INTRODUCTION

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The epidermis is a stratified squamous epithelium that resides at the skin surface. Its proliferative compartment is located in the innermost basal layer, where stem cells and transiently amplifying cells attach to an underlying basement membrane of extracellular matrix (16, 18). In a homeostatic and self-renewing tissue, transiently amplifying basal cells periodically withdraw from the cell cycle, lose their contact with basement membrane, and activate a program of terminal differentiation (16-18). As the cells move upward toward the surface, they progress through three distinct stages: spinous, granular, and stratum corneum (15).

Throughout all but the stratum corneum, unique sets of genes are turned on and off in a growth- and differentiation-specific manner. These include genes encoding structural cytoskeletal proteins, transcription factors, and products that are necessary for the barrier function of skin (16-18). Basal cells transcribe the genes encoding keratins 5 and 14 (K5 and K14), while cells in the spinous layer switch off these genes and induce K1 and K10, required for the marked mechanical resistance of these cells (15). In the granular layer, cells express loricrin, filaggrin, and transglutaminase, involved in assembly of the cornified envelope. This structure forms a sac to contain the keratin filaments and to provide a scaffold upon which specialized lipids are extruded and organized (15, 16, 18). Once the barrier is in place, keratinocytes cease their metabolic activity, becoming dead squames that are eventually sloughed from the skin surface, continually being replaced by inner cells differentiating and transiting outward (18).

Temporally and spatially compartmentalized, the epidermis provides a unique system to study the molecular switches that govern tissue-specific and differentiation-specific gene expression (4, 11). In some studies, the promoters and enhancers of epidermis-specific genes have been examined to elucidate the cis and trans elements that govern gene regulation. In other studies, transcription factors which are preferentially expressed in epidermal keratinocytes have been identified. Most epidermal promoters contain functional binding sites for the AP-2 family of transcription factors expressed not only in epidermis but also in a number of other cell types (5, 6, 26, 30, 32, 41, 48). Although addition of exogenous AP-2 to certain nonkeratinocyte cell types can enable them to activate otherwise epidermally restricted promoters, the AP-2 site in the promoter of at least one epidermal gene, K5, is largely dispensable for tissue-specific gene expression in transgenic mice (5, 6). Together with the broad expression patterns of AP-2 genes (32), these findings suggest that AP-2 alone is unlikely to confer epidermal specificity in vivo.

Functional Sp1 sites are also found in the promoters of epidermally expressed genes, although they do not appear to be involved in cell-type-specific gene expression (4, 5, 28). Additional factors include POU domain proteins, NF-B, AP-1, and ets family members (10, 11, 13, 36, 38, 40, 49). The POU domain proteins Skn-1a/I and Tst-1/Oct-6 seem to suppress suprabasal expression of K5 and K14 genes (1, 12), while NF-B may play a role in the ability of basal keratinocytes to respond to external cues (40). In contrast, AP-1 and ets proteins have been implicated predominantly in regulating suprabasal genes, although some evidence supports the notion that these proteins may also influence expression of basal epidermal genes (7, 24, 28, 34, 38, 44). Whether these findings are physiologically relevant remains unexplored.

Despite extensive research, no transcription factor that is strictly expressed in a keratinocyte-specific fashion has been found, and no transcription factor regulatory motif which on its own can confer keratinocyte-restricted activity to a heterologous promoter in vitro or in vivo has been identified. It could be that such regulatory proteins exist but have yet to be identified. Alternatively, keratinocyte-specific activity could be controlled by combinatorial action of more broadly expressed transcription factors, which in vivo are expressed simultaneously only in keratinocytes.

Since basal epidermal keratinocytes can be easily propagated in culture, our studies have focused on the K5 and K14 genes, which are abundantly transcribed in these cells in vitro (43). Furthermore, K14 and K5 induction in vivo occurs concomitantly with commitment of an embryonic basal cell to an epidermal fate, making them valuable markers of this cell type (6). Previously, we characterized the human K14 and K5 promoters, both of which possess a classical TATA box and contain functional AP-2 and Sp1 sites (5, 27). However, in the course of these analyses, we found that sequences residing within 2 kb 5' upstream of the K14 promoter were also required for appreciable reporter gene activity in SCC-13 squamous cell carcinoma keratinocytes in vitro and in transgenic mice in vivo (27).

