SF-1 Expression Is Mostly Directed by Distal Regulatory Elements.

Abstract

Steroidogenic factor 1 (SF-1/Ad4BP/NR5A1) is a nuclear hormone receptor that has a pivotal role in endocrine regulation and development of the adrenal glands and gonads. Considering the critical nature of SF-1, many studies have focused on uncovering transcriptional mechanisms that drive SF-1 expression. Despite these efforts, little has been revealed regarding the mechanism that direct SF-1 to its appointed cell types, a result largely due to its requirement for distal regulatory sequences in directing tissue-specific expression. Previous transgenic studies identified a 153kb region, containing Nr5a1, that was sufficient for proper SF-1 expression, which provided an important initiation point for the identification of essential regulatory elements. Comparative genomics through the web-based tool ECR browser (http://ecrbrowser.dcode.org/) was performed and identified within the Nr5a1 locus several evolutionarily conserved non-coding regions called "ECR"s. Four of the most conserved ECRs were examined for transcriptional regulatory activity. The ECRs were cloned into a vector that contained SF-1 promoter sequence from -734 to +60 upstream of a luciferase reporter. Transcriptional activity of each ECR was tested individually using transient transfection analysis in different SF-1-expressing cell types. Our results identified one ECR (ECR3) with activity that increased transcription in alpha T3(gonadotrope) and Y-1(adrenal) cells and decreased it in MA-10(Leydig), Myoid, MSC-1(Sertoli), and primary Sertoli cells. The data indicate that ECR3 contains control elements necessary for SF-1 transcriptional induction in the pituitary and adrenal cells and repression in the testis cells. Mutagenesis and DNA/protein interaction studies identified sequences within ECR3 that were important for activity in alpha T3 cells. One such sequence contains an E-box that bound the class A basic-helix-loop-helix (bHLH) protein E2A, suggesting studies will reveal a class B bHLH, which exhibit tissue-specific expression, as its dimeric partner. This Research was supported by a Center Grant in Reproductive Sciences (U54 HD33994 from NICHD to LLH) and a KUMC Biomedical Research Training Grant to R.K.