EAP1 regulation of GnRH promoter activity is important for human pubertal timing

Abstract

The initiation of puberty is orchestrated by an augmentation of gonadotropin-releasing hormone (GnRH) secretion from a few thousand hypothalamic neurons. Recent findings have indicated that the neuroendocrine control of puberty may be regulated by a hierarchically organized network of transcriptional factors acting upstream of GnRH. These include enhanced at puberty 1 (EAP1), which contributes to the initiation of female puberty through transactivation of the GnRH promoter. However, no EAP1 mutations have been found in humans with disorders of pubertal timing. We performed whole-exome sequencing in 67 probands and 93 relatives from a large cohort of familial self-limited delayed puberty (DP). Variants were analyzed for rare, potentially pathogenic variants enriched in case versus controls and relevant to the biological control of puberty. We identified one in-frame deletion (Ala221del) and one rare missense variant (Asn770His) in EAP1 in two unrelated families; these variants were highly conserved and potentially pathogenic. Expression studies revealed Eap1 mRNA abundance in peri-pubertal mouse hypothalamus. EAP1 binding to the GnRH1 promoter increased in monkey hypothalamus at the onset of puberty as determined by chromatin immunoprecipitation. Using a luciferase reporter assay, EAP1 mutants showed a reduced ability to trans-activate the GnRH promoter compared to wild-type EAP1, due to reduced protein levels caused by the Ala221del mutation and subcellular mislocation caused by the Asn770His mutation, as revealed by western blot and immunofluorescence, respectively. In conclusion, we have identified the first EAP1 mutations leading to reduced GnRH transcriptional activity resulting in a phenotype of self-limited DP.

Figure 1

Filtering strategy to identify EAP1 as a candidate gene for self-limited DP, prediction of pathogenicity and conservation across species for EAP1 variants identified, and structural model of the Zn finger domain of EAP1. (A) WES was performed on DNA extracted from peripheral blood leukocytes of 160 individuals from our cohort (67 DP probands, 58 DP relatives and 35 controls). The exome sequences were aligned to the UCSC hg19 reference genome. Picard tools and the genome analysis toolkit were used to mark PCR duplicates, realign around indels, recalibrate quality scores and call variants. Variants were filtered for potential causal variants using filters for quality control, predicted functional annotation, MAF, case–control analysis, variants in multiple families and biological relevance. Targeted exome sequencing using a Fluidigm array of 28 candidate genes identified post-filtering was then performed in a further 42 families from the same cohort (288 individuals, 178 with DP and 110 controls). Variants post-targeted resequencing were filtered using the same criteria as the WES data. RVBT was performed with a multiple comparison adjustment applied post-hoc (17). Screening of 100 further cohort controls was via conventional Sanger sequencing. ^*Data published (15, 16). ^**Excluded due to the presence of variants in multiple controls. (B) Minor allele frequencies for ExAC Finnish population (accessed February 2018), conservation and pathogenicity scores (SIFT (37); Polyphen2 (36); MutationTaster (46)). Multiple sequence alignment (msa) was generated using MutationTaster (46). The p.Ala221 residue is highly conserved among different species, PhyloP score 1.801. The p.Asn770His is highly conserved among different species and the PhyloP score is 4.523. (C) The mutant 3D structure of the Zn finger domain of EAP1 is presented as a cartoon. The Zn atoms are presented as magenta spheres, the conserved C3HC4 residues (which bind Zn atoms) are presented in green and the mutant histidine (H) at position 770 is presented in red. The mutant His770, shown in red, is located on the surface of EAP1 and may be part of a protein–protein interaction site. The position of invariable residues C3HC4 is indicated with green arrows, whereas the position of N770 is indicated with a red arrow. The amino acid numbering and secondary structure is presented above the msa. C indicates cysteine.

