  Article  Google Scholar 

  20. 20

    Schoenhard, J.A. et al. Regulation of the PAI-1 promoter by circadian clock components: differential activation by BMAL1 and BMAL2. J. Mol. Cell. Cardiol. 35, 473–481 (2003).

    CAS  Article  Google Scholar 

  21. 21

    Kwon, I. et al. BMAL1 shuttling controls transactivation and degradation of the CLOCK/BMAL1 heterodimer. Mol. Cell. Biol. 26, 7318–7330 (2006).

    CAS  Article  Google Scholar 

  22. 22

    Kiyohara, Y.B. et al. The BMAL1 C terminus regulates the circadian transcription feedback loop. Proc. Natl. Acad. Sci. USA 103, 10074–10079 (2006).

    CAS  Article  Google Scholar 

  23. 23

    Czarna, A. et al. Structures of Drosophila cryptochrome and mouse cryptochrome1 provide insight into circadian function. Cell 153, 1394–1405 (2013).

    CAS  Article  Google Scholar 

  24. 24

    Czarna, A. et al. Quantitative analyses of cryptochrome-mBMAL1 interactions: mechanistic insights into the transcriptional regulation of the mammalian circadian clock. J. Biol. Chem. 286, 22414–22425 (2011).

    CAS  Article  Google Scholar 

  25. 25

    Sato, T.K. et al. Feedback repression is required for mammalian circadian clock function. Nat. Genet. 38, 312–319 (2006).

    CAS  Article  Google Scholar 

  26. 26

    Takahata, S. et al. Transactivation mechanisms of mouse clock transcription factors, mClock and mArnt3. Genes Cells 5, 739–747 (2000).

    CAS  Article  Google Scholar 

  27. 27

    Heery, D.M., Kalkhoven, E., Hoare, S. & Parker, M.G. A signature motif in transcriptional co-activators mediates binding to nuclear receptors. Nature 387, 733–736 (1997).

    CAS  Article  Google Scholar 

  28. 28

    Radhakrishnan, I. et al. Solution structure of the KIX domain of CBP bound to the transactivation domain of CREB: a model for activator:coactivator interactions. Cell 91, 741–752 (1997).

    CAS  Article  Google Scholar 

  29. 29

    Yuan, Q., Metterville, D., Briscoe, A. & Reppert, S. Insect cryptochromes: gene duplication and loss define diverse ways to construct insect circadian clocks. Mol. Biol. Evol. 24, 948–955 (2007).

    CAS  Article  Google Scholar 

  30. 30

    Wells, M. et al. Structure of tumor suppressor p53 and its intrinsically disordered N-terminal transactivation domain. Proc. Natl. Acad. Sci. USA 105, 5762–5767 (2008).

    CAS  Article  Google Scholar 

  31. 31

    Wang, Y. & Jardetzky, O. Probability-based protein secondary structure identification using combined NMR chemical-shift data. Protein Sci. 11, 852–861 (2002).

    CAS  Article  Google Scholar 

  32. 32

    Wishart, D.S. & Sykes, B.D. The 13C chemical-shift index: a simple method for the identification of protein secondary structure using 13C chemical-shift data. J. Biomol. NMR 4, 171–180 (1994).

    CAS  Article  Google Scholar 

  33. 33

    Chaves, I. et al. Functional evolution of the photolyase/cryptochrome protein family: importance of the C terminus of mammalian CRY1 for circadian core oscillator performance. Mol. Cell. Biol. 26, 1743–1753 (2006).

    CAS  Article  Google Scholar 

  34. 34

    Sugase, K., Dyson, H. & Wright, P. Mechanism of coupled folding and binding of an intrinsically disordered protein. Nature 447, 1021–1025 (2007).

    CAS  Article  Google Scholar 

  35. 35

    Felitsky, D.J., Lietzow, M.A., Dyson, H.J. & Wright, P.E. Modeling transient collapsed states of an unfolded protein to provide insights into early folding events. Proc. Natl. Acad. Sci. USA 105, 6278–6283 (2008).

    CAS  Article  Google Scholar 

  36. 36

    Gustafson, C.L. & Partch, C.L. Emerging models for the molecular basis of mammalian circadian timing. Biochemistry 54, 134–149 (2015).

    CAS  Article  Google Scholar 

  37. 37

    Zhao, W.-N. et al. CIPC is a mammalian circadian clock protein without invertebrate homologues. Nat. Cell Biol. 9, 268–275 (2007).

