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An induction gene trap for identifying a homeoprotein-regulated locus

Nature Biotechnology volume 18, pages 746–749 (2000)Cite this article

Abstract

An important issue in developmental biology is the identification of homeoprotein target genes. We have developed a strategy based on the internalization and nuclear addressing of exogenous homeodomains, using an engrailed homeodomain (EnHD) to screen an embryonic stem (ES) cell gene trap library. Eight integrated gene trap loci responded to EnHD. One is within the bullous pemphigoid antigen 1 (BPAG1) locus, in a region that interrupts two neural isoforms. By combining in vivo electroporation with organotypic cultures, we show that an already identified BPAG1 enhancer/promoter is differentially regulated by homeoproteins Hoxc-8 and Engrailed in the embryonic spinal cord and mesencephalon. This strategy can therefore be used for identifying and mutating homeoprotein targets. Because homeodomain third helices can internalize proteins, peptides, phosphopeptides, and antisense oligonucleotides, this strategy should be applicable to other intracellular targets for characterizing genetic networks involved in a large number of physiopathological states.

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Figure 1: The induction gene trap strategy.
Figure 2: Purified EnHD binds DNA and is internalized by ES cells.
Figure 3: BPAG1 trapped locus is regulated by EnHD.
Figure 4: BPAG1 promoter/enhancer is a neural homeoprotein response element.

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Acknowledgements

We thank Drs C. Théry and M. Wassef for critical reading of the manuscript. We are grateful to Dr. A. Nagy for R1 ES cells, Dr. W. Skarnes for GT1 vector, Dr. S. Saule for providing us with Chick En-2 cDNA, and Dr A. Joyner for many helpful discussions. This work was supported by grants from Association Francaise contre les Myopathies, EC BIOTECH 960146 and HFSPO RG83/96 (to A.P and W.W.), fellowship from EMBO (M.L.M.), and the Deutsche Forschungsgemeinschaft SFP 190 (W.W.).

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Authors and Affiliations

  1. CNRS, UMR 8542, Ecole Normale Supérieure, 46 rue d'Ulm, Paris, 75230 Cedex 05, France

    Gaëll Mainguy, María Luz Montesinos, Brigitte Lesaffre, Alain Prochiantz & Michel Volovitch

  2. Max-Planck Institute of Psychiatry, Kraepelinstrasse 2-10, Munich, 80804, Germany

    Branco Zevnik & Wolfgang Wurst

  3. GSF-Forschungszentrum für Umwelt und Gesundheit, Ingolstädter Landstrasse 1, Oberschleissheim, 85764, Germany

    Mika Karasawa & Wolfgang Wurst

  4. Ottawa General Hospital Research Institute, Center for Molecular Medicine Ottawa, Ontario, Canada

    Rashmi Kothary

  5. University Paris 7, UFR de Biologie, 2 place Jussieu, Paris, 75005, France

    Michel Volovitch

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  1. Gaëll Mainguy
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Correspondence to Wolfgang Wurst or Alain Prochiantz.

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Mainguy, G., Luz Montesinos, M., Lesaffre, B. et al. An induction gene trap for identifying a homeoprotein-regulated locus. Nat Biotechnol 18, 746–749 (2000). https://doi.org/10.1038/77312

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