PARENT SESSION

(163) AR AND GATA FACTORS COLLABORATE TO DRIVE SERTOLI CELL SPECIFIC EXPRESSION OF THE PEM (RHOX-5) PROXIMAL PROMOTER IN THE TESTIS.

Wilkinson, Miles ¹, Bhardwaj, Anjana¹, Rao, Manjeet¹, ¹ UT MD Anderson Cancer Center, Houston, TX

ABSTRACT- How cell type-specific expression is achieved at the transcriptional level remains largely unsolved. In this study, we address the molecular mechanism responsible for Sertoli cell-specific transcription using the Pem (Rhox-5) proximal promoter (Pp)as a model system. Rhox-5 is the founding member of the reproductive homeobox (Rhox) gene cluster, a group of 12 related homeobox genes on the mouse X chromosome. Using transgenic mice, we identified a short region (0.3 kb) immediately upstream of the Pp start site that confers androgen-dependent Sertoli cell-specific transcription within the testis. We found that this androgen-dependent expression is mediated by androgen response elements (AREs), as mutations in any of the four AREs in this region lead to reduced transcription in transfected Sertoli-cell lines. While this indicates that the androgen receptor (AR) contributes to Pp transcription, AR cannot be the only transcription factor involved since the Pp is not expressed in most AR+ cell types, including Leydig and myoid cells in the testis. Because the 0.3-kb Pp minimal promoter harbors three GATA consensus sequences, we hypothesized that GATA transcription factors may also contribute to Sertoli cell-specific transcription. In agreement with this hypothesis, mutation of any of these three GATA sites reduced androgen-induced Pp transcription in the MSC-1 Sertoli-cell line. These GATA sites are in close proximity to the AREs, suggesting the possibility that GATA factors and AR physically collaborate to drive transcription. Consistent with this, overexpression of either GATA4 or GATA6 (both of which are normally expressed in Sertoli cells) rescued transcription from Pp constructs harboring mutations in AREs. We also identified a regulatory region immediately adjacent to the Pp start site that is responsible for silencing Pp transcription in non-Sertoli-cell lines. Deletion of this negative regulatory region allowed transcription of the Pp in non-Sertoli cell lines, an effect that was further enhanced by forced expression of GATA factors. Our study demonstrates that the Rhox-5 Pp is a target of GATA regulation. We also provide evidence that GATA factors are novel coregulators of AR and an interplay of both negative and positive factors drive Sertoli cell-specific transcription.

KEY WORDS: AR, GATA, Rhox-5, Sertoli specific expression