A direct repeat of E-box-like elements is required for cell-autonomous circadian rhythm of clock genes

We report a new cis-element required for cell-autonomous circadian transcription of clock genes. This new element consists of a canonical E-box or a non-canonical E-box and an E-box-like sequence in tandem with the latter with a short interval, 6 base pairs, between them. We demonstrate that both E-box or E-box-like sequences are needed to generate cell-autonomous oscillation. We also verify that the spacing nucleotides with constant length between these 2 E-elements are crucial for robust oscillation. Furthermore, by in silico analysis we conclude that several clock and clock-controlled genes possess a direct repeat of the E-box-like elements in their promoter region.We propose a novel possible mechanism regulated by double E-box-like elements, not to a single E-box, for circadian transcriptional oscillation. The direct repeat of the E-box-like elements identified in this study is the minimal required element for the generation of cell-autonomous transcriptional oscillation of clock and clock-controlled genes.Circadian rhythms, periodicities with an approximate 24-h length, are essential physiological functions in almost all organisms on the earth and are generated by endogenous biological clocks. The biological clocks consist of 3 components: input, pacemaker, and output. The endogenous period length is not exactly 24 h and must be entrained by light. In mammals, other entrainment cues such as food intake or temperature, even social cues, can reset the circadian rhythm. The central oscillator in mammals is located in the suprachiasmatic nuclei (SCN) in the anteroventral hypothalamus. Recent studies revealed that the peripheral clocks reside not only in other brain regions but also in peripheral organs and even in cultured cells [1-4]. The output of the biological clocks includes broad physiological phenomena such as locomotor activity, sleep-wake cycle, hormonal secretion, cardiovascular condition, bowel movement, and even mental states. The disturbance of the bio