A 1-kb Bacteriophage Lambda Fragment Functions as an Insulator to Effectively Block Enhancer–Promoter Interactions in Arabidopsis thaliana

Article

Abstract

Enhancers are known to be capable of overriding the specificity of nearby promoters in a distance-dependent manner, which is problematic when multiple promoters coexist in a single transgene unit. In an attempt to determine whether enhancer activation function is inversely related to its distance from the target promoter, we inserted 1-, 2-, and 4-kb bacteriophage λ fragments, respectively, between the cauliflower mosaic virus 35S enhancer and a flower-specific AGAMOUS second intron-derived promoter (AGIP) fused to the β-glucuronidase (GUS) coding region. In the absence of an insert sequence, the 35S enhancer activates AGIP-driven GUS expression in vegetative tissues of transgenic Arabidopsis thaliana lines. Moreover, neither the 2-kb nor the 4-kb λ fragment was able to block GUS expression in transgenic leaves, implying that the 35S enhancer can override a distance barrier of at least 4 kb in our system. Unexpectedly, insertion of the 1-kb λ insert into the same site resulted in diminished GUS expression in transgenic leaves. Our data indicate that this fragment functions as a true enhancer-blocking insulator that could potentially be utilized to minimize enhancer–promoter interference between multiple transcriptional units within a plasmid vector during plant transformation experiments.

Keywords

Arabidopsis thaliana Bacteriophage lambda Cauliflower mosaic virus 35S promoter Enhancer-blocking insulator Enhancer–promoter interaction 