We now define the nature of these critical upstream sequences and explore more deeply their role in keratinocyte-specific gene expression. Specifically, we have (i) identified the regions of the K14 gene that display altered chromatin structure under conditions where the gene is active but not when it is silent; (ii) elucidated which of these regions are responsible for the bulk of keratinocyte-specific gene activity in vitro and in transgenic mice in vivo; (iii) uncovered a correlation between conservation of K14 gene sequences and keratinocyte-specific alterations in chromatin structure; (iv) demonstrated that a 700-bp regulatory domain encompassing these sequences confers keratinocyte specificity when coupled to a heterologous promoter in vitro and in vivo; (v) showed that this region harbors functional binding sites for the AP-1, AP-2, and ets families of transcription factors, of which the AP-2 and ets sites are key; and (vi) showed that a segment containing functional AP-2 and ets sites can give rise to keratinocyte-preferred gene expression in vivo, whereas an analogous segment in which these sites are mutated cannot. Our findings suggest that basal keratinocyte-specific and differentiation-specific genes are governed in a combinatorial fashion by similar sets of transcription factor families, whose individual members are differentially expressed during terminal differentiation, thereby leading to switches in the expression of structural epidermal genes.

	MATERIALS AND METHODS

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Isolation of nuclei and DNase I HSs analysis. DNase I-hypersensitive sites (HSs) were identified as described by Bellard et al. (3). Cells were scraped from 10 100-cm² plates and subjected to centrifugation at 3,000 × g for 10 min. The pellet was resuspended in 10 ml of buffer A (15 mM Tris HCl, pH 7.5, 60 mM KCl, 15 mM NaCl, 1 mM EDTA [pH 8.0], 1.9 M sucrose, 0.1% Triton X-100, 0.5 mM spermidine), and cells were lysed in a Dounce homogenizer (10 to 15 strokes). After lysis, 10 ml of buffer B (buffer A without Triton X-100) was added, and the suspension was subjected to centrifugation at 12,000 × g for 10 min at 4°C. Nuclei were resuspended in 1.5 ml of buffer C (15 mM Tris HCl [pH 7.5], 60 mM KCl, 15 mM NaCl, 0.34 M sucrose, 0.5 mM spermidine) and then pelleted again in a tabletop centrifuge at 12,000 × g for 5 min. Final pellets were resuspended in a small volume of DNase I assay buffer (40 mM Tris HCl [pH 7.5], 6 mM MgCl₂). (All buffers also contained 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride [PMSF], 10 µg of leupeptin per ml, and 10 µg of pepstatin per ml, which were added fresh for each step.)

Cell culture and transient transfection assays. Human primary keratinocytes were grown in serum-free keratinocyte growth medium (Clonetics) and were split at passage 2 and/or 3 for all experimental purposes. mK, a spontaneously arising immortalized mouse keratinocyte cell line, was grown in low-Ca²⁺ medium (20). All other cells, including HepG2 (American Type Culture Collection, Manassas, Va.), NIH 3T3, and SCC-13 cells, primary human fibroblasts, and HeLa cells, were grown under standard conditions (5, 27). Transient transfections of all DNA constructs were carried out using FuGENE6 transfection reagent (Roche Molecular Biochemicals) according to the manufacturer's instructions. Cells were plated in six-well plates and transfected at 30 to 40% confluence. For each well, 2 µg of the luciferase reporter plasmid and 1 µg of a CMV-lacZ plasmid (internal control) were cotransfected to adjust for variations in the transfection efficiency. Cells were harvested 36 h after transfection, washed with phosphate-buffered saline (PBS), and lysed with luciferase reporter lysis buffer (Promega). Luciferase assays were performed with a luciferase assay system (Promega) using a luminometer. -Galactosidase values were obtained using a chemiluminescence reporter gene assay system (Galactolight kit; Tropix) according to the manufacturer's protocol. Luciferase activity was then normalized against -galactosidase values.

