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Volume 270, Number 13, Issue of March 31, 1995 pp. 7609-7614
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Tissue-specific Expression of the 230-kDa Bullous Pemphigoid Antigen Gene (BPAG1)
IDENTIFICATION OF A NOVEL KERATINOCYTE REGULATORY cis-ELEMENT KRE3 (*)

(Received for publication, July 7, 1994; and in revised form, December 8, 1994)

Katsuto Tamai,  Stephanie A. Silos (§), ,  Kehua Li (¶), ,  Esa Korkeela,  Hiroyasu Ishikawa ,  Jouni Uitto (**)

From the Departments of Dermatology, and Biochemistry and Molecular Biology, Jefferson Medical College and the Section of Molecular Dermatology, Jefferson Institute of Molecular Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The 230-kDa bullous pemphigoid antigen gene (BPAG1) is expressed exclusively in basal keratinocytes of epidermis. In this study, we have identified a novel cis-element, keratinocyte responsive element 3 (KRE3), at position -216 to -197 of the human BPAG1 gene. A promoter-CAT construct containing this element had 50-fold higher expression than a similar construct devoid of this sequence when tested in transient transfections of cultured human keratinocytes. However, there was no effect on the low base-line level of expression in cultured skin fibroblasts. KRE3 contains a palindromic sequence 5`-CAAATATTTG-3`, and mutations in this sequence significantly reduced the promoter activity. Gel mobility shift assays with an oligomer containing KRE3 sequence demonstrated binding activity with nuclear proteins isolated from keratinocytes. One of the DNA/protein complexes was clearly specific, since competition with >12.5-fold excess of the unlabeled oligomer resulted in disappearance of this band. No specific binding activity was noted with nuclear proteins extracted from fibroblasts. Thus, KRE3 appears to serve as the binding site for keratinocyte-specific trans-activating factor(s), and KRE3 may thus confer the tissue-specific expression to the BPAG1 gene.

INTRODUCTION

Bullous pemphigoid (BP) (^1)is a blistering skin disease characterized by circulating IgG autoantibodies which recognize two distinct proteins, the 230-kDa bullous pemphigoid antigen (BPAG1) and the 180-kDa bullous pemphigoid antigen (BPAG2) (Stanley, 1989; Uitto and Christiano, 1992). These two proteins are components of hemidesmosomes, attachment structures anchoring the basal keratinocytes to the underlying cutaneous basement membrane (Uitto and Christiano, 1992). Recent cDNA cloning and chromosomal mapping of the BP antigen genes have clearly demonstrated that BPAG1 and BPAG2 are distinct gene products (Sawamura et al., 1992). Specifically, cloning of BPAG1 has demonstrated that the 230-kDa protein is a non-collagenous intracellular component of hemidesmosomal plaque (Tanaka et al., 1991; Sawamura et al., 1991a, 1991b). In contrast, the 180-kDa BP antigen is a transmembrane collagenous protein (Giudice et al., 1991, 1992; Hopkinson et al., 1992; Li et al., 1992), recently designated as type XVII collagen (Li et al., 1993). In addition to cDNA cloning, the entire gene structure and the intron-exon organization of human BPAG1 has been delineated (Tamai et al., 1993). The gene has been shown to consist of 22 distinct exons spanning 20 kb of the genomic DNA in the short arm of human chromosome 6 (Sawamura et al., 1990; Tamai et al., 1993).

The expression of the BPAG1 in skin, as determined at the mRNA level, has been shown to be limited to keratinocytes with proliferative, basal keratinocyte-like phenotype (Arnemann et al., 1993). The specificity of the expression of this gene has also been demonstrated by transient cell transfections utilizing BPAG1 promoter/chloramphenicol acetyltransferase (CAT) reporter gene constructs (Tamai et al., 1993). The latter studies revealed a marked, >20-fold higher expression in cultured human keratinocytes, as compared with dermal fibroblasts.

Previously, we have identified a specific cis-regulatory element, designated as keratinocyte responsive element 2 (KRE2), with the nucleotide sequence 5`-TGGTTCCCTAAGGCTAGTT-3` (Tamai et al., 1993, 1994). This sequence, which resides in the position -1,786 to -1,778 (upstream from the transcription initiation site) within the BPAG1 promoter, contains an AP2 binding site (underlined in the sequence). Elimination of KRE2 sequence from the BPAG1 promoter/CAT constructs significantly reduced the promoter activity in keratinocytes, whereas cloning of the KRE2 sequence in front of the truncated BPAG1 promoter/CAT construct re-established the high level of keratinocyte-specific expression. It was noted, however, that elimination of the KRE2 sequence resulted only in about 60% reduction in the CAT activity in transient keratinocyte transfections, although this activity was still significantly higher than the corresponding activity noted in fibroblasts transfected in parallel. We concluded, therefore, that additional factors are necessary for keratinocyte-specific expression of the BPAG1 gene.

