BPAG1) Synthesis by Homeoprotein Transcription Factors

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Regulation of Epidermal Bullous Pemphigoid Antigen 1 (BPAG1) Synthesis by Homeoprotein Transcription Factors

Gae¨ll Mainguy,* Henrik Ernø,* Marı´a Luz Montesinos,* Brigitte Lesaffre,* Wolfgang Wurst,†‡ Michel Volovitch,*§ and Alain Prochiantz* *CNRS, UMR 8542, Ecole Normale Supe´rieure, France; †Max-Planck Institute of Psychiatry, Munich, Germany; ‡GSF-Forschungszentrum fu¨r Umwelt und Gesundheit, Oberschleissheim, Germany; §University Paris, UFR de Biologie 2, Paris, France

In a recent gene-trap screen, we identified the gene the cloned BPAG1e promoter, several sites of direct coding for Epidermal Bullous Pemphigoid Antigen 1 interaction with EnHD and Engrailed. Co-transfection (BPAG1) as a putative transcriptional target of experiments in GMA24FIA human keratinocytes, Engrailed and of other homeoproteins with a glutam- COS-7 simian fibroblasts, and CHP-100 human neuro- ine in position 50 of their homeodomain. We now epithelial cells show that Engrailed, Hoxa-5, and show that the nuclear addressing of the homeodomains Hoxc-8 regulate BPAG1e promoter activity and that of Engrailed (EnHD) and Antennapedia (AntpHD) this regulation is context-dependent. Finally, using a upregulates BPAG1e transcription in immortalized mouse line with LacZ inserted within the En1 locus, human keratinocytes (GMA24FIA) expressing En1. we identify the keratinocytes of the ventral paws, including the epithelial cells of the eccrine tubules, as This upregulation is not observed with AntpHD- a strong site of En1 expression throughout adulthood. Q50A, a variant of AntpHD in which a single mutation We therefore propose that BPAG1e, a 230 kDa keratin- abolishes its high-affinity binding to target DNA, binding protein expressed in keratinocytes and parti- thus strongly suggesting that BPAG1e upregulation cipating in the maintenance of hemidesmosomes at homeodomains reflects their specific recognition of the dermis–epidermis border, is directly regulated by homeoprotein-binding sites in the BPAG1e locus. This homeoprotein transcription factors. Keywords: BPAG1/ is further confirmed by DNase I footprinting and Engrailed/hemidesmosomes/homeoproteins/keratinocytes. electrophoretic mobility shift assays that reveal, within J Invest Dermatol 113:643–650, 1999

omeoproteins form a large family of transcription development, and throughout adulthood, our understanding of factors characterized by their highly conserved 60 their precise mode of action at the cellular level is poorly understood. amino-acid DNA-binding domain – the homeodo- This is primarily due to the small number of direct or indirect main (Gehring et al, 1994). Their expression in homeoprotein transcriptional targets, identified so far. It is thus chicken, mouse, and human skin is well docu- obvious that the physiologic significance of homeoprotein expres- mentedH (e.g., Chang et al, 1998; Duboule, 1998) and their sion in the skin and its appendages requires that homeoprotein implication in skin development and continuous regeneration or targets be identified in the latter structures. in appendage formation, proposed by several groups (e.g., Chuong, In several previous studies we have demonstrated that extracellu- 1993; Scott and Goldsmith, 1993), is clearly emerging. For example, larly applied homeodomains gain direct access to the cytoplasm Hoxc-13 is fundamental for hair formation (Godwin and Capecchi, and nucleus of cells in culture where they interfere with the 1998), Dlx-3 plays a key role in keratinocyte terminal differentiation transcriptional activity of endogenous homeoproteins (Joliot et al, (Morasso et al, 1996), and distinct POU domain transcription 1991a; Bloch-Gallego et al, 1993; Le Roux et al, 1993, 1995). factors are important regulators of keratinocyte gene expression Based on the latter properties of homeodomains we have developed (Welter et al, 1996). a