Molecular and General Genetics MGG

, Volume 244, Issue 5, pp 465–473 | Cite as

bHLH proteins encoded by theEnhancer of split complex ofDrosophila negatively interfere with transcriptional activation mediated by proneural genes

  • Nadja Oellers
  • Michaela Dehio
  • Elisabeth Knust
Original Paper


TheEnhancer of split complex [E(SPL)-C] ofDrosophila participates in the control of cell fate choice by uncommitted neuroectodermal cells in the embryo. It encodes seven proteins that belong to the basic helix-loop-helix (bHLH) family, six of which are expressed in very similar patterns in the neuroectoderm. Here we describe experiments aimed at unravelling the molecular basis of their function. We found that two products of the complex, HLH-M5 andEnhancer of split, are capable of binding as homo-and heterodimers to a sequence in the promoters of theEnhancer of split andachaete genes, called the N-box, which differs slightly from the consensus binding site (the E-box) for other bHLH proteins. In transient expression assays in cell culture, both proteins were found to attenuate the transcriptional activation mediated by the proneural bHLH proteinslethal of scute anddaughterless at theEnhancer of split promoter.

Key words

Drosophila Enhancer of split Helix-loop-helix protein Neurogenesis Transcriptional repressors 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Akazawa C, Sasai Y, Nakanishi S, Kageyama R (1992) Molecular characterization of a rat negative regulator with a basic helix-loop-helix structure predominantly expressed in the developing nervous system. J Biol Chem 267:21879–21885Google Scholar
  2. Alonso MC, Cabrera CV (1988) Theachaete-scute gene complex ofDrosophila melanogaster comprises four homologous genes. EMBO J 7:2585–2591Google Scholar
  3. Beckmann H, Su L-K, Kadesch T (1990) TFE3: a helix-loop-helix protein that activates transcription through the immunoglobulin enhancer μE3 motif: Genes Dev 4:167–179Google Scholar
  4. Benezra R, Davis RL, Lockshon D, Turner DL, Weintraub H (1990) The protein Id: a negative regulator of helix-loop-helix DNA binding proteins. Cell 61:49–59Google Scholar
  5. Blackwell TK, Kretzner, L, Blackwood EM, Eisenman, H, Weintraub, H (1990) Sequence-specific DNA-binding of the c-Myc protein. Science 250:1149–1151Google Scholar
  6. Brand M, Campos-Ortega JA (1988) Two groups of interrelated genes regulate early neurogenesis inDrosophila melanogaster. Roux's Arch Dev Biol 197:457–470Google Scholar
  7. Cabrera CV (1990) Lateral inhibition and cell fate during neurogenesis inDrosophila: the interactions betweenscute, Notch andDelta. Development 109:733–742Google Scholar
  8. Cabrera CV (1992) The generation of cell diversity during early neurogenesis inDrosophila. Development 115:893–901Google Scholar
  9. Cabrera CV, Alonso MC (1991) Transcriptional activation by heterodimers of theachaete-scute anddaughterless gene products ofDrosophila. EMBO J 10:2965–2973Google Scholar
  10. Cabrera CV, Martinez-Arias A, Bate M (1987) The expression of three members of theachaete-scute gene complex correlates with neuroblast segregation inDrosophila. Cell 50:425–1133Google Scholar
  11. Campos-Ortega JA (1993) Mechanisms of early neurogenesis inDrosophila melanogaster. Comments Dev Biol 1:287–310Google Scholar
  12. Carroll SB (1990) Zebra pattern in fly embryos: activation of stripes or repression of interstripes? Cell 60:9–16Google Scholar
