(Received for publication, September 20, 1995)
From the
Human involucrin whose gene transcription is directed by a
2456-nucleotide (nt) 5`-noncoding region is a structural component of
the epithelial cornified layer. Transient transfection assays
demonstrated that this region is transcriptionally active in
multiplying keratinocytes and is enhanced by 2 mM CaCl treatment. Calcium-independent transcriptional activity and the
interaction with the AP-1 transcriptional factor was located on the
proximal part (nt -159 to -1) of the 5`-noncoding region.
However, CaCl
responsiveness was mapped to a distal 1185-nt
fragment (nt -2456 to -1272). Moreover, this fragment
potentiated the Herpes simplex thymidine kinase promoter in normal
keratinocytes and is responsive to calcium treatment in a cell
type-specific manner. Interestingly, the absence of a 491-nt fragment
located between the two enhancer domains (nt -651 to -160)
resulted in transcriptional activation in multiplying keratinocytes.
This fragment interacts with AP-1 and the YY1 transcriptional silencer.
It is concluded that human involucrin 5`-noncoding region contains at
least three regulatory domains, a distal CaCl
-responsive
enhancer, a putative transcriptional silencer (that interacts with AP-1
and YY1), and a proximal enhancer/promoter (that interacts with AP-1).
Thus, this study demonstrates the presence of particular
transcriptional factors can potentially regulate the human involucrin
expression.
The differentiation of stratified epithelia requires the harmonious expression of several structural and regulatory proteins. The complex regulatory pathways that direct the transcription of epithelial differentiation-related genes are of particular importance in human disease.
Involucrin, a precursor of the cornified envelope of terminally differentiated keratinocytes(1, 2) , is apparently limited to primates (3) . The involucrin protein has a molecular mass of 68 kDa and possesses a central glutamyl-rich domain formed with 39 repeats of a 10-amino acid cassette (4) which is required for the cross-linking activity of the calcium-dependent epithelial transglutaminase during cornified envelope formation(5, 6, 7, 8) .
The human involucrin gene is about 6000 nt in size composed of two exons of 43 and 2107 nt, respectively, separated by an intron of 1188 nt(4) . A 2456-nt noncoding sequence located 5` of the first involucrin exon has transcriptional regulatory elements that control its transcriptional activity(9, 10, 11) . Analysis of in vitro and in vivo results show that the involucrin gene activation depends on the interaction of transcriptional factors present in the keratinocyte nucleus(9, 10, 11) .
In vitro and in vivo experiments correlate the presence of involucrin transcripts and protein following the progression of keratinocytes from the basal layer to terminal differentiation state(12, 13, 14) . Thus, the transcriptional factors required for specific involucrin gene transcription may also be necessary for expression of other epithelial terminal differentiation-related genes(15, 16, 17) . Interestingly, several genes related to terminal differentiation, such as involucrin, profilaggrin, and loricrin, are located on chromosome 1q21(18) .
The 5`-noncoding region controlled the expression of the involucrin gene in transient transfection of cultured human keratinocytes(9) . This region was divided functionally into two portions: the proximal 900-nt promoter region with the putative TATA box and an upstream 1600-nt region with necessary elements for the proper expression of the involucrin gene(9) . Interestingly, the entire 2456-nt 5`-noncoding segment activity is tissue-specific in transgenic mice, suggesting that the basic regulatory elements of the involucrin gene are widespread in mammals(10, 19) .
The reported sequence of the 900-nt proximal promoter region active
in keratinocytes contains putative target sites for the AP-1 family of
transcriptional factors(9, 11) . Moreover, the
addition of TPA, ()an AP-1 activator, moderately activates
this region in transient transfection assays using cultured rat cells.
The latter suggests that AP-1 could be necessary for involucrin
expression. Furthermore, the proximal 900-nt enhancer was activated by
overexpression of c-fos and c-jun oncogenes,
components of AP-1(11) . Treatment of normal keratinocytes with
calcium, TPA, or vitamin A
depletion(20, 21, 22, 23, 24, 25, 26) are able to increase involucrin mRNA levels. However, how
these compounds directly regulate the involucrin promoter region is not
clear.
