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

1999; Elsevier BV; Volume: 113; Issue: 4 Linguagem: Inglês

10.1046/j.1523-1747.1999.00703.x

1523-1747

Gaëll Mainguy, Henrik Ernø, Marı́a Luz Montesinos, Brigitte Lesaffre, Wolfgang Wurst, Michel Volovitch, Alain Prochiantz,

Wnt/β-catenin signaling in development and cancer

In a recent gene-trap screen, we identified the gene coding for Epidermal Bullous Pemphigoid Antigen 1 (BPAG1) as a putative transcriptional target of Engrailed and of other homeoproteins with a glutamine in position 50 of their homeodomain. We now show that the nuclear addressing of the homeodomains of Engrailed (EnHD) and Antennapedia (AntpHD) upregulates BPAG1e transcription in immortalized human keratinocytes (GMA24FIA) expressing En1. This upregulation is not observed with AntpHD-Q50A, a variant of AntpHD in which a single mutation abolishes its high-affinity binding to target DNA, thus strongly suggesting that BPAG1e upregulation homeodomains reflects their specific recognition of homeoprotein-binding sites in the BPAG1e locus. This is further confirmed by DNase I footprinting and electrophoretic mobility shift assays that reveal, within the cloned BPAG1e promoter, several sites of direct interaction with EnHD and Engrailed. Co-transfection experiments in GMA24FIA human keratinocytes, COS-7 simian fibroblasts, and CHP-100 human neuroepithelial cells show that Engrailed, Hoxa-5, and Hoxc-8 regulate BPAG1e promoter activity and that this regulation is context-dependent. Finally, using a mouse line with LacZ inserted within the En1 locus, we identify the keratinocytes of the ventral paws, including the epithelial cells of the eccrine tubules, as a strong site of En1 expression throughout adulthood. We therefore propose that BPAG1e, a 230 kDa keratin-binding protein expressed in keratinocytes and participating in the maintenance of hemidesmosomes at the dermis–epidermis border, is directly regulated by homeoprotein transcription factors. In a recent gene-trap screen, we identified the gene coding for Epidermal Bullous Pemphigoid Antigen 1 (BPAG1) as a putative transcriptional target of Engrailed and of other homeoproteins with a glutamine in position 50 of their homeodomain. We now show that the nuclear addressing of the homeodomains of Engrailed (EnHD) and Antennapedia (AntpHD) upregulates BPAG1e transcription in immortalized human keratinocytes (GMA24FIA) expressing En1. This upregulation is not observed with AntpHD-Q50A, a variant of AntpHD in which a single mutation abolishes its high-affinity binding to target DNA, thus strongly suggesting that BPAG1e upregulation homeodomains reflects their specific recognition of homeoprotein-binding sites in the BPAG1e locus. This is further confirmed by DNase I footprinting and electrophoretic mobility shift assays that reveal, within the cloned BPAG1e promoter, several sites of direct interaction with EnHD and Engrailed. Co-transfection experiments in GMA24FIA human keratinocytes, COS-7 simian fibroblasts, and CHP-100 human neuroepithelial cells show that Engrailed, Hoxa-5, and Hoxc-8 regulate BPAG1e promoter activity and that this regulation is context-dependent. Finally, using a mouse line with LacZ inserted within the En1 locus, we identify the keratinocytes of the ventral paws, including the epithelial cells of the eccrine tubules, as a strong site of En1 expression throughout adulthood. We therefore propose that BPAG1e, a 230 kDa keratin-binding protein expressed in keratinocytes and participating in the maintenance of hemidesmosomes at the dermis–epidermis border, is directly regulated by homeoprotein transcription factors. apical epidermal ridge Antennapedia bullous pemphigoid antigen 1 epidermal isoform of bullous pemphigoid antigen 1 neural isoform of bullous pemphigoid antigen 1 Engrailed fluorescein isothiocyanate homeodomain Homeoproteins form a large family of transcription factors characterized by their highly conserved 60 amino-acid DNA-binding domain – the homeodomain (Gehring et al., 1994Gehring W.J. Qian Y.Q. Billeter M. et al.Homeodomain-DNA recognition.Cell. 1994; 78: 211-223Abstract Full Text PDF PubMed Scopus (674) Google Scholar). Their expression in chicken, mouse, and human skin is well documented (e.g.,Chang et al., 1998Chang P. Kozono T. Chida K. Kuroki T. Huh N. Differential expression of Hox genes in multistage carcinogenesis of mouse skin.Biochem Biophys Res Commun. 