Figure 4. SATB1 Expression Defines the Silencing Function of Xist in Embryonic Cells.
(A) Combined RNA FISH immunofluorescence analysis of TX/Y R26rtTA/rtTA ES cells shows SATB1 (green) surrounding the characteristic Xist focus (red) after 24 hr of induction with Doxycycline. Scale bar = 5 μm.
(B and C) Western analysis of SATB1 (B) and SATB2 (C) protein during ES cell differentiation shows that SATB1 is lost and SATB2 shifts to a lower molecular weight on day 3, precisely when Xist loses its silencing capability. Asterisk indicates the high molecular weight form of SATB2. Actin serves as loading control.
(D) Repression of the puromycin (puro) marker, which is cointegrated with an Xist transgene on chromosome 11 in clone 36 ES cells, upon Xist induction is reduced by SATB1 or SATB2 RNAi compared to scrambled control. Quantification of puro expression after Xist induction relative to uninduced cells is given.
(E) Western analysis demonstrates strongly reduced SATB1 protein in ES cells treated with SATB1 siRNA but not control siRNA. Western analysis of SATB2 in ES cells treated with scrambles control, SATB2, and SATB1 siRNAs. The higher molecular weight form of SATB2 (asterisk) observed in ES cells is depleted by SATB2 RNAi. The lower molecular weight form of SATB2 is observed in brain. Lamin serves as loading control.
(F) Survival of immortalized fibroblasts established from TX/Y R26rtTA/rtTA embryos and infected with SATB1 and SATB2 expression vector or control empty vector (vector). The percent survival is calculated from the cell number with Xist induction relative to untreated cells (n = 6). Error bars represent standard deviation.
(G) Quantitative RT-PCR showing repression of the X-linked Hprt and Pgk1 genes upon Xist induction in MEFs infected with SATB1 and SATB2 but not empty vector controls. Expression relative to Gapdh is plotted (n = 6). Error bars represent standard deviation.