Generation of transgenic mice and -galactosidase staining. Transgenic mice were generated as before (27, 46). Mice were genotyped by PCR using two specific primers for the -galactosidase gene, and genomic DNA was isolated from tails or toes. Embryos from transgenic females were isolated at embryonic day 15.5 (E15.5) or E16.5, washed in PBS, and fixed in tissue fixation buffer (Specialty Media) for 30 min. Following several washes with PBS, embryos were stained with 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal). Duration of the staining varied from a few hours to overnight, depending on the strength of the signal. No staining was seen in wild-type control embryos. Following staining, the embryos were washed in PBS, postfixed in 4% paraformaldehyde, and embedded in paraffin. Tissues from adult mice were embedded in OCT (optimal cutting temperature) compound and sectioned (10 µm). Frozen sections were fixed in 0.5% glutaraldehyde for 2 min and assayed for -galactosidase activity. Sections were counterstained with eosin.

DNA constructs for transfection and transgenics. Recombinant plasmids were constructed using DNA fragments generated by restriction enzyme digestion or by PCR. Mutants were created by using a two-step PCR approach (45). All junctions or PCR-generated fragments were verified by sequencing. The thymidine kinase (TK) minimal promoter (5) was cloned into the NheI and XhoI sites of the luciferase reporter pGL3-basic vector (Promega) to create the LTK construct. The various fragments of the K14 promoter/enhancer regions were cloned into the KpnI-SacI or KpnI-NheI site of the LTK construct. A 700-bp DNA fragment of the K14 5' region (2000 to 1300) was cloned upstream of the simian virus 40 (SV40) minimal promoter in the pGL3-promoter vector (Promega) and upstream of the K14 minimal promoter (210 to +40) to create 700SV40Luc and 700K14mpLuc, respectively. To facilitate cloning, the vector pNASS (Clontech) was modified to contain several additional restriction enzymes sites. All constructs were then transferred from the LTK constructs using suitable restriction endonucleases to the modified pNASS to create the LTKLacZ plasmids.

Nuclear extract and EMSA. Nuclear extracts were prepared according to Schreiber et al. (39), with slight modifications. Protease inhibitors PMSF (1 mM), leupeptin (10 µg/ml), and pepstatin (10 µg/ml) were added freshly at the beginning of each step of extraction. Nuclear extracts for HeLa cells were purchased from Santa Cruz and Promega. Electrophoretic mobility shift assays (EMSAs) were performed with 4 to 6 µg of nuclear extracts and 3 × 10⁴ cpm of end-labeled double-stranded oligonucleotides. Typically, binding reactions were performed at room temperature for 20 min. All binding reactions were performed in 20 µl of DNA binding buffer (25 mM HEPES [pH 7.9], 75 mM KCl, 1 mM EDTA, 0.1% Nonidet P-40, 10% glycerol, 0.5 mM dithiothreitol, 0.5 mM PMSF); 1 µg of poly(dI-dC) was added to each reaction as nonspecific DNA. For supershift assays, 1 µl of the antibody was added to each reaction. All antibodies were purchased from Santa Cruz Biotechnology. For competition assays, 50- or 100-fold-excess cold oligonucleotides were incubated with each nuclear extract for 10 min before addition of radiolabeled probe. The protein-DNA complexes were resolved by gel electrophoresis on 5% polyacrylamide gels. After electrophoresis, gels were dried and visualized by autoradiography.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Identification of keratinocyte-specific DNase I HSs within the 5' upstream sequence of the human K14 gene. Previously we showed that 2.1 to 2.2 kb of 5' upstream sequence of the human K14 gene was sufficient to target expression of foreign genes to the basal layer of transgenic mouse epidermis (reference 46 and 47 and references therein). To uncover clues as to the possible cis elements needed for K14 gene expression, we began by analyzing its chromatin conformation in two primary diploid cell types: epidermal keratinocytes, which express copious amounts of K14, and fibroblasts, which do not express the K14 gene. Nuclei isolated from cultured human keratinocytes and fibroblasts were treated with increasing amounts of DNase I, and after extraction of proteins, genomic DNAs were further digested into defined fragments by restriction endonuclease treatment.