screen based on an induction gene trap approach (Hill and Wurst, Compared with the amount of information that has accumulated 1993; Forrester et al, 1996) in which internalized homeodomains regarding the role and the importance of homeogenes during are used as chemical inducers. In this screen, which will be described in detail elsewhere (Mainguy et al, in preparation), we have internalized the homeodomain of Engrailed2 (EnHD) into ES cells. Mammalian Engrailed Manuscript received January 14, 1999; revised April 6, 1999; accepted homeoproteins, Engrailed1 and Engrailed2 (En1 and En2), are first for publication May 28, 1999. expressed in the presumptive domain of the midbrain-hindbrain Reprint requests to: Dr. A. Prochiantz, CNRS, UMR 8542, Ecole territory (Davis et al, 1988; Gardner et al, 1988). In addition, En1 Normale Supe´rieure, 46, rue d’Ulm, 75230 Pans Cedex 05, France. Email: [email protected] is expressed in the spinal cord, in the dermomyotome, and in the Abbreviations: AER, apical epidermal ridge; Antp, Antennapedia; ventral ectoderm of the limb bud. BPAG1, bullous pemphigoid antigen 1; BPAG1e, epidermal isoform The use of EnHD as a chemical inducer has allowed us to of bullous pemphigoid antigen 1; BPAG1n, neural isoform of bullous identify Bullous Pemphigoid Antigen 1 (BPAG1) as a candidate pemphigoid antigen 1; En, Engrailed; FITC, fluorescein isothiocyanate; Engrailed transcriptional target locus. In fact, EnHD binds to HD, homeodomain. promoters also regulated by homeoproteins other than En2, includ-

acids 1–8) and a SmaI–EcoRI fragment (amino acids 9–290) taken from the same plasmid as above. Expression and purification on heparin was as described for EnHD. En2 was identified by western blot using 4D9 antibody (Patel et al, 1989). All standard procedures were as in Ausubel et al (1994). Northern blot analysis and in situ hybridization Cells were treated overnight with EnHD (300 ng per 105 cells) with or without 10–6 M cycloheximide. Northern blot and in situ hybridization were performed using standard techniques (Ausubel et al, 1994). Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) served as a loading control. To detect the BPAG1e transcript in keratinocytes, a probe was isolated from GMA24FIA cDNA by RT-PCR, using the following oligonucleotide primers: 5Ј- GGAAGTTCTTCCTCTACT-3Ј and 5Ј-GGTTGAAACTACTCAGAG- 3Ј. The resulting 519 bp amplified DNA product, which extends from position 3048 to position 3567 according to Tamai et al (1993), was cloned in the EcoRV site of pBluescript (Stratagene, La Jolla, USA). Co-transfection assay Starting from the genomic clone mdtl.4.I.2, containing the first BPAG1e exon (cloned by Dr R. Khotary from a wild- type mouse genomic DNA library provided by Drs A. Reaumme and R. Zirngibl), a 7 kb SacI–SalI subfragment containing the epidermal specific promoter was subcloned into the pBluescript vector. To express luciferase under the control of the BPAG1e promoter, a 2.4 kb HindIII fragment was then transferred into the HindIII site of pGL2-Basic (Promega, Lyon, France) leading to the generation of pBP-luc. pBP-luc was co-transfected in GMA24FIA with pTL1Hoxa5m, pTL1Hoxc8m, or pTL1En2m (Joliot et al, 1997, 1998) expressing full-length mouse Hoxa-5, Hoxc-8, and chicken En2. PTL1En2∆N (a deletion in the coding sequence of pTL1En2 up to the BlpI site) encodes a truncated En2 that lacks amino acids 2 to 142. GMA24FIA were transfected using the calcium phosphate precipitation method with a total of 11 µg plasmid DNA, consisting of the amounts of plasmids specified in the figure legends, and filler DNA up to 11 µg (pTL1 vector). COS-7 cells and CHP-100 cells were electroporated in 370 µlof medium and 10 µg of DNA with a gene pulser II apparatus (BioRad, Ivry/Seine, France) at 260 V and 1050 µF. After 48 h, cells were rinsed, lysed, and luciferase activity was measured as in Le Roux et al (1995). Figure 1. GMA24FIA keratinocytes express En1 and internalize