Female Bias in Rhox6 and 9 Regulation by the Histone Demethylase KDM6A (original) (raw)
Figure 3
Kdm6a knockdown causes a female-specific decrease in Rhox6 and 9 expression in ES cells.
(A) Quantitative RT-PCR after Kdm6a knockdown in female PGK12.1 ES cells shows a 75% and a 52% decrease in Kdm6a and Rhox6 and 9 expression, respectively. Expression is shown relative to control levels obtained with scrambled siRNA. Control gene β-actin levels are set to 1. The inset shows a western blot using two different KDM6A antibodies, which confirms a ∼70–90% reduction in protein levels. β-ACTIN is used as a control. (B) Kdm6a knockdown results in a decrease in Rhox6 and 9 expression in female (PGK12.1 and E8) but not male (WD44 and E14) undifferentiated ES cells. Expression measured by array analysis (PGK12.1 and WD44) and qRT-PCR (E8 and E14) is shown as fold change compared to the control gene β-actin. Array results are from four independent experiments and qRT-PCR results are from three independent experiments. (C) The decrease in Rhox6 and 9 expression correlates with the level of Kdm6a knockdown in a dose dependent manner in PGK12.1 ES cells (*p<0.05). Fold changes in levels of Rhox6 and 9 measured by expression arrays are shown relative to fold changes in Kdm6a levels. (D) Kdm6a knockdown results in a significant (*p<0.05) increase in H3K27me3 enrichment at Rhox6 and 9 5′end, gene body, and 3′end. ChIP-qPCR of H3K27me3 enrichment in PGK12.1 ES cells treated with control scrambled siRNA and Kdm6a specific siRNA. Average enrichment for two separate ChIP experiments is shown as ChIP/input.