Rhythmic PER Abundance Defines a Critical Nodal Point for Negative Feedback within the Circadian Clock Mechanism

The suprachiasmatic nucleus (SCN) defines 24 h of time via a transcriptional/posttranslational feedback loop in which transactivation of Per (period) and Cry (cryptochrome) genes by BMAL1-CLOCK complexes is suppressed by PER-CRY complexes. The molecular/structural basis of how circadian protein complexes function is poorly understood. We describe a novel N-ethyl-N-nitrosourea (ENU)-induced mutation, early doors (Edo), in the PER-ARNT-SIM (PAS) domain dimerization region of period 2 (PER2) (I324N) that accelerates the circadian clock of Per2 Edo/Edo mice by 1.5 h. Structural and biophysical analyses revealed that Edo alters the packing of the highly conserved interdomain linker of the PER2 PAS core such that, although PER2 Edo complexes with clock proteins, its vulnerability to degradation mediated by casein kinase 1e (CSNK1E) is increased. The functional relevance of this mutation is revealed by the ultrashort (<19 h) but robust circadian rhythms in Per2 Edo/Edo ; Csnk1e Tau/Tau mice and the SCN. These periods are unprecedented in mice. Thus, Per2 Edo reveals a direct causal link between the molecular structure of the PER2 PAS core and the pace of SCN circadian timekeeping.C ircadian rhythms, as exemplified by the sleep/wake cycle, are the outward manifestation of a 24-h timing mechanism that coordinates many physiological processes (1). The principal circadian clock in mammals is the suprachiasmatic nucleus (SCN). At a molecular level, the SCN clockwork consists of interacting positive and negative transcriptional/translational feedback loops (TTFLs) that drive rhythms in the RNA and protein levels of key clock components. Heterodimers of the PER-ARNT-SIM (PAS) domain-containing transcriptional activators CLOCK and BMAL1 drive rhythmic transcription of Per (period) and Cry (cryptochrome) genes by binding to E-box elements. PER proteins associate with CRY, translocating to the nucleus to inhibit their own transcription by interacting with the CLOCK-BMAL1 complex (2, 3). An additional feedback loop involves the nuclear orphan receptors REV-ERBα (4) and RAR-related orphan receptor A (RORA) (5) that modify the transcription of Bmal1, thereby stabilizing and amplifying the CLOCK-BMAL complex-dependent oscillation. Once suppressed, CLOCK-BMAL complex-mediated transactivation can only reoccur after PER and CRY have been degraded.The circadian TTFL is therefore sustained by timely synthesis and degradation of PER and CRY proteins. Changes in PER stability in flies (6, 7) and changes in PER or CRY stability in mammals lead to a faster or slower clock (8). Phosphorylation-dependent licensing of PER proteins for ubiquitination and subsequent proteasomal degradation is a sensitive checkpoint in setting clock speed (9-11). Moreover, two familial advanced sleep phase syndromes (FASPSs) are associated with site-specific phosphorylation of human period 2 (PER2) (12, 13). Finally, the Tau mutation of casein kinase 1e (Csnk1e) (14) is a hypermorph that accelerates the clock of rodents (15). Thus, phosphorylation, PER protein sta...

Section: Discussionsupporting

confidence: 87%

“…It therefore acts as a gain-of-function mutation. We speculate that this gain of function is due to maintenance of normal interactions of PER2 Edo with other clock proteins (e.g., CRY), whereas its more rapid turnover leads to a change in the repressive "nodal set point" established by PER2 (29).…”

Section: Discussionmentioning

confidence: 97%

Early doors ( Edo ) mutant mouse reveals the importance of period 2 (PER2) PAS domain structure for circadian pacemaking

Militi

Maywood

Sandate

et al. 2016

“…Previous studies showed that FBXL3 interacts with CRY1 and CRY2, promoting the degradation of both these proteins by the ubiquitin/ proteasome system, thus contributing to period length determination (11)(12)(13). However, overexpression of CRY1 protein does not lead to period alteration (14), suggesting that the Fbxl3 mutation might affect additional clock components.…”

mentioning

confidence: 99%

Dual roles of FBXL3 in the mammalian circadian feedback loops are important for period determination and robustness of the clock

