In mammals, the noncoding Xist RNA triggers transcriptional silencing of one of the two X chromosomes in female cells. Here, we report a genetic screen for silencing factors in X chromosome inactivation using haploid mouse embryonic stem cells (ESCs) that carry an engineered selectable reporter system. This system was able to identify several candidate factors that are genetically required for chromosomal repression by Xist. Among the list of candidates, we identify the RNA-binding protein Spen, the homolog of split ends. Independent validation through gene deletion in ESCs confirms that Spen is required for gene repression by Xist. However, Spen is not required for Xist RNA localization and the recruitment of chromatin modifications, including Polycomb protein Ezh2. The identification of Spen opens avenues for further investigation into the gene-silencing pathway of Xist and shows the usefulness of haploid ESCs for genetic screening of epigenetic pathways.

CEITEC Central European Institute of Technology Masaryk University Kamenice 753 5 Brno 62500 Czech Republic

Institute of Molecular Health Sciences Swiss Federal Institute of Technology ETH Hönggerberg Otto Stern Weg 7 8093 Zurich Switzerland

University of Bordeaux European Institute for Chemistry and Biology ARNA Laboratory Bordeaux 33000 France; Institut National de la Sante et de la Recherche Medicale INSERM U869 ARNA Laboratory Bordeaux 33000 France

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