1998; 248: 749-752Crossref PubMed Scopus (18) Google Scholar;Duboule, 1998Duboule D. Hox is in the hair: a break in colinearity.Gens Dev. 1998; 12: 1-4Crossref Scopus (18) Google Scholar) and their implication in skin development and continuous regeneration or in appendage formation, proposed by several groups (e.g.,Chuong, 1993Chuong C.M. The making of a feather: homeoproteins, retinoids and adhesion molecules.Bioessays. 1993; 15: 513-521Crossref PubMed Scopus (96) Google Scholar;Scott and Goldsmith, 1993Scott G.A. Goldsmith L.A. Homeobox genes and skin development: a review.J Invest Dermatol. 1993; 101: 3-8Abstract Full Text PDF PubMed Google Scholar), is clearly emerging. For example, Hoxc-13 is fundamental for hair formation (Godwin and Capecchi, 1998Godwin A.R. Capecchi M.R. Hoxc13 mutant mice lack external hair.Genes Dev. 1998; 12: 11-20Crossref PubMed Scopus (190) Google Scholar), Dlx-3 plays a key role in keratinocyte terminal differentiation (Morasso et al., 1996Morasso M.I. Markova N.G. Sargent T.D. Regulation of epidermal differentiation by a Distal-less homeodomain gene.J Cell Biol. 1996; 135: 1879-1887Crossref PubMed Scopus (102) Google Scholar), and distinct POU domain transcription factors are important regulators of keratinocyte gene expression (Welter et al., 1996Welter J.F. Gali H. Crisch J.F. Eckert R.L. Regulation of human involucrin promoter activity by POU domain proteins.J Biol Chem. 1996; 271: 14727-14733Crossref PubMed Scopus (51) Google Scholar). Compared with the amount of information that has accumulated regarding the role and the importance of homeogenes during development, and throughout adulthood, our understanding of their precise mode of action at the cellular level is poorly understood. This is primarily due to the small number of direct or indirect homeoprotein transcriptional targets, identified so far. It is thus obvious that the physiologic significance of homeoprotein expression in the skin and its appendages requires that homeoprotein targets be identified in the latter structures. In several previous studies we have demonstrated that extracellularly applied homeodomains gain direct access to the cytoplasm and nucleus of cells in culture where they interfere with the transcriptional activity of endogenous homeoproteins (Joliot et al., 1991aJoliot A. Pernelle C. Deagostini-Bazin H. Prochiantz A. Antennapedia homeobox peptide regulates neural morphogenesis.Proc Natl Acad Sci USA. 1991; 88: 1864-1868Crossref PubMed Scopus (511) Google Scholar;Bloch-Gallego et al., 1993Bloch-Gallego E. Le Roux I. Joliot A.H. Volovitch M. Henderson C.E. Prochiantz A. Antennapedia homeobox peptide enhances growth and branching of embryonic chick motoneurons.In Vitro. J Cell Biol. 1993; 120: 485-492Crossref PubMed Scopus (52) Google Scholar;Le Roux et al., 1993Le Roux I. Joliot A.H. Bloch-Gallego E. Prochiantz A. Volovitch M. Neurotrophic activity of the Antennapedia homeodomain depends on its specific DNA-binding properties.Proc Natl Acad Sci USA. 1993; 90: 9120-9124Crossref PubMed Scopus (105) Google Scholar,Le Roux et al., 1995Le Roux I. Duharcourt S. Volovitch M. Prochiantz A. Ronchi E. Promoter-specific regulation of genes expression by an exogenously added homeodomain that promoters neurite growth.FEBS Lett. 1995; 368: 311-314Abstract Full Text PDF PubMed Scopus (24) Google Scholar). Based on the latter properties of homeodomains we have developed a screen based on an induction gene trap approach (Hill and Wurst, 1993Hill D.P. Wurst W. Screening for novel pattern formation genes using trap approaches.Methods Enzymol. 