©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Isolation and Characterization of the Human Tissue Transglutaminase Gene Promoter (*)

Shan Lu , Margaret Saydak , Vittorio Gentile , Joseph P. Stein (1), Peter J. A. Davies (§)

From the (1) Department of Pharmacology, University of Texas Medical School, Houston, Texas 77225 Department of Pharmacology, State University of New York, Syracuse, New York 13210

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Tissue transglutaminase belongs to a family of calcium-dependent enzymes, the transglutaminases that catalyze the covalent cross-linking of specific proteins by the formation of (-glutamyl)lysine isopeptide bonds. The goal of this study has been the isolation and characterization of the human tissue transglutaminase gene promoter. Genomic DNA clones, spanning the 5` region of the gene, were isolated and the structure of the 5`-end of the human tissue transglutaminase gene was determined. 1.74 kilobases of flanking DNA were sequenced and were found to contain a TATA box element (TATAA), a CAAT box element (GGACAAT), a series of potential transcription factor-binding sites (AP1, SP1, interleukin-6 response element), and a glucocorticoid response elements. Transient transfection experiments showed that this DNA fragment included a functional promoter, which is constitutively active in multiple cell types.


INTRODUCTION

Transglutaminases are enzymes that catalyze the covalent cross-linking of proteins by promoting the formation of -(-glutamyl)-lysine isopeptide bonds between selected protein-bound glutamine and lysine residue (1, 2, 3) . Transglutaminase activity has been detected in a variety of tissues and body fluids. It was originally thought that this activity was due to a single transglutaminase enzyme, but it is now clear that transglutaminase activity can be due to a family of related but distinct enzymes differing in their pattern of expression, their substrate specificity, and their physiological regulation (4) . Some transglutaminases, such as Factor XIII and seminal plasma transglutaminase, are extracellular enzymes involved in the cross-linking of aggregated plasma proteins. Other transglutaminases, such as keratinocyte and epidermal transglutaminases, are intracellular enzymes involved in cross-linking of intracellular proteins during the terminal differentiation and cornification of skin cells (5, 6) .

Tissue transglutaminase is a member of this multigene family that is involved in the cross-linking of both intracellular and extracellular proteins. Tissue transglutaminase can be secreted from cells and accumulated in the extracellular matrix (7, 8, 9, 10, 11) . Although the physiological functions of this secreted tissue transglutaminase are not well understood, it may be involved in the specialized processing of the matrix that occurs during bone formation (12) , wound healing (13) , and other remodeling processes. Tissue transglutaminase also appears to be involved in the cross-linking of intracellular protein. Fesus and Thomazy (14) have shown that tissue transglutaminase accumulates in cells undergoing apoptotic cell death. Activation of the enzyme during apoptosis causes the cross-linking of intracellular proteins that, in some cells, may be an integral part of the apoptotic program (15) .

Our laboratory has been interested in the factors that regulate the expression of tissue transglutaminase especially during the process of programmed cell death. Previous studies have demonstrated that retinoids act as direct regulators of tissue transglutaminase gene expression, an effect frequently potentiated by cAMP (16, 17) . Jetten et al. (18) have reported that transforming growth factor- also can induce transglutaminase expression in epithelial cells, and Ikura et al. (19) have shown that IL-6() also induces tissue transglutaminase expression. In order to elucidate the molecular mechanisms that regulate tissue transglutaminase expression, we have initiated studies to establish the structure and function of the human tissue transglutaminase gene. In this paper, we report on the isolation and characterization of 1.74 kb of DNA flanking the 5`-end of this gene. We have demonstrated that this flanking DNA includes a functional promoter with SP1 sites and a CAAT box element that account for its constitutive activity in transient transfection assay.


EXPERIMENTAL PROCEDURES

Cell Lines and Cell Culture

MCF-7 (human breast cancer), SW13 cells (human adrenal adenocarcinoma), and Hela cells were maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (HyClone Laboratories Inc., Logan, UT) at 37 °C in 5% COincubator. 3T3 cells, a Balb/c 3T3 cell line stably transfected with mouse retinoic acid receptor (RAR), were cultured in DMEM supplemented with 10% fetal bovine serum, 5% serum plus(JRH Biosciences, Lenexa, KS), and 200 µg/ml G418 (Life Technologies, Inc.).

Plasmids

Luciferase vectors pXP1-Luc, pXP2-Luc, and pSV2-AL-Luc were kindly provided by Dr. Debbie Wilson, Dept. of Pathology, Texas Children Hospital, Houston (20) .