To investigate the transcriptional regulation of the
involucrin gene, several constructs of the 2456-nt 5`-noncoding region
were transfected into cultured human keratinocytes during
multiplication (0.1 mM CaCl) or differentiation (2
mM CaCl
) conditions. Involucrin transcription is
shown to be regulated by several functional elements: a distal cell
type-specific 1100-nt upstream enhancer (nt -2456 to -1272)
responsive to calcium stimulation and a possible transcriptional
silencer (nt -651 to -160) which in turn is coupled to a
proximal enhancer/promoter (nt -159 to -1/+1)
unaffected by calcium concentration. Further DNA-protein
characterization of the silencer and proximal enhancer/promoter regions
established that AP-1 and YY1 are the main transcriptional factors
interacting with these elements.
Figure 1: Different transcriptional regulatory domains are present in the 2456-nt human involucrin 5`-noncoding region. A, activity of deletion mutants of the involucrin gene 5`-noncoding region. Multiplying normal keratinocytes were transfected with 10 µg of total plasmid DNA from different deletion constructs as described under ``Materials and Methods.'' The average CAT activities relative to the promoterless vector pCAT-basic were obtained from at least three independent experiments 48 h post-transfection. The transcriptional start site is represented by an arrow. The various deleted constructs, the putative TATA box, the restriction sites for ApaI, HindIII, PstI, RsaI, and XbaI, as well as the SV40 minimal promoter (SV) and the herpes simplex type 1 thymidine kinase promoter (TK) are indicated. B, cell type-specific enhancer activity of the human involucrin distal enhancer region. pTKM and p1.1TKM plasmids (10 µg) were transfected into multiplying human keratinocytes, MRC-5 fibroblasts, and C-33A cell line. Cells were harvested 48 h post-transfection. Because of the different transfection efficiencies, the activities are plotted relative to the SV40 enhancer/promoter.
Figure 3: Human involucrin 5`-noncoding region. A, complete nucleotide sequence of the involucrin 2456-nt regulatory region. Nucleotide position number -2456 corresponds to the HindIII site of p2.6CAT plasmid. The sequence segment from nt -784 to 49 was reported previously(12) . Predicted consensus sequences for several transcriptional factors within the proximal 784 nt (underlined) and restriction sites for ApaI, HindIII, and PstI are shown. TATA box and transcription start site are double-underlined. B, plasmid constructs employed for DNase I footprint analysis. The ApaI-XbaI and HindIII-ApaI fragments from p827CAT plasmid are cloned in pIN220 and pIN630 plasmids, respectively. Thick black lines show the position of oligonucleotides employed in gel-shift assays (H1, H2, H3, H4, H4 2072, and H4 2126). The TATA box (vertical box) and ApaI, HindIII, PstI, and XbaI restriction sites are shown.
The complete nucleotide sequence from p2.6CAT insert is recorded in GeneBank(TM) (accession number U23404). All constructs were sequenced using Sequenase (Amersham Corp.) or the chemical degradation method (28) . All oligonucleotides (Table 1) were synthesized in an Applied BioSystems 391 DNA synthesizer.
Confirmation of the above observation was obtained with the p220CAT plasmid containing the ApaI-XbaI fragment from p827CAT, a 6-fold increase in CAT activity relative to p827CAT was observed (Fig. 1A). Therefore, the region upstream the ApaI site, probably has a negative regulatory element. To explore this possibility, the PstI-PstI fragment from p827CAT was cloned in the p610CP plasmid before the SV40 promoter and transfected to multiplying human keratinocytes. However, the observed CAT activity of p610CP was similar to that obtained with the SV40 promoter alone, denotating that other elements present in p827CAT construct are associated with the inhibitory function (Fig. 1A). None of the constructs presented activity when transfected into HeLa cells, which do not express involucrin (data not shown). The p1.1TKM construct was transfected in MRC-5 fibroblasts and C-33A cells to establish the cell type specificity of this enhancer. A mild relative activity of p1.1TKM was noticed in C-33A cells, whereas this same construct remained silent in MRC-5 fibroblasts (Fig. 1B). These results suggest that the elements regulating transcription from the distal enhancer are specific of epithelial-derived cells.
The p2.6CAT plasmid displayed 5-fold
activation in calcium-induced differentiation conditions when
transfected in keratinocytes stimulated to differentiate by increasing
the CaCl concentration to 2 mM in the absence of
epidermal growth factor and bovine pituitary extract (Fig. 2A). These conditions stimulate 3-5-fold
the transcription of the involucrin gene(34) . However,
activity of p97CAT, p220CAT, and p827CAT remained unchanged (Fig. 2A). Therefore, the calcium responsiveness should
reside in the distal 1185-nt enhancer. To test this, the p1.1TKM
construct was also transfected in normal keratinocytes under
multiplying and differentiation conditions resulting in a significant
increase on the p1.1TKM activity in calcium-treated keratinocytes (Fig. 2B).