    CAS  Article  Google Scholar 

  38. 38

    Freedman, S.J. et al. Structural basis for negative regulation of hypoxia-inducible factor-1α by CITED2. Nat. Struct. Biol. 10, 504–512 (2003).

    CAS  Article  Google Scholar 

  39. 39

    Partch, C.L., Card, P.B., Amezcua, C.A. & Gardner, K.H. Molecular basis of coiled coil coactivator recruitment by the aryl hydrocarbon receptor nuclear translocator (ARNT). J. Biol. Chem. 284, 15184–15192 (2009).

    CAS  Article  Google Scholar 

  40. 40

    Partch, C.L. & Gardner, K.H. Coactivators necessary for transcriptional output of the hypoxia inducible factor, HIF, are directly recruited by ARNT PAS-B. Proc. Natl. Acad. Sci. USA 108, 7739–7744 (2011).

    CAS  Article  Google Scholar 

  41. 41

    Etchegaray, J.-P. et al. The polycomb group protein EZH2 is required for mammalian circadian clock function. J. Biol. Chem. 281, 21209–21215 (2006).

    CAS  Article  Google Scholar 

  42. 42

    Akashi, M. et al. A positive role for PERIOD in mammalian circadian gene expression. Cell Reports 7, 1056–1064 (2014).

    CAS  Article  Google Scholar 

  43. 43

    Chen, R. et al. Rhythmic PER abundance defines a critical nodal point for negative feedback within the circadian clock mechanism. Mol. Cell 36, 417–430 (2009).

    Article  Google Scholar 

  44. 44

    Nangle, S.N. et al. Molecular assembly of the period-cryptochrome circadian transcriptional repressor complex. eLife 3, e03674 (2014).

    Article  Google Scholar 

  45. 45

    Padmanabhan, K., Robles, M., Westerling, T. & Weitz, C. Feedback regulation of transcriptional termination by the mammalian circadian clock PERIOD complex. Science 337, 599–602 (2012).

    CAS  Article  Google Scholar 

  46. 46

    Etchegaray, J.-P. et al. Casein kinase 1 delta regulates the pace of the mammalian circadian clock. Mol. Cell. Biol. 29, 3853–3866 (2009).

    CAS  Article  Google Scholar 

  47. 47

    Lee, H.M. et al. The period of the circadian oscillator is primarily determined by the balance between casein kinase 1 and protein phosphatase 1. Proc. Natl. Acad. Sci. USA 108, 16451–16456 (2011).

    CAS  Article  Google Scholar 

  48. 48

    Hirschi, A. et al. An overlapping kinase and phosphatase docking site regulates activity of the retinoblastoma protein. Nat. Struct. Mol. Biol. 17, 1051–1057 (2010).

    CAS  Article  Google Scholar 

  49. 49

    Rossi, F.M., Kringstein, A.M., Spicher, A., Guicherit, O.M. & Blau, H.M. Transcriptional control: rheostat converted to on/off switch. Mol. Cell 6, 723–728 (2000).

    CAS  Article  Google Scholar 

  50. 50

    Forger, D.B. Signal processing in cellular clocks. Proc. Natl. Acad. Sci. USA 108, 4281–4285 (2011).

    CAS  Article  Google Scholar 

  51. 51

    Kim, J.K. & Forger, D.B. A mechanism for robust circadian timekeeping via stoichiometric balance. Mol. Syst. Biol. 8, 630 (2012).

    Article  Google Scholar 

  52. 52

    Lee, Y., Chen, R., Lee, H.-m. & Lee, C. Stoichiometric relationship among clock proteins determines robustness of circadian rhythms. J. Biol. Chem. 286, 7033–7042 (2011).

    CAS  Article  Google Scholar 

  53. 53

    Fuxreiter, M. et al. Malleable machines take shape in eukaryotic transcriptional regulation. Nat. Chem. Biol. 4, 728–737 (2008).

    CAS  Article  Google Scholar 

  54. 54

    Parker, D. et al. Analysis of an activator:coactivator complex reveals an essential role for secondary structure in transcriptional activation. Mol. Cell 2, 353–359 (1998).

    CAS  Article  Google Scholar 

  55. 55

    Parker, D. et al. Role of secondary structure in discrimination between constitutive and inducible activators. Mol. Cell. Biol. 19, 5601–5607 (1999).

    CAS  Article  Google Scholar 

  56. 56

    Ramanathan, C., Khan, S., Kathale, N., Xu, H. & Liu, A. Monitoring cell-autonomous circadian clock rhythms of gene expression using luciferase bioluminescence reporters. J. Vis. Exp. 67, 4234 (2012).

