Genome‑Wide Rnai Screen Identify Melanoma‑Associated Antigen Mageb3 Involved in X Chromosome Inactivation

This document is downloaded from DR‑NTU (https://dr.ntu.edu.sg) Nanyang Technological University, Singapore. Genome‑wide RNAi screen identify melanoma‑associated antigen Mageb3 involved in X chromosome inactivation Li, Wei; Hong, Ru; Lai, Lan‑Tian; Dong, Qiman; Ni, Peiling; Chelliah, Rosi; Huq, Mehnaz; Siti Nadirah Ismail; Chandola, Udita; Ang, Zhiwei; Lin, Bingqing; Chen, Xin; Chen, Lingyi; Zhang, Li‑Feng 2018 Li, W., Hong, R., Lai, L.‑T., Dong, Q., Ni, P., Chelliah, R., . Zhang, L.‑F. (2018). Genome‑wide RNAi screen identify melanoma‑associated antigen Mageb3 involved in X chromosome inactivation. Journal of Molecular Biology, 430(17), 2734‑2746. doi:10.1016/j.jmb.2018.05.031 https://hdl.handle.net/10356/139663 https://doi.org/10.1016/j.jmb.2018.05.031 © 2018 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY‑NC‑ND license (http://creativecommons.org/licenses/by‑nc‑nd/4.0/). Downloaded on 01 Oct 2021 00:29:58 SGT Article Genome-Wide RNAi Screen Identify Melanoma-Associated Antigen Mageb3 Involved in X Chromosome Inactivation Wei Li 1,†, Ru Hong 1,†, Lan-Tian Lai 1, Qiman Dong 3, Peiling Ni 3, Rosi Chelliah 1, Mehnaz Huq 1, Siti Nadirah Binte Ismail 1, Udita Chandola 1, Zhiwei Ang 1, Bingqing Lin 1, Xin Chen 2, Lingyi Chen 3 and Li-Feng Zhang 1 1 - School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 2 - State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Bioactive Materials, Ministry of Education, Tianjin Key Laboratory of Protein Sciences and College of Life Sciences, Nankai University, Tianjin 300071, China 3 - Division of Mathematical Sciences, School of Physical and Mathematical Sciences, Nanyang Technological University, 21 Nanyang Link, Singapore 637371 Correspondence to Lingyi Chen and Li-Feng Zhang: [email protected]; [email protected] https://doi.org/10.1016/j.jmb.2018.05.031 Edited by M Yaniv 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. © 2018 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Introduction However, acting as a recruiter, Xist must work together with other factors to achieve XCI. Currently, a few Xist (inactivated X chromosome specific transcript) epigenetic modifications along the inactive X chromo- RNA is a well-known long noncoding RNA (lncRNA) some (Xi) are known to be associated with Xist, involved in X chromosome inactivation (XCI), a dosage including histone modification such as H3K27me3 compensation mechanism of mammals, whereby one and histone variant such as macroH2A. However, X chromosome in each female cell is epigenetically genetic studies show that none of them are essential for silenced to balance the X-linked gene dosage between XCI [2].WhenXist is conditionally deleted from somatic males and females [1]. At the onset of XCI (~E5.5 cells in which XCI has been fully established, all the mouse embryos), the X-linked Xist gene becomes known Xist-associated epigenetic modifications are allele specifically expressed from one X and the RNA removed together with Xist, but XCI remains largely transcripts coat chromosome territory in cis recruiting intact [2]. These results indicate the existence of other epigenetic factors to establish the chromosome- unknown epigenetic factors or mechanisms, which wide gene silencing [1]. Xist expression triggers XCI. are recruited or established by Xist RNA during early 0022-2836/© 2018 The Author. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). J Mol Biol (2018) 430, 2734–2746 Mageb3 is involved in X chromosome inactivation 2735 embryonic development and functionally essential for common backbone sequence of the lentiviral vector to XCI. Intensive effort of isolating Xist-binding proteins amplify the shRNA sequences. Each PCR product was identified Spen [3–5], which brings histone deacetylase sequenced independently. Thirteen candidate genes activity onto the Xi. Experimental evidence shows Spen werecommonlyfoundinall6PCRsamples(Fig. 1f) is required in XCI during in vitro differentiation of including tumor suppressor P53, which is consistent embryonic stem cells (ES cells) [4–7]. However, Spen with the design of the genetic screen. One hundred is not specifically expressed in early embryonic tissues thirty-four shRNAs can be commonly found in all three [8]. The essential role of Spen in XCI