Regulation Of Fshr And SF-1 In The Hypothalamus-Pituitary-Gonadal (HPG) Axis.

Abstract

Mammalian reproduction is highly dependent on the delicate balance of signals within the hypothalamic-pituitary-gonadal (HPG) axis that maintains proper endocrine environment. One of the key signals is the pituitary glycoprotein follicle-stimulating hormone (FSH) which is critical for gonadal development and fertility. FSH acts via FSHR, a G protein-coupled receptor present on a few distinct cell types, prominently on the Sertoli cells of the testis and granulosa cells of the ovary Prior studies have identified several key regulatory elements required for Fshr transcription but a clear understanding of what controls its limited cell specific expression remains elusive. Comparative genomics identified a number of evolutionary conserved regions (ECR) distal to the proximal promoter, indicating these regions might harbor regulatory elements. One such distal regulatory element known to play a role in transcriptional regulation is the "multivalent" protein CCCTC-binding factor (CTCF). Computational analysis of ECRs identified multiple CTCF binding sites in the intergenic regions of the Fshr locus and depletion of CTCF in rat granulosa cells led to a two-fold increase in Fshr mRNA. These data indicate that CTCF either by itself or in conjunction with other protein complexes might play a role in transcriptional regulation of the Fshr gene. Another important component of the HPG axis is the transcription factor, Steroidogenic factor-1 (SF-1) transcribed from the Nr5a1 gene (also known as Ftz-F1 or Ad4bp) which plays a pivotal role in adrenal and gonadal development and regulates genes at all levels of the axis. Comparative genomics identified two conserved sequences associated with Ftz-F1. One was found to encode a long non-coding RNA and the other a regulatory element important for SF-1 expression in the pituitary. A long non-coding RNA named Fast or Ftz-F1 associated transcript was found transcribed in the opposite direction to SF-1, co-expressed, and co-regulated with SF-1 and hormonally regulated. Knockdown and over-expression of the Fast transcript in MA-10, mouse Leydig cell line, did not alter the transcription or translation of SF-1. Long non-coding RNA have been focus of intense research and have been shown to play diverse role in various physiological and pathological processes. Considering their potential for transcriptional regulation, the identified long non-coding RNA, Fast, might play potentially important role in endocrine development and homeostasis. Comparative sequence analysis identified an evolutionary conserved region, ECR3, approximately 4kb away from the transcriptional start site of SF-1. Transient transfection data revealed ECR3, upregulated SF-1 transcription in alpha T3 (gonadotrope cell line) and Y-1 (adrenal) cells, but downregulated SF-1 in MA-10 (Leydig cell line), MSC-1 (Sertoli cell line) and primary rat peritubular Myoid and Sertoli cells. This region was found to contain an E-box and was bound by upstream stimulatory factors 1 and 2 (USF1 and USF2) and a basic-helix-loop-helix (bHLH) protein E2A. In particular, co-transfection studies identified Inhibitors of DNA binding, Id2 and Id3 to downregulate SF-1 transcription. In summary, this dissertation looks at the regulation of Fshr and SF-1 in the HPG axis and identifies the importance of distal regulatory sequences and non-coding transcripts and its role in gene regulation.