Molecular Vision 2002; 8:94-101 <http://www.molvis.org/molvis/v8/a13/> Received 28 February 2002 | Accepted 3 April 2002 | Published 7 April 2002

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Cis-regulatory elements of the mouse Krt1.12 gene

I-Jong Wang,^1,2 Eric C. Carlson,¹ Chia-Yang Liu,¹ Candace W.-C. Kao,¹ Fung-Rong Hu,² Winston W.-Y. Kao¹
 
(The first two authors contributed equally to this publication)
 
 

¹Department of Ophthalmology, University of Cincinnati, Cincinnati, OH, USA; ²Department of Ophthalmology, National Taiwan University Hospital, Taipei 100, Taiwan

Correspondence to: W. W.-Y. Kao, Department of Ophthalmology, University of Cincinnati Medical Center, Health Professions Building, Suite 350, ML0527, 3223 Eden Avenue, Cincinnati, OH, 45267-0527; Phone: (513) 558-5151; FAX: (513) 558-3108; email: Winston.Kao@uc.edu

Abstract

Purpose: Keratin 12 is a cornea epithelial cell-specific intermediate filament component. To better understand the regulatory mechanism of its expression, the cis-regulatory elements located between the transcription start site and 600 bp upstream of the Krt1.12 gene were determined.

Methods: The promoter activity of reporter gene constructs containing 0.6, 0.4, and 0.2 kb of DNA 5' upstream of Krt1.12 coupled to the lac Z gene were determined in rabbit corneas using Gene Gun technology. DNA foot printing and EMSA (electrophoresis mobility shift assay) were employed to identify putative cis-regulatory elements of the Krt1.12 gene using bovine corneal epithelial cell nuclear extracts.

Results: Enzyme activity assays and histochemical analysis of β-galactosidase from the 0.6, 0.4, and 0.2 kb K12 promoter constructs indicated that the DNA elements between -0.2 and -0.6 kb 5' of the Krt1.12 gene contain cis-regulatory elements for its corneal epithelial cell-specific expression. Foot printing and EMSA showed that the sequences between -181 to -111 and -256 to -193 upstream of the Krt1.12 gene reacted to nuclear proteins isolated from bovine corneal epithelial cells. A Genbank search revealed that these two regions were potential binding sites for many transcription factors such as AP1, c/EBP, and KLF6. Immunofluorescent staining indicated the presence of c-jun and c/EBP transcription factors in the nuclei of corneal epithelial cells.

Conclusions: The data is consistent with the notion that the -182 to -111 and -256 to -193 fragments 5' of the Krt1.12 gene may serve as corneal epithelial cell-specific cis-regulatory elements, and the coordinated interactions of various transcription factors are required for cornea-specific expression of Krt1.12 gene.

Introduction

Keratins are a group of water-insoluble proteins that form the 10 nm intermediate filaments found in epithelial cells [1]. The major function of the cytoskeletal network formed by keratins is to provide a rigid epithelial cell layer that protects underlying tissues from the environment [2,3]. This proposition is substantiated by mutations of K1/K10 and K5/K14 keratins, which manifests as epidermolysis hyperkeratosis and epidermolysis bullosa simplex in skin, respectively [2,4,5]. Mutations of the K12 keratin in human and ablation of the Krt1.12 gene via gene targeting in mice results in fragile corneal epithelium, a clinical manifestation characteristic of Meesmann's corneal dystrophy [6,7].

The approximately thirty different keratin proteins are divided into two groups, an acidic type I and a basic type II [1,8-10]. In vivo, a basic keratin usually heterodimerizes or "pairs" with a particular acidic keratin [1,10-16]. The expression of keratin pairs is tissue-specific, differentiation-dependent, and development regulated. For example, the K5/K14 keratin pair is found in the basal cell layer of all stratified epithelium, whereas the K1/K10 keratin pair is expressed by suprabasal and superficial epidermal epithelial cells [13]. The K3/K12 pair is characteristic of cornea-type epithelial differentiation [17-19]. It should be noted, however, that the expression of K3 is not limited to cornea epithelium; it has also been detected in several other tissues such as conjunctiva [8,11,20-22]. We have cloned the mouse cornea-specific Krt1.12 gene and demonstrated that the expression of K12 is restricted to the corneal epithelium [17,23-25].

