POU6F2 mutation in humans with pubertal failure alters GnRH transcript expression

Abstract

Idiopathic hypogonadotropic hypogonadism (IHH) is characterized by the absence of pubertal development and subsequent impaired fertility often due to gonadotropin-releasing hormone (GnRH) deficits. Exome sequencing of two independent cohorts of IHH patients identified 12 rare missense variants in POU6F2 in 15 patients. POU6F2 encodes two distinct isoforms. In the adult mouse, expression of both isoform1 and isoform2 was detected in the brain, pituitary, and gonads. However, only isoform1 was detected in mouse primary GnRH cells and three immortalized GnRH cell lines, two mouse and one human. To date, the function of isoform2 has been verified as a transcription factor, while the function of isoform1 has been unknown. In the present report, bioinformatics and cell assays on a human-derived GnRH cell line reveal a novel function for isoform1, demonstrating it can act as a transcriptional regulator, decreasing GNRH1 expression. In addition, the impact of the two most prevalent POU6F2 variants, identified in five IHH patients, that were located at/or close to the DNA-binding domain was examined. Notably, one of these mutations prevented the repression of GnRH transcripts by isoform1. Normally, GnRH transcription increases as GnRH cells mature as they near migrate into the brain. Augmentation earlier during development can disrupt normal GnRH cell migration, consistent with some POU6F2 variants contributing to the IHH pathogenesis.

Keywords: GnRH; POU6f2 isoform1; idiopathic hypogonadotropic hypogonadism; puberty; transcription.

Figure 1

The pedigrees of the families with POU6F2 variants. Affected male and female family members are represented by black squares and black circles, respectively. White square symbols indicate unaffected male family members, white circle symbols represent unaffected female family members, and the double line indicates consanguinity. Under each symbol are the genotypes in the same order as the gene and variant descriptions, with WT and M denoting wild type and mutant, respectively. The legend denotes phenotypes as IHH, anosmia, and delayed puberty.

Figure 2

Schematic diagram of human POU6F2 isoforms. Exon-intron structure of human POU6F2 isoforms (middle two schematics) drawn to scale using the Gene Structure Display Server (GSDS 2.0, http://gsds.gao-lab.org). Exons are indicated by boxes to highlight the coding sequence (CDS, pink) and untranslated region (UTR, gray). Introns are indicated by black lines with a shrinked scale (0.01 ratio to scale of exons). Exon 11 is alternatively spliced via two splicing acceptor sites, E11-SA1 and E11-SA2, to generate isoform1 (upper schematic) and isoform2 (lower schematic), respectively. The two conserved DNA-binding domains are indicated by blue boxes and aligned to exons (encoded by exon 10 to 11). Isoform1 has a unique 36aa insertion on POU-specific domain (black box) not found in any other POU protein family members. The amino acid numbers are shown at the start and end point of functional domains. Twelve variants identified from IHH patients are indicated by red arrowheads (upper schematic). Mutation 1 (MT1; c.1801G>A, p.G601R in isoform1; c.1693G>A, p.G565R in isoform2) is in the linker region between the two DNA-binding domains. MT2 (c.1885A>C, p.N629H in isoform1; c.1777A>C, p.N593H in isoform2) is in the POU homeodomain. MT3–MT7, MT9–MT12 are in the Transactivation domain. MT8 (c.1480C>T, p.R494W) is in the POU-specific domain. Orange boxes; Nuclear export signal (NES), green boxes; Nuclear localization signal (NLS).

