An iPSC-based model of 47,XYY Jacobs syndrome reveals a DNA methylation-independent transcriptional dysregulation shared with male X aneuploid cells

NPY genes modulate the expression of their homologs on the X Chromosome. (A,B) Log₂FC of NPY (light blue) and NPX (green) expression in the contrast 47,XYY versus 46,XY in iPSCs (A) and NSCs (B). Expressed NPY–NPX genes are plotted. A single asterisk indicates DEGs with FDR < 0.05 and Log₂FC > |0.58|. (C) Linear regression analysis of TMM normalized gene expression in iPSCs carrying zero (46,XX), one (46,XY), or two (47,XYY) Y Chromosomes. Pearson correlation coefficients (R), P-values, and FDR are indicated. (Top) NPY genes show a positive expression correlation with Y number. (Bottom) NPX homologs show an opposite correlation trend. (D, top) Western blot for UTY and KDM6A expression levels in 47,XYY UTY^−/− and UTY^+/+ iPSCs. (Bottom) Quantification of KDM6A protein expression normalized on GAPDH levels in 47,XYY UTY^−/− and UTY^+/+ iPSCs. Four independent UTY^−/− and two UTY^+/+ (CTR.) clones (cl.) have been tested. The significance of the comparison between wild-type and knockout samples was calculated using the one-way ANOVA and pairwise comparison with post hoc Tukey HSD. (E,F) KDM6A and UTY overexpression in 46,XY H1 hESCs. (E) mRNA levels of KDM6A and UTY normalized to TATA-binding protein (TBP) in nontransfected (NT) cells or cells transfected with 3XT7 empty vector (T7) and 3XT7-UTY. Bars correspond to the average mRNA expression levels measured in triplicates from two independent transfections per sample. (F, top) Protein levels of KDM6A and UTY in cells overexpressing 3XT7-UTY (UTY) and 3XT7-KDM6A (KDM6A). (Lanes 1,2) Basal protein levels of KDM6A and UTY in 46,XX and 46,XY cells. (Lanes 3,4) Protein levels of KDM6A and UTY in 46,XY upon ectopic overexpression of UTY in two independent transfections. (Lanes 5,6) Protein levels of KDM6A and UTY in 46,XY upon ectopic overexpression of KDM6A in two independent transfections. Untransfected female H9 and male H1 hESCs are used for antibody cross-checking. (Bottom) Quantification of the expression levels of KDM6A and UTY proteins normalized to GAPDH, relative to 46,XY levels. (G) Log₂FC of NPX (green) and NPY (light blue) expression in the contrast 47,XXY versus 46,XY in iPSCs. Expressed NPY–NPX genes are plotted, DEGs with FDR < 0.05 and Log₂FC > |0.58| are indicated with asterisks. (H) TaqMan assay for KDM6A and UTY mRNA in 46,XX H9 hESCs transfected with 3XT7 (T7) or 3XT7-UTY. Student's t-test (**) P-value < 0.005.