From the Cell Biology Section, Laboratory of Pulmonary Pathobiology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709
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ABSTRACT |
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The transglutaminase I (TGase I) gene encodes an
enzyme that catalyzes the cross-linking of structural proteins involved
in the formation of the cornified envelope during squamous cell
differentiation. To identify DNA elements important for the
transcriptional control of the TGase I gene, we analyzed the ability of
a 2.9-kilobase pair (kb) upstream regulatory region to control the
expression of a reporter gene in vivo and in
vitro. Transgenic mice bearing the pTG( Transglutaminases
(TGases)1 (EC 2.3.2.13)
constitute a family of Ca2+-dependent enzymes
that covalently cross-link proteins by catalyzing the formation of
isopeptide bonds between the Squamous differentiation is a terminal pathway of differentiation that
occurs as a multistep process (21-23). In epidermal keratinocytes this
differentiation represents a normal process, whereas in
tracheobronchial epithelial cells, it is an aberrant pathway of
differentiation that occurs under conditions of severe vitamin A
deficiency or after injury (20-22). Early during differentiation, cells become committed to irreversible growth arrest that is
accompanied by alterations in the expression of several
growth-regulatory genes (6, 23-25). In subsequent stages in the
differentiation process, cells begin to express a variety of
squamous-specific genes (20-22). In tracheobronchial epithelial cells,
squamous differentiation is associated with induction of keratin 13 (26, 27), relaxin (28), cholesterol sulfotransferase (29), epithelial
membrane protein 1 (30), and several proteins involved in the formation of the cross-linked envelope (31-38).
The formation of the cross-linked envelope is a characteristic event in
the final stages of squamous differentiation. The envelope consists of
a layer of cross-linked protein deposited just beneath the plasma
membrane (5-7, 18). It contributes to the cohesiveness and rigidity of
the cornified layer and provides a vital barrier function. The envelope
is formed by the cross-linking of many precursor proteins, including
involucrin (38), cornifins/small proline-rich proteins (SPRRs) (32, 35,
36), and loricrin (37) catalyzed by TGase I and III (5-7, 17-19).
TGase I is synthesized as a soluble enzyme in the cytosol and becomes
associated with the cell membrane after acylation by palmitic and
myristic acid at a cluster of five cysteines at its amino terminus (39,
40). The TGase I gene has been cloned from various species (33, 41, 42). The human TGase I gene, which spans 14.3 kb and contains 15 exons,
has been mapped to chromosome 14q11 (TGM1 locus) (43-45). The
importance of this enzyme in the morphogenesis and function of the skin
was recently corroborated by studies implicating mutations in the TGase
I gene that result in a lack of TGase I activity in the autosomal
recessive skin disorder lamellar ichthyosis (11, 12). Recent studies
demonstrated that TGase I-null mice exhibit severe deficiencies in
several skin functions, including impairments in cell envelope assembly
and barrier function (46).
Analysis of the TGase I promoter may provide insight not only into the
molecular mechanisms controlling this gene in squamous epithelia and
squamous metaplasia but also into the molecular pathogenesis of
lamellar ichthyosis and lead to new methods of treatment, including
drug and gene therapy. In this study, we analyzed the ability of a
2.9-kb promoter regulatory region of the TGase I gene to control the
expression of a chloramphenicol acetyltransferase (CAT) reporter gene
in vivo and in vitro. We show that this region
directs expression of the transgene to the suprabasal layers,
comprising the late spinous and granular layers, of several squamous
tissues. This pattern of expression correlates well with that reported
for TGase I (47, 48). Deletion analysis identified two DNA elements in
the 2.9-kb regulatory region containing an Sp1- and a CREB/AP-1-like
site that are involved in the transcriptional regulation of TGase I.
Cell Culture--
Rabbit tracheobronchial epithelial (RbTE)
cells were cultured in Ham's F-12 medium supplemented with 10 ng/ml
epidermal growth factor, 5 µg/ml transferrin, and insulin as
described previously (49). Normal human epidermal keratinocytes (NHEK)
were purchased from Clonetics (San Diego, CA) and cultured in KGM
medium (Clonetics). Rabbit tracheal fibroblasts were grown in Ham's
F-12/RPMI 1640 (1:1) supplemented with 10% fetal bovine serum.