View larger version (70K): [in this window] [in a new window]   FIG. 1.   Keratinocyte-specific DNase I HSs in the K14 chromosomal locus. Nuclei from fibroblasts and keratinocytes were treated with increasing amounts of DNase I (ascending triangle; see Materials and Methods for details). Following treatment, DNAs were digested with BglII-SpeI (A) or BglII-EcoRV (B) and subjected to Southern blotting with the radiolabeled probe indicated in panel C. Arrowheads in panels A and B denote the relative positions of HSs. Molecular mass markers (in kilobases) are indicated at the left. Arrows in panel C denote approximate locations of HSs. The transcription initiation site (Tx start) is indicated. Also shown is a partial restriction map of the K14 gene and its 5' upstream regions: EcoRV (1000), HindIII (2200), BamHI (4000), and AflIII (5100). Note that HSsII was faint in panel A and visible clearly only upon longer exposures. HSsII and HSsIII are not well resolved in BglII-EcoRV digests (B).

The K14 promoter encompassing HSsI is dispensable for tissue-specific gene expression in transgenic mice. We next focused on ascertaining the relative contribution of the four DNase HSs to K14 gene activity. Since HSsIV resides at 2800, beyond the 2200 limit sequence necessary for keratinocyte-specific gene expression in transgenic mice (46), we limited the present study to HSsI, -II, and -III. Previously, Leask et al. found that the K14 minimal promoter (450 to +30), which encompasses HSsI, was not sufficient to provide epidermal-specific expression in keratinocytes or in mice (27). To determine if it was dispensable, we replaced it with the heterologous TK promoter, which on its own exhibits no expression in mice (reference 21 and references therein). The stick diagram in Fig. 2 illustrates 1850TKLacZ, the Lacz expression vector engineered for this test.

View larger version (99K): [in this window] [in a new window]   FIG. 2.   Expression of 1850TKLacZ in embryos and adult tissues. The 1850TKLacZ transgene contains the K14 5' upstream sequences (2200 to 350) linked to the heterologous TK minimal promoter. Embryos and skin sections from mice harboring this transgene were processed for -galactosidase activity using X-Gal cleavage as an assay. (A) E15.5 transgenic embryo displaying intense blue staining in skin; (B to F) skin or tongue sections from transgenic animals at ages indicated at the lower right. Tissue area examined is noted on each frame. BL, basal layer; Epi, epidermis; SG, sebaceous gland; ORS, outer root sheath.

A 700-bp enhancer encompassing HSsII and HSsIII is selectively active in keratinocytes. We next examined whether sequences encompassing HSsII and/or HSsIII might harbor clues to keratinocyte-specific gene expression. We began by performing transient transfection studies using K14 reporter gene constructs in cultured keratinocytes. We used three major changes over our past studies. First, recent improvements in gene transfection efficiencies enabled us to use primary keratinocytes, which express five to eight times the level of K14 and K5 proteins over the SCC-13 cell line previously used (25, 43). Second, we used the luciferase reporter gene, more sensitive than the chloramphenicol acetyltransferase gene previously employed. Finally, we used the TK promoter, enabling us to avoid consideration of elements in the K14 minimal promoter that we had already studied and which now appeared dispensable for keratinocyte specificity.

