RNA-Mediated Transcriptional Gene Silencing in Human Cells

  • Kevin V. Morris
Part of the Current Topics in Microbiology and Immunology book series (CT MICROBIOLOGY, volume 320)


The utilization of small interfering RNAs (siRNAs) represents a new paradigm in gene knockout technology. siRNAs can be used to knockdown the expression of a particular gene by targeting the mRNA in a post-transcriptional manner. While there are a plethora of reports applying siRNA-mediated post-transcriptional silencing (PTGS) therapeutically there are apparent limitations such as the duration of the effect and a saturation of the RNA-induced silencing complex (RISC). Recently, data have emerged that indicate an alternative pathway is operative in human cells where siRNAs have been shown, similar to plants, Drosophila, C. elegans, and S. Pombe, to mediate transcriptional gene silencing (TGS). TGS is operative by the antisense strand of the siRNA targeting chromatin remodeling complexes to the specific promoter region(s). This siRNA targeting results in epigenetic modifications that lead to a rewriting of the local histone code, silent state chromatin marks, and ultimately heterochromatization of the targeted gene. The observation that siRNA-directed TGS is operative via epigenetic modifications suggests that similar to plants, and S. Pombe, human genes may also be able to be silenced more permanently or for longer periods following a single treatment and may in fact offer a new therapeutic avenue that could prove robust and of immeasurable therapeutic value in the directed control of target gene expression.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Bernstein E, Caudy AA, Hammond SM, Hannon GJ (2001) Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409:363–366.PubMedCrossRefGoogle Scholar
  2. Boeger H, Bushnell DA, Davis R, Griesenbeck J, Lorch Y, Strattan JS, Westover KD, Kornberg RD (2005) Structural basis of eukaryotic gene transcription. FEBS Lett 579:899–903.PubMedCrossRefGoogle Scholar
  3. Bühler M, Mohn F, Stalder L, Mühlemann O (2005) Transcriptional silencing of nonsense codon-containing immunoglobulin minigenes. Mol Cell 18:307–317.PubMedCrossRefGoogle Scholar
  4. Castanotto D, Tommasi S, Li M, Li H, Yanow S, Pfeifer GP, Rossi JJ (2005) Short hairpin RNA-directed cytosine (CpG) methylation of the RASSF1A gene promoter in HeLa cells. Mol Ther 12:179–183.PubMedCrossRefGoogle Scholar
  5. Chicas A, Forrest EC, Sepich S, Cogoni C, Macino G (2005) Small interfering RNAs that trigger posttranscriptional gene silencing are not required for the histone H3 Lys9 methylation necessary for transgenic tandem repeat stabilization in Neurospora crassa. Mol Cell Biol 25:3793–3801.PubMedCrossRefGoogle Scholar
  6. Dernburg AF, Zalevsky J, Colaiácovo MP, Villeneuve AM (2000) Transgene-mediated cosuppression in the C. elegans germ line. Genes Dev 14:1578–1583.PubMedGoogle Scholar
  7. Fagard M, Boutet S, Morel JB, Bellini C, Vaucheret H (2000) AGO1, QDE-2, and RDE-1 are related proteins required for post-transcriptional gene silencing in plants, quelling in fungi, and RNA interference in animals. Proc Natl Acad Sci U S A 97:11650–11654.PubMedCrossRefGoogle Scholar
  8. Fuks F, Burgers WA, Brehm A, Hughes-Davies L, Kouzarides T (2000) DNA methyltransferase Dnmt1 associates with histone deacetylase activity. Nat Genet 24:88–91.PubMedCrossRefGoogle Scholar
  9. Fuks F, Burgers WA, Godin N, Kasai M, Kouzarides T (2001) Dnmt3a binds deacetylases and is recruited by a sequence-specific repressor to silence transcription. EMBO J 20:2536–2544.PubMedCrossRefGoogle Scholar
  10. Fuks F, Hurd PJ, Deplus R, Kouzarides T (2002) Histone modifications in transcriptional regulation. Curr Opin Genet Dev 12:142–148.CrossRefGoogle Scholar
