Further examination of the Xist promoter-switch hypothesis in X inactivation: Evidence against the existence and function of a P-0 promoter

The onset of X inactivation coincides with accumulation of Xist RNA along t he future inactive X chromosome. A recent hypothesis proposed that accumula tion is initiated by a promoter switch within Xist. in this hypothesis, an upstream promoter (P-0) produces an unstable transcript, while the known do wnstream promoter (P-1) produces a stable RNA. To test this hypothesis, we examined expression and half-life of Xist RNA produced from an Xist transge ne lacking P-0 but retaining P-1. We confirm the previous finding that P-0 is dispensable for Xist expression in undifferentiated cells and that P-1 c an be used in both undifferentiated and differentiated cells. Herein, we sh ow that Xist RNA initiated at P-1 is unstable and does not accumulate. Furt her analysis indicates that the transcriptional boundary at P-0 does not re present the 5' end of a distinct Xist isoform. Instead, P-0 is an artifact of cross-amplification caused by a pseudogene of the highly expressed ribos omal protein S12 gene Rps12. Using strand-specific techniques, we find that transcription upstream of P-1 originates from the DNA strand opposite Xist and represents the 3' end of the antisense Tsix RNA. Thus, these data do n ot support the existence of a P-0 promoter and suggest that mechanisms othe r than switching of functionally distinct promoters control the up-regulati on of Xist.