    Google Scholar 

  57. 57

    Levin, R.D. et al. Circadian function in patients with advanced non-small-cell lung cancer. Br. J. Cancer 93, 1202–1208 (2005).

    CAS  Article  Google Scholar 

  58. 58

    Ozturk, N., Lee, J., Gaddameedhi, S. & Sancar, A. Loss of cryptochrome reduces cancer risk in p53 mutant mice. Proc. Natl. Acad. Sci. USA 106, 2841–2846 (2009).

    CAS  Article  Google Scholar 

  59. 59

    Gekakis, N. et al. Role of the CLOCK protein in the mammalian circadian mechanism. Science 280, 1564–1569 (1998).

    CAS  Article  Google Scholar 

  60. 60

    Partch, C.L., Shields, K.F., Thompson, C.L., Selby, C.P. & Sancar, A. Posttranslational regulation of the mammalian circadian clock by cryptochrome and protein phosphatase 5. Proc. Natl. Acad. Sci. USA 103, 10467–10472 (2006).

    CAS  Article  Google Scholar 

  61. 61

    Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995).

    CAS  Article  Google Scholar 

  62. 62

    Johnson, B.A. Using NMRView to visualize and analyze the NMR spectra of macromolecules. Methods Mol. Biol. 278, 313–352 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. 63

    Shen, Y., Delaglio, F., Cornilescu, G. & Bax, A. TALOS+: a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts. J. Biomol. NMR 44, 213–223 (2009).

    CAS  Article  Google Scholar 

  64. 64

    Farmer, B.T. et al. Localizing the NADP+ binding site on the MurB enzyme by NMR. Nat. Struct. Biol. 3, 995–997 (1996).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank P.E. Wright (Scripps Research Institute) and A. Sancar (University of North Carolina, Chapel Hill) for sharing reagents. We thank J. Miller, R. Palomino and G. Millhauser (University of California, Santa Cruz) for technical assistance and access to instrumentation for automated peptide synthesis and purification, S.M. Rubin (University of California, Santa Cruz) for access to calorimetry instrumentation, critical discussions and advice, and the Chemical Screening Center (University of California, Santa Cruz) for access to other instrumentation. This work was supported by the US National Science Foundation (grant IOS-0920417 to A.C.L.), the US National Institutes of Health (grant GM107069 to C.L.P.) and the University of California, Santa Cruz (startup funds to C.L.P.).

Author information

Affiliations

Authors

Contributions

H.X., C.L.G. and P.J.S. designed the project with A.C.L. and C.L.P.; H.X., C.L.G., P.J.S., S.K.K., C.R., H.-W.L. and C.L.P. performed experiments. N.C.P. contributed new reagents and materials. H.X., C.L.G., P.J.S., A.C.L. and C.L.P. analyzed data. The manuscript was written by H.X., C.L.G., A.C.L. and C.L.P. All authors contributed to editing the manuscript and support the conclusions.

Corresponding authors

Correspondence to Andrew C Liu or Carrie L Partch.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Validation of genetic complementation assay in Bmal1−/– Per2Luc fibroblasts.

(a) Only Bmal1, not Bmal2, rescues circadian rhythms from cells. Cells expressing various Bmal constructs (Flag-tagged at either the N- or- C-terminus) were subjected to cycling analysis: 3xF-Bmal, Bmal tagged with 3xFlag at the N-terminus; Bmal-3xF, 3xFlag tag at the C-terminus; Bmal-1xF, 1xFlag at the C-terminus. A representative western blot of 3xF-BMAL proteins (inset) shows that Bmal1 and Bmal2 were expressed to similar levels off the plasmid. (b) Period length analysis of rescued rhythms in panel (a). Data are plotted as mean ± s.d. error bars from 3 independent clonal lines. Placement of Flag tags seems to impact period length. For this consideration, we tested both 3xF-Bmal and Bmal1-1xF constructs throughout the study and similar results from domain swaps/mutants were obtained. (c) Cyclic expression of Bmal1, but not Bmal2, rescues circadian rhythms in Bmal1−/– Per2Luc fibroblasts. A fragment of the Bmal1 promoter that contains circadian RORE cis-elements was used to drive Bmal transcription. Expression of Bmal2 under control of the rhythmic Bmal1 promoter was unable to rescue rhythms. For consistency, the UBC promoter was used throughout this study. (d) The ability of Bmal1 to rescue rhythms does not depend on the expression vector used. The pLV156 vector expresses an IRES-mediated GFP while the recombination-based pLV7 vector confers high efficiency cloning. Expression of Bmal1 off the pLV7 plasmid reconstituted cycling and was therefore used throughout this study. (e) The ability of Bmal1 to rescue rhythms is dosage independent. Protein expression levels at various levels of infection were determined by Western blot (left panel). While Bmal1 rescued circadian rhythms under all expression regimes tested (center panel), Bmal2 failed to rescue levels even at high levels of expression (right panel). We note that CRY1 displayed a modest dose response to the titration of BMAL1 levels, but demonstrated no effect upon BMAL2 titration.

