1887

Abstract

In the plant pathogen , nuclear integration of transgenic DNA sequences results in internuclear gene silencing of . Although silencing is regulated at the transcriptional level, it also affects transcription from other nuclei within heterokaryotic cells of the mycelium. Here we report experiments exploring the mechanism of internuclear gene silencing in . The DNA methylation inhibitor 5-azacytidine induced reversion of the -silenced state. Also, the histone deacetylase inhibitor trichostatin-A was able to reverse silencing. -expression levels returned to the silenced state when the inhibitors were removed except in non-transgenic -silenced strains that were generated via internuclear gene silencing, where expression was restored permanently. Therefore, -transgenic sequences are required to maintain the silenced state. Prolonged culture of non-transgenic -silenced strains resulted in gradual reactivation of gene expression. Nuclease digestion of -silenced and non-silenced nuclei showed that sequences in silenced nuclei were less rapidly degraded than non-silenced sequences. Bisulfite sequencing of the endogenous locus did not result in detection of any cytosine methylation. Our findings suggest that the -silenced state is based on chromatin remodelling.

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2008-05-01
2024-12-13
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References

  1. Ayer D. E. 1999; Histone deacetylases: transcriptional repression with SINers and NuRDs. Trends Cell Biol 9:193–198
    [Google Scholar]
  2. Baulcombe D. 2002; RNA silencing. Curr Biol 12:R82–R84
    [Google Scholar]
  3. Beclin C., Boutet S., Waterhouse P., Vaucheret H. 2002; A branched pathway for transgene-induced RNA silencing in plants. Curr Biol 12:684–688
    [Google Scholar]
  4. Bird A. 2002; DNA methylation patterns and epigenetic memory. Genes Dev 16:6–21
    [Google Scholar]
  5. Bird A. P., Wolffe A. P. 1999; Methylation-induced repression – belts, braces, and chromatin. Cell 99:451–454
    [Google Scholar]
  6. Brodersen P., Voinnet O. 2006; The diversity of RNA silencing pathways in plants. Trends Genet 22:268–280
    [Google Scholar]
  7. Cerutti H. 2003; RNA interference: traveling in the cell and gaining functions?. Trends Genet 19:39–46
    [Google Scholar]
  8. Chandler V. L., Eggleston W. B., Dorweiler J. E. 2000; Paramutation in maize. Plant Mol Biol 43:121–145
    [Google Scholar]
  9. Cogoni C., Macino G. 1999; Homology-dependent gene silencing in plants and fungi: a number of variations on the same theme. Curr Opin Microbiol 2:657–662
    [Google Scholar]
  10. Cogoni C., Irelan J. T., Schumacher M., Schmidhauser T. J., Selker E. U., Macino G. 1996; Transgene silencing of the al -1 gene in vegetative cells of Neurospora is mediated by a cytoplasmic effector and does not depend on DNA–DNA interactions or DNA. EMBO J 15:3153–3163
    [Google Scholar]
  11. Dalmay T., Hamilton A., Rudd S., Angell S., Baulcombe D. C. 2000; An RNA-dependent RNA polymerase gene in Arabidopsis is required for posttranscriptional gene silencing mediated by a transgene but not by a virus. Cell 101:543–553
    [Google Scholar]
  12. Fire A., Xu S.-Q., Montgomery M. K., Kostas S. A., Driver S. E., Mello C. C. 1998; Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans . Nature 391:806–811
    [Google Scholar]
  13. Fischle W., Wang Y., Allis C. D. 2003; Histone and chromatin cross-talk. Curr Opin Cell Biol 15:172–183
    [Google Scholar]