Plasmids and Reporter Constructs--
The plasmid
pTG( Search for Transcription Factor Binding Sites--
Promoter
flanking regions were analyzed for the presence of potential
transcription factor binding sites using the TFSEARCH Program.2
Transfections and Reporter Gene Assays--
Plasmid DNA used in
transfections were isolated and purified with a Wizard Miniprep kit
(Promega). RbTE cells were grown to confluence in six-well dishes, and
transfections were carried out in triplicate using lipofectamine (Life
Technologies, Inc.) according to the manufacturer's protocol. Cells
were incubated in 1.0 ml of Ham's F-12 medium without growth factors
and antibiotics in the presence of 1.5 µg of total DNA and 5 µl of
lipofectamine. Cotransfection with Electrophoretic Mobility Shift Assays (EMSAs)--
Nuclei were
isolated from differentiated rabbit tracheobronchial epithelial cells,
and nuclear extracts were prepared by the method of Dignam et
al. (50) with slight modifications. Briefly, cells were washed
twice in ice-cold PBS, collected into microtubes, and centrifuged for
10 s in a microcentrifuge. The cell pellet was then resuspended in
1 ml of ice-cold, low salt buffer (10 mM HEPES, pH 7.9, 1 mM EDTA, 0.5 mM EGTA, 10 mM KCl, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM
dithiothreitol, 5 µg/ml leupeptin and aprotinin, 0.1 mM
sodium vanadate). After incubation on ice for 20 min, Nonidet P-40 was
added to a final concentration of 0.5%. The cell suspension was
vigorously vortexed for 20 s followed by centrifugation for 30 s in a microcentrifuge to collect the nuclei. The nuclei were resuspended in 10 volumes of high salt buffer (10 mM HEPES,
pH 7.9, 1 mM EDTA, 0.42 M NaCl, 10% glycerol,
and protease inhibitors) and incubated on ice for 20 min followed by a
10-min centrifugation in a microcentrifuge at 4 °C. Nuclear extracts
were collected and stored in aliquots at
The Sp1 consensus oligonucleotide (CTCCGCCCCGCCCGATCGAAT),
the AP-1 consensus (CGCTTGATGAGTCAGCCGGAA), the CREB consensus (GTACTGGCTGCTGACGTCACCAGCCA), and other
oligonucleotides were synthesized using a model 392 DNA synthesizer
(Applied Biosystems). Oligonucleotides for EMSA were end-labeled with
[ Footprint Analysis--
A TGase I promoter fragment from Generation and Identification of Transgenic Mice--
The
construction of the pTG-( In Situ Hybridization--
Transgenic mice were sacrificed, and
tissues isolated and fixed for 3 h with 4% paraformaldehyde in
phosphate-buffered saline, pH 7.4. After rinsing in phosphate-buffered
saline, tissues were dehydrated in a graded ethanol series, permeated
with xylene, and embedded in paraplast. In situ
hybridization was performed as described (51) with the following minor
modification. Serial sections (5 µm) were mounted on Silane-prepTM
slides (Sigma), deparaffinized, acetylated, and pretreated with 2×
sodium citrate (SCC), 50% formamide, 10 mM dithiothreitol
at 50 °C. Radiolabeled sense and antisense CAT probes were generated
with either T3 or T7 RNA polymerase and 35S-labeled UTP and
CTP (Amersham Pharmacia Biotech; 1000 Ci/mmol), as described (51).
Probes were subjected to limited alkaline hydrolysis to reduce the size
of the transcripts to about 150 bases. After hybridization at 50 °C
for 16 h, the sections were washed and treated with RNase. The
slides were dipped in NTB-2 emulsion (Eastman Kodak Co.) diluted 1:1.5
and exposed for 14 days at 4 °C. Slides were developed and
counterstained with hematoxylin.
A 2.9-kb TGase I Promoter Region Is Sufficient for Tissue- and
Differentiation-specific Expression--
The cloning and sequence of
the 2.9-kb 5'-flanking region of the rabbit TGase I gene has been
reported previously (26). We also mapped the transcription start site
and showed that this promoter region was able to induce transactivation
of the luciferase reporter gene during differentiation of cultured RbTE
cells (26). To determine whether this region is able to control the
expression of TGase I in vivo in a cell type- and
differentiation-specific manner, we constructed an expression vector in
which the CAT reporter is under the control of the 2.9-kb 5'-flanking
region of the rabbit TGase I gene and introduced this plasmid into the
mouse germ line. The presence of this transgene was corroborated by
PCR. Two independent founders, which contained and expressed the
integrated transgene, were selected for subsequent analysis. Protein
extracts prepared from various tissues of control and transgenic mice
were analyzed for CAT protein concentration. As expected, no CAT
protein was detectable in any of the tissues isolated from control mice
(Fig. 1B). In the transgenic
mice, CAT protein was only detected in crude protein extracts from
tissues that normally express TGase I, including skin, tongue, and
esophagus. The highest CAT levels were found in extracts from tongue
and esophagus; lower levels were found in skin. This pattern of CAT
expression is consistent with the known expression levels of TGase I in
these tissues (34). These results demonstrate that this 2.9-kb
5'-flanking region of the TGase I gene contains regulatory elements
that are sufficient to control the transcription of this gene in a
tissue-specific manner.
During squamous differentiation, cells transit from the basal into the
suprabasal layers of the epithelium and begin to express a variety of
differentiation-specific genes. Initiation of TGase I expression occurs
within the late spinous and granular cell layers of the squamous
epithelium. To localize the expression of the transgene in squamous
tissues, we performed in situ hybridization using sense and
antisense riboprobes to the CAT mRNA. A specific hybridization
signal was observed in the suprabasal layers, in particular in the late
spinous and granular layers of the squamous epithelium of the tongue
from transgenic mice, as shown in Fig. 2,
A and B. The hybridization signal in the basal
layer was at background levels. A low background hybridization signal
was observed in squamous epithelia of control mice with no significant
differences between the different layers (Fig. 2, C and
D). These results indicate that the 2.9-kb region contains
DNA elements that are able to regulate transcription of CAT at a
specific stage during differentiation that is similar to the pattern of
endogenous TGase I expression (47).