1993; 225: 664-681Crossref Scopus (29) Google Scholar;Forrester et al., 1996Forrester L.M. Nagy A. Sam M. et al.An induction gene trap screen in embryonic stem cells: Identification of genes that respond to retinoic acid in vitro.Proc Natl Acad Sci USA. 1996; 93: 1677-1682Crossref PubMed Scopus (108) Google Scholar) in which internalized homeodomains 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 homeoproteins, Engrailed1 and Engrailed2 (En1 and En2), are first expressed in the presumptive domain of the midbrain-hindbrain territory (Davis et al., 1988Davis C.A. Noble-Topham S.E. Rossant J. Joyner A.J. Expression of the homeobox containing gene En-2 delineates a specific region of the developing mouse brain.Genes Dev. 1988; 2: 361-371Crossref PubMed Scopus (164) Google Scholar;Gardner et al., 1988Gardner C.A. Darnell D.K. Poole S.J. Ordahl C.P. Barald K.F. Expression of an engrailed-like gene during development of the early embryonic chick nervous system.J Neurosci Res. 1988; 21: 426-437Crossref PubMed Scopus (93) Google Scholar). In addition, En1 is expressed in the spinal cord, in the dermomyotome, and in the ventral ectoderm of the limb bud. The use of EnHD as a chemical inducer has allowed us to identify Bullous Pemphigoid Antigen 1 (BPAG1) as a candidate Engrailed transcriptional target locus. In fact, EnHD binds to promoters also regulated by homeoproteins other than En2, including En1 and, most likely, several homeoproteins of the Q50 family that, as is the case for Engrailed, present a glutamine in position 50 of their homeodomain (Biggin and McGinnis, 1997Biggin M.D. McGinnis W. Regulation of segmentation and segmental identity by Drosophila homeoproteins: the role of DNA binding in functional activity and specificity.Development. 1997; 124: 4425-4433PubMed Google Scholar). Therefore it is obvious that this strategy can also lead to the identification of genes regulated by homeoproteins other than Engrailed. BPAG1 locus codes for two neural (BPAG1n1 and BPAG1n2) and in one epidermal (BPAG1e) proteins. BPAG1e is expressed mainly in epidermal keratinocytes, where it links keratin filaments to a cytoplasmic protein. It is assumed that BPAG1e takes part in the ontogenesis and maintenance of the hemidesmosomal structure at the dermal–epidermal border and is affected in autoimmune skin blistering disease (Guo et al., 1995Guo L. Degenstein L. Dowling J. Yu Q.C. Wollmann R. Perman B. Fuchs E. Gene targeting of BPAG1: abnormalities in mechanical strength and cell migration in stratified epithelia and neurologic degeneration.Cell. 1995; 81: 233-243Abstract Full Text PDF PubMed Scopus (387) Google Scholar). This study was thus undertaken to investigate the regulation of BPAG1e expression by homeoprotein transcription factors. GMA24FIA is a feeder independent immortalized subclone of human keratinocyte GMA (Barandon et al. 1989). GMA24FIA were grown in CFAD medium supplemented with EGF (Rochat et al., 1994Rochat A. Kobayashi K. Barrandon Y. Location of stem cells of human hair follicles by clonal analysis.Cell. 1994; 76: 1063-1073Abstract Full Text PDF PubMed Scopus (447) Google Scholar). CHP-100 cells were grown in RPMI 1640, 15% fetal calf serum, and COS-7 cells were maintained as inJoliot et al., 1997Joliot A. Trembleau A. Raposo G. Calvet S. Volovitch M. Prochiantz A. Association of engrailed homeoproteins with vesicles presenting caveolae-like properties.Development. 1997; 124: 1865-1875Crossref PubMed Google Scholar. The homeobox of chick Engrailed2 (EnHD) was amplified between a NdeI and a BamHI site from a plasmid containing the chick cDNA sequence (gift of Dr S. Saule;Plaza et al., 1997Plaza S. Langlois M.