Many keratin genes are regulated at the transcriptional level in a coordinate manner during keratinocyte differentiation [26-28]. Tissue-specific keratin expression is regulated by a complex collaboration between ubiquitous and tissue-specific transcription factors, and is dependent on the activation and deactivation of a variety of regulatory genes [29-32]. There are several transcriptional factors known for regulation of keratin gene expression. For example, AP-2 and SP-1 have been shown to regulate the expression of K1, K3, K5, K6, and K14 [30,31,33,34]. Furthermore, the regulation of a tissue-specific keratin gene always needs the coordinated expression of several transcription factors [33]. Several extracellular signals, such as vitamin A, and other growth or transcription factors (such as Pax 6, EST 1 and KLF6) are known to regulate keratin expression by epithelial cells [30,31,34].

Recently, we successfully used Gene Gun technology, a particle-mediated gene transfer technique, to deliver K12-promoter reporter genes to rabbit corneal epithelial cells in vivo and further identified a 0.6 kb DNA fragment flanking the 5' end of the Krt1.12 gene which possibly contained corneal epithelial cell-specific regulatory cis-DNA elements [35]. In an attempt to gain a better understanding of the regulatory mechanism(s) involved in Krt1.12 expression and for the maintenance of corneal epithelium integrity, we further examined the 0.6 kb 5' sequence flanking the Krt1.12 gene to determine the cis-regulatory elements accounting for corneal epithelial cell-specific expression, using, in vitro, DNase I footprinting and electrophoretic mobility shift assays (EMSA), and in vivo, Gene Gun technology.

Methods

Plasmids

Two plasmid DNA constructs, pCMVβ and pNASSβ (Clontech, Palo Alto, CA), were used as positive and negative control reporter genes. Three reporter gene constructs, termed 0.2 KZ, 0.4 KZ, and 0.6 KZ, were prepared by cloning 127 bp, 327 bp, and 527 bp upstream sequence, plus 40 bp of exon 1 untranslated region, of the Krt1.12 gene into the pNASSβ plasmid that contained a lacZ reporter gene.

In vivo particle-mediated gene transfer (Gene Gun)

All animal experiments were performed according to the ARVO resolution on the use of animals in vision research. Plasmid DNA purified using Qiagen^TM columns (Qiagen, Chatsworth, CA) was coated onto 0.6 μm, 1.0 μm or 1.6 μm gold particles (5 μg DNA per mg of gold), loaded into Tefzel^TM tubing (Biorad, Hercules, CA), and transiently transfected into New Zealand white rabbit (about 2 kg in weight) corneal epithelium using the Helios^TM Gene Gun System (Biorad) at 150 psi for cornea and conjunctiva, and 400 psi for skin, as described by Shiraishi, et al [35]. Specimens were collected 48 h after delivery, and subjected to β-galactosidase activity analysis.

Preparation of enzyme extracts

Excised tissues were minced with a razor blade, and 0.5 ml of extraction buffer (0.25 M Tris-HCl, pH 7.4, 0.1% Tween 20) was added. The samples were subjected to 3 freeze-thaw cycles, 5 min on dry ice and 5 min at 37 °C. The supernatants were collected by centrifugation at 13,000x g at 4 °C for 10 min.

