title: Screen for reactivation of MeCP2 on the inactive X chromosome identifies the BMP/TGF-β superfamily as a regulator of XIST expression.
abstract: Rett syndrome (RS) is a debilitating neurological disorder affecting mostly girls with heterozygous mutations in the gene encoding the methyl-CpG-binding protein MeCP2 on the X chromosome. Because restoration of MeCP2 expression in a mouse model reverses neurologic deficits in adult animals, reactivation of the wild-type copy of MeCP2 on the inactive X chromosome (Xi) presents a therapeutic opportunity in RS. To identify genes involved in MeCP2 silencing, we screened a library of 60,000 shRNAs using a cell line with a MeCP2 reporter on the Xi and found 30 genes clustered in seven functional groups. More than half encoded proteins with known enzymatic activity, and six were members of the bone morphogenetic protein (BMP)/TGF-β pathway. shRNAs directed against each of these six genes down-regulated X-inactive specific transcript (XIST), a key player in X-chromosome inactivation that encodes an RNA that coats the silent X chromosome, and modulation of regulators of this pathway both in cell culture and in mice demonstrated robust regulation of XIST. Moreover, we show that Rnf12, an X-encoded ubiquitin ligase important for initiation of X-chromosome inactivation and XIST transcription in ES cells, also plays a role in maintenance of the inactive state through regulation of BMP/TGF-β signaling. Our results identify pharmacologically suitable targets for reactivation of MeCP2 on the Xi and a genetic circuitry that maintains XIST expression and X-chromosome inactivation in differentiated cells.
authors.affiliation: Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109.
authors.forename: Smitha
authors.initials: S
authors.lastname: Sripathy
authors.affiliation: Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109.
authors.forename: Vid
authors.initials: V
authors.lastname: Leko
authors.affiliation: Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109.
authors.forename: Robin L
authors.initials: RL
authors.lastname: Adrianse
authors.affiliation: Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109.
authors.forename: Taylor
authors.initials: T
authors.lastname: Loe
authors.affiliation: Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109.
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Foss
authors.affiliation: Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109.
authors.forename: Emily
authors.initials: E
authors.lastname: Dalrymple
authors.affiliation: Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109.
authors.forename: Uyen
authors.initials: U
authors.lastname: Lao
authors.affiliation: Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109.
authors.forename: Tonibelle
authors.initials: T
authors.lastname: Gatbonton-Schwager
authors.affiliation: Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109.
authors.forename: Kelly T
authors.initials: KT
authors.lastname: Carter
authors.affiliation: Howard Hughes Medical Institute, Massachusetts General Hospital, Boston, MA 02114.|Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114.|Department of Genetics, Harvard Medical School, Boston, MA 12115.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Payer
authors.affiliation: Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109.
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Paddison
authors.affiliation: Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109.|Division of Gastroenterology, University of Washington School of Medicine, Seattle, WA 98195.
authors.forename: William M
authors.initials: WM
authors.lastname: Grady
authors.affiliation: Howard Hughes Medical Institute, Massachusetts General Hospital, Boston, MA 02114; lee@molbio.mgh.harvard.edu abedalov@fhcrc.org.|Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114.|Department of Genetics, Harvard Medical School, Boston, MA 12115.
authors.forename: Jeannie T
authors.initials: JT
authors.lastname: Lee
authors.affiliation: Epigenetics Program, Department of Cell and Developmental Biology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104.
authors.forename: Marisa S
authors.initials: MS
authors.lastname: Bartolomei
authors.affiliation: Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109; lee@molbio.mgh.harvard.edu abedalov@fhcrc.org.|Department of Biochemistry and Medicine, University of Washington School of Medicine, Seattle, WA 98195.
authors.forename: Antonio
authors.initials: A
authors.lastname: Bedalov
mesh_terms: D000818:Animals; D055396:Bone Morphogenetic Protein 2 / Q000235:genetics*; D002460:Cell Line; D005260:Female; D020869:Gene Expression Profiling; D015723:Gene Library; D006801:Humans; D051783:Methyl-CpG-Binding Protein 2 / Q000235:genetics*; D051379:Mice; D062085:RNA, Long Noncoding / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics; D015518:Rett Syndrome / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics; D016212:Transforming Growth Factor beta / Q000235:genetics*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics; D049951:X Chromosome Inactivation
chemical_list: D055396:Bone Morphogenetic Protein 2; D051783:Methyl-CpG-Binding Protein 2; D062085:RNA, Long Noncoding; D034741:RNA, Small Interfering; D016212:Transforming Growth Factor beta; C077885:XIST non-coding RNA; C120758:Rlim protein, mouse; D044767:Ubiquitin-Protein Ligases
keywords: BMP/TGF-β; MeCP2; Rett syndrome; X inactivation; XIST