Deletion Analysis of the TGase I Promoter--
In an effort to
locate regulatory sequences important in the transcriptional control of
the TGase I gene, a series of deletion constructs were made using
several unique restriction sites within the 5'-flanking region or
fragments generated by PCR (Fig.
3A). Transcriptional activity
of these pGL2-LUC constructs was analyzed by transient transfection
into primary rabbit tracheobronchial cells cultured under
differentiation-inducing conditions. Compared with the activity induced
by the minimal promoter (bp
Analysis of the promoter activity of TGase I deletion constructs in
differentiated NHEK showed that the relative transcriptional activation
of the reporter gene by the various promoter regions was very similar
to that observed in RbTE cells (Fig. 4).
These results suggest that regulation of TGase I expression during
squamous differentiation of tracheal bronchial epithelial cells and
epidermal keratinocytes appears to occur largely through the same
upstream-regulatory regions. However, deletion of the The Proximal TGase I Promoter Contains a Functional Sp1
Element--
The proximal region was analyzed with TFSearch for the
presence of potential transcriptional factor binding sites. This
computer search indicated that the Deletion Analysis of
Fig. 7 shows that the CREB/AP-1-like
element (TGpal) and the Sp1 site are acting in concert to
optimally increase transcription. Each element used separately with the
minimal promoter increased transactivation of the reporter, while
together (TGpal/
To directly map protein binding sites in the TGase I Promoter Contains a Functional CREB/AP-1-like
Element--
To determine which bases in the TGpal element
are optimal for its transactivation function, mutated oligonucleotides
M1-M8 (Fig. 9A) were inserted
upstream of the
To map the exact position of the enhancer element within the
To determine the identity of some of the proteins in the complexes
binding to TGpal, supershift analysis was carried out with nuclear extracts from squamous differentiated RbTE cells and antibodies against various members of the Jun, Fos, CREB, and ATF families of
transcription factors. These analyses identified CREB1, c-Jun Fra-1,
and c-Fos as part of protein complexes bound to TGpal (Fig. 11A). These proteins were
also found in complexes bound to a consensus CREB oligonucleotide and,
except for CREB1, also in complexes bound to a consensus AP-1
oligonucleotide (Fig. 11, B and C). The anti-c-Fos antibody caused a supershift of almost all of the
protein-AP-1 complexes, while this shift is much less dramatic with
TGpal or CREB oligonucleotides. The supershift with
anti-Fra-1 antibody was most pronounced with AP-1 oligonucleotide and
very weak with TGpal. These differences in the degree of
the supershift are likely to be an indication of the differences in the
affinity of the transcriptional factor for the particular DNA element
and the amount of the protein present in the bound complexes.
Antibodies specific for Ser63-phosphorylated (active) c-Jun
or JunB were also able to cause a supershift of
TGpal-protein complexes (not shown), whereas antibodies against JunD, ATF2, ATF4, Fra-2, and CREB2 did not cause supershifts (not shown).
In this study, we analyzed the promoter activity of the 2.9-kb
5'-flanking region of the rabbit TGase I promoter in vivo
and in vitro. Analysis of the TG( In order to locate the regulatory elements required to drive TGase I
expression during squamous differentiation, a series of deletions were
made to the 2.9-kb 5'-flanking sequence. This analysis mapped the
minimal promoter to The GC-rich region containing the Sp1 consensus element is located just
upstream from the TATA-like box. Point mutations in this element reduce
promoter activity to that of the minimal TGase I promoter (Fig. 5).
Although another consensus Sp1 sequence is present at bp The functionally important CREB/AP-1-like element TGATGTCA is contained
in a 22-bp palindrome that is protected in DNase I footprinting assays.
Point mutations in the CREB/AP-1-like site reduced promoter activity of
this site, supporting the importance of the CREB/AP-1 motif. The
CREB/AP-1-like element has been shown to bind a variety of
transcriptional factors, including members of the CREB, ATF, and
Fos/Jun families (63). Mobility shift assays using an array of
antibodies against different CREB/AP-1-binding proteins demonstrated
that TGpal interacted with protein complexes containing
CREB1, c-Jun, JunB, Fra-1, and c-Fos in nuclear extracts from
differentiated RbTE cells. CREB/AP-1-like elements have been implicated
in the transcriptional regulation of several other squamous marker
genes, including involucrin, profilaggrin, and SPRR-1 genes (64-67).
It is likely that the regulation of squamous-specific genes, which are
induced at different stages during differentiation, involves control by
different CREB/AP-1 protein complexes. The differential expression of
members of the Jun/Fos family during different stages of squamous
differentiation is in agreement with this concept (68). A role for
CREB/AP-1-like elements is further supported by studies showing that
PKC activation, which regulates the expression of Jun/Fos family
members, is an important component of the signaling pathways
controlling squamous differentiation and TGase I (34, 68-71).
Cholesterol sulfate, which is highly induced during squamous
differentiation (71), can function as an endogenous second messenger
and through the activation of different PKC isoforms enhance the
expression of several squamous-specific genes, including TGase I (52,
70).
