©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Proximal Promoter of the Mouse Loricrin Gene Contains a Functional AP-1 Element and Directs Keratinocyte-specific but Not Differentiation-specific Expression (*)

Daniel DiSepio (1), Alma Jones (1), Mary Ann Longley (1), Donnie Bundman (1), Joseph A. Rothnagel (1) (2)(§), Dennis R. Roop (1) (2)(¶)

From the (1) Departments of Cell Biology and (2) Dermatology, Baylor College of Medicine, Houston, Texas 77030

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Loricrin gene expression is limited to terminally differentiating keratinocytes of stratified squamous epithelia. To define the regulatory elements that mediate the expression of the loricrin gene, we replaced the loricrin coding sequences from a 6.5-kilobase genomic fragment with the chloramphenicol acetyltransferase gene and transfected this construct into cultured mouse keratinocytes. High expression levels were observed in both undifferentiated as well as differentiating cells. Transgenic mice bearing a similar construct, but with -galactosidase as the reporter gene, corroborated these in vitro findings and showed tissue- and cell type-specific, but not differentiation-specific expression. Deletion analysis of the promoter region determined that sequences up to -60 base pairs from the start of transcription could be removed without significant loss of promoter activity. Within these proximal 60 base pairs is an evolutionarily conserved AP-1 element that is recognized by both purified c-Jun and AP-1 factors from keratinocytes in vitro. Mutation of this AP-1 site abolished the activity of the loricrin promoter. These studies show that elements directing expression of the loricrin gene to the stratified squamous epithelia are contained within a 6.5-kilobase genomic fragment, and those elements required to restrict expression to differentiated keratinocytes lie outside this region.


INTRODUCTION

Mammalian epidermis undergoes a progressive differentiation to form a mature stratified squamous epithelium. Four histologically distinct cell layers can be identified in the epidermis, each layer expressing a characteristic set of marker proteins specific for the maturation state (Eckert, 1989). Keratinocytes of basal phenotype express keratins K5() and K14 (Fuchs and Green, 1980; Woodcock-Mitchell et al., 1982). In the epidermis, terminal differentiation begins in the basal layer, where once induced to differentiate, keratinocytes down-regulate K5 and K14 and up-regulate several major proteins, including keratins K1 and K10 (Roop et al., 1983). Differentiation continues in sequential order through the spinous and granular layers, where cells express the differentiation-specific proteins filaggrin and loricrin (Rothnagel et al., 1987; Mehrel et al., 1990). Keratinocyte differentiation finally terminates with the cells loosing internal organelles, forming a thickened intracellular cell envelope, and an intercellular lipid multilamellar structure in the stratum corneum prior to desquamation as corneocytes.

Loricrin, a major protein of the cell envelope, is first found in granular cells, and along with filaggrin, is one of the most predominant markers for late epidermal differentiation. In situ hybridization has shown that loricrin transcripts are localized only in the upper spinous and granular layers of the epidermis (Mehrel et al., 1990; Hohl et al., 1991a). In mouse epidermis the loricrin protein is first localized as small, non-membrane bound granules known as L-granules (Steven et al., 1990). Through an unknown mechanism, loricrin is first released from the granular aggregates and is then deposited and cross-linked by transglutaminases as a dense marginal band on the inside of the plasma membrane of the corneocytes (Hohl, 1990).

Recent studies have begun to investigate the mechanisms regulating epidermal differentiation. Calcium is thought to be a major regulator of epidermal differentiation. A calcium gradient has been identified in the epidermis, with the lowest calcium concentrations in basal cells, and concentrations increasing through the spinous and granular layers (Malmqvist et al., 1984; Menon et al., 1985). In vitro studies have shown that keratinocytes maintain their proliferative, basal phenotype when cultured in medium containing less than 0.1 mM calcium. When the medium contained 0.1 mM calcium or higher, cells were induced to differentiate, resulting in the loss of proliferative capacity and an induction of terminal differentiation markers (Hennings et al., 1980), suggesting that calcium concentration could regulate the transcription of epidermal differentiation markers in vitro. More recently loricrin transcription has been shown to be up-regulated by calcium, and repressed by retinoic acid in vitro (Hohl et al., 1991b). These same factors that affect loricrin transcription in vitro also affect the transcription of filaggrin and the differentiation-specific keratins K1 and K10 (Kopan et al., 1987; Roop et al., 1987; Yuspa et al., 1989; Fleckman et al., 1989; Hohl et al., 1991b). Furthermore, the protein kinase C pathway has been implicated in the induction of epidermal differentiation by calcium (Dlugosz and Yuspa, 1993).

