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Genetic and Pharmacological Reactivation of the Mammalian Inactive X Chromosome

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Genetic and Pharmacological Reactivation of the Mammalian Inactive X Chromosome University of Massachusetts Medical School eScholarship@UMMS Program in Gene Function and Expression Publications and Presentations Molecular, Cell and Cancer Biology 2014-09-02 Genetic and pharmacological reactivation of the mammalian inactive X chromosome Sanchita Bhatnagar University of Massachusetts Medical School Et al. Let us know how access to this document benefits ou.y Follow this and additional works at: https://escholarship.umassmed.edu/pgfe_pp Part of the Bioinformatics Commons, Cell Biology Commons, Computational Biology Commons, and the Integrative Biology Commons Repository Citation Bhatnagar S, Zhu X, Ou J, Lin L, Chamberlain L, Zhu LJ, Wajapeyee N, Green MR. (2014). Genetic and pharmacological reactivation of the mammalian inactive X chromosome. Program in Gene Function and Expression Publications and Presentations. https://doi.org/10.1073/pnas.141362011110.1073/ pnas.1413620111. Retrieved from https://escholarship.umassmed.edu/pgfe_pp/265 This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in Program in Gene Function and Expression Publications and Presentations by an authorized administrator of eScholarship@UMMS. For more information, please contact [email protected]. Genetic and pharmacological reactivation of the INAUGURAL ARTICLE mammalian inactive X chromosome Sanchita Bhatnagara,b,c, Xiaochun Zhua,b,c, Jianhong Oub,c, Ling Lina,b,c, Lynn Chamberlaina,b,c, Lihua J. Zhub,c,d, Narendra Wajapeyeee, and Michael R. Greena,b,c,1 aHoward Hughes Medical Institute and Programs in bGene Function and Expression, cMolecular Medicine, and dBioinformatics and Integrative Biology, University of Massachusetts Medical School, Worcester, MA 01605; and eDepartment of Pathology, Yale University School of Medicine, New Haven, CT 06520 This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2014. Contributed by Michael R. Green, July 17, 2014 (sent for review June 25, 2014; reviewed by Gail Mandel and Ben Philpot) X-chromosome inactivation (XCI), the random transcriptional si- Here, we perform a large-scale RNA interference (RNAi) screen lencing of one X chromosome in somatic cells of female mammals, using a genome-wide collection of shRNAs to identify trans- is a mechanism that ensures equal expression of X-linked genes in acting factors that are required for mammalian XCI. both sexes. XCI is initiated in cis by the noncoding Xist RNA, which coats the inactive X chromosome (Xi) from which it is produced. Results However, trans-acting factors that mediate XCI remain largely un- Identification of Factors Required for Mammalian XCI. We used a known. Here, we perform a large-scale RNA interference screen to previously derived female mouse embryonic fibroblast cell line identify trans-acting XCI factors (XCIFs) that comprise regulators of [H4SV (12)] in which genes encoding green fluorescent protein cell signaling and transcription, including the DNA methyltransfer- (GFP) and hypoxanthine guanine phosphoribosyltransferase ase, DNMT1. The expression pattern of the XCIFs explains the se- (HPRT) are present only on the Xi. Knockdown (KD) of a factor lective onset of XCI following differentiation. The XCIFs function, at required for XCI is expected to reactivate expression of the Gfp least in part, by promoting expression and/or localization of Xist to and Hprt reporter genes (Fig. 1A). the Xi. Surprisingly, we find that DNMT1, which is generally a tran- A genome-wide mouse shRNA library comprising 62,400 scriptional repressor, is an activator of Xist transcription. Small-mol- shRNAs (13) was divided into 10 pools, which were packaged CELL BIOLOGY ecule inhibitors of two of the XCIFs can reversibly reactivate the Xi, into retrovirus particles and used to transduce H4SV cells. GFP- which has implications for treatment of Rett syndrome and other positive cells were selected by fluorescence-activated cell sorting dominant X-linked diseases. A homozygous mouse knockout of (FACS) and expanded, and the shRNAs were identified by se- one of the XCIFs, stanniocalcin 1 (STC1), has an expected XCI defect quence analysis. To validate the candidates, single shRNAs di- but surprisingly is phenotypically normal. Remarkably, X-linked Stc1−/− rected against each candidate gene were transduced into H4SV genes are not overexpressed in female mice, revealing cells, and the number of GFP-positive cells was measured by the existence of a mechanism(s) that can compensate for a persis- FACS analysis. The results of these experiments identified 13 tent XCI deficiency to regulate X-linked gene expression. candidate genes whose knockdown resulted in an increased percentage of GFP-positive cells relative to that obtained with MECP2 | RNA FISH | RNA-seq a control, nonsilencing (NS) shRNA (Fig. 1B). The cell viability -chromosome inactivation (XCI), the random transcriptional