Figure 2

Pedigrees of the families with EAP1 mutations with proband growth charts. (A and B) Squares indicate male family members and circles indicate female family members. Black symbols represent clinically affected, gray symbols represent unknown phenotype and clear symbols represent unaffected individuals. ‘P’ indicates the proband in each family and ‘us’ indicates un-sequenced due to lack of DNA from that individual. A horizontal black line above an individual’s symbol indicates they are heterozygous for that mutation as confirmed by either WES or Fluidigm array and verified by Sanger sequencing. (A′, A^″, B^′ and B″) Height and height SDS charts for the probands of each of the two pedigrees. Tanner genital stage (G stage), Tanner pubic hair stage (P stage), testicular volume, standardized (S)-testosterone, LH and FSH values are given for each proband at various time points. Normal values, based on data from >70 000 healthy Finnish children, have been previously published (15). TH means target height based on mid-parental heights. Green dots connected by continuous black lines indicate bone age at the corresponding chronological age (blue dots), as estimated by the Greulich and Pyle method.

Figure 3

Eap1 mRNA is expressed in the hypothalamus of peri-pubertal mice. Peri-pubertal mice hypothalamus sections were used for in situ hybridization and immunohistochemistry to localize Eap1 mRNA (staining in purple) and GnRH neurons (staining in brown), respectively. Eap1 is expressed in the VMH, Arc and Pa nuclei of (A) male and (B) female mice. GnRH neuron bodies are dispersed predominantly in the medial preoptic area (C) in an Eap1 positive milieu. GnRH neuron bodies are shown at higher magnification in (D). GnRH neuron projections are also detected in the ME (A) and at the level of the 3V (C). Sense probe was used as a negative control with no detectable staining (E). Arrows indicate GnRH neuron bodies. ♀ indicates female; ♂, male; 3V, third ventricle; AHC, anterior hypothalamic area, central part; MPA, medial preoptic area; VMPO, ventromedial preoptic nucleus.

Figure 4

EAP1 binding to the GnRH promoter increases at puberty of female rhesus monkeys. An increased association of EAP1 to the rhesus GnRH1 promoter region in the MBH of female monkeys between the EJ and LJ periods is demonstrated. No change in EAP1 binding is detected in intron 2 of the GnRH gene. Inp means Input DNA and bGal means ChIP performed using an antibody to beta-galactosidase (a protein not present in the brain), serves as a negative control. Experiment was performed once, due to the limitation of availability of primate tissue.

Figure 5

EAP1 mutations impair the transcriptional activity of the human GnRH promoter. (A) HEK293T cells were seeded at 17.5 × 10⁴ cells/well onto a 24-well plate and transiently transfected with EAP1 plasmids (WT and mutated; 200 ng/well). Forty-eight hours post-transfection DLR assay was performed. Each transfection was normalized by co-transfecting with Renilla SV-40 vector and was performed in triplicate. EAP1 trans-activating GnRH promoter activity is significantly reduced by the in frame deletion (pAla221del) and missense (Asn770His) mutants compared to the WT. The mutants also cause a dose-dependent reduction of EAP1 WT transcriptional activity. The ablation of the RING finger domain (RINGdel) impairs the ability of trans-activating GnRH promoter and was used as a control, n = 3. (B) HEK293T cells were seeded at 0.3 × 10⁶ cells/well onto a 6-well plate and transiently transfected with EAP1 plasmids (WT and mutated; 1 μg/well). Forty-eight hours post-transfection western blot analysis was performed. Eap1 protein expression is detected at the expected molecular weight (90 kDa) and RINGdel expression is detected at ~80 kDa, as a result of the deletion of the RING finger domain (amino acids 715-762). GAPDH was used as loading control and detected at 37 kDa. HEK293T cells do not express EAP1, n = 3. (C) Quantification of western blot analysis, indicating that Ala221del protein levels are significantly reduced and Asn770His protein levels are significantly increased.

Figure 6

EAP1 p.Asn770His mutant is subcellularly mislocated. Immunofluorescence staining of EAP1 WT and mutated proteins (red) and DAPI (blue) in HEK293T cells. (A, B and C) show EAP1 WT protein expression within the nucleus, co-localizing with DAPI. (D, E and F) show EAP1 Ala221del mutant protein expressed within the nucleus, co-localizing with DAPI. (G, H and I) show EAP1 Asn770His mutant protein not expressed in the nucleus. Images were acquired using a fluorescence microscope (Leica microsystem, Germany) and processed using Adobe Photoshop CS6, n = 2.