Skip to main content
SpringerLink
Log in

Minimizing the unpredictability of transgene expression in plants: the role of genetic insulators

  • Review
  • Published:
Plant Cell Reports Aims and scope Submit manuscript

Abstract

The genetic transformation of plants has become a necessary tool for fundamental plant biology research, as well as the generation of engineered plants exhibiting improved agronomic and industrial traits. However, this technology is significantly hindered by the fact that transgene expression is often highly variable amongst independent transgenic lines. Two of the major contributing factors to this type of inconsistency are inappropriate enhancer-promoter interactions and chromosomal position effects, which frequently result in mis-expression or silencing of the transgene, respectively. Since the precise, often tissue-specific, expression of the transgene(s) of interest is often a necessity for the successful generation of transgenic plants, these undesirable side effects have the potential to pose a major challenge for the genetic engineering of these organisms. In this review, we discuss strategies for improving foreign gene expression in plants via the inclusion of enhancer-blocking insulators, which function to impede enhancer-promoter communication, and barrier insulators, which block the spread of heterochromatin, in transgenic constructs. While a complete understanding of these elements remains elusive, recent studies regarding their use in genetically engineered plants indicate that they hold great promise for the improvement of transgene expression, and thus the future of plant biotechnology.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1

Similar content being viewed by others

Abbreviations

35S :

35S cauliflower mosaic virus promoter/enhancer

EXOB :

1-kb EcoRI/SalI fragment from bacteriophage lambda

MAR:

Matrix attachment region

TBS :

Transformation booster sequence

References

  • Abhyankar MM, Urekar C, Reddi PP (2007) A novel CpG-free vertebrate insulator silences the testis-specific SP-10 gene in somatic tissues: role for TDP-43 in insulator function. J Biol Chem 282:36143–36154

    Article  PubMed  CAS  Google Scholar 

  • Akasaka K, Nishimura A, Takata K, Mitsunaga K, Mibuka F, Ueda H, Hirose S, Tsutsui K, Shimada H (1999) Upstream element of the sea urchin arylsulfatase gene serves as an insulator. Cell Mol Biol (Noisy-le-grand) 45:555–565

    CAS  Google Scholar 

  • Allen GC (2009) The role of nuclear matrix attachment regions in plants. Plant Cell Monogr 14:101–129

    Article  CAS  Google Scholar 

  • Allen GC, Hall GE, Childs LC, Weissinger AK, Spiker S, Thompson WF (1993) Scaffold attachment regions increase reporter gene expression in stably transformed plant cells. Plant Cell 5:603–613

    Article  PubMed  CAS  Google Scholar 

  • Allen GC, Hall GE Jr, Michalowski S, Newman W, Spiker S, Weissinger AK, Thompson WF (1996) High-level transgene expression in plant cells: effects of a strong scaffold attachment region from tobacco. Plant Cell 8:899–913

    Article  PubMed  CAS  Google Scholar 

  • Allen GC, Spiker S, Thompson WF (2000) Use of matrix attachment regions (MARs) to minimize transgene silencing. Plant Mol Biol 43:361–376

    Article  PubMed  CAS  Google Scholar 

  • Antes TJ, Namciu SJ, Fournier REK, Levy-Wilson B (2001) The 5′ boundary of the human apolipoprotein B chromatin domain in intestinal cells. Biochemistry 40:6731–6742

    Article  PubMed  CAS  Google Scholar 

  • Avramova Z, Bennetzen JL (1993) Isolation of matrices from maize leaf nuclei: identification of matrix-binding site adjacent to the Adh1 gene. Plant Mol Biol 22:1135–1143

    Article  PubMed  CAS  Google Scholar 

  • Avramova Z, San Miguel P, Georgieva E, Bennetzen JL (1995) Matrix attachment regions and transcribed sequences within a long chromosomal continuum containing maize Adh1. Plant Cell 7:1667–1680

    Article  PubMed  CAS  Google Scholar 

  • Bae E, Calhoun VC, Levine M, Lewis EB, Drewell RA (2002) Characterization of the intergenic RNA profile at abdominal-A and Abdominal-B in the Drosophila bithorax complex. Proc Natl Acad Sci USA 99:16847–16852

    Article  PubMed  CAS  Google Scholar 

  • Barges S, Mihaly J, Galloni M, Hagstrom K, Muller M, Shanower G, Schedl P, Gyurkovics H, Karch F (2000) The Fab-8 boundary defines the distal limit of the bithorax complex iab-7 domain and insulates iab-7 from initiation elements and a PRE in the adjacent iab-8 domain. Development 127:779–790

