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Abstract

Cell reprogramming promises to make characterization of the impact of human genetic variation on health and disease experimentally tractable by enabling the bridging of genotypes to phenotypes in developmentally relevant human cell lineages. Here we apply this paradigm to two disorders caused by symmetrical copy number variations of 7q11.23, which display a striking combination of shared and symmetrically opposite phenotypes—Williams-Beuren syndrome and 7q-microduplication syndrome. Through analysis of transgene-free patient-derived induced pluripotent stem cells and their differentiated derivatives, we find that 7q11.23 dosage imbalance disrupts transcriptional circuits in disease-relevant pathways beginning in the pluripotent state. These alterations are then selectively amplified upon differentiation of the pluripotent cells into disease-relevant lineages. A considerable proportion of this transcriptional dysregulation is specifically caused by dosage imbalances in GTF2I, which encodes a key transcription factor at 7q11.23 that is associated with the LSD1 repressive chromatin complex and silences its dosage-sensitive targets.

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Change history

  • 09 January 2015

    In the version of this article initially published online, GTF1I knockdown was incorrectly referred to in the legend for Figure 4c. GTF2I is the correct shRNA target in this experiment. The error has been corrected for the print, PDF and HTML versions of this article.

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References

  1. 1.

    & Origins and implications of pluripotent stem cell variability and heterogeneity. Nat. Rev. Mol. Cell Biol. 14, 357–368 (2013).

  2. 2.

    et al. Multiple recurrent de novo CNVs, including duplications of the 7q11.23 Williams syndrome region, are strongly associated with autism. Neuron 70, 863–885 (2011).

  3. 3.

    Williams-Beuren syndrome. N. Engl. J. Med. 362, 239–252 (2010).

  4. 4.

    et al. Severe expressive-language delay related to duplication of the Williams-Beuren locus. N. Engl. J. Med. 353, 1694–1701 (2005).

  5. 5.

    , , & Copy number variants at Williams-Beuren syndrome 7q11.23 region. Hum. Genet. 128, 3–26 (2010).

  6. 6.

    et al. Fourteen new cases contribute to the characterization of the 7q11.23 microduplication syndrome. Eur. J. Med. Genet. 52, 94–100 (2009).

  7. 7.

    Animal models of Williams syndrome. Am. J. Med. Genet. C. Semin. Med. Genet. 154C, 209–219 (2010).

  8. 8.

    et al. Duplication of GTF2I results in separation anxiety in mice and humans. Am. J. Hum. Genet. 90, 1064–1070 (2012).

  9. 9.

    & Global analysis of gene expression in the developing brain of Gtf2ird1 knockout mice. PLoS ONE 6, e23868 (2011).

  10. 10.

    et al. Reduction of NADPH-oxidase activity ameliorates the cardiovascular phenotype in a mouse model of Williams-Beuren syndrome. PLoS Genet. 8, e1002458 (2012).

  11. 11.

    et al. Smaller and larger deletions of the Williams Beuren syndrome region implicate genes involved in mild facial phenotype, epilepsy and autistic traits. Eur. J. Hum. Genet. 22, 64–70 (2014).

  12. 12.

    et al. Brief report: functional MRI of a patient with 7q11.23 duplication syndrome and autism spectrum disorder. J. Autism Dev. Disord. 44, 2608–2613 (2014).

  13. 13.

    et al. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell 7, 618–630 (2010).

  14. 14.

    et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007).

  15. 15.

    et al. Genomic instability in induced stem cells. Cell Death Differ. 18, 745–753 (2011).

  16. 16.

    et al. Induced pluripotent stem cell modeling of multisystemic, hereditary transthyretin amyloidosis. Stem Cell Reports 1, 451–463 (2013).

  17. 17.

    et al. Somatic copy number mosaicism in human skin revealed by induced pluripotent stem cells. Nature 492, 438–442 (2012).

  18. 18.

    et al. Abnormalities in human pluripotent cells due to reprogramming mechanisms. Nature 511, 177–183 (2014).

