Skip to main content
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1997 Jan;17(1):482–494. doi: 10.1128/mcb.17.1.482

Molecular cloning and characterization of a transcription factor for the copia retrotransposon with homology to the BTB-containing lola neurogenic factor.

L Cavarec 1, S Jensen 1, J F Casella 1, S A Cristescu 1, T Heidmann 1
PMCID: PMC231773  PMID: 8972229

Abstract

By transfection experiments, we previously identified a 72-bp enhancer sequence within the Drosophila copia retrotransposon which is involved in the control of the transcription level of this mobile element in cells in culture. Gel shift assays with nuclear extracts from Drosophila hydei-derived DH-33 cells further demonstrated specific interactions of at least two nuclear factors with this enhancer sequence. Using this sequence as a probe for the screening of an expression cDNA library that we constructed from DH-33 cells RNA, we have isolated a cDNA clone encoding a 110-kDa protein with features common to those of known transcription factors; these include a two-zinc-finger motif at the C terminus, three glutamine-rich domains in the presumptive activation domain of the protein, and an N-terminal domain which shares homology with the Bric-à-brac, Tramtrack, and Broad-Complex BTB boxes. The precise DNA recognition sequence for this transcription factor has been determined by both gel shift assays and footprinting experiments with a recombinant protein made in bacteria. The functionality of the cloned element was demonstrated upon transcriptional activation of copia reporter genes, as well as of a minimal promoter coupled with the identified target DNA sequence, in cotransfection assays in cells in culture with an expression vector for the cloned factor. Southern blot and nucleotide sequence analyses revealed a related gene in Drosophila melanogaster (the lola gene) previously identified by a genetic approach as involved in axon growth and guidance. Transfection assays in cells in culture with lola gene expression vectors and in situ hybridization experiments with lola gene mutants finally provided evidence that the copia retrotransposon is regulated by this neurogenic gene in D.melanogaster, with a repressor effect in the central nervous systems of the embryos.

Full Text

The Full Text of this article is available as a PDF (940.0 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bardwell V. J., Treisman R. The POZ domain: a conserved protein-protein interaction motif. Genes Dev. 1994 Jul 15;8(14):1664–1677. doi: 10.1101/gad.8.14.1664. [DOI] [PubMed] [Google Scholar]
  2. Boeke J. D., Garfinkel D. J., Styles C. A., Fink G. R. Ty elements transpose through an RNA intermediate. Cell. 1985 Mar;40(3):491–500. doi: 10.1016/0092-8674(85)90197-7. [DOI] [PubMed] [Google Scholar]
  3. Boulay J. L., Dennefeld C., Alberga A. The Drosophila developmental gene snail encodes a protein with nucleic acid binding fingers. 1987 Nov 26-Dec 2Nature. 330(6146):395–398. doi: 10.1038/330395a0. [DOI] [PubMed] [Google Scholar]
  4. Brown J. L., Sonoda S., Ueda H., Scott M. P., Wu C. Repression of the Drosophila fushi tarazu (ftz) segmentation gene. EMBO J. 1991 Mar;10(3):665–674. doi: 10.1002/j.1460-2075.1991.tb07995.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cavarec L., Heidmann T. The Drosophila copia retrotransposon contains binding sites for transcriptional regulation by homeoproteins. Nucleic Acids Res. 1993 Nov 11;21(22):5041–5049. doi: 10.1093/nar/21.22.5041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cavarec L., Jensen S., Heidmann T. Identification of a strong transcriptional activator for the copia retrotransposon responsible for its differential expression in Drosophila hydei and melanogaster cell lines. Biochem Biophys Res Commun. 1994 Aug 30;203(1):392–399. doi: 10.1006/bbrc.1994.2195. [DOI] [PubMed] [Google Scholar]
