FSHB Transcription is Regulated by a Novel 5' Distal Enhancer With a Fertility-Associated Single Nucleotide Polymorphism

Abstract

The pituitary gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone, signal the gonads to regulate male and female fertility. FSH is critical for female fertility as it regulates oocyte maturation, ovulation, and hormone synthesis. Multiple genome-wide association studies (GWAS) link a 130 Kb locus at 11p14.1, which encompasses the FSH beta-subunit (FSHB) gene, with fertility-related traits that include polycystic ovary syndrome, age of natural menopause, and dizygotic twinning. The most statistically significant single nucleotide polymorphism from several GWAS studies (rs11031006) resides within a highly conserved 450 bp region 26 Kb upstream of the human FSHB gene. Given that sequence conservation suggests an important biological function, we hypothesized that the region could regulate FSHB transcription. In luciferase assays, the conserved region enhanced FSHB transcription and gel shifts identified a binding site for Steroidogenic factor 1 (SF1) contributing to its function. Analysis of mouse pituitary single-cell ATAC-seq demonstrated open chromatin at the conserved region exclusive to a gonadotrope cell-type cluster. Additionally, enhancer-associated histone markers were identified by immunoprecipitation of chromatin from mouse whole pituitary and an immortalized mouse gonadotrope-derived LβT2 cell line at the conserved region. Furthermore, we found that the rs11031006 minor allele upregulated FSHB transcription via increased SF1 binding to the enhancer. All together, these results identify a novel upstream regulator of FSHB transcription and indicate that rs11031006 can modulate FSH levels.

Keywords: FSHB; FSH; Fertility; PCOS; enhancer; gonadotropins.

Figure 1.

Conserved intergenic region enhances FSHB transcription. (A) 100 Vertebrates Basewise Conservation (Cons 100 Verts) by phyloP, UCSC Browser, (GRCh38/hg38 assembly). The y-axis indicates conservation comparing 100 representative vertebrates at each base pair, expressed as -log (p-value of neutral evolution), with positive and negative values respectively assigned to higher or lower conservation than expected by chance due to genetic drift. (B) Luciferase expression from a reporter containing the major allele 450 bp conserved region (Enh) from human upstream of the −1028/+7 FSHB promoter (P) compared with a reporter containing the −1028/+7 FSHB promoter alone transfected into LβT2 mouse gonadotrope cells. The putative enhancer was subcloned in the forward (FW) (hEnh/G) or reverse (RV) (RV-hEnh/G) orientations onto the human FSHB promoter driving Luciferase (Luc) (n = 3). (C) Luciferase expression from the mouse 450 bp conserved region subcloned in the forward (mEnh/G) and reverse (RV-mEnh/G) orientations upstream of the mouse −1000/−1 Fshb promoter compared with a luciferase reporter containing the mouse −1000/−1 Fshb promoter alone (n = 4). (D) Luciferase expression from the human 450 bp conserved region subcloned in the forward and reverse orientations upstream of the −81/+52 TK promoter compared with a reporter containing the TK promoter alone (n = 4). (E) Luciferase expression from the mouse 450 bp conserved region subcloned in the forward and reverse orientations upstream of the TK promoter compared with a reporter containing the TK promoter alone (n = 4). (F) Luciferase expression from the human 450 bp conserved region subcloned in the forward orientation upstream of the mouse −523/+6 Lhb promoter or (G) mouse −493/+9 Gnrhr promoter compared with each promoter alone (n = 4). Luciferase values were normalized to the β-galactosidase internal control and are expressed relative to the empty reporter vector. Values represent mean ± SEM. Data were analyzed by one-way ANOVA, post hoc Dunnett multiple comparisons test (A-E) or Student t test (F and G). A log transform was used prior to statistical analysis for D and E (* P < 0.05, ** P < 0.01, *** P < 0.005). Abbreviations: Enh, enhancer; Luc, luciferase; P, promoter.

Figure 2.

Fshb enhancer is marked by open chromatin exclusively in gonadotropes. scATAC-seq was performed on single cells from adult male pituitary to evaluate chromatin accessibility to transposase digestion. (A) t-SNE analysis identified a cluster of gonadotopes (purple) marked by (B) chromatin accessibility at Lhb, Gnrhr, and (C) Fshb. Fshb enhancer chromatin was open in gonadotropes (boxed in blue) but not in other pituitary cell-types (“other,” depicted in gray).

Figure 3.

The conserved region is marked by the enhancer-specific H3K4me1 histone modification in LβT2 cells and whole pituitary. From LβT2-derived chromatin, enrichment of (A) H3K4Me1 (enhancer-specific marker), (B) H3K27Ac (active marker), and (C) H3K27me3 (repressive marker) at the Fshb enhancer and Fshb proximal promoter are expressed as percent input. For H3K4me1 and H3K27Ac, β-Actin intron 1 (Actin) was used as a positive control, and a gene desert on chromosome 14 (Ch14 desert) was used as a negative control. For H3K27me3, the MyoD proximal promoter was used as a positive control and the β-actin intron 1 was used as a negative control. From whole female pituitary-derived chromatin, enrichment of (D) H3K4me1 or (E) H3K27Ac is expressed as percent input. The POUF1A1 10 Kb enhancer (F) was used as positive control and the chromosome 14 (Ch14 desert) as a negative control. Values represent mean ± SEM. Data from A-E were analyzed by one-way ANOVA, post hoc Tukey HSD. Different letters denote significant differences among groups P < 0.05. Data from F were analyzed by one-way ANOVA, post hoc Dunnett multiple comparisons test (*** P < 0.005). A log transform was used on A-C and F prior to analysis. Enh, enhancer.