View larger version (26K): [in this window] [in a new window]   FIG. 3.   700TKLuc retains strong transcriptional activation in a keratinocyte-specific fashion in vitro (A) Diagram of the five initial luciferase constructs used for experiments shown in panels B to D. The partial restriction map of the K14 5' upstream region shown includes the AvaI (+1), NheI (350), EcoRV (1000), BbsI (1570), and HindIII (2200) sites and the approximate positions of HSsII and HSsIII. (B) Luciferase activity data from primary human keratinocytes transfected with constructs shown in panel A. The value for the TKLuc construct was low and was arbitrarily set at 1. (C) Activity of the 700-bp enhancer (2000 to 1300) in conjunction with different heterologous promoters. Activities of all the minimal promoters were low and set at 1. SV40, major early viral promoter; K14mp, K14 minimal promoter; TK, thymidine kinase minimal promoter. (D) Activity of 700TKLuc in different cell types and/or lines. hK, primary human keratinocytes; mK, immortalized mouse keratinocytes. In all transfection experiments, luciferase activities were corrected for -galactosidase values from an internal control reporter, CMV-lacZ. Note that -galactosidase activities were comparable in all lines tested. Activities represent the mean ± standard deviation of a minimum of three different experiments performed in duplicate.

Delineating the key elements within the 700-bp keratinocyte enhancer. To assess the relative importance of sequences within the 700-bp K14 enhancer element, 5' and 3' deletions were made within the keratinocyte enhancer of 700TKLuc (2000 to 1300), and the smaller segments were tested in human and mouse keratinocytes (Fig. 4). Even small (<100-bp) 3' deletions severely impaired its activity. These small deletions likely removed the sequences contributing to HSsII, which mapped to the 3' end of the enhancer. Larger deletions at this end had little or no additional effect, confirming the presence of key regulatory sequences localized to this end of the enhancer. In contrast, 240 bp could be removed from the 5' end without appreciable loss of K14 enhancer activity. Internal deletions revealed an additional regulatory element, corresponding to the 1760 to 1550 region mapping to HSsIII. The 1760 to 1325 segment had near full activity, whereas the 1500 to 1325 enhancer had less than 25% the activity of the full enhancer.

View larger version (29K): [in this window] [in a new window]   FIG. 4.   Delineating the active elements within the 700-bp K14 enhancer. 700TKLuc was the parental construct used to create all deletion constructs. The partial restriction map of the enhancer indicates approximate locations of HSsII and HSsIII. Each offspring construct is named based on its relative proportion of the 700-bp enhancer. 4×125 indicates four copies of 1450 to 1325 multimerized in a head-to-tail fashion; 4×210 indicates four copies of 1760 to 1550 multimerized in a head-to-tail fashion. Each plasmid was cotransfected with CMV-lacZ plasmid in order to normalize all values against -galactosidase control activity. Transfections were conducted with both primary human keratinocytes (hK) and mouse keratinocytes (mK). Cell extracts were subsequently assayed for luciferase and -galactosidase. Numbers represent the relative luciferase values ± standard deviations of at least three independent experiments. All values are given relative to TKLuc, containing the minimal TK promoter, assigned a value of 1.

The mouse K14 gene contains a DNase I HSs similar to HSsII, which displays significantly greater conservation than most other 5' upstream sequences. Cross-species comparison of cis elements provides a quick and powerful strategy to identify and confirm potential regulatory sequences. We therefore assessed whether the 5' upstream region of the mK14 gene (E. Fogarty and E. Fuchs, unpublished data) possesses keratinocyte-specific, DNase I HSs positioned similarly to those in the human gene. Nuclei from mouse keratinocytes were treated with DNase I, and isolated genomic DNAs were then digested with XbaI (2000) and BglII (600). Southern analysis with a radiolabeled BglII-HindIII (1200) probe identified two HSs located at approximately 1300 and 1700 (Fig. 5A). This corresponded well to the relative positions of HSsII and HSsIII of the human K14 gene. Similar to the human study, an HSs was also detected in the minimal promoter region (data not shown).

View larger version (33K): [in this window] [in a new window]   FIG. 5.   DNase I HSs mapping of the mouse K14 chromosomal locus and sequence comparisons of evolutionarily conserved HSs. (A) Following DNase I treatment of mouse keratinocyte nuclei, genomic DNAs were isolated, digested with XbaI-BglII, and subjected to Southern blot analysis with the radiolabeled probe indicated. Also shown are the relative positions of the two HSs (arrows) corresponding to HSsII and HSsIII of the human K14 gene and the restriction enzymes used to map the region. Molecular markers (in kilobases) are shown at left; the ascending triangle denotes increasing DNase I concentrations used in the assay. (B) Sequence alignment of the human (h) and mouse (m) HSsII regions. The sequence similarity is 80% over this stretch of DNA and 63% in the entire 700-bp enhancer element. Horizontal bars denote possible binding motifs for transcription factor family members.

