Chicken pineal clock genes: implication of BMAL2 as a bidirectional regulator in circadian clock oscillation.
BACKGROUND
In a transcription/translation-based autoregulatory feedback loop of vertebrate circadian clock systems, a BMAL1-CLOCK heterodimer is a positive regulator for the transcription of the negative element gene Per. The chicken pineal gland represents a photosensitive clock tissue, but the pineal clock genes constituting the oscillator loop have been less well characterized.


RESULTS
We identified expression of the Per2, Bmal1, Bmal2 and Clock genes in the chicken pineal gland. Messenger RNA levels of these genes exhibited overt circadian rhythms in the pineal cells, both in vivo and in culture. In vitro functional analyses revealed the formation of cBMAL1-cCLOCK and cBMAL2-cCLOCK heteromers. Both of the cBMAL-cCLOCK heteromers activated E-box element-dependent transcription, which was negatively regulated by cPER2 in luciferase assays. Co-expression of cCLOCK, cBMAL1 and cBMAL2 co-operatively activated E-box element-dependent transcription, and a greater level of expression of cBMAL2 inhibited the activation. In the cultured pineal cells, an over-expression of either cBMAL1 or cBMAL2 disrupted the circadian rhythm of melatonin production.


CONCLUSIONS
The functional characterization of the chicken pineal clock molecules supports the key roles of BMAL1, BMAL2 and CLOCK which contribute to the E-box-dependent transcriptional regulation in the circadian clock system.
Okano
T
T
Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Hongo 7-3-1, Bunkyo-Ku, Tokyo 113-0033, Japan.
Yamamoto
K
K
Okano
K
K
Hirota
T
T
Kasahara
T
T
Sasaki
M
M
Takanaka
Y
Y
Fukada
Y
Y
D056930:ARNTL Transcription Factors; D000595:Amino Acid Sequence; D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins; D015152:Blotting, Northern; D056926:CLOCK Proteins; D018797:Cell Cycle Proteins; D002478:Cells, Cultured; D002645:Chickens / Q000502:physiology*; D002940:Circadian Rhythm / Q000502:physiology*; D017931:DNA Primers / Q000737:chemistry; D024721:E-Box Elements / Q000235:genetics; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D018257:Helix-Loop-Helix Motifs / Q000502:physiology; D008027:Light; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D056950:Period Circadian Proteins; D010870:Pineal Gland / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D056930:ARNTL Transcription Factors; D051792:Basic Helix-Loop-Helix Proteins; D056926:CLOCK Proteins; D018797:Cell Cycle Proteins; D017931:DNA Primers; D005136:Eye Proteins; D005982:Glutathione Transferase; D009687:Nuclear Proteins; D056950:Period Circadian Proteins; D012333:RNA, Messenger; D015534:Trans-Activators; D014157:Transcription Factors; C538961:Bmal1 protein, mouse; C431792:Arntl2 protein, mouse; C513312:Per2 protein, mouse; C538944:Clock protein, mouse