Figure 2:
Activity of the involucrin gene
5`-noncoding region in differentiation-induced keratinocytes. A, differentiation induction does not stimulate human
involucrin 5`-noncoding proximal 784-nt fragment. Normal keratinocytes
were transfected with 10 µg of total plasmid DNAs from p2.6CAT,
p827CAT, p220CAT, and p97CAT constructs. Transfected cells were grown
in multiplying (0.1 mM CaCl) or after
differentiation induction (2 mM CaCl
) conditions
as described under ``Materials and Methods.'' Cell extracts
were obtained 48 h post-transfection. Representative CAT chromatograms
from three independent experiments are shown. pCAT-basic and
pCAT-control vectors were used as negative and positive controls,
respectively. B, calcium responsiveness resides in the distal
enhancer. pTKM and p1.1TKM plasmids (10 µg) were transfected into
keratinocytes and processed as described above. The average CAT
activities relative were obtained from three independent
experiments.
Thus, three transcriptional regulatory domains are established: a distal enhancer with calcium responsiveness located between nt -2456 and -1272, a possible transcriptional silencer located between nt -651 and -160, and a proximal enhancer/promoter located between nt -160 and -1/+1.
Figure 4:
DNase I footprint analysis of the human
involucrin proximal enhancer/promoter region. A, nuclear
extracts (35 µg) from multiplying (Ker) and 2 mM CaCl-induced (Ki) human keratinocytes or HeLa
cells were incubated with the end-labeled EcoRI-HindIII fragment from pIN220 plasmid for DNase
I footprinting as described under ``Materials and Methods''
and electrophoresed in 6% sequencing gels. Brackets show the
regions covered by the HP-1, HP-2, HP-3, and HP-4 footprints in the
upper and lower DNA strands. Numbers on the left side show the nucleotide position in the human involucrin 5`-noncoding
region sequence. F, DNase I digestion pattern of the free
probe. Pu, purine chemical cleavage ladder. Triangles indicate DNase I hypersensitive sites. B, AP-1
competition footprint analysis. The labeled EcoRI-HindIII pIN220 DNA fragment was incubated with
40 µg of human multiplying keratinocytes nuclear extract.
Competition was performed by adding 0.5 and 1.0 µg of nonlabeled
oligonucleotide containing a consensus AP-1 binding site (Table 1) to the binding reaction for 10 min before incubation
with DNase I. The relative location of the HP-2 and HP-3 footprints is
indicated by brackets. Triangles show recovered
sites. Pu, purine sequence ladder.
HP-1 footprint includes the putative TATA box and a consensus sequence for the Sp-1 transcriptional factor 5`-GGAGGG-3`(35) . HP-2 overlaps HP-1 and is located on two putative AP-1 binding sites. The HP-3 footprint is localized over a third AP-1 binding site meanwhile HP-4 is associated to a putative Myb protein binding sequence 5`-CCTAAAG-3` (6) . Footprint assays of pIN220 employing different amounts of a competitor oligonucleotide containing a bona fide AP-1 site from the human papillomavirus type 18 (HPV-18; (31) ), and nuclear extracts from multiplying keratinocytes resulted in a dose-dependent competition of HP-2 and HP-3 footprints, suggesting that the nuclear factor involved is AP-1 (Fig. 4B).
Figure 5:
The human involucrin promoter contains
binding sites recognized by AP-1 factor. A, gel-shift assays
were done incubating the P-end-labeled HindIII-EcoRI fragment from pIN220 plasmid with 8
µg of total nuclear extracts from multiplying (Ker) or 2
mM CaCl
-induced (Ki) human keratinocytes
on ice in the presence of 1 µg of poly[d(I-C)] as
unspecific carrier. Competitions were performed by adding 100 and 200
molar excesses of the indicated nonlabeled competitor oligonucleotides
before electrophoresis in 4% low ionic strength nondenaturing
polyacrylamide gels. Arrows indicate the AP-1-specific
retarded complexes. B, gel supershift experiments were done by
incubating on ice the above described binding reaction mixture with 2
µg of anti-c-jun/AP-1 sc-44 or anti-HPV16 E7 polyclonal
antibodies for 6 h prior electrophoresis. The positions of the AP-1
shifted and supershifted complexes are indicated by arrows.