Screening of Human Tissue Transglutaminase Genomic DNA

An amplified human placental genomic DNA library in Lambda Fixvector (Stratagene Inc., La Jolla, CA) was screened with a 440-bp radiolabeled cDNA probe derived from 5`-end of a human tissue transglutaminase cDNA (hTg-1). Escherichia coli cells (LE392) were infected with recombinant phage, and 1.2 10phage plaques were screened. Thirteen clones were obtained after tertiary screening and rescreened with three oligonucleotide probes whose sequences were derived from different segments of the 5` region of the tissue transglutaminase cDNA. Southern blot analysis of the genomic library by both cDNA and oligonucleotide probes was performed according to the previously described methods. Restriction enzyme mapping of transglutaminase genomic DNA clones was carried out using standard procedures (21) .

DNA Sequence Analysis

DNA flanking the human tissue transglutaminase gene was subcloned into a pSuperlinker 301 vector (Invitrogen Corp., San Diego, CA) for sequencing. Plasmids were sequenced with either T7 or T3 oligonucleotide primers using an automated Taq DyeDeoxyTerminator Cycling sequencing method (Applied Biosystems, Inc., Foster City, CA). Overlapping sequences from 29 fragments of DNA were aligned using the GCG Gelassembly sequence analysis program.

Construction of Tissue Transglutaminase-Luciferase Chimeric Genes

A 4-kb HindIII fragment that included 1.74 kb of DNA upstream of the translation start site of the human tissue transglutaminase gene was subcloned into the HindIII site of both pXP1-Luc and pXP2-Luc vectors. The segment of the human tissue transglutaminase genomic DNA downstream of the translation start site was then deleted by excising a 2.2-kb NcoI/ SalI fragment from both pXP1-Tg-Luc and pXP2-Tg-Luc. These vectors were religated to give constructs HTGP1-Luc and HTGP2-Luc, in which 1.74 kb of the human tissue transglutaminase were inserted in sense (HTGP2-Luc) or antisense (HTGP1-Luc) orientation upstream of the luciferase gene. The deletion mutation constructs HTGP2-mut2-Luc, HTGP2-mut3-Luc, HTGP2-mut5-Luc, HTGP2-mut6-Luc, HTGP2-mut7-Luc, pXP2-TG-Luc, and pXP2-CAAT-Luc were generated by deleting specific DNA fragments according to the restriction map shown in Fig. 5 A. The construct pXP2-5P1-Luc was made by subcloning the PCR fragment spanning the human tissue transglutaminase sequence 40 to +59 bp into the pXP2-Luc vector. The construct pXP2-NF1-Luc was made by subcloning a 39-mer oligonucleotide spanning the human tissue transglutaminase gene 32 to +5 bp into the pXP2-Luc vector.


Figure 5: Functional analysis of the human tissue transglutaminase promoter activity. The recombinant luciferase reporter constructs used in the promoter analysis were shown on the left panel. The luciferase activity was determined by transient transfection assay. 3T3 cells were transfected with 1.5 µg of pSV2-AL-Luc, HTGP1-Luc, HTGP2-Luc, HTGP2-mut2-Luc, HTGP2-mut3-Luc, HTGP2-mut5-Luc, HTGP2-mut6-Luc, and HTGP2-mut7-Luc vector DNA for 48 h, followed by luciferase assay as described under ``Experimental Procedures.''



Transient Transfection Assay

Lipofectin-mediated transfection was used for the transient transfection assays. Lipofectin reagentDNA complex was prepared according to the protocol provided by Life Technologies, Inc. Briefly, plasmid DNA in serum-free DMEM at 10 µg/ml was mixed with the same volume of Lipofectin reagent solution (100 µl/ml Lipofectin reagent in serum-free DMEM). The mixture was incubated at room temperature for 10-15 min allowing the formation of Lipofectin reagentDNA complex. The complex was then mixed with fresh serum-free DMEM at 1:4 (v/v) and was used immediately.

Cells to be transfected were plated in a 35-mm tissue culture dish at a density of 2 10cells/plate in 2 ml of DMEM supplemented with serum. Twenty-four h later, the cells were washed twice with 2 ml of serum-free DMEM. The cells were then incubated with the Lipofectin reagentDNA complex (0.2 ml/plate) for 10 h at 37 °C in 5% CO. The reaction was stopped by replacing the DNA complex-containing DMEM with DMEM containing 10% fetal calf serum and 5% serum plus.

Cell Extract Preparation

Cells in 35-mm plates were washed twice with PBS. After 5 min of incubation with cell lysis buffer (25 mM Tris-phosphate, pH 7.8, 2 mM dithiothreitol, 2 mM EDTA, 10% glycerol, 1% Triton X-100), the cells were scraped with a rubber policeman. The samples were then centrifuged at 4 °C for 5 min, and the supernatants were transferred to fresh vials. The protein concentration of each sample was determined before the assay of luciferase activity.

