A distal region of the human TGM1 promoter is required for expression in transgenic mice and cultured keratinocytes
© Phillips et al; licensee BioMed Central Ltd. 2004
Received: 28 September 2003
Accepted: 05 April 2004
Published: 05 April 2004
TGM1(transglutaminase 1) is an enzyme that crosslinks the cornified envelope of mature keratinocytes. Appropriate expression of the TGM1 gene is crucial for proper keratinocyte function as inactivating mutations lead to the debilitating skin disease, lamellar ichthyosis. TGM1 is also expressed in squamous metaplasia, a consequence in some epithelia of vitamin A deficiency or toxic insult that can lead to neoplasia. An understanding of the regulation of this gene in normal and abnormal differentiation states may contribute to better disease diagnosis and treatment.
In vivo requirements for expression of the TGM1 gene were studied by fusing various lengths of promoter DNA to a reporter and injecting the DNA into mouse embryos to generate transgenic animals. Expression of the reporter was ascertained by Western blotting and immunohistochemistry. Further delineation of a transcriptionally important distal region was determined by transfections of progressively shortened or mutated promoter DNA into cultured keratinocytes.
In vivo analysis of a reporter transgene driven by the TGM1 promoter revealed that 1.6 kilobases, but not 1.1 kilobases, of DNA was sufficient to confer tissue-specific and cell layer-specific expression. This same region was responsible for reporter expression in tissues undergoing squamous metaplasia as a response to vitamin A deprivation. Mutation of a distal promoter AP1 site or proximal promoter CRE site, both identified as important transcriptional elements in transfection assays, did not prevent appropriate expression. Further searching for transcriptional elements using electrophoretic mobility shift (EMSA) and transfection assays in cultured keratinocytes identified two Sp1 elements in a transcriptionally active region between -1.6 and -1.4 kilobases. While mutation of either Sp1 site or the AP1 site singly had only a small effect, mutation of all three sites eliminated nearly all the transcriptional activity.
A distal region of the TGM1 gene promoter, containing AP1 and Sp1 binding sites, is evolutionarily conserved and responsible for high level expression in transgenic mice and in transfected keratinocyte cultures.
Transglutaminases, including the product of the TGM1 gene, catalyze formation of ε-(γ-glutamyl)-lysine crosslinks in proteins and thereby stabilize biological structures . In epidermis, TGM1 is required for the formation of the cross-linked envelope. Point mutations in the gene that cause deficits in enzyme activity can give rise to lamellar ichthyosis. [2–5], a disease characterized by lack of a normal barrier to dehydration . Further analysis of the promoter may assist in evaluating cases where promoter sequence alterations are suspected to yield defective TGM1 expression [7, 8].
TGM1 is normally expressed in the suprabasal cells of stratifying epithelia such as epidermis, the upper digestive tract, the female lower genital tract and in the endometrial epithelium late in pregnancy . It is also expressed as a result of squamous metaplasia in the trachea induced by vitamin A deprivation  and in a number of epithelial cell types, including those from bladder and endometrium, induced by culture on plastic . Transgenes incorporating 2.9 kb of the rabbit  or 2.5 kb of the human  TGM1 promoters have been shown to exhibit appropriate tissue-specific and cell layer-specific expression in mice. Transfection experiments in cultured human, rabbit and rat keratinocytes have identified regions that are important for high transcriptional activity [12, 14, 15]. One of two major regions, at -1.5 kb in the distal promoter, contains a consensus AP1 binding site, and the other region, in the proximal promoter at -0.45 kb, contains a CRE-like binding site. In this study we assessed the in vivo activities of these regions of the TGM1 promoter in transgenic mice fed a normal diet and after vitamin A deprivation that induced squamous metaplasia.