The role of specific CREB/AP-1 complexes in squamous differentiation is
not yet well understood. Recent studies have demonstrated that
expression of c-Fos appears not to be essential for squamous differentiation in the epidermis, since epidermal differentiation is
normal in c-Fos null mice (68). Others have suggested that JunB may be
involved in the induction of early markers, including involucrin and
TGase I, while JunD may play a role in the control of later markers,
such as profilaggrin (65). A role for JunB in TGase I regulation is
supported by immunohistochemical studies showing that JunB is
predominantly present in the granular layer where TGase I is also
expressed (47, 68) and by EMSA demonstrating the presence of JunB in
protein complexes binding TGpal. It is clear that multiple
protein complexes in squamous differentiated cells can compete for
binding to TGpal. Some of these complexes may act as
repressors, while others may function as stimulators of gene
transcription. Therefore, increased transcription of squamous-specific genes may depend on the level and activity of these complexes during
the differentiation process. This may involve changes in heterodimerization partners that could alter the affinity and activity
of the complex. This concept is supported by recent findings showing
that ectopic expression of a dominant-active c-Jun has a repressive
effect on the expression of several squamous-specific genes, including
involucrin and SPRR-1 (73). In addition, it was demonstrated that c-Jun
is expressed predominantly in basal and early spinous layer and that
the level of c-Jun and phosphorylated c-Jun is dramatically
down-regulated during squamous differentiation and is probably related
to the observed reduction in JNK activity (68).3 These findings support
a negative regulatory role of c-Jun in the control of squamous
differentiation. The latter is in agreement with observations showing
that squamous cell markers are not induced in squamous cell carcinoma
cell lines overexpressing c-Jun (73).
In this study, we show that the 2.9-kb 5'-flanking region of the TGase
I gene can regulate this gene in a tissue- and differentiation-specific manner and identify two DNA elements important in its transcriptional control. It is known that certain forms of lamellar ichthyosis are
linked to defects in the expression of the TGase I gene (11, 12). In
some cases, this genetically heterogeneous disease might be caused by
mutations in transcriptionally important promoter regions of TGase I. Therefore, the study of this promoter region could help in
understanding the nature of this disease and provide a tool for gene
therapy of lamellar ichthyosis and other skin disorders.
2.9kb)CAT construct
exhibited the same pattern of tissue-specific expression of CAT as
reported for TGase I. Deletion analysis in transiently transfected
rabbit tracheal epithelial cells indicated that two sequences from bp
490 to
470 and from
54 to
37 are involved in the activation of
TGase I transcription. Point mutation analysis and mobility shift
assays showed that the sequence located between
54 and
37 is a
functional Sp1-like transcription element. Sp1 and Sp3, but not Sp2,
are part of nuclear protein complexes from differentiated RbTE cells
binding to this site. The element TGATGTCA between bp
490 and
470
is contained in a larger 22-bp palindrome and resembles the consensus
cAMP response element-binding protein (CREB)/AP-1 element recognized by
dimeric complexes of members of the CREB, ATF, Fos, and Jun families.
Mutations in this sequence greatly reduced promoter activity.
Supershift analysis identified CREB1, JunB, c-Fos, Fra-1, and c-Jun in
protein complexes isolated from differentiated rabbit tracheal
epithelial cells binding to this site. Our study shows that the Sp1-
and CREB/AP-1-like sites act in concert to stimulate transcription of
the TGase I gene. The 2.9-kb promoter region could guide expression of
specific genes in the granular layer of the epidermis and could be
useful in gene therapy.
INTRODUCTION
Top
Abstract
Introduction
References
-amide group of glutamine and the
-amino group of lysine (1-5). TGases have functions in a variety of
biological processes, such as differentiation, apoptosis, and blood
clotting (4-9), and are implicated in a number of diseases (4,
10-12). Factor XIII catalyzes the cross-linking of a number of
proteins in plasma and plays an important role in fibrinogenesis (1, 2,
4, 5). Tissue TGase (TGase II) has been implicated in the activation of
certain cytokines (13, 14) and cross-linking of specific components of
the extracellular matrix (4). This TGase has also been reported to have
GTPase activity and as such may have a role in signal transduction
(15). In addition, evidence has been provided indicating that TGase II
may catalyze cross-linking of intracellular proteins during apoptosis
(8, 16). TGase I and III are involved in the formation of the
cross-linked envelope during squamous cell differentiation (5-7,17-19).
EXPERIMENTAL PROCEDURES
2.9kb)-LUC containing the luciferase reporter gene under the
control of the
2.9 kb promoter flanking region of the TGase I gene
has been previously described (34). The deletion mutants
pTG(
1.5kb)-LUC and pTG(
350)-LUC were constructed using the
SmaI and ApaI restriction sites (Fig.
1A). The minimal promoter construct pTG(
37)-LUC and
pTG(
54)-LUC were made by cloning the respective PCR products into the
promoterless luciferase vector pGL2-Basic (Promega). Deletion mutants
in region
750 to
350 bp were constructed by PCR-directed cloning
into the construct pTG(
37)-LUC (Fig. 2A). All PCR
reactions were conducted with proofreading Vent DNA polymerase (New
England Biolabs), and the original 2.9-kb construct was used as
template. All primers contained a six-base 5'-leader sequence followed
by a six-base restriction site (KpnI, MluI, or
XhoI) and a 20-base promoter sequence. Point mutation
constructs were made by direct cloning of chemically synthesized
oligonucleotides upstream of pTG(
37)-LUC. The sequences of the PCR
products were checked for accuracy using the dideoxynucleotide chain
termination method and the Sequenase Quick-denature plasmid sequencing
kit (Amersham Pharmacia Biotech).