  7. Chardin P., Courtois G., Mattei M. G., Gisselbrecht S. The KUP gene, located on human chromosome 14, encodes a protein with two distant zinc fingers. Nucleic Acids Res. 1991 Apr 11;19(7):1431–1436. doi: 10.1093/nar/19.7.1431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chen W., Zollman S., Couderc J. L., Laski F. A. The BTB domain of bric à brac mediates dimerization in vitro. Mol Cell Biol. 1995 Jun;15(6):3424–3429. doi: 10.1128/mcb.15.6.3424. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  9. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  10. Church G. M., Gilbert W. Genomic sequencing. Proc Natl Acad Sci U S A. 1984 Apr;81(7):1991–1995. doi: 10.1073/pnas.81.7.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cooley L., Theurkauf W. E. Cytoskeletal functions during Drosophila oogenesis. Science. 1994 Oct 28;266(5185):590–596. doi: 10.1126/science.7939713. [DOI] [PubMed] [Google Scholar]
  12. Courey A. J., Tjian R. Analysis of Sp1 in vivo reveals multiple transcriptional domains, including a novel glutamine-rich activation motif. Cell. 1988 Dec 2;55(5):887–898. doi: 10.1016/0092-8674(88)90144-4. [DOI] [PubMed] [Google Scholar]
  13. Csink A. K., McDonald J. F. copia expression is variable among natural populations of Drosophila. Genetics. 1990 Oct;126(2):375–385. doi: 10.1093/genetics/126.2.375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dhordain P., Albagli O., Ansieau S., Koken M. H., Deweindt C., Quief S., Lantoine D., Leutz A., Kerckaert J. P., Leprince D. The BTB/POZ domain targets the LAZ3/BCL6 oncoprotein to nuclear dots and mediates homomerisation in vivo. Oncogene. 1995 Dec 21;11(12):2689–2697. [PubMed] [Google Scholar]
  15. DiBello P. R., Withers D. A., Bayer C. A., Fristrom J. W., Guild G. M. The Drosophila Broad-Complex encodes a family of related proteins containing zinc fingers. Genetics. 1991 Oct;129(2):385–397. doi: 10.1093/genetics/129.2.385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ding D., Lipshitz H. D. Spatially regulated expression of retrovirus-like transposons during Drosophila melanogaster embryogenesis. Genet Res. 1994 Dec;64(3):167–181. doi: 10.1017/s0016672300032833. [DOI] [PubMed] [Google Scholar]
  17. Dorn R., Krauss V., Reuter G., Saumweber H. The enhancer of position-effect variegation of Drosophila, E(var)3-93D, codes for a chromatin protein containing a conserved domain common to several transcriptional regulators. Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):11376–11380. doi: 10.1073/pnas.90.23.11376. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Echalier G. Drosophila retrotransposons: interactions with genome. Adv Virus Res. 1989;36:33–105. doi: 10.1016/s0065-3527(08)60582-5. [DOI] [PubMed] [Google Scholar]
  19. Echalier G., Ohanessian A. In vitro culture of Drosophila melanogaster embryonic cells. In Vitro. 1970 Nov-Dec;6(3):162–172. doi: 10.1007/BF02617759. [DOI] [PubMed] [Google Scholar]
  20. England B. P., Heberlein U., Tjian R. Purified Drosophila transcription factor, Adh distal factor-1 (Adf-1), binds to sites in several Drosophila promoters and activates transcription. J Biol Chem. 1990 Mar 25;265(9):5086–5094. [PubMed] [Google Scholar]
  21. Fairall L., Harrison S. D., Travers A. A., Rhodes D. Sequence-specific DNA binding by a two zinc-finger peptide from the Drosophila melanogaster Tramtrack protein. J Mol Biol. 1992 Jul 20;226(2):349–366. doi: 10.1016/0022-2836(92)90952-g. [DOI] [PubMed] [Google Scholar]
  22. Farabaugh P. J., Vimaladithan A., Türkel S., Johnson R., Zhao H. Three downstream sites repress transcription of a Ty2 retrotransposon in Saccharomyces cerevisiae. Mol Cell Biol. 1993 Apr;13(4):2081–2090. doi: 10.1128/mcb.13.4.2081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Giniger E., Tietje K., Jan L. Y., Jan Y. N. lola encodes a putative transcription factor required for axon growth and guidance in Drosophila. Development. 1994 Jun;120(6):1385–1398. doi: 10.1242/dev.120.6.1385. [DOI] [PubMed] [Google Scholar]
  24. Godt D., Couderc J. L., Cramton S. E., Laski F. A. Pattern formation in the limbs of Drosophila: bric à brac is expressed in both a gradient and a wave-like pattern and is required for specification and proper segmentation of the tarsus. Development. 1993 Nov;119(3):799–812. doi: 10.1242/dev.119.3.799. [DOI] [PubMed] [Google Scholar]