Significance Xsilencing of one X chromosome in somatic cells of female mammals, is a mechanism that ensures equal expression of X-linked genes in both sexes (1). XCI is initiated by X inactive In somatic cells of female mammals, one of the two X chro- mosomes is randomly silenced, a phenomenon called X-chro- specific transcript (Xist), a 17-kb noncoding RNA whose ex- mosome inactivation (XCI). XCI is initiated in cis by a noncoding pression during early embryogenesis is both necessary and suf- RNA called Xist, but trans-acting factors that mediate XCI re- ficient for silencing (2, 3). Xist represses transcription in cis by main largely unknown. In this study, we perform a large-scale coating only the X chromosome from which it is produced. Once RNA interference screen and identify new trans-acting factors Xist has been up-regulated during early development or differ- that are required for mammalian XCI. Chemical inhibitors of entiation, it continues to be expressed from the inactive X (Xi) some of these factors can reversibly reactivate the inactive X even in fully differentiated somatic cells. Before the initiation of chromosome. Our results have therapeutic implications for XCI, TSIX transcript, XIST antisense RNA (Tsix) an antisense certain human diseases, in particular the neurodevelopmental repressor of Xist,blocksXist up-regulation on the future active X disorder Rett syndrome, which is caused by loss-of-function chromosome (Xa) (4). mutations in the X-linked MECP2 gene. Reactivation of the si- An understanding of the factors and mechanisms involved in lenced wild-type MECP2 allele is a potential strategy for XCI is directly relevant to certain human diseases. In particular, treating the disease. loss-of-function mutations in the X-linked methyl-CpG binding protein 2 (MECP2) gene lead to the neurodevelopmental dis- Author contributions: S.B., N.W., and M.R.G. designed research; S.B., X.Z., L.L., L.C., and order Rett syndrome (RTT) (5–7). Most RTT patients are N.W. performed research; S.B., J.O., L.J.Z., and M.R.G. analyzed data; and S.B. and M.R.G. females who are heterozygous for MECP2 deficiency due to wrote the paper. random XCI. Significantly, in a mouse model of RTT, reac- Reviewers: G.M., Howard Hughes Medical Institute and Oregon Health and Science Uni- versity; and B.P., University of North Carolina at Chapel Hill. tivation of wild-type Mecp2 expression can reverse the disease The authors declare no conflict of interest. phenotype even in late-stage adult animals (8). Thus, reac- Data deposition: The RNA-Seq data reported in this paper have been deposited in the tivation of the silenced wild-type MECP2 allele is a potential Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. strategy for treating RTT. GSE47395). We have previously demonstrated how large-scale short hair- 1To whom correspondence should be addressed. Email: [email protected]. pin RNA (shRNA) screens can be used to identify factors in- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. volved in epigenetic silencing of tumor suppressor genes (9–11). 1073/pnas.1413620111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1413620111 PNAS | September 2, 2014 | vol. 111 | no. 35 | 12591–12598 Fig. 1. Identification of factors required for mam- malian XCI. (A) Schematic summary of the shRNA screen. The Xi is designated as such due to deletion of Xist on the Xa. (B) H4SV cells expressing an shRNA against 1 of the 13 candidates or, as a control, a nonsilencing (NS) shRNA were FACS sorted, and GFP-positive cells were isolated. For each KD cell line, the percentage of GFP-positive cells was ex- pressed as the fold increase relative to that obtained with the NS shRNA, which was set to 1. (C) Two-color RNA FISH monitoring expression of G6pdx (red) and Lamp2 (green; Left) and Pgk1 (red) and Mecp2 (green; Right) in each of the 13 XCIF KD BMSL2 cell lines. DAPI staining is shown in blue. The experiment was performed at least twice, and representative images are shown (Upper) and the results quantified (Lower) from one experiment. assay of Fig. S1A shows that knockdown of each candidate en- The XCIFs Are Required for Initiation of XCI in Mouse Embryonic Stem abled growth in hypoxanthine–aminopterin–thymidine (HAT) Cells. We next asked whether the XCIFs were required to initiate medium, indicating that the Xi-linked Hprt gene was reactivated. XCI in female mouse embryonic stem (ES) cells. Undifferentiated As expected, the mRNA levels of the 13 candidate genes were female mouse PGK12.1 ES cells were transduced with a retrovi- decreased in the corresponding KD H4SV cell line (Fig. S1B). rus expressing an XCIF shRNA. Cells were then treated with To rule out off-target effects, we showed for all 13 candidates retinoic acid (RA), which induces predominantly, but not exclu- that a second, unrelated shRNA also reactivated the Xi-linked sively, neuronal differentiation (17). X-linked gene expression Hprt gene (Fig. S1C) and decreased mRNA levels of the targeted was monitored by two-color RNA FISH.
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