Luciferase Assay and -Gal Assay

Luciferase assays were performed in a Monolight 2010 Luminometer (Analytical Luminescence Laboratory, San Diego, CA). For each assay, cell extract (40 µl) was added into a cuvette, and the reaction started by injection of 200 µl of substrate buffer (27 mM KHPO/KHPOpH 7.8, 42 mM MgSO, 11.2 mM EDTA, 70 mM glycylglycine, 4 mM dithiothreitol, 3.6 mM ATP, 0.4 mM Luciferin) to the cuvette. Each reaction was measured for 10 s in the Luminometer. Luciferase activity was defined as light units/mg protein. -Gal activity was determined using o-nitrophenyl--Dgalactopyranoside as substrate. The reaction was measured at 420 nm according to a standardized protocol (Promega).


RESULTS

Isolation of the Human Tissue Transglutaminase Gene

A 440-bp cDNA probe was used to screen a human placental genomic DNA library. The 440 EcoRI/ PstI fragment was purified from the 5`-end cDNA of the human tissue transglutaminase clone hTg-1 (22) and was labeled by random-prime labeling method. Thirteen clones were obtained after screening 2 10phage plaques. Oligonucleotide probe AS-140, spanning the translation start site, of the human tissue transglutaminase gene, was used to rescreen the positive clones. We found that all the clones hybridized to the oligonucleotide probe, suggesting that the clones span the 5`-end of the human tissue transglutaminase gene. Several of these clones were subjected to more detailed analysis. Fig. 1 shows the restriction map of a 17-kb XhoI fragment of human genomic DNA. Oligo AS-200 is located 60 bp downstream of the initiator ATG codon in the human tissue transglutaminase cDNA and oligo AS-460 is 320 bp downstream of initiator ATG in the cDNA. Southern blot and restriction mapping analysis demonstrated that oligo AS-200 is approximately 6 kb downstream from oligo AS140 in the genomic DNA, indicating the presence of at least one intron. Oligo AS-460 is about 8 kb downstream in the genomic DNA, indicating that further introns separate the cDNA sequences that hybridize to these oligonucleotide probes. Sequence analysis and restriction mapping were used to define precisely the number of introns and the intron-exon boundaries in the 5`-end of the human tissue transglutaminase gene (Fig. 2). As demonstrated in Fig. 2, the initiator ATG codon is included within exon 1. The first intron-exon boundary is within codon 4. The intron 1 is large, 6.1 kb. There is a second intron-exon boundary within codon 64. The second intron is 2.6 kb. The third intron starts within codon 145. The size of this third intron and the structure of the 3`-end of the gene remain to be determined.


Figure 1: Restriction map of a fragment of the human tissue transglutaminase genomic clone. A, the human tissue transglutaminase cDNA. The locations of the translation start site ( ATG), termination codon ( TAA) are marked as are the locations of three oligo nucleotides AS-140, AS-200, and AS-460 ( stippled bars). B, a restriction map of a XhoI fragment of the human tissue transglutaminase gene. The locations of sites hybridizing to the three oligonucleotide probes are indicated by stippled bars.




Figure 2: Organization of the human tissue transglutaminase gene. Sequence analysis and restriction mapping were used to define the exon-intron boundaries. The 5` and 3` splice junctions are located, and the nucleotide sequences of the splice junctions are indicated in lower case letters. The transcription start site is identified as +1.



The Human Tissue Transglutaminase Promoter

The 1.74-kb HindIII/ NcoI fragment located immediately upstream of the translation start site was subcloned and sequenced. Overlapping DNA fragments were used to deduce the sequence of 1.74-kb DNA flanking the 5`-end of the human tissue transglutaminase gene (Fig. 3, A and B). The presumptive cap site is 24 nucleotides downstream from TATA box. The GCAG sequence in this position is identical to the sequence spanning the cap site in both guinea pig (23) and mouse() tissue transglutaminase promoters. Upstream of the TATA box is a potential CAAT box with an intervening GC-rich region (Fig. 3 B). Four SP1 sites (CCGCCC) were found in the proximal promoter region. Two of them are located between the TATA box and the CAAT box, and the other two SP1 sites are located within the 5`-untranslated region (5`-UTR). Four 3` half-sites of NF1 element (CGCCAG) were also found within the 5`-UTR (24) .


Figure 3: Nucleotide sequence of 1.74 kb of 5`- flanking DNA of the human tissue transglutaminase gene. A, 29 DNA fragments were sequenced and aligned by the Gelassembly program. Arrows indicate the direction of sequencing. B, nucleotide sequence of the human transglutaminase gene promoter. The transcription start site is numbered as +1. The translation start codon ATG is marked by asterisks at position +73. TATA box- and CAAT box-like sequences are boxed and located at 29 24 and 99 93. SP1 sites are also boxed at 56 51, 45 40, and +57 +68. Four consensus sequences of NF1 3` half-site are single-underlined within the 5`-UTR. Potential response elements for glucocorticoid response elements (TGTACAGCTTGTTCT), IL-6 (CTGGAAA), AP2 (CCCCAGGG), and AP1 (TGTGTCAG) are indicated by stippled bars at positions 1399, 1190, 634, 183, respectively. The dTdG-rich region located at 581 336 and the dAdG-rich region containing multiple GGATGG elements at 1165 905 are identified by double underlines.



