Abstract
Simple SummaryFoxl2 generally presents a sexually dimorphic expression pattern in animal gonads and is highly expressed in the ovary. However, few studies on the transcriptional regulation of Foxl2 have been documented. To understand the transcriptional regulating of Foxl2 high expression in the ovary, we used the Y1H system, a high throughput approach, for the first time to screen the transcription factors binding to the high transcriptional activity region of Foxl2 promoter in Zhikong scallop (Chlamys farreri) gonads. In the present study, the highly transcriptional activity promoter sequence of Cf-Foxl2 was determined at −1000~−616 bp and 11 candidate factors were verified to involve in Cf-Foxl2 transcriptional regulation. Our findings provided valuable data for better understanding the specific transcriptional regulation mechanism of Foxl2 in the ovary and would further assist in the breeding of aquacultural bivalves.Foxl2 is an evolutionarily conserved female sex gene, which is specifically expressed in the ovary and mainly involved in oogenesis and ovarian function maintenance. However, little is known about the mechanism that regulates Foxl2 specific expression during the ovary development. In the present study, we constructed the gonadal yeast one-hybrid (Y1H) library of Chlamys farreri with ovaries and testes at different developmental stages using the Gateway technology. The library capacity was more than 1.36 × 107 CFU, and the length of the inserted fragment was 0.75 Kb~2 Kb, which fully met the demand of yeast library screening. The highly transcriptional activity promoter sequence of C. farreri Foxl2 (Cf-Foxl2) was determined at −1000~−616 bp by dual-luciferase reporter (DLR) assay and was used as bait to screen possible transcription factors from the Y1H library. Eleven candidate factors, including five unannotated factors, were selected based on Y1H as well as their expressional differences between ovaries and testes and were verified for the first time to be involved in the transcriptional regulation of Cf-Foxl2 by RT-qPCR and DLR. Our findings provided valuable data for further studying the specific regulation mechanism of Foxl2 in the ovary.
Highlights
To screen TFs regulating specific high expression of Foxl2 in the ovary, we identified a promoter sequence of Cf-Foxl2 with high transcriptional activity, constructed C. farreri gonadal yeast one-hybrid (Y1H) library using Gateway technology, and further verified TFs that may bind to this high transcriptional activity sequence through Y1H system
A 1905 bp promoter sequence (−1705/+200 bp) of Cf-Foxl2 was amplified using the primers fwd −1705 and rev +200 (Table S1), and a series of double-luciferase reporter vectors containing the different fragments of Cf-Foxl2 promotor (Figure 1B) were constructed and transiently transfected into HEK293T cells to determine the high transcriptional activity region
The results showed that the transcriptional activities of the promoter fragments, P1 (−1705/+1) and P2 (−1000/+1) were significantly higher than that of P0 (−1705/+200), but no significant difference existed between P1 and P2 (Figure 1C)
Summary
In fish Oreochromis niloticus, Foxl is revealed to involve in the production of estrogen maintaining ovarian differentiation [14], and its targeted deletion causes female-to-male sex reversal [11]. In chicken Gallus gallus, Foxl has been reported to play an important role in ovarian differentiation by antagonizing Sox9 [15]. More studies have focused on downstream genes regulated by FOXL2, and revealed several target genes related to steroidogenesis, proliferation, apoptosis, differentiation, and stress response, such as sex determination gene Sox9 [16], anti-Müllerian hormone [17], estrogen receptor beta Esr2 [18], and steroidogenesis-related gene CYP19A1 [14,19,20], etc. Li et al [21]
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