(Received for publication, May 19, 1995; and in revised form, October 24, 1995)
From the
Tissue transglutaminase (transglutaminase type II) is an
intracellular protein cross-linking enzyme that accumulates in
connective tissue and in cells undergoing apoptosis. Retinoids regulate
the transcription of the mouse tissue transglutaminase gene via
activation of regulatory elements contained within 4 kilobases of the
5`-end of the gene. Co-transfection studies with retinoid receptor
expression vectors in CV-1 cells demonstrated that the mouse tissue
transglutaminase promoter is activated by ligand activation of either
retinoic acid receptor-retinoid X receptor (RARRXR) heterodimers
or RXR homodimers. Optimal induction is achieved with retinoid receptor
panagonists; partial activation can also be achieved with either
RAR-specific or RXR-specific retinoids. Retinoid-dependent activation
of the tissue transglutaminase promoter depends on both a proximal
regulatory region containing sequences highly conserved between the
human and the mouse tissue transglutaminase promoters and a distal
region that includes a 30-base pair retinoid response element
(mTGRRE1). mTGRRE1 contains three hexanucleotide half-sites (two
canonical and one non-canonical) in a DR7/DR5 motif that bind both
RAR
RXR heterodimers and RXR homodimers. These studies suggest
that retinoid-dependent expression of the mouse tissue transglutaminase
gene is mediated by a versatile tripartite retinoid response element
located 1.7 kilobases upstream of the transcription start site.
Tissue transglutaminase is a multifunctional protein involved in a number of distinct biological processes(1, 2) . In addition to its well characterized activity as a catalyst of protein cross-linking reactions, recent studies have shown that tissue transglutaminase also can function as a GTP-dependent regulator of phospholipase C activity(3) . A unique feature of tissue transglutaminase's cross-linking activity is that, unlike other transglutaminases, which are restricted in their activity, tissue transglutaminase cross-links proteins in both intracellular and extracellular compartments(2) . Extracellular protein cross-linking activity occurrs in basement membranes(4) , during chondrogenesis (5) and particularly during wound healing(6) . Tissue transglutaminase-mediated cross-linking of intracellular proteins occurs in cells undergoing apoptotic or programmed cell death(7, 8) . It has been suggested that the induction and activation of tissue transglutaminase in apoptotic cells results in protein cross-linking that stabilizes the apoptotic bodies and prevents the leakage of cytosolic proteins into the extracellular space during cellular fragmentation(9) .
The multiple activities of tissue transglutaminase suggest that its
expression and activation must be tightly regulated. Tissue
transglutaminase has a very selective pattern of tissue-specific
expression, with the highest levels of the enzyme accumulating in
endothelial cells, in erythrocytes, and in cells of the lens epithelium (10, 11) . Retinoids appear to be generalized
regulators of tissue transglutaminase expression. Rats rendered vitamin
A-deficient have a marked depression in the level of tissue
transglutaminase activity in many tissues(12) . This decrease
is rapidly reversed by administration of all-trans retinoic acid
(ATRA). ()Administration of retinoids to rats with normal
retinoid nutritional status causes increased transglutaminase activity
in several tissues (12, 13, 14, 15) . The induction of
tissue transglutaminase in vivo can be reproduced in
vitro. Exposure of macrophages to ATRA results in a dramatic
increase in tissue transglutaminase activity (16) , replicating
the induction that occurs during macrophage activation(17) .
Retinoids also induce the enzyme in human promyelocytic leukemia
(HL-60) cells(18, 19) , replicating the induction of
the enzyme that occurs during myeloid cell apoptosis. TGF-
and
retinoids induce transglutaminase activity in tracheal epithelial
cells(20, 21) , and IL-6 induces the enzyme in
hepatocytes (22) .
The transcriptional effects of retinoids
on gene expression are mediated by at least two families of retinoid
receptors, retinoic acid receptors (RARs) and retinoid X receptors
(RXRs), both of which belong the superfamily of nuclear receptors that
mediate the transcriptional effects of the steroid hormones, vitamin D,
and thyroid hormone(23, 24) . The complexity of
retinoid signaling is further increased by the fact that there are
three distinct genes encoding RARs (RAR, -
, and -
) and
RXRs (RXR
, -
, and -
) that give rise to homologous but
distinct receptor
proteins(25, 26, 27, 28, 29, 30, 31) .