The most studied differentiation-specific epidermal gene is HK1, one of the first genes up-regulated during the induction of terminal differentiation. A region in the 3`-flanking sequence of the HK1 gene was determined to be responsible for the calcium induction of the gene (Huff et al., 1993; Rothnagel et al., 1993). Recent detailed molecular analysis of this 3`-element has identified the cis-elements of the calcium response element (Lu et al., 1994). This element contains a composite AP-1/steroid hormone binding site and functions to modulate the differentiation-specific expression of the HK1 gene in vitro. Since the K1 gene is one of the first genes up-regulated during the differentiation process, we were interested in comparing the regulation of this gene to the regulation of a gene that was induced late in epidermal differentiation. The loricrin gene was a good candidate since it is not transcribed until the cells reach the late spinous and early granular cell layers. The mouse loricrin gene has recently been cloned and characterized (Rothnagel et al., 1994). In order to better understand the regulation of loricrin, a gene up-regulated late in epidermal differentiation, we have created reporter constructs using the chloramphenicol acetyltransferase (CAT) gene and the -galactosidase (-gal) gene linked to 1.5 kb of 5`-flanking sequence and 2.2 kb of 3`-flanking sequence from the mouse loricrin gene. Regulatory elements within these sequences can direct cell- and tissue-specific expression both in vitro and in vivo. We show that loricrin transcription in vitro requires binding of protein factors to an AP-1 consensus site in the loricrin proximal promoter sequences, and that tissue-specific expression can be uncoupled from differentiation-specific expression both in vitro and in vivo.


MATERIALS AND METHODS

Plasmids, Constructs, and PCR Cloning

All transfections employed pSVCAT (Gorman et al., 1982) as the positive control. Fragments to be tested for enhancer activity were cloned into pA10CAT (Laimins et al., 1982). The 3.4-kb 5` BamHI and the 3.1-kb 3` BamHI fragments were cloned separately into the BamHI site of pGEM7Z. These BamHI fragments were also cloned into the BamHI and BglII sites of pA10CAT. Digestion of the clones with HindIII (from the pGEM7Z polylinker) and PstI released 1.9-kb 5` and 2.2-kb 3` fragments, which were cloned into the HindIII and PstI sites of pGEM3Z. Digestion of these clones with BamHI allowed insertion of the fragments into the BamHI and BglII sites of pA10CAT. These 5` BamHI and BamHI/ PstI, and the 3` BamHI and PstI/ BamHI fragments were inserted in both orientations relative to CAT.

The construction of the loricrin reporter constructs were carried out in two parts as follows. A ClaI site was introduced at the stop codon by PCR amplification using the primers 5`-CCATCGATAAGGTCACCGGGTTGC-3` and 5`-CACTTCCCTATCCTTTCT-3`. The PCR product of 1410 bp was digested with PstI and ClaI, yielding a 170-bp fragment which was then cloned into the 3`-flanking sequence of the loricrin gene. Similarly a ClaI site was introduced at the translation start codon by PCR amplification using the primers 5`-CTCTAGATTCCGTAAGGC-3` and 5`-CCATCGATGTTGTTTAAGGAGAAG-3`. The 1470-bp product was cut with ClaI and PstI and the resulting 830-bp product cloned into the 5`-flanking sequences of the loricrin gene. The 3` sequences were cloned adjacent to the 5` sequences creating a loricrin expression vector as shown in Fig. 1 a. A CAT reporter and a -galactosidase reporter with ClaI ends (generated by PCR) was subsequently cloned into the unique ClaI site to create the pML(6.5)CAT vector and the pML(6.5) -gal vector. All 5` deletion constructs were generated utilizing unique restriction sites in the flanking sequence and named by the size of the deletion in base pairs (p990, KpnI; p1480, BclI; p2230, EcoRV; p2610, NsiI).


Figure 1: A, schematic showing the organization of the mouse loricrin gene and the pML(6.5) lacking the loricrin coding sequences. The 6.5-kb genomic loricrin clone contains 1.5 kb of 5`-flanking sequence, a small noncoding exon, a 1.1-kb intron, a single coding exon, and 2.2 kb of 3`-flanking sequence (Rothnagel et al.,1994). Using PCR site-directed mutagenesis techniques, the entire loricrin coding region was deleted from the 6.5-kb genomic fragment and a unique ClaI site was introduced, leaving all other sequences intact. The ClaI restriction site was used to introduce the reporter genes to produce the pML(6.5)CAT and pML(6.5) -gal constructs. B, BamHI; K, KpnI; P, PstI; S, SacI. B, the pML(6.5)CAT reporter was transiently transfected into primary murine keratinocytes in low (0.05 mM) and high (0.35 mM) calcium medium. Lane 1, pSVCAT, high calcium; Lane 2, pA10CAT, high calcium; Lane 3, pML(6.5)CAT, low calcium; Lane 4, pML(6.5)CAT, high calcium. C, expression of pML(6.5) -gal in the epidermis of neonatal transgenic mice. Note expression is present throughout the epidermis, from the basal cell layer through to the cornified layers, but not in the underlying dermis.



