Figure 6. YY1 acts on Xist promoter to control Xist activation.

(a-b) Luciferase reporter assay in female mESC transiently transfected with WT, mutants and reverse human XIST (a) and mouse Xist (b) promoter luciferase constructs (white bars) compared to promoterless control (gray bar). White diamonds represent YY1 sites. “Δ” indicate punctate deletion (10 bp) and “X” point mutations of individual YY1 binding sites. Firefly Luciferase activity is reported after normalization to Renilla activity, considered as internal control for transfection variability. (c) Luciferase reporter assay on pooled clones of female mESC stably transfected with the WT luciferase construct, the construct with deletion of YY1 sites or the control promoterless vector. Normalization is performed to total protein levels. (d) Schematic view of the Xist 5′ region with the position of the region targeted by the two CRISPR RNAs indicated by vertical arrows. The YY1 and CTCF binding sites are represented as diamonds and circles, respectively, and the Xist TSS is indicated by a horizontal arrow. (e) Schematic view of the Xist 5′ region in the four clones analyzed (C1-C4), with the deletion indicated. (f) qRT-PCR analysis of Xist expression in CRIPR/Cas9 targeted clones in ES cells (d0) and at day 2 (d2) and day 4 (d4) of a representative differentiation. Statistical evaluation of the difference between C1, considered here as WT, and clones C2-C4 carrying mutations of YY1 binding sites is provided for each day of differentiation. *** p<0.001; ** p< 0.01; * p<0.05 (Student’s t-test) (g) Representative Xist RNA-FISH images of CRIPR/Cas9 targeted clones at d4 of differentiation. Scale bars represent 5 μm. (h) Quantification of nuclei (n>150) with Xist RNA cloud, pinpoints or with no signal (empty) at d4 of differentiation. (i) Premature Xist expression levels determined by strand-specific qRT-PCR at day 4 of differentiation. (j) Premature Tsix expression levels determined by strand-specific qRT-PCR at day 4 of differentiation.