In this study, we have identified a novel nucleotide sequence within the human BPAG1 promoter in the position -216 to -196. This sequence clearly confers keratinocyte-specific expression to the BPAG1 promoter/CAT construct, in comparison with fibroblasts which do not express the endogenous gene, as determined at the mRNA and protein levels. Furthermore, we provide evidence which suggests that distinct binding proteins are necessary for the KRE3 activity.

MATERIALS AND METHODS

Cell Cultures

Keratinocyte cultures were established from skin samples obtained during cosmetic surgery procedures. The cultures were maintained in serum-free, low calcium (0.15 mM) keratinocyte growth medium which is supplemented with epidermal growth factor, hydrocortisone, insulin, and bovine pituitary extract (KGM, Clonetics Corp., San Diego, CA). The cell cultures were passaged by trypsinization and studied in passage 2. Adult human skin fibroblast cultures were established from similar tissue specimens by explantation method, and the cultures were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calfs serum, 2 mM glutamine, 50 µg/ml streptomycin, and 50 units/ml penicillin. Fibroblast cultures were examined in passages 2-5.

Northern Analyses and Indirect Immunofluorescence

For Northern hybridizations, total RNA was isolated from cultured keratinocytes and fibroblasts by a single-step extraction procedure (Chomczynski and Sacchi, 1987). Total RNA, 30 µg/lane, was fractionated on 1.0% agarose gels and transferred to nitrocellulose filters (Sambrook et al., 1989). The filters were prehybridized and hybridized with the following cDNAs. For detection of BPAG1 mRNA, a 2.3-kb human cDNA (Sawamura et al., 1991a) was used; for detection of type I collagen mRNA, a 1.8-kb alpha2(I) collagen cDNA (Myers et al., 1981) was used; for detection of glyceraldehyde-3-phosphate dehydrogenase mRNA, a 1.3-kb human cDNA (ATCC) was used. The cDNAs were labeled radioactive with [alpha-P] CTP and [alpha-P] GTP by nick translation using a commercial kit (Boehringer Mannheim). The filters were washed to the final stringency of 0.5 times SSC, 0.1% SDS at 65 °C. The filters were then exposed to x-ray films for varying time periods up to 48 h.

For immunostaining, keratinocytes and fibroblasts were cultured on glass chamber slides under conditions described above. The slides were then fixed in cold (-20 °C) ethanol, rinsed with Tris-buffered saline, pH 7.6, and preincubated for 60 min with Tris-buffered saline containing 1% bovine serum albumin. The cells were then exposed to a human monoclonal anti-human 230-kDa BP antigen antibody (Sugi et al., 1989). After overnight incubation at 4 °C, the slides were washed with Tris-buffered saline, and incubated with tetramethylrhodamine isothiocyanate-conjugated anti-human IgG antibody (Miles Laboratories). Control cultures were incubated with the secondary antibody only, which gave essentially negative staining. After a 60-min incubation at room temperature, the slides were washed with Tris-buffered saline for 60 min, rinsed with distilled water, mounted, and examined with a fluorescent microscope.

Transient Cell Transfections

A series of human BPAG1 promoter/CAT reporter gene plasmids, containing a human BPAG1 promoter region of varying size, were used for transient transfections of cultured keratinocytes and fibroblasts. These cultures were also transfected with a human alpha2(I) collagen promoter/CAT construct (Boast et al., 1990), type I collagen being a characteristic gene product of dermal fibroblasts (see Uitto and Chu(1988)). The cells were co-transfected with a RSV/beta-gal construct, which was used as an internal control of transfection efficiency. The transfections of keratinocytes were performed with a commercial kit (DOTAP, Boehringer Mannheim), and fibroblasts were transfected with the calcium phosphate co-precipitation method (Sambrook et al., 1989). After 24 h of incubation, CAT activity was determined in cultures by incubating cell extracts with [^14C]chloramphenicol as substrate, followed by separation of its acetylated and non-acetylated forms by thin layer chromatography (Sambrook et al., 1989). The promoter activity was determined by counting the radioactivity in the acetylated forms of chloramphenicol, expressed as percent of the total radioactivity in the sample, after correction for the beta-galactosidase activity in the same cell extract.