To test the functionality of the AP-1 site in the proximal promoter, it was mutated from TGAGTCA to TCCCGGG, creating a SmaI restriction enzyme site using PCR primers as follows. The 5` reaction used the primer 5`-CCCCGGGATCTATTGATCAGGCACC-3` and a primer 5` to the BclI site, 5`-AGTAAAGTCAGTAGTCAG-3`, generating a 610-bp fragment. The 3` reaction used the primer 5`-CAATAGATCCCGGGG-3` and a primer 3` to the EcoRV site, 5`-CTCCACCAGAGGTCTTTCC-3`, generating a product of 1290 bp. The products of these two reactions were purified and used in a third reaction along with the two external primers to generate a single combined product of 1900 bp. This product was purified and digested with BclI and EcoRV and the fragment containing the mutated AP-1 site re-cloned into the pML(6.5)CAT vector. The resulting mutant promoter was subsequently sequenced to ensure that spacing and all other sequences in this region were intact.

Cell Culture, Transfections, and CAT Assays

Preparation of mouse primary keratinocyte and primary dermal fibroblast cultures and their transfections were carried out as described by Rothnagel et al. (1993). Keratinocytes and fibroblasts (as controls) were transfected with 10 µg of plasmid DNA/60-mm dish, with 20 µg of sonicated salmon sperm as a carrier, with the exception of the 5` RACE experiment, where 50 µg of plasmid p2230 DNA was transfected. Cells were harvested 4 days post-transfection and the CAT assay performed as described previously (Harper et al., 1988), without a sonication step. CAT assays were normalized using an equal amount of protein from each extract. Thin layer chromatography was performed using silica gel chromatogram sheets in 95% chloroform and 5% methanol, followed by autoradiography. Chromatograms were later quantitated using a Betascope blot analyzer (model 603, Betagen Corp., Mountain View, CA). Experiments were repeated at least three times and in duplicate.

5` RACE-PCR Amplification and Sequencing

Four days post-transfection with p2230, total RNA was extracted from the cultures using the RNAzol B method (Biotecx Laboratories, Inc., Houston, TX). The 5` rapid amplification of cDNA ends was carried out utilizing the 5` RACE System (Life Technologies, Inc., Gaithersburg, MD) according to the manufacture's instructions. One µg of total RNA extracted from the transfected cells was used in the first strand synthesis reaction using a primer specific to the CAT coding region (5`-ACGAAATTCCGGATGAGC-3`). The resulting cDNA was then C-tailed. The PCR reaction was carried out using the supplied anchor primer and a primer specific to the loricrin intron sequence (5`-CCATCGATGTTGTTTAAGGAGAAG-3`). The first round of amplification was carried out using 5 µl of C-tailed DNA and 200 ng of each primer in a total 50-µl reaction. The product of this amplification was purified and 1 µl removed and used for an additional round of amplification. In all cases the reaction conditions were 92 °C for 5 min; then 30 cycles of 92 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s, followed by 72 °C for 5 min. A product of approximately 100 bp was cloned into a TA cloning vector (Invitrogen, San Diego, CA) and sequenced using Sequenase 2.0 (U. S. Biochemical Corp., Cleveland, OH) (Fig. 4).


Figure 4: Analysis of the transcription start site of the p2230 deletion construct. The p2230 deletion CAT construct was transiently transfected into primary murine keratinocytes, total RNA isolated, and RACE-PCR used to amplify the 5` ends of p2230 transcripts. The boxed ATG denotes the start of the loricrin coding region. The intron-exon boundary is indicated by the curved arrow. Alignment of the amplified sequence ( underlined) with the loricrin gene showed that transcripts were initiated from within the intron. This analysis revealed a mRNA CAP site (-90 bp relative to the start site), a TATA Box (-128 bp), and a CAAT box (-191 bp) present within the intron of the mouse loricrin gene.



Generation and Identification of Transgenic Mice

The pML(6.5) -gal construct was released from pGem7Z by digestion with ApaI. The fragment was isolated by agarose gel electrophoresis and purified (NA45 paper; Schleicher & Schuell, Keene, NH). Mouse embryos were isolated (Hogan et al., 1986) and the DNA microinjected into the pronuclei of one cell mouse embryos obtained from female ICR (Charles River) mice mated to FVB males (Frederick Cancer Research Facility). The embryos were then transferred to the oviduct of pseudo-pregnant females and normal gestation allowed. Positive transgenic mice were identified by tail DNA isolation (Hogan et al., 1986) and PCR amplification. Positive founder transgenics carrying the construct were detected by PCR analysis of genomic DNA isolated from tail biopsies. Primers 5`-CCCGGGTTCGAATTATTTTTGACACCAGAC-3` and 5`-CTCACGCGTGGCAGC-3` specific to the -galactosidase gene were used for analysis. Expression levels were analyzed by staining ear sections from the transgenics for -gal activity.

-Galactosidase Assay

Tissue samples for sectioning were fixed for 1-2 h at 4 °C in 2% paraformaldehyde, 0.2% glutaraldehyde, 0.01% sodium deoxycholate, 0.02% Nonidet P-40, in 0.05 M sodium phosphate buffer, pH 7.4. The tissue was then washed twice in phosphate-buffered saline containing 2 mM MgCl at room temperature, followed by 5-bromo-4-chloro-3-indoyl -D-galactoside staining at 30 °C in 0.3 M sodium phosphate buffer, pH 7.4, containing 43 mM MgCl, 10 mM KFe(CN), 10 mM KFe(CN), 0.03% sodium deoxycholate, 0.06% Nonidet P-40, 3.3 mg/ml 5-bromo-4-chloro-3-indoyl -D-galactoside. After staining the tissue was washed in phosphate-buffered saline containing 3% MeSO and then stored in phosphate-buffered saline until embedded in paraffin and sectioned.