Both the RAR and RXR receptor subtypes are expressed in specific
temporal and spatial patterns during embryogenesis and in
tissue-specific patterns in adult animals, suggesting that they each
have distinctive biological activities(32) . Vitamin A
(all-trans retinol) does not bind to retinoid receptors, but its
metabolites, such as ATRA and 9-cis-RA, do. ATRA only binds to RARs,
whereas 9-cis-RA binds to and activates both RARs and
RXRs(33, 34) . Retinoid receptors do not bind to DNA
as monomers; they require dimerization for activity. RARs
preferentially dimerize with RXRs to form RAR
RXR heterodimers
that are thought to be obligatory intermediates in the effects of RAR
ligands on gene expression(35, 36) . RXRs can
associate with several other receptors including thyroid hormone
receptors (TR), the vitamin D receptor (VDR), and the receptor for
drugs that induce peroxisomal proliferation (PPARs)(23) . RXRs
also can homodimerize to give transcriptionally active
complexes(37, 38) .
Homo- and heterodimeric
retinoid receptor complexes bind to distinct retinoid response elements
(RREs) embedded in the regulatory regions of retinoid-responsive
genes(23, 39) . Although there is considerable
variability in the sequence and structure of the RREs in
retinoid-regulated genes, they conform to a general canonical sequence
in which two directly repeated receptor-binding hexanucleotide motifs
(consensus (A/G)G(G/T)TCA) are separated by a variable number of
intervening nucleotides(39) . RXR homodimers bind
preferentially to hexanucleotide motifs separated by one nucleotide
(DR1), whereas RXRRAR heterodimers bind to motifs separated by
one, two, or five nucleotides (DR1, DR2, or DR5). Other heterodimeric
complexes (RXR
TR, RXR
VDR, and RXR
PPAR) bind to motifs
with characteristic preferred spacings(39) .
We have been
interested in the molecular mechanisms that contribute to
retinoid-regulated expression of the tissue transglutaminase gene.
Studies in both normal and leukemic myeloid cells have shown that ATRA
acts as an acute and direct activator of tissue transglutaminase gene
expression(40) . Studies with synthetic retinoids that activate
only RARs or RXRs have suggested that ligand activation of both
receptors can induce tissue transglutaminase expression, suggesting
that both RAR and RXR-mediated signaling pathways may regulate
expression of this enzyme(21, 41) . ()Recently we cloned the promoter of the mouse tissue
transglutaminase gene and have shown that, like the endogenous gene,
this promoter is regulated by ligand activation of both RARs and
RXRs.
In the studies reported here, we have isolated and
characterized a retinoid response element embedded in this promoter
fragment. We have found that this retinoid response element is
versatile, capable of binding to and being activated by both
RAR
RXR heterodimer and RXR homodimer receptor complexes. These
observations suggest that induction of tissue transglutaminase
expression may be a response to multiple pathways of retinoid
signaling.