  11. Fuks F, Hurd PJ, Deplus R, Kouzarides T (2003) The DNA methyltransferases associate with HP1 and the SUV39H1 histone methyltransferase. Nucleic Acids Res 31:2305–2312.PubMedCrossRefGoogle Scholar
  12. Grishok A, Pasquinelli AE, Conte D, Li N, Parrish S, Ha I, Baillie DL, Fire A, Ruvkun G, Mello CC (2001) Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell 106:23–34.PubMedCrossRefGoogle Scholar
  13. Grishok A, Sinskey JL, Sharp PA (2005) Transcriptional silencing of a transgene by RNAi in the soma of C. elegans. Genes Dev 19:683–696.PubMedCrossRefGoogle Scholar
  14. Hall IM, Shankaranarayana GD, Noma K, Ayoub N, Cohen A, Grewal SIS (2002) Establishment and maintenance of a heterochromatin domain. Science 297:2232–2237.PubMedCrossRefGoogle Scholar
  15. Hamilton A, Voinnet O, Chappell L, Baulcombe D (2002) Two classes of short interfering RNA in RNA silencing. EMBO J 21:4671–4679.PubMedCrossRefGoogle Scholar
  16. Irvine DV, Zaratiegui M, Tolia NH, Goto DB, Chitwood DH, Vaughn MW, Joshua-Tor L, Martienssen RA (2006) Argonaute slicing is required for heterochromatic silencing and spreading. Science 313:1134–1137.PubMedCrossRefGoogle Scholar
  17. Janowski BA, Huffman KE, Schwartz JC, Ram R, Hardy D, Shames DS, Minna JD, Corey DR (2005) Inhibiting gene expression at transcription start sites in chromosomal DNA with antigene RNAs. Nat Chem Biol 1:210–215.PubMedCrossRefGoogle Scholar
  18. Janowski BA, Huffman KE, Schwartz JC, Ram R, Nordsell R, Shames DS, Minna JD, Corey DR (2006) Involvement of AGO1 and AGO2 in mammalian transcriptional silencing. Nat Struct Mol Biol 13:787–792.PubMedCrossRefGoogle Scholar
  19. Jeffery L, Nakielny S (2004) Components of the DNA methylation system of chromatin control are RNA-binding proteins. J Biol Chem 279:49479–49487.PubMedCrossRefGoogle Scholar
  20. Jenuwein T, Allis CD (2001) The histone code. Science 293:1074–1080.PubMedCrossRefGoogle Scholar
  21. Katayama S, Tomaru Y, Kasukawa T, Waki K, Nakanishi M, Nakamura M, Nishida H, Yap CC, Suzuki M, Kawai J, Suzuki H, Carninci P, Hayashizaki Y, Wells C, Frith M, Ravasi T, Pang KC, Hallinan J, Mattick J, Hume DA, Lipovich L, Batalov S, Engström PG, Mizuno Y, Faghihi MA, Sandelin A, Chalk AM, Mottagui-Tabar S, Liang Z, Lenhard B, Wahlestedt C, et al (2005) Antisense transcription in the mammalian transcriptome. Science 309:1564–1566.PubMedCrossRefGoogle Scholar
  22. Kato H, Goto DB, Martienssen RA, Urano T, Furukawa K, Murakami Y (2005) RNA polymerase II is required for RNAi-dependent heterochromatin assembly. Science 309:467–469.PubMedCrossRefGoogle Scholar
  23. Ketting RF, Plasterk RH (2000) A genetic link between co-suppression and RNA interference in C. elegans. Nature 404:296–298.PubMedCrossRefGoogle Scholar
  24. Kim DH, Villeneuve LM, Morris KV, Rossi JJ (2006) Argonaute-1 directs siRNA-mediated transcriptional gene silencing in human cells. Nat Struct Mol Biol 13:793–797.PubMedCrossRefGoogle Scholar
  25. Langlois MA, Boniface C, Wang G, Alluin J, Salvaterra PM, Puymirat J, Rossi JJ, Lee NS (2005) Cytoplasmic and nuclear retained DMPK mRNAs are targets for RNA interference in myotonic dystrophy cells. J Biol Chem 280:16949–16954.PubMedCrossRefGoogle Scholar
  26. Lee Y, Hur I, Park SY, Kim YK, Suh MR, Kim VN (2006) The role of PACT in the RNA silencing pathway. EMBO J 25:522–532.PubMedCrossRefGoogle Scholar
  27. Lippman Z, May B, Yordan C, Singer T, Martienssen R (2003) Distinct mechanisms determine transposon inheritance and methylation via small interfering RNA and histone modification. PLoS Biol 1:E67.PubMedCrossRefGoogle Scholar