We searched the 1.74 kb upstream DNA segment with the consensus sequences of several well-characterized transcription factor-binding sites and hormone response elements. A potential glucocorticoid response element (TGTACAGCTTGTTCT), IL-6 response element (IL-6-RE, CTGGAAA), transcription factor AP1-binding site (AP1 site, TGTGTCAG), transcription factor AP2-binding site (AP2 site, CCCCAGGG)-like sequences were found (indicated by stippled bars in Fig. 3 B). No consensus sequences for retinoid receptor binding ( i.e. RARE, AGGTCAnnnnnAGGTCA or RXRE, AGGTCAnAGGTCA) were found in the sequence.

Comparison of the Human and Guinea Pig Tissue Transglutaminase Promoter

Recently, Ikura et al. (25) have reported the DNA sequence upstream of the guinea pig tissue transglutaminase gene. The cDNA sequences of the human and guinea pig tissue transglutaminase are conserved (76% identity; 22). The upstream sequence shows a much lower degree of homology (overall 42% identity), and this homology is concentrated in isolated islands of sequence identity. The proximal promoter region, including the TATA box, SP1 sites, and the CAAT box, is conserved between the human and guinea pig tissue transglutaminase gene (Fig. 4 A; 20). Upstream of the core promoter region in both the guinea pig and human tissue transglutaminase promoters, there is a region highly enriched in alternating pyrimidine/purine dinucleotide pairs (dTdG-rich region). Between 350 and 500 bp in the human tissue transglutaminase promoter, the frequency of dTdG dinucleotide pairs ranges from 20-30% (the predicted frequency due to random pairing would be 6%) (Fig. 4 B). This same region includes several extended tracks of dTdG repeats spanning the 402- to 451-bp region. A similar dTdG-rich region can be identified in the guinea pig tissue transglutaminase promoter approximately 300 bp upstream of the cap site.


Figure 4: Analysis of homologies between the human and guinea pig tissue transglutaminase promoters. A, comparison of the core promoter sequences. Proximal regions of the human and guinea pig tissue transglutaminase promoters (23) are aligned to show significant sequence homology. SP1 sites, TATA box element, and transcription start site are underlined. B, analysis of dTdG dinucleotide pairs in the human and guinea pig tissue transglutaminase promoters. The frequency of dTdG dinucleotides is calculated for a 100-nucleotide span whose 3`-boundary is indicated along the abscissa. The expected value for a random distribution is 6%.



A very purine-rich region (comprised of dAdG dinucleotide pairs) is located in the human gene at the position of 900-1165 bp. This region contains multiple repeats of a GGATGG motif (Fig. 3 B). Double GGATGG motifs are found in the corresponding region of the guinea pig gene. However, the biological function of this element remains to be determined. It appears that there are isolated islands of very repetitive DNA conserved in both human and guinea pig transglutaminase promoters. The structural and functional significance of these regions of DNA are not clear.

Analysis of Functional Activity of the Human Tissue Transglutaminase Promoter

The promoter function of the human tissue transglutaminase gene was determined by transient transfection of recombinant reporter genes into cultured cell lines. Reporter genes were constructed by cloning the 1.74-kb HindIII/ NcoI fragment of the human tissue transglutaminase 5`-flanking DNA into a luciferase reporter vector at a position immediately upstream of luciferase gene. 3T3 cells, a Balb/c 3T3 cell line stably transfected with a mouse RAR expression vector, were used for these experiments. Cells, transfected with HTGP1-Luc, which contained the 1.74-kb DNA in the antisense orientation, showed a low level of luciferase activity, similar to that in non-transfected cells (Fig. 5). Cells transfected with the recombinant construct HTGP2-Luc containing the same 1.74-kb DNA in the sense orientation had significant luciferase activity. The level of luciferase activity is about 25% of that detected in cells transfected with the same amount of the pSV2-AL-Luc plasmid DNA, a control vector in which the luciferase gene is placed under control of the SV40 early promoter.

The full 1.74-kb human tissue transglutaminase promoter had a high level of basal expression. To determine the regions of the gene responsible for this constitutive activity, we deleted segments of the promoter from the HTGP2-Luc reporter vector and then measured the activity of the truncated constructs by transient transfection in the 3T3 cells. The intact HTGP2-Luc vector had a high level of constitutive activity. This activity was completely dependent on the core promoter since HTGP2-mut5-Luc, in which the core tissue transglutaminase promoter sequences were deleted, showed very little transcriptional activity (Fig. 5). Progressive deletion of the 5`-end of the human tissue transglutaminase promoter had no adverse effect on basal activity. HTGP2-mut2-Luc and HTGP2-mut3-luc are constructs in which either 0.64 or 1.1 kb, respectively, of the 5`-end of the promoter were deleted. These deletions included removal of part (HTGP2-mut2-Luc) or all (HTGP2-mut3-Luc) of the purine dinucleotide-rich region as well as the consensus glucocorticoid response elements, IL-6-RE, and AP1 elements. In spite of the removal of 65% of the 1.74-kb human tissue transglutaminase promoter, there was no decrease in the constitutive transcriptional activity of the promoter-reporter construct.