Supplementary Figure 2 Domain structure, sequence alignment and structural features of BMAL1 and BMAL2.

(a) Schematic diagram of domain structure of BMAL1 (green) and BMAL2 (blue); cartoons drawn to scale. NRD, N-terminal regulatory domain; NCD, N-terminal core domain; CORD, C-terminal oscillator regulatory domain. The BMAL1-NRD (region A) contains a nuclear localization signal (NLS) that is not conserved in BMAL2. The NCD contains the bHLH DNA-binding domain (region B), PAS-A motif (region C), and PAS-B motif (E). The CORD contains regions G and H. Diamonds indicate boundaries for the protein fragment for which a crystal structure is available (PDB 4F3L)14. Each motif color corresponds to the circadian bioluminescence data for complemented Bmal1−/– Per2Luc fibroblasts in Figure 2b and 2e. (b) Sequence alignment of BMAL1 and BMAL2 demonstrates divergence of C-terminal sequences. Mouse BMAL protein sequences were aligned in Vector NTI. Consensus sequence is shown in black. White barrels indicate JPred-predicted secondary structure (α-helices) in the C-terminus.

Supplementary Figure 3 The C-terminal regulatory domain of BMAL1 is critical for maintaining circadian period length and rhythm amplitude.

(a-c) Analysis of (a) amplitude and (b) damping rates and (c) expression of Bmal1, Bmal2, and chimeric Bmal1-Bmal2 constructs from Figure 2. Data are plotted as mean ± s.d. error bars from 3 independent clonal lines. Relative protein expression levels were determined by western blot using indicated antibodies. (d) Both G and H regions of the BMAL1 C-terminus are required to reconstitute circadian cycling in Bmal1−/− Per2Luc fibroblasts. Experimental details are the same as in Figure 2, except that 3xFlag-Bmal constructs were used here (while Bmal-1xF constructs were used throughout the main figures of the paper). (e) Relative expression levels of mutant chimeric proteins were determined by western blot over a time course after synchronization of clocks by dexamethasone treatment (time = 0). Expression of BMAL1-G2, H2, or G2H2 chimeric proteins was determined using Flag antibody; actin is shown as a loading control. (f-h) Analysis of (f) period length, (g) amplitude, (h) and damping rates for 3xF-Bmal constructs shown in panel (d). Data are plotted as mean ± s.d. error bars from 3 independent clonal lines. (i) Temporal expression profiles of clock gene mRNA in Bmal1−/− Per2Luc fibroblasts expressing Bmal1-G2, H2, or G2H2. Values are expressed as percentage of maximum expression for each gene from 3 independent assays and can be directly compared with those in Figure 1c. Data are plotted as mean ± s.d. of expression levels from two independent cell culture samples (including triplicate technical replicates) for each cell line. Time, hours after dexamethasone synchronization. (j) Western blot analysis of mutant BMAL1 expression in HEK293T cells detected by Flag antibody. Similar results were obtained from Bmal1−/− Per2Luc fibroblasts expressing various forms of the mutant BMAL1 proteins corresponding to cell lines shown in Figure 3 (data not shown). * P < 0.05; ** P < 0.01 compared to Bmal1 by two-tailed paired t test.

Supplementary Figure 4 The BMAL1 TAD interacts directly with transcriptional coregulators and is required for CLOCK–BMAL1 activity.

