title: R-loop formation at Snord116 mediates topotecan inhibition of Ube3a-antisense and allele-specific chromatin decondensation.
abstract: Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are oppositely imprinted autism-spectrum disorders with known genetic bases, but complex epigenetic mechanisms underlie their pathogenesis. The PWS/AS locus on 15q11-q13 is regulated by an imprinting control region that is maternally methylated and silenced. The PWS imprinting control region is the promoter for a one megabase paternal transcript encoding the ubiquitous protein-coding Snrpn gene and multiple neuron-specific noncoding RNAs, including the PWS-related Snord116 repetitive locus of small nucleolar RNAs and host genes, and the antisense transcript to AS-causing ubiquitin ligase encoding Ube3a (Ube3a-ATS). Neuron-specific transcriptional progression through Ube3a-ATS correlates with paternal Ube3a silencing and chromatin decondensation. Interestingly, topoisomerase inhibitors, including topotecan, were recently identified in an unbiased drug screen for compounds that could reverse the silent paternal allele of Ube3a in neurons, but the mechanism of topotecan action on the PWS/AS locus is unknown. Here, we demonstrate that topotecan treatment stabilizes the formation of RNA:DNA hybrids (R loops) at G-skewed repeat elements within paternal Snord116, corresponding to increased chromatin decondensation and inhibition of Ube3a-ATS expression. Neural precursor cells from paternal Snord116 deletion mice exhibit increased Ube3a-ATS levels in differentiated neurons and show a reduced effect of topotecan compared with wild-type neurons. These results demonstrate that the AS candidate drug topotecan acts predominantly through stabilizing R loops and chromatin decondensation at the paternally expressed PWS Snord116 locus. Our study holds promise for targeted therapies to the Snord116 locus for both AS and PWS.
authors.affiliation: Medical Microbiology and Immunology, Genome Center, Medical Investigation of Neurodevelopmental Disorders Institute, and Department of Molecular and Cellular Biology, University of California, Davis, CA 95616, USA.
authors.forename: Weston T
authors.initials: WT
authors.lastname: Powell
authors.forename: Rochelle L
authors.initials: RL
authors.lastname: Coulson
authors.forename: Michael L
authors.initials: ML
authors.lastname: Gonzales
authors.forename: Florence K
authors.initials: FK
authors.lastname: Crary
authors.forename: Spencer S
authors.initials: SS
authors.lastname: Wong
authors.forename: Sarrita
authors.initials: S
authors.lastname: Adams
authors.forename: Robert A
authors.initials: RA
authors.lastname: Ach
authors.forename: Peter
authors.initials: P
authors.lastname: Tsang
authors.forename: Nazumi Alice
authors.initials: NA
authors.lastname: Yamada
authors.forename: Dag H
authors.initials: DH
authors.lastname: Yasui
authors.forename: Frédéric
authors.initials: F
authors.lastname: Chédin
authors.forename: Janine M
authors.initials: JM
authors.lastname: LaSalle
mesh_terms: D017204:Angelman Syndrome / Q000235:genetics*; D000818:Animals; D002843:Chromatin / Q000187:drug effects; D047369:Chromatin Immunoprecipitation; D002884:Chromosomes, Human, Pair 15 / Q000235:genetics*; D005786:Gene Expression Regulation / Q000235:genetics*; D020868:Gene Silencing; D056426:Genetic Loci / Q000235:genetics; D018392:Genomic Imprinting / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D015151:Immunoblotting; D017404:In Situ Hybridization, Fluorescence; D019901:Locus Control Region / Q000235:genetics; D051379:Mice; D018345:Mice, Knockout; D009474:Neurons / Q000378:metabolism; D011218:Prader-Willi Syndrome / Q000235:genetics*; D016372:RNA, Antisense / Q000235:genetics / Q000378:metabolism; D020537:RNA, Small Nucleolar / Q000737:chemistry* / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D018709:Statistics, Nonparametric; D019772:Topotecan / Q000494:pharmacology*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics; D055517:snRNP Core Proteins / Q000235:genetics
chemical_list: D002843:Chromatin; D016372:RNA, Antisense; D020537:RNA, Small Nucleolar; C526417:SNRPN protein, human; D055517:snRNP Core Proteins; D019772:Topotecan; C471639:UBE3A protein, human; D044767:Ubiquitin-Protein Ligases
keywords: HBII85; MBII85; brain; neurodevelopment; snoRNA
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