  28. Lippman Z, Gendrel AV, Black M, Vaughn MW, Dedhia N, McCombie WR, Lavine K, Mittal V, May B, Kasschau KD, Carrington JC, Doerge RW, Colot V, Martienssen R (2004) Role of transposable elements in heterochromatin and epigenetic control. Nature 430:471–476.PubMedCrossRefGoogle Scholar
  29. Liu J, Carmell MA, Rivas FV, Marsden CG, Thomson JM, Song JJ, Hammond SM, Joshua-Tor L, Hannon GJ (2004) Argonaute2 Is the catalytic engine of mammalian RNAi. Science 305:1437–1441.PubMedCrossRefGoogle Scholar
  30. Lusser A (2002) Acetylated, methylated, remodeled: chromatin states for gene regulation. Curr Opin Plant Biol 5:437–443.PubMedCrossRefGoogle Scholar
  31. Maison C, Bailly D, Peters AH, Quivy JP, Roche D, Taddei A, Lachner M, Jenuwein T, Almouzni G (2002) Higher-order structure in pericentric heterochromatin involves a distinct pattern of histone modification and an RNA component. Nat Genet 30:329–334.PubMedCrossRefGoogle Scholar
  32. Matzke MA, Birchler JA (2005) RNAi-mediated pathways in the nucleus. Nat Rev Genet 6:24–35.PubMedCrossRefGoogle Scholar
  33. Matzke MA, Primig M, Trnovsky J, Matzke AJM (1989) Reversible methylation and inactivation of marker genes in sequentially transformed tobacco plants. EMBO J 8:643–649.PubMedGoogle Scholar
  34. Mette MF, Aufsatz W, Van der Winden J, Matzke AJM, Matzke MA (2000) Transcriptional silencing and promoter methylation triggered by double-stranded RNA. EMBO J 19:5194–5201.PubMedCrossRefGoogle Scholar
  35. Morris KV (2005) siRNA-mediated transcriptional gene silencing: the potential mechanism and a possible role in the histone code. Cell Mol Life Sci 62:3057–3066.PubMedCrossRefGoogle Scholar
  36. Morris KV (2006) Therapeutic potential of siRNA-mediated transcriptional gene silencing. Biotechniques [Suppl]:7–13.Google Scholar
  37. Morris KV, Chan SW, Jacobsen SE, Looney DJ (2004) Small interfering RNA-induced transcriptional gene silencing in human cells. Science 305:1289–1292.PubMedCrossRefGoogle Scholar
  38. Motamedi MR, Verdel A, Colmenares SU, Gerber SA, Gygi SP, Moazed D (2004) Two RNAi complexes, RITS and RDRC, physically interact and localize to noncoding centromeric RNAs. Cell 119:789–802.PubMedCrossRefGoogle Scholar
  39. Muchardt C, Guilleme M, Seeler JS, Trouche D, Dejean A, Yaniv M (2002) Coordinated methyl and RNA binding is required for heterochromatin localization of mammalian HP1alpha. EMBO Rep 3:975–981.PubMedCrossRefGoogle Scholar
  40. Pal-Bhadra M, Bhadra U, Birchler JA (2002) RNAi related mechanisms affect both transcriptional and posttranscriptional transgene silencing in Drosophila. Mol Cell 9:315–327.PubMedCrossRefGoogle Scholar
  41. Park CW, Chen Z, Kren BT, Steer CJ (2004) Double-stranded siRNA targeted to the huntingtin gene does not induce DNA methylation. Biochem Biophys Res Commun 323:275–280.PubMedCrossRefGoogle Scholar
  42. Rigoutsos I, Huynh T, Miranda K, Tsirigos A, McHardy A, Platt D (2006) Short blocks from the noncoding parts of the human genome have instances within nearly all known genes and relate to biological processes. Proc Natl Acad Sci U S A 103:6605–6610.PubMedCrossRefGoogle Scholar
  43. Robb GB, Brown KM, Khurana J, Rana TM (2005) Specific and potent RNAi in the nucleus of human cells. Nat Struct Mol Biol 12:133–137.PubMedCrossRefGoogle Scholar
  44. Romano NG (1992) Macino, Quelling: transient inactivation of gene expression in Neurospora crassa by transformation with homologous sequences. Mol Microbiol 6:3343–3353.PubMedCrossRefGoogle Scholar
  45. Ruby JG, Jan C, Player C, Axtell MJ, Lee W, Nusbaum C, Ge H, Bartel DP (2006) Large-scale sequencing reveals 21U-RNAs and additional microRNAs and endogenous siRNAs in C. Elegans. Cell 127:1193–1207.PubMedCrossRefGoogle Scholar
