In the mammalian circadian clockwork, CRY1 and CRY2 repressor proteins are regulated by posttranslational modifications for temporally coordinated transcription of clock genes. Previous studies revealed that FBXL3, an F-box-type E3 ligase, ubiquitinates CRYs and mediates their degradation. Here, we found that FBXL21 also ubiquitinates CRYs but counteracts FBXL3. Fbxl21(-/-) mice exhibited normal periodicity of wheel-running rhythms with compromised organization of daily activities, while an extremely long-period phenotype of Fbxl3(-/-) mice was attenuated in Fbxl3/Fbxl21 double-knockout mice. The double knockout destabilized the behavioral rhythms progressively and sometimes elicited arrhythmicity. Surprisingly, FBXL21 stabilized CRYs and antagonized the destabilizing action by FBXL3. Predominantly cytosolic distribution of FBXL21 contrasts with nuclear localization of FBXL3. These results emphasize the physiological importance of antagonizing actions between FBXL21 and FBXL3 on CRYs, and their combined actions at different subcellular locations stabilize oscillation of the circadian clock.

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In humans, the connection between sleep and mood has long been recognized, although direct molecular evidence is lacking. We identified two rare variants in the circadian clock gene PERIOD3 (PER3-P415A/H417R) in humans with familial advanced sleep phase accompanied by higher Beck Depression Inventory and seasonality scores. hPER3-P415A/H417R transgenic mice showed an altered circadian period under constant light and exhibited phase shifts of the sleep-wake cycle in a short light period (photoperiod) paradigm. Molecular characterization revealed that the rare variants destabilized PER3 and failed to stabilize PERIOD1/2 proteins, which play critical roles in circadian timing. Although hPER3-P415A/H417R-Tg mice showed a mild depression-like phenotype, Per3 knockout mice demonstrated consistent depression-like behavior, particularly when studied under a short photoperiod, supporting a possible role for PER3 in mood regulation. These findings suggest that PER3 may be a nexus for sleep and mood regulation while fine-tuning these processes to adapt to seasonal changes. I n human populations, alterations in circadian timing can result in mood-related problems (1). An example of this is seasonal affective disorder, also known as "winter depression," which is among the most common mood disorders, with a reported prevalence of 1.5-9%, depending on latitude (2). In addition, shift work has been suggested as a risk factor for major depressive disorder (3), and depression severity correlates with the degree of circadian misalignment (4, 5). A number of genetic variants in core clock genes have been reported as statistically associated with mood disorders, including seasonal affective disorder and major depressive disorder (6-14), but to date none has been causally related with an understanding of specific molecular links.Familial advanced sleep phase (FASP) is a human behavioral phenotype defined by early sleep time and early morning awakening (15). We previously identified mutations in core clock genes that cause FASP by linkage analysis/positional cloning (16) and candidate gene sequencing (17, 18). Here we identify two rare missense variants in PER3 (PER3-P415A/H417R) that cause FASP and are associated with elevated Beck Depression Inventory (BDI) and seasonality scores. Transgenic mice carrying human PER3-P415A/H417R exhibit delayed phase in a short photoperiod and a lengthened period of wheel-running rhythms in constant light. At a molecular level, the rare variants lead to decreased PER3 protein levels, likely due to decreased protein stability. Moreover, we found that PER3-P415A/H417R can exert effects on the clock (at least in part) by reducing its stabilizing effect on PER1 and PER2. Although hPER3-P415A/H417R transgenic mice display mild measures of depression-like phenotype, Per3 −/− mice exhibit consistent depression-like behaviors in multiple tests. The differences are particularly evident in short photoperiods, implying a role for PER3 in mood regulation. Taken together, these results support a role for PER3 in ...