  25. Han K., Levine M. S., Manley J. L. Synergistic activation and repression of transcription by Drosophila homeobox proteins. Cell. 1989 Feb 24;56(4):573–583. doi: 10.1016/0092-8674(89)90580-1. [DOI] [PubMed] [Google Scholar]
  26. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  27. Harrison S. D., Travers A. A. The tramtrack gene encodes a Drosophila finger protein that interacts with the ftz transcriptional regulatory region and shows a novel embryonic expression pattern. EMBO J. 1990 Jan;9(1):207–216. doi: 10.1002/j.1460-2075.1990.tb08097.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Heidmann O., Heidmann T. Retrotransposition of a mouse IAP sequence tagged with an indicator gene. Cell. 1991 Jan 11;64(1):159–170. doi: 10.1016/0092-8674(91)90217-m. [DOI] [PubMed] [Google Scholar]
  29. Kassis J. A., Poole S. J., Wright D. K., O'Farrell P. H. Sequence conservation in the protein coding and intron regions of the engrailed transcription unit. EMBO J. 1986 Dec 20;5(13):3583–3589. doi: 10.1002/j.1460-2075.1986.tb04686.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kuff E. L., Lueders K. K. The intracisternal A-particle gene family: structure and functional aspects. Adv Cancer Res. 1988;51:183–276. doi: 10.1016/s0065-230x(08)60223-7. [DOI] [PubMed] [Google Scholar]
  31. Laloux I., Dubois E., Dewerchin M., Jacobs E. TEC1, a gene involved in the activation of Ty1 and Ty1-mediated gene expression in Saccharomyces cerevisiae: cloning and molecular analysis. Mol Cell Biol. 1990 Jul;10(7):3541–3550. doi: 10.1128/mcb.10.7.3541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Laloux I., Jacobs E., Dubois E. Involvement of SRE element of Ty1 transposon in TEC1-dependent transcriptional activation. Nucleic Acids Res. 1994 Mar 25;22(6):999–1005. doi: 10.1093/nar/22.6.999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Miyake T., Mae N., Shiba T., Kondo S. Production of virus-like particles by the transposable genetic element, copia, of Drosophila melanogaster. Mol Gen Genet. 1987 Apr;207(1):29–37. doi: 10.1007/BF00331487. [DOI] [PubMed] [Google Scholar]
  35. Mount S. M., Rubin G. M. Complete nucleotide sequence of the Drosophila transposable element copia: homology between copia and retroviral proteins. Mol Cell Biol. 1985 Jul;5(7):1630–1638. doi: 10.1128/mcb.5.7.1630. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Numoto M., Niwa O., Kaplan J., Wong K. K., Merrell K., Kamiya K., Yanagihara K., Calame K. Transcriptional repressor ZF5 identifies a new conserved domain in zinc finger proteins. Nucleic Acids Res. 1993 Aug 11;21(16):3767–3775. doi: 10.1093/nar/21.16.3767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Parkhurst S. M., Corces V. G. Developmental expression of Drosophila melanogaster retrovirus-like transposable elements. EMBO J. 1987 Feb;6(2):419–424. doi: 10.1002/j.1460-2075.1987.tb04771.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Parkhurst S. M., Harrison D. A., Remington M. P., Spana C., Kelley R. L., Coyne R. S., Corces V. G. The Drosophila su(Hw) gene, which controls the phenotypic effect of the gypsy transposable element, encodes a putative DNA-binding protein. Genes Dev. 1988 Oct;2(10):1205–1215. doi: 10.1101/gad.2.10.1205. [DOI] [PubMed] [Google Scholar]
  39. Payre F., Noselli S., Lefrère V., Vincent A. The closely related Drosophila sry beta and sry delta zinc finger proteins show differential embryonic expression and distinct patterns of binding sites on polytene chromosomes. Development. 1990 Sep;110(1):141–149. doi: 10.1242/dev.110.1.141. [DOI] [PubMed] [Google Scholar]
  40. Read D., Manley J. L. Alternatively spliced transcripts of the Drosophila tramtrack gene encode zinc finger proteins with distinct DNA binding specificities. EMBO J. 1992 Mar;11(3):1035–1044. doi: 10.1002/j.1460-2075.1992.tb05142.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Schneider I. Cell lines derived from late embryonic stages of Drosophila melanogaster. J Embryol Exp Morphol. 1972 Apr;27(2):353–365. [PubMed] [Google Scholar]
