title: DICER/AGO-dependent epigenetic silencing of D4Z4 repeats enhanced by exogenous siRNA suggests mechanisms and therapies for FSHD.
abstract: Facioscapulohumeral muscular dystrophy (FSHD) is caused by the aberrant expression of the DUX4 transcription factor in skeletal muscle. The DUX4 retrogene is encoded in the D4Z4 macrosatellite repeat array, and smaller array size or a mutation in the SMCHD1 gene results in inefficient epigenetic repression of DUX4 in skeletal muscle, causing FSHD1 and FSHD2, respectively. Previously we showed that the entire D4Z4 repeat is bi-directionally transcribed with the generation of small si- or miRNA-like fragments and suggested that these might suppress DUX4 expression through the endogenous RNAi pathway. Here we show that exogenous siRNA targeting the region upstream of the DUX4 transcription start site suppressed DUX4 mRNA expression and increased both H3K9 methylation and AGO2 recruitment. In contrast, similarly targeted MOE-gapmer antisense oligonucleotides that degrade RNA but do not engage the RNAi pathway did not repress DUX4 expression. In addition, knockdown of DICER or AGO2 using either siRNA or MOE-gapmer chemistries resulted in the induction of DUX4 expression in control muscle cells that normally do not express DUX4, indicating that the endogenous RNAi pathway is necessary to maintain repression of DUX4 in control muscle cells. Together these data demonstrate a role of the endogenous RNAi pathway in repeat-mediated epigenetic repression of the D4Z4 macrosatellite repeat, and show that enhancing the activity of this pathway by supplying exogenous siRNA oligonucleotides represents a potential therapeutic approach to silencing DUX4 in FSHD.
authors.affiliation: Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.
authors.forename: Jong-Won
authors.initials: JW
authors.lastname: Lim
authors.affiliation: Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.
authors.forename: Lauren
authors.initials: L
authors.lastname: Snider
authors.affiliation: Allen Brain Institute, Seattle, WA 98103, USA.
authors.forename: Zizhen
authors.initials: Z
authors.lastname: Yao
authors.affiliation: Department of Neurology, University of Rochester, Rochester, NY 14642, USA.
authors.forename: Rabi
authors.initials: R
authors.lastname: Tawil
authors.affiliation: Department of Human Genetics, Leiden University Medical Center, Leiden 2333 ZA, The Netherlands and.
authors.forename: Silvère M
authors.initials: SM
authors.lastname: Van Der Maarel
authors.affiliation: Isis Pharmaceuticals, Carlsbad, CA 92010, USA.
authors.forename: Frank
authors.initials: F
authors.lastname: Rigo
authors.affiliation: Isis Pharmaceuticals, Carlsbad, CA 92010, USA.
authors.forename: C Frank
authors.initials: CF
authors.lastname: Bennett
authors.affiliation: Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA, gfilippo@fhcrc.org stapscot@fhcrc.org.
authors.forename: Galina N
authors.initials: GN
authors.lastname: Filippova
authors.affiliation: Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA, gfilippo@fhcrc.org stapscot@fhcrc.org.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Tapscott
mesh_terms: D060565:Argonaute Proteins / Q000378:metabolism*; D002460:Cell Line; D002843:Chromatin / Q000235:genetics / Q000378:metabolism; D044127:Epigenesis, Genetic; D005786:Gene Expression Regulation; D020868:Gene Silencing; D006657:Histones / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D018895:Microsatellite Repeats; D020391:Muscular Dystrophy, Facioscapulohumeral / Q000235:genetics* / Q000378:metabolism* / Q000628:therapy; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D043244:Ribonuclease III / Q000378:metabolism*; D024363:Transcription Initiation Site
chemical_list: D060565:Argonaute Proteins; D002843:Chromatin; C121257:DUX4L1 protein, human; D006657:Histones; D018398:Homeodomain Proteins; D034741:RNA, Small Interfering; D043244:Ribonuclease III