R-loop formation at Snord116 mediates topotecan inhibition of Ube3a-antisense and allele-specific chromatin decondensation

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.

[1]  P. Tsang,et al.  A Prader–Willi locus lncRNA cloud modulates diurnal genes and energy expenditure , 2013, Human molecular genetics.

[2]  J. Manley,et al.  Inactivation of the SR Protein Splicing Factor ASF/SF2 Results in Genomic Instability , 2005, Cell.

[3]  Ping Fang,et al.  De novo truncating mutations in E6-AP ubiquitin-protein ligase gene (UBE3A) in Angelman syndrome , 1997, Nature Genetics.

[4]  R. Ratliff,et al.  Evidence from CD spectra that d(purine).r(pyrimidine) and r(purine).d(pyrimidine) hybrids are in different structural classes. , 1994, Nucleic acids research.

[5]  P. Hieter,et al.  R-loop-mediated genome instability in mRNA cleavage and polyadenylation mutants. , 2012, Genes & development.

[6]  C. Brannan,et al.  Regulation of the large (∼1000 kb) imprinted murine Ube3a antisense transcript by alternative exons upstream of Snurf/Snrpn , 2004 .

[7]  M. Lalande,et al.  UBE3A/E6-AP mutations cause Angelman syndrome , 1996, Nature Genetics.

[8]  A. Beaudet.  Angelman syndrome: Drugs to awaken a paternal gene , 2012, Nature.

[9]  Andrés Aguilera,et al.  R loops: from transcription byproducts to threats to genome stability. , 2012, Molecular cell.

[10]  P. Hanawalt,et al.  Anchoring nascent RNA to the DNA template could interfere with transcription. , 2011, Biophysical journal.

[11]  P. Hanawalt,et al.  Mechanisms and implications of transcription blockage by guanine-rich DNA sequences , 2010, Proceedings of the National Academy of Sciences.

[12]  J. Brosius,et al.  Deletion of the MBII-85 snoRNA Gene Cluster in Mice Results in Postnatal Growth Retardation , 2007, PLoS genetics.

[13]  C. Brannan,et al.  Regulation of the large (approximately 1000 kb) imprinted murine Ube3a antisense transcript by alternative exons upstream of Snurf/Snrpn. , 2004, Nucleic acids research.

[14]  J. Cavaille,et al.  Long nuclear-retained non-coding RNAs and allele-specific higher-order chromatin organization at imprinted snoRNA gene arrays , 2010, Journal of Cell Science.

[15]  Lance Stewart,et al.  The mechanism of topoisomerase I poisoning by a camptothecin analog , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[16]  D. Tollervey,et al.  Loss of Topoisomerase I leads to R-loop-mediated transcriptional blocks during ribosomal RNA synthesis. , 2010, Genes & development.

[17]  F. Pauler,et al.  Airn Transcriptional Overlap, But Not Its lncRNA Products, Induces Imprinted Igf2r Silencing , 2012, Science.

[18]  Marwan Shinawi,et al.  Prader-Willi phenotype caused by paternal deficiency for the HBII-85 C/D box small nucleolar RNA cluster , 2008, Nature Genetics.

[19]  Yves Pommier,et al.  Topoisomerase I suppresses genomic instability by preventing interference between replication and transcription , 2010, Nature Cell Biology.

[20]  F. Watrin,et al.  Dynamic developmental regulation of the large non-coding RNA associated with the mouse 7C imprinted chromosomal region. , 2005, Developmental biology.

[21]  Konstantina Skourti-Stathaki,et al.  Human Senataxin Resolves RNA/DNA Hybrids Formed at Transcriptional Pause Sites to Promote Xrn2-Dependent Termination , 2011, Molecular cell.

[22]  J. Rosenfeld,et al.  Paternally inherited microdeletion at 15q11.2 confirms a significant role for the SNORD116 C/D box snoRNA cluster in Prader–Willi syndrome , 2010, European Journal of Human Genetics.

[23]  David Tollervey,et al.  Distinguishing the Roles of Topoisomerases I and II in Relief of Transcription-Induced Torsional Stress in Yeast rRNA Genes , 2010, Molecular and Cellular Biology.

[24]  U. Francke,et al.  SnoRNA Snord116 (Pwcr1/MBII-85) Deletion Causes Growth Deficiency and Hyperphagia in Mice , 2008, PloS one.

[25]  Michael L. Gonzales,et al.  Phosphorylation of Distinct Sites in MeCP2 Modifies Cofactor Associations and the Dynamics of Transcriptional Regulation , 2012, Molecular and Cellular Biology.

[26]  J. Griffith,et al.  The presence of RNA in a double helix inhibits its interaction with histone protein. , 1980, Nucleic acids research.

[27]  L. Pevny,et al.  Isolation, culture, and differentiation of progenitor cells from the central nervous system. , 2008, CSH protocols.

[28]  A. Hüttenhofer,et al.  The IC-SNURF-SNRPN transcript serves as a host for multiple small nucleolar RNA species and as an antisense RNA for UBE3A. , 2001, Human molecular genetics.

[29]  J. LaSalle,et al.  Imprinting regulates mammalian snoRNA-encoding chromatin decondensation and neuronal nucleolar size , 2009, Human molecular genetics.

[30]  C. Moore,et al.  Unravelling the means to an end: RNA polymerase II transcription termination , 2011, Nature Reviews Molecular Cell Biology.

[31]  I. Korf,et al.  R-loop formation is a distinctive characteristic of unmethylated human CpG island promoters. , 2012, Molecular cell.

[32]  Jian Jin,et al.  Topoisomerase inhibitors unsilence the dormant allele of Ube3a in neurons , 2011, Nature.

[33]  D. J. Driscoll,et al.  Prader-Willi syndrome. , 1984, Current problems in pediatrics.

[34]  B. Philpot,et al.  Angelman syndrome: insights into genomic imprinting and neurodevelopmental phenotypes , 2011, Trends in Neurosciences.

[35]  S. O’Rahilly,et al.  A deletion of the HBII-85 class of small nucleolar RNAs (snoRNAs) is associated with hyperphagia, obesity and hypogonadism. , 2009, Human molecular genetics.

[36]  J. Wang,et al.  Supercoiling of the DNA template during transcription. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[37]  A. Beaudet,et al.  Ube3a-ATS is an atypical RNA polymerase II transcript that represses the paternal expression of Ube3a. , 2012, Human molecular genetics.

[38]  S. Chamberlain,et al.  Angelman Syndrome, a Genomic Imprinting Disorder of the Brain , 2010, The Journal of Neuroscience.