-C. Turque N. et al.The homeobox-containing Engrailed (En-1) product down-regulates the expression of Pax-6 through a DNA binding-independent mechanism.Cell Growth Diff. 1997; 8: 1115-1125Google Scholar) and introduced into the expression vector pET-3a. EnHD was produced in BL21(DE3)pLysS bacteria as inStudier et al., 1990Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Use of T7 RNA polymerase to direct expression of cloned genes.Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (5905) Google Scholar and purified on heparin-sepharose (Pharmacia, Orsay, France) (Perez et al., 1994Perez F. Lledo P.-M. Karagogeos D. Vincent J.D. Prochiantz A. Ayala J. Rab3A and Rab3B carboxy-terminal peptides are potent and specific inhibitors of prolactin release by rat cultured anterior pituitary cells.Mol Endocrinol. 1994; 8: 1278-1287Crossref PubMed Scopus (43) Google Scholar). FITC-labelling was performed as inJoliot et al., 1991aJoliot A. Pernelle C. Deagostini-Bazin H. Prochiantz A. Antennapedia homeobox peptide regulates neural morphogenesis.Proc Natl Acad Sci USA. 1991; 88: 1864-1868Crossref PubMed Scopus (511) Google Scholar. Full-length chick En2 coding sequence was reconstructed in pET-3a between the NdeI site and BamHI sites using a synthetic oligonucleotide (amino 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., 1989Patel N.H. Martin B.E. Coleman K.G. Poole S.J. Ellis M.C. Kornberg T.B. Goodman C.S. Expression of engrailed proteins in arthropods, annelids, and chordates.Cell. 1989; 58: 955-968Abstract Full Text PDF PubMed Scopus (796) Google Scholar). All standard procedures were as inAusubel et al., 1994Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley, 1994Google Scholar. 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., 1994Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley, 1994Google Scholar). 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: 5′-GGAAGTTCTTCCTCTACT-3′ and 5′-GGTTGAAACTACTCAGAG-3′. The resulting 519 bp amplified DNA product, which extends from position 3048 to position 3567 according toTamai et al., 1993Tamai K. Sawamura D. Do H.C. Tamai Y. Li K. Uitto J. The human 230-kD bullous pemphigoid antigen gene (BPAG1). Exon-intron organization and identification of regulatory tissue specific elements in the promoter region.J Clin Invest. 1993; 92: 814-822Crossref PubMed Scopus (34) Google Scholar, was cloned in the EcoRV site of pBluescript (Stratagene, La Jolla, USA). 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., 1997Joliot A. Trembleau A. Raposo G. Calvet S. Volovitch M. Prochiantz A. Association of engrailed homeoproteins with vesicles presenting caveolae-like properties.Development. 1997; 124: 1865-1875Crossref PubMed Google Scholar,Joliot et al., 1998Joliot A. Maizel A. Rosenberg D. Trembleau A. Dupas S. Volovitch M. Prochiantz A. Identification of a signal sequence necessary for the unconventional secretion of Engrailed homeoprotein.Curr Biol. 1998; 8: 856-863Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar) expressing full-length mouse Hoxa-5, Hoxc-8, and chicken En2. PTL1En2ΔN (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 μl of 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 inLe Roux et al., 1995Le Roux I. Duharcourt S. Volovitch M. Prochiantz A. Ronchi E. Promoter-specific regulation of genes expression by an exogenously added homeodomain that promoters neurite growth.FEBS Lett. 1995; 368: 311-314Abstract Full Text PDF PubMed Scopus (24) Google Scholar. DNA fragments were end-labeled by polynucleotide kinase and [γ-32P]ATP. Extracts from E.coli expressing EnHD or En2 protein and control E.coli extracts were added to the labelled probes in 20 mM Tris–HCl pH 7.9, 35 mM KCl, 2.5 mM MgCl2, 1 μg poly-dIdC, and 15% 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 