Gel Mobility Shift Assays

For DNA binding assays, nuclear proteins were isolated from cultured keratinocytes and fibroblasts using a small-scale preparation technique (Schreiber et al., 1989). For the binding assay, double-stranded oligomers were end labeled with P, and aliquots containing approximately 5 times 10^4 cpm was incubated with 8 µg of nuclear protein extracts, as described previously (Tamai et al., 1994). In some reactions, a competing oligomer in 12.5-100-fold excess was added as a competitor. After the binding reaction, DNA-protein complexes were fractionated on 4% polyacrylamide gels under nondenaturing conditions, as described elsewhere (Tamai et al., 1994). The gels were dried, and autoradiographs were developed by exposure to x-ray films with intensifying screens at -70 °C.

RESULTS

Keratinocyte-specific Expression of BPAG1

Previous studies have suggested that the BPAG1 gene is specifically expressed in epidermal keratinocytes (see Stanley(1989)). In this study, we have examined the specificity of the keratinocyte expression by parallel comparison of cutaneous epidermal keratinocytes and dermal fibroblasts. To verify the cell type-specific expression in the cultures of these cells, immunofluorescence and Northern analyses were performed on normal human keratinocytes and fibroblasts (Fig. 1, A and B). Northern analyses were performed with a BPAG1 cDNA, as well as with an alpha2(I) collagen cDNA. As shown in Fig. 1, two different cell strains of normal human keratinocytes clearly expressed the BPAG1 mRNA with the apparent size of 9 kb, whereas no evidence of the corresponding mRNA in two different normal human fibroblast strains was noted. In contrast, type I collagen mRNAs (5.8 and 4.8 kb transcripts) were evident in fibroblast cultures, whereas there was no expression of this gene in keratinocytes (Fig. 1). The presence of glyceraldehyde-3-phosphate dehydrogenase mRNA was noted in both types of cells (Fig. 1B). The selective expression of the BPAG1 gene in keratinocytes was confirmed at the protein level by indirect immunofluorescence using a monoclonal antibody recognizing a 230-kDa BP antigen epitope. Bright immunofluorescence staining was noted on keratinocytes, whereas fibroblasts displayed only background staining (Fig. 1A), similar to that observed in negative controls stained with secondary antibody alone (not shown).

Figure 1: Tissue-specific expression of the BPAG1 gene. A, the expression of 230-kDa bullous pemphigoid antigen at the protein level was examined by indirect immunofluorescence of normal human keratinocytes (NHK) and normal human fibroblasts (NHF) with human anti-human monoclonal antibody. Note strongly positive immunofluorescence in NHK cultures, whereas NHF cultures are negative. B, Northern analysis of RNA isolated from NHK or NHF cultures. Total RNA, 30 µg/lane, was electrophoresed on 1.0% agarose gels, and the Northern filters were hybridized successively with human BPAG1, alpha2(I) collagen, or with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNAs. Note that two separate NHK cultures depict a clear signal of 9 kb with BPAG1 cDNA, whereas the NHF mRNA is devoid of the corresponding transcript. The latter cells clearly demonstrate the presence of the alpha2(I) collagen mRNA transcripts. The mRNA for glyceraldehyde-3-phosphate dehydrogenase, a ubiquitously expressed housekeeping gene, is present in both types of cultures.


To demonstrate the selective expression of these genes at the transcriptional level, keratinocytes and fibroblasts were transfected with a BPAG1- or an alpha2(I)-collagen-promoter/CAT reporter gene construct in parallel. The BPAG1/CAT construct used for transfection contained 296 bp of 5`-flanking sequence upstream from the transcription initiation site. Keratinocytes clearly expressed the BPAG1/CAT construct, whereas no evidence for CAT activity was noted in fibroblast cultures (Fig. 2). Conversely, activity of the alpha2(I) collagen-promoter/CAT containing 3.2 kb of 5`-flanking DNA was clearly detected in fibroblasts, whereas no evidence for the expression of this construct in keratinocytes was noted (Fig. 2). Thus, normal human keratinocytes in culture selectively express the BPAG1 promoter, as compared with normal human fibroblasts which do not express this gene at the mRNA or protein levels. These results also indicate that the cis-elements conferring keratinocyte-specific expression to the BPAG1 gene reside within 0.3 kb of the 5`-flanking DNA.

Figure 2: Keratinocyte-specific expression of the BPAG1 promoter in transient transfections. NHF or NHK cultures were transfected in parallel with a BPAG1 promoter (pBP296CAT), human alpha2(I) collagen promoter, or SV2 promoter CAT constructs. The constructs were co-transfected with a RSV-betagal construct. A parallel culture was transfected with the promoterless pBS0CAT construct (C). The promoter activities were expressed as percent acetylation by determining the radioactivity in acetylated forms of [^14C]chloramphenicol as a percent of the total radioactivity in the sample and corrected for transfection efficiencies by determination of the activity of beta-galactosidase in the same specimens. The values on the right (B) are the mean of two parallel determinations shown in the autoradiograms on the left (A). The results indicate that the relative activity of the BPAG1 promoter in NHK cultures is 52.0 times higher than that of alpha2(I) collagen, after correction for SV2 promoter activity and transfection efficiency by beta-galactosidase determination. Also, the BPAG1 expression in NHK cultures was 52.0 times higher than that in NHF cultures.