Shi

Xing

Liu

et al. 2013

The mammalian circadian clock is composed of interlocking feedback loops. Cryptochrome is a central component in the core negative feedback loop, whereas Rev-Erbα, a member of the nuclear receptor family, is an essential component of the interlocking loop. To understand the roles of different clock genes, we conducted a genetic interaction screen by generating single-and double-mutant mice. We found that the deletion of Rev-erbα in F-box/leucine rich-repeat protein (Fbxl3)-deficient mice rescued its long-circadian period phenotype, and our results further revealed that FBXL3 regulates RevErb/retinoic acid receptor-related orphan receptor-binding element (RRE)-mediated transcription by inactivating the Rev-Erbα:histone deacetylase 3 corepressor complex. By analyzing the Fbxl3 and Cryptochrome 1 double-mutant mice, we found that FBXL3 also regulates the amplitudes of E-box-driven gene expression. These two separate roles of FBXL3 in circadian feedback loops provide a mechanism that contributes to the period determination and robustness of the clock.C ircadian rhythms control many physiological processes in almost all eukaryotic organisms (1-6). The current mammalian clock model comprises a core negative feedback loop that includes the Per-Arnt-Sim (PAS) domain-containing helix-loophelix transcription factors Clock and Bmal1, Period genes (Per1, Per2, and Per3), and Cryptochrome genes (Cry1 and Cry2). The CLOCK:BMAL1 complex activates the transcription of the Period and Cryptochrome genes by binding to E-boxes in their promoters, whereas the PER:CRY complex closes the negative feedback loop by repressing the activity of CLOCK:BMAL1, resulting in endogenous circadian oscillations of Per and Cry mRNA (3, 5, 7). The nuclear receptors Rev-Erbα and RORα are components of another feedback loop that is interlocked with the core negative loop. These receptors function by competitively binding to the Rev-Erb/ROR-binding element (RRE) of Bmal1 to regulate its rhythmic transcription (8-10).Mutation of FBXL3 (C358S or I364T), a component of a SKP1-CUL1-F-box (SCF) E3 ubiquitin ligase complex, results in ∼26-hperiod phenotypes in mice, indicating that FBXL3 plays an important role in circadian period determination (11-13). Previous studies showed that FBXL3 interacts with CRY1 and CRY2, promoting the degradation of both these proteins by the ubiquitin/ proteasome system, thus contributing to period length determination (11-13). However, overexpression of CRY1 protein does not lead to period alteration (14), suggesting that the Fbxl3 mutation might affect additional clock components.To gain further insights into the mammalian clock network, we studied the genetic interactions between different clock genes in the mouse. Our screens revealed an unexpected genetic interaction between Rev-erbα and Fbxl3. Further biochemical analysis showed that FBXL3 regulates the Rev-Erbα-dependent histone deacetylase 3 (HDAC3) repressor complex, suggesting that the action of FBXL3 on Rev-Erbα:HDAC3 is crucial for clock function. Our study further ...

“…The PAS domains mediate homo-and heterodimeric interactions between the mPER homologues (5)(6)(7)(8) as well as interactions of the mPERs with mBMAL1/2, mCLOCK, and NPAS2 (9)(10)(11)(12). These interactions regulate the stability and cellular localization of the mPERs and modulate the activity of the mBMAL1/mCLOCK transcription factor complex.…”

mentioning

confidence: 99%

Unwinding the differences of the mammalian PERIOD clock proteins from crystal structure to cellular function

Kucera

Schmalen

Hennig

et al. 2012

The three PERIOD homologues mPER1, mPER2, and mPER3 constitute central components of the mammalian circadian clock. They contain two PAS (PER-ARNT-SIM) domains (PAS-A and PAS-B), which mediate homo-and heterodimeric mPER-mPER interactions as well as interactions with transcription factors and kinases. Here we present crystal structures of PAS domain fragments of mPER1 and mPER3 and compare them with the previously reported mPER2 structure. The structures reveal homodimers, which are mediated by interactions of the PAS-B β-sheet surface including a highly conserved tryptophan (Trp448 mPER1 , Trp419 mPER2 , Trp359 mPER3 ). mPER1 homodimers are additionally stabilized by interactions between the PAS-A domains and mPER3 homodimers by an N-terminal region including a predicted helix-loop-helix motive. We have verified the existence of these homodimer interfaces in solution and inside cells using analytical gel filtration and luciferase complementation assays and quantified their contributions to homodimer stability by analytical ultracentrifugation. We also show by fluorescence recovery after photobleaching analyses that destabilization of the PAS-B/tryptophan dimer interface leads to a faster mobility of mPER2 containing complexes in human U2OS cells. Our study reveals structural and quantitative differences between the homodimeric interactions of the three mouse PERIOD homologues, which are likely to contribute to their distinct clock functions.circadian clock | PAS domains | PERIOD proteins | protein interactions