TET1 regulates fibroblast growth factor 8 transcription in gonadotropin releasing hormone neurons

Abstract

Fibroblast growth factor 8 (FGF8) is a potent morphogen that regulates the ontogenesis of gonadotropin-releasing hormone (GnRH) neurons, which control the hypothalamus-pituitary-gonadal (HPG) axis, and therefore reproductive success. Indeed, FGF8 and FGFR1 deficiency severely compromises vertebrate reproduction in mice and humans and is associated with Kallmann Syndrome (KS), a congenital disease characterized by hypogonadotropic hypogonadism associated with anosmia. Our laboratory demonstrated that FGF8 signaling through FGFR1, both of which are KS-related genes, is necessary for proper GnRH neuron development in mice and humans. Here, we investigated the possibility that non-genetic factors, such as the epigenome, may contribute to KS onset. For this purpose, we developed an embryonic explant model, utilizing the mouse olfactory placode (OP), the birthplace of GnRH neurons. We show that TET1, which converts 5-methylcytosine residues (5mC) to 5-hydroxymethylated cytosines (5hmC), controls transcription of Fgf8 during GnRH neuron ontogenesis. Through MeDIP and ChIP RT-qPCR we found that TET1 bound to specific CpG islands on the Fgf8 promoter. We found that the temporal expression of Fgf8 correlates with not only TET1 binding, but also with 5hmC enrichment. siRNA knockdown of Tet1 reduced Fgf8 and Fgfr1 mRNA expression. During this time period, Fgf8 also switched histone status, most likely via recruitment of EZH2, a major component of the polycomb repressor complex-2 (PRC2) at E13.5. Together, these studies underscore the significance of epigenetics and chromatin modifications to temporally regulated genes involved in KS.

Fig 1

A) Schematic of transient Fgf8 transcription during GnRH neuronal emergence in the embryonic mouse OP. B) Fgf8 and GnRH mRNA expression at E10.5 (n = 2) or at E10.5+3 days in-vitro (DIV) (n = 3) in the mouse OP. p < 0.0005, p < 0.01; Student’s t-test C) MethPrime CpG prediction of CpG islands on mouse Fgf8 promoter with relative locations of CpG1-4 primers [30]. * indicates p <0.05.

Fig 2. AZA induced Fgf8 expression in the OP.

A) RT-qPCR for Fgf8, GnRH and HDAC1 mRNA in vehicle (n = 6) vs 1 μM AZA-treated E10.5 mouse OP explants (n = 6) for 3 DIV B) Two representative 5mC dot blots in GT1-7 neurons treated with AZA (Vehicle, 0.01, 0.1, or 1 μM) for 3 days. Note that 5mC is virtually absent at the 1 μM concentration only. * indicates p < 0.05; Student’s t-test.

Fig 3. TET or DNMT expression in the OP.

A) Tet 1, 2, 3 or B) Dnmt1, 3a, 3b, and HDAC1 mRNA expression in E10.5 or E13.5 mouse OP (n = 4). C) Tet 1, 2, 3 mRNA expression in GN11 and GT1-7 GnRH neurons (n = 4). D) 5hmC dot blot quantification of E10.5 (n = 4) versus E13.5 (n = 4) in 75 ng of OP genomic DNA and original dot blot image. * indicates p < 0.05; Student’s t-test.

Fig 4. DNMT3b binds to the Fgf8 promoter early in OP development.

DNMT3b ChIP-RT-qPCR of 6 pooled E9.5 mouse OPs on the Fgf8 promoter (n = 4). * indicates p < 0.05. Student’s t-test.

Fig 5. 5hmC accumulation on the Fgf8 promoter is driven in a time-dependent fashion.

MeDIP RT-qPCR in 3–5 pooled mouse OPs at E9.5 (n = 4), E10.5 (n = 4), E13.5 (n = 4) along the Fgf8 promoter. CpG islands are indicated in numbers 1–4. * indicates p < 0.05 compared to IgG; Student’s t-test. ** indicates p < 0.05 5hmC enrichment on CpG 1 and 3 between E9.5 and E10.5 or E13.5; One-way ANOVA followed by Holm-Sidak post hoc.

Fig 6. Epigenetic switch on the Fgf8 promoter in the E9.5 and E13.5 OP.

A) At E9.5 (n = 4), TET1 was enriched at CpG 1 and 3. B) At E13.5 (n = 4), TET1 was enriched at CpG 1 and 3, while EZH2 was enriched at all 3 CpG sites. * indicates p < 0.05; Student’s t-test.

Fig 7. Fgf8 histone modifications during GnRH neuron ontogenesis.

A) ChIP for H3K4me3/H3K27me3 of 3–5 pooled E9.5 (n = 4) and B) 3–5 pooled E13.5 OP (n = 4) on CpG 3. E9.5 OPs are enriched for H3K4me3 and H3K27me3, whereas only H3K27me3 was detected in E13.5. * indicates p < 0.05; Student’s t-test.

Fig 8. Tet1 siRNA knockdown in GT1-7 neurons.

A) Tet1 siRNA (n = 4) did not affect other TET or DNMT mRNA expression, and reduced Tet1 mRNA expression compared to non-targeting controls (n = 4). B) Tet1 knockdown reduced Fgf8, Fgfr1, and Fgf2 mRNA (n = 4). * indicates p < 0.05; Student’s t-test.

Fig 9. Schematic model of age-dependent Fgf8 transcriptional control in the embryonic mouse OP.

A) At time (t) = 0, TET1 interaction with the Fgf8 promoter maintains its hypomethylated, and H3K4me3/H3K27me3 bivalent state, thereby inducing Fgf8 transcription B) In contrast at t = 1, EZH2 recruitment maintained H3K27 trimethylation, while H3K4me3 was lost, which represses Fgf8 transcription. Closed circles = methylated, open circles = demethylated.