Analysis of the elements within the 125-bp HSsII segment of the K14 enhancer: binding of keratinocyte nuclear proteins and their preliminary characterization. A computer analysis (35) of the 1325 to 1450 HSsII enhancer element revealed a number of putative sequence motifs for known classes of DNA binding proteins, including c-Myb, GATA, AP-1, AP-2, and ets family members (Fig. 5B). To explore whether these sites bind nuclear proteins, we conducted EMSAs using radiolabeled oligonucleotides corresponding to each of these sites in the human sequence and nuclear extracts from different cell types. Three oligonucleotides bound specific DNA-protein complexes and were chosen for further study.

View larger version (122K): [in this window] [in a new window]   FIG. 6.   EMSAs of nuclear extracts showing binding of AP-1 to a motif within the 125-bp HSsII K14 enhancer. (A) The wild-type oligonucleotide (WT) corresponds to the human sequence and is shown at the top, with the GATA and AP-1 sites underlined. Mutant oligonucleotides are shown below, with mutations in bold. M1, GATA mutant; M2, AP-1 mutant; M3, corresponding WT mouse sequence (Fig. 5B); AP-1 and Sp1 are consensus oligonucleotides for these two transcription factor family members and were purchased from Promega. (B) EMSAs were performed with radiolabeled WT oligonucleotide and nuclear extracts (4 to 6 mg) from the cell type indicated above each lane. Arrowhead and asterisk denote broadly expressed and keratinocyte-specific AP-1 complexes, respectively; note that all complexes within this mobility range were supershifted with AP-1 antibodies (D). (C) EMSAs were performed as for panel B, this time using radiolabeled WT oligonucleotide and a 100-fold excess of the unlabeled double-stranded oligonucleotide indicated above each lane. Note that the AP-1 complexes were specifically competed only by oligonucleotides encompassing an AP-1 binding site, whereas most other complexes were competed nonspecifically with all oligonucleotides. (D) EMSAs were performed as for panel B, but anti-c-Jun and anti-c-Fos were added to the complexes prior to gel electrophoresis. The arrow indicates supershifts, reflective of complexes between AP-1, DNA, and antibody.

View larger version (111K): [in this window] [in a new window]   FIG. 7.   EMSAs of nuclear extracts showing binding of ets family members to a motif within the K14 enhancer. (A) The wild-type oligonucleotide (WT-2) corresponding to the relevant sequence of the human enhancer is shown at the top, with the ets site underlined. The mutant oligonucleotides are shown below, indicating residues that were either deleted (TTCC in M4; dashed lines) or mutated (TTC>GAG; shown in bold in M5). M6, the WT oligonucleotide to the corresponding mouse sequence (Fig. 5B). (B) EMSAs were performed by incubating radiolabeled WT-2 oligonucleotide with nuclear extracts (4 to 6 µg) from the cell line indicated above each lane. The four DNA-protein complexes are denoted I, II, II, and IV (complex I was often faint). (C) EMSAs were performed as for panel B, this time adding a 50- or 100-fold excess of unlabeled competitor oligonucleotide as indicated above each lane. The arrow denotes complex IV, competed by human and mouse WT oligonucleotides. (D) EMSAs performed with radiolabeled WT-2, M4, and M5 oligonucleotides. Note that complex IV (arrow) is absent from the EMSAs conducted with mutants M4 and M5 (compare lane 1 with lanes 2 and 3).