In addition, gel supershift experiments were
performed with nuclear extracts from multiplying keratinocytes to
confirm the identity of the observed AP-1-specific complexes in the
pIN220 fragment. A decrease in the intensity of the specific
DNA-protein retarded complex was observed in the presence of a specific
rabbit polyclonal anti-c-jun/AP-1 antibody with the appearance
of a clear supershifted band (Fig. 5B). In contrast, a
heterologous rabbit polyclonal antibody directed against the human
papillomavirus type 16 E7 protein did not affect the retarded complexes (Fig. 5B). Similar results were obtained with nuclear
extracts from HeLa cells and CaCl-treated keratinocytes
(data not shown). Thus, it is concluded that AP-1 is the nuclear factor
from normal keratinocytes associated with the proximal 159-nt
enhancer/promoter.
Figure 6:
Footprint analysis of the human involucrin
transcriptional silencer. Nuclear extracts (30 µg) from multiplying (Ker) and 2 mM CaCl-treated (Ki)
keratinocytes and HeLa cells were incubated with the end-labeled EcoRI-HindIII fragment from pIN630 plasmid as in Fig. 4A. Footprints H1, H2, H3, and H4 in upper and
lower DNA strands are defined with brackets. Triangles indicate DNase I hypersensitivity sites (closed) and
changes in footprint pattern (open). The nucleotide sequence
number is on the left side. Free probe DNase I digestion
patterns for 60 and 70 s are shown in F and F` lanes
of the upper DNA strand, respectively. Pu, purine chemical
cleavage ladders.
Figure 7:
DNA-binding proteins interaction with the
human involucrin transcriptional silencer. Gel-shift assays were
performed incubating nuclear extracts from multiplying (Ker)
and 2 mM CaCl-treated (Ki) keratinocytes
with 1 ng of end-labeled oligonucleotides containing the H1, H2, H3,
and H4 footprint sequences (Table 1) from pIN630 (panels H1,
H2, H3, and H4, respectively). The binding reactions were
done as described in the legend to Fig. 5A.
Competitions were performed with 100 molar excess of competitor
oligonucleotide before electrophoresis 6% nondenaturing low ionic
strength polyacrylamide gels. Arrows indicate the position of
specific retarded complexes.
H3 and H4 oligonucleotides
produced a more elaborated gel-shift pattern. H3 presented at least two
specific retarded complexes which were increased in nuclear extracts
from CaCl-treated keratinocytes (Fig. 7, panel
H3). H4 oligonucleotide had two specific DNA-protein complexes
with either nuclear extract, suggesting the interaction of multiple
nuclear factors with this sequence (Fig. 7, panel H4).
Cell type specificity was tested using nuclear extracts from HeLa cells. The similarity of the gel-shift pattern with H1 and H2 suggests that nuclear factors are shared by HeLa cells and keratinocytes (Fig. 8, panels H1 and H2). In contrast, differences were observed between HeLa cells and keratinocytes with the H3 and H4 oligonucleotides. H3 had an extra upper DNA-protein complex with HeLa nuclear extracts. The common complexes seem to be produced by a nuclear protein more abundant in HeLa cells than in keratinocytes (Fig. 8, panel H3). For H4 oligonucleotide, at least one DNA-protein complex was absent from keratinocyte nuclear extracts, suggesting that in HeLa cells additional factors may interact with this region (Fig. 8, panel H4).
Figure 8:
Differential DNA-protein binding between
keratinocytes and HeLa cells. Nuclear extracts of multiplying (Ker) and CaCl-treated keratinocytes (Ki)
or HeLa cells were used in gel-shift assays with the H1, H2, H3, and H4
oligonucleotides as described in the legend to Fig. 5A (panels H1, H2, H3, and H4, respectively). The
keratinocyte (black arrows) and the differential HeLa cells (open arrows) complexes are shown.
Figure 9:
The H2 footprint corresponds to AP-1
transcriptional factor. A, gel-shift assays were performed
using 1 ng of end-labeled H2 oligonucleotide as described in the legend
to Fig. 5A(-) or with 30- and 100-fold molar
excesses of the indicated competitor oligonucleotides and nuclear
extracts from multiplying human keratinocytes. The arrow indicates the specific AP-1-retarded complex. B, gel
supershift assays were performed as described in the legend to Fig. 5B using nuclear extracts from multiplying (Ker) and 2 mM CaCl-treated keratinocytes (Ki) and HeLa cells with the H2-end-labeled oligonucleotide in
the presence of rabbit polyclonal sc-44 (anti-c-jun/AP-1) or
anti-HPV-16 E7 antibodies. The arrows show the position of the
H2-AP-1 and supershifted complexes.