by incubating the DNA fragments (200 ng) with 10–50 ng EnHD in a final volume of 50 μl containing 20 mM Tris–HCl pH 7.9, 35 mM KCl, 2.5 mM MgCl2, 1 μg poly-dIdC, 4% polyvinylalcohol, and 15% glycerol, for 10 min on ice and then adding an equal volume of DNase I in 10 mM MgCl2, 4 mM CaCl2 for 2 min. The DNA was extracted with phenol and chloroform, ethanol precipitated and analysed on a 6% sequencing gel. The sequences of the oligonucleotides used in gel-shift assays correspond to protected areas FP1 (–2225 to –2180) and FP2 (–694 to –665) in the footprint experiment. Volar epidermis of En1lki/+ mice (Hanks et al., 1995Hanks M. Wurst W. Anson Cartwright L. Auerbach A.B. Joyner A.L. Rescue of the En-1 Mutant Phenotype by replacement of En-1 with.En-2. Science. 1995; 269: 679-682Google Scholar) were dissected in phosphate-buffered saline, cryoprotected in phosphate-buffered saline 15% sucrose, embedded in tissue-tek (Gassalem, Limeil-Brevannes, France), sectioned (14 μm), air dried, and stored at –20°C. After fixation in 4% paraformaldehyde, β-galactosidase activity was revealed using standard procedures at 30°C for 24 h and sections counterstained with Ehrlich’s hematoxylin (BDH) were mounted in glycerol:gelatin (1:1). To determine whether the expression of BPAG1e is under the control of Engrailed ex vivo, we took advantage of a human immortalized basal keratinocyte cell line (GMA24FIA) (Barrandon et al., 1989Barrandon Y. Morgan J.R. Mulligan R.C. Green H. Restoration of growth potential in paraclones of human keratinocytes by a viral oncogene.Proc Natl Acad Sci USA. 1989; 86: 4102-4106Crossref PubMed Scopus (46) Google Scholar) that expresses Engrailed1 (En1). As shown in Figure 1(a), αEnhb-1, a polyclonal antibody recognizing both En1 and En2 (Davis et al., 1991Davis C.A. Holmyard D.P. Millen K.J. Joyner A.J. Examining pattern formation in mouse, chicken and frog embryos with an En-specific antiserum.Development. 1991; 111: 287-298PubMed Google Scholar), stains the cell nuclei. The same antibody recognizes a single 52 kDa protein on western blots (not shown), demonstrating that these cells express En1. The expression of En1 by GMA24FIA does not result from immortalization because the original GMA cells also express this transcription factor (not shown). By northern blot analysis a single transcript of 9 kb corresponding to BPAG1e mRNA was detected but not the 15 kb transcripts corresponding to BPAG1n (not shown). To demonstrate that EnHD is taken up by GMA24FIA cells in culture, fluorescein isothiocyanate (FITC) labelled EnHD (Joliot et al., 1991aJoliot A. Pernelle C. Deagostini-Bazin H. Prochiantz A. Antennapedia homeobox peptide regulates neural morphogenesis.Proc Natl Acad Sci USA. 1991; 88: 1864-1868Crossref PubMed Scopus (511) Google Scholar) was added to cells in culture for 2 h. The confocal sections of Figure 1(b) show the EnHD is efficiently taken up by GMA24FIA cells and accumulates in their nuclei. Similar results were observed by immunocytochemical detection of EnHD (not shown). We then analysed the effect of EnHD internalization on BPAG1e expression. As demonstrated by northern blot (Figure 2a) and in situ hybridization (Figure 2c), the addition of EnHD leads to a strong increase in the amount of BPAG1e mRNA (5-fold in Figure 2a). BPAG1e mRNA increase was also observed in the presence of cycloheximide (Figure 2c), strongly suggesting that regulation by EnHD is direct. Finally, GMA24FIA cells were incubated overnight with or without EnHD in the presence of cycloheximide, washed and further incubated for 2 h without cycloheximide allowing de novo protein synthesis. As illustrated in Figure 2(e), EnHD has a strong positive effect on BPAG1e synthesis, demonstrating that the induced mRNA is functional. Collectively, these data demonstrate that BPAG1e expression is increased by EnHD in GMA24FIA human keratinocytes. Homeoprotein target sequences are often promiscuous and different homeoproteins expressed in GMA24FIA cells may regulate the same genes, in particular members of the Q50 family that have a glutamine in position 50 of their homeodomain (Biggin and McGinnis, 1997Biggin M.D. McGinnis W. Regulation of segmentation and segmental identity by Drosophila homeoproteins: the role of DNA binding in functional activity and specificity.Development. 