The preceding studies indicated that the high constitutive activity of the human tissue transglutaminase promoter depended on the proximal promoter region. This region included the dTdG-rich region and a consensus AP2 site. To test the activity of these elements, a construct HTGP2-mut6-Luc was prepared in which a segment of the promoter spanning the dTdG-rich region and AP2 site was deleted. Removal of this region resulted in no loss of basal activity (Fig. 5). Finally, we deleted the segment of the core promoter sequence upstream of the SP1 sites in the proximal GC-rich region of the promoter (HTGP2-mut7-Luc). This construct again had high basal transcriptional activity. These studies suggest that the high constitutive transcriptional activity of the human tissue transglutaminase promoter depends only on the core promoter sequences (TATA box, four SP1 sites, and four potential NF1 sites) located in the 134 bp upstream of the translation start site.

To identify the contribution of the core promoter regulatory elements, a series of reporter genes was constructed in which fragments of the core tissue transglutaminase promoter were inserted upstream of the luciferase gene (Fig. 6). The intact core promoter (entire 5`-UTR, TATA box, SP1 sites, and CAAT box) was included in the pXP2-TG-Luc construct (122 to +72). This construct, when transfected into SW13 cells, showed the same high constitutive activity as the other reporter constructs that contained more of the upstream sequences. Removal of the CAAT box from this construct ( pXP2-CAAT-Luc) resulted in a significant decrease in the transcriptional activity of the promoter. However, the residual promoter still showed significant constitutive activity suggesting that the CAAT box element was functional and enhanced the activity of the core promoter. Removal of the four SP1 sites ( pXP2-SP1-Luc) resulted in a marked decrease in the transcriptional activity of the core promoter. The residual activity in this construct was only 8-fold more active than the ``promoterless'' control vector (pXP2-Luc). Deletion of the 5`-UTR ( pXP2-NF1-Luc) that included the putative NF1-binding sites resulted in even less basal activity. These studies suggested that the high level of constitutive activity of the tissue transglutaminase promoter was attributable to the four SP1 sites that flank the TATA box element. The presence of a functional CAAT box element also contributed to this high level of basal transcriptional activity. The potential NF1 sites at the 5`-UTR showed a weak activity.


Figure 6: Analysis of the core promoter activity of the human tissue transglutaminase gene. SW13 cells were transfected with 1 µg of pXP2-Luc, pXP2-TG-Luc, pXP2-CAAT-Luc, pXP2-SP1-Luc, and pXP2-NF1-Luc vector DNAs for 48 h, and the luciferase activity was determined as described under ``Experimental Procedures.'' The relative activities of the transglutaminase reporter constructs compared to pXP2-Luc are indicated. Open boxes indicate DNA upstream of the cap site, and hatched boxes indicate transglutaminase DNA downstream of the cap site (5`-UTR).



Tissue transglutaminase is expressed in a very cell- and tissue-specific manner. To determine if the tissue transglutaminase promoter shows comparable specificity, we compared the activity of the 1.74-kb human tissue transglutaminase promoter-reporter (HTGP2-Luc) construct (measured as fold increase over the activity of a control HTGP2-mut5-Luc plasmid) with the basal level of endogenous transglutaminase activity (). In all the cell lines tested, the full promoter construct HTGP2-Luc was 30 60-fold more active than the control vector. This relative activity was independent of the basal level of transglutaminase activity, suggesting that cell type-specific regulation of the endogenous promoter is not reflected in the activity of the 1.74-kb promoter fragment.

Retinoic acid (1 µM) can increase the tissue transglutaminase activity of 3T3 cells 5-fold; however, there was no retinoid-dependent induction of luciferase activity following transfection of HTGP2-Luc into 3T3 cells. This observation suggested that the retinoic acid regulatory elements of the human tissue transglutaminase gene are not located within the 1.74-kb 5`-flanking DNA fragment we had cloned.


DISCUSSION

The goal of this study has been to identify the functional elements associated with the DNA sequences upstream of the human tissue transglutaminase transcription unit. To address this issue we have isolated and characterized the 5`-end of the human tissue transglutaminase gene and have analyzed its activity in transfected cells. These studies have provided novel insights into both the structural and functional aspects of the human tissue transglutaminase gene.

