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Evidences for insulator activity of the 5′UTR of the Drosophila melanogaster LTR-retrotransposon ZAM

Abstract

Insulators or chromatin boundary are DNA elements that organize the genome into discrete regulatory domains by limiting the actions of enhancers and silencers through a “positional-blocking mechanism”. The role of these sequences, both in modulation of the enhancers range of action (enhancer–promoter selectivity) and in the organization of the chromatin in functional domains, is emerging strongly in these last years. There is a great interest in identifying new insulators because deeper knowledge of these elements can help understand how cis-regulatory elements coordinate the expression of the target genes. However, while insulators are critical in gene regulation and genome functioning, only a few have been reported so far. Here, we describe a new insulator sequence that is located in the 5′UTR of the Drosophila retrotransposon ZAM. We have used an “enhancer–blocking assay” to test its effects on the activity of the enhancer in transiently transfected Drosophila S2R+ cell line. Moreover, we show that the new insulator is able to affect significantly the enhancer–promoter interaction in the human cell line HEK293. These results suggest the possibility of employing the ZAM insulator in gene transfer protocols from insects to mammals in order to counteract the transgene positional and genotoxic effects.

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Abbreviations

HP1:

Heterochromatin protein 1

5′UTR:

Untranslated region

Su(Hw):

Suppressor of Hairy-wing

LTR:

Long terminal repeat

MAR/SAR:

Matrix/scaffold attachment region

L:

Gene reporter luciferase

P:

TATA-box sequence of the human cytomegalovirus immediate early promoter

EA :

Enhancer sequence of the regulative region of beta-actin

Yii:

The fragment obtained from the second exon of the yellow gene

G:

Gene reporter enhanced GFP

EC :

Enhancer of human cytomegalovirus immediate early promoter

References

  1. Blackwood EM, Kadonaga JT (1998) Going the distance: a current view of enhancer action. Science 281:60–63

  2. Cai HN, Shen P (2001) Effects of cis arrangement of chromatin insulators on enhancer-blocking activity. Science 291:493–495

  3. Conte C, Dastugue B, Vaury C (2002) Coupling of enhancer and insulator properties identified in two retrotransposons modulates their mutagenic impact on nearby genes. Mol Cell Biol 22:1767–1777

  4. Elnitski L, Li J, Noguchi CT, Miller W, Hardison R (2001) A negative cis-element regulates the level of enhancement by hypersensitive site 2 of the beta-globin locus control region. J Biol Chem 276:6289–6298

  5. Gasser SM, Laemmli UK (1986) Cohabitation of scaffold binding regions with upstream/enhancer elements of three developmentally regulated genes of D. melanogaster. Cell 46:521–530

  6. Gdula DA, Gerasimova TI, Corces VG (1996) Genetic and molecular analysis of the gypsy chromatin insulator of Drosophila. Proc Natl Acad Sci USA 93:9378–9383

  7. Gerasimova TI, Lei EP, Bushey AM, Corces VG (2007) Coordinated control of dCTCF and gypsy chromatin insulators in Drosophila. Mol Cell 28:761–772

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

  9. Minervini CF, Marsano RM, Casieri P, Fanti L, Caizzi R, Pimpinelli S, Rocchi M, Viggiano L (2007) Heterochromatin protein 1 interacts with 5’UTR of transposable element ZAM in a sequence-specific fashion. Gene 393:1–10

  10. Morcillo P, Rosen C, Dorsett D (1996) Genes regulating the remote wing margin enhancer in the Drosophila cut locus. Genetics 144:1143–1154

  11. 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

  12. Namciu SJ, Fournier RE (2004) Human matrix attachment regions are necessary for the establishment but not the maintenance of transgene insulation in Drosophila melanogaster. Mol Cell Biol 24:10236–10245

  13. Pai CY, Lei EP, Ghosh D, Corces VG (2004) The centrosomal protein CP190 is a component of the gypsy chromatin insulator. Mol Cell 16:737–748

  14. Palla F, Melfi R, Anello L, Di Bernardo M, Spinelli G (1997) Enhancer blocking activity located near the 3′ end of the sea urchin early H2A histone gene. Proc Natl Acad Sci USA 94:2272–2277

  15. Parkhurst SM, Corces VG (1986) Interactions among the gypsy transposable element and the yellow and the suppressor of hairy-wing loci in Drosophila melanogaster. Mol Cell Biol 6:47–53

  16. Phillips JE, Corces VG (2009) CTCF: master weaver of the genome. Cell 137:1194–1211

  17. Polikanov YS, Rubtsov MA, Studitsky VM (2007) Biochemical analysis of enhancer-promoter communication in chromatin. Methods 41:250–258

  18. 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

  19. Szutorisz H, Dillon N, Tora L (2005) The role of enhancers as centres for general transcription factor recruitment. Trends Biochem Sci 30:593–599

  20. Tjian R, Maniatis T (1994) Transcriptional activation: a complex puzzle with few easy pieces. Cell 77:5–8

  21. Valenzuela L, Kamakaka RT (2006) Chromatin insulators. Annu Rev Genet 40:107–138

  22. van der Vlag J, den Blaauwen JL, Sewalt RG, van Driel R, Otte AP (2000) Transcriptional repression mediated by polycomb group proteins and other chromatin-associated repressors is selectively blocked by insulators. J Biol Chem 275:697–704

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

  24. Walters MC, Fiering S, Bouhassira EE, Scalzo D, Goeke S, Magis W, Garrick D, Whitelaw E, Martin DI (1999) The chicken beta-globin 5′HS4 boundary element blocks enhancer-mediated suppression of silencing. Mol Cell Biol 19:3714–3726

  25. Wei W, Brennan MD (2001) The gypsy insulator can act as a promoter-specific transcriptional stimulator. Mol Cell Biol 21:7714–7720

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

  27. Zhao H, Dean A (2005) Organizing the genome: enhancers and insulators. Biochem Cell Biol 83:516–524

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Acknowledgments

We thank Prof. Ruggiero Caizzi, for the critical reviewing and helpful discussion during the preparation of the manuscript. This work was supported by “Progetti di Ateneo” 2008 to L.V.

Author information

Correspondence to Luigi Viggiano.

Additional information

Communicated by G. Reuter.

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Minervini, C.F., Ruggieri, S., Traversa, M. et al. Evidences for insulator activity of the 5′UTR of the Drosophila melanogaster LTR-retrotransposon ZAM. Mol Genet Genomics 283, 503–509 (2010). https://doi.org/10.1007/s00438-010-0529-4

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Keywords

  • Insulator
  • LTR-retrotransposon
  • Gene expression
  • Drosophila melanogaster