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Tissue- and age-specific DNA replication patterns at the CTG/CAG-expanded human myotonic dystrophy type 1 locus

Nature Structural & Molecular Biology volume 17, pages 1079–1087 (2010)Cite this article

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Abstract

Myotonic dystrophy, caused by DM1 CTG/CAG repeat expansions, shows varying instability levels between tissues and across ages within patients. We determined DNA replication profiles at the DM1 locus in patient fibroblasts and tissues from DM1 transgenic mice of various ages showing different instability. In patient cells, the repeat is flanked by two replication origins demarcated by CTCF sites, with replication diminished at the expansion. In mice, the expansion replicated from only the downstream origin (CAG as lagging template). In testes from mice of three different ages, replication toward the repeat paused at the earliest age and was relieved at later ages—coinciding with increased instability. Brain, pancreas and thymus replication varied with CpG methylation at DM1 CTCF sites. CTCF sites between progressing forks and repeats reduced replication depending on chromatin. Thus, varying replication progression may affect tissue- and age-specific repeat instability.

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Figure 1: The human DM1 locus.
Figure 2: Replication activity at the human DM1 locus.
Figure 3: Tissue- and age-specific variations in replication activity.
Figure 4: Altered DNA replication at the DM1 locus in patient cells.
Figure 5: The effects of CTCF on replication efficiency.
Figure 6: CpG methylation at the DM1 CTCF binding sites and flanking sequence.
Figure 7: Models of replication-associated repeat instability.

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References

  1. Pearson, C.E., Nichol Edamura, K. & Cleary, J.D. Repeat instability: mechanisms of dynamic mutations. Nat. Rev. Genet. 6, 729–742 (2005).

    Article  CAS  Google Scholar 

  2. Thornton, C.A., Johnson, K. & Moxley, R.T. III. Myotonic dystrophy patients have larger CTG expansions in skeletal muscle than in leukocytes. Ann. Neurol. 35, 104–107 (1994).

    Article  CAS  Google Scholar 

  3. Panigrahi, G.B., Lau, R., Montgomery, S.E., Leonard, M.R. & Pearson, C.E. Slipped (CTG)*(CAG) repeats can be correctly repaired, escape repair or undergo error-prone repair. Nat. Struct. Mol. Biol. 12, 635–637 (2005).

    Article  Google Scholar 

  4. Cleary, J.D. & Pearson, C.E. The contribution of cis-elements to disease-associated repeat instability: clinical and experimental evidence. Cytogenet. Genome Res. 100, 25–55 (2003).

    Article  CAS  Google Scholar 

  5. Ansved, T., Lundin, A. & Anvret, M. Larger CAG expansions in skeletal muscle compared with lymphocytes in Kennedy disease but not in Huntington disease. Neurology 51, 1442–1444 (1998).

    Article  CAS  Google Scholar 

  6. Wohrle, D. et al. Heterogeneity of DM kinase repeat expansion in different fetal tissues and further expansion during cell proliferation in vitro: evidence for a casual involvement of methyl-directed DNA mismatch repair in triplet repeat stability. Hum. Mol. Genet. 4, 1147–1153 (1995).

    Article  CAS  Google Scholar 

  7. Martorell, L. et al. Progression of somatic CTG repeat length heterogeneity in the blood cells of myotonic dystrophy patients. Hum. Mol. Genet. 7, 307–312 (1998).

    Article  CAS  Google Scholar 

  8. Cleary, J.D., Nichol, K., Wang, Y.H. & Pearson, C.E. Evidence of cis-acting factors in replication-mediated trinucleotide repeat instability in primate cells. Nat. Genet. 31, 37–46 (2002).

    Article  CAS  Google Scholar 

  9. Yang, Z., Lau, R., Marcadier, J.L., Chitayat, D. & Pearson, C.E. Replication inhibitors modulate instability of an expanded trinucleotide repeat at the myotonic dystrophy type 1 disease locus in human cells. Am. J. Hum. Genet. 73, 1092–1105 (2003).

    Article  CAS  Google Scholar 

  10. Farrell, B.T. & Lahue, R.S. CAG*CTG repeat instability in cultured human astrocytes. Nucleic Acids Res. 34, 4495–4505 (2006).

