A counting process senses the X chromosome/autosome ratio and ensures that X 
chromosome inactivation (XCI) initiates in the early female (XX) embryo and in 
differentiating female ES cells but not in their male (XY) counterparts. 
Counting depends on the X inactivation center (Xic), which contains the Xist 
gene encoding a nuclear RNA, which coats the inactive X chromosome and induces 
gene silencing. A 37-kb sequence lying 3' to the Xist gene is known to prevent 
initiation of XCI in male differentiating ES cells. This region contains the 
major and minor promoters of the Tsix gene, which runs antisense to Xist, and 
the DXPas34 tandem repeat lying close to the Tsix major promoter. We have 
addressed the role of these elements in counting by using male ES cells. 
Targeted deletion of DXPas34 impaired recruitment of RNA-polymerase II and TFIIB 
at the Tsix major promoter, resulting in low levels of Tsix expression in ES 
cells and moderate ectopic initiation of XCI upon differentiation. A deletion 
extending 3' to Xist and including the Tsix major promoter resulted in almost 
complete impairment of Tsix transcription and in efficient ectopic XCI upon 
differentiation of male ES cells. Internal deletions within the Tsix gene did 
not affect significantly the level of antisense transcription within Xist and 
had only minor effects upon differentiation. Our results identify a function for 
DXPas34 in murine XCI and demonstrate the critical role of Tsix transcription in 
preventing XCI in differentiating male ES cells and in normal functioning of the 
counting pathway.