Tissue Transglutaminase Gene Structure

Transglutaminases are a family of enzymes that share the same basic enzymatic activity, but whose members are adapted to a number of specialized functions. The organization of the genes for these transglutaminases (and transglutaminase-like proteins) reflects both their conserved enzymatic activity and their specialized functional attributes.

All the transglutaminases share a highly conserved active site sequence that flanks the reactive cysteine and histidine residues critical to their enzymatic activity (in band 4.2 the cysteine is replaced with an alanine and histidine is replaced with a glutamine but the organization of the flanking sequences is preserved). Although there is considerable sequence divergence in other regions of the molecules, the overall organization of exons and introns within the transglutaminase genes is strikingly conserved (28, 29, 30) . The genes for Factor XIIIa, keratinocyte transglutaminase, and erythrocyte band 4.2 have similar-sized exons encoding homologous segments of the proteins (26, 27, 28, 29, 30) . Fig. 7 compares the structure of the 5`-end of the human tissue transglutaminase gene with the corresponding region of several other human transglutaminase genes that have recently been reported in the literature. In all four genes, the two distal exons (exons II and III for tissue transglutaminase and band 4.2, exons III and IV for keratinocyte transglutaminase and Factor XIIIa) are of similar in size and show significant sequence homology. These exons represent the 5`-end of the core structural unit conserved among the different members of the transglutaminase multigene family, and it is clear that tissue transglutaminase preserves this structural motif.


Figure 7: The structure of the 5`-end of the transglutaminase genes. Comparison of the structure of the human transglutaminase genes. Exons and introns are identified by Roman and Arabic numbers, respectively. Sizes of exon and introns are in base pairs. The size of the introns in Factor XIIIa subunit has not been determined. Exon I of band 4.2 is subjected to alternative splicing.



The diversity in the members of the transglutaminase gene family is reflected in the diversity in the organization of the 5`-ends of the genes for these enzymes. As can be seen in Fig. 7, the tissue transglutaminase gene appears to represent the simplest member of this series. In this gene the entire 5`-untranslated region, the translation start site, and the first four codons are included in a single exon (exon I). This exon is juxtaposed directly to the core transglutaminase sequence represented by exon II. This simple gene structure suggests that tissue transglutaminase may be the simplest of the transglutaminase enzymes. Tissue transglutaminase is a ubiquitous enzyme, expressed in many cells and tissues. It is also a cytosolic enzyme that does not associate with specific subcellular compartments. Furthermore, it is translated as a fully active enzyme, and there is no evidence of proteolytic activation for this transglutaminase. There appear to be no specific regulatory functions associated with the amino terminus of the protein and therefore no specialized functional domains associated with the 5`-end of the tissue transglutaminase gene.

The structure and the function of the other transglutaminases is considerably more complex. Erythrocyte band 4.2 actually includes two polypeptides generated by alternative splicing of the primary transcript (30, 31) . This heterogeneity is derived from the use of alternative splice sites within exon I. The structure of the 5`-end of the keratinocyte and Factor XIIIa genes are also complex. In both genes, the first exon contains only untranslated sequences. The translation start site is embedded in the second exon (26, 27, 28, 29) . In the human keratinocyte transglutaminase gene, this second exon is large, encoding a long polypeptide sequence that directs the myristylation of the amino-terminal end of the molecule (32) . In Factor XIIIa, the second exon encodes a thrombin-sensitive peptide bond (33, 34, 35) . Thus in both genes, the second exon encodes function that gives the particular enzymes their distinctive regulatory properties.

It has been speculated that the introduction of specialized functions into the primordial transglutaminase gene occurred by the apposition of exons encoding specific activities ( i.e. susceptibility to myristylation, thrombin activation, etc.) to the 5`-end of the gene. Comparison of the gene structures shown in Fig. 7suggests that such diversity might have arisen from replacement of the simple first exon structure in the tissue transglutaminase gene with a variety of more complex structural motifs. Introduction of alternative splicing sites, membrane-anchoring domains, or proteolytic activation domains could have contributed to the generation of a multigene family of similar enzymes with diverse and specialized regulatory functions.

The diversity in the transglutaminase gene family is not restricted to the organization of the 5`-exon-intron boundaries but also extends upstream into the promoter sequences as well. Tissue transglutaminase appears to have a typical type II promoter structure; there is a well-defined TATA box element located 24 nucleotide upstream from a canonical cap site. The promoter includes an extensive GC-rich region upstream from the TATA box element that encodes at least two consensus SP1-binding sites, and located further upstream is a canonical CAAT box element (Fig. 3 B). The keratinocyte transglutaminase also has a TATA box-like motif (ATAAA) upstream of the cap site, and this gene also contains two SP1 sites. These sites are, however, located considerable further upstream of the TATA box element than the SP1 sites, are in the human tissue transglutaminase gene. The keratinocyte transglutaminase promoter also lacks a putative CAAT box element (27, 29) . The band 4.2 promoter has a TATA box-like element located upstream of the putative cap site, but this promoter lacks a clearly defined GC-rich region or consensus SP1 sites (30) . The Factor XIIIa subunit promoter is a TATA box-less promoter that lacks both TATA box element or clearly defined SP1-binding sites (26) .