Figure 3

Expression of Pou6f2 isoforms in mouse and bioinformatic prediction of POU6F2 isoforms bound to a DNA octamer. (A) Exon-intron structure of mouse Pou6f2 (GSDS 2.0, http://gsds.gao-lab.org). In mice, only one isoform has been reported that is composed of nine exons and corresponds to isoform1 of human POU6F2. Primers used for PCR are shown as arrows on exon 8 and 9. (B) Gel image of RT-PCR analysis performed in mouse tissue. Top band (447 bp) shows isoform1 and bottom band (339bp) shows isoform2, which is skipping 108 bp by alternative splicing on exon 9. (C) Gel image of RT-PCR analysis of Pou6f2 isoforms (top and middle gel) in GnRH single cells (bottom gel). A robust signal for GnRH was detected in three cells, and in two of these cells, only isoform1 was detected. Isoform2 was not detected in any of the GnRH cells. (D) Upper Left, Superimposition of isoform1 (purple) and isoform2 (gray) structures predicted by C-I-TASSER. The location of MT1 and MT2 is indicated by boxes. Upper Right, HDOCK prediction of POU6F2 binding to the OCT1 DNA consensus site (5’-ATGCAAAT-3’). Template-free docking was used to prevent simulation bias. Lower Left and Right, Structural representation of the interaction between each isoform and dsDNA octamers. Two-dimensional cartoon illustrating the molecular interactions between each POU domain and their predicted binding sites. Satisfactory (for isoform2) and unsatisfactory (for isoform1) binding modes are indicated.

Figure 4

Structural analysis of IHH variants MT1 and MT2 on POU6F2 isoform1. (A) OCT1 consensus-like site (5’-ATGCTTTT-3’) is identified in human GnRH1 promoter (-98 to -88). Binding site in 3D modeling uses POU_S to ATGC, and the POU_H is predicted to insert into a groove between both faces of the dsDNA, thus contacting both TTTT and AAAA. (B) HDOCK prediction of POU6F2 isoform1 binding to the OCT1 consensus-like site. Template-free docking was used to prevent simulation bias. (C) DynaMut prediction of WT and mutant proteins for isoform1. Individual amino acid substitutions are indicated in cyan. (D) Structural evaluation scores indicating how MT1 and MT2 affect POU6F2 isoform1 protein folding (DynaMut), natural protein flexibility (CABS-flex), and DNA binding (SAMPDI). DynaMut and CABS-flex represent changes in the individual protein structures, whereas SAMPDI represents changes in the affinity of POU6F2 isoform1 to bind the OCT1 consensus-like site (5’-ATGCTTTT-3’). Characterization of stabilizing or destabilizing effects are indicated. CABS-flex values analyzed using a paired t-test.

Figure 5

In vitro transcription assay of isoform1 in immortalized human GnRH cells. (A) Expression of POU6F2 isoforms in human brain and FNC-B4-hTERT cells. RT-PCR analysis performed in human brain and immortalized human GnRH cells (with or without GnRH stimulation). Top band (281 bp) shows isoform1 in all tissue samples. In human brain, a bottom band (173 bp) is detected, isoform2 that is skipping 108 bp by alternative splicing on exon 11. Primers used for PCR are shown as arrows on exon 10 and 11. (B) Nested RT-PCR analysis performed using isoform2-specific primers (shown as arrows on the junction of exon 10–11 and exon 11). Consistent with the first run, isoform2 (126 bp) was only detected in human brain not in the FNC-B4-hTERT cells. (C) Quantitative RT-PCR of GnRH1 in FNC-B4-hTERT cells transfected with POU6F2 isoform1s (WT, MT1, MT2). The expression of GnRH1 was normalized to each experimental Mock group (plasmid only, relative expression level = 1), and the relative values of the other three groups are shown in the bar graph. MT1 significantly increased GnRH1 transcript compared to both WT and MT2 groups but was not significantly different from the Mock group. (D) RT-PCR for POU6F2 isoform2 in FNC-B4-hTERT cells transfected with POU6F2 isoform1 WT, MT1, and MT2. Consistent with non-transfected FNC-B4-hTERT cells (A), each of the experimental groups expressed only isoform1. Since endogenous POU6F2 was still present in the transfected cells, our results suggest that overexpression of MT11 had a dominant-negative effect. cDNA in other lanes: hBr = human brain, M = mock, W = water. Arrow on left pointing to 600-bp band on ladder. (E) Schematic summary of isoform1 as a transcriptional regulator generated by Biorender (https://biorender.com/).