  14. Grewal S. I. S., Elgin S. C. R. 2002; Heterochromatin: new possibilities for the inheritance of structure. Curr Opin Genet Dev 12:178–187
    [Google Scholar]
  15. Henikoff S. 1998; Conspiracy of silence among repeated transgenes. Bioessays 20:532–535
    [Google Scholar]
  16. Hollick J. B., Dorweiler J. E., Chandler V. L. 1997; Paramutation and related allelic interactions. Trends Genet 13:302–308
    [Google Scholar]
  17. Judelson H. S., Whittaker L. S. 1995; Inactivation of transgenes in Phytophthora infestans is not associated with their deletion, methylation, or mutation. Curr Genet 28:571–579
    [Google Scholar]
  18. Kass S. U., Pruss D., Wolffe A. P. 1997; How does DNA methylation repress transcription?. Trends Genet 13:444–449
    [Google Scholar]
  19. Katan-Khaykovich Y., Struhl K. 2002; Dynamics of global histone acetylation and deacetylation in vivo : rapid restoration of normal histone acetylation status upon removal of activators and repressors. Genes Dev 16:743–752
    [Google Scholar]
  20. Kelly W. G., Fire A. 1998; Chromatin silencing and the maintenance of a functional germline in Caenorhabditis elegans . Development 125:2451–2456
    [Google Scholar]
  21. Kooter J. M., Matzke M. A., Meyer P. 1999; Listening to the silent genes: transgene silencing, gene regulation and pathogen control. Trends Plant Sci 4:340–347
    [Google Scholar]
  22. Lu R., Martin-Hernandez A. M., Peart J. R., Malcuit I., Baulcombe D. C. 2003; Virus-induced gene silencing in plants. Methods 30:296–303
    [Google Scholar]
  23. Lyko F. 2001; DNA methylation learns to fly. Trends Genet 17:169–172
    [Google Scholar]
  24. Lyko F., Ramsahoye B. H., Jaenisch R. 2000; DNA methylation in Drosophila melanogaster . Nature 408:538–540
    [Google Scholar]
  25. Mette M. F., Aufsatz W., van der Winden J., Matzke M. A., Matzke A. J. 2000; Transcriptional silencing and promoter methylation triggered by double-stranded RNA. EMBO J 19:5194–5201
    [Google Scholar]
  26. Morris K. V., Chan S. W., Jacobsen S. E., Looney D. J. 2004; Small interfering RNA-induced transcriptional gene silencing in human cells. Science 305:1289–1292
    [Google Scholar]
  27. Nakayashiki H. 2005; RNA silencing in fungi: mechanisms and applications. FEBS Lett 579:5950–5957
    [Google Scholar]
  28. Nykänen A., Haley B., Zamore P. D. 2001; ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell 107:309–321
    [Google Scholar]
  29. Pickford A. S., Cogoni C. 2003; RNA-mediated gene silencing. Cell Mol Life Sci 60:871–882
    [Google Scholar]
  30. Plasterk R. H., Ketting R. F. 2000; The silence of the genes. Curr Opin Genet Dev 10:562–567
    [Google Scholar]
  31. Raeder U., Broda P. 1985; Rapid preparation of DNA from filamentous fungi. Lett Appl Microbiol 1:17–20
    [Google Scholar]
  32. Reinhart B. J., Bartel D. P. 2002; Small RNAs correspond to centromere heterochromatic repeats. Science 297:1831
    [Google Scholar]
  33. Roth S. Y. 1995; Chromatin-mediated transcriptional repression in yeast. Curr Opin Genet Dev 5:168–173
    [Google Scholar]
  34. Ruiz M. T., Voinnet O., Baulcombe D. C. 1998; Initiation and maintenance of virus-induced gene silencing. Plant Cell 10:937–946
    [Google Scholar]
  35. Ryner L. C., Swain A. 1995; Sex in the '90s. Cell 81:483–493
    [Google Scholar]
  36. Selker E. U. 1997; Epigenetic phenomena in filamentous fungi: useful paradigms or repeat-induced confusion. Trends Genet 13:296–301
    [Google Scholar]