The differences among the structures of the promoters for the members of the transglutaminase gene family argue against the suggestion that the 5`-exons were mere inserted downstream from a core transglutaminase promoter sequenced. The diversity suggests rather that key regulatory sequences and promoter elements were appended upstream of the core transglutaminase gene to generate a family of enzymes with divergent transcriptional control as well as unique structural and regulatory features.

Functions of the Tissue Transglutaminase Promoter

Tissue transglutaminase is expressed in cells and tissues in a highly regulated manner. Many cells, such as endothelial cells, vascular smooth muscle cells, platelets, and epithelial cells of the lens, express the enzyme constitutively and accumulate high levels of active enzyme (3) . In other cells, such as monocytes and tissue macrophages, the enzyme is inducible (16, 17, 36) . Basal expression of the enzyme is very low, but the enzyme is dramatically induced following exposure to an inflammatory stimulus. In a few cells, particularly neurons and skeletal muscle cells, tissue transglutaminase expression is very low and very little active enzyme accumulates in these cells under normal conditions. These cells, and many other cells, do accumulate high levels of the enzyme if they enter into the pathway of programmed cell death. In view of the evidence for the tight physiological control of tissue transglutaminase expression in vivo, it was surprising to us to discover that the tissue transglutaminase core promoter is constitutively active in many different cell types. This high level of basal activity appears to be attributable to the presence of a CAAT box as well as the four SP1 sites, two located directly upstream of the TATA box element and the other two at the 5`-UTR. This strong constitutive activity is not unique to the human tissue transglutaminase promoter. Ikura et al. (23) have reported that the guinea pig tissue transglutaminase promoter, which shows similar transcription factor-binding sites, also shows strong constitutive activity.

The presence of a constitutively active promoter in a gene subject to complex regulation suggests that important negative or tissue-specific regulatory elements must control the activity of this gene in many cells and tissues. Further analysis of the human transglutaminase gene will be required to identify and characterize the cis-regulatory regions that are responsible for the cell- and tissue-specific gene expression. The rapid increase in the expression of the enzyme that occurs in many cells undergoing programmed cell death may not be due to a specific induction of the enzyme but rather may be due to the loss of factors that normally suppress its expression. Cell death may serve to unmask the activity of the constitutive core promoter of the tissue transglutaminase gene, and this in turn may lead to the marked accumulation of the enzyme that occurs in many dying cells.

Several studies have demonstrated that the expression of tissue transglutaminase can be regulated by retinoids (3, 17, 36) . Retinoids did not increase the transcriptional activity of the 1.74-kb tissue transglutaminase promoter fragment when it was transfected into a retinoid-responsive (3T3) cell line. This finding suggests that retinoid regulation of the human transglutaminase promoter is conferred by DNA sequences that lie outside the proximal 1.74-kb promoter fragment analyzed in these studies.

  
Table: Constitutive activity of the human tissue transglutaminase promoter in SW13, 3T3, MCF-7, and Hela cells

Cells were transfected with 1.5 µg of HTGP2-mut5-Luc or HTGP2-Luc for 48 h, followed by luciferase assay.



FOOTNOTES

*
This work was supported in part by research Grant DK27078 from the National Institute of Health. 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) U13920 and Z46905.

§
To whom correspondence should be addressed.

The abbreviations used are: IL-6, interleukin 6; 5`-UTR, 5`-untranslated region; kb, kilobase(s); DMEM, Dulbecco's modified Eagle's medium; bp, base pair(s).

M. Saydak and P. J. A. Davies, unpublished observation.


ACKNOWLEDGEMENTS

We gratefully thank the excellent technical assistance of Mary Sobieski and Nancy Shipley and the secretarial assistance of Joan Jennings in support of these studies.