1997; 124: 4425-4433PubMed Google Scholar). Because several Hox genes are expressed in adult skin (Chang et al., 1998Chang P. Kozono T. Chida K. Kuroki T. Huh N. Differential expression of Hox genes in multistage carcinogenesis of mouse skin.Biochem Biophys Res Commun. 1998; 248: 749-752Crossref PubMed Scopus (18) Google Scholar), we used the homeodomain of Antennapedia (AntpHD), a Drosophila Hox homologue, to interfere with transcription. We also used AntpHD-Q50A (50A), a mutated form of AntpHD in which the glutamine in position 50 of the homeodomain was replaced by an alanine. Both peptides are taken up by cells in culture, but only AntpHD is able to interfere efficiently with endogenous homeoproteins due to the lower affinity of 50A for homeoprotein cognate binding sites (Le Roux et al., 1993Le Roux I. Joliot A.H. Bloch-Gallego E. Prochiantz A. Volovitch M. Neurotrophic activity of the Antennapedia homeodomain depends on its specific DNA-binding properties.Proc Natl Acad Sci USA. 1993; 90: 9120-9124Crossref PubMed Scopus (105) Google Scholar,Le Roux et al., 1995Le Roux I. Duharcourt S. Volovitch M. Prochiantz A. Ronchi E. Promoter-specific regulation of genes expression by an exogenously added homeodomain that promoters neurite growth.FEBS Lett. 1995; 368: 311-314Abstract Full Text PDF PubMed Scopus (24) Google Scholar). Figure 2(b) shows that AntpHD has an effect similar to that of EnHD on BPAG1e mRNA accumulation both with or without cycloheximide (Figure 2d). As expected, 50A had no clear effect on BPAG1e expression as illustrated by in situ hybridization (Figure 2d). These data demonstrate that AntpHD directly upregulates BPAG1e expression and that this effect requires that AntpHD specific DNA binding properties be preserved. They therefore support the hypothesis of a transcriptional regulation of BPAG1e by homeoproteins of the Q50 family. The promoter of BPAG1e has been characterized (Tamai et al., 1995Tamai K. Silos S.A. Li K. Korkeel E. Ishikawa H. Uitto J. Tissue-specific expression of the 230-kDa bullous pemphigoid antigen gene (BPAG1). Identification of a novel keratinocyte regulatory cis-element KRE3.J Biol Chem. 1995; 270: 7609-7614Crossref PubMed Scopus (17) Google Scholar;Sawamura et al., 1994Sawamura D. Sato T. Kon A. et al.Mouse 230-kDa bullous pemphigoid antigen gene: structural and functional characterization of the 5′-flanking region and interspecies conservation of the deduced amino-terminal peptide sequence of the protein.J Invest Dermatol. 1994; 103: 651-655Crossref PubMed Scopus (9) Google Scholar) (Figure 3d), thus allowing us to investigate its potential interactions with Engrailed. Electrophoretic mobility shift assays were performed using labelled BstNI restriction endonuclease fragments of the promoter region (–2466 to –89) and partially purified En2. Fragments I (–810 to –89) and II (–2226 to –1569), which shift in the presence of En2 (Figure 3a), were analysed by DNase I footprinting with EnHD. Footprinting experiments performed with their labelled strand gave identical results and revealed several protected areas (Figure 3b). Most of them (capitals in Figure 3d) harbour homeoprotein cognate binding sequences (Catron et al., 1993Catron K.M. Iler N. Abate C. Nucleotides flanking a conserved TAAT core dictate the DNA binding specificity of three murine homeodomain proteins.Mol Cell Biol. 1993; 13: 2354-2365Crossref PubMed Scopus (176) Google Scholar). Interestingly, several protected areas extend broadly (e.g., 107 bp protected in II from –1966 to –1860) suggesting homomeric cooperative binding, a hallmark of homeoproteins DNA interaction (Biggin and McGinnis, 1997Biggin M.D. McGinnis W. Regulation of segmentation and segmental identity by Drosophila homeoproteins: the role of DNA binding in functional activity and specificity.Development. 