The TGM1 promoter/human involucrin reporter fusion genes used to generate transgenic mice were made by first cutting the human involucrin genomic clone pλI-3H6B  with HinCII, which cleaves the gene within the second exon at 15 bp 5' to the translation start site. The DNA was ligated to a Bgl II linker and subsequently cut with Bgl II and BamH I to excise a 2.5 kb piece of DNA containing the entire involucrin coding region. This DNA was gel purified and subcloned into the Bgl II and BamH I sites of the pGL3 Basic vector (Promega) to generate the reporter plasmid, pINV, with involucrin coding sequence substituted for luciferase. The TGM1 promoter was amplified with Pfu polymerase (Stratagene) using human TGM1 clone TGI  as template and an upstream primer, 5' base at -2200 (containing an untemplated Sal I restriction site) and a downstream primer, 3' base at +67 (containing an untemplated Bgl II restriction site). The promoter PCR product was completely sequenced to verify that no errors had been introduced by PCR, then cut with Sal I and Bgl II and ligated to pINV which had been cut with Xho I and Bgl II. Transgenes were excised from the plasmid by cutting with Kpn I/Sal I, Spe I/Sal I or Hind III/Sal I to generate TG2.2, TG1.1 and TG0.3, respectively. TG1.6 was constructed by substituting a sequence verified KpnI/EcoRV restriction fragment from TG1.6 in pGL3 (described below) for the KpnI/EcoRV fragment of the TG2.2 transgene. TG2.2/AP1m and TG/2.2CREm with mutated AP1 and CRE sites, respectively, were made by subcloning restriction fragments containing the mutations (mutagenesis described in ) into TG2.2. The regions containing the subcloned restriction fragments were sequence verified. DNA for microinjection was purified by agarose gel electrophoresis and Schleicher and Schuell Elutip columns. Transgenic mice were created using standard pronuclear injection techniques. Multiple founders were obtained for each transgene construct and all resulting progeny were maintained in a purebred FVB strain. The F1 or later generations were used for analysis.
Presence of the transgene was detected by PCR amplification of a portion of the chimeric sequences from mouse genomic DNA. The mouse DNA was prepared by a standard method . Briefly, approximately 5 mm of mouse tail biopsy was digested overnight at 55°C in lysis buffer (50 mM Tris, pH 8.0, 100 mM EDTA, 0.125% SDS, 1 mg/ml proteinase K). Samples were extracted twice with phenol/chloroform (1:1), once with chloroform and precipitated with ethanol. Each sample was reconstituted in 200 μl of 10 mM Tris buffer (pH 8) containing 1 mM EDTA resulting in a final DNA concentration of 50 to 150 ng/μl. One μl of genomic DNA was used as the template for PCR. Primers were designed to anneal to the TGM1 promoter at -0.2 kb (forward primer 5'-GGTGCCAGGGGCCATCACAG) and to the 5' end of the involucrin coding region (reverse primer 5'-GGCATGGGGGAGGCAGTGG), resulting in a 460 bp product, identified by agarose gel electrophoresis.
Tissues were homogenized in 0.5 ml of lysis buffer (20 mM Tris, pH 7.5, 20 mM EDTA, 0.1% SDS) in a ground glass homogenizer. After centrifugation for 15 min at 10,000 × g at 4°C, the supernatant was collected and stored at -80°C. Protein concentration was determined using Coomassie G-250 (Biorad) , and 5 μg of protein from each tissue were separated by SDS polyacrylamide gel electrophoresis. Proteins were electrophoretically transferred to a nitrocellulose membrane using a semi-dry blotter (Biorad). The human involucrin reporter was detected using rabbit anti-human involucrin antiserum and ECL Plus chemiluminescence kit (Amersham).
Mouse tissues were fixed in phosphate buffered saline containing 10% formalin, embedded in paraffin and sectioned. The sections were incubated with rabbit anti-human involucrin antiserum, followed by biotin-labeled anti-rabbit antibody, then streptavidin linked to horseradish peroxidase. Sections were stained with 3,3'-diaminobenzidine (Biorad)  and counter-stained with hematoxylin. Two to five independent founder lines were examined for each construct. Use of human involucrin as a reporter is a departure from the commonly used β-galactosidase, luciferase, or green fluorescent protein. This protein was chosen since very specific antibodies to it are available . The antiserum recognized the antigen in fixed, paraffin-embedded tissues, did not cross-react with the mouse involucrin protein, and reacted with a single protein in western blotting. Immunohistochemistry showed that, like endogenous transglutaminase 1 protein , the involucrin reporter is expressed in the suprabasal layers of the epidermis and increased as the cells moved toward the surface. The observed staining pattern resembled that of involucrin in human epidermis in that the cornified layers did not stain well, presumably because most of the involucrin immunoreactivity was masked by cross-linking.