The tissues analyzed for -gal activity by chemiluminescence assay were excised from transgenic or non-transgenic control animals, frozen in liquid nitrogen, and ground using a mortar and pestle. The cells were lysed by three rounds of freeze/thaw, followed by vortexing. The samples were centrifuged and the protein extract concentrations measured using the Bradford assay. For each tissue, 10 µg of protein extract was used in the Galacto-Light (Tropix, Inc., Bedford, MA) reporter assay system without modification. Samples were analyzed in a Lumat LB 9501-1 with built-in injector (Wallac Inc., Gaithersburg, MD).

Preparation of Oligonucleotides and Probes for Binding Assays

The 27-bp probe containing the AP-1 site from the loricrin promoter used in the gel shift assays was generated using an oligo synthesizer. The top strand, 5`-GATCTATAGATGAGTCAGAGCAG-3`, was annealed to the lower strand, 5`-GATCCTGCTCTGACTCATCTATA-3`, and the overlapping ends filled in with P-radionucleotides using Klenow enzyme. The 200-bp probe encompassing the loricrin proximal promoter region used in the DNase I footprinting assays was generated by PCR using primers with either BamHI (5`) or BglII (3`) restriction enzyme sites. After amplification, the PCR product was cut with either BamHI or BglII and labeled at one end with P-radionucleotides by Klenow fill-in. All probes were purified by native polyacrylamide gel electrophoresis prior to analysis.

Preparation of Whole Cell Protein Extracts

Primary murine keratinocytes were prepared and cultured as described above. Whole cell extracts were prepared using a modified procedure of Zimarino and Wu (1987). Frozen cell pellets were resuspended and lysed in 1 volume of Wu buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl , 0.1 mM EGTA, 0.5 mM dithiothreitol, 5% glycerol) with protease inhibitors, containing 400 mM KCl and centrifuged at 14,000 g for 30 min at 4 °C. One ml of the supernatant was added to 250 µl of saturated (at 0 °C) ammonium sulfate, and incubated on ice for 20 min, then centrifuged 14,000 g for 10 min. The supernatant was removed, and 400-µl aliquots were added to 600 µl of saturated ammonium sulfate, incubated on ice for 20 min, and centrifuged at 14,000 g for 10 min. The pellet was resuspended in 100 mM KCl/Wu buffer and dialyzed 1 h in a microdialysis unit (Life Technologies, Inc.) versus 1 liter of 100 mM KCl/Wu buffer.

Electrophoretic Mobility Shift Assays (EMSA) and DNase I Footprinting

All steps were carried out at room temperature unless noted otherwise. For gel shift assays, whole cell extracts, purified c-Jun protein (Promega, Madison, WI), and HeLa cell nuclear extracts (Promega) were preincubated with 2.5 µg of poly(dI-dC) (Pharmacia) and unlabeled competitor oligonucleotides for 10 min. Ten microliters of binding mixture (2.5 mg of bovine serum albumin/ml, 8% Ficoll, 0.1 mg of pd(N)/ml (Pharmacia)) containing 50,000 cpm of P-labeled probe was added and binding allowed to proceed for 10 min. For supershift assays, rabbit polyclonal anti-JunD antibodies (Santa Cruz Biotechnology) were then added and binding allowed to proceed for an additional 20 min. Samples were analyzed on a 6% native polyacrylamide gel run at 200 V, then dried for 45 min at 80 °C onto Whatman 3MM paper followed by autoradiography.

For DNase I footprint analysis, all initial binding steps were carried out as above with the exception that only 100 ng of poly(dI-dC) was added to the reaction. After the 10-min incubation with protein, the probe was digested for 30 s with 0.5-15 µg of DNase I (Worthington Biochemical Corp., Freehold NJ). Digestion was stopped by the addition of 100 µl of 10 mM EDTA, 0.1% SDS, 50 µg/ml proteinase K, and 1.5 µg of sonicated salmon sperm, followed by a 30-min incubation at 37 °C. Samples were extracted with phenol/chloroform followed by ethanol precipitation. Samples were resuspended in 3 µl of formamide, heated to 90 °C for 5 min, then loaded on a 7% denaturing polyacrylamide gel and run at 65 watts. Gels were dried onto Whatman 3MM paper for 1 h at 80 °C followed by autoradiography.


