A Cryptochrome 2 mutation yields advanced sleep phase in humans.

Arisa Hirano,Thomas Mcmahon,Louis J Ptáček,Len A Pennacchio,Ying Xu,Maya Yamazaki,William C Hallows,Guangsen Shi,Ying-Hui Fu,Anna Lipzen,Christopher R Jones

doi:10.7554/elife.16695

Abstract

Familial Advanced Sleep Phase (FASP) is a heritable human sleep phenotype characterized by very early sleep and wake times. We identified a missense mutation in the human Cryptochrome 2 (CRY2) gene that co-segregates with FASP in one family. The mutation leads to replacement of an alanine residue at position 260 with a threonine (A260T). In mice, the CRY2 mutation causes a shortened circadian period and reduced phase-shift to early-night light pulse associated with phase-advanced behavioral rhythms in the light-dark cycle. The A260T mutation is located in the phosphate loop of the flavin adenine dinucleotide (FAD) binding domain of CRY2. The mutation alters the conformation of CRY2, increasing its accessibility and affinity for FBXL3 (an E3 ubiquitin ligase), thus promoting its degradation. These results demonstrate that CRY2 stability controlled by FBXL3 plays a key role in the regulation of human sleep wake behavior.

Highlights

Ala260 is located in the flavin adenine dinucleotide (FAD) binding domain of Cryptochrome 2 (CRY2) and it is highly conserved in CRY1 and CRY2 proteins of various species (Figure 1B). 123 Familial Advanced Sleep Phase (FASP) in mouse model carrying A260T mutation To test whether the A260T mutation causes FASP and has a dominant effect on the circadian sleep-wake cycle, we generated wild-type hCRY2 and mutant hCRY2 human BAC transgenic (Tg) mice (Figure 2-supplement 1A)
Circadian period shortening by the A260T mutation was found using mouse embryonic fibroblasts (MEFs) derived from mice with a mutant vs. WT transgene on both WT and mCry2 knockout backgrounds (Figure 3D, E)
These results indicate that the conformation of the phosphate loop may play critical roles in regulating CRY2 stability and repressor activity. 220 A260T mutation affects FBXL3-CRY2 interaction 221 FBXL3 primarily localizes in the nucleus and promotes proteasomal degradation of CRY2, having a strong impact on the circadian period of mice

Summary

Introduction

Sleep-wake timing is regulated by the internal biological clock driving physiological rhythms with a period of approximately 24 hours (Takahashi, 1995). The circadian clock is composed of interlocked transcriptional and translational negative feedback loops (Lowrey and Takahashi, 2004; Reppert and Weaver, 2001). A CLOCK-BMAL1 heterodimer binds to E-boxes and activates gene expression of the Period (Per) and Cryptochrome (Cry) genes. Translated PERs and CRYs proteins form a complex that enters the nucleus to inhibit their own transcription through direct interaction with CLOCK-BMAL1 heterodimers. PER and CRY proteins accumulating in the nucleus are degraded over time. As protein levels fall (depending on rate of degradation), the transcription-translation feedback loop begins anew

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Results

Conclusion