β-Galactosidase activity assay and histochemical staining

Aliquots of supernatant were incubated in a 0.3 ml mixture containing 50 mM 2-mercaptoethanol, 1 mM MgCl₂, 1.33 mg/ml o-nitrophenyl β-galactopyranoside and 0.1 M phosphate buffer, pH 7.0 at 37 °C for 1 to 5 h. A 0.7 ml aliquot of 1 M Na₂CO₃ was added to terminate the reaction. Enzyme activity was determined by comparing the optical density at 460 nm to that of purified β-galactosidase (Boehringer Mannheim, Indianapolis, IN). The promoter activity of KZ reporter gene constructs was calculated as a fold-increase of the enzyme activities derived from the promoter-less pNASSβ construct [35].

For whole mount X-gal staining, the eyeball was enucleated, fixed immediately with 4% paraformaldehyde in PBS at 4 °C for 2 h, and washed with PBS buffer (pH 7.0) containing 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, and 1.4 mM KH₂PO₄. Staining was carried out at 30 °C for 16 h in a solution of 5-bromo-4-chloro-3-indolyl-β-galactopyronoside (X-gal, Sigma, St. Louis, MO) at a final concentration of 0.4 mg/ml in PBS. Following staining, eyeballs were rinsed with PBS and photographed as whole mounts as previously described [35].

Preparation of nuclear extracts

Nuclear extracts were prepared as described by Andrews and Faller [36]. Briefly, bovine corneal epithelium was scraped from enucleated eyes into ice-cold PBS, and then homogenized by Teflon-glass in homogenization buffer (2 ml/gm tissue) containing 0.1% Triton X-100, 10 mM HEPES (pH 7.6), 25 mM KCl, 1 mM EDTA, 2 M sucrose, 0.5 mM spermidine, 0.15 mM spermine, 10% (v/v) glycerol in DEPC H₂O. The homogenate was then diluted 3-fold in the same buffer without glycerol and centrifuged at 104,000x g for 30 min on a 10 ml cushion of homogenization buffer. Pellets were then combined and resuspended in 15-20 ml of a 9:1 (v/v) mixture of homogenization buffer and glycerol, using a Teflon-glass homogenizer. This homogenate was layered on a 10 ml cushion of the 9:1 mixture and centrifuged as above. The nuclei were homogenized in cold lysis buffer containing 10 mM HEPES (pH 7.6), 0.1 M KCl, 3 mM MgCl₂, 0.1 mM EDTA, 10% glycerol, 1 mM DTT, and 0.1 mM PMSF in DEPC H₂0 with a glass-glass homogenizer. The absorbance was then checked with a spectrophotometer at 260 nm, and the specimens were diluted in lysis buffer to 10 A₂₆₀ unit/ml. The nuclear extract was subjected to two steps of ammonium sulfate fractionation by adding a 10% volume of 4 M (NH₄)₂SO₄ (pH 7.9) and incubation at 4 °C for 30 min, followed by centrifugation (100,000x g for 60 min). The nuclear proteins in the supernatant were then precipitated by adding solid (NH₄)₂SO₄ (0.39 g/ml) and collected by centrifugation (100,000x g, 60 min). The nuclear protein extract was resuspended and dialyzed in buffer containing 25 mM, 10 mM HEPES, 40 mM KCl, 10% glycerol, 0.1 mM EDTA, 1 mM DTT, in DEPC H₂O. The extract was then stored at -70 °C until use.

DNase I footprinting analysis

Three DNA probes (-332 to -109 bp, -394 to -131 bp, and -599 to -375 bp upstream of the Krt1.12 gene) were prepared via PCR (primers used are listed in Table 1) and 5' labeled with [γ-³²P] ATP using T4 polynucleotide kinase according recommendation of the manufacturer (New England Biolab, Beverly, MA). A 50 μl mixture containing 10 mM Tris-HCl (pH 8.0), 5 mM MgCl₂, 5 mM CaCl₂, 50 mM KCl, 0.5 mM dithiothreitol (DTT), 0.05 mg/ml bovine serum albumin, and 40.5 ng/μl poly-dIdC was incubated with crude nuclear extracts (75 μg) on ice for 15 min. The mixture was then incubated for an additional 45 min with 100,000 cpm of DNA probe. Variable amounts of DNase I were added to the mixture and incubated at 25 °C for 1.5 min. This reaction was stopped with 50 μl of stop buffer (0.2 M NaCl, 30 mM EDTA, 1% SDS, and 0.1 mg/ml yeast tRNA). Digested DNA probes were purified by phenol extraction and ethanol precipitation and separated on 6.5% denaturing polyacrylamide gels. Dried gels were exposed to Kodak XAR film with intensifying screens for 24 h at -80 °C. G and G+A sequencing reactions were performed to determine the positions of the protected regions. Negative control reactions were performed in the absence of nuclear protein extracts. Regions protected by nuclear proteins were numbered according to the nucleotide positions relative to the transcription start site of the Krt1.12 gene.

