Dax1 modulates ERα-dependent hypothalamic estrogen sensing in female mice

Abstract

Coupling the release of pituitary hormones to the developmental stage of the oocyte is essential for female fertility. It requires estrogen to restrain kisspeptin (KISS1)-neuron pulsatility in the arcuate hypothalamic nucleus, while also exerting a surge-like effect on KISS1-neuron activity in the AVPV hypothalamic nucleus. However, a mechanistic basis for this region-specific effect has remained elusive. Our genomic analysis in female mice demonstrate that some processes, such as restraint of KISS1-neuron activity in the arcuate nucleus, may be explained by region-specific estrogen receptor alpha (ERα) DNA binding at gene regulatory regions. Furthermore, we find that the Kiss1-locus is uniquely regulated in these hypothalamic nuclei, and that the nuclear receptor co-repressor NR0B1 (DAX1) restrains its transcription specifically in the arcuate nucleus. These studies provide mechanistic insight into how ERα may control the KISS1-neuron, and Kiss1 gene expression, to couple gonadotropin release to the developmental stage of the oocyte.

Fig. 1. ERα binds to different genomic loci in the arcuate and AVPV nuclei.

A Schematic representation of integrated analysis of ERα ChIP-seq data and nucleus-specific transcriptomic data. B ERα occupancy in identified by ChIP-seq peaks, categorized according to Arcuate- or AVPV-enrichment. C Venn diagram of ERα-enriched peaks in both locations. D Genomic annotation of ERα peaks separated by enrichment category. E Selected GO-term enrichment p-values in genes proximal to ERα peaks.

Fig. 2. ERα binds in the proximity of genes that coordinate Kiss1-neuron pulsatility in the arcuate nucleus.

A Genes found in proximity to ERα binding sites and affected by estrogen in the arcuate nucleus. B Expression of Kiss1, Kor, Pdyn, and Tac2 genes in the arcuate and AVPV^Kiss1 nuclei upon E2 treatment. C Expression of Kiss1 and Golt1a in the arcuate and AVPV^Kiss1 (n = 3-6). Error bars represent SEM. DeseqFDR **p < 0.01, ***p < 0.001, ns not significant. D ERα occupancy at the Golt1a/Kiss1 locus in the arcuate and AVPV nuclei (http://genome-euro.ucsc.edu/). E ERE sequence detected for the Greb1 (positive control), and the peaks identified proximal to Kiss1, Pdyn, and Tac2. Gene expression data were obtained from previously published studies^,118.

Fig. 3. Dax1 is enriched in the arcuate hypothalamus and can repress Kiss1-transcription.

A qPCR screening of 84 nuclear receptor co-regulators in the mouse arcuate and AVPV hypothalamus under estrogen stimulation (n = 4). B Confirmation of Dax1 expression in the arcuate and AVPV hypothalamus using biological replicates under estrogen stimulation (n = 4) *p < 0.05 by t-test error bars represent SEM. C Detection of the DAX1 protein (red) by immunofluorescence in the whole arcuate and in Kiss1-neurons (green) in intact Kiss1-Cre-GFP mice in diestrous (n = 3) *p < 0.05 by t-test Error bars represent SEM. Scale bar 50 µm (left), 20 µm (right). D DAX1 and POMC in human hypothalamus (n = 3). Error bars represent SEM. E expression of POMC and DAX1 in human arcuate nucleus (n = 3) Error bars represent SEM. F CRISPR-activation of the Golt1a promotor in C2C12 cells transfected with mERα or/and mDax1. Representative data from an experiment conducted twice (n = 4 wells per group) *p < 0.05 compared to Golt1a by Holm-Sidak multiple t-tests. Error bars represent SD. Source data are provided as a Source Data file.

Fig. 4. Mice lacking DAX1 in Kiss1-neurons have abnormal Kiss1 gene regulation.

A quantification of Cre-mediated deletion of DAX1-protein in arcuate Kiss1-neurons (n = 3 mice). B Expression of Kiss1, Tac2, and Pdyn in the arcuate nucleus of intact mice at 9am on the first day of diestrous (n = 8 Dax1^tm1, n = 12 Dax1^th1(Kiss1) *p < 0.05. C Cycle length (n = 8), Offipring/3-months (n = 8 Dax1^tm1, n = 10 Dax1^th1(Kiss1)) *p < 0.05 by t-test, Tertiary follicles per ovary (n = 5). (D) Plasma FSH levels at 9am on the first day of diestrous (n = 7). E Representative LH profiles starting at 9am on the first day of diestrous. Peak LH (n = 8) an inter-pulse interval (n = 5 Dax1^tm1, n = 4 Dax1^th1(Kiss1)). F Kiss1-expression in the AVPV and arcuate following estrogen stimulation (n = 8) *p < 0.05 compared to vehicle 2-way ANOVA with Tukey’s multiple test correction. G Schematic representation of ERα function during negative feedback in arcuate Kiss1-neurons (Created with BioRender.com). Error bars represent SEM. Source data are provided as a Source Data file.