EnHD. (A) Immunocytochemistry of GMA24FIA keratinocytes in culture Electrophoretic mobility shift assay and DNase I footprinting using αEnhb1 antibody (left panel, control without primary antibody). Scale analysis DNA fragments were end-labeled by polynucleotide kinase and bar:10µm. (B) GMA24FIA keratinocytes were incubated 2 h with FITC- [γ-32P]ATP. Extracts from E.coli expressing EnHD or En2 protein and EnHD (30 ng per 104 cells) (left) or FITC alone (right). Confocal sections control E.coli extracts were added to the labelled probes in 20 mM Tris– µ show that FITC-EnHD is internalized and accumulates in the nuclei. Scale HCl pH 7.9, 35 mM KCl, 2.5 mM MgCl2,1 g poly-dIdC, and 15% bar:5µm. glycerol, incubated for 10 min on ice and then analysed on native 6% polyacrylamide gels. For DNase I footprints the promoter fragments were end-labelled using Klenow enzyme. Footprinting assays were performed ing En1 and, most likely, several homeoproteins of the Q50 family by incubating the DNA fragments (200 ng) with 10–50 ng EnHD in a that, as is the case for Engrailed, present a glutamine in position final volume of 50 µl containing 20 mM Tris–HCl pH 7.9, 35 mM KCl, µ 50 of their homeodomain (Biggin and McGinnis, 1997). Therefore 2.5 mM MgCl2,1 g poly-dIdC, 4% polyvinylalcohol, and 15% glycerol, it is obvious that this strategy can also lead to the identification of for 10 min on ice and then adding an equal volume of DNase I in 10 mM genes regulated by homeoproteins other than Engrailed. MgCl2, 4 mM CaCl2 for 2 min. The DNA was extracted with phenol and chloroform, ethanol precipitated and analysed on a 6% sequencing gel. BPAG1 locus codes for two neural (BPAG1n1 and BPAG1n2) The sequences of the oligonucleotides used in gel-shift assays correspond and in one epidermal (BPAG1e) proteins. BPAG1e is expressed to protected areas FP1 (–2225 to –2180) and FP2 (–694 to –665) in the mainly in epidermal keratinocytes, where it links keratin filaments footprint experiment. to a cytoplasmic protein. It is assumed that BPAG1e takes part in lki/ϩ the ontogenesis and maintenance of the hemidesmosomal structure Histology Volar epidermis of En1 mice (Hanks et al, 1995) were at the dermal–epidermal border and is affected in autoimmune skin dissected in phosphate-buffered saline, cryoprotected in phosphate-buffered saline 15% sucrose, embedded in tissue-tek (Gassalem, Limeil-Brevannes, blistering disease (Guo et al, 1995). This study was thus undertaken to France), sectioned (14 µm), air dried, and stored at –20°C. After fixation investigate the regulation of BPAG1e expression by homeoprotein in 4% paraformaldehyde, β-galactosidase activity was revealed using standard transcription factors. procedures at 30°C for 24 h and sections counterstained with Ehrlich’s hematoxylin (BDH) were mounted in glycerol:gelatin (1:1). MATERIALS AND METHODS Cell culture GMA24FIA is a feeder independent immortalized subclone RESULTS of human keratinocyte GMA (Barandon et al, 1989). GMA24FIA were Regulation of BPAG1e expression by EnHD in human grown in CFAD medium supplemented with EGF (Rochat et al, 1994). CHP-100 cells were grown in RPMI 1640, 15% fetal calf serum, and keratinocytes To determine whether the expression of BPAG1e COS-7 cells were maintained as in Joliot et al (1997). is under the control of Engrailed ex vivo, we took advantage of a human immortalized basal keratinocyte cell line (GMA24FIA) Expression and purification of EnHD and Engrailed2 The homeobox (Barrandon et al, 1989) that expresses Engrailed1 (En1). As shown of chick Engrailed2 (EnHD) was amplified between a NdeI and a BamHI in Fig 1(A), αEnhb-1, a polyclonal antibody recognizing both En1 site from a plasmid containing the chick cDNA sequence (gift of Dr S. and En2 (Davis et al, 1991), stains the cell nuclei. The same Saule; Plaza et al, 1997) and introduced into the expression vector pET- 3a. EnHD was produced in BL21(DE3)pLysS bacteria as in Studier et al antibody recognizes a