    Article  CAS  Google Scholar 

  11. Claassen, D.A. & Lahue, R.S. Expansions of CAG.CTG repeats in immortalized human astrocytes. Hum. Mol. Genet. 16, 3088–3096 (2007).

    Article  CAS  Google Scholar 

  12. Mirkin, S.M. & Smirnova, E.V. Positioned to expand. Nat. Genet. 31, 5–6 (2002).

    Article  CAS  Google Scholar 

  13. Cleary, J.D. & Pearson, C.E. Replication fork dynamics and dynamic mutations: the fork-shift model of repeat instability. Trends Genet. 21, 272–280 (2005).

    Article  CAS  Google Scholar 

  14. Samadashwily, G.M., Raca, G. & Mirkin, S.M. Trinucleotide repeats affect DNA replication in vivo . Nat. Genet. 17, 298–304 (1997).

    Article  CAS  Google Scholar 

  15. Dere, R., Napierala, M., Ranum, L.P. & Wells, R.D. Hairpin structure-forming propensity of the (CCTG.CAGG) tetranucleotide repeats contributes to the genetic instability associated with myotonic dystrophy type 2. J. Biol. Chem. 279, 41715–41726 (2004).

    Article  CAS  Google Scholar 

  16. Rindler, P.M., Clark, R.M., Pollard, L.M., De Biase, I. & Bidichandani, S.I. Replication in mammalian cells recapitulates the locus-specific differences in somatic instability of genomic GAA triplet-repeats. Nucleic Acids Res. 34, 6352–6361 (2006).

    Article  CAS  Google Scholar 

  17. Nichol Edamura, K., Leonard, M.R. & Pearson, C.E. Role of replication and CpG methylation in fragile X syndrome CGG deletions in primate cells. Am. J. Hum. Genet. 76, 302–311 (2005).

    Article  Google Scholar 

  18. Nenguke, T., Aladjem, M.I., Gusella, J.F., Wexler, N.S. & Arnheim, N. Candidate DNA replication initiation regions at human trinucleotide repeat disease loci. Hum. Mol. Genet. 12, 1021–1028 (2003).

    Article  CAS  Google Scholar 

  19. Gray, S.J., Gerhardt, J., Doerfler, W., Small, L.E. & Fanning, E. An origin of DNA replication in the promoter region of the human fragile X mental retardation (FMR1) gene. Mol. Cell. Biol. 27, 426–437 (2007).

    Article  CAS  Google Scholar 

  20. Chastain, P.D. II, Cohen, S.M., Brylawski, B.P., Cordeiro-Stone, M. & Kaufman, D.G. A late origin of DNA replication in the trinucleotide repeat region of the human FMR2 gene. Cell Cycle 5, 869–872 (2006).

    Article  CAS  Google Scholar 

  21. Brylawski, B.P., Chastain, P.D. II, Cohen, S.M., Cordeiro-Stone, M. & Kaufman, D.G. Mapping of an origin of DNA replication in the promoter of fragile X gene FMR1. Exp. Mol. Pathol. 82, 190–196 (2007).

    Article  CAS  Google Scholar 

  22. Takano, H. et al. Somatic mosaicism of expanded CAG repeats in brains of patients with dentatorubral-pallidoluysian atrophy: cellular population-dependent dynamics of mitotic instability. Am. J. Hum. Genet. 58, 1212–1222 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Curtis, M.A. et al. Increased cell proliferation and neurogenesis in the adult human Huntington's disease brain. Proc. Natl. Acad. Sci. USA 100, 9023–9027 (2003).

    Article  CAS  Google Scholar 

  24. Pearson, C.E. Slipping while sleeping? Trinucleotide repeat expansions in germ cells. Trends Mol. Med. 9, 490–495 (2003).

    Article  CAS  Google Scholar 

  25. Seznec, H. et al. Transgenic mice carrying large human genomic sequences with expanded CTG repeat mimic closely the DM CTG repeat intergenerational and somatic instability. Hum. Mol. Genet. 9, 1185–1194 (2000).

    Article  CAS  Google Scholar 

  26. Giacca, M., Pelizon, C. & Falaschi, A. Mapping replication origins by quantifying relative abundance of nascent DNA strands using competitive polymerase chain reaction. Methods 13, 301–312 (1997).

    Article  CAS  Google Scholar 

  27. Todorovic, V., Falaschi, A. & Giacca, M. Replication origins of mammalian chromosomes: the happy few. Front. Biosci. 4, D859–D868 (1999).

