Figures and data in Quantification of protein abundance and interaction defines a mechanism for operation of the circadian clock

Version of Record: April 5, 2022
Accepted Manuscript: March 14, 2022

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Alex A Koch

James S Bagnall

Nicola J Smyllie

Nicola Begley

Antony D Adamson

Jennifer L Fribourgh

David G Spiller

Qing-Jun Meng

Carrie L Partch

Korbinian Strimmer

Thomas A House

Michael H Hastings

Andrew SI Loudon

(2022)
Quantification of protein abundance and interaction defines a mechanism for operation of the circadian clock

eLife 11:e73976.

https://doi.org/10.7554/eLife.73976
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Figures
Tables
Additional files

7 figures, 4 tables and 1 additional file

Figures

Figure 1 with 2 supplements

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Short-lived DNA binding of BMAL1 and CLOCK.

(A) Schematic representation of parameters regulating CLOCK:BMAL1 dimers binding to target DNA sites. (B) NIH/3T3 cells are either singularly or sequentially transduced to express fluorescent …

Figure 1—source data 1 BMAL1 residence times.: https://cdn.elifesciences.org/articles/73976/elife-73976-fig1-data1-v2.csv
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Figure 1—figure supplement 1

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Ectopically expressed mRNA is the major form in a lentivirus transduced system.

(A) NIH/3T3 cells were stained for CLOCK mRNA by single-molecule fluorescent in situ hybridisation (smFISH). (B) Mature mRNA was counted from images of many single cells to determine the mRNA …

Figure 1—figure supplement 1—source data 1 Summary statistics.: https://cdn.elifesciences.org/articles/73976/elife-73976-fig1-figsupp1-data1-v2.xlsx
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Figure 1—figure supplement 2

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Binding plays a significant role in BMAL1 mobility.

Fluorescence recovery after photobleaching for NIH/3T3 cells transduced with tagRFP::CLOCK and BMAL1:EGFP. (A) Imaging protocol was performed on BMAL1::EGFP signal. Regions of photobleaching are …

Figure 2 with 2 supplements

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Live-cell interaction measurements demonstrate BMAL1 and CLOCK mobility is regulated by dimerisation and DNA binding.

(A) Schematic of confocal volume used in FCCS with corresponding photon count traces. Interaction may be seen by correlation between both channels. Representative auto- and cross- correlation data …

Figure 2—source data 1 BMAL1 and CLOCK FCS diffusion rates.: https://cdn.elifesciences.org/articles/73976/elife-73976-fig2-data1-v2.csv
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Figure 2—source data 2 BMAL1 and CLOCK paired FCS concentrations.: https://cdn.elifesciences.org/articles/73976/elife-73976-fig2-data2-v2.csv
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Figure 2—figure supplement 1

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Anomalous diffusion best fits protein movement.

(A) Representative normal and anomalous model fits for BMAL1::EGFP FCS data sets for cells transduced with lentivirus to express BMAL1, CLOCK or control fluorescent proteins. (B) Summary data sets …

Figure 2—figure supplement 2

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Fluorescent BMAL1 and CLOCK proteins behave similarly when colours are swapped.

(A) Confocal images of NIH/3T3-LV2 EGFP::CLOCK-BMAL1::tagRFP cells. (B) Average auto- and cross- correlation curves shown as mean (line) and standard deviation (error envelope) for NIH/3T3 cells …

Figure 3 with 3 supplements

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A rhythmic and strong interaction observed between slow-diffusing BMAL1 and CRY1 facilitates repression.

