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.

Cryptochrome proteins regulate the circadian intracellular behavior and localization of PER2 in mouse suprachiasmatic nucleus neurons

“…3 I ). We found that this threshold represented ∼50% of endogenous CRY1 levels (at CT12) and approximately one-third of peak (CT18) endogenous CRY1 levels, as determined from a CRY1::mRuby3 knock-in mouse ( 23 ) ( SI Appendix , Fig. S5 G and H ).…”

Combined multiple transcriptional repression mechanisms generate ultrasensitivity and oscillations

“…facilitating the sequestration), and compensating for the heterogeneous binding affinity of NF- B to the promoter of I B [ 64 ]. Furthermore, a recent study of the transcriptional NFL of the mammalian circadian clock found that the displacement of the transcriptional activator (BMAL1:CLOCK) by its repressor (PER:CRY) can facilitate the mobility of the BMAL1:CLOCK to its various target sites, pointing out the hidden role of PER:CRY [ 65 ]. While PER:CRY dissociates and sequesters CLOCK:BAML1 from E-box (i.e.…”

Combined multiple transcriptional repression mechanisms generate ultrasensitivity and oscillations

“…Z. Wang et al found that the displacement can enhance NF-𝜅B oscillation by dissociating the NF-𝜅B from decoy sites and promoting its nuclear export (i.e., facilitating the sequestration), and compensating for the heterogeneous binding affinity of NF-𝜅B to the promoter of I𝜅B𝛼 [63]. Furthermore, a recent study of the transcriptional NFL of the mammalian circadian clock found that the displacement of the transcriptional activator (BMAL1:CLOCK) by its repressor (PER:CRY) can facilitate the mobility of the BMAL1:CLOCK to its various target sites, pointing out the hidden role of PER:CRY [64]. While PER:CRY dissociates and sequesters CLOCK:BAML1 from E-box (i.e., sequestration-and displacement-type), CRY blocks the transcriptional activity of CLOCK:BMAL1 (i.e., blocking type) [15][16][17].…”