Figure 4.

Analysis of PIC recruitment to Xist promoter in differentiating Tsix-truncated male ES cells. (A) Analysis of RNA Polymerase (n = 2) and TFIIB (n = 2) binding at the two Xist promoters (P1 and P2), the Tsix promoter, and three control promoters (ArpoP0, β-Actin, and Oct3/4) after 4 d of differentiation of wild-type (white bars) and mutant (black bars) male ES cells. (B) Extensive analysis across the Xist 5′ region of RNA Polymerase II distribution after 4 d of differentiation in control (dotted lines) and Tsix-truncated (black lines) male ES cells. For A and B, the graph shows the fold variation in binding calculated by dividing the percent immunoprecipitation obtained at day 4 by the percent immunoprecipitation obtained at day 0. Ratios >100% indicate an increase in binding, and ratios <100% indicate a release of the analyzed factor. (C) Analysis of RNA levels in wild-type (white bars) and Tsix-truncated (black bars) male ES cells differentiated for 4 d. Spliced RNA for β-Actin and Oct3/4 are shown as controls. Four independent primer pairs (Xist intron 1a, 1b, and 1c and Xist intron 3) were used to evaluate the fold variation of primary, unspliced Xist RNA levels in Tsix-truncated ES cells. Note that in wild-type ES cells, the four primers mapping within Xist introns also amplify Tsix RNA. The ratios between day 4 and day 0 of differentiation were calculated after standardizing each amplification against Arpo P0 mRNA levels. (D) A model for the role of Tsix in programming Xist transcription. The chromatin structure of the Xist 5′ region is controlled by Tsix. Continuous transcription of Tsix in undifferentiated ES cells maintains the region in a heterochromatic-like structure (characterized by H3K9 trimethylation and CpG methylation, black box). In the absence of Tsix, the region adopts a pseudoeuchromatic structure (characterized by H3K4 di/ trimethylation and H3K9 acetylation, gray box). CTCF likely participates in the definition of the region submitted to such a Tsix-dependent transition. Depending on these chromatic states, the Xist promoter will be differentially regulated during differentiation. When a euchromatic conformation is acquired early enough during (or before) differentiation, the Xist P1 promoter will recruit the transcriptional machinery to produce high levels of Xist RNA and therefore induce X inactivation, whether in female or male ES cells. In contrast, when silent epigenetic marks characterize the Xist 5′ region exclusively, the Xist promoter is unable to efficiently recruit the transcriptional machinery, preventing Xist up-regulation and X inactivation.