Positive Autoregulation Delays the Expression Phase of Mammalian Clock Gene Per2
(A) Schematics of Per1::luc, Per2::luc and delta-Per2 promoter driving luciferase reporter (delta-Per2::luc). Delta-Per2::luc does not contain the region between two E-box like elements (115-35 bp upstream from transcription start site  ) that contributes to positive feedback regulation. (B) PER1 and PER2 co-transfection with CLOCK and BMAL1 induced only Per2 promoter activity. Left: Per1::luc, middle: Per2::luc, and right: delta-Per2::luc. Induction intensities of Per1::luc by CLOCK-BMAL1 were 6.86±0.38 without PER1/2, 6.78±0.44 with PER1, and 4.64±0.31 with PER2 in reference to the basal promoter activity. Induction intensities of Per2::luc by CLOCK-BMAL1 were 2.52±0.08 without PER1/2, 6.57±0.47 with PER1, and 7.53±0.39 with PER2 in reference to the basal promoter activity. Both PER1 and PER2 proteins significantly induced the Per2 promoter in the presence of CLOCK-BMAL1, but not Per1 promoter (Student's t-test, P<0.01). Induction intensities of delta-Per2::luc by CLOCK-BMAL1 were 6.32±0.19 without PER1/2, 5.27±0.14 with PER1, and 5.76±0.03 with PER2 in reference to the basal promoter activity, and there were no significant differences. Normalization was conducted with a pCIneo vector co-expression. Error bars indicate SEM determined from independent experiments in triplicate. (C) Representative bioluminescence oscillations of Per1::luc (square), Per2::luc (filled circle), and deleted-Per2::luc (open circle). The time difference from the Per1::luc to the Per2::luc expression peaks was 3.88±0.14 hours (Student's t-test, P<0.005). The phase of delta-Per2::luc was advanced by 2.86±0.39 hours (Student's t-test, P<0.01) compared with wild-type Per2::luc. Statistical data for the period and phase are described in the text and Table 1.