Electrophoretic mobility shift assays

Gel-shift analysis of the potential tissue specific region of the Krt1.12 promoter was performed with ³²P labeled double-stranded synthetic oligonucleotide probes (-182 to -111 bp and -256 to -193 bp, upstream of the Krt1.12 gene Table 2), and crude nuclear extracts prepared from bovine corneal epithelial cells as described above. Binding reactions were performed in a volume of 20 μl, which contained 10 μg of nuclear proteins, 20,000 cpm double strand oligonucleotides end-labeled with [γ-³²P] ATP, 12 mM HEPES (pH 7.9), 10% glycerol, 4 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.3 mg/ml bovine serum albumin, 1 mM DTT, 0.1 mg/ml poly-dAdT, and 60 mM KCl. Each labeled probe was competed with an excess of the same unlabeled double-stranded synthetic oligonucleotide. Binding mixtures were separated on a nondenaturing 5% polyacrylamide gel and exposed to Kodak XAR film.

Immunohistochemistry

Paraffin sections of mouse eyes were deparaffinized and incubated in PBS for 30 min. The tissue section was then blocked with 1% BSA or 3% nonfat milk in PBS for 60 min at room temperature. Rabbit antibodies against mouse c-jun/AP-1 and c/EBPβ (Santa Cruz Biotech. Inc., Santa Cruz, CA) were diluted 25 fold in the blocking solution and incubated on the tissue sections at 4 °C overnight. The sections were then washed in PBS. A goat anti-rabbit Alexa Fluor 488 (Molecular Probes Inc. Eugene, OR) secondary antibody-conjugate was used for immunoreactivity detection using fluorescence microscopy.

Results

Promoter activities in the 5'-flanking region of the Krt1.12 gene

It was previously demonstrated that the 0.6 kb 5' flanking DNA fragment of the Krt1.12 gene could direct cornea-specific expression of a lac Z gene in vivo [35]. In an attempt to identify the region where cornea-specific 5' flanking cis-regulatory elements might exist in the Krt1.12 gene, we tested the 0.2 KZ, 0.4 KZ, and 0.6 KZ constructs in vivo. Rabbit corneas, conjunctivas, and skin were transiently transfected with the 0.2, 0.4, and 0.6 KZ constructs using the Gene Gun. Figure 1 shows that all three promoter constructs yielded 7-10 fold β-galactosidase (β-gal) activities above the promoter-less pNASSβ construct in the cornea. In skin and conjunctiva, the 0.4 and 0.6 KZ constructs yielded insignificant β-gal activities in comparison to those of promoter-less pNASSβ, however, the 0.2 KZ construct produced a 3-fold β-gal activity above pNASSβ. These observations suggested that the 0.2 KZ construct does not have cornea-specific cis-regulatory elements for the expression of the β-gal reporter gene. The 0.4 KZ and 0.6 KZ constructs did show cornea-specific β-gal activities when compared to that of conjunctiva and skin. To further elucidate this suggestion, whole mount X-gal staining was performed with transfected rabbit eyes. Figure 2 demonstrates that the 0.2 KZ construct (Figure 2A) shows β-gal expression in both the cornea and conjunctiva. The 0.4 KZ (Figure 2B) and 0.6 KZ construct [35] show reporter gene expression exclusively in the cornea. Figure 2C shows the negative control (promoterless pNASSβ construct). This observation suggested that a cornea-specific regulatory element(s) might exist in the -327 to +40 bp region of the Krt1.12 gene.