Gel supershift
assays confirmed the AP-1 identity of the H2 complex using nuclear
extracts from multiplying and CaCl-treated keratinocytes
and HeLa cells nuclear extracts. The H2 complex intensity was
simultaneously reduced with the appearance of a supershifted complex
only after addition of an anti-AP-1 antibody to the binding mixture,
verifying that the H2 footprint indeed corresponds to AP-1 (Fig. 9B).
Two different potential YY1 binding sites coincide with the position of H4 footprint, 5`-TTTCCATTTCA-3` and 5`-TCATTTTGAA-3` at nt -383 and -329, respectively (Fig. 3A). These sequences share homology with the 5`-CAT-3` motif present in the YY1 binding sites from several genes (Fig. 10A). To test if these sequences indeed bind the YY1 transcriptional factor, competitive gel-shift assays were performed using the end-labeled H4 2072 and H4 2126 oligonucleotides (flanking the H4 footprint) and the P5+1 and P5+1 mutant from AAV (38) and YY1 (40, 41) nonlabeled competitors with nuclear extracts from HeLa cells and multiplying keratinocytes. Specific complexes for H4 2072 and H4 2126 were efficiently competed with both P5+1 and YY1 oligonucleotides, but not with a P5+1 mutant, which does not bind YY1 (Fig. 10B). Additionally, cross-competition between H4 2072 and H4 2126 oligonucleotides indicates that YY1 interacts with both sequences.
Figure 10: YY1 transcriptional factor binds to the human involucrin silencer region. A, comparison of homologous YY1 sequences from different promoters. Boxes show conserved sequences. H4 2072 and H4 2126 are referred to oligonucleotides containing the 5` and 3` ends of the H4 footprint, respectively. B, YY1 interacts with the human involucrin putative transcriptional silencer. Gel-shift assays were done employing 1 ng of end-labeled H4 2072 or H4 2126 oligonucleotides (containing the putative YY1 sites from H4 footprint) and nuclear extracts from multiplying keratinocytes as described in the legend to Fig. 5A. For specific competition, 100- and 200-fold molar excess of the indicated competitor oligonucleotide was used prior electrophoresis through 4% nondenaturing low ionic strength polyacrylamide gels.
The human involucrin 5`-noncoding region contains several binding sites for transcriptional regulatory proteins. The present results describe the presence of three functional domains, one enhancer/promoter and a transcriptional silencer domains located within a 784-nt fragment proximal to the transcription start site as well as a far upstream 1185-nt enhancer domain (Fig. 11).
Figure 11: Summary of transcriptional factors interacting with the proximal enhancer/promoter and silencer domains within the human involucrin 5`-noncoding region. Footprint sites in upper and lower DNA strands are indicated by cross-hatched boxes. The TATA box position is represented by a vertical box. Identified YY1 and AP-1 sites are shown as pentagons and hexagons, respectively. The arrow shows the direction and transcription start site. Restriction sites are provided as a reference.
The full-length 2456-nt involucrin upstream regulatory region displayed significant activity in multiplying normal keratinocytes, while the proximal 784-nt fragment did not. Interestingly, only the p2.6CAT construct with the intact 5`-noncoding region and the distal 1185-nt enhancer were activated after calcium induction of differentiation, suggesting that factors associated with calcium activation interact within this last region. The distal enhancer was ineffective in fibroblasts indicating cell type specificity of this enhancer function. Nevertheless, the proximal enhancer/promoter and the transcriptional silencer were not altered by differentiation induction. No transcriptional activity was noticed with any of these functional regions in non-involucrin expressing cells, such as HeLa or fibroblasts, indicating that cell type specificity could be dependent either on several factors or a single transcriptional factor associated with all the three regulatory domains.
