System-level identification of transcriptional circuits underlying mammalian circadian clocks

Mammalian circadian clocks consist of complexly integrated regulatory loops1,2,3,4,5, making it difficult to elucidate them without both the accurate measurement of system dynamics and the comprehensive identification of network circuits6. 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 elements7 and RevErbA/ROR binding elements8 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.