Figure 7:
Binding of Retinoid Receptor Complexes to
mTGRRE1. A, specificity of heterodimer binding. Nuclear
extracts from Sf-1 cells transfected with an RAR or an RXR
baculovirus expression vector were combined (1:1 ratio) and incubated
with radiolabeled mTGRRE1 alone (lane 2), mTGRRE1 plus a
100-fold molar excess of a nonspecific unlabeled competitor
oligonucleotide (Nsp; lane 3), or mTGRRE1 plus a
100-fold molar excess of unlabeled mTGRRE1 (Sp; lane
4) under conditions described under ``Experimental
Procedures.'' The mixture was fractionated on a 5% polyacrylamide
gel, and the mobility of the radiolabeled oligonucleotide was
identified by autoradiography. Lane 1 is radiolabeled probe
alone. The open arrow identifies the mobility of the
radiolabeled probe, and the filled arrow identifies the band
of shifted electrophoretic mobility. B, effect of RXR
on
RAR
binding. Varying amounts of RAR
nuclear extracts were
combined with an RXR
-containing extract and then incubated with
radiolabeled mTGRRE1 under conditions described in the legend to Fig. 6A. Lane 1, probe alone; lane 2,
RAR
alone; lane 3, RAR
and RXR
(2:1 ratio), lane 4, RAR
and RXR
(4:1 ratio); lane 5,
RAR
and RXR
(8:1 ratio). The open arrow identifies
the mobility of the radiolabeled probe, and the filled arrows identify bands of shifted electrophoretic mobility. C,
specificity of RXR
binding. An RXR
containing nuclear extract
was incubated with radiolabeled mTGRRE1 alone (lane 2),
mTGRRE1 plus a 100-fold molar excess of a nonspecific unlabeled
competitor oligonucleotide (Nsp; lane 3), or mTGRRE1
plus a 100-fold molar excess of unlabeled mTGRRE1 (Sp; lane 4) under conditions described under ``Experimental
Procedures.'' The open arrow identifies the mobility of
the radiolabeled probe, and the filled arrows identify the
bands of shifted electrophoretic mobility.
Figure 6:
Comparison of the retinoid-inducible
activity of pmTG3.8-Luc and pmTG3.8-mTGRRE1-Luc. A,
retinoid receptor panagonists. CV-1 cells were co-transfected with
RAR
(0.1 µg) and RXR
(0.1 µg) expression vectors,
pmTG3.8-Luc (0.5 µg; filled symbols) or
pmTG3.8-
mTGRRE1-Luc (0.5 µg; open symbols), and
pSV2-
-gal (0.5 µg). Transfected cells were treated for 20 h
with either 9-cis-RA (open and filled
squares) or an equimolar mixture of TTNPB and LG100069 (open and filled triangles). Cells were lysed and assayed for
luciferase activity, and the -fold induction was calculated as
described in the legend to Fig. 1. Values shown are the mean
± S.D. of triplicate determinations. B,
receptor-selective retinoids. CV-1 were cells co-transfected with
pmTG3.8-Luc (filled symbols) or pmTG3.8-
mTGRRE1-Luc (open symbols) as described in the legend to Fig. 5A. Transfected cells were treated for 20 h with
either TTNPB (open and filled squares) or LG100069 (open and filled circles), and the -fold induction of
luciferase activity was calculated as described in Fig. 5A. Values shown are the means ± S.D. of
triplicate determinations.
Figure 1:
Structure and activity
of the mouse tissue transglutaminase reporter gene. A,
transglutaminase gene and promoter/reporter gene structures. The
structure of genomic clone mTG-GC3 including exons I and II,
introns, and the 5`-flanking DNA are shown. The structure of
pmTG3.8-LacF including the pUC18 plasmid (thin line), tissue
transglutaminase promoter (-3800 to +66, striped
box),
-galactosidase structural gene (open box), and
an intron and polyadenylation signal from the murine protamine-1 gene (dotted box) are shown. B,
-galactosidase
activity. HeLa cells transiently transfected with pLacF or pmTG3.8-LacF
and the control vector pSV2-CAT were cultured in control media or media
containing ATRA for 48 h, and then
-galactosidase and CAT activity
was measured as described under ``Experimental Procedures.''
Values shown are the mean ± S.D. of triplicate
determinations.
Figure 5:
Deletion analysis of the mouse tissue
transglutaminase promoter. A, retinoid responsiveness of
deletion constructs. Fragments of the mouse tissue transglutaminase
promoter linked to CAT were generated as described under
``Experimental Procedures.'' The deletion constructs were
transiently transfected into RAR-3T3 cells, and the ATRA-inducible
CAT activity was measured as described in the legend to Fig. 3.