-actin-CAT reporter plasmid was
carried out to correct for differences in transfection efficiency.
After a 5-h incubation, medium was replaced with complete F-12 or KGM medium. Cells were collected 48 h after transfection and assayed for reporter activity. Whole cell extracts were prepared with the lysis
buffer included in the CAT ELISA kit (Boehringer Mannheim) according to
the manufacturer's protocol and used in CAT and luciferase assays.
Luciferase assays were performed with the Luciferase Assay Kit from
Promega. The relative luciferase activity was normalized for CAT
reporter activity.
70 °C.
-32P]ATP by T4 polynucleotide kinase (Promega) and
purified with NAP-5 columns (Amersham Pharmacia Biotech). Approximately
0.1 ng (50,000 cpm) of the oligonucleotide probe and 0.5 µg of
nuclear extract were used in the binding reaction in buffer containing 20 mM HEPES, pH 7.9, 50 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride,
10% glycerol. To prevent nonspecific binding, 1 µg of poly(dI-dC)
and 0.2 µg of salmon sperm DNA were included in the reaction. The
binding reactions were carried out at room temperature for 25 min in
the presence or absence of a 10- or 100-fold molar excess of
oligonucleotide competitors. For supershift analysis, various
antibodies were included in the incubation mixture. DNA-protein complexes were separated on 5% nondenaturing polyacrylamide gel electrophoresis at room temperature in 0.5× TBE. Antibodies against Sp1, Sp2, Sp3, c-Jun, Ser63-phosphorylated c-Jun, JunB,
JunD, Fra-1, Fra-2, c-Fos, CREB1, CREB2, ATF-2, and ATF-4 were obtained
from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
750
to
350 bp was generated by PCR with proofreading Vent DNA polymerase
(NE Biolabs). One of the primers used for PCR was labeled with T4
polynucleotide kinase (Promega). The binding mixture contained 20 mM HEPES (pH 8.0), 10% glycerol, 50 mM NaCl,
10 mM MgCl2, 10-20 µg of nuclear extract, 1 ng of labeled DNA, and 1 µg of poly(dI-dC). The binding reaction was
carried out for 25 min at room temperature and then treated with
different concentrations of DNase I. After 1 min of incubation with
DNase I, 1 volume of stop buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, and 1% SDS) was added. DNA was purified by phenol
extraction and concentrated with Microcon-30 (Amicon). DNA fragments
were separated on a 6% sequencing gel.
2.9)CAT reporter was carried out by cloning
the 2.9-kb promoter fragment (KpnI/NheI
restriction fragment) from pTG(
2.9)LUC into the XbaI site
of the promoterless pCAT reporter plasmid (Promega). Compatible end
ligation of the NheI site into the XbaI site was
followed by incubation with T4 DNA polymerase (Amersham Pharmacia
Biotech). The resulting blunt ended XbaI and KpnI
sites were then autoligated. Orientation of the clones was confirmed by
sequencing. This plasmid was linearized, and a 4.5-kb
HindIII/BamHI DNA fragment containing the
2873
to +89 bp regulatory region of TGase I gene and the CAT reporter excised. The fragment was purified and microinjected into the pronuclei
of one-cell mouse embryos obtained from C57BL/6J females mated with SJL
F2 males. The embryos were then transferred to the oviduct of
pseudopregnant females, and normal gestation was allowed. Positive
founder transgenic mice carrying the construct were identified by PCR
analysis of genomic DNA isolated from tail biopsies. A rabbit TGase I
promoter-specific primer, 5'-TCGGCCCCGCCCTCCCCA, and a CAT-specific
primer, 5'-AACGGTGGTATATCCAGTGA, were used for the analysis. Tissues
for CAT assay, RNA isolation, and in situ hybridization were
taken from 3- or 12-month-old mice.
RESULTS
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Fig. 1.
A, Schematic view of the pTG( 2.9)-CAT
construct used in the TGase promoter analysis in transgenic mice. The
TATA-box-like sequence CATAA is indicated. B, relative CAT
activity in various tissues from transgenic mice expressing
pTG(
2.9)-CAT. The relative CAT activity in protein extracts from
various tissues was determined by enzyme-linked immunosorbent assay.
Tissues from normal (1) and two transgenic mice of two
different lines (2 and 3) were analyzed. Only
skin, tongue, and esophagus, which normally express TGase I, contained
CAT protein. The assay was done in triplicate, and the S.D. was less
then 10%. Sk, skin; Es, esophagus;
To, tongue; Lu, lung; Ki, kidney;
Li, liver; Te, testis; Mu, muscle;
Br, brain.
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Fig. 2.
Localization of CAT reporter mRNA in the
stratified, squamous-differentiated epithelium of the tongue from
transgenic mice containing pTG( 2.9)-CAT. Sections prepared from
the tongue of transgenic (A and B) and control
mice (C and D) were analyzed by in
situ hybridization using radiolabeled antisense CAT probe to
detect expression of CAT mRNA. A and C,
bright field; B and D, dark field exposures. The
dotted line indicates the position of the
basement membrane.