    Article  CAS  Google Scholar 

  28. Zentilin, L. & Giacca, M. Competitive PCR for precise nucleic acid quantification. Nat. Protoc. 2, 2092–2104 (2007).

    Article  CAS  Google Scholar 

  29. Kumar, S. et al. Utilization of the same DNA replication origin by human cells of different derivation. Nucleic Acids Res. 24, 3289–3294 (1996).

    Article  CAS  Google Scholar 

  30. Filippova, G.N. et al. CTCF-binding sites flank CTG/CAG repeats and form a methylation-sensitive insulator at the DM1 locus. Nat. Genet. 28, 335–343 (2001).

    Article  CAS  Google Scholar 

  31. Aladjem, M.I. Replication in context: dynamic regulation of DNA replication patterns in metazoans. Nat. Rev. Genet. 8, 588–600 (2007).

    Article  CAS  Google Scholar 

  32. Zuhlke, C. et al. Homozygous myotonic dystrophy: clinical findings in two patients and review of the literature. Am. J. Med. Genet. A. 143, 2058–2061 (2007).

    Article  Google Scholar 

  33. Savouret, C. et al. MSH2-dependent germinal CTG repeat expansions are produced continuously in spermatogonia from DM1 transgenic mice. Mol. Cell. Biol. 24, 629–637 (2004).

    Article  CAS  Google Scholar 

  34. Lia, A.S. et al. Somatic instability of the CTG repeat in mice transgenic for the myotonic dystrophy region is age dependent but not correlated to the relative intertissue transcription levels and proliferative capacities. Hum. Mol. Genet. 7, 1285–1291 (1998).

    Article  CAS  Google Scholar 

  35. Guiraud-Dogan, C. et al. DM1 CTG expansions affect insulin receptor isoforms expression in various tissues of transgenic mice. Biochim. Biophys. Acta 1772, 1183–1191 (2007).

    Article  CAS  Google Scholar 

  36. Aladjem, M.I., Rodewald, L.W., Kolman, J.L. & Wahl, G.M. Genetic dissection of a mammalian replicator in the human β-globin locus. Science 281, 1005–1009 (1998).

    Article  CAS  Google Scholar 

  37. Foiry, L., Megret, J., Junien, C. & Gourdon, G. A simple and fast method for cell recovery and DNA content analysis from various mouse tissues by flow cytometry. Cytotechnology 52, 107–112 (2006).

    Article  CAS  Google Scholar 

  38. Girard-Reydet, C., Gregoire, D., Vassetzky, Y. & Mechali, M. DNA replication initiates at domains overlapping with nuclear matrix attachment regions in the Xenopus and mouse c-myc promoter. Gene 332, 129–138 (2004).

    Article  CAS  Google Scholar 

  39. Kalejta, R.F. et al. Distal sequences, but not ori-β/OBR-1, are essential for initiation of DNA replication in the Chinese hamster DHFR origin. Mol. Cell 2, 797–806 (1998).

    Article  CAS  Google Scholar 

  40. Kitsberg, D., Selig, S., Keshet, I. & Cedar, H. Replication structure of the human β-globin gene domain. Nature 366, 588–590 (1993).

    Article  CAS  Google Scholar 

  41. Stillman, B. DNA replication. Replicator renaissance. Nature 366, 506–507 (1993).

    Article  CAS  Google Scholar 

  42. Martorell, L., Martinez, J.M., Carey, N., Johnson, K. & Baiget, M. Comparison of CTG repeat length expansion and clinical progression of myotonic dystrophy over a five year period. J. Med. Genet. 32, 593–596 (1995).

    Article  CAS  Google Scholar 

  43. Vergouwen, R.P., Jacobs, S.G., Huiskamp, R., Davids, J.A. & de Rooij, D.G. Proliferative activity of gonocytes, Sertoli cells and interstitial cells during testicular development in mice. J. Reprod. Fertil. 93, 233–243 (1991).

    Article  CAS  Google Scholar 

  44. Cho, D.H. et al. Antisense transcription and heterochromatin at the DM1 CTG repeats are constrained by CTCF. Mol. Cell 20, 483–489 (2005).

    Article  CAS  Google Scholar 

  45. Libby, R.T. et al. CTCF cis-regulates trinucleotide repeat instability in an epigenetic manner: a novel basis for mutational hot spot determination. PLoS Genet. 4, e1000257 (2008).

