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Targeting the histone methyltransferase G9a activates imprinted genes and improves survival of a mouse model of Prader–Willi syndrome

Nature Medicine volume 23, pages 213222 (2017) | Download Citation

Abstract

Prader–Willi syndrome (PWS) is an imprinting disorder caused by a deficiency of paternally expressed gene(s) in the 15q11–q13 chromosomal region. The regulation of imprinted gene expression in this region is coordinated by an imprinting center (PWS-IC). In individuals with PWS, genes responsible for PWS on the maternal chromosome are present, but repressed epigenetically, which provides an opportunity for the use of epigenetic therapy to restore expression from the maternal copies of PWS-associated genes. Through a high-content screen (HCS) of >9,000 small molecules, we discovered that UNC0638 and UNC0642—two selective inhibitors of euchromatic histone lysine N-methyltransferase-2 (EHMT2, also known as G9a)—activated the maternal (m) copy of candidate genes underlying PWS, including the SnoRNA cluster SNORD116, in cells from humans with PWS and also from a mouse model of PWS carrying a paternal (p) deletion from small nuclear ribonucleoprotein N (Snrpn (S)) to ubiquitin protein ligase E3A (Ube3a (U)) (mouse model referred to hereafter as m+/pΔS−U). Both UNC0642 and UNC0638 caused a selective reduction of the dimethylation of histone H3 lysine 9 (H3K9me2) at PWS-IC, without changing DNA methylation, when analyzed by bisulfite genomic sequencing. This indicates that histone modification is essential for the imprinting of candidate genes underlying PWS. UNC0642 displayed therapeutic effects in the PWS mouse model by improving the survival and the growth of m+/pΔS−U newborn pups. This study provides the first proof of principle for an epigenetics-based therapy for PWS.

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References

  1. 1.

    & Prader–Willi syndrome. Eur. J. Hum. Genet. 17, 3–13 (2009).

  2. 2.

    Prader-Willi syndrome and Angelman syndrome. Am. J. Med. Genet. C. Semin. Med. Genet. 154C, 365–376 (2010).

  3. 3.

    et al. Prader-Willi phenotype caused by paternal deficiency for the HBII-85 C/D box small nucleolar RNA cluster. Nat. Genet. 40, 719–721 (2008).

  4. 4.

    et al. A deletion of the HBII-85 class of small nucleolar RNAs (snoRNAs) is associated with hyperphagia, obesity and hypogonadism. Hum. Mol. Genet. 18, 3257–3265 (2009).

  5. 5.

    et al. Paternally inherited microdeletion at 15q11.2 confirms a significant role for the SNORD116 C/D box snoRNA cluster in Prader-Willi syndrome. Eur. J. Hum. Genet. 18, 1196–1201 (2010).

  6. 6.

    et al. Highly restricted deletion of the SNORD116 region is implicated in Prader-Willi Syndrome. Eur. J. Hum. Genet. 23, 252–255 (2015).

  7. 7.

    et al. Clinical phenotypes of MAGEL2 mutations and deletions. Orphanet J. Rare Dis. 9, 40 (2014).

  8. 8.

    et al. Truncating mutations of MAGEL2 cause Prader-Willi phenotypes and autism. Nat. Genet. 45, 1405–1408 (2013).

  9. 9.

    et al. A paternal deletion of MKRN3, MAGEL2 and NDN does not result in Prader-Willi syndrome. Eur. J. Hum. Genet. 17, 582–590 (2009).

  10. 10.

    et al. The IC-SNURF-SNRPN 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).

  11. 11.

    , , & Small evolutionarily conserved RNA, resembling C/D box small nucleolar RNA, is transcribed from PWCR1, a novel imprinted gene in the Prader-Willi deletion region, which Is highly expressed in brain. Am. J. Hum. Genet. 67, 1067–1082 (2000).

  12. 12.

    , , & Evidence for the role of PWCR1/HBII-85 C/D box small nucleolar RNAs in Prader-Willi syndrome. Am. J. Hum. Genet. 71, 669–678 (2002).

  13. 13.

    et al. Minimal definition of the imprinting center and fixation of chromosome 15q11-q13 epigenotype by imprinting mutations. Proc. Natl. Acad. Sci. USA 93, 7811–7815 (1996).

  14. 14.

    & Association of acetylated histones with paternally expressed genes in the Prader–Willi deletion region. Hum. Mol. Genet. 10, 645–652 (2001).

  15. 15.

    , & Parent-specific complementary patterns of histone H3 lysine 9 and H3 lysine 4 methylation at the Prader-Willi syndrome imprinting center. Am. J. Hum. Genet. 69, 1389–1394 (2001).

  16. 16.

    & Parent-of-origin specific histone acetylation and reactivation of a key imprinted gene locus in Prader-Willi syndrome. Am. J. Hum. Genet. 66, 1958–1962 (2000).

