Figure 5. Homologous Xic pairing can occur in the absence of biallelic Tsix expression.

A) Schematic representation of a female ESC line with a doxycycline-inducible promoter regulating the expression of one endogenous Xist allele (TX1072). B) Schematic representation of the initiation of differentiation and induction of Xist expression by addition of doxycycline in TX1072 cells. C) RNA FISH for nascent Xist (red, red arrowheads) and Tsix (white, white arrowheads) transcripts as well as processed and accumulating Xist RNA (green) in undifferentiated TX1072 or TX1072 cells differentiated for 2 days by LIF withdrawal either in the presence or absence of doxycycline in the culture medium. Depicted are representative cells for each condition. D) Quantification of the fraction of cells with monoallelic Tsix expression and monoallelic Xist (intron 1) expression in differentiating TX1072 either not induced with doxycycline or with Xist expression induced one day prior to the initiation of differentiation. E) DNA FISH for the Xist/Tsix region (red, BAC 8) in induced and uninduced TX1072 ESCs and at day 1.5 of differentiation after LIF withdrawal. Depicted are representative cells for each condition. F) Fraction of cells with Xic at pairing distance in differentiating induced and uninduced TX1072 cells. 3D distance was determined after DNA FISH for the Xist/Tsix region (BAC 8) as depicted in D). (n = number of cells, scale bar = 2 µm)