Figure 4.

Enhancer mapping identifies putative transcription factor binding sites necessary for enhancing FSHB transcription. (A) Luciferase expression after transfection into LβT2 cells from subregions (halves or quarters) of the human 450 bp enhancer subcloned upstream of the human −1028/+7 FSHB promoter. A subregion spanning 216-341 was sufficient for enhancer function (n = 3). (B) Luciferase expression from the human 216-341 subregion cloned in the reverse orientation (RV 216-341) compared with the forward orientation (FW 216-341) and human −1028/+7 FSHB promoter alone (n = 5). (C) From the 450-base-pair enhancer subcloned upstream of the human −1028/+7 FSHB promoter, a series of constructs were created with 10-base-pair deletions spanning 216-345. Δ indicates the span of the 10-base-pair deletion in each construct. Expression levels from four deletion constructs were significantly different from the full-length construct (n = 5). Putative transcription factor binding sites were identified from the TRANSFAC database using the Match program and each consensus sequence is shown compared with the mouse and human genomic sequences at the corresponding region. The consensus motif is underlined in each genomic sequence. Bases that differ from the consensus are bolded. Luciferase values were normalized to the β-galactosidase internal control and are expressed relative to the empty reporter vector. Values represent mean ± SEM. Data were analyzed by one-way ANOVA, post hoc Dunnett multiple comparisons test. A square transform was used on data from A and a log transform on B prior to statistical analysis (* P < 0.05, *** P < 0.005). Abbreviation: P, promoter.

Figure 5.

SF1 binds to the FSHB enhancer and rs11031006 G>A increases SF1 binding to the enhancer. (A) The major and minor allele sequences from human and mouse at the putative SF1 binding site are compared with the SF1 consensus sequence. The consensus motif is underlined. Bases that differ from the consensus are bolded. The 2-base-pair mutation used in Fig. 5D and Fig. 6 is also shown. (B) Gel shift using 30-base-pair radiolabeled oligonucleotide probes from human containing the rs11031006 site major (G) and minor (A) alleles, or the equivalent region from mouse. Probes were incubated with LβT2 nuclear extracts and no antibody (-), normal rabbit IgG (IgG), or rabbit anti-SF1 antibody (SF1) (exposure: RT for one week). (C) The radiolabeled SF1 consensus probe (Gonadotrope-specific element, abbreviated GSE, from the human CGA promoter) (77) was competed by excess cold rs11031006 human major and minor alleles and mouse equivalent probes. (exposure: −80 °C, 4 days.) (D) Gel shift with probes containing the wild-type major allele or a 2-base-pair mutation in the SF1 consensus sequence (exposure: −80 °C, 1 day). Supershifted bands and complexes containing SF1 are noted with arrows. Images are representative of at least three experiments. Abbreviations: Ab, antibody; cold comp., cold competitor; comp. conc., competitor concentration; GSE, gonadotrope specific element (SF1 consensus).

Figure 6.

rs11031006 G>A regulates FSHB expression through an SF1 binding site. (A) Luciferase expression from the forward human enhancer containing the minor allele (hEnh/A) upstream of human −1028/+7 FSHB promoter, compared with the major (hEnh/G) allele and promoter alone (n = 6). (B) Luciferase expression from the reversed mouse enhancer with a (G>A) mutation equivalent to rs11031006 minor allele (RV-mEnh/A) upstream of the mouse −1000/−1 Fshb promoter, compared with the wild-type reversed enhancer (RV-mEnh/G) and promoter alone. The reversed enhancer from mouse was chosen as it showed higher expression in Fig. 1C. (C) Luciferase expression from the human enhancer upstream of human −1028/+7 FSHB promoter (n = 4) or (D) mouse enhancer upstream of −1000/−1 Fshb promoter (n = 6) with a 2-base-pair mutation in the SF1 consensus site (same mutation as in Fig. 5A, D). (E) Luciferase expression from the hEnh/G:TK reporter in 3T3 cells transfected with SF1 expression vector or pcDNA3 backbone (Empty vector). Luciferase values were normalized to the β-galactosidase internal control for all experiments and are expressed relative to the empty pGL3 reporter vector (A-D) or as a ratio of normalized luciferase expression from the enhancer-promoter construct divided by the promoter alone (E). Values represent mean ± SEM. Data were analyzed by one-way ANOVA, post hoc Tukey HSD (A-D) or Student t test (E) (** P < 0.01). Different letters denote significant differences among groups P < 0.05. Abbreviations: Enh, enhancer; Luc, luciferase; P, promoter.