Multitissue Circadian Proteome Atlas of WT and Per1-/-/Per2-/- Mice.

The molecular basis of circadian rhythm, driven by core clock genes such as Per1/2, has been investigated on the transcriptome level, but not comprehensively on the proteome level. Here we quantified over 11,000 proteins expressed in eight types of tissues over 46 h with an interval of 2 h, using WT and Per1/Per2 double knockout mouse models. The multitissue circadian proteome landscape of WT mice shows tissue-specific patterns and reflects circadian anticipatory phenomena, which are less obvious on the transcript level. In most peripheral tissues of double knockout mice, reduced protein cyclers are identified when compared with those in WT mice. In addition, PER1/2 contributes to controlling the anticipation of the circadian rhythm, modulating tissue-specific cyclers as well as key pathways including nucleotide excision repair. Severe intertissue temporal dissonance of circadian proteome has been observed in the absence of Per1 and Per2. The γ-aminobutyric acid might modulate some of these temporally correlated cyclers in WT mice. Our study deepens our understanding of rhythmic proteins across multiple tissues and provides valuable insights into chronochemotherapy. The data are accessible at https://prot-rhythm.prottalks.com/.

Rapid assessment of circadian behavior in mice.

Circadian rhythms have a cycle length of about 24 hours, i.e. a 24-hour internal clock. In order to adapt to the periodic changes of the circadian environment, almost all organisms on the earth, including algae, bacteria, plants, animals, etc., have evolved a special system-the circadian clock. It helps organisms to adapt to the daily changes in the environment and maintains the physiological process and the behavior in synchronization with the environment changes. Circadian rhythms are composed of an intracellular feedback loop that drives the expression of molecular components and their constitutive protein products to oscillate over a period of about 24 hours. Almost every aspect of the body's functions, including behavior and physiology, is regulated by the circadian clock, and shows obvious daily rhythms, such as sleep and wakefulness, alertness, body temperature fluctuations, urinary system, hormone secretion, immune regulation, and cytokine release. Circadian factors are also increasingly recognized for potentially affecting the occurrence, progression, treatment, and prognosis of a variety of diseases. This paper discusses several methods for measuring circadian behavior disorders in mice for different purposes, and shares experimental operations and analysis ideas, including the use of metabolism cage, wheel running activity, jet lag, lengthened light, bones photoperiod, as well as the T7-cycle. In addition, this paper also studies the possible reasons for variations caused by genetic backgrounds and light conditions. Given these methods, researchers can choose appropriate experiments to evaluate the influence of genetic factors, environmental factors or diseases on circadian behavior.

Long-term SCN calcium signal recording in freely moving mice.

The suprachiasmatic nucleus (SCN) is the master circadian pacemaker of the mammalian biological clock. Here, we provide a detailed protocol for long-term recording of calcium signals in SCN neurons of freely moving mice through a multichannel optical fiber recording system. This system can simultaneously collect calcium signals from up to seven animals. The calcium signals can be visualized by the appropriate software and code. This protocol can be used to explore the long-term response of SCN to external environmental stimulation. For complete details on the use and execution of this protocol, please refer to Zhai et al. (2022).

Time-restricted feeding entrains long-term behavioral changes through the IGF2-KCC2 pathway.

The suprachiasmatic nucleus (SCN) integrates light and systemic signals from peripheral tissues to coordinate physiology and behavior daily rhythms. However, the contribution that nutrients and feeding patterns provide to the SCN network regulation remains controversial. Here, we found that time-restricted feeding (TRF) in ZT0-4 (Zeitgeber Time) generates a robust and long-term shift in locomotor behavior and increased wakefulness. Intracellular Ca2+ signals in SCN GABAergic neurons of freely moving mice showed significant activation after ZT0-4 TRF treatment. Furthermore, RNA-seq profiling of SCN showed that TRF during ZT0-4 increased Insulin-like Growth Factor 2 (Igf2) expression and dysregulated ion transporters, including the downregulation of Kcc2. SCN neuron-specific loss of function of Kcc2 amplified ZT0-4 TRF induced aftereffect. Moreover, overexpression of IGF2 in SCN GABAergic neurons extended the locomotion range, mirroring the TRF aftereffect. In summary, our study showed that the IGF2-KCC2 pathway plays an important role for TRF induced behavior changes.

Human-specific gene CT47 blocks PRMT5 degradation to lead to meiosis arrest.

Exploring the functions of human-specific genes (HSGs) is challenging due to the lack of a tractable genetic model system. Testosterone is essential for maintaining human spermatogenesis and fertility, but the underlying mechanism is unclear. Here, we identified Cancer/Testis Antigen gene family 47 (CT47) as an essential regulator of human-specific spermatogenesis by stabilizing arginine methyltransferase 5 (PRMT5). A humanized mouse model revealed that CT47 functions to arrest spermatogenesis by interacting with and regulating CT47/PRMT5 accumulation in the nucleus during the leptotene/zygotene-to-pachytene transition of meiosis. We demonstrate that testosterone induces nuclear depletion of CT47/PRMT5 and rescues leptotene-arrested spermatocyte progression in humanized testes. Loss of CT47 in human embryonic stem cells (hESCs) by CRISPR/Cas9 led to an increase in haploid cells but blocked the testosterone-induced increase in haploid cells when hESCs were differentiated into haploid spermatogenic cells. Moreover, CT47 levels were decreased in nonobstructive azoospermia. Together, these results established CT47 as a crucial regulator of human spermatogenesis by preventing meiosis initiation before the testosterone surge.