    Article  Google Scholar 

  46. Vostrov, A.A., Taheny, M.J. & Quitschke, W.W. A region to the N-terminal side of the CTCF zinc finger domain is essential for activating transcription from the amyloid precursor protein promoter. J. Biol. Chem. 277, 1619–1627 (2002).

    Article  CAS  Google Scholar 

  47. Cereghini, S. & Yaniv, M. Assembly of transfected DNA into chromatin: structural changes in the origin-promoter-enhancer region upon replication. EMBO J. 3, 1243–1253 (1984).

    Article  CAS  Google Scholar 

  48. Li, J.J. & Kelly, T.J. Simian virus 40 DNA replication in vitro . Proc. Natl. Acad. Sci. USA 81, 6973–6977 (1984).

    Article  CAS  Google Scholar 

  49. Panigrahi, G.B., Slean, M.M., Simard, J.P., Gileadi, O. & Pearson, C.E. Isolated short CTG/CAG DNA slipouts are repaired efficiently by hMutSβ, but clustered slip-outs are poorly repaired. Proc. Natl. Acad. Sci. USA 107, 12593–12598 (2010).

    Article  CAS  Google Scholar 

  50. Musova, Z. et al. Highly unstable sequence interruptions of the CTG repeat in the myotonic dystrophy gene. Am. J. Med. Genet. A. 149A, 1365–1374 (2009).

    Article  CAS  Google Scholar 

  51. Braida, C. et al. Variant CCG and GGC repeats within the CTG expansion dramatically modify mutational dynamics and likely contribute toward unusual symptoms in some myotonic dystrophy type 1 patients. Hum. Mol. Genet. 19, 1399–1412 (2010).

    Article  CAS  Google Scholar 

  52. Romero, J. & Lee, H. Asymmetric bidirectional replication at the human DBF4 origin. Nat. Struct. Mol. Biol. 15, 722–729 (2008).

    Article  CAS  Google Scholar 

  53. Bergstrom, R., Whitehead, J., Kurukuti, S. & Ohlsson, R. CTCF regulates asynchronous replication of the imprinted H19/Igf2 domain. Cell Cycle 6, 450–454 (2007).

    Article  Google Scholar 

  54. Rajcan-Separovic, E., Barcelo, J.M. & Korneluk, R.G. Fluorescence in situ hybridization analysis of the replication properties of the myotonic dystrophy protein kinase (DMPK) gene region. Cytogenet. Cell Genet. 82, 247–250 (1998).

    Article  CAS  Google Scholar 

  55. Terret, M.E., Sherwood, R., Rahman, S., Qin, J. & Jallepalli, P.V. Cohesin acetylation speeds the replication fork. Nature 462, 231–234 (2009).

    Article  CAS  Google Scholar 

  56. Rubio, E.D. et al. CTCF physically links cohesin to chromatin. Proc. Natl. Acad. Sci. USA 105, 8309–8314 (2008).

    Article  CAS  Google Scholar 

  57. Farrar, D. et al. Mutational analysis of the poly(ADP-ribosyl)ation sites of the transcription factor CTCF provides an insight into the mechanism of its regulation by poly(ADP-ribosyl)ation. Mol. Cell. Biol. 30, 1199–1216 (2010).

    Article  CAS  Google Scholar 

  58. Loukinov, D.I. et al. BORIS, a novel male germ-line-specific protein associated with epigenetic reprogramming events, shares the same 11-zinc-finger domain with CTCF, the insulator protein involved in reading imprinting marks in the soma. Proc. Natl. Acad. Sci. USA 99, 6806–6811 (2002).

    Article  CAS  Google Scholar 

  59. Monk, M., Hitchins, M. & Hawes, S. Differential expression of the embryo/cancer gene ECSA(DPPA2), the cancer/testis gene BORIS and the pluripotency structural gene OCT4, in human preimplantation development. Mol. Hum. Reprod. 14, 347–355 (2008).

    Article  CAS  Google Scholar 

  60. Rein, T., Kobayashi, T., Malott, M., Leffak, M. & DePamphilis, M.L. DNA methylation at mammalian replication origins. J. Biol. Chem. 274, 25792–25800 (1999).

