The DXPas34 repeat regulates random and imprinted X inactivation

X chromosome inactivation (XCI) is initiated by expression of the noncoding Xist RNA in the female embryo. Tsix, the antisense noncoding partner of Xist, serves as its regulator during both imprinted and random XCI. Here, we show that Tsix in part acts through a 34mer repeat, DXPas34. DXPas34 contains bidirectional promoter activity, producing overlapping forward and reverse transcripts. We generate three new Tsix alleles in mouse embryonic stem cells and show that, while the Tsix promoter is unexpectedly dispensable, DXPas34 plays dual positive-negative functions. At the onset of XCI, DXPas34 stimulates Tsix expression through its enhancer activity. Once XCI is established, DXPas34 becomes repressive and stably silences Tsix. Germline transmission of the DXPas34 mutation demonstrates its necessity for both random and imprinted XCI in mice. Intriguingly, sequence analysis suggests that DXPas34 could potentially have descended from an ancient retrotransposon. We hypothesize that DXPas34 was acquired by Tsix to regulate antisense function.