| Popis: |
Additional file 2: Fig. S2. Diploid gene expression changes in Kdm6a KO BC and CB clones. (A) Principal component analysis (PCA) based on diploid expression values for all transcribed genes from RNA-seq in wt and Kdm6a KO clones derived from the BC and CB crosses. BC clones include two male wt, two female wt, three male KO (Kdm6aΔ/Y), while CB clones include two male wt and three male KO (Kdm6aΔ/Y). Clone identifiers are included in the plot. (B) Hierarchal clustering of the clones described in (A). The color scale represents the sample-to-sample distance. (C) Expression fold changes between BC derived Kdm6aΔ/Y clones (Kdm6aΔE1, Kdm6aΔE3, and Kdm6aΔP4) and wt measured by RNA-seq show a significant decrease in Kdm6a expression, but no significant decrease in expression of pluripotent genes (Nanog, Pou5f1, Sox2, Cd9, Stat3, Fut4) (**p < 0.01 using a student’s t-test). (D) Deficiencies in ES cell differentiation potential following Kdm6a KO were tested by removal of LIF and by all-trans retinoic acid (RA) treatment. Smaller and less dense embryoid bodies were observed 6 days after removal of LIF in Kdm6aΔE1. In the presence of RA, wt cells show morphological signs of differentiation after 8 days, while Kdm6aΔE1 cells remain similar in morphology to day 0 controls. (E) Expression fold changes between male BC Kdm6aΔ/Y clones (Kdm6aΔE1, Kdm6aΔE3, and Kdm6aΔP4) and wt measured by RNA-seq confirm decreased expression of known KDM6A target genes Wt1, Bcar3, Foxh1, Dnmt3a, Sox3, Hsd17b11. Expression is based on diploid analysis at **FDR < 0.001. (F) Expression fold changes between male BC Kdm6aΔ/Y clones (Kdm6aΔE1, Kdm6aΔE3, and Kdm6aΔP4) and wt measured by quantitative RT-PCR analysis confirm downregulation of Vrtn, Bcar3, and Sall2 (*p < 0.01 using a student’s t-test) (Additional file 9A). Expression is normalized to Actb. (G) Venn diagrams to compare the number of downregulated and upregulated DEGs in male BC Kdm6aΔ/Y clones (Kdm6aΔE1, Kdm6aΔE3, and Kdm6aΔP4) and male CB Kdm6aΔ/Y clones (Kdm6aΔE2.5, Kdm6aΔE2.7, Kdm6aΔP2.1). DESeq2 was employed to identify differentially expressed genes (DEGs) in each cross using an FDR cutoff of < 0.05 and a ≥ 1.5 fold-change. |
