Molecular Analysis Of The Circadian Clock In Mice

Almost all organisms, from cyanobacteria to vertebrates, show endogenous circadian rhythms. It accommodates organisms' physiology and behavior to surrounding environmental changes, thereby conferring an adaptive advantage, such as avoidance of predators, balance of metabolic processes, and regulation of cell cycle.The core feature of circadian oscillator is based on interlocked auto-regulatory positive and negative transcriptional/translational feedback loops. Positive elements CLOCK and BMAL1 proteins form heterodimers, bind on E-box cis-element, and activate the transcription of Pers (Per1, Per2, and Per3) and Crys(Cry1, Cry2) genes. Then, PER and CRY proteins inhibit their own transcriptions by interacting with BMAL1-CLOCK complexes. Moreover, nuclear receptors REV-ERBs and RORs strictly control the transcription of Bmall by competitively binding on RRE-box cis-element. The circadian clock system in mammals has three parts:inputs, circadian oscillator{suprachiasmatic nucleus (SCN)}, and outputs.In the section one including chapter 2,3 and 4, we focused on studying the function of zinc finger protein gene Zbtb20 in circadian clock, and found that Zbtb20 plays a crucial role in the regulation of bimodal activities in mice. We generated conditional Zbtb20 knockout mice. Mice loss of Zbtb20 exhibited a burst in morning activity, while evening activity was missing. When exposed to different light-dark cycles, these mice showed impaired entrainment, especially through the early evening hours. The switch of bimodal to unimodal activity was also found to compromise activity-dependent downstream rhythms, such as metabolism and body temperature rhythms.Then, Zbtb20-deficient mice resembled phenotypes of Prok2/Prokr2-knockout mice and exhibited a pronounced decrease in the expression of Prokr2. ZBTB20 could directly bind in the promoter region of Prokr2, and enhance transcriptional activity repeatable with P300 on a Prokr2 promoter reporter. Furthermore, injection of adeno-associated virus expressing PROK.R2 in suprachiasmatic nucleus could partly restore evening activity in Nestin-Cre; Zbtb20fl/fl mice, demonstrating that ZBTB20-mediated PROKR2 signaling was critical for the evening behavioral rhythms. In addition, using a combination of RNA sequencing and mass spectra identification, we revealed that ZBTB20 targeted multiple pathways, providing a model that integrates circadian output signals in neurodegenerative disorders.In the section two, chapter 5, the roles of CLOCK-Interacting Protein, Circadian (CIPC) were characterized in circadian clock at the animal level. CIPC has been indicated as an additional negative-feedback regulator of the circadian clock in vitro, although its physiological roles in circadian clock are unknown. Here, we generated Cipc homozygous knockout (Cipc-/-) mice and assessed the resultant circadian phenotypes. Surprisingly, the mRNA expression profiles of core clock genes in the liver of Cip-/- mice showed no significant differences from that in wild-type mice except for Perl. Cipc-/- mice displayed normal locomotor rhythm and entrained activity pattern in both 12:12 light-dark cycle and constant dark cycle. Furthermore, deletion of Cipc in lungs and adipose tissues did not influence their peripheral clock.The results from this work provided more conclusive data suggesting that CIPC is not critically required for basic clock function.