single 52 kDa protein on western blots (not (1990) and purified on heparin-sepharose (Pharmacia, Orsay, France) (Perez shown), demonstrating that these cells express En1. The expression et al, 1994). FITC-labelling was performed as in Joliot et al (1991a). Full- of En1 by GMA24FIA does not result from immortalization because length chick En2 coding sequence was reconstructed in pET-3a between the original GMA cells also express this transcription factor (not the NdeI site and BamHI sites using a synthetic oligonucleotide (amino shown). By northern blot analysis a single transcript of 9 kb VOL. 113, NO. 4 OCTOBER 1999 BPAG1e AS A HOMEOPROTEIN TARGET PROTEIN 645

Figure 2. BPAG1e expression is directly regulated by EnHD and AntpHD in GMA24FIA keratinocytes. (A, B) Northern blot analysis showing that GMA24FIA cells treated overnight with EnHD (A) or AntpHD (B) present a 5- fold increase in BPAG1e transcript levels. (C, D) BPAG1e transcripts detected by in situ hybridization on GMA24FIA cells incubated overnight with or without EnHD, AntpHD, or AntpHD-Q50A (50A) in the presence of cycloheximide. Scale bar:25µm. (E) Cells were treated overnight with cycloheximide plus or minus EnHD and the protein synthesis inhibitor was removed for 2 h before BPAG1e immunostaining (BP patient autoantisera, Dr C. Prost, Paris). Scale bar: 5 µm.

corresponding to BPAG1e mRNA was detected but not the 15 kb main of Antennapedia (AntpHD), a Drosophila Hox homologue, transcripts corresponding to BPAG1n (not shown). to interfere with transcription. We also used AntpHD-Q50A (50A), To demonstrate that EnHD is taken up by GMA24FIA cells in a mutated form of AntpHD in which the glutamine in position 50 culture, fluorescein isothiocyanate (FITC) labelled EnHD (Joliot of the homeodomain was replaced by an alanine. Both peptides et al, 1991a) was added to cells in culture for 2 h. The confocal are taken up by cells in culture, but only AntpHD is able to sections of Fig 1(B) show the EnHD is efficiently taken up by interfere efficiently with endogenous homeoproteins due to the GMA24FIA cells and accumulates in their nuclei. Similar results lower affinity of 50A for homeoprotein cognate binding sites (Le were observed by immunocytochemical detection of EnHD (not Roux et al, 1993, 1995). Figure 2(B) shows that AntpHD has an shown). effect similar to that of EnHD on BPAG1e mRNA accumulation We then analysed the effect of EnHD internalization on BPAG1e both with or without cycloheximide (Fig 2D). As expected, 50A expression. As demonstrated by northern blot (Fig 2A) and in situ had no clear effect on BPAG1e expression as illustrated by in situ hybridization (Fig 2C), the addition of EnHD leads to a strong hybridization (Fig 2D). These data demonstrate that AntpHD increase in the amount of BPAG1e mRNA (5-fold in Fig 2A). directly upregulates BPAG1e expression and that this effect requires BPAG1e mRNA increase was also observed in the presence of that AntpHD specific DNA binding properties be preserved. They cycloheximide (Fig 2C), strongly suggesting that regulation by therefore support the hypothesis of a transcriptional regulation of EnHD is direct. Finally, GMA24FIA cells were incubated overnight BPAG1e by homeoproteins of the Q50 family. with or without EnHD in the presence of cycloheximide, washed and further incubated for 2 h without cycloheximide allowing Engrailed binding sites are present within the promoter of de novo protein synthesis. As illustrated in Fig 2(E), EnHD has a BPAG1e The promoter of BPAG1e has been characterized (Tamai strong positive effect on BPAG1e synthesis, demonstrating that the et al, 1995; Sawamura et al, 1994) (Fig 3D), thus allowing us to induced mRNA is functional. Collectively, these data demonstrate investigate its potential interactions with Engrailed. Electrophoretic that BPAG1e expression is increased by EnHD in GMA24FIA mobility shift assays were performed using labelled BstNI restriction human