Identification of Keratinocyte-responsive Element 3 (KRE3)

To pinpoint the sequences responsible for the keratinocyte-specific expression of the BPAG1 gene, 5` deletion libraries of the promoter/CAT construct were developed by polymerase chain reaction amplification. Truncation of the clone pBP296 CAT from the 5` end to -176 resulted in complete loss of keratinocyte-specific expression, and this loss of specificity was similarly evident in three additional clones with their 5` ends at -106, -56 and -26, examined under identical conditions (Fig. 3). To define more precisely the keratinocyte-specific element(s) within the region extending from -296 to -176, several additional 5` deletion clones were developed. As shown in Fig. 4, the relative CAT activity remained essentially unaltered as a result of truncation of the construct pBP296CAT down to -216. However, further deletion of 20 bp, to generate the clone pBP196CAT, resulted in complete loss of the CAT activity (Fig. 4). This segment was designated as keratinocyte responsive element 3 (KRE3).

Figure 3: Transient transfections of NHK and NHF cultures with 5`-deletion BPAG1 promoter/CAT reporter gene constructs. A series of BPAG1 promoter constructs with the size of the promoter region indicated on the left were transfected in parallel to NHK and NHF cultures, and CAT activity was determined as shown in Fig. 2. As shown on the right, significant CAT activity was noted in NHK cultures with the construct pBP296CAT, whereas the four shorter deletion clones did not demonstrate similar activity. Furthermore, none of the five clones demonstrated detectable activity in NHF cultures.


Figure 4: Fine mapping of the region conferring keratinocyte-specific expression to the BPAG1 promoter in transient transfections of NHK cultures. Deletion clones between the region extending from -296 to -176, the area which was shown in Fig. 3to contain the putative keratinocyte-specific elements, were developed. The constructs were transfected to NHK cultures in parallel, and CAT activity was determined as shown in Fig. 3. The relative CAT activity was determined as percent acetylation as described in Fig. 2, and the activity noted with the construct pBP296 CAT was set as 100%. Examination of the CAT activity indicates that deletion of the 20-bp segment between -216 and -196 results in essentially complete loss of expression. The sequence between -216 and -177 is shown below the figure, and the segment containing the putative keratinocyte-responsive element, KRE3, is underlined. Note also the presence of CAAT and SP1 consensus sequences (asterisks).


Examination of the nucleotide sequence between -216 and -197 revealed the presence of a palindromic sequence, 5`-CAAATATTTG-3`. This sequence was just upstream from the canonical CAAT box, 5`-CCAAT-3`, and an overlapping SP1 site, 5`-CGCCC-3` (asterisks in Fig. 4). To further characterize the role of the KRE3 region in providing keratinocyte-specific expression to the BPAG1 promoter, nucleotide substitutions were introduced to this region by polymerase chain reaction amplification using primers with altered sequence. The mutated promoter constructs were then tested in transient transfections of keratinocyte cultures in parallel. One of the mutated constructs (M5), containing two adjacent nucleotide substitutions, was expressed at the same level as the control promoter construct, pBP216 CAT (Fig. 5). Substitution of the two most 5` nucleotides (M1) or central nucleotides (M4) resulted in 48 and 24% inhibition of the activity, respectively (Fig. 5). However, substitution of two As by Ts (construct M2) or two Ts by As (construct M3) significantly (78 and 68%, respectively) decreased the promoter activity (Fig. 5). The nucleotide substitutions in the M2 and M3 constructs were within the palindromic 10-bp segment (Fig. 5, underlined). Thus, this core sequence within KRE3 may play a critical role in providing tissue specificity to the BPAG1 expression.

Figure 5: Transient transfections with the BPAG1 promoter/CAT construct pBP216 CAT containing the KRE3 element, as well as five additional similar constructs, M1-M5, into which two base pair mutations were introduced (outlined) within the KRE3 region. The relative CAT activity was determined as in Fig. 2Fig. 3Fig. 4. As indicated, the 2-base pair substitutions in the construct M5 did not alter the promoter activity while 2-base pair substitutions in M1, M2, M3, and M4 resulted in a marked reduction of the promoter activity. The values are means of duplicate assays. Note that the substitutions in M2 and M3 are within the palindromic sequence 5`-CAAATATTTG-3` within the KRE3 element (underlined).