References

  1. Bao L, Zhou M, Cui Y (2008) CTCFBSDB: a CTCF-binding site database for characterization of vertebrate genomic insulators. Nucleic Acids Res 36:D83–D87CrossRefPubMedGoogle Scholar
  2. Belostotsky DA, Meagher RB (1996) A pollen-, ovule-, and early embryo-specific poly(A) binding protein from Arabidopsis complements essential functions in yeast. Plant Cell 8:1261–1275CrossRefPubMedGoogle Scholar
  3. Benfey PN, Ren L, Chua NH (1989) The CaMV 35S enhancer contains at least two domains which can confer different developmental and tissue-specific expression patterns. EMBO J 8:2195–2202PubMedGoogle Scholar
  4. Benfey PN, Ren L, Chua NH (1990) Tissue-specific expression from CaMV 35S enhancer subdomains in early stages of plant development. EMBO J 9:1677–1684PubMedGoogle Scholar
  5. Benson G (1999) Tandem repeats finder: a program to analyze DNA sequences. Nucleic Acids Res 27:573–580CrossRefPubMedGoogle Scholar
  6. Bevan M (1984) Agrobacterium vectors for plant transformation. Nucleic Acids Res 12:8711–8721CrossRefPubMedGoogle Scholar
  7. 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 74:505–514CrossRefPubMedGoogle Scholar
  8. Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743CrossRefPubMedGoogle Scholar
  9. Dunn KL, Zhao H, Davie JR (2003) The insulator binding protein CTCF associates with the nuclear matrix. Exp Cell Res 288:218–223CrossRefPubMedGoogle Scholar
  10. Geyer PK, Spana C, Corces VG (1986) On the molecular mechanism of gypsy-induced mutations at the yellow locus of Drosophila melanogaster. EMBO J 5:2657–2662PubMedGoogle Scholar
  11. Goderis IJWM, De Bolle MFC, François IEJA, Wouters PFJ, Broekaert WF, Cammue BPA (2002) A set of modular plant transformation vectors allowing flexible insertion of up to six expression units. Plant Mol Biol 50:17–27CrossRefPubMedGoogle Scholar
  12. Grissa I, Vergnaud G, Pourcel C (2007) CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats. Nucleic Acids Res 35:W52–W57. doi:10.1093/nar/gkm360:1-6 CrossRefPubMedGoogle Scholar
  13. Hajdukiewicz P, Svab Z, Maliga P (1994) The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol 25:989–994CrossRefPubMedGoogle Scholar
  14. Hebbes TR, Clayton AL, Thorne AW, Crane-Robinson C (1994) Core histone hyperacetylation co-maps with generalized DNase I sensitivity in the chicken β-globin chromosomal domain. EMBO J 13:1823–1830PubMedGoogle Scholar
  15. Hily JM, Liu Z (2009) A simple and sensitive high-throughput GFP screening in woody and herbaceous plants. Plant Cell Rep 28:493–501CrossRefPubMedGoogle Scholar
  16. 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(7):1095–1104CrossRefPubMedGoogle Scholar
  17. Hirner B, Fischer WN, Rentsch D, Kwart M, Frommer WB (1998) Developmental control of H+/amino acid permease gene expression during seed development of Arabidopsis. Plant J 14:535–544CrossRefPubMedGoogle Scholar
  18. 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 Breeding 8:11–23CrossRefGoogle Scholar
  19. Kellum R, Schedl P (1991) A position-effect assay for boundaries of higher order chromosomal domains. Cell 64:941–950CrossRefPubMedGoogle Scholar
  20. Kellum R, Schedl P (1992) A group of scs elements function as domain boundaries in an enhancer-blocking assay. Mol Cell Biol 12:2424–2431PubMedGoogle Scholar
  21. 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 128:1231–1245CrossRefPubMedGoogle Scholar
  22. Kobayashi N, Horikoshi T, Katsuyama H, Handa T, Takayanagi K (1998) A simple and efficient DNA extraction method for plants, especially woody plants. Plant Tissue Cult Biotechnol 4:76–80Google Scholar
  23. Koltunow AM, Truettner J, Cox KH, Wallroth M, Goldberg RB (1990) Different temporal and spatial gene expression patterns occur during anther development. Plant Cell 2:1201–1224CrossRefPubMedGoogle Scholar
  24. Lanfranco L (2003) Engineering crops, a deserving venture. Riv Biol 96:31–54PubMedGoogle Scholar
  25. Li Z, Jayasankar S, Gray DJ (2001) Expression of a bifunctional green fluorescent protein (GFP) fusion marker under the control of three constitutive promoters and enhanced derivatives in transgenic grape (Vitis vinifera). Plant Sci 160:877–887CrossRefPubMedGoogle Scholar
  26. Liu Z, Liu Z (2008) The second intron of AGAMOUS drives carpel- and stamen-specific expression sufficient to induce complete sterility in Arabidopsis. Plant Cell Rep 27:855–863CrossRefPubMedGoogle Scholar
  27. Nabirochkin S, Ossokina M, Heidmann T (1998) A nuclear matrix/scaffold attachment region co-localizes with the gypsy retrotransposon insulator sequence. J Biol Chem 273:2473–2479CrossRefPubMedGoogle Scholar
  28. Namciu SJ, Blochlinger KB, Fournier REK (1998) Human matrix attachment regions insulate transgene expression from chromosomal position effects in Drosophila melanogaster. Mol Cell Biol 18:2382–2391PubMedGoogle Scholar
  29. 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 10:263–272CrossRefGoogle Scholar
  30. Ouwerkerk PBF, de Kam RJ, Hoge JHC, Meijer AH (2001) Glucocorticoid-inducible gene expression in rice. Planta 213:370–378CrossRefPubMedGoogle Scholar
  31. Prestridge DS (1995) Predicting pol II promoter sequences using transcription factor binding sites. J Mol Biol 249:923–932CrossRefPubMedGoogle Scholar
  32. Rosso MG, Li Y, Strizhov N, Reiss B, Dekker K, Weisshar B (2003) An Arabidopsis thaliana T-DNA mutagenized population (GABI-Kat) for flanking sequence tag-based reverse genetics. Plant Mol Biol 53:247–259CrossRefPubMedGoogle Scholar
  33. Savidge B, Rounsley SD, Yanofsky MF (1995) Temporal relationship between the transcription of two Arabidopsis MADS box genes and the floral organ identity genes. Plant Cell 7:721–733CrossRefPubMedGoogle Scholar
  34. Singh GB, Kramer JA, Krawetz SA (1997) Mathematical model to predict regions of chromatin attachment to the nuclear matrix. Nucleic Acids Res 25:1419–1425CrossRefPubMedGoogle Scholar
  35. Smith DL, Fedoroff NV (1995) LRP1, a gene expressed in lateral and adventitious root primordia of Arabidopsis. Plant Cell 7:735–745CrossRefPubMedGoogle Scholar
  36. Stief A, Winter DM, Strätling WH, Sippel AE (1989) A nuclear DNA attachment element mediates elevated and position-independent gene activity. Nature 341:343–345CrossRefPubMedGoogle Scholar
  37. van der Geest AHM, Hall TC (1997) The β-phaseolin 5′ matrix attachment region acts as an enhancer facilitator. Plant Mol Biol 33:553–557CrossRefPubMedGoogle Scholar
  38. 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 221:523–530CrossRefPubMedGoogle Scholar
  39. 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 26:1195–1203CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Stacy D. Singer
    • 1
    • 2
  • Jean-Michel Hily
    • 1
    • 2
  • Zongrang Liu
    • 1
  1. 1.USDA-ARS Appalachian Fruit Research StationKearneysvilleUSA
  2. 2.Department of Plant Pathology, New York State Agricultural Experiment StationCornell UniversityGenevaUSA

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