  19. 19.

    et al. Partial 7q11.23 deletions further implicate GTF2I and GTF2IRD1 as the main genes responsible for the Williams-Beuren syndrome neurocognitive profile. J. Med. Genet. 47, 312–320 (2010).

  20. 20.

    et al. An atypical deletion of the Williams-Beuren syndrome interval implicates genes associated with defective visuospatial processing and autism. J. Med. Genet. 44, 136–143 (2007).

  21. 21.

    et al. Williams syndrome deficits in visual spatial processing linked to GTF2IRD1 and GTF2I on chromosome 7q11.23. Genet. Med. 5, 311–321 (2003).

  22. 22.

    , , & Williams-Beuren syndrome–associated transcription factor TFII-I regulates osteogenic marker genes. J. Biol. Chem. 284, 36234–36239 (2009).

  23. 23.

    et al. Association of GTF2i in the Williams-Beuren syndrome critical region with autism spectrum disorders. J. Autism Dev. Disord. 42, 1459–1469 (2012).

  24. 24.

    et al. Haploinsufficiency of Gtf2i, a gene deleted in Williams Syndrome, leads to increases in social interactions. Autism Res. 4, 28–39 (2011).

  25. 25.

    , , & Calcium metabolism in Williams-Beuren syndrome. J. Pediatr. 121, 902–907 (1992).

  26. 26.

    , , , & Hyperacusis in Williams syndrome: characteristics and associated neuroaudiologic abnormalities. Neurology 66, 390–395 (2006).

  27. 27.

    et al. The pattern of sensory processing abnormalities in autism. Autism 10, 480–494 (2006).

  28. 28.

    , , , & Incidence and spectrum of renal abnormalities in Williams-Beuren syndrome. Am. J. Med. Genet. 63, 301–304 (1996).

  29. 29.

    , , , & Parent-of-origin effects of FAS and PDLIM1 in attention-deficit/hyperactivity disorder. J. Psychiatry Neurosci. 37, 46–52 (2012).

  30. 30.

    , , , & Characterization of CLP36/Elfin/PDLIM1 in the nervous system. J. Neurochem. 111, 790–800 (2009).

  31. 31.

    et al. Nonmuscle myosin heavy-chain gene MYH14 is expressed in cochlea and mutated in patients affected by autosomal dominant hearing impairment (DFNA4). Am. J. Hum. Genet. 74, 770–776 (2004).

  32. 32.

    , & Transcriptome profile in Williams-Beuren syndrome lymphoblast cells reveals gene pathways implicated in glucose intolerance and visuospatial construction deficits. Hum. Genet. 128, 27–37 (2010).

  33. 33.

    et al. An atypical 7q11.23 deletion in a normal IQ Williams-Beuren syndrome patient. Eur. J. Hum. Genet. 18, 33–38 (2010).

  34. 34.

    et al. GTF2I hemizygosity implicated in mental retardation in Williams syndrome: genotype-phenotype analysis of five families with deletions in the Williams syndrome region. Am. J. Med. Genet. A. 123A, 45–59 (2003).

  35. 35.

    & Molecular basis of Williams-Beuren syndrome: TFII-I regulated targets involved in craniofacial development. Cleft Palate Craniofac. J. 48, 109–116 (2011).

  36. 36.

    & ZNF198 stabilizes the LSD1-CoREST-HDAC1 complex on chromatin through its MYM-type zinc fingers. PLoS ONE 3, e3255 (2008).

  37. 37.

    , , , & A candidate X-linked mental retardation gene is a component of a new family of histone deacetylase–containing complexes. J. Biol. Chem. 278, 7234–7239 (2003).

  38. 38.

    et al. Cellular reprogramming: recent advances in modeling neurological diseases. J. Neurosci. 31, 16070–16075 (2011).

  39. 39.

    et al. RCOR2 is a subunit of the LSD1 complex that regulates ESC property and substitutes for SOX2 in reprogramming somatic cells to pluripotency. Stem Cells 29, 791–801 (2011).