  17. 17.

    et al. Role of histone methyltransferase G9a in CpG methylation of the Prader-Willi syndrome imprinting center. J. Biol. Chem. 278, 14996–15000 (2003).

  18. 18.

    et al. EHMT2 directs DNA methylation for efficient gene silencing in mouse embryos. Genome Res. 26, 192–202 (2016).

  19. 19.

    et al. Deletions of a differentially methylated CpG island at the SNRPN gene define a putative imprinting control region. Nat. Genet. 8, 52–58 (1994).

  20. 20.

    et al. Dynamic developmental regulation of the large non-coding RNA associated with the mouse 7C imprinted chromosomal region. Dev. Biol. 286, 587–600 (2005).

  21. 21.

    , & Deficiency of Rbbp1/Arid4a and Rbbp1l1/Arid4b alters epigenetic modifications and suppresses an imprinting defect in the PWS/AS domain. Genes Dev. 20, 2859–2870 (2006).

  22. 22.

    et al. Topoisomerase inhibitors unsilence the dormant allele of Ube3a in neurons. Nature 481, 185–189 (2011).

  23. 23.

    et al. A chemical probe selectively inhibits G9a and GLP methyltransferase activity in cells. Nat. Chem. Biol. 7, 566–574 (2011).

  24. 24.

    et al. Discovery of an in vivo chemical probe of the lysine methyltransferases G9a and GLP. J. Med. Chem. 56, 8931–8942 (2013).

  25. 25.

    et al. Optimization of cellular activity of G9a inhibitors 7-aminoalkoxy-quinazolines. J. Med. Chem. 54, 6139–6150 (2011).

  26. 26.

    et al. Reversal of H3K9me2 by a small-molecule inhibitor for the G9a histone methyltransferase. Mol. Cell 25, 473–481 (2007).

  27. 27.

    et al. Lysine methyltransferase G9a is required for de novo DNA methylation and the establishment, but not the maintenance, of proviral silencing. Proc. Natl. Acad. Sci. USA 108, 5718–5723 (2011).

  28. 28.

    et al. Imprinted expression of UBE3A in non-neuronal cells from a Prader-Willi syndrome patient with an atypical deletion. Hum. Mol. Genet. 23, 2364–2373 (2014).

  29. 29.

    et al. The human MAGEL2 gene and its mouse homologue are paternally expressed and mapped to the Prader-Willi region. Hum. Mol. Genet. 8, 2497–2505 (1999).

  30. 30.

    et al. Induced pluripotent stem cell models of the genomic imprinting disorders Angelman and Prader-Willi syndromes. Proc. Natl. Acad. Sci. USA 107, 17668–17673 (2010).

  31. 31.

    , , , & 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).

  32. 32.

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

  33. 33.

    , & Ube3a-ATS is an atypical RNA polymerase II transcript that represses the paternal expression of Ube3a. Hum. Mol. Genet. 21, 3001–3012 (2012).

  34. 34.

    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).

  35. 35.

    et al. De novo DNA methylation promoted by G9a prevents reprogramming of embryonically silenced genes. Nat. Struct. Mol. Biol. 15, 1176–1183 (2008).

  36. 36.

    , , , & G9a/GLP complexes independently mediate H3K9 and DNA methylation to silence transcription. EMBO J. 27, 2681–2690 (2008).

  37. 37.

    et al. Reactivation of maternal SNORD116 cluster via SETDB1 knockdown in Prader-Willi syndrome iPSCs. Hum. Mol. Genet. 23, 4674–4685 (2014).

  38. 38.

    et al. G9a histone methyltransferase plays a dominant role in euchromatic histone H3 lysine 9 methylation and is essential for early embryogenesis. Genes Dev. 16, 1779–1791 (2002).

  39. 39.

    et al. G9a selectively represses a class of late-replicating genes at the nuclear periphery. Proc. Natl. Acad. Sci. USA 106, 19363–19368 (2009).

  40. 40.

    & H3K9 methyltransferase G9a and the related molecule GLP. Genes Dev. 25, 781–788 (2011).

  41. 41.

    et al. The ankyrin repeats of G9a and GLP histone methyltransferases are mono- and dimethyllysine binding modules. Nat. Struct. Mol. Biol. 15, 245–250 (2008).

  42. 42.

    & A case study in cross-talk: the histone lysine methyltransferases G9a and GLP. Nucleic Acids Res. 38, 3503–3511 (2010).

  43. 43.

    , , , & Role of distinct surfaces of the G9a ankyrin repeat domain in histone and DNA methylation during embryonic stem cell self-renewal and differentiation. Epigenetics Chromatin 7, 27 (2014).

  44. 44.

    et al. A histone H3K36 chromatin switch coordinates DNA double-strand break repair pathway choice. Nat. Commun. 5, 4091 (2014).

  45. 45.

    et al. Neurons but not glial cells show reciprocal imprinting of sense and antisense transcripts of Ube3a. Hum. Mol. Genet. 12, 837–847 (2003).