Evidence for Keratinocyte-specific trans-Acting Factors

To examine the putative functionality of the KRE3 sequence, nuclear proteins were isolated from keratinocytes, and gel mobility shift assays with the 20-bp oligonucleotide corresponding to KRE3 were performed, as shown in Fig. 6. Incubation of the oligomer extending from -216 to -196 with the nuclear protein extracts from keratinocytes resulted in the formation of three distinct DNA/protein complexes (Fig. 6, second lanes). The binding in band a was specific, since competition with a 100-fold excess of the same unlabeled oligomer resulted in disappearance of this band (Fig. 6, third lanes). This displacement by competitor was noted in separate experiments with as little as 12.5-fold excess of the same oligomer (not shown). However, the DNA/protein complexes b and c were still detectable after competition, suggesting less specific binding. Furthermore, a 100-fold excess of an oligomer overlapping with the 3` end of KRE3 but devoid of the palindromic sequence as a competitor failed to abolish the formation of the complex a (Fig. 6, fourth lanes).

Figure 6: Gel mobility shift assay with a 20-bp oligomer containing the KRE3 sequence with nuclear proteins extracted from NHK or NHF cultures. Radiolabeled oligomer, 50 ng containing 5 times 10^4 cpm, was incubated with 8 µg of the nuclear protein. In some reactions, a competing oligonucleotide KRE3 was added in 100-fold excess or a 20-bp oligomer (DS) containing the last 10 bp of the KRE3 sequence and 10 downstream nucleotides was added as competitor. Autoradiogram indicates three radioactive DNA/protein bands in NHK (a-c), and the binding activity in band a was specific since it could be competed with excess of unlabeled KRE3. Downstream oligomer DS as a competitor did not displace the binding activity. Note that proteins extracted from NHF cultures (on the right) did demonstrate nonspecific binding activity only.


Similar gel mobility shift assays were performed with the 20-bp KRE3 oligomer using nuclear extracts from cultured dermal fibroblasts (NHF). No specific binding activity similar to the bands noted with keratinocyte protein extracts could be detected in fibroblasts (Fig. 6). Thus, keratinocytes appear to contain specific binding protein(s) which interact with the KRE3 within the BPAG1 promoter.

DISCUSSION

Expression of the BPAG1 gene, encoding the 230-kDa bullous pemphigoid antigen, is highly restricted, as determined both at the protein and the mRNA levels (Jordan et al., 1967, 1971; Mutasim et al., 1985; Sawamura et al., 1991a, 1991b; Tamai et al., 1993). Specifically, this gene is expressed only in stratifying squamous epithelia, such as the epidermis, and its expression within the epidermis is limited to basal keratinocytes with mitotic phenotype. We have previously identified a cis-acting element, KRE2, which resides within the BPAG1 promoter region at position -1786 to -1778 (Tamai et al., 1993, 1994). We demonstrated that elimination of KRE2 from the promoter region cloned in CAT constructs significantly reduced the expression of the BPAG1 promoter in transient transfection of keratinocytes, whereas it had no effect on the low level of expression in fibroblasts. The KRE2 had sequence similarity with the AP2 consensus binding site, and in fact, AP2 trans-activating factor was able to bind to an oligomer containing the KRE2 sequence in gel mobility shift assays (Tamai et al., 1994). More importantly, however, we were able to demonstrate a novel keratinocyte-specific binding protein, KTP-1, which was binding to the KRE2 sequence. Utilizing gel mobility shift assays, UV cross-linking studies, and Southwestern analysis, we clearly demonstrated that KTP-1 is distinct from AP2 in molecular size and subunit composition, but has similar binding activities (Tamai et al., 1994).

In this study, we have identified a novel keratinocyte-responsive element, KRE3, a 20-bp segment at -216 to -196, which appears to be necessary for keratinocyte-specific expression of the gene. Elimination of KRE3 from BPAG1 promoter/CAT constructs abolished the promoter activity in keratinocytes, and similarly, introduction of distinct point mutations within the KRE3 region significantly suppressed the promoter activity. The critical region within KRE3 appeared to consist of a palindromic sequence, 5`-CAAATATTTG-3`. In this context, it is of interest that several regulatory cis-elements, such as the binding sites for AP1 and AP2, similarly demonstrate a palindromic sequence (Angel et al., 1987; Williams and Tjian, 1991). Gel mobility shift assays demonstrated three bands with binding activity, one of them being specific, as illustrated by competition assays. The specific nature of this binding activity is currently unknown, but the KRE3 binding site is clearly distinct from that for AP2. Thus, it is conceivable that KRE3 plays a critical role in providing tissue-specific expression to the BPAG1 gene.

