Multiomic analysis implicates nuclear hormone receptor signalling in clustering epilepsy

Abstract

Clustering Epilepsy (CE) is an epileptic disorder with neurological comorbidities caused by heterozygous variants of the X chromosome gene Protocadherin 19 (PCDH19). Recent studies have implicated dysregulation of the Nuclear Hormone Receptor (NHR) pathway in CE pathogenesis. To obtain a comprehensive overview of the impact and mechanisms of loss of PCDH19 function in CE pathogenesis, we have performed epigenomic, transcriptomic and proteomic analysis of CE relevant models. Our studies identified differential regulation and expression of Androgen Receptor (AR) and its targets in CE patient skin fibroblasts. Furthermore, our cell culture assays revealed the repression of PCDH19 expression mediated through ERα and the co-regulator FOXA1. We also identified a protein-protein interaction between PCDH19 and AR, expanding upon the intrinsic link between PCDH19 and the NHR pathway. Together, these results point to a novel mechanism of NHR signaling in the pathogenesis of CE that can be explored for potential therapeutic options.

Fig. 1. DNA methylation analysis of CE skin fibroblasts.

A Comparison of the number of DMRs in each group. B Venn diagram showing the overlap of genes with differentially methylated promoter and/or gene body in AFs vs FCs and TMs vs MCs comparisons. C Hierarchical clustering of samples by DMPs identified in AFs vs. FCs. D KEGG pathway and TF binding enrichment analysis of the genes with differentially methylated promoter/gene body identified in AFs (ShinyGO, TF.Target.ChEA.2016 database- FDR cut-off 0.1) [47]. Top 10 most enriched pathways are shown. AF affected female, FC female control, TM transmitting male, MC male control, TF transcription factor.

Fig. 2. Gene expression analysis of CE skin fibroblasts.

A Comparison of the number of DEGs in each group. B Venn diagram showing the overlap in dysregulated genes when comparing AFs vs. FCs and TMs vs. MCs. C Hierarchical clustering of samples by DEGs for AFs vs. FCs. D Biological Process enrichment analysis of AF vs. FC DEGs (ShinyGO, Biological Process, FDR cut-off 0.05) [47]. E Validation of altered expression of NOVA1, RUFY3, TET3, AR, ERα and PGR expression in FC (n = 3) and AF (n = 14) skin fibroblasts by RT-qPCR. Statistical analysis was performed using unpaired t-test with Welch’s correction. *p ≤ 0.05, **p ≤ 0.01. AF affected female, FC female control, TM transmitting male, MC male control.

Fig. 3. PCDH19 expression is regulated by steroid treatment.

A T47D cells were cultured in hormone-depleted conditions for 48 h and then treated with 10 nM E2, 10 nM P4, 10 nM DHT or vehicle (V) for 4, 6, 16, 24 and 48 h. PCDH19 expression was assayed by RT-qPCR. n ≥ 3 biological replicates. Statistical analysis was performed using one-way ANOVA for each timepoint. B Pcdh19 mRNA expression in female (n = 3) or C male (n $\ge$ 3) E18.5 mouse cortical neurons treated with 10 nM E2, 10 nM P4, 10 nM DHT or vehicle (V) for 24 h. Statistical analysis was performed using one-way ANOVA. D ERα, PGR and AR were KD in T47D cells with two siRNA molecules and PCDH19 expression was determined by RT-qPCR. n $\ge$ 3. Statistical analysis performed using one-way ANOVA comparing against SCR. E FOXA1 was KD in T47D cells with two siRNA molecules and treated with 10 nM E2 or vehicle. PCDH19 expression was determined by RT-qPCR. n = 3. Statistical analysis performed using one-way ANOVA comparing against SCR V. Schematic diagram of the location of two FOXA1 binding sites in the PCDH19 promoter is also shown. F MCF-7 cells were transfected with pGL2-TK2 (EV), pGL2-TK2-BS1 + BS2 WT or pGL2-TK2-BS1 + BS2 MT reporter constructs and treated with 10 nM E2 or vehicle. Firefly Luciferase values were normalized to Renilla Luciferase transfection control and expressed as Relative Light Units (RLU). Each experiment was performed in four technical and three biological replicates. Statistical analysis was performed using one-way ANOVA. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Fig. 4. Androgen Receptor interacts with PCDH19.

A Myc-PCDH19 was immunoprecipitated with anti-Myc magnetic beads. Inputs and IP samples were western blotted to detect Myc-PCDH19 and FLAG-AR. B Myc-PCDH19 isoforms (+Ex2 or -Ex2) and pathogenic missense variants were immunoprecipitated with anti-Myc magnetic beads. Inputs and IP samples were western blotted to detect Myc-PCDH19 and FLAG-AR. C FLAG-AR was immunoprecipitated with anti-FLAG agarose beads. Inputs and IP samples were western blotted to detect FLAG-AR, HA-PCDH10 and HA-PCDH12.