View larger version (117K): [in this window] [in a new window]   FIG. 8.   EMSAs of nuclear extracts showing binding of AP-2 family members to a motif within the K14 enhancer. (A) The wild-type oligonucleotide (WT-3) corresponding to the relevant sequence of the human enhancer is shown at the top, with the AP-2 site underlined. An equivalent oligonucleotide (M7) mutated for the AP-2 site is shown below, with mutant residues in bold. AP-2, a consensus AP-2 oligonucleotide, purchased from Promega. (B) EMSAs were performed by incubating radiolabeled WT-3 oligonucleotide with either nuclear extracts (4 to 6 µg) from human hepatocytes (HepG2) or keratinocytes (hK) or recombinant AP-2 protein (rAP-2; Promega). The asterisk denotes an AP-2 complex, known to be absent in HepG2 cells (6). (C) EMSAs were performed as for panel B, this time adding a 50- or 100-fold excess of unlabeled competitor oligonucleotide as indicated by the triangle over each lane. (D) EMSAs performed with radiolabeled WT-3 as in panel B, this time adding AP-2 antibody to the complexes prior to gel electrophoresis. The arrow denotes supershifts.

Functional analysis of the sequences within the 125-bp K14 enhancer reveals a key role for the ets site. We next mutagenized sequences across the 125-bp K14 enhancer and assessed the consequences of these mutations to keratinocyte-specific enhancer function. The underlying rationale for our mutagenesis was based on (i) whether a specific sequence had been shown by EMSA to bind a protein from keratinocyte nuclear extracts and (ii) whether a specific sequence was highly conserved between mouse and human (Fig. 9). The mutants spanned the entire 125-bp segment, enabling us to broadly assess the importance of elements within the enhancer. All mutations were tested in the context of the 125-bp enhancer, which with the TK promoter was used to drive luciferase reporter gene expression. Wherever mutations were also tested in the context of the 700-bp enhancer, the effects of the mutations were similar with the two constructs (data not shown).

View larger version (32K): [in this window] [in a new window]   FIG. 9.   Functional analysis of the HSsII region. The 125-bp HSsII enhancer was scanned for (i) sequences conserved between mouse and human and (ii) putative binding motifs for any of the known transcription factor families. Based on this information, we engineered mutant 125-bp K14 enhancer constructs harboring mutations in one of the conserved sequences (Con Seq) within this region. Areas mutated are denoted by the circumscribed X's, with the mutated residues indicated above. Wild-type and mutant plasmids were transfected into human and mouse keratinocytes (hK and mK) along with a CMV-lacZ reporter construct, and luciferase activities were determined and normalized against the -galactosidase values. Each experiment was repeated at least three times, and data represent averages with standard deviations. The activities of the 700TKLuc (not shown) and 125TKLuc were set at 100%.

Analysis of the upstream enhancer in transgenic mice. To assess whether our fine mapping of the K14 enhancer element in vitro was relevant to keratinocyte-specific gene expression in vivo, we engineered a series of additional constructs, depicted in Fig. 10, and used these to generate transgenic mice. Both 1200TKLacZ and 700TKLacZ showed marked LacZ gene expression in the skin of mouse embryos (Fig. 11A to D). In both cases, expression was largely if not solely restricted to keratinocytes. Due to variability in transgene site integration, determining or comparing levels of expression was not possible. However, the percentages of mice expressing transgenes were similar for the 1850TKLacZ, 1200TKLacZ, and the 700TKLacZ animals (Fig. 10).

View larger version (17K): [in this window] [in a new window]   FIG. 10.   Summary of -galactosidase expression in mice and/or embryos harboring various TKLacZ transgenes. The nomenclature of the transgenes is described in the text. The HSs are denoted by arrows, and the presence of AP-1, ets and AP-2, sites is indicated to the right of each stick diagram. Transgenic embryos (Tgs) were scored as positive or negative after overnight incubation with X-Gal and visual inspection for blue staining. Embryos displaying transgene expression were sectioned to evaluate expression patterns in more detail. Wherever observed, ectopic expression was always in the CNS, cartilage, bone, and mesenchyme underlying the epidermis. Shown are the number of embryos and/or mice that expressed LacZ versus the total number of transgenic mice generated for each construct.