The keratinocyte lines used were normal human epidermal cells (hEp) derived from foreskin, the spontaneously immortalized SIK line, derived from human foreskin epidermis , the human squamous cell carcinoma line SCC9 , the rEp line derived from rat epidermis , and the rB line from rat bladder epithelium . Cells were cultured in the presence of a lethally irradiated 3T3 feeder layer  using a 3:1 mixture of Dulbecco's modified Eagle's and Ham's F-12 media, supplemented with 5% fetal bovine serum, 0.4 μg/ml cortisol, 5 μg/ml insulin and transferrin, 20 pM triiodothyronine and 0.18 mM adenine . Medium for hEp and SIK cultures was also supplemented with 10 ng/ml cholera toxin upon inoculation and 10 ng/ml epidermal growth factor starting at the first medium change until confluence. All cell lines express easily detectable levels of TGM1 mRNA on Northern blots ( and our unpublished results).
Cells were inoculated in 12-well plates and transfected two to four days later, when the small colonies covered about 50% of the surface of the wells. SIK, hEp and rEp cultures were transfected with calcium phosphate-DNA coprecipitates  using 2.5 μg of reporter DNA, 2.5 μg of sonicated salmon sperm DNA and 1 ng of pRL-cytomegalovirus (Promega) per well. The precipitates were incubated with the cells overnight, then the medium was changed. Cell extracts were prepared four days later and stored frozen. The rB cell line was transfected using Fugene reagent (Roche) according to the manufacturer's protocol with 1.5 μl Fugene reagent, 0.5 μg of reporter DNA and 10 ng of pRL-cytomegalovirus per well in 1 ml of serum free DMEM. After 3–4 hours the medium was replaced with growth medium. After two days, the cells were harvested.
The reporter DNAs contained pieces of the TGM1 promoter which were amplified by PCR using primers with added restriction sites at the 5' end, and cloned into the pGL3 vector (Promega). All terminated at the 3' end at +67 relative to the transcription start site. Internal deletions were made by fusing PCR products of different amplified regions of the promoter to a TGM1 minimal promoter (-90 to +67). Promoters containing mutated transcription factor binding sites were made using the Stratagene QuikChange Site-directed Mutagenesis kit. Mutated sites were identified by sequencing and the promoters were excised and subcloned back into the pGL3 vector. Firefly luciferase (driven by the TGM1 promoter) and Renilla luciferase (used to normalize for differing transfection efficiencies) were assayed with the dual luciferase kit from Promega.
Electrophoretic mobility shift assay (EMSA)
Nuclear extracts were prepared by minor modification of a standard method [29, 30]. Probes were made by labeling annealed oligonucleotides containing a single added 5' G overhang with the Klenow fragment of DNA polymerase I (New England Biolabs) in the presence of 32P dCTP (Perkin Elmer) or, for the consensus Sp1 site (Stratagene), by end-labelling with 32P ATP and T4 polynucleotide kinase. Binding reactions (20 μl) contained 5–10 μg nuclear extract protein and 500 ng of poly [d(I-C)-poly(dI-C)] in a solution of 12.5 mM HEPES, pH 7.9 (at 4°C); 0.2 mM EDTA; 0.01% NP-40; 0.5 mM dithiothreitol; 100 mM NaCl and 10% glycerol. After a 10 minute incubation at room temperature, 10 fmol of labeled probe was added and incubation was continued for 10 minutes. DNA protein complexes were separated on 4% polyacrylamide gels in 0.5X TBE buffer (final concentrations: 22.5 mM Tris-borate, 0.5 mM EDTA). In some experiments competitor oligonucleotides were added at 100 fold molar excess. Antibody competition for Sp1 and Sp3 binding was done by adding 1 or 2 μg of antibody (Santa Cruz, sc-59X or sc-644) to binding reactions, followed by incubation on ice for 1 hr before probe addition.
