From the
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
Transglutaminases are enzymes that catalyze the covalent
cross-linking of proteins by promoting the formation of
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-
Cells to be transfected were plated in a 35-mm tissue
culture dish at a density of 2
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
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 (
Retinoic acid (1
µM) can increase the tissue transglutaminase activity of
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.
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.
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.
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
(
Cells were transfected with 1.5 µg of HTGP2-mut5-Luc or
HTGP2-Luc for 48 h, followed by luciferase assay.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank/EMBL Data Bank with accession number(s) U13920 and Z46905.
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.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
-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.
-(
-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) .
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.
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
10
phage 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 reagent
DNA complex. The complex was
then mixed with fresh serum-free DMEM at 1:4 (v/v) and was used
immediately.
10
cells/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 reagent
DNA 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
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
KH-Gal Assay
PO
/K
HPO
pH 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).
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
10
phage 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.
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.
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.
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.
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.
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.
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.
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
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.