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Laboratoire de Biologie Moléculaire de la Cellule, CNRS UMR 5161, INRA LA 1237, IFR 128 BioSciences Lyon-Gerland, Ecole Normale Supérieure de Lyon, 46 allée d’Italie, 69364 Lyon cedex, France

(Requests for offprints should be addressed to V Laudet; Email: vincent.laudet{at}ens-lyon.fr)

T Kakizawa and S-i Nishio contributed equally to this work

The orphan nuclear receptor Rev-erb (NR1D1) plays an important role in the regulation of the circadian pacemaker and its expression has been shown to be regulated with a robust circadian rhythm in zebrafish and mammals. In addition, in zebrafish its expression has been shown to be developmentally regulated. In order to analyze the mechanisms of the zfRev-erb gene regulation, we have isolated its 5'-upstream region. We found that two promoters control the zfRev-erb expression. The first one (ZfP1) is characterized by a very high degree of sequence identity with the mammalian P1 promoter and contains, as the mammalian P1, a functional Rev-erb-binding site (RevDR2). Inhibition of zfRev-erb activity in zebrafish embryos using antisense-morpholino knockdown results in an increase of zfRev-erb gene expression suggesting that zfRev-erb is repressing its own transcription in vivo. In addition, we show that ROR orphan receptors also regulate in vitro and in vivo zfRev-erb gene expression through the same RevDR2 element. In contrast, the second promoter ZfP2 is strikingly different from the mammalian P2: its sequence is not conserved between zebrafish and mammals and is not regulated by the same transcription factors. Together, these data suggest that ZfP1 is orthologous to the mammalian P1 promoter, whereas zebrafish ZfP2 has no mammalian ortholog and does not function like ZfP1 to control Rev-erb expression.

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