REFERENCES
  1. Folk, J. E. (1980) Annu. Rev. Biochem. 49, 517-31 [CrossRef][Medline] [Order article via Infotrieve]
  2. Ichinose, A., Bottenus, R. E., and Davie, E. W. (1990) J. Biol. Chem. 265, 13411-13414 [Free Full Text]
  3. Greenberg, C. S., Birckbichler, P. J., and Rice, R. H. (1991) FASEB J. 5, 3071-3077 [Abstract/Free Full Text]
  4. Aeschlimann, D., and Paulsson, M. (1994) Thrombo. Hemostasis 71, 402-415
  5. Rice, R. H., and Green, H. (1978) J. Cell Biol. 76, 705-711 [Abstract/Free Full Text]
  6. Thacher, S. M., and Rice, R. H. (1985) Cell 40, 685-695 [Medline] [Order article via Infotrieve]
  7. Slife, C. W., Dorsett, M. D., and Tillotson, M. L. (1986) J. Biol. Chem. 261, 3452-3456
  8. Bowness, J. M., Folk, J. E., and Timpl, R. (1987) J. Biol. Chem. 262, 1022-1024 [Abstract/Free Full Text]
  9. Kinsella, M. G., and Wight, T. N. (1990) J. Biol. Chem. 265, 17891-17898 [Abstract/Free Full Text]
  10. Martinez, J., Rich, E., and Barsigian, C. (1989) J. Biol. Chem. 264, 20502-20508 [Abstract/Free Full Text]
  11. Gentile, V., Thomazy, V., Piacentini, M., Fesus, L., and Davies, P. J. A. (1992) J. Cell Biol. 119, 463-474 [Abstract]
  12. Aeschlimann, D., Wetterwald, A., Fleisch, H., and Paulsson, M. (1993) J. Cell Biol. 120, 1461-1470 [Abstract]
  13. Upchurch, H. F., Conway, E., Patterson, M. K., and Maxwell, M. D. (1991) J. Cell. Physiol. 149, 375-382 [Medline] [Order article via Infotrieve]
  14. Fesus, L., and Thomazy, V. (1988) Adv. Exp. Med. Biol. 231, 119-134
  15. Fesus, L. (1992) Immunol. Today 13, A16-A17
  16. Murtaugh, M. P., Arend, W. P., and Davies, P. J. A. (1984) J. Exp. Med. 159, 114-125 [Abstract]
  17. Antonio Chiocca, E., Davies, P. J. A., and Stein, J. P. (1988) J. Biol. Chem. 263, 11584-11589 [Abstract/Free Full Text]
  18. George, M. D., Vollberg, T. M., Floyd, E. E., Stein, J. P., and Jetten, A. M. (1990) J. Biol. Chem. 265, 11098-11104 [Abstract/Free Full Text]
  19. Suto, N., Ikura, K., and Sasaki, R. (1993) J. Biol. Chem. 268, 7469-7473 [Abstract/Free Full Text]
  20. Nordeen, S. K. (1988) BioTechniques 6, 454-456 [Medline] [Order article via Infotrieve]
  21. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  22. Gentile, V., Saydak, M., Chiocca, E. A., Akande, O., Birckbichler, P. J., Lee, K. N., Stein, J. P., and Davies, P. J. A. (1991) J. Biol. Chem. 266, 478-483 [Abstract/Free Full Text]
  23. Suto, N., Ikura, K., Shinagawa, R., and Sasaki, R. (1993) Biochim. Biophys. Acta 1172, 319-322 [Medline] [Order article via Infotrieve]
  24. Chang, T. S., and Shapiro, D. J. (1990) J. Biol. Chem. 265, 8176-8182 [Abstract/Free Full Text]
  25. Ikura, K., Nasu, T., Yokota, H., Tsuchiya, Y., Sasaki, R., and Chiba, H. (1988) Biochemistry 27, 2898-2905 [Medline] [Order article via Infotrieve]
  26. Ichinose, A., and Davie, E. W. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 5829-5833 [Abstract]
  27. Yamanishi, K., Inazawa, J., Liew, F. M., Nonomura, K., Ariyama, T., Yasuno, H., Abe, T., Doi, H., Hirano, J., and Fukushima, S. (1992) J. Biol. Chem. 267, 17858-17863 [Abstract/Free Full Text]
  28. Phillips, M. A., Stewart, B. E., Qin, Q., Chakravarty, R., Floyd, E. E., and Jetten, A. G. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 9333-9337 [Abstract]
  29. Phillips, M. A., Stewart, B. E., and Rice, R. H. (1992) J. Biol. Chem. 267, 2282-2286
  30. Korsgren, C., and Cohen, C. M. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 4840-4844 [Abstract]
  31. Sung, L. A., Chien, S., Fan, Y. S., Lin, C. C., Lambert, K., Zhu, L. Y., Lam, J. S., and Chang, L. S. (1992) Blood 79, 2763-2770 [Abstract]
  32. Phillips, M. A., Qin, Q., Mehrpouyan, M., and Rice, R. H. (1993) Biochemistry 32, 11057-11063 [Medline] [Order article via Infotrieve]
  33. Grundmann, U., Amann, E., Zettlmeissl, G., and Kupper, H. A. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 8024-8028 [Abstract]
  34. Ichinose, A., Hendrickson, L. E., Fujikawa, K., and Davie, E. W. (1986) Biochemistry 25, 6900-6906 [Medline] [Order article via Infotrieve]
  35. Takahashi, N., Takahashi, Y., and Putnam, F. W. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 8019-8023 [Abstract]
  36. Moore, W. T., Michael, P., Murtaugh, M. P., and Davies, P. J. A. (1984) J. Biol. Chem. 259, 12794-12802 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.