Cell type-specific functions of period genes revealed by novel adipocyte and hepatocyte circadian clock models.
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.
Department of Biological Sciences, University of Memphis, Memphis, Tennessee, United States of America.
Chidambaram
C
Ramanathan
Department of Biological Sciences, University of Memphis, Memphis, Tennessee, United States of America.
Haiyan
H
Xu
Department of Biological Sciences, University of Memphis, Memphis, Tennessee, United States of America.
Sanjoy K
SK
Khan
Department of Biological Sciences, University of Memphis, Memphis, Tennessee, United States of America.
Yang
Y
Shen
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.
Paula J
PJ
Gitis
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; Veterans Affairs San Diego Healthcare System, San Diego, California, United States of America.
David K
DK
Welsh
Department of Pharmacology and Institute for Translational Medicine and Therapeutics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America.
John B
JB
Hogenesch
Department of Biological Sciences, University of Memphis, Memphis, Tennessee, United States of America; Feinstone Genome Research Center, University of Memphis, Memphis, Tennessee, United States of America.
Andrew C
AC
Liu
D017667:Adipocytes / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D057906:Circadian Clocks / Q000235:genetics*; D002940:Circadian Rhythm / Q000235:genetics*; D022781:Hepatocytes / Q000378:metabolism*; D008156:Luciferases / Q000235:genetics; D051379:Mice; D018345:Mice, Knockout; D041681:NIH 3T3 Cells; D009474:Neurons / Q000378:metabolism; D056950:Period Circadian Proteins / Q000235:genetics / Q000378:metabolism
D056950:Period Circadian Proteins; D008156:Luciferases