Cell Type-Specific Functions of Period Genes Revealed by Novel Adipocyte and Hepatocyte Circadian Clock Models

Abstract

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.

Author Summary

Various aspects of our daily rhythms in physiology and behavior such as the sleep-wake cycle are regulated by endogenous circadian clocks that are present in nearly every cell. It is generally accepted that these oscillators share a similar biochemical negative feedback mechanism, consisting of transcriptional activators and repressors. In this study, we developed cell-autonomous, metabolically relevant clock models in mouse hepatocytes and adipocytes. Each clock model has an integrated luciferase reporter that allows for kinetic luminescence recording with an inexpensive microplate reader and thus is feasible for most laboratories. These models are amenable to high throughput screening of small molecules or genomic entities for impacts on cell-autonomous clocks relevant to metabolism. We validated these new models by RNA interference via lentivirus-mediated knockdown of known clock genes. As expected, we found that many core clock components have similar functions across cell types. To our surprise, however, we also uncovered previously under-appreciated cell type-specific functions of core clock genes, particularly Per1, Per2, and Per3. Because the circadian system is integrated with, and influenced by, the local physiology that is under its control, our studies provide important implications for future studies into cell type-specific mechanisms of various circadian systems.