1997; 124: 4425-4433PubMed Google Scholar). One EnHD footprint does not contain any ATTA sequences. Footprinted sites corresponding to oligonucleotide (–2225 to –2180 that contains three bona fide ATTA motifs and oligonucleotide (–694 to –665) that lacks any ATTA sequence (underlined in Figure 3d), are also recognized by full-length En2 in a gel-shift assay (Figure 3c). Taken together, these data demonstrate that two functional clusters of Engrailed binding sites are present in the BPAG1e promoter. A 2.4 kb HindIII/BamHI BPAG1e genomic fragment (position –2463 to –89), which contains all Engrailed binding sites in the promoter (Figure 3c) and confers specific expression in keratinocytes (Sawamura et al., 1994Sawamura D. Sato T. Kon A. et al.Mouse 230-kDa bullous pemphigoid antigen gene: structural and functional characterization of the 5′-flanking region and interspecies conservation of the deduced amino-terminal peptide sequence of the protein.J Invest Dermatol. 1994; 103: 651-655Crossref PubMed Scopus (9) Google Scholar), was placed in front of a luciferase reported gene (pBP-luc). This construct was expressed in GMA24FIA cells together with plasmids encoding full-length En2 or En2 with a deleted N-terminal region (En2ΔN, Figure 4a). En2ΔN lacks the domains characterized for mediating active repression (Jiménez et al., 1997Jiménez G. Paroush Z. Ish-Horowicz D. Groucho acts as a corepressor for a subset of negative regulators, including Hairy and Engrailed.Genes Dev. 1997; 11: 3072-3082Crossref PubMed Scopus (206) Google Scholar;Tolkunova et al., 1998Tolkunova E.N. Fujioka M. Kobayashi M. Deka D. Jaynes J.B. Two distinct types of repression domain in Engrailed: one interacts with the Groucho corepressor and is preferentially active on integrated target genes.Mol Cell Biol. 1998; 18: 2804-2814Crossref PubMed Scopus (127) Google Scholar) and is therefore expected to act like EnHD. Co-transfecting pBP-luc with increasing amounts of the plasmid expressing En2ΔN resulted in an increase in promoter activity, whereas full-length En2 has the opposite (and dose-dependent) effect (Figure 4b). En2 is thus capable of actively repressing BPAG1e promoter activity in GMA24FIA cells and En2ΔN has the opposite effect by derepression. 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., 1998Stelnicki E. Komuves L. Kwong A. et al.HOX homeobox genes exhibit spatial and temporal changes in expression during human skin development.J Invest Dermatol. 1998; 110: 110-115Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). 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 inPinsonneault et al., 1997Pinsonneault J. Florence B. Vaessin H. McGinnis W. A model for extradenticle function as a switch that changes HOX proteins from repressors to activators.EMBO J. 1997; 16: 2032-2042Crossref PubMed Scopus (121) Google Scholar;Mann and Affolter, 1998Mann R.S. Affolter M. Hox proteins meet more partners.Curr Opin Gene Dev. 1998; 8: 423-429Crossref PubMed Scopus (317) Google Scholar). 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 Figure 4 and Figure 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. 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., 1996Loomis C.A. Harris E. Michaud J. Wurst W. Hanks M. Joyner A.L. The mouse Engrailed-1 gene and ventral limb patterning.Nature. 1996; 382: 360-363Crossref PubMed Scopus (243) Google Scholar) that, in mice deleted for En1, most of the typical palm structures are missing. This corresponds to a do