System-level identification of transcriptional circuits underlying mammalian circadian clocks

Mammalian circadian clocks consist of complexly integrated regulatory loops(1-5), making it difficult to elucidate them without both the accurate measurement of system dynamics and the comprehensive identification of network circuits(6). Toward a system-level understanding of this transcriptional circuitry, we identified clock-controlled elements on 16 clock and clock-controlled genes in a comprehensive surveillance of evolutionarily conserved cis elements and measurement of their transcriptional dynamics. Here we report the roles of E/E' boxes, DBP/E4BP4 binding elements(7) and RevErbA/ROR binding elements(8) in nine, seven and six genes, respectively. Our results indicate that circadian transcriptional circuits are governed by two design principles: regulation of E/E boxes and RevErbA/ROR binding elements follows a repressor-precedes-activator pattern, resulting in delayed transcriptional activity, whereas regulation of DBP/E4BP4 binding elements follows a repressor-antiphasic-to-activator mechanism, which generates high-amplitude transcriptional activity. Our analysis further suggests that regulation of E/E boxes is a topological vulnerability in mammalian circadian clocks, a concept that has been functionally verified using in vitro phenotype assay systems.