(Received for publication, July 7, 1994; and in revised form, December 8, 1994)
From the
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.
Bullous pemphigoid (BP) ()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.
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.
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,
2(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
2(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 2(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
2(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
2(I) collagen promoter, or SV2 promoter CAT constructs. The
constructs were co-transfected with a RSV-
gal 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 [
C]chloramphenicol as a percent of the
total radioactivity in the sample and corrected for transfection
efficiencies by determination of the activity of
-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
2(I) collagen, after correction for SV2 promoter
activity and transfection efficiency by
-galactosidase
determination. Also, the BPAG1 expression in NHK cultures was
52.0 times higher than that in NHF
cultures.
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).
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
10
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.
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 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
-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.
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