    PubMed  CAS  Google Scholar 

  • Bell AC, Felsenfeld G (2000) Methylation of a CTCF-dependent boundary controls imprinted expression of the Igf2 gene. Nature 405:482–485

    Article  PubMed  CAS  Google Scholar 

  • Bell AC, West AG, Felsenfeld G (1999) The protein CTCF is required for the enhancer blocking activity of vertebrate insulators. Cell 98:387–396

    Article  PubMed  CAS  Google Scholar 

  • Bernstein BE, Mikkelsen TS, Xie X, Kamal M, Huebert DJ, Cuff J, Fry B, Meissner A, Wernig M, Plath K, Jaenisch R, Wagschal A, Feil R, Schreiber SL, Lander ES (2006) A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell 125:315–326

    Article  PubMed  CAS  Google Scholar 

  • Bhat SR, Srinivasan S (2002) Molecular and genetic analyses of transgenic plants: considerations and approaches. Plant Sci 164:673–681

    Article  Google Scholar 

  • Bi X, Broach JR (2006) UASrpg can function as a heterochromatin boundary element in yeast. Genes Dev 13:1089–1101

    Article  Google Scholar 

  • Blanton J, Gaszner M, Schedl P (2003) Protein-protein interactions and the pairing of boundary elements in vivo. Genes Dev 17:664–675

    Article  PubMed  CAS  Google Scholar 

  • Bode J, Benham C, Knopp A, Mielke C (2000) Transcriptional augmentation: modulation of gene expression by scaffold/matrix-attached regions (S/MAR elements). Crit Rev Eukaryot Gene 10:73–90

    CAS  Google Scholar 

  • Brasset E, Hermant C, Jensen S, Vaury C (2010) The Idefix enhancer-blocking insulator also harbors barrier activity. Gene 450:25–31

    Article  PubMed  CAS  Google Scholar 

  • Breyne P, van Montagu M, Depicker A, Gheysen G (1992) Characterization of a plant scaffold attachment region in a DNA fragment that normalizes transgene expression in tobacco. Plant Cell 4:463–471

    Article  PubMed  CAS  Google Scholar 

  • Brouwer C, Bruce W, Maddock S, Avramova Z, Bowen B (2002) Suppression of transgene silencing by matrix attachment regions in maize: a dual role for the maize 5′ ADH1 matrix attachment region. Plant Cell 14:2251–2264

    Article  PubMed  CAS  Google Scholar 

  • Buising CM, Benbow RM (1994) Molecular analysis of transgenic plants generated by microprojectile bombardment: effect of petunia transformation booster sequence. Mol Gen Genet 243:71–81

    Article  PubMed  CAS  Google Scholar 

  • Butaye KMJ, Goderis IJWM, Wouters PFJ, Pues JM-TG, Delauré SL, Broekaert WF, Depicker A, Cammue BPA, De Bolle MFC (2004) Stable high-level transgene expression in Arabidopsis thaliana using gene silencing mutants and matrix attachment regions. Plant J 39:440–449

    Article  PubMed  CAS  Google Scholar 

  • Butaye KMJ, Cammue BPA, Delauré De, Bolle MFC (2005) Approaches to minimize variation of transgene expression in plants. Mol Breed 16:79–91

    Article  Google Scholar 

  • Carabana J, Watanabe A, Hao B, Krangel MS (2011) A barrier-type insulator forms a boundary between active and inactive chromatin at the murine TCRβ locus. J Immunol 186:3556–3562

    Article  PubMed  CAS  Google Scholar 

  • Cheng Z, Targolli J, Wu R (2001) Tobacco matrix attachment region sequence increased transgene expression levels in rice plants. Mol Breed 7:317–327

    Article  CAS  Google Scholar 

  • Chinn AM, Comai L (1996) The heat shock cognate 80 gene of tomato is flanked by matrix attachment regions. Plant Mol Biol 32:959–968

    Article  PubMed  CAS  Google Scholar 

  • Chung JH, Whiteley M, Felsenfeld G (1993) A 5′ element of the chicken β-globin domain serves as an insulator in human erythroid cells and protects against position effect in Drosophila. Cell 74:505–514

    Article  PubMed  CAS  Google Scholar 

  • Chung JH, Bell AC, Felsenfeld G (1997) Characterization of the chicken β-globin insulator. Proc Natl Acad Sci USA 94:575–580

    Article  PubMed  CAS  Google Scholar 

  • Conkling MA, Cheng C-L, Yamamoto YT, Goodman HM (1990) Isolation of transcriptionally regulated root-specific genes from tobacco. Plant Physiol 93:1203–1211

