Screen for reactivation of MeCP2 on the inactive X chromosome identifies the BMP/TGF-β superfamily as a regulator of XIST expression

Significance Rett syndrome is a neurodevelopmental disorder in girls who are heterozygous for a mutation in the X-linked gene MeCP2. Because cells in these individuals will be missing MeCP2 function only when the wild-type copy of the gene is on the inactive X, reactivation of the silenced copy of MeCP2 presents a potential therapeutic strategy. To identify genes that silence MeCP2 on the inactive X and that could therefore prove valuable as therapeutic targets, we carried out a screen for genes whose down-regulation reactivated a MeCP2 reporter on the inactive X. The 30 genes we have identified reveal a genetic circuitry required for maintenance of X-chromosome inactivation in differentiated cells and a large number of targets suitable for pharmacologic intervention. Rett syndrome (RS) is a debilitating neurological disorder affecting mostly girls with heterozygous mutations in the gene encoding the methyl-CpG–binding protein MeCP2 on the X chromosome. Because restoration of MeCP2 expression in a mouse model reverses neurologic deficits in adult animals, reactivation of the wild-type copy of MeCP2 on the inactive X chromosome (Xi) presents a therapeutic opportunity in RS. To identify genes involved in MeCP2 silencing, we screened a library of 60,000 shRNAs using a cell line with a MeCP2 reporter on the Xi and found 30 genes clustered in seven functional groups. More than half encoded proteins with known enzymatic activity, and six were members of the bone morphogenetic protein (BMP)/TGF-β pathway. shRNAs directed against each of these six genes down-regulated X-inactive specific transcript (XIST), a key player in X-chromosome inactivation that encodes an RNA that coats the silent X chromosome, and modulation of regulators of this pathway both in cell culture and in mice demonstrated robust regulation of XIST. Moreover, we show that Rnf12, an X-encoded ubiquitin ligase important for initiation of X-chromosome inactivation and XIST transcription in ES cells, also plays a role in maintenance of the inactive state through regulation of BMP/TGF-β signaling. Our results identify pharmacologically suitable targets for reactivation of MeCP2 on the Xi and a genetic circuitry that maintains XIST expression and X-chromosome inactivation in differentiated cells.