Constructs giving a reproducible >2-fold induction of CAT activity
were scored as responsive (+), and those with consistently lower
levels of activation were scored as unresponsive(-). B,
nucleotide sequence of segments of the mouse tissue transglutaminase
promoter necessary for retinoid-inducible activation. HR-1,
homology region I. Shown is a comparison of the nucleotides of mouse (upper sequence, -1088 to -1152) and human (lower sequence, -1597 to -1662). Identical
nucleotides are boxed. mTGRRE1, mouse tissue
transglutaminase retinoid response element 1. Shown are both strands of
mTGRRE1 (-1703 to -1751); candidate hexanucleotide
half-sites are highlighted (identified as A, B, and C); and the orientation and spacing of the half-sites are
identified by the arrows and spacers. DR7 is direct
repeat with a 7-nucleotide spacer, and DR5 is a direct repeat with a
5-nucleotide spacer.
Figure 3:
Effect of receptor-selective retinoids on
the activation of pmTG3.8-Luc in RARRXR
co-transfected
CV-1 cells. CV-1 cells co-transfected with RAR
(0.1 µg) and
RXR
(0.1 µg) expression vectors, pmTG3.8-Luc (0.5 µg), and
pSV2-
-gal (0.5 µg) were treated with varying concentrations of
9-cis-RA (filled squares), TTNPB (filled
triangles), LG100069 (open triangles), or solvent control
(<0.1% ethanol) for 20 h. Cells were lysed and assayed for
luciferase activity, and the -fold induction was calculated as
described in the legend to Fig. 1. Values shown are the means
± S.D. of triplicate determinations.
Figure 2:
Retinoid-dependent activation of
pmTG3.8-Luc cotransfected with RAR and RXR
expression
vectors. CV-1 cells were transiently transfected with either a
combination of RAR
(0.1 µg) and RXR
(0.1 µg)
expression vectors or either expression vector alone, pmTG3.8-Luc (0.5
µg), and pSV2-
-gal (0.5 µg) under conditions described
under ``Experimental Procedures.'' Cells were treated with
9-cis-RA (1 µM) or solvent (<0.1% ethanol) for
20 h and then lysed, and luciferase activity was assayed. Luciferase
activity was normalized for transfection efficiency
(
-galactosidase activity). The -fold induction was calculated as
the ratio of the normalized luciferase activity in cells treated with
retinoid to that in control cells. Values shown are the mean ±
S.D. of triplicate determinations. The inset shows a schematic
representation of pmTG3.8-Luc. The open box denotes the mouse
tissue transglutaminase promoter, the striped box the
luciferase structural gene, and the filled box the
3`-untranslated region, intron, and polyadenylation signal from the
SV40 vector. The thin line represents the remainder of the
pGL2 cloning vector.
Both RAR and RXR receptors are
encoded by multigene families(23) . To assess the role of the
individual RAR and RXR subtypes in retinoid-dependent transcription of
the tissue transglutaminase gene, CV-1 cells were co-transfected with
the transglutaminase promoter/reporter construct and combinations of
receptor expression vectors (Table 1). None of the RAR receptors
alone induced significant retinoid-dependent promoter activation (1.6,
1.1, and 1.8-fold activations for RAR, -
, and -
,
respectively). RXR
and RXR
increased promoter activity 2.9-
and 2.8-fold, respectively, whereas RXR
was inactive (1.2-fold).
Cells transfected with RXRs plus RARs had higher levels of promoter
activation than cells transfected with RXRs alone.The lack of
retinoid-dependent activity of the transglutaminase promoter in
RXR
-transfected cells was promoter-specific since co-transfection
of the same cells with the RXR
expression vector and another
RXR-responsive reporter construct, RXRE-TK-luciferase, showed
9-cis-RA-dependent transcriptional activation (data not
shown).
Figure 4:
Effect of ATRA on the transcriptional
activity of pmTG1.8-CAT transiently transfected into RAR-3T3
cells. A, CAT assay. Equivalent amounts (2.5 µg) of
pBasic-CAT, pmTG1.8-CAT, or RARE-TK-CAT and pSV
-
-Gal
(1.5 µg) were transiently transfected into RAR
-3T3 cells under
conditions described under ``Experimental Procedures.'' Cells
were treated for 24 h in the presence or absence of ATRA (1
µM); then they were lysed, and CAT activity was determined
by enzymatic assay. Shown is the autoradiograph of the chromatogram of
[
C]chloramphenicol and its acetylated
derivatives. Lane 1 is from cells transfected with pCAT-Basic. Lanes 2 and 3 are from cells transfected with
pmTG1.8-CAT and treated with control media (lane 3) or media
containing 1 µM ATRA (lane 4). Lanes 5 and 6 are from cells transfected with RARE-TK-CAT and
treated with control media (lane 4) or media containing 1
µM ATRA (lane 5). B, normalized CAT
activity. Acetylated [
C]chloramphenicol was
quantitated and normalized for transfection efficiency
(
-galactosidase activity). Values shown are the means of duplicate
determinations.
