Genome-Wide RNAi Screen Identify Melanoma-Associated Antigen Mageb3 Involved in X Chromosome Inactivation

Abstract

Xist (inactivated X chromosome specific transcript) is a prototype long noncoding RNA in charge of epigenetic silencing of one X chromosome in each female cell in mammals. In a genetic screen, we identify Mageb3 and its homologs Mageb1 and Mageb2 as genes functionally required for Xist-mediated gene silencing. Mageb1-3 are previously uncharacterized genes belonging to the MAGE (melanoma-associated antigen) gene family. Mageb1-3 are expressed in undifferentiated ES cells and early stages of in vitro differentiation, a critical time window of X chromosome inactivation. Mageb3 showed both cytoplasmic and nuclear localization without enrichment on the inactive X (Xi). Mageb3 interacted with Polycomb group ring finger 3 (Pcgf3), a RING finger protein involved in recruiting Polycomb activities onto Xi. Mageb3 overexpression stabilized Pcgf3 protein. Mageb1-3 gene knockout affected H3K27me3 enrichment and the spreading of gene silencing along Xi. These data suggested that Mageb3 might regulate the recruitment of the Polycomb complex onto Xi and subsequent H3K27me3 modification through Pcgf3. Moreover, the nucleolar enrichment of Mageb3 was diminished when nuclear matrix factor hnRNP U is overexpressed, implying the interaction between Mageb3 and nuclear matrix, which is another possible mechanism for Mageb3 to regulate X chromosome inactivation.

Xmas ESC: A new female embryonic stem cell system that reveals the BAF complex as a key regulator of the establishment of X chromosome inactivation

“…We chose to use this system to screen for genes that regulate the establishment of XCI during normal female ESC differentiation. Due to the major issues maintaining XX ESCs, all previous screens for XCI regulators have been performed either in differentiated cells for factors that alter maintenance of XCI (Bhatnagar et al, 2014;Chan et al, 2011;Keniry et al, 2016;Lessing et al, 2016;Li et al, 2018;Minajigi et al, 2015;Minkovsky et al, 2015;Minkovsky et al, 2014;Sripathy et al, 2017), or using non-native (though cunning) systems that instead induce Xist out of context, where Xist is often in a different chromosomal location in male cells, or in female cells but not induced during the exit from pluripotency (Chu et al, 2015;McHugh et al, 2015;Moindrot et al, 2015;Monfort et al, 2015). Our Xmas ESC system has enabled us to overcome the challenges of working with female ESCs and perform the first screen for regulators of the establishment of XCI in its near-native context.…”