Two teleost specific usf2 are involved in fshr regulation in european sea bass

Abstract

[Introduction] Upstream Stimulatory Factors (USFs) are Helix-Loop-Helix transcription factors involved in a wide range of biological processes. We recently demonstrated that Human Upstream Stimulatory Factors (USFs) could bind to European sea bass follicle stimulating hormone receptor (fshr) 5¿ UTR through an E-box to activate its expression. Next, we cloned for the first time in a teleost species, three partial sequences coding for three distinct usfs (namely usf1, usf2a and usf2b) in sea bass (Dicentrarchus labrax), and described their ubiquitous expression. However, no information is available about their origin or their function in teleosts.

[Methods] We isolated sea bass usf2a and usf2b full length cDNAs from ovarian follicular cells and introduced them in pcDNA3. CHO cells were transfected with these expression plasmids i) to determine by immunohistochemistry the specificity of a heterologous antibody designed for Human USF2 (IgG Z). ii) to produce nuclear extracts enriched in sea bass Usf2a, Usf2b or Usf2a/Usf2b heterodimers. iii) to determine by luciferase assays their effect on fshr promoter. EMSAs were performed with fshr promoter probes and ovary or nuclear extracts from Usfs- transfected CHO cells.

[Results and Discussion] The isolated full length cDNAs code for two nuclear proteins, Usf2a and Usf2b, that can be recognized by the heterologous antibody IgG Z in sea bass follicular cells. Phylogenetic analysis indicates that these genes originated from the teleost-specific whole genome duplication. These two proteins can physically bind to E-boxes located in the fshr 5¿UTR as demonstrated by EMSA. However, we could not appreciate the formation of Usf2a/Usf2b heterodimers. Finally, luciferase assays show both Usf2a and Usf2b could activate fshr promoter through this E-box.

[Conclusion] Usf2a and Usf2b are two teleost-specific proteins present in sea bass follicular cells that can activate the fshr promoter by binding through an E-box (CACGTG). Although these interactions have to be confirmed in vivo these two transcription factors could play a role in teleost reproduction success.