  46. 46.

    , , & Differential regulation of non-protein coding RNAs from Prader-Willi Syndrome locus. Sci. Rep. 4, 6445 (2014).

  47. 47.

    , , , & Large histone H3 lysine 9 dimethylated chromatin blocks distinguish differentiated from embryonic stem cells. Nat. Genet. 41, 246–250 (2009).

  48. 48.

    et al. Chemical modification-assisted bisulfite sequencing (CAB-Seq) for 5-carboxylcytosine detection in DNA. J. Am. Chem. Soc. 135, 9315–9317 (2013).

  49. 49.

    et al. Oxidative bisulfite sequencing of 5-methylcytosine and 5-hydroxymethylcytosine. Nat. Protoc. 8, 1841–1851 (2013).

  50. 50.

    et al. The behaviour of 5-hydroxymethylcytosine in bisulfite sequencing. PLoS One 5, e8888 (2010).

  51. 51.

    et al. Randomized open-label phase II study of decitabine in patients with low- or intermediate-risk myelodysplastic syndromes. J. Clin. Oncol. 31, 2548–2553 (2013).

  52. 52.

    , & Predicting response to epigenetic therapy. J. Clin. Invest. 124, 47–55 (2014).

  53. 53.

    et al. A maternal hypomethylation syndrome presenting as transient neonatal diabetes mellitus. Hum. Genet. 120, 262–269 (2006).

  54. 54.

    et al. Hypomethylation of multiple imprinted loci in individuals with transient neonatal diabetes is associated with mutations in ZFP57. Nat. Genet. 40, 949–951 (2008).

  55. 55.

    et al. CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol. 7, R100 (2006).

  56. 56.

    , , , & Transcriptional and functional complexity of Shank3 provides a molecular framework to understand the phenotypic heterogeneity of SHANK3 causing autism and Shank3 mutant mice. Mol. Autism 5, 30 (2014).

  57. 57.

    et al. Development of SHIRPA to characterise the phenotype of gene-targeted mice. Behav. Brain Res. 125, 43–47 (2001).

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Acknowledgements

We thank A. Beaudet at Baylor College of Medicine for providing the Snrpn-EGFP mice and some of the PWS cell lines; B. Philpot and M. Zlyka (University of North Carolina at Chapel Hill) for discussion. K. Konze for his advice on the ChIP experiment; S.-O. Han for his advice on blood-sample collection; C. Means and R. Rodriguiz for their assistance with neurological screening; and A. Bey for proofreading and discussion. This study is supported by grants from the US National Institutes of Health (HD077197 to Y.-H.J. and R01GM103893 to J.J.). Y.-H. Jiang is also supported by a grant from the Foundation for Prader–Willi Syndrome Research (FPWR). We thank the International Rett Syndrome Foundation and GlaxoSmithKline for providing the CNS-penetrating drug library (SMART library) and Published Kinase Inhibitor Set (PKIS), the NIMH Psychoactive Drug Screening Program (B.L. Roth) and the Michael Hooker Chair of Translational Proteomics (B.L. Roth).

Author information

Author notes

    • Yuna Kim
    •  & Hyeong-Min Lee

    These authors contributed equally to this work.

Affiliations

  1. Department of Pediatrics, School of Medicine, Duke University, Durham, North Carolina, USA.

    • Yuna Kim
    • , Xinyu Cao
    •  & Yong-hui Jiang
  2. Department of Cell Biology and Physiology, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA.

    • Hyeong-Min Lee
  3. Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA.

    • Hyeong-Min Lee
    • , Noah Sciaky
    •  & Bryan L Roth
  4. Departments of Pharmacological Sciences and Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, New York, USA.

    • Yan Xiong
    •  & Jian Jin
  5. Department of Neurobiology, School of Medicine, Duke University, Durham, North Carolina, USA.

    • Samuel W Hulbert
    •  & Yong-hui Jiang
  6. Department of Pathology, School of Medicine, Duke University, Durham, North Carolina, USA.

    • Jeffrey I Everitt
  7. Program in Neuroscience, Division of Chemical Biology and Medicinal Chemistry, and NIMH Psychoactive Drug Screening Program, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA.

    • Bryan L Roth
  8. Program in Genetics and Genomics, School of Medicine, Duke University, Durham, North Carolina, USA.

    • Yong-hui Jiang

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Contributions

Y.K. and H.-M.L. designed and performed the experiments and wrote the manuscript. Y.X. and J.J. provided G9a inhibitors and epigenetic-small-molecule libraries. N.S. performed the Cell Profiler. B.L.R. supervised the high-content screening and provided small-molecule libraries. X.C. supported cell culture and mouse-colony maintenance. S.W.H. supported mouse-colony maintenance and neurological analysis. J.I.E. performed histopathological analysis. Y.J. designed the experiments and wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Bryan L Roth or Yong-hui Jiang.

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DOI

https://doi.org/10.1038/nm.4257

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