Figure 6

Model of RA-induced Xist expression in Tsix -mutant male cells. A. Undifferentiated wild-type X/Y cells maintain high levels of Oct4, Sox2, and Nanog, which bind to Xist intron 1. This synergistic binding -- in addition to high Tsix RNA expression -- effectively suppresses Xist. Under the EB differentiation method (left), Oct4 expression remains high early during differentiation, allowing continued binding of Oct4, Sox2, and Nanog to keep Xist off. Although Oct4 levels and binding decrease during late differentiation, Xist continues to remain repressed as the Xist-dependent phase of XCI has passed. Under the RA differentiation method (right), RA binds to its receptor to form RAR complexes, which then bind to and negatively regulate Oct4. However, persistent Tsix expression maintains stable Xist repression. B. In undifferentiated X^Δ/Y cells, high levels of Oct4, Sox2, and Nanog are sufficient to repress Xist in the absence of Tsix. Under the EB differentiation method (left), Oct4 expression remains high early during differentiation, allowing continued binding of Oct4, Sox2, and Nanog to maintain low Xist expression even in the absence of Tsix transcription. Although Oct4 mRNA levels and binding to Xist intron 1 decrease during late differentiation, Xist is stably repressed as the Xist-dependent phase has passed. However, in the RA differentiated state (right), RAR complexes bind to Oct4, causing premature Oct4 downregulation; loss of Oct4 binding to Xist intron 1 and absence of Tsix lead to moderate Xist upregulation during the early (Xist-dependent) stages of differentiation. Due to Xist derepression during this early stage and lack of antisense Tsix transcription, Xist RNA starts to accumulate and diffusely coats the chromosome in cis, leading to formation of visible Xist RNA clusters and incomplete silencing. DE and PE refer to distal and proximal Oct4 enhancers, respectively.