Figure 5. X-linked Dusp9 gene regulates global DNA hypomethylation in female stem cells.

(A) Upregulated X-linked genes in XX EGC (n=2) and ESC (n=3) lines relative to XY counterparts (3 EGC and 3 ESC lines).

(B) List of candidate X-linked genes and their expression levels in XX stem cells compared to XY stem cells. FC, fold-change.

(C) Four candidate genes (Rhox5, Klf8, ERas; left) and (Dusp9; right) from Figure 5B were individually overexpressed in a XY ESC line and global DNA methylation levels were subsequently examined by 5mC dot blot analysis. Global DNA methylation levels of each line were normalized to parental, wild-type ESCs.

(D) Western blot analysis for DUSP9 in isogenic XX and XY ESCs/EGCs. See Figure S6A for quantification. Blue color represents XY cells, red color represents XX cells, green (*) represents XO cell line

(E) Mass-spectrometric quantification of DUSP9 protein levels in XY (blue; 2 replicates) and XX (red; 2 replicates) ESC lines.

(F) (Top) Schematic of doxycycline-inducible Dusp9 allele introduced into KH2 (male) ESCs; these cells also carry a ROSA26-M2rtTA allele. (Bottom) Western blot analysis for DUSP9 in Dusp9-overexpressing KH2 cells treated with or without doxycycline (left), plus quantification (right).

(G) Global methylation analysis of Dusp9-overexpressing KH2 cells (with or without additional Dusp9 lentiviral overexpression construct) by RRBS using violin plot representation. XX and XY ESCs were included as controls.

(H) (Top) Schematic of heterozygous deletion of Dusp9 alleles in a female 129xCast hybrid ESC line using CRISPR/Cas9 technology. Western blot analysis for DUSP9 in Dusp9^+/+ and Dusp9^+/− ESC lines (left), plus quantification (right).

(I) Global methylation analysis of the Dusp9^+/+ and Dusp9^+/− ESC lines by RRBS using violin plot representation. Wild-type 129xB6 XX and XY ESC lines were included as controls.

See also Figure S6 and Table S5.