37 to +60), the full-length 2.9-kb region
was able to increase luciferase activity by about 30-50-fold,
depending on the experiment. Deletions up to bp
750 had only a small
effect (10% reduction) on luciferase activity, indicating that the
region between bp
2874 and
750 does not contribute significantly to
differentiation-specific activation when transfected into
squamous-differentiated RbTE cells (Fig. 3). In contrast, deletion of
the region spanning bp
750 to
348 resulted in a 70% decrease in
luciferase activity. Further deletions of the region up to bp
54 had
only minor effects; however, deletion of the region between bp
54 and
37 caused an additional 90% reduction in promoter activity (Fig.
3B). As demonstrated in Fig. 3C, the minimal
TGase I promoter (pTG(
37/+60)LUC) containing the TATA box-like
sequence CATAA still exhibited transactivating activity compared with
the promoterless pGL2-LUC. Deletion of this sequence (pTG(0/+60bp)LUC)
or the region downstream of CATAA (pTG(
37/0)LUC) completely abolished
the ability to activate transcription (Fig. 3C).
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Fig. 3.
Analysis of the promoter activity of the
5'-regulatory region of the rabbit TGase I gene. A series of 5'
deletion mutants were made to the pTG( 2.9)-LUC reporter construct and
analyzed by transient transfection into differentiating RbTE cells. The
constructs are named by the size of the regulatory region.
A, the relative luciferase activity for each construct was
calculated and plotted. B, -fold increase in relative
luciferase activity is shown on the right in A.
C, effect of deletions within the minimal promoter region
(
37/+60) on promoter activity. The 0/+60 construct contains the
region downstream from the TATA-box-like sequence CATTAA, while the
37/0 construct contains the upstream region including the TATA
box.
2.9 kb to
750
bp region caused a consistently greater reduction (45%) in promoter
activity in NHEK than RbTE cells, indicating species- or cell
type-specific differences in the promoter activity of DNA elements in
this region. In contrast to RbTE and NHEK, rabbit tracheal fibroblasts
transfected with several deletion constructs showed very low levels of
luciferase activity, and no significant difference in promoter activity
was observed between the various regions (Fig. 4). This finding
supports the conclusion that this upstream-regulatory region of the
TGase I promoter is important for squamous-specific regulation of TGase I in keratinocytes and is in agreement with previous findings (34,
52-54).
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Fig. 4.
Comparison of TGase I promoter activity in
NHEK, RbTE, and rabbit tracheobronchial fibroblasts
(RbF). Cells were transfected with -actin-CAT
and either pTG(
54)-LUC, pTG(
37)-LUC, pTG(
750)-LUC, pTG(750*)-LUC,
or pTG(
2.9)-LUC reporter plasmid DNA as described under
"Experimental Procedures" and 48 h later assayed for
luciferase activity and CAT protein levels. The relative luciferase
activity for each construct was calculated and plotted. pTG(750*)-LUC
is identical to pTG(750)-LUC except that it does not contain the region
between bp
37 and
350.
54/
37 region responsible for
5-10-fold transactivation of the basal TGase I promoter contains an
Sp1 consensus sequence at
47 to
40 (Fig.
5A). A two-base mutation in
this Sp1 element reduced transcriptional activity of the
54/+60 fragment to that of the minimal promoter (
37/+60), indicating that
the increase in transcriptional activation is related to this sequence
(Fig. 5B). Gel shift assays, using a 32P-labeled
oligonucleotide TG-Sp1 corresponding to the
54/
37 region and
nuclear protein extracts from squamous differentiated RbTE cells,
revealed the formation of several specific DNA-protein complexes.
Unlabeled TG-Sp1 was able to compete for these complexes, while the
mutated oligonucleotide TG-mtSp1 competed much less efficiently (Fig.
5C). A commercially available Sp1 consensus oligonucleotide
(Sp1cons.) also competed well for binding. An antibody
against Sp1 protein caused a supershift of the upper band, indicating
that Sp1 is a major part of this protein complex (Fig. 5D).
The second and third faster migrating bands were supershifted by
antibodies against Sp3. Antibodies against Sp2 or Egr-1 (not shown), a
transcription factor that binds the consensus sequence GCGGGGGCG,
failed to interact with any of the protein complexes binding
TG-Sp1.
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Fig. 5.
The proximal TGase I promoter contains a
functional Sp1 element. A, sequence of the
oligonucleotide TG-Sp1 encoding the region from 54 to
35 bp. The
location of the two mutations in the oligonucleotide TG-mtSp1 or
reporter construct pTG-(
mt54)-LUC are indicated. The sequence of the
commercial Sp1 oligonucleotide (Sp1cons.) is also shown.
B, RbTE cells were transfected with pTG(
37)-LUC
(
37), pTG(
54)-LUC (
54) or pTG(
mt54)-LUC
(
54mt) and 48 h later collected and assayed for
luciferase activity as described under "Experimental Procedures."
The data shown are the mean of triplicate dishes. C, EMSA
was carried out with 32P-labeled TG-Sp1 and nuclear
extracts from squamous-differentiated RbTE cells in the presence or
absence of a 100-fold excess of unlabeled TG-Sp1, TG-mtSp1, or the
commercial Sp1 oligonucleotide. D, supershift
(arrow) of protein complexes was carried out by including
specific antibodies against Sp1, -2, or -3 in the incubation
mixture.