keratinocytes. endonuclease fragments of the promoter region (–2466 to –89) and partially purified En2. Fragments I (–810 to –89) and II Regulation of BPAG1e expression by AntpHD in human (–2226 to –1569), which shift in the presence of En2 (Fig 3A), keratinocytes Homeoprotein target sequences are often promis- were analysed by DNase I footprinting with EnHD. Footprinting cuous and different homeoproteins expressed in GMA24FIA cells experiments performed with their labelled strand gave identical may regulate the same genes, in particular members of the Q50 results and revealed several protected areas (Fig 3B). Most of family that have a glutamine in position 50 of their homeodomain them (capitals in Fig 3D) harbour homeoprotein cognate binding (Biggin and McGinnis, 1997). Because several Hox genes are sequences (Catron et al, 1993). Interestingly, several protected areas expressed in adult skin (Chang et al, 1998), we used the homeodo- extend broadly (e.g., 107 bp protected in II from –1966 to 646 MAINGUY ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Figure 3. Engrailed binding sites are present in the BPAG1e promoter. (A) Electrophoretic mobility shift assay of Engrailed on BstNI fragments from the BPAG1e promoter. Lane 1, control (2 µgofE.coli extract); lane 2,2µg of En2-containing extract; fragments I (–810 to –89 and II (–2226 to –1569, minus strand) show high-affinity binding activity. (B) DNase footprint analysis of shifting fragments using purified 50 ng EnHD. The two rightmost lanes correspond to fragment II (–2226 to –1569) and the remaining four lanes to fragment I (–810 to –89) with a long run (left) and a short run (centre). Footprints are indicated by black bars. (C) Gel shift analysis of two footprinted areas (underlined in D) with full-length En2. The electrophoretic mobility shift assays in the right panel were performed with oligonucleotide (–2225 to –2180) that contains three bona fide ATTA motifs and in the left panel with oligonucleotide (–694 to –665) that lacks any ATTA sequence. 0, no extract; C, 2 µg E.coli extract; En, 2 µg En2-expressing E.coli extract. (D) Results of the DNase I footprint analysis of fragments I and II (A) using 50 ng purified EnHD; protected regions are in capitals.

–1860) suggesting homomeric cooperative binding, a hallmark of assay (Fig 3C). Taken together, these data demonstrate that two homeoproteins DNA interaction (Biggin and McGinnis, 1997). functional clusters of Engrailed binding sites are present in the One EnHD footprint does not contain any ATTA sequences. BPAG1e promoter. Footprinted sites corresponding to oligonucleotide (–2225 to –2180 that contains three bona fide ATTA motifs and oligonucleo- BPAG1e promoter activity is regulated by Engrailed, Hoxa- tide (–694 to –665) that lacks any ATTA sequence (underlined in 5, and Hoxc-8 A 2.4 kb HindIII/BamHI BPAG1e genomic Fig 3D), are also recognized by full-length En2 in a gel-shift fragment (position –2463 to –89), which contains all Engrailed VOL. 113, NO. 4 OCTOBER 1999 BPAG1e AS A HOMEOPROTEIN TARGET PROTEIN 647 binding sites in the promoter (Fig 3C) and confers specific active repression (Jimeńez et al, 1997; Tolkunova et al, 1998) and expression in keratinocytes (Sawamura et al, 1994), was placed in is therefore expected to act like EnHD. Co-transfecting pBP-luc front of a luciferase reported gene (pBP-luc). This construct was with increasing amounts of the plasmid expressing En2∆N resulted expressed in GMA24FIA cells together with plasmids encoding in an increase in promoter activity, whereas full-length En2 has full-length En2 or En2 with a deleted N-terminal region (En2∆N, the opposite (and dose-dependent) effect (Fig 4B). En2 is thus Fig 4A). En2∆N lacks the domains characterized for mediating capable of actively repressing BPAG1e promoter activity in GMA24FIA cells and En2∆N has the opposite effect by dere- pression. Hoxc-8 is expressed in murine skin in a graded distribution from posterior to