    Article  PubMed  CAS  Google Scholar 

  • D’Apolito D, Baiamonte E, Bagliesi M, Di Marzo R, Calzolari R, Ferro L, Franco V, Spinelli G, Maggio A, Acuto S (2009) The sea urchin sns5 insulator protects retroviral vectors from chromosomal position effects by maintaining active chromatin structure. Mol Ther 17:1434–1441

    Article  PubMed  CAS  Google Scholar 

  • de Bolle MFC, Butaye KMJ, Coucke WJW, Goderis IJWM, Wouters PFJ, van Boxel N, Broekaert WF, Cammue BPA (2003) Analysis of the influence of promoter elements and a matrix attachment region on the inter-individual variation of transgene expression in populations of Arabidopsis thaliana. Plant Sci 165:169–179

    Article  CAS  Google Scholar 

  • Dickson J, Gowher H, Strogantsev R, Gaszner M, Hair A, Felsenfeld G, West AG (2010) VEZF1 elements mediate protection from DNA methylation. PLoS Genet 6:e10000804

    Article  CAS  Google Scholar 

  • Donze D, Kamakaka RT (2001) RNA polymerase III and RNA polymerase II complexes are heterochromatin barriers in Saccharomyces cerevisiae. EMBO J 20:520–531

    Article  PubMed  CAS  Google Scholar 

  • Dorsett D (1999) Distant liaisons: long-range enhancer-promoter interactions in Drosophila. Curr Opin Genet Dev 9:505–514

    Article  PubMed  CAS  Google Scholar 

  • Drewell RA, Bae E, Burr J, Lewis EB (2002) Transcription defines the embryonic domains of cis-regulatory activity at the Drosophila bithorax complex. Proc Natl Acad Sci USA 99:16853–16858

    Article  PubMed  CAS  Google Scholar 

  • Engelbrecht CC, Schoof H, Böhm S (2004) Conservation, diversification and expression of C2H2 zinc finger proteins in the Arabidopsis thaliana genome. BMC Genomics 5:1–17

    Article  Google Scholar 

  • Filippova GN, Fagerlie S, Klenova EM, Myers C, Dehner Y, Goodwin G, Neiman PE, Collins SJ, Lobanenkov VV (1996) An exceptionally conserved transcriptional repressor, CTCF, employs different combinations of zinc fingers to bind diverged promoter sequences of avian and mammalian c-myc oncogenes. Mol Cell Biol 16:2802–2813

    PubMed  CAS  Google Scholar 

  • Francis KE, Spiker S (2005) Identification of Arabidopsis thaliana transformants without selection reveals a high occurrence of silenced T-DNA integrations. Plant J 41:464–477

    Article  PubMed  CAS  Google Scholar 

  • Gaszner M, Felsenfeld G (2006) Insulators: exploiting transcriptional and epigenetic mechanisms. Nat Rev Genet 7:703–713

    Article  PubMed  CAS  Google Scholar 

  • Gaszner M, Vazquez J, Schedl P (1999) The Zw5 protein, a component of the scs chromatin domain boundary, is able to block enhancer-promoter interaction. Genes Dev 13:2098–2107

    Article  PubMed  CAS  Google Scholar 

  • Georgiev PG, Gerasimova TI (1989) Novel genes influencing the expression of the yellow locus and mdg4 (gypsy) in Drosophila melanogaster. Mol Gen Genet 220:121–126

    Article  PubMed  CAS  Google Scholar 

  • Gerasimova TI, Gdula DA, Gerasimov DV, Simonova O, Corces VG (1995) A Drosophila protein that imparts directionality on a chromatin insulator is an enhancer of position-effect variegation. Cell 82:587–597

    Article  PubMed  CAS  Google Scholar 

  • Geyer PK (1997) The role of insulator elements in defining domains of gene expression. Curr Opin Genet Dev 7:242–248

    Article  PubMed  CAS  Google Scholar 

  • Geyer PK, Spana C, Corces VG (1986) On the molecular mechanism of gypsy-induced mutations at the yellow locus of Drosophila melanogaster. EMBO J 5:2657–2662

    PubMed  CAS  Google Scholar 

  • Ghirlando R, Felsenfeld G (2008) Hydrodynamic studies on defined heterochromatin fragments support a 30-nm fiber having six nucleosomes per turn. J Mol Biol 376:1417–1425

    Article  PubMed  CAS  Google Scholar 

  • Golovnin A, Melnikova L, Volkov I, Kostuchenko M, Galkin AV, Georgiev P (2008) ‘Insulator bodies’ are aggregates of proteins but not of insulators. EMBO Rep 9:440–445

    Article  PubMed  CAS  Google Scholar 

  • Gudynaite-Savitch L, Johnson DA, Miki BLA (2009) Strategies to mitigate transgene-promoter interactions. Plant Biotechnol J 7:472–485