750/
350 Region of the TGase I
Promoter--
To identify elements in the
750/
350 region that are
involved in the regulation of the TGase I gene, a series of overlapping deletion mutants was constructed by inserting various segments of this
regulatory region into pTG(-37)-LUC (Fig.
6A). Transient transfection
analysis showed that the region most important for transactivation was
located between bp
490 and
470 (Fig. 6B). Deletion of
this sequence within the bp
750 to
350 fragment decreased
transactivation by more than 80%. When ligated to the minimal TGase I
promoter (D10; Fig. 6B) this fragment increased the level of
reporter gene activity by more than 10-fold. The
490 to
470
sequence consists of the almost perfect palindromic structure
CTGGCTGCTGATGTCACCAGCCAG (referred to as TGpal) containing a CREB/AP-1-like sequence (TGATGTCA) in the middle (Fig.
6C). It is interesting to note that this palindromic region including the CREB/AP-1-like element is highly conserved in the corresponding 5'-flanking region of the human TGase I gene (Fig. 6C).
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Fig. 6.
Functional analysis of various deletions of
the 750/
350 5'-flanking region of the TGase I gene.
A, schematic view of the various deletions D1-D11 made in
the
750 to
350 upstream flanking region. B, analysis of
the ability of the various 5'-flanking regions to regulate the
transcription of the luciferase reporter gene in differentiating RbTE
cells. The relative luciferase activity for each construct was
calculated and plotted. The -fold induction from the activity of the
minimal promoter is shown on the right in A.
C, comparison of the nucleotide sequence of the
corresponding 5'-flanking regulatory region containing the
TGpal (underlined) and CREB/AP-1-like site
(boldface type) in the rabbit and human TGase I gene. The
arrows indicate palindromes.
54) they synergistically enhanced
transactivation to a level slightly less than that of the full 2.9-kb
5'-flanking region.
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Fig. 7.
The Sp1 and CREB-like site act in concert to
enhance transcription of the TGase I gene. Reporter plasmids
containing the LUC reporter gene under the control of the minimum TGase
I promoter ( 37), the
54 to +60 promoter region including
the Sp1 site (
54), the minimal promoter plus
TGpal (TGpal/
37), the
54
to +60 promoter region plus TGpal
(TGpal/
54), or the
2.9 kb regulatory
region (
2.9K) were transfected into differentiated RbTE
cells. After 48 h, cells were assayed for reporter gene
activities, and the relative LUC activity was calculated.
750/
350 region of the
TGase I promoter, nuclear extracts from squamous differentiated RbTE
cells and a 400-bp DNA fragment (
750/
350) were used for DNase I
footprint analysis. Although several DNA elements were found to be
protected, the region between bp
490 and
470 containing the
TGpal was one of the most strongly protected sequences
(Fig. 8).
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Fig. 8.
DNase I footprint analysis of the 350/
450
promoter flanking region of the TGase I gene. Footprint analysis
was carried out as described under "Experimental Procedures."
Lane 1, no nuclear extract; lane 2,
nuclear extract from squamous differentiated RbTE cells. The protected
region coincides with that of TGpal as indicated by the
sequence on the right.
37/+60 basic promoter region of pTG(
37)-LUC. The
transcriptional activity of these constructs was tested in
differentiating RbTE cells by transient transfection assays. As
demonstrated in Fig. 9B, transactivation of the LUC reporter
under the control of TGpal was increased about 10-fold
compared with the activity of the basic promoter. The mutations
(M2-M7) that disrupt transcriptional activation most are within the
CREB/AP-1-like sequence (TGATGTCA).
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Fig. 9.
Effect of various mutations on the ability of
TGpal to increase transcription of the luciferase
reporter. A, sequence of TGpal
(wt) and mutated oligonucleotides M1-M8. B, the
different oligonucleotides were inserted into pTG( 37)-LUC just
upstream from the minimal promoter, and their ability to regulate the
transcription of the luciferase reporter gene was analyzed.
490/
470 region of the TGase I promoter, we performed EMSA with a
32P-labeled TGpal oligonucleotide and nuclear
extracts isolated from differentiated RbTE. Several oligonucleotides
(M1-M8; Fig. 9A), containing different point mutations in
this region, were tested for their ability to compete with
32P-labeled TGpal for protein binding. The
oligonucleotide M1 competed as effectively as the unlabeled
TGpal itself (Fig.
10A). The oligonucleotides M2-M4 and M8 were less effective competitors than unlabeled
TGpal, while M6, M7, and to a lesser degree M5 did not
compete well for binding. These results indicate that TCA within the
CREB-like element is optimal for the formation of DNA-protein
complexes. Since TGpal contains an element that shows
similarity to CREB and AP-1 sites, we assessed the ability of consensus
AP-1 (TGAGTCA) and CREB (TGACGTCA) oligonucleotides to compete
with 32P-labeled TGpal for binding to
nuclear proteins from squamous differentiated RbTE cells. As shown in
Fig. 10B, both the consensus AP-1 and CREB oligonucleotides
were able to compete with TGpal; however, they were less
efficient than TGpal itself.