(a) Deletion of the BMAL1 TAD by truncation at residue 584 (584X) eliminates CLOCK–BMAL1 activation of a Per1-luciferase reporter gene in HEK293T cells. Data are plotted as mean relative luminescence counts (scaled down by 103) ± s.d. error bars from one representative assay of three independent replicates. (b) The BMAL1 TAD is required to generate circadian rhythms. Representative bioluminescence records from Bmal1−/− Per2Luc fibroblasts complemented with the indicated Bmal1 constructs (ΔTAD indicates truncation of H region). (c) Conservation of TAD primary sequence and secondary structure across metazoan species with a vertebrate-like clock29. Blue barrel, predicted α-helix (JPred); black line, no predicted structure. (d) The TAD has a modest propensity for α-helix formation, demonstrated by comparison of Cα (black) or Cβ (gray) chemical shifts to the chemical shift index of random coil shifts (Δδ)31,32. Residues correspond to alignment and secondary structure prediction in panel (c). Dashed line: significance cutoff for helical prediction from Δδ. (e) Central region of the 15N HSQC spectrum of the 15N BMAL1 TAD displays the modest chemical shift dispersion typical of unfolded/partly helical proteins. Assignments for residues of the IxxLL motif in the α-helix are indicated. p.p.m., parts per million. (f) Modular domain architecture of CBP(p300) (green) and CRY1 (red), with domains used in the present study underlined. (g) Crystal structure of mouse CRY1 (PDB 4K0R)23 displayed with domain coloring from panel (f). The PHR domain comprises most of CRY1, followed by the CC helix (orange), which forms the final α-helix before the disordered C-terminus (yellow dashed line). (h-i) The CBP(p300) KIX domain and CRY1 CC helix bind overlapping regions on the BMAL1 TAD. A highlighted region of 15N HSQC spectra of 15N BMAL1 TAD titrated with increasing concentrations of either p300 KIX (h) or the monomeric CRY1 CC peptide (i) show perturbation of the same residues. CBP KIX induces the same perturbations as p300 KIX (data not shown). Quantification of chemical shift perturbations at the 1:1 molar ratio for p300 KIX and CRY1 CC are depicted in Figure 4a and b, respectively.

Supplementary Figure 5 The TAD undergoes dynamic structural rearrangement upon binding to the p300 KIX domain and CRY1 CC peptide to couple the TAD helix and C terminus.

(a-b) Covalent coupling of the MTS-EDTA moiety at a C-terminal cysteine (TAD–EDTA) does not perturb interactions with p300 KIX or CRY1 CC. Correlation of chemical shift perturbations in WT TAD or TAD-EDTA 15N BMAL1 TAD upon complex formation at 1:1 stoichiometry with p300 KIX domain (a) or CRY1 CC peptide (b). Each point represents the chemical shift perturbation of a different backbone amide. Goodness-of-fit for linear regression (R2) is shown for both correlations. (c-d) 15N HSQC spectra of 15N TAD–EDTA ± Mn2+ titrated with p300 KIX (c) or CRY1 CC (d) up to 1:1 stoichiometry. Inset legends represent various steps of relative stoichiometry of p300 KIX or CRY CC to 15N BMAL1 TAD–EDTA in the absence (left panel) or presence (right panel) of chelated Mn2+. Arrow lengths and orientation are maintained within all panels to illustrate changes (or lack thereof) in chemical shift perturbation upon chelation of paramagnetic Mn2+. Dashed arrow for residue Ser620 illustrates a residue in the TAD C-terminus that undergoes complex-independent broadening due to its proximity to the C-terminal PRE label (see Figure 4d). (e) 15N HSQC spectra of 15N TAD 619X–EDTA ± Mn2+ titrated with CRY1 CC peptide demonstrate the lack of CRY1-dependent broadening at the TAD helix in the absence of the C-terminal seven residues.

Supplementary Figure 6 Comparison of BMAL1 and BMAL2 TAD domains by NMR spectroscopy.

(a) 15N HSQC spectra of 15N BMAL1 TAD (green) or 15N BMAL2 TAD (blue) demonstrate minimal overlap of chemical shifts. Dashed circles indicate the only residues that have similar chemical shifts; these residues represent the conserved N-terminal vector artifact left after TEV cleavage. (b) The BMAL2 TAD has a modest propensity for α-helix formation, demonstrated by comparison of Cα (black) or Cβ (gray) chemical shifts to the chemical shift index of random coil shifts (Δδ)31,32. Dashed line: significance cutoff for helical prediction from Δδ analysis. (c-d) Comparison of BMAL TAD interactions with the CBP(p300) KIX domain and CRY1 CC peptide. Chemical shift changes (ΔδTOT) on 15N BMAL2 TAD for the 1:1 molar ratio of either CBP(p300) KIX domain (c) or the CRY1 CC peptide (d). Dashed line: significance cutoff of 0.04 p.p.m. for chemical shift perturbations. The BMAL TAD sequences are aligned underneath NMR data with predicted secondary structure: barrel, predicted α-helix; red line, C-terminal seven residues; black line, no predicted structure. (e) Mutations in BMAL1 TAD induce local chemical shift perturbations. Bar graphs plot total chemical shift perturbation (ΔδTOT) of backbone shifts from 15N HSQC spectra of mutant TADs compared to WT TAD. Mutations in the α-helix largely perturb just the helix and truncation of the C-terminal seven residues gives rise to large chemical shift perturbations only at the new C-terminus.

