Transcriptional regulation of the FSH receptor: new perspectives

Abstract

The cell-surface receptor for the gonadotropin follicle-stimulating hormone (FSH) is expressed exclusively on Sertoli cells of the testis and granulosa cells of the ovary. FSH signal transduction through its receptor (Fshr) is critical for the timing and maintenance of normal gametogenesis in the mammalian gonad. In the 13 years since the gene encoding Fshr was first cloned, the mechanisms controlling its transcription have been extensively examined, but a clear understanding of what drives its unique cell-specificity remains elusive. Current knowledge of basal Fshr transcription highlights the role of an E-box in the proximal promoter which is bound by the basic helix-loop-helix transcription factors upstream stimulatory factor 1 (Usf1) and Usf2. Recent studies utilizing knockout mice and chromatin immunoprecipitation validated the importance of Usf to Fshr transcription and demonstrated a sexually dimorphic requirement for the Usf proteins to maintain normal Fshr expression. Studies have also shown that the promoter region itself is insufficient for appropriate Fshr expression in transgenic mice, indicating Fshr transcription depends on regulatory elements that lie outside of the promoter. Identification of such elements has been propelled by recent availability of genome sequence data, which facilitated studies using comparative genomics, DNase I hypersensitivity mapping, and transgenic analysis with large fragments of DNA. This review will focus on the current understanding of transcriptional regulatory processes that control expression of rat Fshr, including recent advances from our laboratory.

Figure 1. Fshr gene structure and chromosomal location

(A) The exon-intron structure of Fshr corresponds to the domain structure of the protein. Exons 1 through 9 code for the extracellular ligand binding domain while exon 10 codes for the transmembrane and intracellular domains. Each of the small exons 2 through 8 encode individual leucine rich repeats and exon 9 codes for 2. The size of each exon in base pairs is shown above the diagram and the intron sizes are noted for rat (above) and human (below) between each exon. (B) In addition to the gene structure, synteny in the chromosomal environment surrounding Fshr is conserved between species. The size of Fshr and neighboring genes in humans and rats is shown above each gene and intergenic distances are noted. Adapted with permission from Heckert, 2005 (Copyright 2005, Elsevier Academic Press).

Figure 2. Transcriptional activity of the Fshr promoter

Promoter regions containing sequentially shorter amounts of Fshr 5′ flanking sequence, ranging from −5000bp through −100bp relative to the first Fshr transcriptional start site and extending to +123bp were placed immediately 5′ to the firefly luciferase reporter gene in pGL3-Basic. At the top, the longest promoter construct −5000bp/+123bp is shown as an example. Each of the constructs was transfected into the mouse Sertoli cell line MSC-1 along with a control plasmid (RSV-βgalactosidase), and the luciferase/βgalactosidase activity of each plasmid is shown relative to the luciferase/βgalactosidase activity of promoter-less pGL3-Basic. As little as 223bp of promoter sequence overlapping the transcriptional start sites was required to maintain transcriptional activity in transient transfections (−100bp/+123bp). Deletion mutagenesis also revealed two repressive regions are present in the promoter (−100 to −220 and −2.7kb to −3.6kb). Reprinted with permission from Heckert et al., 1998 (Copyright 1998, The Endocrine Society).