Several other genes, in addition to BPAG1, are also expressed exclusively in the basal keratinocytes of the epidermis, including the BPAG2 gene encoding the 180-kDa bullous pemphigoid antigen, as well as keratin 5 and 14 genes (KRT5 and KRT14). In case of the genes encoding epidermal keratins, there is considerable evidence for the role of AP2 in participating in the keratinocyte-specific expression (Leask et al., 1990, 1991; Snape, 1990, 1991; Byrne and Fuchs, 1993). In particular, in KRT14 promoter region, an AP2 site within -220 plays a critical role in providing tissue-specific expression, and elimination of this element reduces the overall activity of the promoter. However, an additional contribution to the tissue-specificity is provided by an upstream distal element between -2,100 and -1,700. This distal element appears to act in concert with the proximal AP2 element in the KRT14 promoter to confer keratinocyte specificity (Leask et al., 1990). In case of KRT5, deletion of an AP2 binding site at position -104 to -94, as well as mutations in this cis-element, reduced the overall level of KRT5 expression in cultured keratinocytes (Byrne and Fuchs, 1993). However, in either case was the cell type specificity preserved, a result which was confirmed in transgenic mice. Thus, it appears that in case of KRT5 gene expression, AP2 is implicated primarily in regulating the level of gene expression, but this regulatory element does not contribute appreciably to the epithelial cell-specific expression which is displayed by the 90-bp segment of promoter in transgenic animals. Collectively, it appears that the epithelial cell-specific expression of the KRT5 gene is regulated by two protein complexes (see Byrne and Fuchs(1993)). One of them, complex 1-2, binds close to the transcription initiation site, whereas the other, complex 4, binds in the vicinity of the TATA box.

As indicated above, BPAG1, as well as KRT5 and KRT14, are expressed exclusively in the basal keratinocytes of the epidermis in vivo, and their expression is not at all detectable at the suprabasal level. At the same time, the terminal differentiation of the epidermal keratinocytes involves induction of keratin 1 and 10 gene expression (Fuchs and Green, 1980; Moll et al., 1982). Furthermore, the expression of other suprabasal genes, including desmosomal proteins and the cornified envelope genes, will be activated (Steinert and Roop, 1988; Huff et al., 1993; Saunders et al., 1993). The mechanisms for suppression of BPAG1 gene expression in the suprabasal keratinocytes are currently not known. However, since inactivation of KRE3 results in complete loss of BPAG1 gene expression, the lack of trans-activation of KRE3 could potentially explain down-regulation of this gene in suprabasal levels.

Of particular interest to the epidermal differentiation process is a recent discovery of a gene family characterized by a bipartite binding motif referred to as the POU domain (Sturm et al., 1988; Ko et al., 1988; Clerc et al., 1988). These domains serve as binding sites for cell-specific transcription factors, many of which have been suggested to be important in terminal differentiation of neuronal, pituitary, and B lymphocyte cell types, respectively (see Andersen et al., 1993). These differentiation processes have been suggested to be mediated by cell-specific trans-activating factors, including Oct-2 (see Schöler, 1991). In addition, Oct-1 is a ubiquitous activator of gene programs required for cell proliferation and may also play a cell-specific role (Fletcher et al., 1987; Tanaka et al., 1988; Verrijzer et al., 1990; Luo et al., 1992). Recently, two functionally distinct trans-activating factors, Skn-1a and Skn-1i, which are highly related to Oct-2, have been shown to be selectively expressed in terminally differentiating epidermis (Anderson et al., 1993). The two factors are members of the POU domain family of transcriptional regulators. One of these factors, Skn-1i, contains an amino-terminal domain that inhibits DNA binding and can inhibit trans-activation of genes by Oct-1. The second form, Skn-1a, contains an alternative amino terminus and serves specifically to activate keratin 10 gene expression (Anderson et al., 1993). These two factors are products of alternative RNA splicing, and Skn-1a contains 113 unique amino acids at the NH(2) terminus replacing 31 amino acid residues in Skn-1i. Expression of a mutant Skn-1i lacking 60 amino acids from the amino terminus also activated the KRT10 promoter. It is likely, therefore, that the critical function of NH(2)-terminal sequences of Skn-1a is to relieve the action of the Skn-1i inhibitory domain, rather than to serve as a trans-activation domain (Anderson et al., 1993). Collectively, these data suggest that the Skn-1a/i system represents tissue-restricted POU domain factors that exert selective activating and inhibiting functions in developing epidermis. Similar mechanisms may be operative in allowing expression of other suprabasal genes, such as KRT1, desmosomal cadherins, and the cornified envelope proteins. Conversely, an analogous system could be regulating the expression of BPAG1, as well as KRT5 and KRT14, by limiting their expression to the basal keratinocytes.

