Quantification of circadian interactions and protein abundance defines a mechanism for operational stability of the circadian clock
Abstract
The mammalian circadian clock exerts substantial control of daily gene expression through cycles of DNA binding. Understanding of mechanisms driving the circadian clock is hampered by lack of quantitative data, without which predictive mathematical models cannot be developed. Here we develop a quantitative understanding of how a finite pool of BMAL1 protein can regulate thousands of target sites over daily time scales. We have used fluorescent correlation spectroscopy (FCS) to track dynamic changes in CRISPR-modified fluorophore-tagged proteins in time and space in single cells across SCN and peripheral tissues. We determine the contribution of multiple rhythmic processes in coordinating BMAL1 DNA binding, including the roles of cycling molecular abundance, binding affinities and two repressive modes of action. We find that nuclear BMAL1 protein numbers determine corresponding nuclear CLOCK concentrations through heterodimerization and define a DNA residence time of 2.6 seconds for this complex. Repression of CLOCK:BMAL1 is in part achieved through rhythmic changes to BMAL1:CRY1 affinity as well as a high affinity interaction between PER2:CRY1 which mediates CLOCK:BMAL1 displacement from DNA. Finally, stochastic modelling of these data reveals a dual role for PER:CRY complexes in which increasing concentrations of PER2:CRY1 promotes removal of BMAL1:CLOCK from genes consequently enhancing ability to move to new target sites.
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Evaluation Summary:
Koch et al. quantified the abundance of the core clock molecules and their binding affinities, thereby providing critical information for our quantitative understanding of the core transcriptional negative feedback loop of the mammalian circadian clock. Furthermore, they used mathematical modeling to incorporate the quantified information and identified the hidden role of PER:CRY complex, enhancing the mobility of BMAL1:CLOCK to new target sites. The work makes the important contribution that the displacement type repression frees CLOCK-BMAL1 to bind to other targets and activate several sets of genes. This is an important insight, but some of the data need further explanation and some statements ought to change to improve the manuscript.
(This preprint has been reviewed by eLife. We include the public reviews from …
Evaluation Summary:
Koch et al. quantified the abundance of the core clock molecules and their binding affinities, thereby providing critical information for our quantitative understanding of the core transcriptional negative feedback loop of the mammalian circadian clock. Furthermore, they used mathematical modeling to incorporate the quantified information and identified the hidden role of PER:CRY complex, enhancing the mobility of BMAL1:CLOCK to new target sites. The work makes the important contribution that the displacement type repression frees CLOCK-BMAL1 to bind to other targets and activate several sets of genes. This is an important insight, but some of the data need further explanation and some statements ought to change to improve the manuscript.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
The authors provide quantitative-image data and stochastic modelling to address the detail of CLOCK nucleus mobility regulated by BMAL1, CLOCK-BMAL1-E-box binding time, BMAL1/CRY1 affinity, PER2-CRY1 mediated CLOCK-BMAL1 displacement from E-box and then visiting more new target sites. Based on their mathematic modelling data, the model is interesting and makes sense that they find CLOCK-BMAL1 move to new sites after removal by PER-CRY. Their quantitative biology and mathematic modelling method provide new insight, which are complementary with biochemistry and structure in the circadian field. The findings are consistent with current circadian model from the Sancar lab: CRY1 blocks CLOCK-BMAL1 activity independent of PER and PER-CRY-CK1 complex displace CLOCK-BMAL1 from E-box, and the authors go a step …
Reviewer #1 (Public Review):
The authors provide quantitative-image data and stochastic modelling to address the detail of CLOCK nucleus mobility regulated by BMAL1, CLOCK-BMAL1-E-box binding time, BMAL1/CRY1 affinity, PER2-CRY1 mediated CLOCK-BMAL1 displacement from E-box and then visiting more new target sites. Based on their mathematic modelling data, the model is interesting and makes sense that they find CLOCK-BMAL1 move to new sites after removal by PER-CRY. Their quantitative biology and mathematic modelling method provide new insight, which are complementary with biochemistry and structure in the circadian field. The findings are consistent with current circadian model from the Sancar lab: CRY1 blocks CLOCK-BMAL1 activity independent of PER and PER-CRY-CK1 complex displace CLOCK-BMAL1 from E-box, and the authors go a step further from the Sancar lab regarding BMAL1 activator is inhibition by CRY1/CRY2 by "blocking type" repression or by CRY-PER complex by "displacement type" repression. This manuscript makes the important contribution that the displacement type repression frees CLOCK-BMAL1 to bind to other targets and activate several sets of genes. This is an important insight. However, some data need explaining and some statements need to change to improve the manuscript.
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Reviewer #2 (Public Review):
The transcriptional negative feedback loop of the mammalian circadian clock is mainly regulated by interactions among BMAL1, CLOCK, PER1/2 and CRY1/2 in the nucleus. While the binding of CRY with BMAL1:CLOCK is known to block the transcriptional activity of BMAL1:CLOCK and the binding of PER:CRY dissociates BMAL1:CLOCK from DNA have been known, our understanding is limited in qualitative level. Koch et al. quantified the dynamic interactions among the core clock molecules such as their diffusion coefficients, binding affinity, and abundances in the nucleus. This will greatly improve our understanding of the mammalian circadian clock. Importantly, this dynamic information is incorporated via a mathematical model to understand BMAL1-CLOCK binding to the target site (e.g., circadian proteins operate within an …
Reviewer #2 (Public Review):
The transcriptional negative feedback loop of the mammalian circadian clock is mainly regulated by interactions among BMAL1, CLOCK, PER1/2 and CRY1/2 in the nucleus. While the binding of CRY with BMAL1:CLOCK is known to block the transcriptional activity of BMAL1:CLOCK and the binding of PER:CRY dissociates BMAL1:CLOCK from DNA have been known, our understanding is limited in qualitative level. Koch et al. quantified the dynamic interactions among the core clock molecules such as their diffusion coefficients, binding affinity, and abundances in the nucleus. This will greatly improve our understanding of the mammalian circadian clock. Importantly, this dynamic information is incorporated via a mathematical model to understand BMAL1-CLOCK binding to the target site (e.g., circadian proteins operate within an optimal range to modulate E-box binding). Certainly, this work is novel and highly impactful. However, the description of how the quantified information can be incorporated into the mathematical modeling is unclear. There is also uncertainly in the identified parameters of the models.
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