    Article  PubMed  CAS  Google Scholar 

  • Halweg C, Thompson WF, Spiker S (2005) The Rb7 matrix attachment region increases the likelihood and magnitude of transgene expression in tobacco cells: a flow cytometric study. Plant Cell 17:418–429

    Article  PubMed  CAS  Google Scholar 

  • Han K-H, Ma C, Strauss SH (1997) Matrix attachment regions (MARs) enhance transformation frequency and transgene expression in poplar. Transgenic Res 6:415–420

    Article  CAS  Google Scholar 

  • Hark AT, Schoenherr CJ, Katz DJ, Ingram RS, Levorse JM, Tilghman SM (2000) CTCT mediates methylation-sensitive enhancer-blocking activity at the H19/Igf2 locus. Nature 405:486–489

    Article  PubMed  CAS  Google Scholar 

  • Harrison DA, Gdula DA, Coyne RS, Corces VG (1993) A leucine zipper domain of the suppressor of Hairy-wing protein mediates its repressive effect on enhancer function. Genes Dev 7:1966–1978

    Article  PubMed  CAS  Google Scholar 

  • Heard E, Bickmore W (2007) The ins and outs of gene regulation and chromosome territory organisation. Curr Opin Cell Biol 19:311–316

    Article  PubMed  CAS  Google Scholar 

  • Hily JM, Singer SD, Yang Y, Liu Z (2009) A transformation booster sequence (TBS) from Petunia hybrida functions as an enhancer-blocking insulator in Arabidopsis thaliana. Plant Cell Rep 28:1095–1104

    Article  PubMed  CAS  Google Scholar 

  • Hohn B, Puchta H (2003) Some like it sticky: targeting of the rice gene Waxy. Trends Plant Sci 8:51–53

    Article  PubMed  CAS  Google Scholar 

  • Holmes-Davis R, Comai L (1998) Nuclear matrix attachment regions and plant gene expression. Trends Plant Sci 3:91–97

    Article  Google Scholar 

  • Huang S, Xingguo Li, Yusufzai TM, Qiu Y, Felsenfeld G (2007) USF1 recruits histone modification complexes and is critical for maintenance of a chromatin barrier. Mol Cell Biol 27:7991–8002

    Article  PubMed  CAS  Google Scholar 

  • Jack J, Dorsett D, DeLotto Y, Liu S (1991) Expression of the cut locus in the Drosophila wing margin is required for cell type specification and is regulated by a distant enhancer. Development 113:735–747

    PubMed  CAS  Google Scholar 

  • Jagannath A, Bandyopadhyay P, Arumugam N, Gupta V, Kumar P, Pental D (2001) The use of a Spacer DNA fragment insulates the tissue-specific expression of a cytotoxic gene (barnase) and allow high-frequency generation of transgenic male sterile lines in Brassica juncea L. Mol Breed 8:11–23

    Article  CAS  Google Scholar 

  • Kadauke S, Blobel GA (2009) Chromatin loops in gene regulation. Biochim Biophys Acta 1789:17–25

    PubMed  CAS  Google Scholar 

  • Kellum R, Schedl P (1991) A position-effect assay for boundaries of higher order chromosomal domains. Cell 64:941–950

    Article  PubMed  CAS  Google Scholar 

  • Kellum R, Schedl P (1992) A group of scs elements function as domain boundaries in an enhancer-blocking assay. Mol Cell Biol 12:2424–2431

    PubMed  CAS  Google Scholar 

  • Kim T, Abdullaev Z, Smith A, Ching K, Loukinov D, Green R, Zhang M, Lobanenkov V, Ren B (2007) Analysis of the vertebrate insulator protein CTCF-binding sites in the human genome. Cell 128:1231–1245

    Article  PubMed  CAS  Google Scholar 

  • Kurshakova M, Maksimenko O, Golovnin A, Pulina M, Georgieva S, Georgiev P, Krasnov A (2007) Evolutionarily conserved E(y)2/Sus1 protein is essential for the barrier activity of Su(Hw)-dependent insulators in Drosophila. Mol Cell 27:332–338

    Article  PubMed  CAS  Google Scholar 

  • Lanfranco L (2003) Engineering crops, a deserving venture. Riv Biol 96:31–54

    PubMed  Google Scholar 

  • Lauber AH, Barrett TJ, Subramaniam M, Schuchard M, Spelsberg TC (1997) A DNA-binding element for a steroid receptor-binding factor is flanked by dual nuclear matrix DNA attachment sites in the c-myc gene promoter. J Biol Chem 272:24657–24665

