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The orphan nuclear receptor Rev-erbalpha (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-erbalpha gene regulation, we have isolated its 5'-upstream region. We found that two promoters control the zfRev-erbalpha 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-erbalpha-binding site (RevDR2). Inhibition of zfRev-erbalpha activity in zebrafish embryos using antisense-morpholino knockdown results in an increase of zfRev-erbalpha gene expression suggesting that zfRev-erbalpha is repressing its own transcription in vivo. In addition, we show that ROR orphan receptors also regulate in vitro and in vivo zfRev-erbalpha 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-erbalpha expression.

Two differentially active alternative promoters control the expression of the zebrafish orphan nuclear receptor gene Rev-erbα