SMCX & Random X-inactivation
Lack of Xist RNA induction upon SMCX loss in mouse EpiLCs
When pluripotent female epiblast cells differentiate, one of their two X-chromosomes is inactivated in a random manner. Despite much progress, the factors and mechanisms that cause random X-inactivation selectively in females remain obscure. We recently postulated that genes that escape X-inactivation function as dose-sensitive factors that induce X-inactivation selectively in females (Gayen et al., 2016). X-inactivation escapees are expressed from both X-chromosomes in females, including from the otherwise inactivated X-chromosome. As a result, the expression of X-inactivation escapees is higher in females compared to males.
We nominated the X-inactivation escapee Smcx / Kdm5c as a candidate inducer of Xist and X-linked gene silencing. SMCX demethylates histone H3 di- and tri- methylated at lysine 4 (H3K4me2 and H3K4me3), which are chromatin marks associated with active gene expression. Additionally, hypomethylation of H3K4 is associated with Xist induction and silencing of X-linked genes on the inactive-X. Finally, unlike many other escapees, Smcx escapes X-inactivation in both mouse and human, suggesting an evolutionarily conserved dose-dependent function.
In testing a role for SMCX in X-inactivation, we found that SMCX is indeed required for both Xist RNA induction and for the silencing of a subset or more of all X-linked genes (in the image above, Xist RNA coating is seen as a green 'cloud', the Xist antisense RNA is detected as a green pinpoint, and RNA from the X-linked gene Pgk1 is in red). When mouse embryonic stem cells (ESCs) are differentiated into epiblast-like cells (EpiLCs), the absence of SMCX results in the lack of Xist RNA induction. These and other results suggest that SMCX lies at or near the top of the molecular hierarchy that causes X-inactivation selectively in females and not in males (see model below and Gayen et al., 2017 for more information).
Ongoing and future work aims to dissect the evolutionarily conserved, dose-dependent function of the histone demethylase SMCX in inducing random X-inactivation in mammalian females via both in vitro and in vivo approaches. We also aim to inhibit SMCX as a means to reactivate silenced X-linked genes, which has therapeutic implications for X-linked gene disorders in females (e.g, Rett Syndrome).