    Article  CAS  Google Scholar 

  61. Hansen, R.S., Canfield, T.K., Lamb, M.M., Gartler, S.M. & Laird, C.D. Association of fragile X syndrome with delayed replication of the FMR1 gene. Cell 73, 1403–1409 (1993).

    Article  CAS  Google Scholar 

  62. Dion, V. & Wilson, J.H. Instability and chromatin structure of expanded trinucleotide repeats. Trends Genet. 25, 288–297 (2009).

    Article  CAS  Google Scholar 

  63. Gourdon, G. et al. Moderate intergenerational and somatic instability of a 55-CTG repeat in transgenic mice. Nat. Genet. 15, 190–192 (1997).

    Article  CAS  Google Scholar 

  64. Kramer, P.R., Pearson, C.E. & Sinden, R.R. Stability of triplet repeats of myotonic dystrophy and fragile X loci in human mutator mismatch repair cell lines. Hum. Genet. 98, 151–157 (1996).

    Article  CAS  Google Scholar 

  65. Abdurashidova, G. et al. Start sites of bidirectional DNA synthesis at the human lamin B2 origin. Science 287, 2023–2026 (2000).

    Article  CAS  Google Scholar 

  66. Frommer, M. et al. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc. Natl. Acad. Sci. USA 89, 1827–1831 (1992).

    Article  CAS  Google Scholar 

  67. Panigrahi, G.B., Cleary, J.D. & Pearson, C.E. In vitro (CTG)*(CAG) expansions and deletions by human cell extracts. J. Biol. Chem. 277, 13926–13934 (2002).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank K.N. Edamura, R. Lau and J. Simard for technical assistances and support. This work was supported by grants from Canadian Institutes of Health Research (C.E.P.), the Muscular Dystrophy Association, USA (C.E.P.), Canadian Institutes of Health Research International Opportunities (C.E.P. and G.G.), University of Rochester Paul Wellstone Muscular Dystrophy Cooperative Research Center with support from the US National Institutes of Health (U54NS48843) (C.E.P.); France-Canada Research Fund (C.E.P. and G.G.); Institut national de santé et de la recherche médicale (G.G.), the Association Française contre les Myopathies (G.G.), the Université René-Descartes Paris V (G.G.), a Canadian Institutes of Health Research fellowship (J.D.C.); The Hospital for Sick Children Research Training Centre (K.A.H.) and an American Society of Human Genetics Trainee Award (K.A.H.).

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Authors and Affiliations

  1. Program of Genetics & Genome Biology, The Hospital for Sick Children, Toronto, Ontario, Canada

    John D Cleary, Arturo López Castel, Gagan B Panigrahi, Katharine A Hagerman, David Chitayat & Christopher E Pearson

  2. Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada

    John D Cleary, Katharine A Hagerman, David Chitayat & Christopher E Pearson

  3. Institut national de la santé et de la recherche médicale, U781, Hôpital Necker-Enfants Malades, Université René Descartes Paris V, Paris, France

    Stéphanie Tomé, Laurent Foiry & Geneviève Gourdon

  4. Department of Obstetrics and Gynecology, The Prenatal Diagnosis and Medical Genetics Program, Mount Sinai Hospital, Toronto, Ontario, Canada

    Hana Sroka & David Chitayat

  5. Department of Pediatrics, Division of Clinical and Metabolic Genetics, The Hospital for Sick Children, Toronto, Ontario, Canada

    David Chitayat

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Contributions

J.D.C. prepared nascent DNAs, assessed the replication profiles and performed small-pool PCR and replication efficiency analyses; S.T. and L.F. performed mice handling and tissue isolation; G.B.P. performed in vitro replication reactions; A.L.C. performed methylation analysis; K.A.H. constructed efficiency templates; H.S. and D.C. obtained human patient samples; C.E.P., J.D.C. and G.G. designed the study; C.E.P., J.D.C., G.B.P., A.L.C., S.T. and G.G. interpreted the results; J.D.C. and C.E.P. wrote the paper.

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Correspondence to Christopher E Pearson.

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Cleary, J., Tomé, S., López Castel, A. et al. Tissue- and age-specific DNA replication patterns at the CTG/CAG-expanded human myotonic dystrophy type 1 locus. Nat Struct Mol Biol 17, 1079–1087 (2010). https://doi.org/10.1038/nsmb.1876

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