    Article  PubMed  CAS  Google Scholar 

  • Li X, Zhu Z, Junwang X, Qian W, Honglin X (2001) Isolation of pea matrix attachment region and study on its function in transgenic tobaccos. Sci China C 44:400–408

    Article  CAS  Google Scholar 

  • Lin N, Li X, Cui K, Chepelev I, Tie F, Liu B, Li G, Harte P, Zhao K, Huang S, Zhou L (2011) A barrier-only boundary element delimits the formation of facultative heterochromatin in Drosophila melanogaster and vertebrates. Mol Cell Biol 31:2729–2741

    Article  PubMed  CAS  Google Scholar 

  • Liu J, Bramblett D, Zhu Q, Lozano M, Kobayashi R, Ross SR, Dudley JP (1997) The matrix attachment region-binding protein SATB1 participates in negative regulation of tissue-specific gene expression. Mol Cell Biol 17:5275–5287

    PubMed  CAS  Google Scholar 

  • Liu Z, Zhou C, Wu K (2008) Creation and analysis of a novel chimeric promoter for the complete containment of pollen- and seed-mediated gene flow. Plant Cell Rep 27:995–1004

    Article  PubMed  CAS  Google Scholar 

  • Lunyak VV (2008) Boundaries. Boundaries…Boundaries? Curr Opin Cell Biol 20:281–287

    Article  PubMed  CAS  Google Scholar 

  • Mankin SL, Allen GC, Phelan T, Spiker S, Thompson WF (2003) Elevation of transgene expression level by flanking matrix attachment regions (MAR) is promoter dependent: a study of the interactions of six promoters with the RB7 3′ MAR. Transgenic Res 12:3–12

    Article  PubMed  CAS  Google Scholar 

  • Matzke AJ, Matzke MA (1998) Position effects and epigenetic silencing of plant transgenes. Curr Opin Plant Biol 1:142–148

    Article  PubMed  CAS  Google Scholar 

  • Maximova S, Miller C, Antúnez de Mayolo G, Pishak S, Young A, Guiltinan MJ (2003) Stable transformation of Theobroma cacao L. and influence of matrix attachment regions on GFP expression. Plant Cell Rep 21:872–883

    PubMed  CAS  Google Scholar 

  • Mlynárová L, Loonen A, Heldens J, Jansen RC, Keizer P, Stiekema WJ, Nap JP (1994) Reduced position effect in mature transgenic plants conferred by the chicken lysozyme matrix-associated region. Plant Cell 6:417–426

    Article  PubMed  Google Scholar 

  • Moon H, Filippova G, Loukinov D, Pugacheva E, Chen Q, Smith ST, Munhall A, Grewe B, Bartkuhn M, Arnold R, Burke LJ, Renkawitz-Pohl R, Ohlsson R, Zhou J, Renkawitz R, Lobanenkov V (2005) CTCF is conserved from Drosophila to humans and confers enhancer blocking of the Fab-8 insulator. EMBO Rep 6:165–170

    Article  PubMed  CAS  Google Scholar 

  • Morris JR, Chen J-I, Geyer PK, Wu C-t (1998) Two modes of transvection: enhancer action in trans and bypass of a chromatin insulator in cis. Proc Natl Acad Sci USA 95:10740–10745

    Article  PubMed  CAS  Google Scholar 

  • Mutskov V, Raaka BM, Felsenfeld G, Gershengom MC (2007) The human insulin gene displays transcriptionally active epigenetic marks in islet-derived mesenchymal precursor cells in the absence of insulin expression. Stem Cells 25:3223–3233

    Article  PubMed  CAS  Google Scholar 

  • Nabirochkin S, Ossokina M, Heidmann T (1998) A nuclear matrix/scaffold attachment region co-localizes with the gypsy retrotransposon insulator sequence. J Biol Chem 273:2473–2479

    Article  PubMed  CAS  Google Scholar 

  • Nagaya S, Yoshida K, Kato K, Akasaka K, Shinmyo A (2001) An insulator element from the sea urchin Hemicentrotus pulcherrimus suppresses variation in transgene expression in cultured tobacco cells. Mol Genet Genomics 265:405–413

    Article  PubMed  CAS  Google Scholar 

  • Namciu SJ, Blochlinger KB, Fournier REK (1998) Human matrix attachment regions insulate transgene expression from chromosomal position effects in Drosophila melanogaster. Mol Cell Biol 18:2382–2391

    PubMed  CAS  Google Scholar 

  • Nishihara H, Smit AFA, Okada N (2006) Functional noncoding sequences derived from SINEs in the mammalian genome. Genome Res 16:864–874