  13. Carthew RW, Chodosh LA, Sharp PA (1985) An RNA polymerase transcription factor binds to an upstream element in the adenovirus major late promoter. Cell 43:439–448Google Scholar
  14. Caudy M, Vässin H, Brand M, Tuma R, Jan LY, Jan YN (1988)daughterless, a gene essential for both neurogenesis and sex determination inDrosophila, has sequence similarities tomyc and theachaete-scute complex. Cell 55:1061–1067Google Scholar
  15. Cronmiller C, Schedl P, Cline TW (1988) Molecular characterization ofdaughterless, aDrosophila sex determination gene with multiple roles in development. Genes Dev 2:1666–1676Google Scholar
  16. Davis RL, Cheng PF, Lassar AB, Weintraub H (1990) The MyoD DNA-binding domain contains a recognition code for muscle-specific gene activation. Cell 60:733–746Google Scholar
  17. Delidakis C, Artavanis-Tsakonas S (1992) The Enhancer of split [E(spl)] locus ofDrosophila encodes seven independent helix-loop-helix proteins. Proc Nat Acad Sci USA 89:8731–8735Google Scholar
  18. Di Nocera PP, David IB (1983) Transient expression of genes introduced into cultured cells ofDrosophila. Proc Natl Acad Sci USA 80:7095–7098Google Scholar
  19. Driever W, Nüsslein-Volhard C (1989) Thebicoid protein is a positive regulator ofhunchback transcription in the earlyDrosophila embryo. Nature 337:138–143Google Scholar
  20. Dynan WS, Tjian R (1983) The promoter-specific transcription factor Spl binds to upstream sequences in the SV40 early promoter. Cell 35:79–87Google Scholar
  21. Ellis HM, Spann DR, Posakony JW (1990) extramacrochaetae, a negative regulator of sensory organ development inDrosophila, defines a new class of helix-loop-helix proteins. Cell 61:27–38Google Scholar
  22. Fisher DE, Carr CS, Parent LA, Sharp PA (1991) TFEB has DNA-binding and oligomerization properties of a unique helix-loop-helix/leucine-zipper family. Genes Dev. 5:2342–2352Google Scholar
  23. Garrell J, Campuzano S (1991) The helix-loop-helix domain: a common motif for bristles, muscles and sex. Bioessays 13:493–498Google Scholar
  24. Garrell J, Modolell J (1990) TheDrosophila extramacrochaetae locus, an antagonist of proneural genes that, like these genes, encodes a helix-loop-helix protein. Cell 61:39–48Google Scholar
  25. Ghysen A, Dambly-Chaudière C, Jan LY, Jan Y-N (1993) Cell interactions and gene interactions in peripheral neurogenesis. Genes Dev. 7:723–733Google Scholar
  26. Gonzalez F, Romani S, Cubas P, Modolell J, Campuzano S (1989) Molecular analysis ofasense, a member of theachaete-scute complex ofDrosophila melanogaster, and its novel role in optic lobe development. EMBO J 8:3553–3562Google Scholar
  27. Gorman CF, Moffat LF, Howard BH (1982) Reconbinant genomes which express chloramphenicolacetyltransferase in mammalian cells. Mol. Cell Biol 2:1044–1051Google Scholar
  28. Gregor PD, Sawadogo M, Roeder RG (1990) The adenovirus major late transcription factor USF is a member of the helix-loop-helix group of regulatory proteins and binds to DNA as dimer. Genes Dev 4:1730–1740Google Scholar
  29. Hinz U, Giebel B, Campos-Ortega JA (1994) The basic-helix-loop-helix domain ofDrosophila LETHAL OF SCUTE protein is sufficient for proneural function and activates neurogenic genes. Cell 76 77–87Google Scholar
  30. Ishibashi M, Sasai Y, Nakanishi S, Kageyama R (1993) Molecular characterization of HES-2, a mammalian helix-loop-helix factor structurally related toDrosophila hairy andEnhancer of split. Eur J Biochem 215:645–652Google Scholar
