Site-specific phosphorylation of ERα determines sex-dependent metabolic, reproductive, and body compositional phenotypes in mice

Estrogen receptor alpha (ERα) phosphorylation modulates receptor activity and downstream signaling. Previous studies identified human phosphorylation sites, serine-167 (S167) and serine-212 (S212) as critical regulators of ERα-mediated gene expression. Serine(S) to alanine(A) phospho knock-in mouse models of the corresponding murine sites, S171 and S216, were generated to assess physiological impact. Compared to WT mice, ERα protein levels in tissues were altered in a sex- and phosphorylation site-dependent manner. S216A males exhibited reduced adipose and increased lean mass. S216A females exhibited decreased femur bone density whereas S171A females exhibited increased bone density. S216A females were subfertile with fewer pups/litter. Single-cell and spatial transcriptomics revealed markedly altered E2-regulated uterine gene expression in both models. S216A mice exhibited reduced plasma metabolites in several key metabolic pathways. This study identifies critical roles for site-specific ERα phosphorylation in modulating receptor level/activity and gene expression that has profound impact on murine body composition, fertility, and metabolism. This study employed NanoString CosMx spatial transcriptomics to analyze formalin-fixed, paraffin-embedded (FFPE) uterine sections from ovariectomized mice treated with either vehicle or estradiol (E2). A 1000-plex gene panel was used to capture spatial gene expression. Machine learning-based cell segmentation enabled the identification of individual cells. Data normalization, principal component analysis (PCA), and uniform manifold approximation and projection (UMAP) clustering delineated distinct cell populations. Differential gene expression analysis and module scoring revealed cell type-specific transcriptional changes between wild-type and ERα phosphorylation mutants (S171A and S216A).