  40. 40.

    , , & Identification of the TFII-I family target genes in the vertebrate genome. Proc. Natl. Acad. Sci. USA 105, 9006–9010 (2008).

  41. 41.

    et al. Diversity and complexity in chromatin recognition by TFII-I transcription factors in pluripotent embryonic stem cells and embryonic tissues. PLoS ONE 7, e44443 (2012).

  42. 42.

    et al. Genomic and proteomic analysis of transcription factor TFII-I reveals insight into the response to cellular stress. Nucleic Acids Res. 42, 7625–7641 (2014).

  43. 43.

    , & Case report: autistic disorder and chromosomal abnormality 46, XX duplication (4) p12-p13. Eur. Child Adolesc. Psychiatry 9, 307–311 (2000).

  44. 44.

    et al. Genetic variability in the regulation of gene expression in ten regions of the human brain. Nat. Neurosci. 17, 1418–1428 (2014).

  45. 45.

    et al. An anatomically comprehensive atlas of the adult human brain transcriptome. Nature 489, 391–399 (2012).

  46. 46.

    et al. Follow-up association studies of chromosome region 9q and nonsyndromic cleft lip/palate. Am. J. Med. Genet. A. 152A, 1701–1710 (2010).

  47. 47.

    , , , & Human cerebral cortex development from pluripotent stem cells to functional excitatory synapses. Nat. Neurosci. 15, 477–486 (2012).

  48. 48.

    , & Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks. Nat. Protoc. 7, 1836–1846 (2012).

  49. 49.

    et al. Directed differentiation of human pluripotent cells to neural crest stem cells. Nat. Protoc. 8, 203–212 (2013).

  50. 50.

    et al. Bmp signaling regulates a dose-dependent transcriptional program to control facial skeletal development. Development 139, 709–719 (2012).

  51. 51.

    et al. Sonic hedgehog signalling inhibits palatogenesis and arrests tooth development in a mouse model of the nevoid basal cell carcinoma syndrome. Dev. Biol. 331, 38–49 (2009).

  52. 52.

    et al. Mutations in Hedgehog acyltransferase (Hhat) perturb Hedgehog signaling, resulting in severe acrania-holoprosencephaly-agnathia craniofacial defects. PLoS Genet. 8, e1002927 (2012).

  53. 53.

    et al. SATB2 is a multifunctional determinant of craniofacial patterning and osteoblast differentiation. Cell 125, 971–986 (2006).

  54. 54.

    , , & Disrupting hedgehog and WNT signaling interactions promotes cleft lip pathogenesis. J. Clin. Invest. 124, 1660–1671 (2014).

  55. 55.

    et al. Patched1 is required in neural crest cells for the prevention of orofacial clefts. Hum. Mol. Genet. 22, 5026–5035 (2013).

  56. 56.

    , , & Molecular profiles of mitogen activated protein kinase signaling pathways in orofacial development. Birth Defects Res. A Clin. Mol. Teratol. 79, 35–44 (2007).

  57. 57.

    et al. The role of SATB2 in skeletogenesis and human disease. Cytokine Growth Factor Rev. 25, 35–44 (2014).

  58. 58.

    & Molecular mechanisms of cranial neural crest cell migration and patterning in craniofacial development. Development 137, 2605–2621 (2010).

  59. 59.

    et al. Neural crest cell survival is dependent on Rho kinase and is required for development of the mid face in mouse embryos. PLoS ONE 7, e37685 (2012).

  60. 60.

    et al. Modeling supravalvular aortic stenosis syndrome with human induced pluripotent stem cells. Circulation 126, 1695–1704 (2012).

  61. 61.

    et al. Williams syndrome transcription factor is critical for neural crest cell function in Xenopus laevis. Mech. Dev. 129, 324–338 (2012).

  62. 62.

    et al. The H3K27 demethylase JMJD3 is required for maintenance of the embryonic respiratory neuronal network, neonatal breathing, and survival. Cell Rep. 2, 1244–1258 (2012).

