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Screen for Reactivation of Mecp2 on the Inactive X Chromosome Identifies the BMP/TGF-Β Superfamily As a Regulator of XIST Expression

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Screen for Reactivation of Mecp2 on the Inactive X Chromosome Identifies the BMP/TGF-Β Superfamily As a Regulator of XIST Expression Screen for reactivation of MeCP2 on the inactive X chromosome identifies the BMP/TGF-β superfamily as a regulator of XIST expression Smitha Sripathya, Vid Lekoa,1, Robin L. Adriansea, Taylor Loea, Eric J. Fossa, Emily Dalrymplea, Uyen Laoa, Tonibelle Gatbonton-Schwagera, Kelly T. Cartera, Bernhard Payerb,c,d, Patrick J. Paddisone, William M. Gradya,f, Jeannie T. Leeb,c,d,2, Marisa S. Bartolomeig, and Antonio Bedalova,h,2 aClinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109; bHoward Hughes Medical Institute, Massachusetts General Hospital, Boston, MA 02114; cDepartment of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114; dDepartment of Genetics, Harvard Medical School, Boston, MA 12115; eHuman Biology Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109; fDivision of Gastroenterology, University of Washington School of Medicine, Seattle, WA 98195; gEpigenetics Program, Department of Cell and Developmental Biology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104; and hDepartment of Biochemistry and Medicine, University of Washington School of Medicine, Seattle, WA 98195 Contributed by Jeannie T. Lee, December 31, 2016 (sent for review November 13, 2016; reviewed by Michael R. Green and Ben Philphot) Rett syndrome (RS) is a debilitating neurological disorder affecting KIAA2022 (5), USP9X (6), CDKL5 (7), HDAC8 (8), and mostly girls with heterozygous mutations in the gene encoding SMC1A (9). Rett syndrome, one of the most common hereditary the methyl-CpG–binding protein MeCP2 on the X chromosome. forms of severe cognitive impairment in girls, is caused by a Because restoration of MeCP2 expression in a mouse model re- heterozygous mutation in the MeCP2 gene (3), which encodes a verses neurologic deficits in adult animals, reactivation of the methyl-DNA–binding protein essential for normal development wild-type copy of MeCP2 on the inactive X chromosome (Xi) pre- and function of the neurons (10). Because restoration of MeCP2 sents a therapeutic opportunity in RS. To identify genes involved expression in a mouse model for Rett syndrome reverses neurologic in MeCP2 silencing, we screened a library of 60,000 shRNAs using a deficits, both in young and adult animals (11), reactivation of the cell line with a MeCP2 reporter on the Xi and found 30 genes wild-type MeCP2 allele from the inactive X presents a potential GENETICS clustered in seven functional groups. More than half encoded pro- therapeutic strategy. teins with known enzymatic activity, and six were members of the Here we report results of a large-scale genetic screen for regu- bone morphogenetic protein (BMP)/TGF-β pathway. shRNAs di- lators of MeCP2 on the Xi. We generated a cell line harboring a rected against each of these six genes down-regulated X-inactive MeCP2-linked reporter on the Xi, using a transgenic mouse we specific transcript (XIST), a key player in X-chromosome inactiva- described previously (12), and exposed it to a library of 60,000 tion that encodes an RNA that coats the silent X chromosome, and different small hairpin RNAs (shRNAs). We identified 30 genes modulation of regulators of this pathway both in cell culture and whose down-regulation led to reactivation of the silent MeCP2 on in mice demonstrated robust regulation of XIST. Moreover, we the Xi. These genes fell into several functional groups, including show that Rnf12, an X-encoded ubiquitin ligase important for ini- mitotic kinases, PI3K/AKT signaling, chromatin components, sister tiation of X-chromosome inactivation and XIST transcription in ES chromatid cohesion, acyl-pool biosynthesis, and bone morphogenetic cells, also plays a role in maintenance of the inactive state through regulation of BMP/TGF-β signaling. Our results identify pharmaco- Significance logically suitable targets for reactivation of MeCP2 on the Xi and a genetic circuitry that maintains XIST expression and X-chromosome inactivation in differentiated cells. Rett syndrome is a neurodevelopmental disorder in girls who are heterozygous for a mutation in the X-linked gene MeCP2. XIST | X inactivation | MeCP2 | Rett syndrome | BMP/TGF-β Because cells in these individuals will be missing MeCP2 func- tion only when the wild-type copy of the gene is on the in- active X, reactivation of the silenced copy of MeCP2 presents a -chromosome inactivation (XCI) is a dosage-compensation potential therapeutic strategy. To identify genes that silence Xphenomenon in which one of the two X chromosomes in MeCP2 on the inactive X and that could therefore prove valu- female cells becomes transcriptionally silent. This process assures able as therapeutic targets, we carried out a screen for genes that the ratio of X to autosome expression in females mimics that in whose down-regulation reactivated a MeCP2 reporter on the males, who have only a single X chromosome. One of the two X inactive X. The 30 genes we have identified reveal a genetic chromosomes is inactivated during the random XCI stage after circuitry required for maintenance of X-chromosome inactiva- implantation, and that X chromosome remains stably inactivated tion in differentiated cells and a large number of targets suit- (1, 2). One of the initiating events in XCI is random expression of able for pharmacologic intervention. XIST (X-inactive specific transcript) from the X chromosome to be inactivated. XIST is a noncoding RNA that coats that chromosome Author contributions: S.S., V.L., and A.B. designed research; S.S., V.L., R.L.A., T.L., E.D., U.L., T.G.