RESULTS

Analysis of the Loricrin Promoter in Cultured Cells

The cloning and characterization of the 6.5-kb genomic loricrin fragment which were used in these studies have been reported elsewhere (Rothnagel et al., 1994). This fragment contains the entire loricrin coding sequence, a 1.1-kb intron located within the 5` noncoding region, 1.5 kb of 5`-flanking and 2.2 kb of 3`-flanking sequences (Fig. 1 A). In an initial survey for the putative regulatory regions involved in loricrin expression, various subfragments of the genomic clone were introduced into the enhancerless reporter construct pA10CAT. Individual fragments consisted of the 5` BamHI- PstI, and BamHI- BamHI; and the 3` BamHI- BamHI and PstI- BamHI fragments (see Fig. 1A). These fragments contained flanking sequences as well as intronic and coding sequences and were cloned into BglII and BamHI sites of pA10CAT in both orientations relative to CAT (see ``Materials and Methods''). Interestingly, none of these fragments were able to appreciably enhance CAT activity in primary mouse keratinocytes (data not shown).

Since neither the 5`- nor 3`-flanking region could enhance CAT expression from a heterologous promoter, a CAT reporter gene was introduced into the 6.5-kb fragment, replacing the loricrin coding sequences and leaving both 5`- and 3`-flanking and noncoding sequences intact (Fig. 1 A). This construct, pML(6.5)CAT, was analyzed by transient transfection into primary murine keratinocytes. High levels of CAT activity were observed in cells cultured under conditions that promote differentiation (0.35 mM calcium) as well as in undifferentiated cells maintained in low calcium (0.05 mM) culture conditions (Fig. 1 B). The high activity of this construct in low calcium does not correspond to the expression of the endogenous loricrin gene, which has been shown previously to be expressed only in differentiated keratinocytes cultured in media with calcium levels of 0.12 mM and higher (Hohl et al., 1991b). Significantly, the pML(6.5)CAT reporter construct was not active in transiently transfected primary murine dermal fibroblasts (data not shown).

Analysis of the Loricrin Promoter in Transgenic Mice

To determine the in vivo expression pattern of the loricrin expression vector derived from the 6.5-kb genomic fragment, a -gal reporter gene was inserted into this fragment at the unique ClaI site and introduced into the germline of transgenic mice (Fig. 1 A). Four founder lines which expressed high levels of the pML(6.5) -gal reporter transgene were identified and used for subsequent analysis. An examination of -gal-stained epidermal sections from these mice revealed strong expression of the transgene not only in the differentiated suprabasal layers but also in undifferentiated basal layer keratinocytes (Fig. 1 C). This expression pattern was consistent with the in vitro data which showed that the 6.5-kb loricrin construct was expressed in undifferentiated keratinocytes. Expression of the -gal transgene was only detected in tissues normally expressing loricrin, including skin, tongue, and forestomach. The expression in tongue and forestomach was focal and relatively weak compared to the expression detected in the epidermis. This may indicate that additional cis-elements located outside the 6.5-kb fragment are required for complete expression in these tissues. Notably, -gal staining was limited to these epithelial tissues and not detected in any other tissues. Expression of the -gal transgene did not produce any observable pathology. Control epidermis from a non-transgenic sibling did not stain for -gal activity. An additional tissue survey of transgene expression was performed using a more sensitive chemiluminescence assay, found significantly higher -gal activity in crude protein extracts from transgenic skin and tongue, but not in tissues that do not express loricrin such as brain, liver, kidney, muscle, or heart (Fig. 2). Therefore, the regulatory elements present within the 6.5-kb loricrin construct could direct tissue-specific expression of the transgene, but is missing those elements which direct expression to the correct cell layer within the epidermis.


Figure 2: Relative -gal activity in various tissues from transgenics expressing pML(6.5) -gal. Using a chemiluminescence assay, the relative -gal activity of protein extracts from: H, heart; M, muscle; K, kidney; L, liver; B, brain; S, skin; and T, tongue from pML(6.5) -gal transgenic mice were compared to non-transgenic control mice. Only skin and tongue, which normally express loricrin, showed a significant increase in -gal activity over control tissues. Values shown are an average of three normal and three control mice, with standard deviations less than 20%.



Analysis of Deletion Constructs

In an effort to locate important regulatory sequences involved in the expression of loricrin in keratinocytes in vitro, a series of 5` deletions were made to the pML(6.5)CAT construct using unique restriction sites within the 5`-flanking region and designated by the number of bases removed (Fig. 3). These constructs were analyzed by transient transfection into primary murine keratinocytes cultured in both undifferentiating (0.05 mM Ca) and differentiating (0.35 mM Ca) culture conditions. This analysis revealed that deletions up to 60 bp 5` from the transcriptional start site were well tolerated and retained very high levels of promoter activity (p990 and p1480 in Fig. 3 ). In fact, CAT activity actually increased with these deletions, indicating the possibility that some repressor or controlling elements may be located in these deleted sequences. Not unexpectedly, deletions that extended past the TATA box and into the intron resulted in a substantial reduction in CAT activity.


Figure 3: Using convenient restriction sites, a series of 5` deletions were made to the pML(6.5)CAT reporter ( pMLC) and analyzed by transient transfection into primary murine keratinocytes in low ( L, 0.05 mM) and high ( H, 0.35 mM) calcium medium. The constructs are named by the size of the deletion in base pairs. The arrow indicates to position of the TATA box in each of the deletions. Percent CAT activity is relative to the highest expressing construct.



