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Preventing gene silencing with human replicators

Nature Biotechnology volume 24, pages 572–576 (2006)Cite this article

Abstract

Transcriptional silencing, one of the major impediments to gene therapy in humans, is often accompanied by replication during late S-phase. We report that transcriptional silencing and late replication were prevented by DNA sequences that can initiate DNA replication (replicators). When replicators were included in silencing-prone transgenes, they did not undergo transcriptional silencing, replicated early and maintained histone acetylation patterns characteristic of euchromatin. A mutant replicator, which could not initiate replication, could not prevent gene silencing and replicated late when included in identical transgenes and inserted at identical locations. These observations suggest that replicators introduce epigenetic chromatin changes that facilitate initiation of DNA replication and affect gene silencing. Inclusion of functional replicators in gene therapy vectors may provide a tool for stabilizing gene expression patterns.

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Figure 1: A mammalian experimental system in which replication timing could be altered in a controlled manner.
Figure 2: A replication delay precedes transcriptional silencing of gene expression and chromatin condensation in a transgene inserted at the RL4 site.
Figure 3: Replication timing of transgenes at the RL4 site in MEL cells.
Figure 4: Replicators can prevent chromatin condensation at the RL4 site.

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References

  1. Gilbert, D.M. Replication timing and transcriptional control: beyond cause and effect. Curr. Opin. Cell Biol. 14, 377–383 (2002).

    Article  CAS  Google Scholar 

  2. Azuara, V. et al. Heritable gene silencing in lymphocytes delays chromatid resolution without affecting the timing of DNA replication. Nat. Cell Biol. 5, 668–674 (2003).

    Article  CAS  Google Scholar 

  3. Simon, I. et al. Developmental regulation of DNA replication timing at the human beta globin locus. EMBO J. 20, 6150–6157 (2001).

    Article  CAS  Google Scholar 

  4. Dhar, V., Skoultchi, A.I. & Schildkraut, C.L. Activation and repression of a beta-globin gene in cell hybrids is accompanied by a shift in its temporal replication. Mol. Cell. Biol. 9, 3524–3532 (1989).

    Article  CAS  Google Scholar 

  5. Ermakova, O.V. et al. Evidence that a single replication fork proceeds from early to late replicating domains in the IgH locus in a non-B cell line. Mol. Cell 3, 321–330 (1999).

    Article  CAS  Google Scholar 

  6. Mostoslavsky, R. et al. Asynchronous replication and allelic exclusion in the immune system. Nature 414, 221–225 (2001).

    Article  CAS  Google Scholar 

  7. Wutz, A. & Jaenisch, R. A shift from reversible to irreversible X inactivation is triggered during ES cell differentiation. Mol. Cell 5, 695–705 (2000).

    Article  CAS  Google Scholar 

  8. Lin, C.M., Fu, H., Martinovsky, M., Bouhassira, E. & Aladjem, M.I. Dynamic alterations of replication timing in mammalian cells. Curr. Biol. 13, 1019–1028 (2003).

    Article  CAS  Google Scholar 

  9. Feng, Y.Q. et al. The human beta-globin locus control region can silence as well as activate gene expression. Mol. Cell. Biol. 25, 3864–3874 (2005).

    Article  CAS  Google Scholar 

  10. Feng, Y.Q., Lorincz, M.C., Fiering, S., Greally, J.M. & Bouhassira, E.E. Position effects are influenced by the orientation of a transgene with respect to flanking chromatin. Mol. Cell. Biol. 21, 298–309 (2001).

    Article  CAS  Google Scholar 

  11. Aladjem, M.I. et al. Participation of the human beta-globin locus control region in initiation of DNA replication. Science 270, 815–819 (1995).

    Article  CAS  Google Scholar 

  12. Kalejta, R.F. et al. Distal sequences, but not ori-beta/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 

  13. Wang, L. et al. The human beta-globin replication initiation region consists of two modular independent replicators. Mol. Cell. Biol. 24, 3373–3386 (2004).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  15. Epner, E., Forrester, W.C. & Groudine, M. Asynchronous DNA replication within the human beta-globin gene locus. Proc. Natl. Acad. Sci. USA 85, 8081–8085 (1988).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  17. Paixao, S. et al. Modular structure of the human lamin B2 replicator. Mol. Cell. Biol. 24, 2958–2967 (2004).

    Article  CAS  Google Scholar 

  18. Aladjem, M.I. & Fanning, E. The replicon revisited: an old model learns new tricks in metazoan chromosomes. EMBO Rep. 5, 686–691 (2004).

    Article  CAS  Google Scholar 

  19. Kim, C.G., Epner, E.M., Forrester, W.C. & Groudine, M. Inactivation of the human beta-globin gene by targeted insertion into the beta-globin locus control region. Genes Dev. 6, 928–938 (1992).

    Article  CAS  Google Scholar 

  20. Saha, S., Shan, Y., Mesner, L.D. & Hamlin, J.L. The promoter of the Chinese hamster ovary dihydrofolate reductase gene regulates the activity of the local origin and helps define its boundaries. Genes Dev. 18, 397–410 (2004).

    Article  CAS  Google Scholar 

  21. Ghosh, M., Liu, G., Randall, G., Bevington, J. & Leffak, M. Transcription factor binding and induced transcription alter chromosomal c-myc replicator activity. Mol. Cell. Biol. 24, 10193–10207 (2004).