DNase I footprinting assay

Since the in vivo data indicated that the 0.4 KZ reporter gene construct has cornea-specific promoter activity, DNase I footprinting was performed to further identify the DNA sequences that may account for cornea-specific Krt1.12 gene expression. Two footprinting probes (-394 to -131 bp and -332 to -109 bp) were used to identify the possible transcription factor binding site(s) that the previously described data suggested exist in this region. Bovine epithelial nuclear extract protection was seen for a 72 bp fragment in the -182 to -110 bp region of the Krt1.12 gene (Figure 3). Also, protection existed in 2 other regions of the Krt1.12 gene, from -231 to -193 bp and from -256 to -243 bp (Figure 3).

Electrophoretic mobility shift assays (EMSA)

EMSAs were performed to further identify the 5' flanking region of Krt1.12, which may bind to nuclear factors. Synthetic oligonucleotides used in EMSAs (Table 2) were designed from the regions protected by epithelial nuclear proteins in the footprinting assays. EMSA of site from -182 to -111 and -256 to -193 showed a binding complex with nuclear proteins from bovine corneal epithelial cells, but the complex could also be detected at lower affinity with nuclear proteins of retinal cells (Figure 4). The radioactive labeled complex was competed and eliminated by a 400- or 800-fold molar excess of the unlabeled oligonucleotides, termed self-competitor. The observation suggests that these regions may contain binding sites of common transcription factors. Sequence analysis indicates that several transcriptional factors can potentially bind to theses regions of the of Krt1.12 promoter, which include c-jun/AP-1, c/EBPβ and KLF6 binding sites (Figure 5).

Immunohistochemistry

Consensus sequence analysis revealed potential c-jun/AP-1 and c/EBPβ binding sites in the 5' flanking Krt1.12 gene region. Immunohistochemistry was performed to determine if the expression of these two transcription factors may be present in corneal epithelial cells, but absent in limbal basal epithelial cells and can be correlated with that of keratin 12 expression. Figure 6 demonstrates the presence of c-jun/AP-1 and c/EBPβ in the nucleus of all mouse corneal epithelial cell layers. However, c/EBPβ appears to have a significantly lower expression level in the basal limbal epithelial cells as compared to c-jun/AP-1. This observation is consistent with an expression pattern of keratin 12 such that keratin 12 is expressed by corneal and suprabasal limbal epithelial cells, but not by the limbal basal cells [17,25].

Discussion

Although many keratin genes have been cloned, the 5'-upstream sequences of only a few of them have been found to function as tissue-specific promoters by in vitro analysis of cultured keratinocytes and in vivo in transgenic mice [28,33,35,37-40]. In the present study, we discovered the 0.4 kb 5' flanking region of the mouse Krt1.12 gene appeared to contain the cis-regulatory elements necessary for cornea-specific expression as demonstrated by in vivo transfection of lac Z reporter gene constructs with the Gene Gun. Analyses using foot printing and EMSA further identifies two regions -256 to -193 bp and -182 to -111 bp 5' to the Krt1.12 transcription initiation site that may play a role in Krt1.12 gene expression by corneal epithelial cells.

Recently, Szabowski et al demonstrated that c-jun is essential for fibroblast-mediated paracrine control of epidermal keratinocytes proliferation and differentiation [41]. Analysis of sequence comparison to the NCBI GenBank database (Blast web client software) revealed several possible transcription factor binding sites including, AP-1 (-240 to -250 bp and -124 to -133 bp) and c/EBPβ (-133 to -147 bp, -229 to -242 bp, and -226 to -243 bp) and many others in the sense and antisense orientation as shown in Figure 5. We also tested other transcription factors, via immunohistochemistry, having consensus sequences in the sense or antisense orientation of Krt1.12, but no clear nuclear signal was detected (data not shown).

