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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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.


  1. 1

    Kishino, T., Lalande, M. & Wagstaff, J. UBE3A/E6-AP mutations cause Angelman syndrome. Nature Genet. 15, 70–73 (1997)

    CAS  Article  Google Scholar 

  2. 2

    Matsuura, T. et al. De novo truncating mutations in E6-AP ubiquitin-protein ligase gene (UBE3A) in Angelman syndrome. Nature Genet. 15, 74–77 (1997)

    CAS  Article  Google Scholar 

  3. 3

    Sutcliffe, J. S. et al. The E6-Ap ubiquitin-protein ligase (UBE3A) gene is localized within a narrowed Angelman syndrome critical region. Genome Res. 7, 368–377 (1997)

    CAS  Article  Google Scholar 

  4. 4

    Albrecht, U. et al. Imprinted expression of the murine Angelman syndrome gene, Ube3a, in hippocampal and Purkinje neurons. Nature Genet. 17, 75–78 (1997)

    CAS  Article  Google Scholar 

  5. 5

    Rougeulle, C., Glatt, H. & Lalande, M. The Angelman syndrome candidate gene, UBE3A/E6-AP, is imprinted in brain. Nature Genet. 17, 14–15 (1997)

    CAS  Article  Google Scholar 

  6. 6

    Vu, T. H. & Hoffman, A. R. Imprinting of the Angelman syndrome gene, UBE3A, is restricted to brain. Nature Genet. 17, 12–13 (1997)

    CAS  Article  Google Scholar 

  7. 7

    Mabb, A. M., Judson, M. C., Zylka, M. J. & Philpot, B. D. Angelman syndrome: insights into genomic imprinting and neurodevelopmental phenotypes. Trends Neurosci. 34, 293–303 (2011)

    CAS  Article  Google Scholar 

  8. 8

    Peters, S. U. et al. Double-blind therapeutic trial in Angelman syndrome using betaine and folic acid. Am. J. Med. Genet. 152A, 1994–2001 (2010)

    CAS  Article  Google Scholar 

  9. 9

    Chamberlain, S. J. & Brannan, C. I. The Prader–Willi syndrome imprinting center activates the paternally expressed murine Ube3a antisense transcript but represses paternal Ube3a. Genomics 73, 316–322 (2001)

    CAS  Article  Google Scholar 

  10. 10

    Landers, M. et al. Regulation of the large (approximately 1000 kb) imprinted murine Ube3a antisense transcript by alternative exons upstream of Snurf/Snrpn. Nucleic Acids Res. 32, 3480–3492 (2004)

    CAS  Article  Google Scholar 

  11. 11

    Numata, K., Kohama, C., Abe, K. & Kiyosawa, H. Highly parallel SNP genotyping reveals high-resolution landscape of mono-allelic Ube3a expression associated with locus-wide antisense transcription. Nucleic Acids Res. 39, 2649–2657 (2011)

    CAS  Article  Google Scholar 

  12. 12

    Nakatani, J. et al. Abnormal behavior in a chromosome-engineered mouse model for human 15q11-13 duplication seen in autism. Cell 137, 1235–1246 (2009)

    Article  Google Scholar 

  13. 13

    Jiang, Y. H. et al. Mutation of the Angelman ubiquitin ligase in mice causes increased cytoplasmic p53 and deficits of contextual learning and long-term potentiation. Neuron 21, 799–811 (1998)

    CAS  Article  Google Scholar 

  14. 14

    Miura, K. et al. Neurobehavioral and electroencephalographic abnormalities in Ube3a maternal-deficient mice. Neurobiol. Dis. 9, 149–159 (2002)

    CAS  Article  Google Scholar 

  15. 15

    Dindot, S. V., Antalffy, B. A., Bhattacharjee, M. B. & Beaudet, A. L. The Angelman syndrome ubiquitin ligase localizes to the synapse and nucleus, and maternal deficiency results in abnormal dendritic spine morphology. Hum. Mol. Genet. 17, 111–118 (2008)

    CAS  Article  Google Scholar 

  16. 16

    Rougeulle, C., Cardoso, C., Fontes, M., Colleaux, L. & Lalande, M. An imprinted antisense RNA overlaps UBE3A and a second maternally expressed transcript. Nature Genet. 19, 15–16 (1998)

    CAS  Article  Google Scholar 

  17. 17

    Runte, M. et al. The IC-SNURFSNRPN transcript serves as a host for multiple small nucleolar RNA species and as an antisense RNA for UBE3A. Hum. Mol. Genet. 10, 2687–2700 (2001)

    CAS  Article  Google Scholar 

  18. 18

    Watanabe, Y. et al. Genome-wide analysis of expression modes and DNA methylation status at sense-antisense transcript loci in mouse. Genomics 96, 333–341 (2010)

    CAS  Article  Google Scholar 

  19. 19

    Pommier, Y. Topoisomerase I inhibitors: camptothecins and beyond. Nature Rev. Cancer 6, 789–802 (2006)

