Fig. 1. Two RREs in the 5’-UTR of Bmal1 are essential for rhythmic expression of Bmal1.

a A model for the mammalian circadian clock composed of two interlocking loops through DNA cis-elements, E-box and RRE. CLOCK and BMAL1 activate the E-box-mediated transcription of a series of genes such as Per, Cry, and Rev-erb genes, and the transactivation is suppressed by translated PER and CRY proteins. REV-ERB proteins repress the RRE-mediated transcription of a series of genes such as Bmal1. b The genome structure of the Bmal1 gene locus (Upper panel) and the sequences of ΔRRE cell line #1 and ΔRRE mouse line #1 (Lower). Thick and thin lines indicate exons and introns of Bmal1, respectively. RRE elements are indicated by two boxes in purple (Upper) or bold italic letters in purple (Lower). c Temporal profiles of Bmal1 mRNA levels. Fibroblasts (NIH3T3 cells), livers, kidneys, hearts and epididymal white adipose tissues (eWAT) were harvested at 4-hr intervals, followed by quantitative RT-PCR. The mRNA levels of Bmal1 were normalized by Rps29. Data are means ± SEM (n = 3 for WT, n = 4 for ΔRRE). Blue and black lines indicate the ΔRRE mutants and the littermate controls, respectively.