Journal of Steroid Biochemistry & Molecular Biology 86 (2003) 381–386 Analysis of zebrafish cyp19 promoters  Sok-Keng Tong, Bon-chu Chung ∗ a Institute of Molecular Biology 48, Academia Sinica, Nankang, Taipei 115, Taiwan, ROC Abstract Cyp19 encodes P450 aromatase, the key enzyme catalyzing the conversion of androgens into estrogens. Estrogens play a crucial role in the anatomical, functional and behavioral characteristics of sexually dimorphic development. In zebrafish, two cyp19 genes, cyp19a and cyp19b, expressed in ovary and brain, respectively, were found. We have isolated the promoter regions of the zebrafish cyp19 genes from a bacterial artificial chromosome library to search for regulatory sequences that bind to transcription factors. Sequences like arylhydrocarbon receptor (AhR) recognition site, estrogen receptor recognition half sites (1/2ERE) and c-AMP responsive elements were found in the 5 ′ -flanking regions of both cyp19 genes. For ovarian-specific expression, we found binding sites for steroidogenic factor-1 (SF-1), GATA transcription factor 4 (GATA-4) and Wilm tumor 1 (WT1–KTS) on the promoter region of cyp19a but not cyp19b. For brain-specific expression of the cyp19b gene, sequences for recognition of chicken ovalbumin upstream promoter-transcription factor (COUP) and Ptx-1 were detected in the promoter. The importance of these putative control elements in ovary and brain-specific promoter has been assessed by sequence comparison among various species. © 2003 Elsevier Ltd. All rights reserved. Keywords: Ovary; Brain; Promoter; Aromatase; cyp19; P450aro; Zebrafish 1. Introduction Estrogens play crucial roles in the development of sex- ually dimorphic, anatomical, functional and behavioral characteristics that are vital for reproduction in vertebrates. Estrogen biosynthesis is catalyzed by an enzyme complex, one of which components is P450 aromatase encoded by the cyp19 gene. The cyp19 gene is highly conserved through- out the vertebrate phylum, such as human [1], mouse [2], rat [3], cow [4], birds like chicken [5] and zebra finch [6], reptile [7], and fishes like zebrafish [8] and medaka [9]. In mammals, cyp19 is expressed in several types of tissues including gonad, skin, adipose tissue, placenta, and brain [10], while in fishes it is expressed mainly in the brain and the gonad. Except for pig [11] and fish [12,13], where multiple cyp19 genes have been identified, there is only a single gene in most species. The tissue-specific expressions of cyp19 in human [10], sheep [14], rabbit [15] and zebra finch [16] are achieved by the use of alternative transcrip- tion start sites that arise as a consequences of the use of tissue-specific promoters and alternative splicing.  Presented at the VI International Aromatase Conference: ARO- MATASE 2002, Kyoto, Japan, 26–30 October 2002. ∗ Corresponding author. Tel.: +886-2-2789-9215; fax: +886-2-2788 3464. E-mail address: mbchung@sinica.edu.tw (B.-c. Chung). So far, six promoter regions have been identified in hu- man CYP19 gene, namely, Ia, Ib, Ic, Id (II), Ie, and If; they are expressed in placenta, fetal liver/skin fibroblast, ovary, ovary/testis, placenta and brain, respectively [10,17–21]. The gonad-specific promoter is highly conserved in human, bovine, and even in avian [6,22,23], so is the brain-specific promoter sequence [23]. It indicates the conservation of regulatory elements that control expression of cyp19 in gonad and brain across species. Extensive cyp19 promoter analysis has been carried out for better understanding of the complex regulation of its expression in various tissues. In both ovary and testis, the gonadotropins FSH and LH act through increasing concentrations of intracellular cyclic-AMP to induce expression [24]. A steroidogenic factor-1 (SF-1) binding site and a c-AMP responsive el- ement located within 278 bp upstream of exon II were essential for the basal ovarian-specific transcriptional ac- tivity in human [25]. SF-1, also termed NR5A1 [26], is important for the expression of many steroidogenic genes. The gonad-specific promoter has been extensively studied because over-expression of CYP19 mRNA in breast cancer and endometriosis is a consequence of the unusual utiliza- tion of promoter II [27,28]. Transcription factors WT1 and DAX1 inhibit CYP19 expression in human endometriotic stroma cells [29]. Aside from CRE, which is required for basal cAMP induction, CCAAT/enhancer binding proteins, C/EBP is essential for ovarian follicle development in 0960-0760/$ – see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0960-0760(03)00347-9