    Article  PubMed  CAS  Google Scholar 

  • Odell JT, Knowlton S, Lin W, Mauvais J (1988) Properties of an isolated transcription stimulating sequence derived from the cauliflower mosaic virus 35S promoter. Plant Mol Biol 10:263–272

    Article  CAS  Google Scholar 

  • Oh S-J, Jeong JS, Kim EH, Yi NR, Yi S-I, Jang I-C, Kim YS, Suh S-C, Nahm BH, Kim J-K (2005) Matrix attachment region from the chicken lysozyme locus reduces variability in transgene expression and confers copy number-dependence in transgenic rice plants. Plant Cell Rep 24:145–154

    Article  PubMed  CAS  Google Scholar 

  • Parkhurst SM, Harrison DA, Remington MP, Spana C, Kelley RL, Coyne RS, Corces VG (1988) The Drosophila su(Hw) gene, which controls the phenotypic effect of the gypsy transposable element, encodes a putative DNA-binding protein. Genes Dev 2:1205–1215

    Article  PubMed  CAS  Google Scholar 

  • Petersen K, Leah R, Knudsen S, Cameron-Mills V (2002) Matrix attachment regions (MARs) enhance transformation frequencies and reduce variance of transgene expression in barley. Plant Mol Biol 49:45–58

    Article  PubMed  CAS  Google Scholar 

  • Phi-Van L, Strätling WH (1988) The matrix attachment regions of the chicken lysozyme genes co-map with the boundaries of the chromatin domain. EMBO J 7:655–664

    CAS  Google Scholar 

  • Phi-Van L, Strätling WH (1996) Dissection of the ability of the chicken lysozyme gene 5′ matrix attachment to stimulate transgene expression and to dampen position effects. Biochemistry 35:10735–10742

    Article  PubMed  CAS  Google Scholar 

  • Phi-Van L, von Kries JP, Ostertag W, Strätling WH (1990) The chicken lysozyme 5′ matrix attachment region increases transcription from a heterologous promoter in heterologous cells and dampens position effects on the expression of transfected genes. Mol Cell Biol 10:2302–2307

    PubMed  CAS  Google Scholar 

  • Pikaart MJ, Recillas-Targa F, Felsenfeld G (1998) Loss of transcriptional activity of a transgene is accompanied by DNA methylation and histone deacetylation and is prevented by insulators. Genes Dev 12:2852–2862

    Article  PubMed  CAS  Google Scholar 

  • Ptashne M (1986) Gene regulation by proteins acting nearby and at a distance. Nature 322:697–701

    Article  PubMed  CAS  Google Scholar 

  • Raab JR, Kamakaka RT (2010) Insulators and promoters: closer than we think. Nat Rev Genet 11:439–446

    Article  PubMed  CAS  Google Scholar 

  • Recillas-Targa F, Pikaart MJ, Burgess-Beusse B, Bell AC, Litt MD, West AG, Gaszner M, Felsenfeld G (2002) Position-effect protection and enhancer blocking by the chicken β-globin insulator are separable activities. Proc Natl Acad Sci USA 99:6883–6888

    Article  PubMed  CAS  Google Scholar 

  • Ren S, Johnston JS, Shippen DE, McKnight T (2004) TELOMERASE ACTIVATOR1 induces telomerase activity and potentiates responses to auxin in Arabidopsis. Plant Cell 16:2910–2922

    Article  PubMed  CAS  Google Scholar 

  • Román AC, González-Rico FJ, Moltó E, Hernando H, Neto A, Vicente-Garcia C, Ballestar E, Gómez-Skarmeta JL, Vavrova-Anderson J, White RJ, Montoliu L, Fernández-Salguero PM (2011) Dioxin receptor and SLUG transcription factors regulate the insulator activity of B1 SINE retrotranspososons via an RNA polymerase switch. Genome Res 21:422–432

    Article  PubMed  CAS  Google Scholar 

  • Schöffl F, Schröder G, Kliem M, Rieping M (1993) An SAR sequence containing 395 bp DNA fragment mediates enhanced, gene-dosage-correlated expression of a chimaeric heat shock gene in transgenic tobacco plants. Transgenic Res 2:93–100

    Article  PubMed  Google Scholar 

  • Scott KS, Geyer PK (1995) Effects of the su(Hw) insulator protein on the expression of the divergently transcribed Drosophila yolk protein genes. EMBO J 14:6258–6267

    PubMed  CAS  Google Scholar 

  • She W, Lin W, Zhu Y, Chen Y, Jin W, Yang Y, Han N, Bian H, Zhu M, Wang J (2010) The gypsy insulator of Drosophila melanogaster, together with its binding protein suppressor of hairy-wing, facilitate high and precise expression of transgenes in Arabidopsis thaliana. Genetics 185:1141–1150

