Further examination of the Xist promoter-switch hypothesis in X inactivation: Evidence against the existence and function of a P₀ promoter

Abstract

The onset of X inactivation coincides with accumulation of Xist RNA along the future inactive X chromosome. A recent hypothesis proposed that accumulation is initiated by a promoter switch within Xist. In this hypothesis, an upstream promoter (P₀) produces an unstable transcript, while the known downstream promoter (P₁) produces a stable RNA. To test this hypothesis, we examined expression and half-life of Xist RNA produced from an Xist transgene lacking P₀ but retaining P₁. We confirm the previous finding that P₀ is dispensable for Xist expression in undifferentiated cells and that P₁ can be used in both undifferentiated and differentiated cells. Herein, we show that Xist RNA initiated at P₁ is unstable and does not accumulate. Further analysis indicates that the transcriptional boundary at P₀ does not represent the 5′ end of a distinct Xist isoform. Instead, P₀ is an artifact of cross-amplification caused by a pseudogene of the highly expressed ribosomal protein S12 gene Rps12. Using strand-specific techniques, we find that transcription upstream of P₁ originates from the DNA strand opposite Xist and represents the 3′ end of the antisense Tsix RNA. Thus, these data do not support the existence of a P₀ promoter and suggest that mechanisms other than switching of functionally distinct promoters control the up-regulation of Xist.