(A) Schematic of triple-labelled mice from which primary lung fibroblasts were isolated (B) Confocal images of two cells shown for Venus::BMAL1 and CRY1::mRuby3 over time. FCS determined measurement …

Figure 3—source data 1 CRY1 FCS diffusion rates.: https://cdn.elifesciences.org/articles/73976/elife-73976-fig3-data1-v2.csv
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Figure 3—source data 2 BMAL1 FCS diffusion rates.: https://cdn.elifesciences.org/articles/73976/elife-73976-fig3-data2-v2.csv
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Figure 3—source data 3 BMAL1 FCS concentration.: https://cdn.elifesciences.org/articles/73976/elife-73976-fig3-data3-v2.csv
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Figure 3—source data 4 CRY1 FCS concentration.: https://cdn.elifesciences.org/articles/73976/elife-73976-fig3-data4-v2.csv
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Figure 3—figure supplement 1

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BMAL1 concentration and DNA binding parameters minimally vary across cell types.

(A) Confocal microscopy images of primary cultures isolated from Venus::BMAL1 mice. (B) Characteristic bound time calculated from FRAP measurements of primary cultures from A. Chondrocyte data was …

Figure 3—figure supplement 1—source data 1 CRY1::mRuby3 mouse genotyping.: https://cdn.elifesciences.org/articles/73976/elife-73976-fig3-figsupp1-data1-v2.pdf
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Figure 3—figure supplement 2

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Generation of CRY1::mRuby3 mouse line.

(A) Genotyping of pups by PCR. Images are PCR reactions run on Qiaxcel with red arrows indicating correct HDR product size, blue asterisks indicate mice in which InDels resulting from NHEJ are …

Figure 3—figure supplement 3

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Triple endogenous labelled mice used to assay rhythms in SCN and peripheral lung fibroblasts.

(A) Schematic representation of newly made transgenic mouse engineered to express CRY1::mRuby3, Venus::BMAL1, and PER2::LUC. (B) Confocal microscopy image of SCN organotypic slice expressing …

Figure 4 with 1 supplement

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PER2 modulates CRY1 mobility via a high-affinity association.

(A) Confocal images of transduced NIH/3T3 cells that either solo- or co- express PER2 and CRY1. (B) FCS data showing diffusion for PER2 and CRY1 in solo- and co-expressed conditions (n = 165, 174, …

Figure 4—source data 1 PER2 and CRY1 FCS diffusion rates.: https://cdn.elifesciences.org/articles/73976/elife-73976-fig4-data1-v2.csv
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Figure 4—figure supplement 1

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CRY1 mobility is affected by co-expression with PER2.

(A) Average auto- and cross- correlation curves shown as mean (line) and standard deviation (error envelope) for NIH/3T3 cells transduced to express EGFP::PER2 and CRY1::tagRFP. Measurements were …

Figure 5 with 1 supplement

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PER2 acts via CRY1 to mediate rhythmic displacement of CLOCK:BMAL1 from DNA.

(A) Schematic representation of model topology used for the deterministic model of CLOCK:BMAL1 DNA binding. (B) Primary lung fibroblasts from BMAL1 x CRY1 x PER2 mice were synchronised with …

Figure 5—figure supplement 1

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ODE model of CLOCK:BMAL1 DNA binding using measured inputs and modelled perturbations.

(A) Normalised model simulated FRAP using ODE model by counting recovery of site bound BMAL1 species after removal. (B) Model determined residence time of DNA bound CLOCK:BMAL1 across different …

Figure 6

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Circadian proteins operate within an optimal range to modulate E-Box binding.

Sensitivity analysis of the deterministic binding model showing relationship of measured parameters (bottom) against model for occupancy of active BMAL1:CLOCK on target sites (top). (A) Changing …

Figure 6—source data 1 Model OAT outputs.: https://cdn.elifesciences.org/articles/73976/elife-73976-fig6-data1-v2.xlsx
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Figure 7 with 1 supplement

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Mathematical modelling demonstrates dual function of PER:CRY mediated repression.

