Identification of regulatory elements in the Cyp19 proximal promoter in rat luteal cells

Abstract

The cytochrome P450 aromatase (Cyp19) gene encodes an enzyme of crucial importance in the synthesis of estradiol. Estradiol is luteotropic in the rat. In this species, luteal Cyp19 expression increases progressively during pregnancy and falls before parturition. The mechanisms that control these changes are unknown. Using gel shift assays, we sought to identify the promoter regions that control Cyp19 expression in the rat corpus luteum (CL). The Cyp19 promoter contains a cAMP response element-like sequence (CLS), two nuclear receptor elements half sites (NREs), a GATA binding site, a Yin Yang-1 (YY1) response element, and an activation protein 3 (AP3) binding site. Nuclear extracts were obtained from CL of rats on days 4, 15, and 23 of pregnancy and from the ovaries of immature rats treated with vehicle or a hormone that induces Cyp19 expression in the follicles. CLS was active in immature ovaries but inactive in the CL of pregnant rats, whereas binding to NREs and GATA was observed in both tissues. YY1 was inactive in all samples tested. In the CL, AP3 binding was higher on day 15 of pregnancy when compared with day 4 and day 23 but it was absent in ovaries of immature rats, whereas luteinization increased AP3 binding activity. Mutation of the AP3 site blunted the stimulation of Cyp19 promoter activity in granulosa cells. Our results indicate that CLS is active only in follicles; whereas in the CL, binding to the GATA, NRE, and AP3 sites associates with changes in Cyp19 expression, suggesting that they control Cyp19 promoter activity in luteal cells.

Aromatase expression in the ovary: Hormonal and molecular regulation

“…5). In PMSG-treated immature rats, AP-3 binding activity increases after treatment with hCG, suggesting that AP-3 is active only in luteal cells [107]. In pregnant rats, luteal protein binding to AP-3 correlates with variations in aromatase expression (Fig.…”

“…AP-3 binding sites are also present in the promoter of humans and mice, and at least in rats, it seems to be necessary for full activation of the aromatase promoter [107]. In rats, this AP-3 binding site interacts with luteal nuclear extracts obtained from pregnant rats but not with ovarian extracts of immature rats or of immature rats treated with PMSG (Fig.…”

“…As shown in Figure 4, within this region a cAMP-responsive element-like sequence (CLS) [52] and two binding sites for members of the nuclear receptors 5A family of transcription factors (NREa and NREb) are present [53,55,99,[102][103][104]. The proximal promoter also contains one response element for members of the zinc finger family of transcription factors known as GATA [70,105,106] and one AP-3 binding site [107]. These binding sites are highly conserved between species.…”

“…AP-3 (TAACCACA)-We recently described the presence of an AP-3 binding site in the proximal promoter of the rat aromatase gene [107]. AP-3 binding sites are also present in the promoter of humans and mice, and at least in rats, it seems to be necessary for full activation of the aromatase promoter [107].…”

“…These results suggest that AP-3 activation may play a role in the regulation of luteal aromatase expression. The protein that recognizes AP-3 has a molecular weight of approximately 48 kDa [107]; however, its identity is not known yet.…”