Figure 1.

Experimental systems for analysis of the repressive phase of the mammalian clock TTFL. (A) Canonical model of the mammalian circadian clock. In this highly simplified model, only the core TTFL is shown: The CLOCK–BMAL1 heterodimer binds to the E-boxes in Per and Cry genes and activates their transcription. The CRY and PER proteins dimerize in the cytoplasm and, after a time lag, enter the nucleus, bind to CLOCK–BMAL1, and inhibit their own transcription. The cycle starts over after CRY and PER levels decrease by proteolysis. (B) Targeted nuclear delivery system for analysis of CRY1 and PER2 functions. CRY1–ER* or PER2–ER* are expressed in Cry1/2^−/− or Per1/2^−/− cells, respectively. The fusion proteins are retained in the cytoplasm in complex with heat-shock protein (HSP). Upon addition of 4-hydroxytamoxifen (4-OHT), HSP dissociates, and the fusion proteins enter the nucleus. (C,D) 4-OHT-induced nuclear entry of CRY1–ER* and PER2–ER* analyzed by immunofluorescence microscopy (C) and immunoblotting analysis of nuclear extracts (D). Red arrows point to nuclei. A nonspecific band (N.S.) and tubulin protein are shown for loading controls.