Supplementary Figure 7 Quantitative analysis of BMAL TAD interactions with transcriptional coregulators.

(a-b) Data from isothermal titration calorimetry (ITC) experiments of BMAL TAD (BMAL1, BMAL2, or BMAL1 mutants, as indicated above) with either CBP KIX domain (a) or the CRY1 CC peptide (b). All ITC experiments were set up with 15-30 μM TAD in the cell and 220-250 μM KIX or CRY1 CC peptide in the syringe (details for each experiment can be found in Online Methods) and run at 25°C. Data shown are representative of at least two independent ITC runs. ITC data were fit to a one-site binding model (Origin) to derive parameters that populate Table 1, with representative N values of 0.7-1.0 to indicate 1:1 stoichiometry. (c) Fluorescence polarization-based titration of rhodamine-labeled CRY1 CC peptide against BMAL1, BMAL2 or BMAL1 mutant TADs. Data are shown as mean change in milli-polarization units (Δmp) from free CC peptide ± s.d. error bars from four replicates in a representative experiment (n = 3 independent experiments). Curves were fit by non-linear regression to a one-site binding model (GraphPad Prism) to determine KD values in Table 1. (d) Relative protein expression of BMAL1 WT and mutant constructs within the complemented Bmal1−/− Per2Luc lines were determined by western blot using indicated antibodies.

Supplementary Figure 8 CRY1 regulation of CLOCK–BMAL1 requires interaction with both CLOCK and BMAL1.

(a) Wild-type and mutant Flag-Bmal1 clones are expressed to similar levels in HEK293T cells as determined by western blot using anti-Flag antibody. *, non-specific band that serves as a loading control. (b) Relative expression levels of CLOCK, BMAL1 and CRY1 with plasmid ratios used in luciferase assays. HEK293T cells were transfected with plasmid ratios used in Per1-luciferase assay (100 ng each Clock and Bmal1, and Cry1 plasmid as indicated, scaled 4X for increase in culture dish area). Relative protein expression levels are shown by western blotting using indicated antibodies. Under these experimental conditions, BMAL1 expression is just at the detection limit with the anti-Flag antibody. Expression of CLOCK and BMAL1 was also verified by blotting with anti-CLOCK and anti-BMAL1 antibodies, which provide greater sensitivity of detection compared to the Flag antibody. **, migration of the CLOCK-dependent phosphorylated BMAL1 species in the anti-Flag blot relative to its detection with the anti-BMAL1 antibody. (c) Mutation of the CLOCK PAS-B HI loop (Q361P W362R) disrupts interaction with CRY1 by anti-Flag co-IP when co-expressed with Flag-BMAL1 in HEK293T cells. The CRY1 CC mut (R501Q K503R) (Ozber, N., Baris, I., Tatlici, G., Kilinc, S., Unal, E.B. & Kavakli, I.H. Identification of two amino acids in the C-terminal domain of mouse CRY2 essential for PER2 interaction. BMC Mol. Biol. 11, 69 (2010)) does not affect interaction of CRY1 with co-expressed Flag-CLOCK and Flag-BMAL1. (d) The CRY1 CC helix does not bind directly to the CLOCK PAS-B domain. 15N HSQC spectra of 60 μM 15N CLOCK PAS-B in the absence (black) and presence (orange) of 240 μM CRY1 CC peptide show no chemical shift perturbations upon addition of CRY1 C peptide. Dashed box: tryptophan indole region shown in Figure 6g. Assignment of the CLOCK PAS-B W362 tryptophan indole peak was made by mutagenesis (data not shown).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 (PDF 1252 kb)

Supplementary Data Set 1

Uncropped autoradiographs and blots (PDF 2417 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Xu, H., Gustafson, C., Sammons, P. et al. Cryptochrome 1 regulates the circadian clock through dynamic interactions with the BMAL1 C terminus. Nat Struct Mol Biol 22, 476–484 (2015). https://doi.org/10.1038/nsmb.3018

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing