KDM6A facilitates Xist upregulation at the onset of X inactivation

Abstract Background: X chromosome inactivation (XCI) is a female-specific process in which one X chromosome is silenced to balance X-linked gene expression between the sexes. XCI is initiated in early development by upregulation of the lncRNA Xist on the future inactive X (Xi). A subset of X-linked genes escape silencing and thus have higher expression in females, suggesting female-specific functions. One of these genes is the highly conserved gene Kdm6a, which encodes a histone demethylase that removes methyl groups at H3K27 to facilitate gene expression. KDM6A mutations have been implicated in congenital disorders such as Kabuki Syndrome, as well as in sex differences in development and cancer. Methods: Kdm6a was knocked out (KO) using CRISPR/Cas9 gene editing in hybrid female mouse embryonic stem cells derived from a 129 x Mus castaneus (cast) cross in which a transcriptional stop signal is inserted onto the 129 allele of Tsix, resulting in completely skewed silencing of the 129 X chromosome upon differentiation. Allelic RNA-seq was done to compare gene expression between wild-type and Kdm6aKO clones. The effects of Kdm6a KO on Xist expression during the onset of XCI and the resulting changes in XCI potency were investigated using allele-specific RNA-seq and RNA FISH. Changes in H3K27me3 enrichment on the Xi in KO cells were investigated by CUT&RUN followed by allelic analysis. KDM6A binding to the Xist gene during the onset of XCI in wild-type cells was characterized by CUT&RUN. Results:We observed impaired upregulation of Xist during early stages of differentiation in hybrid mouse ES cells following CRISPR/Cas9 knockout of Kdm6a. This is associated with reduced Xist RNA coating of the Xi, suggesting diminished XCI potency. Indeed, Kdm6aknockout results in aberrant overexpression of genes from the Xi after differentiation. Consistent with a direct role in Xist regulation, KDM6A binds to the Xist promoter and knockout cells show an increase in H3K27me3 at Xist. Conclusions:These results reveal a novel female-specific role for the X-linked histone demethylase, KDM6A in the initiation of XCI through histone demethylase-dependent activation of Xistduring early differentiation.