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Topoisomerase inhibitors unsilence the dormant allele of Ube3a in neurons


Angelman syndrome is a severe neurodevelopmental disorder caused by deletion or mutation of the maternal allele of the ubiquitin protein ligase E3A (UBE3A)1,2,3. In neurons, the paternal allele of UBE3A is intact but epigenetically silenced4,5,6, raising the possibility that Angelman syndrome could be treated by activating this silenced allele to restore functional UBE3A protein7,8. Using an unbiased, high-content screen in primary cortical neurons from mice, we identify twelve topoisomerase I inhibitors and four topoisomerase II inhibitors that unsilence the paternal Ube3a allele. These drugs included topotecan, irinotecan, etoposide and dexrazoxane (ICRF-187). At nanomolar concentrations, topotecan upregulated catalytically active UBE3A in neurons from maternal Ube3a-null mice. Topotecan concomitantly downregulated expression of the Ube3a antisense transcript that overlaps the paternal copy of Ube3a9,10,11. These results indicate that topotecan unsilences Ube3a in cis by reducing transcription of an imprinted antisense RNA. When administered in vivo, topotecan unsilenced the paternal Ube3a allele in several regions of the nervous system, including neurons in the hippocampus, neocortex, striatum, cerebellum and spinal cord. Paternal expression of Ube3a remained elevated in a subset of spinal cord neurons for at least 12 weeks after cessation of topotecan treatment, indicating that transient topoisomerase inhibition can have enduring effects on gene expression. Although potential off-target effects remain to be investigated, our findings suggest a therapeutic strategy for reactivating the functional but dormant allele of Ube3a in patients with Angelman syndrome.

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Figure 1: A small-molecule screen identifies a topoisomerase inhibitor that unsilences the paternal allele of Ube3a in neurons.
Figure 2: Topotecan unsilences the paternal allele of Ube3a and the unsilenced protein is catalytically active.
Figure 3: Topotecan enduringly unsilences the paternal allele of Ube3a in vivo.


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We thank A. Beaudet and Y.-h. Jiang for providing Ube3a–YFP and Ube3am–/p+ mice; T. Riday and J. E. Han for assistance in i.c.v. mini-osmotic pump infusion; A. Burns for assistance in maintaining mouse colonies; V. Gukassyan for help with the Surveyor and confocal imaging systems; K. McNaughton for help with tissue sectioning; W. Zamboni for providing belotecan, rubitecan and silatecan; and W. Janzen and the Center of Integrative Chemical Biology and Drug Discovery for providing the epigenetic library. B.D.P., M.J.Z. and B.L.R. were supported by the Simons Foundation Autism Research Initiative (SFARI) and by the Angelman Syndrome Foundation. B.D.P. and M.J.Z. were supported by the National Institute of Mental Health (NIMH) (R01MH093372). B.D.P. was supported by the National Eye Institute (R01EY018323) and NC TraCS (50KR41016). M.J.Z. was supported by the National Institute of Neurological Disorders and Stroke (NINDS) (R01NS060725, R01NS067688). B.L.R. was supported by national Institutes of Health (NIH) HHSN-271-2008-00025-C, the NIMH Psychoactive Drug Screening Program, the Michael Hooker Distinguished Chair of Pharmacology, and grants from NIMH and the National Institute on Drug Abuse (NIDA). H.-S.H. was supported by a NARSAD grant from the Brain and Behavior Research Foundation Young Investigator Award and NC TraCS (10KR20910). J.A.A. was supported by NIH T32HD040127-07, the University of North Carolina-Carolina Institute for Developmental Disabilities, and an Autism Concept Award AR093464 from the US Department of Defense. A.M.M. was supported by a National Research Service Award from NINDS (5F32NS067712). I.F.K. was supported by a Joseph E. Wagstaff Postdoctoral Research Fellowship from the Angelman Syndrome Foundation. Assay development costs were partially supported by NINDS (5P30NS045892). Confocal and montage imaging was performed at the University of North Carolina at Chapel Hill Confocal and Multiphoton Imaging Facility, which is co-funded by grants from NINDS (5P30NS045892) and the National Institute of Child Health and Human Development (NICHD) (P30HD03110).

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H.-S.H., J.A.A., A.M.M., I.F.K., M.J.Z., B.L.R. and B.D.P. conceived and designed experiments, and wrote the manuscript. All authors reviewed and edited the manuscript. H.-S.H., J.A.A., J.M. and H.-M.L. performed drug screens and validations. H.-S.H., B.T.-B., J.M. and J.W.D. performed in vivo studies and immunofluorescence. H.-S.H. and A.S.B. performed pharmacokinetic analyses. A.M.M. performed tests of UBE3A functionality. I.F.K. performed quantitative PCR with reverse transcription and methylation analyses. N.S. oversaw high content screening instrumentation and implemented image processing algorithms. X.C. and J.J. synthesized the lactam E ring inactive camptothecin analogue and the three indenoisoquinoline derivatives. The laboratories of M.J.Z., B.L.R. and B.D.P. contributed equally to this work.

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Correspondence to Mark J. Zylka, Bryan L. Roth or Benjamin D. Philpot.

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Huang, HS., Allen, J., Mabb, A. et al. Topoisomerase inhibitors unsilence the dormant allele of Ube3a in neurons. Nature 481, 185–189 (2012).

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