    Article  PubMed  CAS  Google Scholar 

  • Singer SD, Cox KD, Liu Z (2010a) Both the constitutive cauliflower mosaic virus 35S and tissue-specific AGAMOUS enhancers activate transcription autonomously in Arabidopsis thaliana. Plant Mol Biol 74:293–305

    Article  PubMed  CAS  Google Scholar 

  • Singer SD, Hily J-M, Liu Z (2010b) A 1 kb bacteriophage lambda fragment functions as an insulator to effectively block enhancer-promoter interactions in Arabidopsis thaliana. Plant Mol Biol Rep 28:69–76

    Article  CAS  Google Scholar 

  • Singer SD, Cox KD, Liu Z (2011a) Enhancer-promoter interference and its prevention in transgenic plants. Plant Cell Rep 30:723–731

    Article  PubMed  CAS  Google Scholar 

  • Singer SD, Hily J-M, Cox KD (2011) Analysis of the enhancer-blocking function of the TBS element from Petunia hybrida in transgenic Arabidopsis thaliana and Nicotiana tabacum. Plant Cell Rep. doi:10.1007/s00299-011-1109-8 (in press)

  • Spana C, Harrison DA, Corces VG (1988) The Drosophila melanogaster suppressor of Hairy-wing protein binds to specific sequences of the gypsy retrotransposon. Genes Dev 2:1414–1423

    Article  PubMed  CAS  Google Scholar 

  • Steinwaerder DS, Lieber A (2000) Insulation from viral transcriptional regulatory elements improves inducible transgene expression from adenovirus vectors in vitro and in vivo. Gene Ther 7:556–567

    Article  PubMed  CAS  Google Scholar 

  • Stief A, Winter DM, Strätling WH, Sippel AE (1989) A nuclear DNA attachment element mediates elevated and position-independent gene activity. Nature 341:343–345

    Article  PubMed  CAS  Google Scholar 

  • Tao Y, Shang-long Z, Jing-mei L, Da-ming C (2006) Approaches to improve heterogeneous gene expression in transgenic plants. Chin J Agric Biotechnol 3:75–81

    Article  CAS  Google Scholar 

  • Torney F, Partier A, Says-Lesage V, Nadaud I, Barret P, Beckert M (2004) Heritable transgene expression pattern imposed onto maize ubiquitin promoter by maize adh-1 matrix attachment regions: tissue and developmental specificity in maize transgenic plants. Plant Cell Rep 22:931–938

    Article  PubMed  CAS  Google Scholar 

  • Twyman RM, Stoger E, Schillberg S, Christou P, Fischer R (2003) Molecular farming in plants: host systems and expression technology. Trends Biotechnol 21:570–578

    Article  PubMed  CAS  Google Scholar 

  • Ülker B, Allen GC, Thompson WF, Spiker S, Weissinger AK (1999) A tobacco matrix attachment region reduces the loss of transgene expression in the progeny of transgenic tobacco plants. Plant J 18:253–263

    Article  Google Scholar 

  • Vain P, Worland B, Kohli A, Snape JW, Christou P, Allen GC, Thompson WF (1999) Matrix attachment regions increase transgene expression levels and stability in transgenic rice plants and their progeny. Plant J 18:233–242

    Article  CAS  Google Scholar 

  • van der Geest AHM, Hall TC (1997) The β-phaseolin 5′ matrix attachment region acts as an enhancer facilitator. Plant Mol Biol 33:553–557

    Article  PubMed  Google Scholar 

  • van der Geest AHM, Hall GE Jr, Spiker S, Hall TC (1994) The β-phaseolin gene is flanked by matrix attachment regions. Plant J 6:413–423

    Article  Google Scholar 

  • Verma D, Verma M, Dey M, Jain RK, Wu R (2005) Molecular dissection of the tobacco Rb7 matrix attachment region (MAR): effect of 5′ half on gene expression in rice. Plant Sci 169:704–711

    Article  CAS  Google Scholar 

  • Volfson D, Marciniak J, Blake WJ, Ostroff N, Tsimring LS, Hasty J (2006) Origins of extrinsic variability in eukaryotic gene expression. Nature 439:861–864

    Article  PubMed  CAS  Google Scholar 

  • Wallace JA, Felsenfeld G (2007) We gather together: insulators and genome organization. Curr Opin Genet Dev 17:400–407

    Article  PubMed  CAS  Google Scholar 

  • Wang Y, Tang X, Cheng Z, Mueller L, Giovannoni J, Tanksley SD (2006) Euchromatin and pericentromeric heterochromatin: comparative composition in the tomato genome. Genetics 172:22529–22540