In conclusion, our data on BPAG1, together with previous studies on other epidermal-specific genes, such as keratins (Fuchs and Green, 1980), suggest that the mechanisms governing the cell-specific and differentiation-specific gene expression are complex, and both positive and negative regulatory systems may explain the tightly controlled expression during developmental organogenesis and physiologic terminal differentiation of epidermis.

FOOTNOTES

*
This study was supported by United States Public Health Service/National Institutes of Health Grants PO1-AR38923 and T32-AR07561 and by the Dermatology Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
M.D.-Ph.D. student supported by the Foerderer Foundation.

Recipient of the Dermatology Foundation Research Fellowship.

**
To whom correspondence should be addressed: Dept. of Dermatology, Jefferson Medical College, 233 South 10th St., Rm. 450, Philadelphia, PA 19107. Tel.: 215-955-5785; Fax: 215-955-5788.

(^1)
The abbreviations used are: BP, Bullous pemphigoid; KRE, keratinocyte responsive element; KTP, keratinocyte transcriptional protein; CAT, chloramphenicol acetyltransferase; kb, kilobase pair(s); bp, base pair(s); NHK, normal human keratinocyte; NHF, normal human fibroblast.

ACKNOWLEDGEMENTS

We thank Lin Lin and Tamara Alexander for expert assistance. Drs. James W. Fox IV and John H. Moore, Jr., Division of Plastic Surgery, Jefferson Medical College, provided tissue for cell cultures.