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Fig. 10.
Interaction of nuclear proteins isolated
from squamous-differentiated RbTE cells with oligomers spanning the
TGpal regulatory element. EMSA was carried out using
32P-labeled TGpal and nuclear extracts from
squamous differentiated RbTE cells. Incubations were carried out in the
presence or absence of a 100-fold excess of unlabeled
TGpal, oligonucleotides M1-M8 (A) (see Fig.
9A) or the consensus AP-1 site or consensus CREB-site
(B).
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Fig. 11.
EMSA characterization of protein
interactions with TGpal (A), consensus
AP-1 (B), and consensus CREB (C)
oligonucleotides. Nuclear extracts from differentiated RbTE cells
were preincubated with 2 µg of each antibody as indicated, followed
by incubation with 32P-labeled TGpal, AP-1, or
CREB. The protein-DNA complexes were separated by polyacrylamide gel
electrophoresis.
DISCUSSION
2.9)-CAT transgenic mice
revealed that CAT mRNA was only expressed in squamous tissues and
was restricted to the suprabasal layers comprising the late spinous and
granular cell layers. This pattern of transgene expression correlates
well with that reported for endogenous TGase I mRNA (47). These
results demonstrate that this 2.9-kb region contains elements that are sufficient to direct the tissue- and differentiation-specific expression of TGase I in vivo and are in agreement with a
recent report on the promoter activity of the human TGase I in
transgenic mice (54). This conclusion is supported by observations in
cultured cells showing that the 2.9-kb region of the TGase I promoter
is able to control the transcription of a reporter gene in a cell type-
and differentiation-specific manner (Fig. 4; Refs. 34 and 52).
37/+60 and identified two major sites that are
important in the transcriptional control of TGase I: a palindromic
sequence from
490 to
470 bp (TGpal) containing a
CREB/AP-1-like site in the middle and the region from bp
53 to
37
consisting of a functional Sp1 site. We showed that these two elements
work in concert to regulate the basal promoter activity of the TGase I
gene. Previous studies implicated several AP-2-like sites in the
transcriptional regulation of human TGase I in NHEK cells (51); these
sites may cooperate with the Sp1 and TGpal elements.
194, no
evidence was obtained indicating that this site is important in the
regulation of the TGase I gene. Sp1 elements have been reported to be
able to bind a number of transcriptional factors including members of
the Sp1 family (56). Sp1 contains a DNA-binding domain consisting of
three zinc fingers and binds the consensus sequence 5'-GGGCGG. In
addition to Sp1, three related genes (Sp2, -3, and -4) have been
identified (56, 57). These transcription factors can act alone or in
cooperation with other transcription factors and can enhance or repress
transcription depending on promoter context and cell type. Sp1 elements
have been linked to the regulation of several genes during squamous differentiation. The Sp1 site is needed for optimal transcriptional activation of the involucrin gene, and protein complexes bound to this
site were found to contain Sp1 but not Sp2, -3, or -4 (57). Sp1 sites
have also been implicated in the regulation of SPRR-2A (59) and TGase
III (60). Our mobility shift analysis demonstrated that in
differentiated RbTE cells, Sp1 and to a minor extent Sp3 are part of
protein complexes that interact with the TG-Sp1 element. A recent study
has demonstrated that the ratio of Sp1 to Sp3 is low in
undifferentiated and high in squamous-differentiated NHEK cells and
that Sp1 is involved in the differentiation-specific regulation of
HPV-16 genes (61). This differential expression may hint at a possible
role for Sp1-Sp3 antagonism during squamous differentiation. Antagonism
of Sp1-mediated transcriptional activation by Sp3 has been demonstrated
in several other cell systems (56). In contrast to these observations,
the differentiation-dependent, transient induction of the
cyclin-dependent kinase inhibitor p21WAF1/Cip1
during early stages of squamous differentiation in NHEK cells has been
reported to depend on the interaction of Sp3 rather than Sp1 with a
GC-box in the proximal promoter region of p21WAF1/Cip1
(62). The reported increase in the Sp1/Sp3 ratio during differentiation may be a later event and be part of the down-regulation of
p21WAF1/Cip1. Moreover, the activation or repressor
function of Sp3 may be determined by the sequence or context of the
GC-box.
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ACKNOWLEDGEMENTS |
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We thank Dr. T. Yamaai for advice on the in situ hybridization technique.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Dept. of Medicine, University of Queensland,
Princess Alexandra Hospital, Brisbane 4102, Australia.
§ To whom correspondence should be addressed. Tel.: 919-541-2768; Fax: 919-541-4133; E-mail: jetten{at}niehs.nih.gov.
The abbreviations used are: TGase, transglutaminase; CAT, chloramphenicol acetyltransferase; RbTE, rabbit tracheobronchial epithelial; NHEK, normal human epidermal keratinocyte(s); LUC, luciferase; CREB, cAMP response element-binding protein; SPRR, small proline-rich protein; EMSA, electrophoretic mobility shift assay; PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase pair(s).
2 This program is available on the World Wide Web at http://pdap1.trc.rwcp.or.jp/research/db/TFSEARCH.html.
3 H. Adachi and A. M. Jetten, unpublished observations.
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REFERENCES |
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