Stochastic binding model outputs using parameters corresponding to T28, T32 or T40 post dexamethasone BMAL1 x CRY1 data sets. (A) Shows a promoter corresponding to the average binding rate of …

Figure 7—figure supplement 1

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Stochastic binding model using experimentally measured parameters (A) Stochastic model showing the average binding (with SD) of CLOCK:BMAL1 bound target sites using input measurements from all time points for both WT and without PER2 simulations.

(B) The time to visit every E-Box site once for T28 showing fit. (C) Model simulation plots showing CLOCK:BMAL1 visits to a single promoter over time. Red and blue lines show simulations using …

Tables

Table 1

Summary of ordinary differential equation model parameters.

Model fit $χ^{2} = 7.46$ .

Input parameters
Parameter	Unit	Description	Value±SD
$K_{D} (C:B - E-Box)$	nM	CLOCK:BMAL1 - E-Box dissociation constant	10 (Huang et al., 2012)
$K_{D} (C-B)$	nM	CLOCK - BMAL1 dissociation constant	147.6 ± 9.8
$K_{D} (B-C1)$	nM	BMAL1 - CRY1 dissociation constant	Time-point dependent, Figure 3F
$K_{D} (C1-P2)$	nM	CRY1 - PER2 dissociation constant	81.8 ± 4.9
Fitted parameters
Parameter	Unit	Description	Value±SD (Inverse Hessian eigenvalue of fit)
$k_{ON}$	${n m}^{- 1} s^{- 1}$	CLOCK:BMAL1 - DNA binding on rate	$0.027 \pm 1.034 (1.96)$
$d_{ON}$	${n m}^{- 1} s^{- 1}$	BMAL1 - CRY1 binding rate	$0.237 \pm 1.003 (3.42 \times 10^{- 2})$
$a_{ON}$	${n m}^{- 1} s^{- 1}$	PER2 - CRY1 binding rate	$6.34 \pm 1.00 (6.79 \times 10^{- 8})$
$R_{OFF}$	$s^{- 1}$	CLOCK:BMAL1:CRY1:PER2 - DNA unbinding rate	$(1.23 \pm 0.33) \times 10^{1} (1.00)$
$b_{ON}$	${n m}^{- 1} s^{- 1}$	CLOCK - BMAL1 binding rate	$9.17 \pm 1.29 (1.00)$
Derived parameters
Parameter	Unit	Description	Value±SD
$k_{OFF}$	$s^{- 1}$	CLOCK:BMAL1 - DNA binding unbinding rate	$k_{ON} \times K_{D} (C:B - E-Box) = 0.27 \pm 10.34$
$b_{OFF}$	$s^{- 1}$	CLOCK - BMAL1 unbinding rate	$b_{ON} \times K_{D} (C-B) = (1.35 \pm 0.21) \times 10^{3}$
$d_{OFF}$	${n m}^{- 1} s^{- 1}$	BMAL1 - CRY1 unbinding rate	Time-point dependent, $d_{ON} \times K_{D} (B:C1)$
$a_{OFF}$	$s^{- 1}$	PER2 - CRY1 unbinding rate	$a_{ON} \times K_{D} (C1-P2) = (5.19 \pm 0.88) \times 10^{2}$

Table 2

BMAL1 ChIP reports.

No.	Tissue	BMAL1 peaks	Reference
1	Liver	2049	Rey et al., 2011
2	Liver	5952	Koike et al., 2012
3	U2OS	2001	Wu et al., 2017
4	PECS	2026	Oishi et al., 2017
5	Liver	4813	Beytebiere et al., 2019
6	Kidney	4034	Beytebiere et al., 2019
7	Heart	2520	Beytebiere et al., 2019
8	NIH3T3	4740	Chiou et al., 2016
9	Skeletal muscle	2787	Dyar et al., 2018
	Mean average	3436