    Google Scholar 

  • West AG, Gaszner M, Felsenfeld G (2002) Insulators: many functions, many mechanisms. Genes Dev 16:271–288

    Article  PubMed  CAS  Google Scholar 

  • Weterings K, Schrauwen J, Wullems G, Twell D (1995) Functional dissection of the promoter of the pollen-specific gene NTP303 reveals a novel pollen-specific, and conserved cis-regulatory element. Plant J 8:55–63

    Article  PubMed  CAS  Google Scholar 

  • Willoughby DA, Vilalta A, Oshima RG (2000) An Alu element from the K18 gene confers position-independent expression in transgenic mice. J Biol Chem 275:759–768

    Article  PubMed  CAS  Google Scholar 

  • Xu Q, Li M, Adams J, Cai HN (2004) Nuclear location of a chromatin insulator in Drosophila melanogaster. J Cell Sci 117:1025–1032

    Article  PubMed  CAS  Google Scholar 

  • Xue H, Yang Y-T, Wu C-A, Yang G-D, Zhang M-M, Zheng C-C (2005) TM2, a novel strong matrix attachment region isolated from tobacco increases transgene expression in transgenic rice calli and plants. Theor Appl Genet 110:620–627

    Article  PubMed  CAS  Google Scholar 

  • Yang Y, Singer SD, Liu Z (2010) Evaluation and comparison of the insulation efficiency of three enhancer-blocking insulators in plants. Plant Cell Tissue Organ 105:405–414

    Article  CAS  Google Scholar 

  • Ye X, Liang M, Meng X, Ren X, Chen H, Li ZY, Ni S, Lieber A, Hu F (2003) Insulation from viral transcriptional regulatory elements enables improvement to hepatoma-specific gene expression from adenovirus vectors. Biochem Biophys Res Commun 307:759–764

    Article  PubMed  CAS  Google Scholar 

  • Yoo SY, Bomblies K, Yoo SK, Yang JW, Choi MS, Lee JS, Weigel D, Ahn JH (2005) The 35S promoter used in a selectable marker gene of a plant transformation vector affects the expression of the transgene. Planta 221:523–530

    Article  PubMed  CAS  Google Scholar 

  • Yusufzai TM, Hagami H, Nakatani Y, Felsenfeld G (2004) CTCF tethers an insulator to subnuclear sites, suggesting shared insulator mechanisms across species. Mol Cell 13:291–298

    Article  PubMed  CAS  Google Scholar 

  • Zhao K, Hart CM, Laemmli UK (1995) Visualization of chromosomal domains with boundary element-associated factor BEAF-32. Cell 81:879–889

    Article  PubMed  CAS  Google Scholar 

  • Zheng X, Deng W, Luo K, Duan H, Chen Y, McAvoy R, Song S, Pei Y, Li Y (2007) The cauliflower mosaic virus (CaMV) 35S promoter sequence alters the level and patterns of activity of adjacent tissue- and organ-specific gene promoters. Plant Cell Rep 26:1195–1203

    Article  PubMed  CAS  Google Scholar 

  • Zhong X-P, Krangel MS (1997) An enhancer-blocking element between α and δ gene segments within the human T cell receptor α/δ locus. Proc Natl Acad Sci USA 94:5219–5224

    Article  PubMed  CAS  Google Scholar 

  • Zhu X, Ling J, Zhang L, Pi W, Wu M, Tuan D (2007) A facilitated tracking and transcription mechanism of long-range enhancer function. Nucleic Acids Res 35:5532–5544

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

The authors wish to thank Sara Villani for her invaluable assistance. Support was provided by state, federal, and institutional funds appropriated to the New York State Agricultural Experiment Station, Cornell University.

Author information

Authors and Affiliations

  1. Department of Plant Pathology and Plant-Microbe Biology, New York State Agricultural Experiment Station, Cornell University, 630 West North Street, Geneva, NY, 14456, USA

    Stacy D. Singer & Kerik D. Cox

  2. USDA-ARS Appalachian Fruit Research Station, 2217 Wiltshire Road, Kearneysville, WV, 25430, USA

    Zongrang Liu

Authors
  1. Stacy D. Singer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zongrang Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kerik D. Cox
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kerik D. Cox.

Additional information

Communicated by R. Reski.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Singer, S.D., Liu, Z. & Cox, K.D. Minimizing the unpredictability of transgene expression in plants: the role of genetic insulators. Plant Cell Rep 31, 13–25 (2012). https://doi.org/10.1007/s00299-011-1167-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-011-1167-y

Keywords

Search

Navigation