TGM1 promoter activity in transgenic mice
Summary of tissue reactivity with reporter antibody on western blots1
Endometrial epithelium of the uterus has been shown to express TGM1 late in pregnancy in the rat . This was also observed for reporter expression in the transgenic mice. The general promoter regions required for endometrial expression were the same as those required for expression in stratified squamous epithelia (2.2 and 1.6 kb, but not shorter promoters).
TGM1 promoter in squamous metaplasia
Delineating regions of TGM1 distal promoter required for transcriptional activity
Comparison of TGM1 promoter activities in various cultured keratinocytes
Cell Line Transfected
112 ± 26
86 ± 24
85 ± 19
85 ± 18
46 ± 6
27 ± 8
59 ± 7
56 ± 17
4 ± 4
7 ± 6
12 ± 1
9 ± 5
54 ± 1
81 ± 12
49 ± 17
82 ± 5
51 ± 6
55 ± 13
69 ± 37
15 ± 11
7 ± 9
6 ± 4
Detection of DNA binding proteins by EMSA
Mutational analysis of Sp1-like sites
Binding of proteins to TGM1 promoter Sp1-like elements during differentiation in culture
Completion of the human genome sequence has generated renewed interest in determining which elements in the vast stretches of intergenic DNA are important for regulation of transcription. So far this is still not possible to predict based on sequence alone. Rather, experiments are required to test the abilities of different regions of gene promoters to regulate transcription appropriately. Common patterns may then emerge to explain such features as cell-specific expression. Studying the regulation of genes expressed specifically in keratinocytes has led to the identification of several transcription factors commonly associated with this process . As yet, no predictable patterns have emerged, necessitating the continued use of a gene by gene approach for the elucidation of keratinocyte-specific gene regulatory regions.
We have concentrated on regulation of the TGM1 gene in keratinocyte cultures and in transgenic mice. While our work was in progress, reports appeared that a 2.5 kb region upstream of the human TGM1 coding region was sufficient to confer appropriate expression of a linked reporter in transgenic mice  and a 2.9 kb upstream region of the rabbit TGM1 gene acted similarly . Our further studies have narrowed the minimum length of necessary promoter DNA to between 1.6 and 1.1 kb upstream of the transcription start site. That the required promoter region is rather short probably reflects the close proximity of the upstream Rab geranylgeranyl transferase gene [35, 36]. Alignments of human, rabbit, rat and mouse promoters show several regions that are well conserved. These include regions containing previously identified CRE [12, 14] and AP1  sites. Mutation of either of these sites to sequences shown in vitro to destroy DNA binding activity did not prevent appropriate expression of the reporter gene in most of the founder lines harboring the mutations. This finding suggests that, although these sites contribute to transcription of the TGM1 gene in transfection assays with cultured cells [12, 14], there must be compensatory mechanisms for their loss. This is in contrast to the loss of expression due to mutation of AP1 sites in the loricrin  and involucrin  gene promoters. Although some transgenic founder lines harboring the mutated TGM1 promoters exhibited correct expression patterns, this was not the case for all of the founder lines and is in contrast to the TG2.2 mice in which high levels of tissue specific expression were seen in 6 of 6 founder lines. Only 1 of 3 TG2.2AP1mut founder lines exhibited high levels of reporter expression, leaving open the possibility that this element contributes to transcriptional efficiency of the promoter. This is difficult to conclude decisively in transgenic animals due to the contribution of the integration site to transcription levels even when the copy number inserted is known and rearrangements are not evident. The CRE mutation, on the other hand, did not appear to affect levels of transcription, although low levels of ectopic expression were noted in the mutants. Therefore, that element may have a role in preventing gene expression in tissues where it is not normally expressed.
TGM1 is expressed normally during the process of squamous differentiation [9, 39] and in addition is one of the markers associated with squamous metaplasia, a process that occurs in several tissues as a result of pathological processes, including vitamin A deficiency . Since sites of metaplasia in regions of toxic insult can progress to neoplasias (e.g., in the upper airways as a result of smoking), understanding of the metaplastic process may assist in cancer prevention, diagnosis and treatment. In addition to identifying genes that are induced during squamous metaplasia, we are now in a position to determine the transcriptional mechanisms that lead to adoption of the metaplastic phenotype. As a beginning, we asked whether the same TGM1 promoter elements that drive reporter expression in normal stratified epithelia of transgenic mice would be sufficient for expression in metaplastic tissue as well. The results suggest that the regulation of the metaplastic process involves use of the same response elements and perhaps even induction of the same transcription factors that are present in normal stratified tissues. This was not necessarily predictable as the process might have occurred using different transcription factors acting at distinct promoter elements.