  63. 63.

    et al. Lineage-specific polycomb targets and de novo DNA methylation define restriction and potential of neuronal progenitors. Mol. Cell 30, 755–766 (2008).

  64. 64.

    et al. The histone H3 lysine 27–specific demethylase Jmjd3 is required for neural commitment. PLoS ONE 3, e3034 (2008).

  65. 65.

    et al. A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell 125, 315–326 (2006).

  66. 66.

    et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat. Biotechnol. 25, 681–686 (2007).

  67. 67.

    et al. High-throughput chromatin immunoprecipitation for genome-wide mapping of in vivo protein-DNA interactions and epigenomic states. Nat. Protoc. 8, 539–554 (2013).

  68. 68.

    , & Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat. Protoc. 2, 1896–1906 (2007).

  69. 69.

    et al. LSD1 regulates the balance between self-renewal and differentiation in human embryonic stem cells. Nat. Cell Biol. 13, 652–659 (2011).

  70. 70.

    et al. (Hetero)aryl cyclopropylamine compounds as LSD1 inhibitors. International Bureau of the World Intellectual Property Organization patent number WO2013057322 (2013).

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Acknowledgements

We thank the AFSW (Associazione Famiglie Sindrome di Williams) and AISW (Associazione Italiana Sindrome di Williams) for agreeing to participate and making this study possible and the Genomic and Genetic Disorder Biobank, Galliera Genetic Bank and members of the Telethon Network of Genetic Biobanks (project numbers GTB12001G and GTB12001A), along with the EuroBioBank network, for providing us with specimens. We also thank scientists at the Drug Discovery Unit, Drug Development Program (DDU-DDP) of the European Institute of Oncology (IEO) for sharing with us the two LSD1 inhibitors used in this study; A. Bachi, A. Cattaneo and P. Soffiantini from the Mass Spectrometry service of the FIRC (Fondazione Italiana per la Ricerca sul Cancro) Institute of Molecular Oncology (IFOM); F. Pisati for processing of the teratomas; P. Andrews (University of Sheffield) for sharing two control iPSC lines (CTL2-C1 and CTL2-C2; reprogrammed from CRL-2429 fibroblasts); G. Mostoslavsky and the Center for Regenerative Medicine of Boston University for sharing the BU1Cr3-1 line; G. Barbagiovanni for help with FACS profiling and analysis; and L. Marelli along with all other members of the Testa laboratory for discussion. This work was funded by the European Research Council (consolidator grant number 616441-DISEASEAVATARS to G.T.), the Italian Ministry of Health (Ricerca Corrente to G.T. and G.M. and Bando Giovani Ricercatori 2008 and 2009 to G.T.), the EPIGEN Flagship Project of the Italian National Research Council (G.T.), the Jerome-Lejeune Foundation (G.T. and G.M.), the ERA-NET Neuron Program (G.T.), the Umberto Veronesi Foundation (S.A. and G.D.) and the Federation of European Biochemical Societies (FEBS; fellowship awarded to A.A. to work in the laboratory of G.T.).

Author information

Author notes

    • Antonio Adamo
    • , Sina Atashpaz
    •  & Pierre-Luc Germain

    These authors contributed equally to this work.

Affiliations

  1. Department of Experimental Oncology, European Institute of Oncology (Istituto di Ricovero e Cura a Carattere Scientifico, IRCCS), Milan, Italy.

    • Antonio Adamo
    • , Sina Atashpaz
    • , Pierre-Luc Germain
    • , Matteo Zanella
    • , Giuseppe D'Agostino
    • , Veronica Albertin
    • , Giancarlo Pruneri
    •  & Giuseppe Testa
  2. Lieber Institute for Brain Development, Baltimore, Maryland, USA.

    • Josh Chenoweth
    •  & Ronald McKay
  3. Medical Genetics Unit, IRCCS Casa Sollievo della Sofferenza Hospital, San Giovanni Rotondo, Italy.