AP-1 (activation protein 1) is a transcription factor that exists in many cells and has been well known to be a regulator of many keratin genes including K1, K3, K5, K8, K10, and K18, which are expressed by suprabasal and superficial epithelial cells of stratified epithelium, as well as single cell-layer epithelium [34,42-46]. Immunohistochemistry detected AP-1 in all cell layers of the cornea, which is consistent with the expression patterns of keratinocyte differentiation. The retina was also positive for AP-1 (data not shown), which in parts explains the EMSA data in which the nuclear extract of retina also interacts with the oligonucleotides (Figure 3).

Expression of K12 keratin is limbal basal negative until after migration of the epithelial cells from the limbal basal layer [17,25]. Our c/EBPβ and AP-1 transcription factor immunostaining, shown in Figure 6, was positive in the nuclei of corneal epithelial cells, but expression appears down regulated in the limbal basal region, especially with respect to c/EBPβ. C/EBP family members (α, β, γ, and δ) contain the bZIP region. The bZIP region is characterized by two motifs, one of which is involved in DNA binding, and the other a leucine zipper involved in dimerization. c/EBPβ can homodimerize or heterodimerizes with other family members and other transcription factors (NFκB, p65, p50, and rel family members) [47]. The interaction of c/EBPβ is believed to be a gene regulation mechanism involving an interaction of various transcription factors. This provides further evidence of a multimeric transcription factor complex regulating tissue-specific gene expression. The presence of KLF6 in human corneal epithelial cells was recently demonstrated, along with its ability to activate Krt1.12 transcription in those cells (manuscript submitted). The Krt1.12 binding region of this transcription factor falls in the same region we define as being essential for corneal expression. It should be noted that transcript factors such as c/EBP, AP 1, or KLF 6, for instance, are often found in various cell types. Thus, tissue specific expression of genes is likely governed by the collaboration of many transcription factors as well as chromatin structures within a given cell type [47-50].

In order to replenish the corneal epithelial cell population, limbal stem cells exist in the periphery of the cornea that proliferate, migrate, and then differentiate into corneal epithelial cells after migrating from the limbal region to the cornea [51]. Traditionally the hallmark step of a limbal stem cell differentiating into a corneal epithelial cell has been the initiation of K12 expression [17,35,52]. Identifying the key factors in the limbal stem cell differentiation process provides insight on the mechanism of stem cell differentiation. Understanding this process will lead to identification of those factors involved in maintaining the multipotency of stem cells as well as those promoting terminal differentiation.

Further characterization of the corneal epithelial cell specific Krt1.12 gene promoter coupled with in vivo gene delivery techniques such as the Gene Gun, in vivo electroporation, and retroviral vectors provides a strong potential for gene therapy treatments. Localized gene delivery with a specific tissue expression pattern may serve as a treatment regimen for ocular surface diseases in the future. It is with further research into tissue specific gene expression and pinpointing the origin of such diseases that gene therapy techniques can be implemented to treat human disease.

This study identifies the 5' flanking region of the Krt1.12 gene, which contributes to tissue-specific expression, provides evidence of transcription factor cooperation for tissue-specific gene expression, and potentially elucidates an in vivo transfection technique for controlled gene delivery and expression. This study further indicates that regulation of Krt1.12 gene expression may result from a coordinated function of several transcriptional factors including KLF6, AP-1/c-jun and c/EBPβ [26-28].

Acknowledgements

The Study was in part supported by NIH grants: EY11845, EY10556, EY12486, Challenge grants of Research to Prevent Blindness, New York, and Unrestricted grants of Ohio Lions' Eye Research Foundation, Columbus, OH, as well as grants from the Taiwan Government to I-J Wang and H-R Hu, NSC-88-2314B-002-372, -239 and -481.

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