Molecular daily rhythms can be captured by precisely timed tissue harvests from groups of animals. This protocol will allow the investigator to identify transcriptional rhythms in the mouse liver while also providing a template for similar analyses in other whole metabolic organs. We describe steps for mouse entrainment, liver dissection, and rhythmicity analysis from total RNA sequencing data. The resulting rhythmic transcriptome will provide the user with a starting point for defining specific biological processes that undergo daily rhythms. For complete details on the use and execution of this protocol, please refer to Koronowski et al. (2019). A similar protocol for interfollicular epidermal cells is demonstrated in Welz et al. (2019). ; The IRB Barcelona is a Severo Ochoa Center of Excellence (MINECO award SEV-2015-0505). K.B.K. was supported by NIH-NIDDK (award F32DK121425). T.M. received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 754510.
The mammalian circadian clock, expressed throughout the brain and body, controls daily metabolic homeostasis. Clock function in peripheral tissues is required, but not sufficient, for this task. Because of the lack of specialized animal models, it is unclear how tissue clocks interact with extrinsic signals to drive molecular oscillations. Here, we isolated the interaction between feeding and the liver clock by reconstituting Bmal1 exclusively in hepatocytes (Liver-RE), in otherwise clock-less mice, and controlling timing of food intake. We found that the cooperative action of BMAL1 and the transcription factor CEBPB regulates daily liver metabolic transcriptional programs. Functionally, the liver clock and feeding rhythm are sufficient to drive temporal carbohydrate homeostasis. By contrast, liver rhythms tied to redox and lipid metabolism required communication with the skeletal muscle clock, demonstrating peripheral clock cross-talk. Our results highlight how the inner workings of the clock system rely on communicating signals to maintain daily metabolism. ; C.M.G. was supported by the National Cancer Institute of the National Institutes of Health (NIH) under award number T32CA009054 and by the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement 749869. K.B.K. was supported by NIH-NINDS F32DK121425, and J.G.S. was supported by the Zymo-CEM Postdoctoral Fellowship (Zymo Research). P.P. was supported by a scholarship from the Wenner-Gren Foundations. C.V. and M.M.S. are supported by NIH grants DK20AU4084 and HL138193.The work of S.C., M.S., and P.B. was in part supported by NIH grant GM123558 to P.B. Work in the W.L. laboratory was supported by NIH grants R01HG007538, R01CA193466, and R01CA228140. Work in the P.S.-C. laboratory is supported by NIH grants R21DK114652 and R21AG053592, a Challenge Grant from the Novo Nordisk Foundation (NNF-202585), and through access to the Genomics High Throughput Facility Shared Resource of the Cancer Center Support Grant (CA-62203) at the UCI and NIH-shared instrumentation grants 1S10RR025496-01, 1S10OD010794-01, and 1S10OD021718-01. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. P.-S.W. is supported by a "Ramon y Cajal" contract (RYC2019-026661-I) from the Spanish Ministry of Science and Innovation (MICINN). Research in the P.M.-C. laboratory is supported by MINECO-Spain (RTI2018-096068), ERC-2016-AdG-741966, AFM, MDA-USA, La Marató/TV3 Foundation, LaCaixa-HEALTH-HR17-00040, and UPGRADE-H2020-825825 and María-de-Maeztu-Program for Units of Excellence to UPF (MDM-2014-0370) and Severo-Ochoa-Program for Centers of Excellence to CNIC (SEV-2015-0505). Research in the S.A.B. laboratory is supported by the European Research Council (ERC), the Government of Cataluña (SGR grant), the Government of Spain (MINECO), the La Marató/TV3 Foundation, and The Worldwide Cancer Research Foundation (WCRF). Author contributions: C.M.G., K.B.K., J.G.S., P.B., S.M., S.A.B., P.M.-C., and P.S.-C. conceived and designed the study. C.M.G., K.B.K., J.G.S., S.A.B., P.M.-C., and P.S.-C. wrote and edited the manuscript. C.M.G., K.B.K., J.G.S., P.-S.W., V.M.Z., T.M., K.S., T.S., P.P., S.K.C., and K.A.D. performed experiments. O.D., A.K. and M.V.-D. provided technical support. D.L., J.M.M.K., C.V., and M.M.S. performed bioinformatic correlation analyses. S.C., M.S., P.K., R.C., J.S., and W.L. performed sequencing analysis.