  37. Selker E. U. 1998; Trichostatin A causes selective loss of DNA methylation in Neurospora . Proc Natl Acad Sci U S A 95:9430–9435
    [Google Scholar]
  38. Struhl K. 1998; Histone acetylation and transcriptional regulatory mechanisms. Genes Dev 12:599–606
    [Google Scholar]
  39. Tyler J. K., Kadonaga J. T. 1999; The ‘dark side’ of chromatin remodelling: repressive effects on transcription. Cell 99:443–446
    [Google Scholar]
  40. Unkles S. E., Moon R. P., Hawkins A. R., Duncan J. M., Kinghorn J. R. 1991; Actin in the oomycetous fungus Phytophthora infestans is the product of several genes. Gene 100:105–112
    [Google Scholar]
  41. Vaistij F. E., Jones L., Baulcombe D. C. 2002; Spreading of RNA targeting and DNA methylation in RNA silencing requires transcription of the target gene and a putative RNA-dependent RNA polymerase. Plant Cell 14:857–867
    [Google Scholar]
  42. van Blokland R., ten Lohuis M., Meyer P. 1997; Condensation of chromatin in transcriptional regions of an inactivated plant transgene: evidence for an active role of transcription in gene silencing. Mol Gen Genet 257:1–13
    [Google Scholar]
  43. van der Krol A. R., Mur L. A., Beld M., Mol J. N., Stuitje A. R. 1990; Flavonoid genes in petunia: addition of a limited number of gene copies may lead to a suppression of gene expression. Plant Cell 2:291–299
    [Google Scholar]
  44. van West P., de Jong A. J., Judelson H. S., Emons A. M. C., Govers F. 1998; The ipiO gene of Phytophthora infestans is highly expressed in invading hyphae during infection. Fungal Genet Biol 23:126–138
    [Google Scholar]
  45. van West P., Kamoun S., van't Klooster J. W., Govers F. 1999; Internuclear gene silencing in Phytophthora infestans . Mol Cell 3:339–348
    [Google Scholar]
  46. Vaucheret H., Beclin C., Elmayan T., Feuerbach F., Godon C., Morel J. B., Mourrain P., Palauqui J. C., Vernhettes S. 1998; Transgene-induced gene silencing in plants. Plant J 16:651–659
    [Google Scholar]
  47. Vermaak D., Ahmad K., Henikoff S. 2003; Maintenance of chromatin states: an open-and-shut case. Curr Opin Cell Biol 15:266–274
    [Google Scholar]
  48. Vogelauer M., Wu J., Suka N., Grunstein M. 2000; Global histone acetylation and deacetylation in yeast. Nature 408:495–498
    [Google Scholar]
  49. Volpe T. A., Kidner C., Hall I. M., Teng G., Grewal S. I., Martienssen R. A. 2002; Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi. Science 297:1833–1837
    [Google Scholar]
  50. Wada H., Kagoshima M., Ito K., Barnes P. J., Adcock I. M. 2005; 5-Azacytidine suppresses RNA polymerase II recruitment to the SLPI gene. Biochem Biophys Res Commun 331:93–99
    [Google Scholar]
  51. Waterborg J. H. 1998; Dynamics of histone acetylation in Chlamydomonas reinhardtii . J Biol Chem 273:27602–27609
    [Google Scholar]
  52. Weissmann F., Lyko F. 2003; Cooperative interactions between epigenetic modifications and their function in the regulation of chromosome architecture. Bioessays 25:792–797
    [Google Scholar]
  53. Whisson S. C., Avrova A. O., van West P., Jones J. T. 2005; A method for double-stranded RNA-mediated transient gene silencing in Phytophthora infestans . Mol Plant Pathol 6:153–163
    [Google Scholar]
  54. Wozniak R. J., Klimecki W. T., Lau S. S., Feinstein Y., Futscher B. W. 2006; 5-Aza-2-deoxycytidine-mediated reductions in G9A histone methyltransferase and histone H3 K9 di-methylation levels are linked to tumor suppressor gene reactivation. Oncogene 26:77–90
    [Google Scholar]
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