The interaction of AP-1 with the proximal enhancer/promoter and the putative transcriptional silencer reported here suggests that this transcriptional factor may be primarily responsible for specific involucrin transcriptional activity in normal keratinocytes. The similarity between the footprint and gel-shift patterns observed with both regions independent of the differentiation state of normal keratinocytes supports this notion. AP-1, a transcriptional factor integrated by dimerization of products from fos and jun oncogene families(42) , activates genes with the 5`-TGANTC/AA-3` consensus motif in response to compounds such as TPA that activate the protein kinase C(36, 37) . The results presented here agree with previous reports showing that TPA treatment or fos and jun overexpression activates transcription from the proximal 784-nt fragment(11) . A recent report (43) demonstrates that AP-1 sites present in the proximal enhancer/promoter (nt -124 to -118) and in the transcriptional silencer (nt -288 to -282) are important for the TPA responsiveness of the human involucrin gene. Accordingly, these AP-1 sites coincide with the position of HP-3 and H3 footprints. Furthermore, the present results provide evidence of the presence of an extra AP-1 site (H2) at positions -263 to -255.
Calcium-induced differentiation of transfected keratinocytes did not affect the activity of either the proximal promoter/enhancer or the transcriptional silencer regions, both being capable of interacting with AP-1. Additionally, no activity was registered with any reporter construct in fibroblasts, a cell type that contains AP-1. Furthermore, the p220CAT construct was inactive in transfected HeLa cells despite the interaction of AP-1 (data not shown), suggesting that a particular combination of AP-1 may be implicated in involucrin gene transcription. In agreement with this, Welter et al. 1995 (43) established that Fra1, JunB, and JunD are the factors associated to the enhancer/promoter region. Thus, the sum of the results suggests the existence of two different control mechanisms for involucrin gene transcription, one dependent on AP-1 activation and the other associated with calcium-dependent pathways.
Consistent with this hypothesis, the intact 2456-nt
noncoding region is more efficient in the presence of 2 mM
CaCl. The enhanced activity requires the far upstream 1648
nt that includes the distal 1185-nt enhancer domain described in this
work. Thus, the AP-1- and calcium-dependent involucrin regulatory
pathways are apparently functionally and physically separable within
the 5`-noncoding region.
Several putative binding sites for transcriptional factors are located within the 1185-nt distal enhancer. A detailed analysis is needed to establish the interaction and functional value of each of these factors in the context of calcium-induced differentiation.
Ying-Yang 1 or YY1 is a zinc finger
protein related to the Krüppel family of
transcriptional regulators of Drosophila melanogaster, with
the unusual property of being able to activate or repress transcription
initiation depending on the cellular context. Moreover, YY1 binding
sites vary among cellular and viral promoters (44) . On one
hand, YY1 activates transcription of c-myc(45) ,
ribosomal proteins L30 and L32(40) , and cytochrome c oxidase genes (46) and the leaky late promoter of herpes
simplex (47) and the P6 promoter of B19
parvovirus(48) . On the other hand, YY1 represses the
regulatory regions from c-fos(49) , the skeletal
-actin(50) , human immunodeficiency virus type
1(51) , HPV-18 long control region(52) , and the human
cytomegalovirus major immediate early enhancer/promoter(53) .
The ambivalent nature of YY1 as an activator or a silencer led to a hypothesis concerning the importance of this factor for human involucrin transcription. Although the abundance and function of YY1 in human keratinocytes are not known, the current results suggest that this factor may repress human involucrin transcription in multiplying and calcium-treated keratinocytes. Both, the 1185-nt distal enhancer and the 159-nt proximal enhancer/promoter are active, lacking the 624-nt fragment that contains the YY1 binding sites. Because YY1 physically interacts with other proteins(39, 54) , it is possible that the mechanism of YY1 repression in the involucrin gene could be the association of YY1 with the Sp-1 basal transcription factor, whose putative binding site is present within the 159-nt enhancer/promoter. Accordingly, the substitution of the native involucrin TATA box with the SV40 promoter in the p610CP plasmid (that contains several Sp-1 sites in the 21-nt repeats) showed no increase in activity when compared with the control despite the presence of four AP-1 sites (Fig. 1A). Site-directed mutagenesis experiments will be required to verify such interaction.
The association between two apparently antagonistic transcriptional factors such as AP-1 and YY1 with the involucrin 5`-noncoding region resembles the epithelial-specific HPV-18 long control region, which also interacts with both factors in similar tissue-specific enhancer (AP-1) and transcriptional silencer (YY1) functions(31, 52) . A particular combination of AP-1 containing junB is responsible for the HPV-18 tissue-trophism(31) . Interestingly, oligonucleotides containing an AP-1 site from HPV-18 efficiently competed for the involucrin AP-1 complexes from the 159-nt promoter/enhancer. It has been shown that JunB is associated with involucrin transcription(43) . Therefore, functional association between YY1 and junB can be proposed as a possible regulatory mechanism for epithelial expressed genes.