A mouse model of Weaver syndrome displays overgrowth and excess osteogenesis reversible with KDM6A/6B inhibition
Christine W. Gao, WanYing Lin, Ryan C. Riddle, Priyanka Kushwaha, Leandros Boukas, Hans T. Björnsson, Kasper D. Hansen, Jill A. Fahrner
Christine W. Gao, WanYing Lin, Ryan C. Riddle, Priyanka Kushwaha, Leandros Boukas, Hans T. Björnsson, Kasper D. Hansen, Jill A. Fahrner
View: Text | PDF
Research Article Genetics

A mouse model of Weaver syndrome displays overgrowth and excess osteogenesis reversible with KDM6A/6B inhibition

Abstract

Weaver syndrome is a Mendelian disorder of the epigenetic machinery (MDEM) caused by germline pathogenic variants in EZH2, which encodes the predominant H3K27 methyltransferase and key enzymatic component of Polycomb repressive complex 2 (PRC2). Weaver syndrome is characterized by striking overgrowth and advanced bone age, intellectual disability, and distinctive facies. We generated a mouse model for the most common Weaver syndrome missense variant, EZH2 p.R684C. Ezh2R684C/R684C mouse embryonic fibroblasts (MEFs) showed global depletion of H3K27me3. Ezh2R684C/+ mice had abnormal bone parameters, indicative of skeletal overgrowth, and Ezh2R684C/+ osteoblasts showed increased osteogenic activity. RNA-Seq comparing osteoblasts differentiated from Ezh2R684C/+, and Ezh2+/+ BM-mesenchymal stem cells (BM-MSCs) indicated collective dysregulation of the BMP pathway and osteoblast differentiation. Inhibition of the opposing H3K27 demethylases KDM6A and KDM6B substantially reversed the excessive osteogenesis in Ezh2R684C/+ cells both at the transcriptional and phenotypic levels. This supports both the ideas that writers and erasers of histone marks exist in a fine balance to maintain epigenome state and that epigenetic modulating agents have therapeutic potential for the treatment of MDEMs.

Authors

Christine W. Gao, WanYing Lin, Ryan C. Riddle, Priyanka Kushwaha, Leandros Boukas, Hans T. Björnsson, Kasper D. Hansen, Jill A. Fahrner

×

Figure 5

The KDM6A/6B inhibitor GSK-J4 substantially reverses the Ezh2R684C/+ osteogenic phenotype and transcriptomic profile.

Options: View larger image (or click on image) Download as PowerPoint
The KDM6A/6B inhibitor GSK-J4 substantially reverses the Ezh2R684C/+ ost...
(A) Balance hypothesis (1). Left: loss of EZH2 in Weaver syndrome allows for unopposed demethylase activity by KDM6A/6B. Right: inhibition of KDM6A/6B by GSK-J4 restores balance to the chromatin state. (B) Alizarin red staining of female Ezh2R684C/+ and Ezh2+/+ osteoblasts treated with 2 μM GSK-J4 or vehicle (DMSO). Cells were differentiated for 21 days from BM-MSCs. Representative whole-well images shown. (C) GSK-J4 treatment decreases Alizarin red staining in Ezh2R684C/+ osteoblasts, as quantified by absorbance at 405 nm. Ezh2R684C/+ osteoblasts continue to have higher absorbance than Ezh2+/+. No significant difference between Ezh2+/+ vehicle-treated and Ezh2R684C/+ GSK-J4–treated osteoblasts. Blue circles: Ezh2+/+ females, n = 12. Red triangles: Ezh2R684C/+ females, n = 12. *P < 0.05, **P < 0.01, 2-way ANOVA with Tukey’s multiple-comparison test. Data represent mean ± 1 SD. (D) Volcano plot of day 21 RNA-Seq, displaying log2 fold changes in the Ezh2R684C/+ DMSO versus Ezh2+/+ DMSO contrast. Blue: MGI osteoblast differentiation genes. Magenta: MGI BMP pathway genes. FDR = 0.1 (red dashed line). (E) Conditional P value density plot displaying P values from the day 14 untreated RNA-Seq, stratified by significance at day 21 in the Ezh2R684C/+ DMSO versus Ezh2+/+ DMSO contrast (orange line, Padj < 0.1) or not (black line, Padj > 0.1). (F) Principal component analysis of Ezh2+/+ (blue circles, n = 6) and Ezh2R684C/+ (red triangles, n = 6) RNA-Seq samples at day 21 of osteoblast differentiation, treated either with vehicle (filled icons) or GSK-J4 (open icons). Corresponding Alizarin red staining images shown for representative samples. (G) Volcano plot of day 21 RNA-Seq, displaying log2 fold changes in the Ezh2R684C/+ GSK-J4 versus Ezh2R684C/+ DMSO contrast. FDR = 0.1 (red dashed line). (H) Scatter plot comparing log2 fold changes in the Ezh2R684C/+ DMSO versus Ezh2+/+ DMSO contrast and corresponding log2 fold-changes in the Ezh2R684C/+ GSK-J4 versus Ezh2R684C/+ DMSO contrast. Only genes meeting an adjusted P value threshold corresponding to FDR < 0.1 in both contrasts are shown (n = 1,075). Blue: MGI osteoblast differentiation genes. Magenta: MGI BMP pathway genes.