The importance of the human TGM1 promoter region from -1.6 to -1.1 kb for expression in transgenic mice led us to focus on identifying transcription factors that bind to this region. This is most easily done by first using transfections of cultured cells, then re-testing more precisely defined promoters in transgenic mice. The availability of cell culture models that exhibit many of the features of the normal differentiation process is essential for this work. In this case we have primarily used a spontaneously immortalized human keratinocyte line (SIK) and a line of keratinocytes derived from rat bladder epithelium (rB). The latter have the advantage that they are easily transfected and express high levels of reporter activity. The major conclusions are the same for both cell lines: (1) we have identified a 200 bp region, from -1.6 to -1.4 that is required for 40 to 100% of the activity of the 1.6 kb promoter in rB and SIK cells, respectively; and (2) mutational analysis showed that AP1 and Sp1 binding sites account for most of the transcriptional activity of that region. Some quantitative differences were noted in the effects of certain deletions and mutations in the two cell lines, e.g. mutation of the TG-A site suppresses activity in rB cells but not in the SIK line (Fig 7); however, these differences do not affect our overall conclusions.
AP1 and Sp1 family transcription factor binding sites are commonly found in keratinocyte-specific genes. Since members of these transcription factor families are widely expressed, it is not yet understood how keratinocyte-specific expression is achieved, although it should be noted that keratinocytes have unusually high levels of both factors and that overexpression of Sp1 in fibroblasts reportedly activates expression of a transfected involucrin promoter . Furthermore, we found that binding of keratinocyte nuclear proteins to TGM1 Sp1-like binding sites increased as the cells progressed in their differentiation program with time in culture, as might be expected if the abundance of these proteins is critical for initiating TGM1 transcription. This was most dramatic in SCC9 cells, which exhibit a strong induction of differentiation markers (including TGM1) at confluence ( and earlier work). Our results agree with others who have observed sustained or increased Sp1/Sp3 binding activity upon differentiation of keratinocytes derived from mouse epidermis, rabbit cornea and the human epidermal-derived HaCat cell line [41–46]. Specificity of gene expression may lie in the arrangement of sites or in cooperation with other as yet unidentified keratinocyte-specific factors. It may be noteworthy that, although various computer algorithms for predicting transcription factor binding sites identified the AP1 and Sp1 sites, other regions of sequence conservation were not identified as binding sites for known factors and remain to be explored as potential response elements for keratinocyte-specific factors.
Alignment of TGM1 promoters from four species also points to the -1.6 to -1.4 kb region as potentially important for transcriptional regulation since much of the sequence in that region is conserved. However, an important difference in the location of Sp1 binding sites between rodents and human and rabbit is evident. The distal Sp1 site has been eliminated in the rodent lineage and the location of the proximal site is slightly shifted. The importance of these differences has yet to be explored, but may contribute to differences we have seen in some aspects of regulation of the endogenous TGM1 gene in human and rat cell lines (BAJ, unpublished data).
The distal region of the TGM1 gene promoter was shown to be required for appropriate expression of a linked reporter in transgenic mice both in normal squamous tissues and in squamous metaplasia. A 200 bp region from -1.6 to -1.4 kb upstream of the transcription start site was determined to be responsible for a majority of the transcriptional activity of the promoter in transfection assays and is largely conserved in sequence across four mammalian species. This region contains AP1 and Sp1 transcription factor binding sites, and mutation of all three sites destroys nearly all the transcriptional activity.
We thank the UC Davis Targeted Genomics Lab for preparing some transgenic mice, Dr. Charles Stephensen for advice on vitamin A deprivation and Ms. Lauren Stevens for assisting in mouse handling. This research was supported by USPHS grants R01 AR27130, T32 ES07059, P42 ES04699 and P30 ES05707.
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