-S., in cis, recruits additional epigenetic modifiers, and renders it tran- and K.T.C. performed research; B.P., J.T.L., P.J.P., W.M.G., and M.S.B. contributed new reagents/ scriptionally inactive. As a result, in any female cell, only one of two analytic tools; S.S., E.J.F., M.S.B., and A.B. analyzed data; and S.S., E.J.F., and A.B. wrote the paper. alleles on the X chromosome is transcriptionally active. Reviewers: M.R.G., University of Massachusetts Medical School; and B.P., University of When an X-linked disease gene is heterozygous, it can be North Carolina at Chapel Hill. critically important whether the wild-type allele is on the active The authors declare no conflict of interest. or inactive X: When the mutant allele is on the active X, tran- Data deposition: The sequencing data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE90685). scriptional inactivation of the remaining wild-type allele can cause a 1Present address: Hematology Branch, National Heart, Lung and Blood Institute, National complete loss of gene function. However, the resulting disease state Institutes of Health, Bethesda, MD 20892. could potentially be ameliorated by reactivation of the wild- 2 To whom correspondence may be addressed. Email: [email protected] or type allele on the inactive X chromosome (Xi). Several neuro- [email protected]. developmental disorders in females are caused by heterozygous This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. mutations of X-linked genes including MeCP2 (3), DDX3X (4), 1073/pnas.1621356114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1621356114 PNAS Early Edition | 1of6 protein (BMP)/TGF-β signaling pathway. We found that the BMP and subjected it to high-throughput sequencing. The frequency signaling pathway is required for maintenance of XIST expression. of hairpins in the pre- and postselection specimens was com- Furthermore, we show that Rnf12, a known regulator of BMP pared with the calculated enrichment for each hairpin (Dataset signaling (13), modulates XIST expression in adult cells, suggesting S1). To reduce the false positive rate, we performed the screen in that the role of Rnf12 as a regulator of XIST during initiation of quadruplicate. The most enriched hairpins in all of the screens X-chromosome inactivation in ES cells (14, 15) is retained in dif- were four hairpins targeting XIST, with enrichment varying from ferentiated cells through control of BMP signaling. 2.4- to 220-fold in different screens (Table S1). Identification of the four hairpins targeting XIST as the most enriched hairpins Results validated our screening strategy for identifying genes involved in We previously described a transgenic mouse model for monitoring silencing of MeCP2 on the Xi. MeCP2 expression with a fusion of the MeCP2 gene with firefly We next tested the selected hairpins for down-regulation of luciferase and hygromycin resistance genes (MeCP2-FL-HR) (12). their intended target genes and activation of the luciferase re- Whereas the fusion protein proved unstable, resulting in a MeCP2 porter. We used several criteria for choosing enriched hairpins loss-of-function phenotype in mice, the reporter expression pattern for further analysis, including enrichment in at least two of the paralleled the expression of the native MeCP2 gene (12). To de- four screens, the magnitude of the enrichment, and the presence velop clonal cell lines that have the transgene on either the active of multiple hairpins targeting the same gene. In instances where (Xa) or inactive X chromosome, we immortalized tail tendon fi- a gene was targeted with a single hairpin, we separately tested broblasts from heterozygous MeCP2-FL-HR/MeCP2 female mice. additional hairpins targeting the same gene. This approach One cell line with the reporter gene on the Xi (Xi-8) and one with confirmed reactivation of the luciferase reporter for the 30 target the reporter gene on the Xa (Xa-3) were chosen for further studies. genes listed in Table 1. These could be classified in several Xi-8 cells had no detectable luciferase activity (Fig. 1A)but functional categories, including the TGF-β family, PI3K/AKT expressed the wild-type MeCP2 (Fig. 1B), whereas Xa-3 exhibited signaling pathway, chromatin-modifying enzymes and compo- the opposite properties.
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