    CAS  Article  Google Scholar 

  20. 20

    Hertzberg, R. P. et al. Modification of the hydroxy lactone ring of camptothecin: inhibition of mammalian topoisomerase I and biological activity. J. Med. Chem. 32, 715–720 (1989)

    CAS  Article  Google Scholar 

  21. 21

    Plaschkes, I., Silverman, F. W. & Priel, E. DNA topoisomerase I in the mouse central nervous system: age and sex dependence. J. Comp. Neurol. 493, 357–369 (2005)

    CAS  Article  Google Scholar 

  22. 22

    Scheffner, M., Nuber, U. & Huibregtse, J. M. Protein ubiquitination involving an E1–E2–E3 enzyme ubiquitin thioester cascade. Nature 373, 81–83 (1995)

    ADS  CAS  Article  Google Scholar 

  23. 23

    Beaudenon, S., Dastur, A. & Huibregtse, J. M. Expression and assay of HECT domain ligases. Methods Enzymol. 398, 112–125 (2005)

    CAS  Article  Google Scholar 

  24. 24

    Kumar, S., Kao, W. H. & Howley, P. M. Physical interaction between specific E2 and Hect E3 enzymes determines functional cooperativity. J. Biol. Chem. 272, 13548–13554 (1997)

    CAS  Article  Google Scholar 

  25. 25

    Bressler, J. et al. The SNRPN promoter is not required for genomic imprinting of the Prader-Willi/Angelman domain in mice. Nature Genet. 28, 232–240 (2001)

    CAS  Article  Google Scholar 

  26. 26

    Reik, W. Stability and flexibility of epigenetic gene regulation in mammalian development. Nature 447, 425–432 (2007)

    ADS  CAS  Article  Google Scholar 

  27. 27

    Gammon, D. C. et al. Intrathecal topotecan in adult patients with neoplastic meningitis. Am. J. Health Syst. Pharm. 63, 2083–2086 (2006)

    CAS  Article  Google Scholar 

  28. 28

    Greer, P. L. et al. The Angelman syndrome protein Ube3A regulates synapse development by ubiquitinating arc. Cell 140, 704–716 (2010)

    CAS  Article  Google Scholar 

  29. 29

    Lyu, Y. L. et al. Role of topoisomerase IIβ in the expression of developmentally regulated genes. Mol. Cell. Biol. 26, 7929–7941 (2006)

    CAS  Article  Google Scholar 

  30. 30

    Bomgaars, L., Berg, S. L. & Blaney, S. M. The development of camptothecin analogs in childhood cancers. Oncologist 6, 506–516 (2001)

    CAS  Article  Google Scholar 

  31. 31

    Cushman, M. et al. Synthesis of new indeno[1,2-c]isoquinolines: cytotoxic non-camptothecin topoisomerase I inhibitors. J. Med. Chem. 43, 3688–3698 (2000)

    CAS  Article  Google Scholar 

  32. 32

    Nagarajan, M. et al. Synthesis and evaluation of indenoisoquinoline topoisomerase I inhibitors substituted with nitrogen heterocycles. J. Med. Chem. 49, 6283–6289 (2006)

    CAS  Article  Google Scholar 

  33. 33

    Abramoff, M. D., Magelhaes, P. J. & Ram, S. J. Image processing with ImageJ. Biophotonics International 11, 36–42 (2004)

    Google Scholar 

  34. 34

    Lamprecht, M. R., Sabatini, D. M. & Carpenter, A. E. CellProfiler: free, versatile software for automated biological image analysis. Biotechniques 42, 71–75 (2007)

    CAS  Article  Google Scholar 

  35. 35

    Tsai, T. F., Jiang, Y. H., Bressler, J., Armstrong, D. & Beaudet, A. L. Paternal deletion from Snrpn to Ube3a in the mouse causes hypotonia, growth retardation and partial lethality and provides evidence for a gene contributing to Prader-Willi syndrome. Hum. Mol. Genet. 8, 1357–1364 (1999)

    CAS  Article  Google Scholar 

  36. 36

    Peery, E. G., Elmore, M. D., Resnick, J. L., Brannan, C. I. & Johnstone, K. A. A targeted deletion upstream of Snrpn does not result in an imprinting defect. Mamm. Genome 18, 255–262 (2007)

    CAS  Article  Google Scholar 

  37. 37

    Fairbanks, C. A. Spinal delivery of analgesics in experimental models of pain and analgesia. Adv. Drug Deliv. Rev. 55, 1007–1041 (2003)

    CAS  Article  Google Scholar 

  38. 38

    Pierce, A. A. & Xu, A. W. De novo neurogenesis in adult hypothalamus as a compensatory mechanism to regulate energy balance. J. Neurosci. 30, 723–730 (2010)

    CAS  Article  Google Scholar 

Download references


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).

Author information




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.

Corresponding authors

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

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

The file contains Supplementary Figures 1-17 with legends and Supplementary Table 1. (PDF 4895 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.