Surprisingly though, the p2230 deletion was still able to direct significant CAT expression in the absence of the TATA box and CAP site. Note that CAT expression was not abolished until most of the intron was removed, suggesting that a cryptic promoter may be located within the intron sequence. In an effort to account for this activity, 5` RACE-PCR was performed on total RNA obtained from keratinocytes transiently transfected with the p2230 construct. Alignment of the PCR generated sequence with the mouse loricrin gene revealed that transcription from the p2230 construct was initiated from a region within the intron that encodes a weak cryptic promoter complete with a TATA box, CAAT box, and an mRNA CAP site (Fig. 4). Comparison of this region with the human gene revealed similar motifs, suggesting that these sequences may be functionally significant. However, in a survey of the 5` ends of loricrin transcripts we were only able to detect the native transcript (as determined by Mehrel et al. (1990)) in RNA isolated from normal mouse epidermis (data not shown), suggesting that the weak activity of the p2230 construct is most likely an in vitro phenomenon of the deletion and not relevant in vivo.

The Loricrin Promoter Contains a Functional AP-1 Element

Sequence analysis of loricrin promoter revealed a perfect AP-1 consensus sequence (TGAGTCA) present 15 bp upstream of the TATA box (Rothnagel et al., 1994). This sequence is conserved in both human and mouse genes and is retained in the most active p1480 CAT construct (see Fig. 5 A). To characterize the AP-1 element in the loricrin promoter and to determine if the element could interact with AP-1 transcription factors, both electrophoretic mobility shift assays and DNase I footprint analysis were performed. In mobility shift experiments on a 27-bp fragment encoding the AP-1 element from the loricrin proximal promoter, purified c-Jun (Promega) produced a specific retarded complex (Fig. 5 B, lanes 2 and 3) that could be inhibited by preincubation with a 100-fold molar excess of an unlabeled oligonucleotide containing the AP-1 consensus sequence (Promega) (Fig. 5 B, lane 4). To demonstrate the interaction of keratinocyte factors with this region, whole cell extracts were used and either preincubated with an excess of unlabeled AP-1 competitor or supershifted with polyclonal antibodies to JunD. As shown in Fig. 5C, a specific retarded complex formed with keratinocyte extracts interacting with the 27-bp fragment ( lane 2), and formation of this complex was completely abolished using a 100-fold molar excess of AP-1 competitor ( lane 1), or partially supershifted with JunD antibodies ( lane 3), demonstrating that this site is capable of binding members of the AP-1 family of transcription factors present in keratinocytes. To define precisely the sites of interaction between keratinocyte nuclear factors and the proximal promoter, a 200-bp fragment encompassing the promoter region and including the AP-1 site was used in DNase I footprint analyses. The results in Fig. 6show that keratinocyte extracts produced a complex footprint with several protected regions within the loricrin promoter, including the same DNA region protected by purified c-Jun protein. The identity of the other sites protected within the loricrin promoter, and their potential functions, were not determined in this study.


Figure 5: A, a comparison of the promoter region of the mouse and human loricrin genes shows the conserved AP-1 element upstream of the TATA box. The conserved CAP site is also shown as is the BclI site used to make the p1480 deletion. B, electrophoretic mobility shift analysis of the AP-1 element with purified c-Jun protein showed a specific complex formed ( arrow) using 0.2 µl ( Lane 2) and 0.5 µl ( Lane 3) of protein (1 foot printing unit/µl). This complex could be competed using a 100-fold molar excess of an unlabeled AP-1 oligo ( Lane 4). Lane 1 is a control lane with probe alone. C, mobility shift analysis of the AP-1 element with keratinocyte whole cell extracts show a single complex formed with the probe ( Lane 2). The formation of this complex could be inhibited using a 100-fold molar excess of an unlabeled AP-1 competitor ( Lane 1). In addition, incubation with polyclonal antibodies to JunD partially supershifted the complex ( Lane 3, open arrow), indicating that AP-1 factors from cultured keratinocytes interact with the probe. Lane 4 is a control lane with probe alone.




Figure 6: DNase I footprint analysis of the loricrin promoter. Lane 1, no protein; Lane 2, 5 µg of keratinocyte whole cell extract; Lane 3, 10 µg of keratinocyte whole cell extract; Lane 4, 1 footprinting unit of c-Jun protein; Lane 5, 2 footprinting units of c-Jun protein; Lane 6, no protein. Keratinocyte extracts protected the same region of the promoter as the c-Jun protein. The protected region coinciding with the AP-1 element is indicated.



Mutation of the AP-1 Site Abolishes Transcriptional Activation

The conservation of the AP-1 element in both mouse and human genes (Rothnagel et al., 1994) and the observation that both purified c-Jun and keratinocyte AP-1 factors bind to it, strongly suggests an important role for this element in the regulation of the loricrin gene. To test the functionality of this element, the site was mutated from TGAGTCA to TCCCGGG, leaving the spacing between the other promoter elements and adjacent sequences intact. As can be seen in Fig. 7, mutation of this AP-1 element essentially abolishes the activation of the CAT reporter in primary keratinocytes. Promoter activity was reduced 93% in proliferating keratinocytes and 85% in differentiated cells, indicating that this AP-1 element is required for activity of the loricrin promoter in vitro.