is not fully rounds of genetic screen (Supplementary Table 1). supported by the mutant phenotype of Spen knockout Unfortunately, we did not identify any known XCI factor mouse embryos [8]. in the screening results, which may relate with the quality of individual shRNAs in the library or the sensitivity of cells to the dosage change of an individual Results gene (shRNAs are not able to completely knockout a gene). From these data and three rounds of pilot screen To search for novel genes involved in XCI, we carried (data not shown), we selected, validated using the cell out a genetic screen using a genome-wide short hairpin survival assay and ruled out 52 individual shRNAs RNA (shRNA) library. A male ES cell line with an (Supplementary Table 2). Furthermore, we validated inducible Xist transgene (iXist) was established for the using the cell survival assay and ruled out 6 candidate genetic screen. iXist was established from Ainv15 cells, genes using the CRISPR/Cas9 approach (Supplemen- a male mouse ES cell line carrying an engineered tary Table 3, Supplementary Fig. 2). From the 13 cassette upstream of the X-linked hypoxanthine candidate genes, which were commonly found in all 6 guanine phosphoribosyl transferase (Hprt) gene PCR samples (Fig. 1f), Mageb3 was successfully (Fig. 1a). Through Cre-mediated gene targeting, a validated as a gene functionally important for XCI full-length Xist transgene including its polyA signal was (Figs. 1fand2). inserted downstream of the tetracycline response To validate Mageb3, we knocked out the gene in iXist element of Ainv15 cells, restoring the neomycin (iXist-KO1) using CRISPR/Cas9 with a nucleotide oligo resistance (Fig. 1a). Moreover, a red fluorescent protein as the repair template. The mutated Mageb3 allele (tdTomato) was included as a reporter gene (Fig. 1a). contains a premature stop codon 3 bp downstream of Both neomycin resistance and tdTmomato could be the gene's start codon (Fig. 2a). A HindIII restriction site used as reporters to indicate status of XCI. The cell was created for the genotyping purpose (Fig. 2a). line's genotype was confirmed by polymerase chain Moreover, a reading-frame shift was left in the rest of reaction (PCR; Supplementary Fig. 1a) and DNA thecodingregion(Fig. 2a). The Mageb3 (−/−) fluorescence in situ hybridization (FISH) (Fig. 1b). Its genotype of iXist-KO1 cells was confirmed by HindIII karyotype was confirmed as 40XY (Supplementary digestion and DNA sequencing (Supplementary Fig. 1c). Induced Xist RNA transcripts coated (Fig. 1c) Fig. 3). Mageb3 is highly homologous with two other thesingleXchromosomeinthe male cells and silenced genes (Mageb1 and Mageb2) in the gene family its genes (Supplementary Fig. 1b) causing cell growth (Supplementary Fig. 5). In the mouse genome, retardation and cell death (Fig. 1d). Although induced Mageb1 and Mageb2 are X-linked genes encoding Xist expression resulted in XCI in both undifferentiated identical proteins [9] (Supplementary Fig. 5) and and differentiating cells, the killing effect of induced XCI Mageb3 is located on chromosome 2 (Fig. 2b). The is significantly stronger in differentiating cells in the sequence of the shRNA clone (TRCN0000112714) presence of geneticin (G418) (Supplementary Fig. 1b). cannot distinguish the three genes, although the target We established a protocol (Fig. 1e) whereby more than oftheshRNAcloneislistedasMageb3 in the shRNA 99% of the iXist cells were killed by induced XCI in the library design. In this study, for genotyping PCR and presence of G418 (Fig. 1d) and applied this protocol to allele-specific reverse transcription PCR (RT-PCR), three independent rounds of genetic screen using a allele-specific primers could only be designed to pooled lentiviral shRNA library. The genomic DNA distinguish Mageb3 from Mageb1 and Mageb2. We isolated from the surviving cells of each round of screen coin the term Mageb1–3 referring to all three genes and was subjected to PCR reactions in duplicate using the the term “Mageb1&2” to discuss the experimental Fig. 1. The genetic screen. (a) iXist, the transgenic male mouse ES cell line engineered for the genetic screen. (b) DNA FISH on a metaphase chromosome spread of an iXist cell. DNA was stained by DAPI (blue). Chromosome X was detected by a Cy3-labeled chromosome paint (red).

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