To locate the sites of retinoid regulation within pmTG1.8-CAT we
made several deletions in the reporter construct (Fig. 5A).
Removal of a 1-kb segment of the promoter immediately upstream of the
core promoter region (pmTG1.8-SmaI-CAT) did not alter its
retinoid-dependent activity. The removal of an additional 439 bp
(pmTG1.8-
SmaI
SphI-CAT) also had no effect
on the retinoid inducibility of the construct. This residual
retinoid-responsive reporter construct contained only three small
fragments of the 3.8-kb tissue transglutaminase promoter, the 60-bp
core promoter, a 113-bp SphI-SmaI fragment, and a
125-bp PstI-SphI fragment. Removal of either of the
two fragments upstream of the core promoter resulted in a complete loss
of retinoid-inducible activity. These observations suggested that
retinoid-dependent activation of the core tissue transglutaminase
promoter depended on the cooperation of two small upstream regulatory
regions.
Sequence analysis of the two fragments of the tissue
transglutaminase promoter necessary for retinoid-dependent activation (Fig. 5B) suggests that they both may have important
regulatory functions. The proximal regulatory sequence (SphI-SmaI fragment, -1207 to -1094)
contains an unusual set of DNA motifs that is highly conserved between
the human and mouse tissue transglutaminase
promoters(53) . This 67-bp segment of DNA (homology
region I), which is located far upstream in both the mouse (-1087
to -1152) and the human (-1597 to -1682) promoters,
includes 45/67 nucleotides that are identical in the two promoters (Fig. 5B). The function of this segment is unknown, but
its conservation between species and its role in the retinoid-dependent
activation of the transglutaminase promoter suggests that it may have
an important regulatory role.
The distal regulatory element (PstI-SphI) includes three directly repeated hexanucleotide motifs (highlighted in Fig. 5B), separated by 7 and 5 nucleotides (DR7/DR5). Two of these motifs (the A and C motifs in Fig. 5B) conform to the canonical retinoid receptor binding motif, (A/G)G(G/T)TCA), and one (the B motif) differs in the substitution of a C residue for an A in position -1734. This distal regulatory fragment of the mouse transglutaminase promoter is a potential tissue transglutaminase retinoid response element (mTGRRE1).
To assess the
contribution of mTGRRE1 to retinoid regulation of the tissue
transglutaminase promoter we deleted it from the 3.8-kb promoter
fragment and compared the retinoid-dependent activation of the modified
promoter (pmTG3.8-mTGRRE1-Luc) with that of the intact promoter
(pmTG3.8-Luc) (Fig. 6). The intact and modified reporter
constructs were co-transfected with RAR
and RXR
expression
vectors into CV-1 cells. In cells treated with either 9-cis-RA
or equimolar concentrations of TTNPB and LGD1069, deletion of the
DR7/DR5 response element caused a much lower retinoid-dependent
induction of luciferase activity than in cells transfected with the
intact promoter (Fig. 6A). To determine if the loss in
response to 9-cis-RA reflected a selective loss in ligand
activation of either RAR or RXR receptors, we also measured the
induction of luciferase activity in cells treated with either TTNPB or
LGD1069 alone (Fig. 6B). TTNPB alone increased
luciferase activity to the same degree regardless of whether the
promoter did or did not include the DR7/DR5 element. The same was true
for LGD1069. Although the LGD1069 dose-response curve was shifted to
the right of the TTNPB curve, it induced a similar modest increase in
promoter activity in both the intact and modified reporter constructs.