  31. Jiménez F, Campos-Ortega JA (1990) Defective neuroblast commitment in mutants of theachaete-scute complex and adjacent genes ofDrosophila melanogaster. Neuron 5:81–89Google Scholar
  32. Jiménez F, Modolell J (1993) Neural fate specification inDrosophila. Curr Op Genetics Dev 3:626–632Google Scholar
  33. Kerkhoff E, Bister K (1991)Myc protein structure: localisation of DNA-binding and protein dimerization domains. Oncogene 6:93–102Google Scholar
  34. Klämbt C, Knust E, Tietze K, Campos-Ortega JA (1989) Closely related transcripts encoded by the neurogenic gene complexEnhancer of split ofDrosophila melanogaster. EMBO J 8:203–210Google Scholar
  35. Knust E, Bremer KA, Vässin H, Ziemer A, Tepaß U, Campos-Ortega JA (1987a) TheEnhancer of split locus and neurogenesis inDrosophila melanogaster. Dev Biol 122:262–273Google Scholar
  36. Knust E, Tietze K, Campos-Ortega JA (1987b) Molecular analysis of the neurogenic locusEnhancer of split ofDrosophila melanogaster. EMBO J 6:4113–4123Google Scholar
  37. Knust E, Schrons H, Grawe F, Campos-Ortega JA (1992) Seven genes of theEnhancer of split complex ofDrosophila melanogaster encode helix-loop-helix proteins. Genetics 132:505–518Google Scholar
  38. Krasnow MA, Saffman EE, Kornfeld K, Hogness DS (1989) Transcriptional activation and repression byUtrabithorax proteins in culturedDrosophila cells. Cell 57:1031–1043Google Scholar
  39. Martín-Bermudo MD, Martínez C, Jiménez F (1991) Distribution and function of thelethal of scute gene product during early neurogenesis inDrosophila. Development 113:445–454Google Scholar
  40. Martín-Bermudo MD, González F, Domínguez M, Rodríguez I, Ruiz-Gómez M, Romani S, Modolell J, Jiménez F (1993) Molecular characterization of thelethal of scute genetic function. Development 118:1003–1012Google Scholar
  41. Martinéz C, Modolell J (1991) Cross-regulatory interactions between the proneuralachaete andscute genes ofDrosophila. Science 251:1485–1487Google Scholar
  42. Martínez C, Modolell J, Garrell J (1993) Regulation of the proneural geneachaete by helix-loop-helix proteins. Mol Cell Biol 13:351Google Scholar
  43. Maxam AM, Gilbert W (1980) Sequencing end-labeled DNA with base-specific chemical cleavages. Meth Enzym 65:499–560Google Scholar
  44. Moscoso del Prato J, Garcia-Bellido A (1984) Genetic regulation of theachaete-scute complex ofDrosophila melanogaster. Roux's Arch. Dev. Biol. 193:242Google Scholar
  45. Murre C, Schonleber McCaw P, Baltimore D (1989a) The amphipathic helix-loop-helix: a new DNA-binding and dimerization motif in immunglobulin enhancer binding, daughterless, MyoD and myc proteins. Cell 56:777–783Google Scholar
  46. Murre C, Schonleber McCaw P, Vaessin H, Candy M, Jan LY, Jan YN, Cabrera CV, Buskin JN, Hauschka SD, Lassar AB, Weintraub H, Baltimore D (1989b) Interactions between heterologous helix-loop-helix proteins generate complexes that bind specifically to a common DNA sequence. Cell 58:537–544Google Scholar
  47. Olson EN (1990) MyoD family: a paradigm for development? Genes Dev 4:1454–1461Google Scholar
  48. Pankratz MJ, Jäckle H (1990) Making stripes in theDrosophila embryo. Trends Genet 6:287–292Google Scholar
  49. Prendergast GC, Lawe D, Ziff EB (1991) Association of Myn, the murine homolog of Max, with cMyc stimulates methylation-sensitive DNA-binding and Ras cotransformation. Cell 65:395–407Google Scholar
  50. Romani S, Campuzano S, Modolell J (1987) Theachaete-scute complex is expressed in neurogenic regions ofDrosophila embryos. EMBO J 6:2085–2092Google Scholar