View larger version (88K): [in this window] [in a new window]   FIG. 11.   Expression of 1200TKLacZ, 700TKLacZ, and 125×4TKLacZ in embryos. Transgene expressed is indicated in the upper right of each frame, and animal age and tissue are indicated in the box on each frame. Animals were either processed intact for -galactosidase activity or sectioned prior to processing (for methods, see reference 6). Note that animal panel A was a mosaic, as denoted by the patchy striped pattern of X-Gal staining. The embryo in panel C shows stronger expression over the nose, limbs, and ears than other body sites; however, sectioning revealed skin-specific expression with no CNS or bone or cartilage staining. Note that embryo in panel E shows signs of ectopic expression in the brain and bone or cartilage. This was confirmed in embryo sections (not shown). Abbreviations: BL, basal layer; Epi, epidermis.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

An epidermal enhancer with keratinocyte-specific chromatin structure. We examined for the first time the relation between keratinocyte-specific changes in chromatin structure, specific functional regulatory sequences, and transcription factor family members that bind to these sequences. DNase I HS mapping has long provided a means of detecting perturbations in nucleosome arrangement that typically arise when nonhistone proteins bind to DNA upon transcriptional activation (9, 14). Our DNase I HSs mapping led to identification of a novel 700-bp segment encompassing two HSs, HSsII and HSsIII, which together are sufficient to confer epidermis-specific gene expression in transgenic mice in vivo and in primary keratinocytes in vitro. The importance of this segment had not been revealed from earlier mutagenesis and transfection studies, presumably due to the lack of sensitivity in measuring K14 transcriptional activity in SCC-13 cells and the absence of a systematic procedure to identify potentially key regulatory domains.

Specific roles of HSsI, HSsII, and HSsIII in epidermal specificity and in differentiation-specific gene expression in vitro and in vivo. By replacing the K14 minimal promoter encompassing HSsI with one of several generic promoters, we have discovered that it plays a role in differentiation-specific regulation of gene expression in vivo and in vitro. Since the heterologous TK promoter otherwise functioned equivalently, the K14 minimal promoter is not required for epidermis-specific gene expression.

Functional elements of the HSsII enhancer. A number of other studies have implicated AP-1, AP-2, and ets family members in regulating the expression of viral and endogenous genes in various cultured keratinocyte cell lines (4, 10, 11, 24, 25, 26, 28, 41, 42, 49). Curiously, the genes studied in those reports include both basal and differentiation-specific epidermal genes. Our findings here establish a role for these factors in epidermis-specific gene expression in vivo. Our most persuasive evidence was the near quantitative loss of keratinocyte enhancer activity when all three sites were mutated and, conversely, near full enhancer activity when a 125-bp fragment encompassing these three motifs was multimerized. Additionally, our studies reveal an interesting correlation between keratinocyte-specific changes in chromatin structure and a DNA segment containing the functional AP-1, AP-2, and ets sites. Finally, our data strengthen the view that epidermis-specific gene expression is governed by multiple transcriptional regulatory sequences and factors.

Summary. We have identified structural differences in chromatin that exist within the locus of an epidermally expressed gene in primary keratinocytes but not in other cell types in vitro. Such differences provided a powerful method for identifying an epidermis-specific enhancer that is both necessary and sufficient for keratinocyte-specific gene expression. The enhancer contains AP-2, AP-1, and ets sites that all contribute to epidermis-specific and/or differentiation-specific transcriptional activity in keratinocytes in vitro and in vivo. No one factor or element is on its own sufficient for the faithful restriction of transcription to the epidermis. Rather, these regulatory sequences act combinatorially to confer keratinocyte-specific expression. In turn, it is the specific factors that bind to these sequences, or the specific context of the sequence motifs themselves, that influence the differential activity of these genes during the terminal differentiation process. Of the factors involved, our studies illuminate the importance of ets, AP-2, and AP-1 in conferring preferential epidermis expression both in vitro and in vivo. This study now paves the way for future studies aimed at elucidating the specific family members and the intricacies of the underlying mechanisms involved in this process.