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Genetic reagent (M. musclus)	C57BL/6 Venus::BMAL1	Yang et al., 2020		Venus sequence inserted before BMAL1 start codon.
Genetic reagent (M. musclus)	C57BL/6 Cry1::mRuby3	This paper		CRY1 stop codon replaced with mRuby3
Genetic reagent (M. musclus)	C57BL/6 Venus::BMAL1 x CRY1::mRuby3	This paper		Crossed from Venus::BMAL1 and CRY1::mRuby3 mice
Cell line (M. musculus)	NIH/3T3	ATCC	CRL-1658
Transfected construct (M. musculus)	pLNT-NLS::EGFP	Vector Builder VB900119-0501njq		Lentiviral construct to express nuclear EGFP.
Transfected construct (M. musculus)	pLNT-BMAL1::EGFP or pLNT-BMAL1::RFP	This paper	NCBI reference: NM_007489.4	Lentiviral construct to express fluorescent BMAL1.
Transfected construct (M. musculus)	pLNT-BMAL1-L95E::EGFP	This paper	NCBI reference: NM_007489.4	Lentiviral construct to express fluorescent BMAL1 L95E mutant.
Transfected construct (M. musculus)	pLNT-BMAL1-V435R::EGFP	This paper	NCBI reference: NM_007489.4	Lentiviral construct to express fluorescent BMAL1 V435R mutant.
Transfected construct (M. musculus)	pLNT-EGFP::CLOCK or pLNT-RFP::CLOCK	This paper	NCBI reference: NM_007715.6	Lentiviral construct to express fluorescent CLOCK.
Transfected construct (M. musculus)	pLNT-EGFP::PER2	This paper	NCBI reference: NM_011066	Lentiviral construct to express fluorescent PER2.
Transfected construct (M. musculus)	pLNT-CRY1::RFP	This paper	NCBI reference: NM_007771.3	Lentiviral construct to express fluorescent CRY1.
Chemical compound, drug	Dexamethasone	Sigma Aldrich	D4902
Software, algorithm	GraphPad Prism	GraphPad Prism	Version 9
Software, algorithm	FCCS analysis pipeline	This paper	https://github.com/LoudonLab/FcsAnalysisPipeline,(copy archived at swh:1:rev:b12e9007ed7f8a033485e57c8605e27c67df74f1; Koch, 2021)

Table 3

Stochastic model reactions and propensities.

Counter for arrivals by CLOCK:BMAL1 ( $C B$ ) without CRY1 ( $C 1$ ) to previously unbound sites $S$ converting them to S₀ given by $A_{C B}$ as well as counters for marked site $M$ binding represented by $B_{X}$ , and …

No.	Reaction	Propensity
1	$C B + S ⟶ C B S + A_{C B}$	$(k_{ON} / Ω) \cdot C B \cdot S$
2	$C B + S_{0} ⟶ C B S$	$(k_{ON} / Ω) \cdot C B \cdot S_{0}$
3	$C B S ⟶ C B + S_{0}$	$k_{OFF} \cdot C B S$
4	$C B C 1 + S ⟶ C B C 1 S$	$(k_{ON} / Ω) \cdot C B C 1 \cdot S$
5	$C B C 1 S ⟶ C B C 1 + S$	$k_{OFF} \cdot C B C 1 S$
6	$C B C 1 + S_{0} ⟶ C B C 1 S_{0}$	$(k_{ON} / Ω) \cdot C B C 1 \cdot S_{0}$
7	$C B C 1 S_{0} ⟶ C B C 1 + S_{0}$	$k_{OFF} \cdot C B C 1 S_{0}$
8	$C B + M ⟶ C B M + B_{C B}$	$(k_{ON} / Ω) \cdot C B \cdot M$
9	$C B M ⟶ C B + M + U_{C B}$	$k_{OFF} \cdot C B M$
10	$C B C 1 + M ⟶ C B C 1 M + B_{C B C 1}$	$(k_{ON} / Ω) \cdot C B C 1 \cdot M$
11	$C B C 1 M ⟶ C B C 1 + M + U_{C B C 1}$	$k_{OFF} \cdot C B C 1 M$