The induction by either TTNPB alone or LGD1069 alone was similar to the
induction by either 9-cis-RA or TTNPB plus LGD1069 in cells
transfected with pmTG3.8-
mTGRRE1-Luc. This induction was much
lower than the maximal induction of pmTG3.8-Luc by 9-cis-RA or
TTNPB plus LGD1069 (compare Fig. 6, A and B).
These studies suggest that the small activation of the mouse
transglutaminase promoter generated by either TTNPB or LGD1069 alone
does not depend on the presence of the DR7/DR5 response element,
whereas the significantly larger stimulation of activity generated by
either 9-cis-RA or TTNPB plus LGD1069 does.
Figure 8:
Competitive binding analysis of the
specificity of RARRXR
binding to mTGRRE1. A,
sequence of the oligonucleotides used in the competitive binding
studies. B, comparison of displacement by mTGRRE1 and DR1,
DR4, and DR5. A combination of RAR
RXR
nuclear extracts
(1:1 ratio) was incubated with radiolabeled mTGRRE1 and varying amounts
of unlabeled mTGRRE1 (filled squares), unlabeled DR1 (open
circles), unlabeled DR5 (open squares), unlabeled DR4 (closed circle), or the nonspecific oligonucleotide (
)
under conditions described in the legend to Fig. 6A.
The intensity of the shifted band was quantitated by densitometry of
the autoradiograph as described under ``Experimental
Procedures.'' C, comparison of displacement by mTGRRE1
and half-site rearranged oligos. A combination of
RAR
RXR
nuclear extracts (1:1 ratio) was incubated with
radiolabeled mTGRRE1 and varying amounts of the following unlabeled
oligonucleotides, unlabeled mTGRRE1 (filled squares), MUT A1 (open squares), MUT C1 (
), and MUT B1 (open
circles) under conditions described in the legend to Fig. 6, A and B.
Heterodimers of RAR and RXR bind with high affinity to directly
repeated consensus hexanucleotide motifs separated by one, two, or five
nucleotides(39) . To compare the affinity of RARRXR
heterodimers for mTGRRE1 with their affinity for either DR1 or DR5
consensus RREs (Fig. 8A), we measured the competitive
inhibition of radiolabeled mTGRRE1 binding to RAR
RXR
by
unlabeled oligonucleotides, mTGRRE1, DR1, DR4, and DR5 (Fig. 8B). Both DR1 and DR5 competed equivalently with
mTGRRE1 for binding to RAR
RXR
, whereas DR4 and the
nonspecific oligonucleotide did not compete.
The oligonucleotide
mTGRRE1 is comprised of three hexanucleotide half-sites (A, B, and C)
separated by seven and five nucleotides (Fig. 8A). The
consensus retinoid receptor binding motifs GGGTCA (sites A and C) and
the variant motif AGGTCC (site B) are located on the
``antisense'' DNA strand, such that A (GGGTCA) is 7 bp
upstream of B (AGGTCC), which is in turn 5 bp upstream of C (GGGTCA).
To assess the contribution of each half-site to receptor binding we
synthesized oligonucleotides in which nucleotides within the A, B, or C
half-sites were rearranged (Fig. 8A). We then compared
the ability of these mutant oligonucleotides to compete with mTGRRE1
for binding to RARRXR
(Fig. 8C).
Rearrangement of nucleotides in the A half-site (RRE-MUT A1) gave an
oligonucleotide that competed very well for RAR
RXR binding to
mTGRRE1. The affinity of receptors for this oligonucleotide appears to
be greater than their affinity for the mTGRRE1, since the competition
curve of the RRE-MUT A1 oligonucleotide is shifted to the left of the
competition curve of unlabeled mTGRRE1 (Fig. 8C).
Rearrangement of the nucleotides in the C half-site (RRE-MUT C1) gave
an oligonucleotide that competes weakly with mTGRRE1 for receptor
binding. A point mutation in the B half-site was made by replacing the
C residue in the position corresponding to nucleotide -1734 with
an A. This mutation converts the non-canonical hexanucleotide motif
AGGTCC to a canonical retinoid receptor binding motif AGGTCA (Fig. 8A). This mutant oligonucleotide (RRE-MUT B1) was
a very poor competitor of receptor binding to mTGRRE1. These studies
suggested that perturbations in the B and C hexanucleotide motifs
disrupted RAR
RXR
binding to the mTGRRE1, whereas
perturbations in the A motif had no deleterious effect.