  46. Sijen T, Vign I, Rebocho A, et al (2001) Transcriptional and posttranscriptional gene silencing are mechanistically related. Curr Biol 11:436–440.PubMedCrossRefGoogle Scholar
  47. Song JJ, Smith SK, Hannon GJ, Joshua-Tor L (2004) Crystal structure of Argonaute and its implications for RISC slicer activity. Science 305:1434–1437.PubMedCrossRefGoogle Scholar
  48. Strahl BD, Ohba R, Cook RG, Allis CD (1999) Methylation of histone H3 at lysine 4 is highly conserved and correlates with transcriptionally active nuclei in tetrahymena. Proc Natl Acad Sci U S A 96:14967–14972.PubMedCrossRefGoogle Scholar
  49. Suzuki K, Shijuuku T, Fukamachi T, Zaunders J, Guillemin G, Cooper D, Kelleher A (2005) Prolonged transcriptional silencing and CpG methylation induced by siRNAs targeted to the HIV-1 promoter region. J RNAi Gene Silencing 1:66–78.PubMedGoogle Scholar
  50. Svoboda P, Stein P, Filipowicz W, Schultz RM (2004) Lack of homologous sequence-specific DNA methylation in response to stable dsRNA expression in mouse oocytes. Nucleic Acids Res 32:3601–3606.PubMedCrossRefGoogle Scholar
  51. Tabara H, Sarkissian M, Kelly WG, Fleenor J, Grishok A, Timmons L, Fire A, Mello CC (1999) The rde-1 gene, RNA interference, and transposon silencing in C. elegans. Cell 99:123–132.PubMedCrossRefGoogle Scholar
  52. Tijsterman M, Ketting RF, Plasterk RH (2002) The genetics of RNA silencing. Annu Rev Genet 36:489–519.PubMedCrossRefGoogle Scholar
  53. Ting AH, Schuebel KE, Herman JG, Baylin SB (2005) Short double-stranded RNA induces transcriptional gene silencing in human cancer cells in the absence of DNA methylation. Nat Genet 37:906–910.PubMedCrossRefGoogle Scholar
  54. Turner BM (2000) Histone acetylation and an epigenetic code. BioEssays 22:836–845.PubMedCrossRefGoogle Scholar
  55. Vastenhouw NL, Brunschwig K, Okihara KL, Muller F, Tijsterman M, Plasterk RH (2006) Gene expression: long-term gene silencing by RNAi. Nature 442:882.PubMedCrossRefGoogle Scholar
  56. Verdel A, Jia S, Gerber S, Sugiyama T, Gygi S, Grewal SI, Moazed D (2004) RNAi-mediated targeting of heterochromatin by the RITS complex. Science 303:672–676.PubMedCrossRefGoogle Scholar
  57. Viré E, Brenner C, Deplus R, Blanchon L, Fraga M, Didelot C, Morey L, Van Eynde A, Bernard D, Vanderwinden JM, Bollen M, Esteller M, Di Croce L, de Launoit Y, Fuks F (2005) The Polycomb group protein EZH2 directly controls DNA methylation. Nature 439:871–874.PubMedCrossRefGoogle Scholar
  58. Volpe TA, Kidner C, Hall IM, Teng G, Grewal SIS, Martienssen RA (2002) Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi. Science 297:1833–1837.PubMedCrossRefGoogle Scholar
  59. Wassenegger M, Graham MW, Wang MD (1994) RNA-directed de novo methylation of genomic sequences in plants. Cell 76:567–576.PubMedCrossRefGoogle Scholar
  60. Weinberg MS, Villeneuve LM, Ehsani A, Amarzguioui M, Aagaard L, Chen Z, Riggs AD, Rossi JJ, Morris KV (2005) The antisense strand of small interfering RNAs directs histone methylation and transcriptional gene silencing in human cells. RNA 12:256–262.PubMedCrossRefGoogle Scholar
  61. Zhang M, Ou H, Shen YH, Wang J, Wang J, Coselli J, Wang XL (2005) Regulation of endothelial nitric oxide synthase by small RNA. Proc Natl Acad Sci U S A 102:16967–16972.PubMedCrossRefGoogle Scholar
  62. Zilberman D, Cao X, Jacobsen SE (2003) Argonaute4 control of locus-specific siRNA accumulation and DNA and histone methylation. Science 299:716–719.PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2008

Authors and Affiliations

  • Kevin V. Morris
    • 1
  1. 1.Department of Molecular and Experimental MedicineThe Scripps Research InstituteLa JollaUSA

Personalised recommendations