Ramanathan Chidambaram, Xu Haiyan, Khan Sanjoy K, Shen Yang, Gitis Paula J, Welsh David K, Hogenesch John B, Liu Andrew C
Department of Biological Sciences, University of Memphis, Memphis, Tennessee, United States of America.
Department of Psychiatry, University of California, San Diego, La Jolla, California, United States of America; Center for Chronobiology, University of California, San Diego, La Jolla, California, United States of America.
PLoS Genet. 2014 Apr 3;10(4):e1004244. doi: 10.1371/journal.pgen.1004244. eCollection 2014 Apr.
In animals, circadian rhythms in physiology and behavior result from coherent rhythmic interactions between clocks in the brain and those throughout the body. Despite the many tissue specific clocks, most understanding of the molecular core clock mechanism comes from studies of the suprachiasmatic nuclei (SCN) of the hypothalamus and a few other cell types. Here we report establishment and genetic characterization of three cell-autonomous mouse clock models: 3T3 fibroblasts, 3T3-L1 adipocytes, and MMH-D3 hepatocytes. Each model is genetically tractable and has an integrated luciferase reporter that allows for longitudinal luminescence recording of rhythmic clock gene expression using an inexpensive off-the-shelf microplate reader. To test these cellular models, we generated a library of short hairpin RNAs (shRNAs) against a panel of known clock genes and evaluated their impact on circadian rhythms. Knockdown of Bmal1, Clock, Cry1, and Cry2 each resulted in similar phenotypes in all three models, consistent with previous studies. However, we observed cell type-specific knockdown phenotypes for the Period and Rev-Erb families of clock genes. In particular, Per1 and Per2, which have strong behavioral effects in knockout mice, appear to play different roles in regulating period length and amplitude in these peripheral systems. Per3, which has relatively modest behavioral effects in knockout mice, substantially affects period length in the three cellular models and in dissociated SCN neurons. In summary, this study establishes new cell-autonomous clock models that are of particular relevance to metabolism and suitable for screening for clock modifiers, and reveals previously under-appreciated cell type-specific functions of clock genes.
在动物体内，生理和行为的昼夜节律源于大脑中的生物钟与全身其他生物钟之间协调一致的节律性相互作用。尽管存在许多组织特异性生物钟，但目前对分子核心生物钟机制的了解大多来自于对下丘脑视交叉上核（SCN）和其他几种细胞类型的研究。在此，我们报告了三种细胞自主型小鼠生物钟模型的建立及其遗传学特征：3T3成纤维细胞、3T3-L1脂肪细胞和MMH-D3肝细胞。每个模型在遗传学上都易于操作，并且具有一个整合的荧光素酶报告基因，可使用廉价的现成微孔板读数器对节律性生物钟基因表达进行纵向发光记录。为了测试这些细胞模型，我们针对一组已知的生物钟基因生成了一个短发夹RNA（shRNA）文库，并评估了它们对昼夜节律的影响。在所有三种模型中，敲低Bmal1、Clock、Cry1和Cry2均导致相似的表型，这与先前的研究一致。然而，我们观察到生物钟基因的Period家族和Rev-Erb家族存在细胞类型特异性的敲低表型。特别是，在敲除小鼠中具有强烈行为效应的Per1和Per2，在调节这些外周系统的周期长度和振幅方面似乎发挥着不同的作用。在敲除小鼠中行为效应相对较小的Per3，在三种细胞模型和分离的SCN神经元中对周期长度有显著影响。总之，本研究建立了与代谢特别相关且适合筛选生物钟调节剂的新的细胞自主型生物钟模型，并揭示了生物钟基因此前未被充分认识的细胞类型特异性功能。