Figure 7: The AP-1 element is necessary for loricrin promoter activity. The AP-1 element in the proximal promoter was mutated from TGAGTCA to TCCCGGG in the context of the pML(6.5)CAT vector and analyzed by transient transfection in primary murine keratinocytes cultured in either 0.05 mM calcium ( lo) or 0.35 mM calcium ( hi). Mutation of the element resulted in a 93% reduction in low calcium and a 85% reduction in high calcium medium. Relative CAT activity was calculated as expression of the AP-1 minus promoter relative to the native promoter over an average of three experiments. Standard deviation was less than 20%.




DISCUSSION

From this study, we can conclude that the correct transcription of the mouse loricrin gene in vitro and in vivo requires the synergistic effects of elements in both the 5`-proximal promoter and a distal element(s) not present in the 6.5-kb sequences. In addition, the elements which direct cell type specificity of the promoter can be uncoupled from those regulatory elements required to give differentiation-specific expression. The elements required to give cell and tissue type specificity are contained within a 6.5-kb genomic fragment, but this fragment lacks the elements required to direct correct expression with respect to differentiation state. In addition, the loricrin proximal promoter contains an evolutionarily conserved, functional AP-1 element which is absolutely necessary for the transcription of the loricrin gene in vitro.

Previous work has shown that transcription of the loricrin gene in vitro can be regulated by calcium, retinoic acid, and cell density (Hohl et al., 1991b). To gain a greater understanding of this regulation at a molecular level, various 5` and 3` fragments from a 6.5-kb mouse genomic loricrin construct were cloned into a heterologous SV40 promoter CAT construct and assayed by transient transfection into primary murine keratinocytes. These fragments were unable to enhance significant CAT expression from the heterologous promoter. Since previous studies on the HK1 gene suggested that both 5` and 3` elements are necessary for efficient expression of the reporter gene in vitro (Rothnagel et al., 1993), and work on the HK14 gene showed the importance of distal elements in the regulation of this gene (Leask et al., 1990), we constructed a composite pML(6.5)CAT reporter containing both 5`- and 3`-flanking sequences to ensure expression in the event that transcription of the loricrin gene required synergistic effects of both 5` and 3` regulatory elements. When this expression vector containing a CAT reporter was transfected into primary keratinocytes and analyzed in low and high calcium medium, we observed surprisingly high activity in cells cultured to promote both basal and differentiated phenotypes. Previous reports, examining endogenous loricrin expression in vitro, showed that loricrin transcription was only activated when cells were induced to differentiate, at calcium levels above 0.12 mM (Hohl et al., 1991b). In addition, in situ hybridization and immunofluorescence data has previously shown that the loricrin gene is expressed in the granular layer of the epidermis in vivo (Mehrel et al., 1990). When a -gal gene was cloned into the ClaI site of the reporter, the pML(6.5) -gal vector was expressed in a tissue-specific manner, however, the reporter was expressed throughout the entire epidermis, including the basal and spinous layers, supporting what was observed in vitro with the CAT reporter. Therefore, this 6.5-kb construct contains those elements which direct expression of loricrin to the epidermis, but must lack the regulatory elements necessary to confine loricrin transcription to differentiated keratinocytes.

Recent work on the human loricrin gene by Yoneda and Steinert (1993) has shown that 9 kb of 5`-flanking sequence and 9 kb of 3`-sequence could direct tissue and differentiation specific expression of a human transgene in transgenic mice. It should also be noted that data from our laboratory, which will be reported elsewhere, indicates that the distal element(s) which restricts expression of the mouse loricrin gene to the granular layer is located within additional sequences found in a larger 14-kb mouse genomic fragment.

In order to localize the regulatory elements required to drive expression in keratinocytes, a series of deletions were made to the 5`-flanking sequences. These 5` deletions showed that deleting up to 60 bp 5` from the initiation of transcription in the basal promoter resulted in no reduction of activity. It was not until the remaining 60 bp of 5`-flanking sequence, including the TATA box and the first exon were removed by deleting into the intron sequence, that a reduction in CAT activity was observed (Fig. 3).

