Identification of functional clock-controlled elements involved in differential timing of Per1 and Per2 transcription.
It has been proposed that robust rhythmic gene expression requires clock-controlled elements (CCEs). Transcription of Per1 was reported to be regulated by the E-box and D-box in conventional reporter assays. However, such experiments are inconclusive in terms of how the CCEs and their combinations determine the phase of the Per1 gene. Whereas the phase of Per2 oscillation was found to be the most delayed among the three Period genes, the phase-delaying regions of the Per2 promoter remain to be determined. We therefore investigated the regulatory mechanism of circadian Per1 and Per2 transcription using an in vitro rhythm oscillation-monitoring system. We found that the copy number of the E-box might play an important role in determining the phase of Per1 oscillation. Based on real-time bioluminescence assays with various promoter constructs, we provide evidence that the non-canonical E-box is involved in the phase delay of Per2 oscillation. Transfection experiments confirmed that the non-canonical E-box could be activated by CLOCK/BMAL1. We also show that the D-box in the third conserved segment of the Per2 promoter generated high amplitude. Our experiments demonstrate that the copy number and various combinations of functional CCEs ultimately led to different circadian phases and amplitudes.
Yamajuku
Daisuke
D
Pharmacology Research Laboratories, Astellas Pharma, Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan. daisuke.yamajuku@jp.astellas.com
Shibata
Yasutaka
Y
Kitazawa
Masashi
M
Katakura
Toshie
T
Urata
Hiromi
H
Kojima
Tomoko
T
Nakata
Osamu
O
Hashimoto
Seiichi
S
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D057906:Circadian Clocks / Q000235:genetics*; D002940:Circadian Rhythm / Q000235:genetics*; D017124:Conserved Sequence; D024721:E-Box Elements; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D056950:Period Circadian Proteins / Q000096:biosynthesis / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D016415:Sequence Alignment; D014158:Transcription, Genetic
C421902:PER2 protein, human; C539023:Per1 protein, mouse; C513312:Per2 protein, mouse; C512645:Per3 protein, mouse; D056950:Period Circadian Proteins; D012333:RNA, Messenger