Figure 3. Fshr promoter activity requires the E-box and Usf1/2

(A) Systematic block-replacement mutations were introduced into the Fshr (−220/+123) luciferase promoter construct and each mutant promoter construct was transfected into MSC-1 cells along with the control plasmid (RSV-βgalactosidase). The luciferase/βgalactosidase activity of each construct is shown normalized to the luciferase/βgalactosidase activity of the wildtype promoter. A diagram of the promoter below notes the position of each mutation directly below the bar showing transcriptional activity of that mutant. Bent arrows signify the two transcriptional start sites. Asterisks above the bars indicate a statistically significant change in promoter activity. Activity of the Fshr promoter was largely contained within a small region 20bp upstream of the transcriptional start site (mutant 9) which contained a conserved E-box element. Adapted with permission from Heckert et al., 1998 (Copyright 1998, The Endocrine Society) (B) Electrophoretic mobility shift assay was performed with nuclear proteins isolated from immature mouse testes and a radiolabeled probe containing the Fshr E-box (5′-TCTTGGTGGGTCACGTGACTTTGCCCGT-3′) as described (Heckert et al., 2000). When included in the reactions, antibodies against Usf1 and Usf2 cross-reacted with the major specific binding complex to form supershifted complexes (*), implicating Usf1 and Usf2 as components of the E-box binding complex (arrow). Homologous (E-box) or nonspecific competitors (NS) competitors were added at 100-fold molar excess and 2μg of antibody was included in noted reactions (see Heckert et al., 2000). (C) A model of Fshr promoter function emphasizes Usf1/2 binding to the E-box and includes contributions from E2F, GATA and AP-1 elements. Sequences of the E-box (underlined), InR (GATA site bolded), and E2F site are shown. Bolded bases in the E2F site are most functionally relevant. The relative abundance of Usf hetero- and homodimeric binding complexes in the testis is noted (unpublished data). Adapted with permission from Heckert, 2005 (Copyright 2005, Elsevier Academic Press).

Figure 4. Usf binds the Fshr promoter, in vivo

Chromatin immunoprecipitation was employed to analyze Usf1 and Usf2 binding to the Fshr E-box. Sertoli cells were isolated from mice and used to generate the chromatin employed in the assay (Heckert et al., 2000; Hermann and Heckert, 2005). Immunoprecipitation was performed either with an antibody to Usf1 or normal IgG, which was included as a negative control. Precipitated chromatin was PCR-amplified with primers to detect either the Fshr promoter or a region located 5kb upstream of Fshr exon 1. Samples containing a portion of the chromatin input served as positive controls. The rabbit anti-Usf1(C-20X) IgG was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Figure 5. Fshr regulatory elements revealed by comparative genomics and DHS mapping

(A) At the top, the chromosomal environment of Fshr and its neighboring genes (Nrxn1 and Lhcgr) is shown. Below, a portion of this region from the rat and human FSHR loci was compared by pairwise sequence analysis to identify conserved sequenced (≥75% sequence identity over 100bp or more; Hermann and Heckert, 2005). Identified conserved sequences are noted by numbered balloons. The Fshr coding region is noted (shaded rectangle), white lines in the base sequence represent repeats, and the black bar below indicates the region shown in part B. (B) DNase I hypersensitivity (DHS) mapping performed across a 45kb segment of the 5′flanking sequence of the gene using Sertoli cells and non-expressing peritubular myoid cells identified four hypersensitive sites (DHS1-4; Hermann and Heckert, 2005). DHS1, 2 and 4 were specific to Sertoli cells, while DHS3 was observed in both cell types. The position of the DHS sites (numbered boxes) are shown on the sequence (black line), and a percent identity curve above the line shows the percent identity between human and rat Fshr (vertical axis) at any given position on the sequence (horizontal axis), illustrating the position of the evolutionary conserved sequences over this segment of Fshr. The curve was generated by the VISTA genome browser by comparing human and rat Fshr (http://pipeline.lbl.gov; Couronne et al., 2003). Shading beneath the curve indicates ≥75% sequence identity and the position of conserved sites identified by pairwise analysis are noted by numbered circles (Hermann and Heckert, 2005). The position of exon 1 is noted.

Figure 6. Proposed model of Fshr transcription

Transcription factors (shaded objects) bind cis-regulatory elements (rectangles) on Fshr locus DNA (black line). Within the promoter region, Usf1 and Usf2 bind to the E-box and SF-1 binds to a region upstream of the transcriptional start site (bent arrow). Other distally-located elements are proposed to reside within evolutionarily conserved regions (ECRs). While some transcription factors bind to DNA, others form bridges between bound transcription factors. In Sertoli and granulosa cells, proteins bound to distal enhancers might interact with transcription factors bound to the Fshr promoter via chromatin loops, inducing secondary modifications to the local chromatin environment and interactions with the transcriptional apparatus to activate Fshr transcription. Proteins bound to distal insulators might protect Fshr from the surrounding heterochromatin environment or block action of neighboring genes’ enhancers.