To confirm
the conclusions reached from the competitive binding assays we also
measured the ability of the RARRXR
receptor combination
to bind directly to the oligonucleotides containing modifications in
the A, B, or C half-sites (Fig. 9). The receptors bound very
well to the RRE-MUT A1 oligonucleotide, producing a shifted band with
the same electrophoretic mobility as the complex formed on the mTGRRE1 (Fig. 9, lane 5 versus lane 2). The increased intensity
of the shifted band in the sample containing the RRE-MUT A1
oligonucleotide supports the conclusion from the competitive binding
studies that the RAR
RXR
receptor complexes have a
higher affinity for this oligonucleotide than they do for mTGRRE1. The
same band-shifted complex is not seen when receptors are combined with
either the RRE-MUT C1 or RRE-MUT B1 oligonucleotides (lanes 8 and 11). Some low affinity complexes with anomalous
mobility are formed on these oligonucleotides, but the single
RAR
RXR-dependent complex formed on the mTGRRE1 and RRE-MUT A1
oligonucleotides is not detected. These studies support the conclusion
of the competitive binding studies that RAR
RXR complexes
preferentially bind the oligonucleotides in which the B and C sites are
intact. It appears that replacement of a single nucleotide in the B
hexanucleotide half-site is sufficient to disrupt receptor binding to
mTGRRE1.
Figure 9:
Binding of RARRXR
to
mTGRRE1 and mutated oligonucleotides. Radiolabeled mTGRRE1 (lanes
1-3), MUT A1 (lanes 4-6), MUT B1 (lanes
7-9) and MUT C1 (lanes 10-12) were incubated
with RAR
RXR
nuclear extracts (1:1 ratio) alone (lanes 2, 5, 8, 11) or in the
presence of a 100-fold molar excess of unlabeled oligonucleotide (Sp; lanes 3, 6, 9, and 12) under conditions described under ``Experimental
Procedures'' and in the legend to Fig. 6. The radiolabeled
oligonucleotides alone were run in lanes 1, 4, 7, 10.
Studies of receptor binding to the native and
mutant mTGRRE1s demonstrated that disruption of the A hexanucleotide
half-site increased receptor binding. To determine if this increased
affinity for receptors improved the transcriptional enhancer properties
of the oligonucleotide, we measured its transcriptional activity in the
CV-1 cell co-transfection assay (Table 3). The mutant enhancer
element (mTGRRE1-MUTA1-TK-Luc) showed no retinoid-dependent
transcriptional activation in cells transfected with either
RARRXR
or RXR
alone. Thus, although this mutated
oligonucleotide binds receptors better than mTGRRE1, it is much less
efficient in its ligand-dependent transcriptional enhancer activity.
Our approach has been
to reduce the size of the transglutaminase promoter and then to
identify residual segments essential for retinoid-dependent activation.
Using this approach we have identified two regions of the promoter
critical for this activation. One of these, the most upstream, contains
a triplicate retinoid receptor binding motif that functions as a
ligand-dependent enhancer element. Although this retinoid-response
element (mTGRRE1) can activate a heterologous promoter such as the
thymidine kinase promoter, it cannot by itself confer ligand-dependent
transcriptional activity on the mouse tissue transglutaminase promoter.
For this activity it must be coupled with a second short DNA segment
(HR-1) that is located 1 kb upstream from the transcription start site.
This segment of DNA is unusual because it is one of the very few
segments of DNA that is highly conserved between the human and the
mouse tissue transglutaminase promoters(53) . Within the 67-bp span of HR-1 the sequence homology is striking;
not only are there no gaps in the optimal nucleotide alignment, but
homologous sequences are grouped into colinear segments of 5-15
nucleotides. This conservation of sequence appears to reflect important
functional properties of the promoter, since combination of this
element with the upstream retinoid-responsive motif is permissive for
retinoid-dependent transcriptional activation.
