Figure 5

CLOCK/BMAL1 heterodimers activate the expression of mir-142. (A) The schematic representation for the luciferase reporter constructs. The upstream regulatory sequence of mir-142 together with pre-mir-142 was inserted upstream of the luciferase reporter gene. ‘E’ represents the conserved canonical E-box (CACGTG). ‘MT’ and ‘DEL’ denote the mutation (GTCGAC) and deletion of the E-box, respectively. (B) The over-expression of Clock and Bmal1 in NIH3T3 cells was confirmed by western blotting. Bmal1-3.1 (Bmal1-pcDNA3.1) and Clock-3.1 (Clock-pcDNA3.1) denote the Bmal1 and Clock expression plasmids. (C) A luciferase reporter assay was performed to determine whether CLOCK and BMAL1 heterodimers could enhance the transcription of the luciferase reporters (mean ± SD, n = 4). (D) After over-expressing Clock and Bmal1 in NIH3T3, the level of mir-142-3p was determined by quantitative RT-PCR (mean ± SD, n = 3). ***P < 0.001. (E) A simple model indicating a potential negative feedback loop involving both the activation of mir-142 transcription by CLOCK/BMAL1 heterodimers and the ensuing inhibition of Bmal1 expression by mir-142-3p.