anterior, and Hoxa-5 has recently been identified in human skin (Stelnicki et al, 1998). We therefore co-transfected pBP-luc with increasing amounts of the plasmids expressing Hoxa-5 or Hoxc-8. Figure 4(C) illustrates that both proteins downregulate BPAG1e promoter activity in GMA24FIA. Due to their interactions with several cofactors, homeoprotein activity is highly context- dependent (reviewed in Pinsonneault et al, 1997; Mann and Affoltter, 1998). Therefore we compared the regulation of BPAG1e promoter in human keratinocytes (GMA24FIA), COS-7 cells, and a human neuroepithelial cell line (CHP-100 cells)). As shown in Figs 4 and 5, En2, Hoxa-5, and Hoxc-8 effects differ very much between he different cell types. It is, in particular, noteworthy that En2, which represses the BPAG1e promoter in human keratinocytes, has the opposite activating effect in fibroblasts and neuroepithelial cells. Hoxc-8 and Hoxa-5, also repressors in keratinocytes, activate BPAG1e promoter activity in COS-7 and CHP-100 cells, respectively. Engrailed expression in a specific subpopulation of keratinocytes is compatible with a regulation of BPAG1e The experiments described above demonstrate that BPAG1e expression can be regulated by several homeoproteins, including Engrailed. A direct in vivo regulation of BPAG1e by En1 requires that the latter transcription factor be expressed in basal keratinocytes. It has been recently reported (Loomis et al, 1996) that, in mice deleted for En1, most of the typical palm structures are missing. This corresponds to

Figure 5. Engrailed, Hoxa-5, and Hoxc-8 regulation of BPAG1e promoter activity is context dependent. COS-7 simian ﬁbroblasts (A) or CHP-100 human neuroepithelial cells (B) were electroporated with pTL1 vector alone or 10 µg of plasmids expressing En2, Hoxa-5, or Hoxc- 8. *pϽ0.005, **pϽ0.0005.

Figure 4. Engrailed represses BPAG1e promoter activity in GMA24FIA keratinocytes. (A) Scheme of the two different constructs (En2 full-length and En2∆N) used in co-transfection with pBP-luc; 42– 68 box, Groucho interacting domain that mediates repression; hatched box, homeodomain (HD). (B, C)Twoϫ105 GMA24FIA keratinocytes were transfected with 1 µg pBP-luc plasmid and increasing doses (none, 1 µg, 3 µg, and 10 µg) of plasmids coding for En2 and En2∆N(B) or for Hoxc-8 and Hoxa-5 (C). After 48 h luciferase activity was measured in cell lysates. Each value corresponds to the average of four independent wells. *pϽ0.005; **pϽ0.0005 (Bonferroni/Dunn test). 648 MAINGUY ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY a dorsalization of the limb as suggested by the presence of strain; Hanks et al, 1995), we analysed the post-natal expression supernumerary hair follicles and by the loss of distal pads and pattern of En1 in the volar epidermis. We ﬁnd that LacZ is strongly associated eccrine glands. Accordingly, En1 is expressed in the expressed from birth until, at least, post-natal day 60 (P60) in all epidermis at early developmental stages when the fundamental traits keratinocytes of the pad epidermis and in the underlying eccrine of dorsal and ventral regions are established. However, the in vitro glands. Figure 6 illustrates LacZ expression at P8 and P28 and regulation of BPAG1e expression by Engrailed in adult human demonstrates that the levels of expression decrease from the apex keratinocytes (GMA24FIA) suggested that En1 might be expressed of the pad towards its borders and, therefore, correlates with the in vivo throughout adulthood. Using heterozygous mice with a thickness of the epidermis. Interestingly, P8 and P28 mice present LacZ reporter gene inserted within the En1 locus (En1lki mouse very similar levels of LacZ expression (Fig 6A, B), consistent with a maintenance of En1 expression in the adult. Moreover, Fig 6(B, C) also illustrates how LacZ expression is progressively lost as hair follicles appear at the transition between naked and hairy regions of the ventral hindlimb epithelium.

