746 NATURE BIOTECHNOLOGY VOL 18 JULY 2000 http://biotech.nature.com RESEARCH ARTICLES Homeogenes code for homeoproteins, a large family of transcrip- tion factors characterized by their highly conserved 60-amino acid DNA-binding homeodomain 1 . They are highly expressed in the developing and adult nervous system, where they have been linked to many normal and pathological 2 processes. Phenotypic analyses of gene-targeted mouse embryos have been invaluable for understand- ing homeogene functions during development, but defining their precise mode of action requires that their target genes be identified. To systematically identify target genes of mammalian homeobox genes, we have developed an approach that combines induction gene trapping of embryonic stem (ES) cells 3,4 with the ability of extracellu- larly applied homeodomains to gain direct access to the cytoplasm and nucleus of cells in culture 5–7 and to interfere with the transcriptional activity of endogenous homeoproteins 8–11 . Since Engrailed-2 (En-2) is expressed by ES cells 12 , we used the En-2 homeodomain (EnHD) as a chemical inducer of engrailed targets. However, we anticipated that the use of EnHD as an inducer would permit identification of targets not only of Engrailed but also of homeoproteins that are expressed in ES cells and that, like Engrailed, have a glutamine in position 50 of their homeodomain (the Q50 family) 13 . Indeed, this position is very impor- tant in the determination of the DNA-binding properties of home- odomains 14 . The results presented below and identifying the BPAG1 locus as a homeoprotein target validate this strategy. Results Experimental strategy. Figure 1 illustrates the strategy developed in this study. Embryonic stem cells transformed either with PT2 or GT1 (Fig. 1B) were selected with neomycin. Because of the splice acceptor site, integrated vectors are controlled by endogenous regulatory ele- ments (Fig. 1C). Recombinant clones were then incubated with EnHD, which, once internalized, should compete with endogenous Engrailed (Fig. 1D) and modify lacZ expression when lacZ is fused to an Engrailed regulated exon. Since Engrailed acts primarily as a repres- sor 15 , ES cells were first transformed with PT2, a bicistronic vector allowing gancyclovir selection against integrations into constitutively active genes. In other experiments GT1 was used to identify genes con- stitutively active in ES cells and activated or repressed by Engrailed. This strategy requires that EnHD be internalized by a large number of ES cells. To demonstrate internalization, pure recombinant EnHD (Fig. 2A) showing efficient DNA binding in vitro (Fig. 2B) was labeled with fluorescein isothiocyanate (FITC) 5 and added to ES cultures for 2 h. Confocal sections show that EnHD is efficiently taken up by ES cells (Fig. 2C). Similar results were observed by immunocytochemical detection of EnHD (data not shown). Figure 2D illustrates lacZ induc- tion, following EnHD entry, in one of the responding clones. Identification of putative targets. A library of 30,000 indepen- dent integrations in ES cells was screened with the PT2 vector. In the case of GT1, 2,000 clones were expanded and individually plated in 96-well culture dishes. Table 1 summarizes the results obtained in the two independent screens. As anticipated, the four candidate tar- gets obtained with PT2 are upregulated by EnHD; however, the use of the GT1 vector allowed us to identify two upregulated and two downregulated candidate targets. Among the identified clones (see Table 1 for details), we decided to focus primarily on BPAG1 (clone C3) on the basis of its functions at the cellular and intercellular levels and because it has been involved in two important pathologies, dys- tonia musculorum and bullous pemphigoid. Indeed, the DNA sequence of the C3 integration (Fig. 3A) revealed that lacZ was fused to the dystonin neural isoform, also called BPAG1n (for bullous pemphigoid antigen 1 neural isoform). The organization of the BPAG1 locus has been partially elucidated 16 and is shown in Figure 3A with the lacZ gene integration. It encodes at least three neuronal isoforms (collectively BPAG1n), and one iso- form expressed in keratinocytes of the epidermis basal layer An induction gene trap for identifying a homeoprotein-regulated locus Gaëll Mainguy 1 , María Luz Montesinos 1 , Brigitte Lesaffre 1 , Branco Zevnik 2 , Mika Karasawa 3 , Rashmi Kothary 4 , Wolfgang Wurst 2,3 *, Alain Prochiantz 1 *, and Michel Volovitch 1,5 1 CNRS, UMR 8542, Ecole Normale Supérieure, 46 rue d’Ulm, 75230 Paris Cedex 05 France. 2 Max-Planck Institute of Psychiatry, Kraepelinstrasse 2-10, 80804 Munich, Germany. 3 GSF-Forschungszentrum für Umwelt und Gesundheit, Ingolstädter Landstrasse 1, Oberschleissheim, 85764, Germany. 4 Ottawa General Hospital Research Institute, Center for Molecular Medicine Ottawa, Ontario, Canada. 5 University Paris 7, UFR de Biologie, 2 place Jussieu, 75005 Paris, France. Corresponding authors (wurst@mpipsykl.mpg.de) and (prochian@wotan.ens.fr). Received 18 February 2000; accepted 2 May 2000 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 home- odomains, 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 regu- lated 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. Keywords: gene trap, nervous system, electroporation, homeoprotein, BPAG1 © 2000 Nature America Inc. • http://biotech.nature.com © 2000 Nature America Inc. • http://biotech.nature.com