FTO-mediated m6A demethylation regulates GnRH expression in the hypothalamus via the PLCß3/Ca2+/CAMK signalling pathway.

N6-methyladenosine (m6A) plays a crucial role in the development and functional homeostasis of the central nervous system. The fat mass and obesity-associated (FTO) gene, which is highly expressed in the hypothalamus, is closely related to female pubertal development. In this study, we found that m6A methylation decreased in the hypothalamus gradually with puberty and decreased in female rats with precocious puberty. FTO expression was increased at the same time. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) showed that the m6A methylation of PLCß3, a key enzyme of the Ca2+ signalling pathway, was decreased significantly in the hypothalamus in precocious rats. Upregulating FTO increased PLCß3 expression and activated the Ca2+ signalling pathway, which promoted GnRH expression. Dual-luciferase reporter and MeRIP-qPCR assays confirmed that FTO regulated m6A demethylation of PLCß3 and promoted PLCß3 expression. Upon overexpressing FTO in the hypothalamic arcuate nucleus (ARC) in female rats, we observed advanced puberty onset. Meanwhile, PLCß3 and GnRH expression in the hypothalamus increased significantly, and the Ca2+ signalling pathway was activated. Our study demonstrates that FTO enhances GnRH expression, which promotes puberty onset, by regulating m6A demethylation of PLCß3 and activating the Ca2+ signalling pathway.

Transcriptome-scale spatial gene expression in rat arcuate nucleus during puberty.

BACKGROUND: A variety of neurons in hypothalamus undergo a complicated regulation on transcription activity of multiple genes for hypothalamic-pituitary-gonadal axis activation during pubertal development. Identification of puberty-associated cell composition and characterization of the unique transcriptional signatures across different cells are beneficial to isolation of specific neurons and advanced understanding of their functions. METHODS: The hypothalamus of female Sprague-Dawley rats in postnatal day-25, 35 and 45 were used to define the dynamic spatial atlas of gene expression in the arcuate nucleus (ARC) by 10× Genomics Visium platform. A surface protein expressed selectively by kisspeptin neurons was used to sort neurons by flow cytometric assay in vitro. The transcriptome of the isolated cells was examined using Smart sequencing. RESULTS: Four subclusters of neurons with similar gene expression signatures in ARC were identified. Only one subcluster showed the robust expression of Kiss1, which could be isolated by a unique membrane surface biomarker Solute carrier family 18 member A3 (SLC18A3). Moreover, genes in different subclusters presenting three expression modules distinctly functioned in each pubertal stage. Different types of cells representing distinct functions on glial or neuron differentiation, hormone secretion as well as estradiol response precisely affect and coordinate with each other, resulting in a complicated regulatory network for hypothalamic-pituitary-gonadal axis initiation and modulation. CONCLUSION: Our data revealed a comprehensive transcriptomic overview of ARC within different pubertal stages, which could serve as a valuable resource for the study of puberty and sexual development disorders.

The epigenetic role of HTR1A antagonist in facilitaing GnRH expression for pubertal initiation control.

Serotonin (5-hydroxytryptamine [5-HT]), a metabolite of tryptophan, acts on the components of the hypothalamus-hypophysis-gonad axis and induces puberty delay in mammals via 5-HT receptor 1A (HTR1A). However, the roles of HTR1A in the hypothalamus in pubertal regulation of gene expression are not fully understood. In the current study, the upregulated gonadotropin-releasing hormone (GnRH) expression in GT1-7 GnRH neuronal cells induced by the HTR1A antagonist WAY-100635 maleate was observed in vitro. Furthermore, RNA sequencing (RNA-seq) showed decreased expression of chromobox 4 (CBX4), a member of the polycomb-repressive complex 1 (PRC1), and the loss of RING2 and YY1 interaction with CBX4, suggesting the degradation of the PRC1 in GT1-7 cells treated with maleate. Chromatin immunoprecipitation sequencing (ChIP-seq) showed that the genome-wide occupancy of CBX4 and histone H2A lysine-119 ubiquitination (H2AK119ub) was compromised, especially on the promoter of GnRH. Finally, we determined that inactivation of phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) contributed to CBX4 downregulation. Taken together, we concluded that HTR1A antagonists could enhance GnRH transcription via PRC1 degradation and H2AK119ub loss driven by reduced CBX4 expression through PI3K/Akt and MAPK/ERK pathway suppression in GT1-7 cells and provided a potential epigenetic mechanism of action of HTR1A on GnRH gene expression for mammalian puberty onset.

Downregulation of TTF1 in the rat hypothalamic ARC or AVPV nucleus inhibits Kiss1 and GnRH expression, leading to puberty delay.

BACKGROUND: TTF1 is a transcription factor that is expressed in the hypothalamus after birth and plays crucial roles in pubertal development. TTF1 may regulate the expression of the Kiss1 gene, which may drive puberty onset in the hypothalamic arcuate (ARC) and anterior ventral paraventricular (AVPV) nuclei. METHODS: A dual-luciferase reporter assay was used to detect binding between TTF1 and the Kiss1 gene promoter. To investigate the effects of TTF1, we modified TTF1 expression in cell lines and in the ARC or AVPV nucleus of 21-day-old female rats via lentivirus infection. TTF1 and other puberty onset-related genes were detected by qRT-PCR and western blot analyses. RESULTS: The in vitro data indicated that TTF1 knockdown (KD) significantly reduced Kiss1 and GnRH expression. Overexpression (OE) of TTF1 promoted Kiss1 expression. In vivo, the expression of Kiss1 and GnRH decreased significantly in the rats with hypothalamic ARC- or AVPV-specific TTF1 KD. The TTF1-KD rats showed vaginal opening delay. H&E staining revealed that the corpus luteum was obviously reduced at the early puberty and adult stages in the rats with ARC- or AVPV-specific TTF1 KD. CONCLUSION: TTF1 bound to the promoter of the Kiss1 gene and enhanced its expression. For 21-day-old female rats, decreased TTF1 in the hypothalamic ARC or AVPV nucleus resulted in delayed vaginal opening and ovarian abnormalities. These observations suggested that TTF1 regulates puberty onset by promoting the expression of Kiss1 and plays an important role in gonad development.