CHRONO participates in multi-modal repression of circadian transcriptional complexes

Abstract

The mammalian protein CHRONO was previously identified to be a rhythmically expressed repressor of the circadian transcriptional activator complex CLOCK:BMAL1. Mice and cells lacking CHRONO display a lengthened circadian period and altered circadian gene expression. Currently, however, we lack specific mechanistic understanding of CHRONO′s activity and function. Here we define an evolutionarily conserved minimal repressive domain (MRD) of CHRONO and demonstrate this domain′s capacity to repress CLOCK:BMAL1 activity through interaction with the BMAL1 C-terminal transactivation domain (TAD). Notably, this binding region overlaps with the binding site for CRY and coactivators CBP/p300, with CHRONO capable of competing with both of these classical regulators of BMAL1 for TAD binding, highlighting this as a hotspot for BMAL1 regulation. Additionally, we investigate the previously unexplored interaction between CHRONO and another major circadian repressor, PER2. We show that CHRONO reduces PER2 stability through interaction between the CHRONO C-terminus and the Casein Kinase 1 (CK1)-binding domain of PER2. This results in competition between CHRONO and CK1 for binding at this site on PER2, adding another layer to our understanding of PERIOD protein regulation. Taken together, these data show a more substantive role for CHRONO within molecular circadian timekeeping than previously posited and provide a platform for further investigation into CHRONO′s role within the circadian repressive complex.

Hierarchical and scaffolded phosphorylation of two degrons controls PER2 stability

“…Our data as well as that of others suggest that PER2 is phosphorylated in the repressor complex both in cis and in trans through scaffolding of CK1 (17, 43). This scaffolded phosphorylation did not enable D1 activation in PER2, while it did allow D1 activation in PER1.…”

“…This implies that steric considerations may allow CK1 greater access in trans to PER2 D2 than to D1 and that could contribute to the observed differences in activity between the degrons. This trans phosphorylation of D2 may drive PER degradation even when CK1 binding to one copy of PER is inhibited by various mechanisms, e.g., by competitive binding of CHRONO (43). Elucidating the mechanism of cis versus trans CK1 activity could be important in identifying new means of modulating circadian rhythms.…”

A conserved chronobiological complex timesC. elegansdevelopment

“…Alternatively, this result could reflect substrate inhibition, where increasing amounts of LIN-42 CK1BD+Tail result in a non-productive enzyme-substrate complex. Such substrate inhibition has also been reported in several recent studies of CK1 showing phosphorylated substrates binding the enzyme and regulating its activity (Gebel et al , 2020; Cullati et al , 2022; Harold et al , 2023; Crosby et al , 2023). Further enzymatic and structural studies will be required to distinguish between these modes of regulation.…”

“…Mammalian CK1 binds PER through its kinase domain, a region that is highly conserved, with CK1δ and KIN-20 exhibiting 79% and 100% sequence identity within the kinase domain and active sites, respectively (Fig. S3)(Dahlberg et al , 2009; Crosby et al , 2023). By contrast, the CK1BD is less well conserved in LIN-42 with only ∼30% sequence identity and a different spacing of the putative LIN-42 CK1BD-A and -B sites (Fig.…”

“…The LIN-42 SYQ-LT regions form a functional CK1BD capable of binding mammalian CK1 Mammalian CK1 binds PER through its kinase domain, a region that is highly conserved, with CK1δ and KIN-20 exhibiting 79% and 100% sequence identity within the kinase domain and active sites, respectively (Fig. S3) (Dahlberg et al, 2009;Crosby et al, 2023).…”