The observation that
retinoid-dependent promoter activity requires the cooperation of two
distinct regulatory elements resembles the situation in the mouse CRABP
II promoter(58) . In both promoters two distant and distinct cis-regulatory elements are required for efficient ligand
dependent transactivation. In the case of the mouse CRABP II promoter,
both elements bind retinoid receptors. In the mouse transglutaminase
promoter the upstream element binds retinoid receptors; the downstream
element does not contain sequences that match a canonical retinoid
receptor binding motif, although recent studies suggest that it too may
have retinoid receptor binding activity. ()Detailed
characterization of the proteins associated with both the upstream and
downstream regulatory elements will be required to explain the
cooperation between these two regulatory regions.
Activation of the tissue transglutaminase promoter by retinoid receptor panagonists appears to be linked to the activity of a specific retinoid response element (mTGRRE1) that is located 1.7 kb upstream of the transcription start site. In promoter constructs from which this element was excised, RAR- or RXR-agonists could induce a slight activation of the promoter (suggesting residual or cryptic retinoid response elements within the promoter) but there was no greater activation of the promoter if the RAR- and RXR-specific retinoids were combined or if the promoter was stimulated with a panagonist such as 9-cis-RA. In promoter constructs in which the mTGRRE1 was intact, RAR- and RXR-specific retinoids induced some activation but it was much less than that achieved either by panagonists or by combinations of receptor-selective retinoids. It appears that mTGRRE1 has the pharmacologic properties of a retinoid receptor panagonist response element.
The configuration of mTGRRE1 is
complex and includes three directly repeated receptor-binding motifs
(half-sites). This is a pattern recognizable in a number of response
elements from hormone- regulated
genes(67, 68, 69, 70) . The
configuration of the MTGRRE1 is strikingly similar to the thyroid
hormone response element (TRE) of the Moloney leukemia virus long
terminal repeat promoter (Fig. 10,
MoMLV-LTR)(42, 72) . Both response elements include an
upstream canonical half-site located either 7 or 11 nucleotides
upstream from a core receptor binding sequence. The MoMLV-LTR TRE
element includes a GGTCA motif separated by four nucleotides from an
AGGTCC motif. In mTGRRE1 the same half-sites are present, but their
order is reversed. The AGGTCC motif is located five nucleotides
upstream of the canonical GGTCA halfsite. The MoMLV-LTR TRE binds
thyroid hormone receptors with high affinity and does not bind retinoid
receptors(72) . The mTGRRE1 core motif binds retinoid receptors
(both RARRXR heterodimers and RXR homodimers), but it does not
bind thyroid hormone receptors.
In both cases the sequence
of the non-canonical half-site plays a critical role in receptor
binding. In the context of the MoMLV-LTR-TRE, conversion of the sixth
position C into an A (AGGTCC to AGGTCA) increases retinoid receptor
binding(42) , whereas in the context of mTGRRE1 the same
conversion of the same nucleotide abolishes retinoid receptor binding.
It appears that both the spacing and nucleotide sequence of the
receptor binding half-sites dictate receptor specificity in these
naturally occurring hormone response elements.
Figure 10: Comparison of mTGRRE1 and the TRE of the MoMLV-LTR
The multiplicity of half-sites in mTGRRE1 is a common feature of naturally occurring hormone response elements(23) . Furthermore, there is evidence that cooperation between these half-sites contributes to the transactivation process. The TRE of the rat growth hormone promoter contains three half-sites. Mutation of any of these half-sites abolishes the transcriptional activity of the entire element(70) . Similarly the half-sites of the rat CRBPI RXRE cooperate(69) . mTGRRE1 appears also to conform to this type of regulation. Even though the distal two half-sites (B and C) are sufficient for retinoid receptor binding, the intact tripartite element is required for efficient transcriptional activity. We do not know the molecular basis for the cooperation between the half-sites in mTGRRE1. The upstream A motif could contribute an additional site for retinoid receptor binding in vivo or it could be a site of binding for one or more additional transcription activating factors.
Figure 11: A model of retinoid-dependent activation of the mouse tissue transglutaminase gene promoter.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U24148[GenBank].