    Article  CAS  Google Scholar 

  22. Aggarwal, B.D. & Calvi, B.R. Chromatin regulates origin activity in Drosophila follicle cells. Nature 430, 372–376 (2004).

    Article  CAS  Google Scholar 

  23. Danis, E. et al. Specification of a DNA replication origin by a transcription complex. Nat. Cell Biol. 6, 721–730 (2004).

    Article  CAS  Google Scholar 

  24. Zhou, J. et al. The origin of a developmentally regulated Igh replicon is located near the border of regulatory domains for Igh replication and expression. Proc. Natl. Acad. Sci. USA 99, 13693–13698 (2002).

    Article  CAS  Google Scholar 

  25. Ellis, J. Silencing and variegation of gammaretrovirus and lentivirus vectors. Hum. Gene Ther. 16, 1241–1246 (2005).

    Article  CAS  Google Scholar 

  26. Bestor, T.H. Gene silencing as a threat to the success of gene therapy. J. Clin. Invest. 105, 409–411 (2000).

    Article  CAS  Google Scholar 

  27. Yao, S. et al. Retrovirus silencing, variegation, extinction, and memory are controlled by a dynamic interplay of multiple epigenetic modifications. Mol. Ther. 10, 27–36 (2004).

    Article  CAS  Google Scholar 

  28. Lorincz, M.C., Schubeler, D. & Groudine, M. Methylation-mediated proviral silencing is associated with MeCP2 recruitment and localized histone H3 deacetylation. Mol. Cell. Biol. 21, 7913–7922 (2001).

    Article  CAS  Google Scholar 

  29. Ellis, J. & Pannell, D. The beta-globin locus control region versus gene therapy vectors: a struggle for expression. Clin. Genet. 59, 17–24 (2001).

    Article  CAS  Google Scholar 

  30. Lorincz, M.C., Schubeler, D., Hutchinson, S.R., Dickerson, D.R. & Groudine, M. DNA methylation density influences the stability of an epigenetic imprint and Dnmt3a/b-independent de novo methylation. Mol. Cell. Biol. 22, 7572–7580 (2002).

    Article  CAS  Google Scholar 

  31. Mutskov, V. & Felsenfeld, G. Silencing of transgene transcription precedes methylation of promoter DNA and histone H3 lysine 9. EMBO J. 23, 138–149 (2004).

    Article  CAS  Google Scholar 

  32. Aladjem, M.I. et al. Replication initiation patterns in the beta-globin loci of totipotent and differentiated murine cells: evidence for multiple initiation regions. Mol. Cell. Biol. 22, 442–452 (2002).

    Article  CAS  Google Scholar 

  33. Bielinsky, A.K. & Gerbi, S.A. Discrete start sites for DNA synthesis in the yeast ARS1 origin. Science 279, 95–98 (1998).

    Article  CAS  Google Scholar 

  34. Kobayashi, T., Rein, T. & DePamphilis, M.L. Identification of primary initiation sites for DNA replication in the hamster dihydrofolate reductase gene initiation zone. Mol. Cell. Biol. 18, 3266–3277 (1998).

    Article  CAS  Google Scholar 

  35. Forsberg, E.C. et al. Developmentally dynamic histone acetylation pattern of a tissue-specific chromatin domain. Proc. Natl. Acad. Sci. USA 97, 14494–14499 (2000).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank Barbara J. Taylor, FACS Core Facility, Center for Cancer Research (CCR), National Cancer Institute (NCI), National Institutes of Health (NIH), for expert help with cell sorting experiments, Joseph O. Lopriaeto and Carla Valenzuela for help with plasmid construction, Kurt W. Kohn, Yves G. Pommier, William M. Bonner and Sohyoung Kim for critical reading of the manuscript, and James Ellis, Mel DePamphilis, Carl Schildkraut, Tal Kafri, Chava Kimchi-Zarfaty, Tsutomu Shimura, Jung-Hyun Kim and Elliot Epner for helpful discussions. This study was supported by the Intramural Research Program of the NIH, NCI, CCR (M.I.A.), by NIH grants R01 DK56845, P01 HL55435 (E.E.B.) and by the Howard Hughes Medical Institute/Montgomery County Public Schools internship program (S.S.).

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Author notes

  1. Haiqing Fu and Lixin Wang: These authors contributed equally to this work.

Authors and Affiliations

  1. Laboratory of Molecular Pharmacology, NCI, Bethesda, 20892, Maryland, USA

    Haiqing Fu, Lixin Wang, Chii-Mei Lin, Sumegha Singhania & Mirit I Aladjem

  2. Albert Einstein College of Medicine, Yeshiva University, Bronx, 10461, New York, USA

    Eric E Bouhassira

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  1. Haiqing Fu
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Correspondence to Mirit I Aladjem.

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Supplementary information

Supplementary Fig. 1

Time course of GFP expression of transgenes inserted into the RL4 site. (PDF 403 kb)

Supplementary Fig. 2

Replicators can prevent gene silencing at a the RL5 site on chromosome 4. (PDF 338 kb)

Supplementary Table 1

Primers and probes used in this study (PDF 99 kb)

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Fu, H., Wang, L., Lin, CM. et al. Preventing gene silencing with human replicators. Nat Biotechnol 24, 572–576 (2006). https://doi.org/10.1038/nbt1202

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