DISCUSSION The use of homeodomain translocation as a means to interfere specifically with the transcriptional activity of endogenous homeoproteins has been legitimized following the observation that AntpHD is internalized by cells in culture and addressed to their nuclei (Joliot et al, 1991a,b). Indeed, AntpHD internalized by fibroblasts in culture downregulates the transcriptional activity of Hoxd-9 on its own Hox-Responsive-Element (HRE) and this repression depends on the high-affinity binding properties of AntpHD on the HRE as illustrated by the lack of activity of the 50A mutant (Le Roux et al, 1995). Similarly, AntpHD, but not 50A, has a neurite growth promoting effect on cortical neurons and purified motor-neurons (Bloch-Gallego et al, 1993; Le Roux et al, 1993). The specificity of the transcriptional regulation by a distinct homeodomain is high in the sense that the presence of the glutamine in 50 is very important, but, conversely, is low as a homeodomain with a glutamine in position 50 is likely to interfere with the transcriptional activity of a large number of homeoproteins belonging to the Q50 family (Biggin and McGinnis, 1997). Thus, the gene trap screen in which EnHD was used to identify genes in the Engrailed genetic pathway is likely also to identify genes in the genetic pathway of other homeogenes, in particular Hox genes. This is indeed the case for the BPAG1 locus and, within this locus, for BPAG1e. The fact that BPAG1e expression can be regulated at the transcriptional level both by AntpHD and by EnHD is clearly demonstrated by the experiment in which the homeodomains are introduced into GMA24FIA cells. Both homeodomains upregulate the expression of BPAG1e by 5-fold and this upregulation is still observed in the presence of cycloheximide, thus in the absence of protein synthesis. The latter experiment, although it does not totally eliminate an effect of the homeodomains on the stability of the transcripts, strongly suggests that AntpHD and EnHD act directly at the transcriptional level and not through the induction of intermediate proteins. The previous identification of the promoter region of BPAG1e made it simple to verify the presence of homeoprotein binding sites and to localize them by gel shift and footprinting experiments. In these experiments we used EnHD and Engrailed and not any protein or homeodomain of the Hox family. This choice is justified by the lack of specificity among Q50 homeoproteins, at least in the conditions of such in vitro experiments in which naked DNA is used and which preclude complex protein–protein interactions. The physiologic interactions between the BPAG1e promoter and Engrailed or Hox proteins, here Hoxc-8 and Hoxa-5, was further verified by co-transfecting the three transcription factors and the luciferase reporter under the control of the BPAG1e promoter. In human GMA24FIA keratinocytes, the three proteins downregulate luciferase expression. Interestingly, a truncated form of Engrailed slightly larger than the homeodomain and deprived Figure 6. En1 is expressed in basal keratinocytes of volar epidermis. of the repressor domain enhances reporter activity. Because this Fourteen micrometre cryostat sections of volar epidermis of P8 (A) or P28 domain is unlikely to bear, by itself, any transcriptional activity, it (B, C) En1lki/ϩ hindlimb footpads stained for β-galactosidase activity. is likely that its action reflects its capability to antagonize the Forelimbs present similar patterns of expression (not shown). de, dermis; repressor activity of endogenous homeoproteins, including ep, epidermis; egt, eccrine gland tubules; hf, hair follicles. Engrailed. Thus, the fact that the homeodomain and the homeo- VOL. 113, NO. 4 OCTOBER 1999 BPAG1e AS A HOMEOPROTEIN TARGET PROTEIN 649 protein have opposite effects on the regulation of BPAG1e promoter and Dr R. Khotary for sending the genomic clone mdtI.4.I.2. We also express our activity further supports the strategy developed in this study and gratitude to Drs A. Rochat and Y. Barrandon for their help in designing and gives weight to the proposal that BPAG1e is a true homeoprotein interpreting several experiments. This work was supported by CNRS (A.P.), target. MENRT (A.P.), EC BIOTECH 960146 (to A.P. and W.W.), HFSPO The expression of homeoproteins in the dermis and the epidermis RG83/96 (to A.P. and W.W.), the Deutsche Forschungsgemeinschaft SFP 190 of developing skin is now well established. Among them are (W.W.) and fellowships from EMBO (H.E. and M.L.M.) proteins of the Hox, Msx, Dlx, Otx, Engrailed, or Pou families (Kanzler et al, 1994; Morasso et al, 1996; Welter et al, 1996; Stelnicki et al, 1997, 1988; Loomis, 1997; Chang et al, 1998). REFERENCES Expression is in the dermis and/or epidermis and is temporally and Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K: spatially regulated. 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