  42. Schwartz H. E., Lockett T. J., Young M. W. Analysis of transcripts from two families of nomadic DNA. J Mol Biol. 1982 May 5;157(1):49–68. doi: 10.1016/0022-2836(82)90512-5. [DOI] [PubMed] [Google Scholar]
  43. Seeger M. A., Kaufman T. C. Molecular analysis of the bicoid gene from Drosophila pseudoobscura: identification of conserved domains within coding and noncoding regions of the bicoid mRNA. EMBO J. 1990 Sep;9(9):2977–2987. doi: 10.1002/j.1460-2075.1990.tb07490.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Seeger M., Tear G., Ferres-Marco D., Goodman C. S. Mutations affecting growth cone guidance in Drosophila: genes necessary for guidance toward or away from the midline. Neuron. 1993 Mar;10(3):409–426. doi: 10.1016/0896-6273(93)90330-t. [DOI] [PubMed] [Google Scholar]
  45. Simon J. A., Lis J. T. A germline transformation analysis reveals flexibility in the organization of heat shock consensus elements. Nucleic Acids Res. 1987 Apr 10;15(7):2971–2988. doi: 10.1093/nar/15.7.2971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Singh H., LeBowitz J. H., Baldwin A. S., Jr, Sharp P. A. Molecular cloning of an enhancer binding protein: isolation by screening of an expression library with a recognition site DNA. Cell. 1988 Feb 12;52(3):415–423. doi: 10.1016/s0092-8674(88)80034-5. [DOI] [PubMed] [Google Scholar]
  47. Sneddon A., Flavell A. J. The transcriptional control regions of the copia retrotransposon. Nucleic Acids Res. 1989 Jun 12;17(11):4025–4035. doi: 10.1093/nar/17.11.4025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Soeller W. C., Oh C. E., Kornberg T. B. Isolation of cDNAs encoding the Drosophila GAGA transcription factor. Mol Cell Biol. 1993 Dec;13(12):7961–7970. doi: 10.1128/mcb.13.12.7961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Sondermeijer P. J., Derksen J. W., Lubsen N. H. New cell line: established cell lines of Drosophila hydei. In Vitro. 1980 Nov;16(11):913–914. doi: 10.1007/BF02619327. [DOI] [PubMed] [Google Scholar]
  50. Tautz D., Pfeifle C. A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma. 1989 Aug;98(2):81–85. doi: 10.1007/BF00291041. [DOI] [PubMed] [Google Scholar]
  51. Whiteley M., Noguchi P. D., Sensabaugh S. M., Odenwald W. F., Kassis J. A. The Drosophila gene escargot encodes a zinc finger motif found in snail-related genes. Mech Dev. 1992 Feb;36(3):117–127. doi: 10.1016/0925-4773(92)90063-p. [DOI] [PubMed] [Google Scholar]
  52. Xiong W. C., Montell C. tramtrack is a transcriptional repressor required for cell fate determination in the Drosophila eye. Genes Dev. 1993 Jun;7(6):1085–1096. doi: 10.1101/gad.7.6.1085. [DOI] [PubMed] [Google Scholar]
  53. Yoshioka K., Honma H., Zushi M., Kondo S., Togashi S., Miyake T., Shiba T. Virus-like particle formation of Drosophila copia through autocatalytic processing. EMBO J. 1990 Feb;9(2):535–541. doi: 10.1002/j.1460-2075.1990.tb08140.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Zeng C., Pinsonneault J., Gellon G., McGinnis N., McGinnis W. Deformed protein binding sites and cofactor binding sites are required for the function of a small segment-specific regulatory element in Drosophila embryos. EMBO J. 1994 May 15;13(10):2362–2377. doi: 10.1002/j.1460-2075.1994.tb06520.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Zollman S., Godt D., Privé G. G., Couderc J. L., Laski F. A. The BTB domain, found primarily in zinc finger proteins, defines an evolutionarily conserved family that includes several developmentally regulated genes in Drosophila. Proc Natl Acad Sci U S A. 1994 Oct 25;91(22):10717–10721. doi: 10.1073/pnas.91.22.10717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. von Kalm L., Crossgrove K., Von Seggern D., Guild G. M., Beckendorf S. K. The Broad-Complex directly controls a tissue-specific response to the steroid hormone ecdysone at the onset of Drosophila metamorphosis. EMBO J. 1994 Aug 1;13(15):3505–3516. doi: 10.1002/j.1460-2075.1994.tb06657.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES