Abstract

X-chromosome inactivation (XCI) depends on the noncoding Xist gene. Xist transcription is negatively regulated by its antisense partner Tsix, whose disruption results in nonrandom XCI in females. However, males can maintain Xist in a repressed state without Tsix, indicating participation of additional factor(s) in the protection of the single male X from inactivation. Here, we provide evidence that the histone methyltransferase Eed is also involved in the process. Male embryonic stem cells with Eed-null and Tsix mutations (XY Eed-/-) showed Xist hyperactivation upon differentiation, whereas cells with either mutation alone did not. Impaired X-linked gene expression was observed in the XY Eed-/- ES cells at the onset of differentiation. The Xist promoter in the XY Eed-/- cells showed elevated histone H3-dimethyl lysine 4 modifications and lowered CpG methylation, which are characteristics of open chromatin. Hence, we identified Eed as an additional major player in the regulation of Xist expression. The synergy of Polycomb group proteins and antisense Tsix transcription in Xist gene regulation explains why males can repress Xist without Tsix.