REFERENCES

  1. Andersen, B., Schonemann, M. D., Flynn, S. E., Pearse, R. V., II, and Rosenfeld, M. B. (1993) Science 260, 78-82 [Abstract/Free Full Text]
  2. Angel, P., Imagawa, M., Chiu, R., Stein, B., Imbra, R. J., Rahmsdorff, H. J., Jonat, C., Herrlich, P., and Karin, M. (1987) Cell 49, 729-739 [CrossRef][Medline] [Order article via Infotrieve]
  3. Arnemann, J., Sullivan, K. H., Magee, A. I., King, I. A., and Buxton, R. S. (1993) J. Cell Sci. 104, 741-750 [Abstract]
  4. Boast, S., Su, M.-W., Ramirez, F., Sanchez, M., and Avvedimento, E. V. (1990) J. Biol. Chem. 265, 13351-13356 [Abstract/Free Full Text]
  5. Byrne, C., and Fuchs, E. (1993) Mol. Cell Biol. 13, 3176-3190 [Abstract/Free Full Text]
  6. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159 [Medline] [Order article via Infotrieve]
  7. Clerc, R. G., Corcoran, L. M., LeBowits, J. H., Baltimore, D., and Sharp, P. A. (1988) Genes & Dev. 2, 1570-1581
  8. Fletcher, C., Heintz, N., and Roeder, R. G. (1987) Cell 51, 773-781 [CrossRef][Medline] [Order article via Infotrieve]
  9. Fuchs, E., and Green, H. (1980) Cell 19, 1033-1042 [CrossRef][Medline] [Order article via Infotrieve]
  10. Giudice, G. J., Squiquera, H. L., Elias, P. M., and Diaz, L. A. (1991) J. Clin. Invest. 87, 734-738
  11. Giudice, G. J., Emery, D. J., and Diaz, L. A. (1992) J. Invest. Dermatol. 99, 243-250 [CrossRef][Medline] [Order article via Infotrieve]
  12. Hopkinson, S. B., Riddelle, K. S., and Jones, J. C. R. (1992) J. Invest. Dermatol. 99, 264-270 [CrossRef][Medline] [Order article via Infotrieve]
  13. Huff, C. A., Yuspa, S. H., and Rosenthal, D. (1993) J. Biol. Chem. 268, 377-384 [Abstract/Free Full Text]
  14. Jordan, R. E., Buetner, E. H., Witebsky, E., Blumenthal, G., Hale, W. L., and Lever, W. F. (1967) J. Am. Med. Assoc. 200, 91-96
  15. Jordan, R. E., Triftshauser, C. T., and Schroeter, A. L. (1971) Arch. Dermatol. 103, 486-491 [CrossRef][Medline] [Order article via Infotrieve]
  16. Ko, H.-S., Fast, P., McBride, W., and Staudt, L. M. (1988) Cell 55, 135-144 [CrossRef][Medline] [Order article via Infotrieve]
  17. Leask, A., Rosenberg, M., Vassar, R., and Fuchs, E. (1990) Genes & Dev. 4, 1985-1988
  18. Leask, A., Byrne, C., and Fuchs, E. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 7948-7952 [Abstract/Free Full Text]
  19. Li, K., Giudice, G. J., Tamai, K., Do, H. C., Sawamura, D., Diaz, L. A., and Uitto, J. (1992) J. Invest. Dermatol. 99, 258-263 [CrossRef][Medline] [Order article via Infotrieve]
  20. Li, K., Tamai, K., Tan, E. M. L., and Uitto, J. (1993) J. Biol. Chem. 268, 8825-8834 [Abstract/Free Full Text]
  21. Luo, Y., Fujii, H., Gerster, T., and Roeder, R. G. (1992) Cell 71, 231-241 [CrossRef][Medline] [Order article via Infotrieve]
  22. Moll, R., Franke, W. W., Schiller, D. L., Geiger, B., and Krepler, R. (1982) Cell 31, 11-24 [CrossRef][Medline] [Order article via Infotrieve]
  23. Mutasim, D. F., Takahashi, Y., Labib, R. S., Anhalt, G. J., Patel, H. P., and Diaz, L. A. (1985) J. Invest. Dermatol. 84, 47-53 [CrossRef][Medline] [Order article via Infotrieve]
  24. Myers, J. C., Chu, M. L., Faro, S. H., Clark, W. J., Prockop, D. J., and Ramirez, F. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 3516-3520 [Abstract/Free Full Text]
  25. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  26. Saunders, N. A., Bernacki, S. H., Vollberg, T. M., and Jetten, A. M. (1993) Mol. Endocrinol. 7, 387-398 [Abstract]
  27. Sawamura, D., Nomura, K., Sugita, Y., Mattei, M.-G., Chu, M.-L., Knowlton, R., and Uitto, J. (1990) Genomics 8, 722-726 [CrossRef][Medline] [Order article via Infotrieve]
  28. Sawamura, D., Li, K., Chu, M.-L., and Uitto, J. (1991a) J. Biol. Chem. 266, 17784-17790 [Abstract/Free Full Text]
  29. Sawamura, D., Li, K., Nomura, K., Sugita, Y., Christiano, A. M., and Uitto, J. (1991b) J. Invest. Dermatol. 96, 908-915 [CrossRef][Medline] [Order article via Infotrieve]
  30. Sawamura, D., Li, K., and Uitto, J. (1992) J. Invest. Dermatol. 98, 942-943 [CrossRef][Medline] [Order article via Infotrieve]
  31. Schöler, H. R. (1991) Trends Genet. 7, 323-328 [Medline] [Order article via Infotrieve]
  32. Schreiber, E., Matthias, P., Müller, M. M., and Schaffner, W. (1989) Nucleic Acids Res. 17, 6419 [Free Full Text]
  33. Snape, A. M., Jonas, E. A., and Sargent, T. D. (1990) Development (Camb.) 109, 157-165 [Abstract]
  34. Snape, A. M., Winning, R. S., and Sargent, T. D. (1991) Development (Camb.) 113, 283-293 [Abstract]
  35. Stanley, J. R. (1989) J. Clin. Invest. 83, 1443-1448
  36. Steinert, P. M., and Roop, D. R. (1988) Annu. Rev. Biochem. 57, 593-625 [CrossRef][Medline] [Order article via Infotrieve]
  37. Sturm, R. A., Das, G., and Herr, W. (1988) Genes & Dev. 2, 1582-1599
  38. Sugi, T., Hashimoto, T., Hibi, T., and Nishikawa, T. (1989) J. Clin. Invest. 84, 1050-1055
  39. Tamai, K., Sawamura, D., Do, H. C., Tamai, Y., Li, K., and Uitto, J. (1993) J. Clin. Invest. 92, 814-822
  40. Tamai, K., Li, K., and Uitto, J. (1994) J. Biol. Chem. 269, 493-502 [Abstract/Free Full Text]
  41. Tanaka, M., Grossniklaus, U., Herr, W., and Hernandez, N. (1988) Genes & Dev. 2, 1764-1778
  42. Tanaka, T., Parry, D. A. D., Klaus-Kovtun, V., Steinert, P. M., and Stanley, J. R. (1991) J. Biol. Chem. 266, 12555-12559 [Abstract/Free Full Text]
  43. Uitto, J., and Christiano, A. M. (1992) J. Clin. Invest. 90, 687-692
  44. Uitto, J., and Chu, M.-L. (1988) in Collagen (Olsen, B. R., and Nimni, M. E., eds) pp. 109-124, Vol. IV, CRC Press, Boca Raton, FL _
  45. Verrijzer, C. P., Kal, A. J., and Van der Vliet, C. (1990) EMBO J. 9, 1883-1888 [Medline] [Order article via Infotrieve]
  46. Williams, T., and Tjian, R. (1991) Genes & Dev. 5, 670-682
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