  51. Ruiz-Gómez M, Ghysen A (1993) The expression and role of a proneural gene,achaete, in the development of the larval nervous system ofDrosophila. EMBO J 12:1121–1130Google Scholar
  52. Ruiz-Gómez M, Modolell J (1987) Deletion analysis of theachaete-scute locus ofDrosophila melanogaster. Genes Dev 1:1238–1246Google Scholar
  53. Rushlow CA, Hogan A, Pinchin SM, Howe KM, Lardelli M, Ish-Horowicz D (1989) TheDrosophila hairy protein acts in both segmentation and bristle patterning and shows homology to N-myc. EMBO J 8:3095–3103Google Scholar
  54. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-termination inhibitors. Proc Natl Acad Sci USA 74:5463–5467Google Scholar
  55. Sasai Y, Kageyama R, Tagawa Y, Shigemoto R, Nakanishi S (1992) Two mammalian helix-loop-helix factors structurally related toDrosophila hairy andEnhancer of split. Genes Dev 6:2620–2634Google Scholar
  56. Sawadogo M, Roeder RG (1985) Interaction of a gene-specific upstream stimulatory transcription factor with the adenovirus major late promoter upstream of the TATA box region. Cell 43:165–175Google Scholar
  57. Schneider I (1972) Cell lines derived from late embryonic stages ofDrosophila melanogaster. J Embryol Exp Morph 27:353–365Google Scholar
  58. Skeath JB, Carroll SB (1992) Regulation of proneural gene expression and cell fate during neuroblast segregation in theDrosophila embryo. Development 114:939–946Google Scholar
  59. Studier FW, Moffat BA (1986). Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189:113–130Google Scholar
  60. Thummel CS, Boulet AM, Lipshitz HD (1988) Vectors forDrosophila P element-mediated transformation and tissue culture transfection. Gene 74:445–450Google Scholar
  61. Tietze K, Oellers N, Knust E (1992)Enhancer of split D, a dominant mutation ofDrosophila and its use in the study of functional domains of a helix-loop-helix protein. Proc Nat Acad Sci USA 89:6152–6156Google Scholar
  62. Tietze K, Schrons H, Campos-Ortega JA, Knust E (1993) A functional analysis of the genesEnhancer of split andHLH-m5 during early neurogenesis ofDrosophila melanogaster. Roux's Arch Dev Biol 203:10–17Google Scholar
  63. Van Doren M, Ellis HM, Posakony JW (1991) TheDrosophila extramachrochaetae protein antagonizes sequence-specific DNA-binding bydaughterless/achaete-scute protein complexes. Development 113:245–255Google Scholar
  64. Van Doren M, Powell PA, Pasternak D, Singson A, Posakony JW (1992) Spatial regulation of proneural gene activity: auto and cross-activation ofachaete is antagonized byextramacrochatae. Genes Dev 6:2592–2605Google Scholar
  65. Villares R, Cabrera CV (1987) Theachaete-scute gene complex ofDrosophila melanogaster: conserved domains in a subset of genes required for neurogenesis and their homology tomyc. Cell 50:415–424Google Scholar
  66. Winslow GM, Hayashi S, Krasnow M, Hogness DS, Scott MP (1989) Transcriptional activation by the Antennapedia and fushi tarazu proteins in cultured Drosophila cells. Cell 57:1017–1030Google Scholar
  67. Younger-Shephard S, Vässin H, Bier E, Jan LY, Jan YN (1992)deadpan, an essential pan-neural gene encoding an HLH protein, acts as denominator inDrosophila sex determination. Cell 70:911–922Google Scholar

Copyright information

© Springer-Verlag 1994

Authors and Affiliations

  • Nadja Oellers
    • 1
  • Michaela Dehio
    • 1
  • Elisabeth Knust
    • 1
  1. 1.Institut für EntwicklungsbiologieUniversität zu KölnKölnGermany

Personalised recommendations