    • Lucia Micale
    • , Carmela Fusco
    • , Bartolomeo Augello
    • , Orazio Palumbo
    • , Massimo Carella
    •  & Giuseppe Merla
  4. Department of Biomedical Sciences, University of Sheffield, Sheffield, UK.

    • Christian Unger
  5. Stemgent, Cambridge, Massachusetts, USA.

    • Brad Hamilton
  6. Medical Genetics Unit, Hospital Santa Maria della Misericordia, University of Perugia, Perugia, Italy.

    • Emilio Donti
    •  & Paolo Prontera
  7. Unità Operativa Semplice (UOS) Genetica Clinica Pediatrica, Fondazione Monza e Brianza per il Bambino e la sua Mamma (Fondazione MBBM), Azienda Ospedaliera San Gerardo, Monza, Italy.

    • Angelo Selicorni
  8. Department of Pediatrics, University of Turin, Turin, Italy.

    • Elisa Biamino
  9. Department of Health Sciences, University of Milan, Milan, Italy.

    • Giuseppe Testa

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Contributions

S.A. initiated this project and set up human iPSC reprogramming and culture, including mRNA-based reprogramming. S.A. and A.A. reprogrammed the lines presented in this study. A.A., S.A., G.D. and M.Z. cultured and characterized iPSC lines and profiled transcriptomes. A.A. performed the biochemical characterization of the GTF2I complex and GTF2I and LSD1 ChIP-seq. A.A. performed the Nanostring experiment. A.A. and V.A. generated the GTF2I RNAi lines. S.A. established human iPSC differentiation into the cortical neural and neural crest lineages. S.A. and A.A. differentiated human iPSCs into cortical neural progenitors. S.A. analyzed NPCs and NCSCs by microarray. S.A. and M.Z. differentiated human iPSCs into NCSCs and MSCs. P.-L.G. performed the computational analysis for the microarray, Nanostring, RNA-seq and ChIP-seq data sets. P.-L.G. created the WikiWilliams-7qGeneBase web platform. G.M. organized the recruitment of patients, including molecular diagnostics and derivation of fibroblast cultures (with L.M., C.F. and B.A.). O.P., M.C. and G.M. performed aCGH analysis. G.P. performed histopathological analysis of teratomas. A.S. diagnosed and recruited patient AtWBS1, E.B. diagnosed and recruited patient WBS4, and P.P. and E.D. diagnosed and recruited patient 7dupASD1. R.M. and J.C. performed RNA-seq on a subset of samples. C.U. provided two control iPSC lines. B.H. provided mRNA reprogramming kits and expertise. P.-L.G., S.A., A.A. and G.T. wrote the manuscript. G.T. conceived, designed and supervised the study.

Competing interests

B.H. is the director of research and development for Stemgent and Asterand. All other authors declare no competing financial interests.

Corresponding author

Correspondence to Giuseppe Testa.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–9 and Supplementary Tables 1 and 10

Excel files

  1. 1.

    Supplementary Table 2

    Summary of the copy number variations (CNVs) identified through aCGH.

  2. 2.

    Supplementary Table 3

    GO biological processes enriched among linear DEGs, defined as mean (control) within a 20–80% range between mean (WBS) and mean (7dupASD), and abs(Pearson correlation) > 0.5 with WBS copy number.

  3. 3.

    Supplementary Table 4

    Proportion of DEGs, in each comparison between genotypes, attributable to GTF2I.

  4. 4.

    Supplementary Table 5

    GTF2I interactors identified through mass spectrometry analysis.

  5. 5.

    Supplementary Table 6

    GTF2I target classification according to ChIP analysis.

  6. 6.

    Supplementary Table 7

    GO biological processes enriched among the union of NCSC DEGs.

  7. 7.

    Supplementary Table 8

    Comparison of GO biological processes enriched among MSC DEGs and in MSC shuffling.

  8. 8.

    Supplementary Table 9

    List of performed experiments (Nat. Biotechnol. 25, 681–686, 2007).

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https://doi.org/10.1038/ng.3169

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