It was interesting to find that the 5` promoter contained an AP-1 site which is perfectly conserved between the human and mouse loricrin genes (Rothnagel et al., 1994). This AP-1 site is retained in the most active 5` deletion (p1480) containing 30 bp of sequence 5` to the TATA box. This site was of great interest since we have previously determined that the calcium responsive induction of HK-1 is directly regulated by a composite AP-1/steroid hormone binding site located in the 3`-flanking sequence of the HK-1 gene (Lu et al., 1994). In addition, functional AP-1 sites have been identified in other epidermal-specific genes including keratins K8 and K18 (Tamai et al., 1991; Oshima et al., 1990), the bovine keratin 5 gene (Casatorres et al., 1994), transglutaminase I (Liew and Yamanishi, 1992), and in the promoters of keratinocyte-specific human papilloma viruses types 16 and 18 (Cripe et al., 1990; Offord and Beard, 1990; Mack and Laimins, 1991; Thierry et al., 1992). Furthermore, the involucrin gene, another cornified envelope protein, has been found to be activated by 12- O-tetradecanoylphorbol-13-acetate (Takahashi and Iizuka, 1993), a tumor promoter known to stimulate the activity of genes regulated by AP-1 (Boyle et al., 1991; Angel et al., 1987; Lee et al., 1987). Furthermore, in vitro studies have determined that co-transfection of AP-1 factors c- fos and c- jun could also mimic 12- O-tetradecanoylphorbol-13-acetate activation of the involucrin gene (Takahashi and Iizuka, 1993). Likewise, loricrin gene transcription could be induced in vitro by 12- O-tetradecanoylphorbol-13-acetate treatment (Dlugosz and Yuspa, 1993). It is interesting to speculate that the loricrin and involucrin genes may be regulated by very similar regulatory mechanisms since both genes have almost identical gene structures and are components of the cornified envelope, both genes are expressed in the differentiated layers of the epidermis, and both genes are induced by 12- O-tetradecanoylphorbol-13-acetate.

While some data point to AP-2 as well as other transcription factors such as Sp1 and the Pou proteins as regulators of keratinocyte gene expression (Leask et al., 1990, 1991; Andersen et al., 1993; Magnaldo et al., 1993a, 1993b; Yukawa et al., 1993; Faus et al., 1994;), and recent reports indicate the possible presence of a keratinocyte-specific transcription factor termed basonuclin (Tseng and Green, 1992, 1994), several lines of independent data are now pointing to the AP-1 family of transcription factors as important regulators of epidermal differentiation-specific gene expression. The identification of functional AP-1 elements in genes of differentiation- specific expression coincides well with the demonstrated expression of AP-1 factors within the epidermis. Subpopulations of basal cells in the epidermis have been shown to express c- jun mRNA, while the differentiation of the epidermis is accompanied by an induction of c-Fos and JunB expression (Wilkenson et al., 1989; Fisher et al., 1991; Smeyne et al., 1992). These observations suggest the differential expression of these and possibly other AP-1 transcription factors are involved in regulating the expression of differentiation-specific genes in the epidermis.

As previously stated, the K1 gene is normally expressed in the differentiated suprabasal cells of the epidermis. Like the loricrin promoter, we have shown that a portion of the HK1 promoter can direct tissue-specific expression of a reporter gene, yet the expression of the HK1 promoter is promiscuous and present in a portion of basal cells (Chung et al., 1994). This laboratory has evidence to show that the elements that repress HK1 expression in basal cells and restrict expression to cells above the basal layer are responsive to retinoic acid and these elements lie some 6 kb upstream from the HK1 promoter.() Likewise, elements specifying the differentiation-specific expression of loricrin lie in the additional upstream sequences present in a larger 14-kb genomic construct. Because it is known that retinoic acid also plays a role in the suppression of loricrin transcription in vitro (Hohl et al., 1991b), the elements responsible for loricrin suppression in basal cells may also be retinoic acid-responsive elements. Whether this is a direct cis-regulation by the binding of a retinoic acid receptor to the distal elements of the loricrin gene, an indirect trans-regulation, or a direct interaction between the retinoic acid receptors and the AP-1 factors regulating loricrin in the epidermis remains to be determined.

While the most intuitive argument would be the additional elements located outside the 6.5-kb clone are acting as repressors (the gene is on by default and off in basal and spinous cells in the presence of additional sequences), this may not necessarily be the case. Byrne and Fuchs (1993) have published data on the expression of a -galactosidase reporter driven by a truncated form of the human K5 promoter in transgenic mice. Normally the keratin 5 gene is only expressed in basal cells in the epidermis. When the cells are induced to differentiate, the gene is then down-regulated. It was reported that while 6 kb of 5`-promoter could direct basal cell-specific expression in transgenic mice, a deletion containing only 90 bp of promoter sequence switched differentiation specificity. This 90-bp promoter, while largely retaining its tissue specificity, was now expressed only in differentiated cells in the epidermis.

Clearly, a complex interplay of different transcription factors are involved in the regulation and expression of epidermal genes during both proliferation and differentiation, and as more information is gathered, it will be interesting to compare the tissue- and differentiation-specific elements regulating these epidermal genes.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant AR40240. 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.

§
Recipient of a Dermatology Foundation Career Development Award sponsored by Ortho Pharmaceutical Corp.

To whom correspondence should be addressed: Dept. of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Tel.: 713-798-4966; Fax: 713-798-0545.

The abbreviations used are: K1, K5, etc., keratin 1, keratin 5, etc.; -gal, -galactosidase; CAT, chloramphenicol acetyltransferase; RACE, rapid amplification of cDNA ends; HK1, human keratin 1; HK14, human keratin 14; kb, kilobase pair(s); bp, base pairs(s); PCR, polymerase chain reaction.

J. A. Rothnagel and D. R. Roop, unpublished results.


ACKNOWLEDGEMENTS

We thank Rachel Welborn for her help with figure preparation, and Bo Lu for his expert advise.


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