From the Department of Biochemistry, University of Iowa,
Iowa City, Iowa 52242
Transcription of the gene for malic enzyme in
chick embryo hepatocytes is stimulated about 30-fold by
triiodothyronine (T3). T3 responsiveness is mediated by seven direct
repeat hexamers that resemble T3 response elements (T3REs); these
elements are located far upstream in the 5
-flanking DNA (Hodnett,
D. W., Fantozzzi, D. A., Thurmond, D. C., Klautky,
S. A., MacPhee, K. G., Estrem, S. T., Xu, G., and
Goodridge, A. G. (1996) Arch. Biochem. Biophys. 334, 309-324). In transiently transfected hepatocytes, single copies of six
of these elements conferred varying degrees of T3 responsiveness to
linked reporter genes. In gel electrophoretic mobility shift analyses,
the T3REs bound retinoid X receptor (RXR)-T3 receptor (TR) heterodimers
and non-RXR/TR factors present in nuclear extracts prepared from
hepatocytes. Binding of the non-RXR/TR factors was specific to
individual T3REs and was unaffected by antibodies to TR or RXR.
Mutagenesis of binding sites for proteins specific for T3REs 2-5
altered binding of the proteins and T3 responsiveness. These factors
appear to bind to and alter function of T3REs without binding directly
to TR, differentiating their actions from other TR cofactors; they were
tentatively characterized as co-repressors, inhibitors, and activators
of T3RE function. Together with RXR and TR, they modulate T3
responsiveness of the gene for chicken malic enzyme.
 |
INTRODUCTION |
Triiodothyronine (T3)1
binds to the nuclear thyroid hormone receptor (TR) and regulates
transcription of T3-responsive genes (1). TR binds to thyroid hormone
response elements (T3REs) in the regulatory regions of T3-responsive
genes. T3REs consist of pairs of hexameric repeats organized as
palindromes, everted repeats or direct repeats, or as extended single
copies of the hexamer (2). In the consensus hexameric T3RE half-site,
RGGWMA, guanosines in positions II and III of the hexamer are critical for DNA-protein interactions (3-5). TR is a member of the superfamily of steroid/thyroid hormone receptors and interacts in solution with
itself and other family members to form dimers that bind to the T3RE
(6-8). Heterodimers of retinoid X receptors (RXR) and TR bind to T3REs
with higher affinity and transactivate more effectively than TR/TR
homodimers (9, 10). The RXR/TR heterodimers bind to direct repeat T3REs
with defined polarity; RXR and TR bind to the upstream and downstream
half-sites, respectively (11, 12). RXR/TR dimers require a 4-bp spacer
region between the half-sites (12, 13). The nucleotide sequence of the
half-sites and flanking DNA and the spacing of the half-sites influence
binding of the RXR/TR heterodimers and their interactions with other
proteins. Consequently, the structure of the T3RE influences the
transactivation potential of the bound heterodimer (4, 14-17).
However, the mechanisms by which the transactivation signal is
transmitted to the basal transcription machinery and by which
T3-dependent transactivation is modulated by other factors
remain poorly understood.
Several cDNAs have been cloned using strategies that rely on the
ability of the encoded proteins to bind to TR and RXR; the proteins
encoded by these proteins may be involved in transmission of the
transactivation signal. In the absence of T3, co-repressors such as
silencing mediator for retinoic acid and thyroid hormone receptors (18)
and NCoR (19, 20) augment the silencing function of TR (21). In the
presence of T3, transactivation is co-activated by steroid receptor
co-activator 1 (22, 23) and inhibited by short heterodimer partner
(24). CREB-binding protein and p300 bind to RXR and function as
bridging proteins between components of this and other signal
transduction pathways (25, 26). Similar bridging proteins (TAFII40 and
TAFII110) are present in Drosophila tissues; this
nonvertebrate also expresses transcription factors that belong to the
steroid/thyroid superfamily of receptors (27, 28). These and other
reports suggest that the binding of such proteins to RXR and TR
influences transactivation.
Understanding of the binding of TR and RXR to T3REs is based primarily
on studies that use characterized response elements and bacterially
expressed or in vitro translated proteins. Functional analyses have used a variety of cell lines, many of which are normally
unresponsive to T3. These experiments have profoundly increased our
understanding of the mechanisms by which T3 regulates transcription.
Nevertheless, results of functional analyses are sometimes consistent
with results of in vitro binding experiments and sometimes
not. Binding may not correlate with function, and functions deduced
from experiments with one cell line may differ from those deduced from
experiments using a different cell line. To determine physiologically
relevant mechanisms, we must use T3REs of natural genes and test
hypotheses about binding and function in physiologically relevant cell
systems.
The gene for chicken malic enzyme is an appropriate model system for
testing hypotheses about the relationship(s) between function of T3REs
and binding of proteins directly to T3REs, to flanking DNA, or to
T3RE-bound receptors. Malic enzyme is involved in the de
novo synthesis of long chain fatty acids and responds to
nutritional and hormonal signals in intact animals (29); these
responses can be mimicked by manipulating the hormone and fatty acid
milieu of chick-embryo hepatocytes in culture (30, 31). In hepatocytes
transfected with chimeric genes containing 5
-flanking DNA of the malic
enzyme gene linked to the gene for chloramphenicol acetyltransferase
(CAT), a robust T3-induced increase in CAT activity is elicited without
overexpressing TR or other protein (31, 32).
The gene for chicken malic enzyme contains at least six putative T3REs;
five are clustered in a 130-bp T3 response unit (T3RU) (32). The T3RU
is centered about 3.8 kilobase pairs upstream of the start site for
transcription and is complemented by an additional T3RE that is about
800 bp downstream of the T3RU. These T3REs resemble degenerate DR4-type
elements; their contributions to T3 responsiveness of the T3RU vary
considerably (32). We report here that, in addition to the nucleotides
within the half-sites, nucleotides flanking each T3RE play critical
roles in defining the differential function of each T3RE; they appear
to do so by binding novel proteins.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Restriction enzymes were obtained from Life
Technologies Inc., New England Biolabs, U.S. Biochemical Corp., and
Boehringer Mannheim. Other enzymes were obtained from the indicated
sources: T4 polynucleotide kinase and the Klenow fragment of
Escherichia coli DNA polymerase I (Boehringer Mannheim) and
Bst polymerase (Bio-Rad). Nucleotides were purchased from
Sigma, Pharmacia Biotech, Inc., or Life Technologies. Radiolabeled
nucleotides and Hyperfilm-HP were obtained from Amersham.
D-Threo-[dichloroacetyl-1,2,14C]chloramphenicol
was purchased from NEN Life Science Products. LipofectACE, Waymouth MD
705/1 medium, and competent E. coli cells of strain DH5
were obtained from Life Technologies. SeaKem LE agarose, NuSieve GTG
agarose, and SpinBind DNA isolation columns were purchased from FMC
Corp. Columns (BioSpin 6) for purifying DNA probes used in gel
electrophoretic mobility shift assays were obtained from Bio-Rad.
Hormones were from Sigma. Polyclonal antibody (IgG) against chicken
TR
was from Santa Cruz Biotechnologies and recognizes both
and
forms. Monoclonal antibody to chicken RXR was provided by Pierre
Chambon (Strasbourg, France) and recognizes
,
, and
forms.
Other chemicals were reagent grade or the best quality commercially
available.
Plasmids--
Construction of pRSV-LUC (luciferase) was
described (32). Bruno Luckow and Gunter Schutz (Heidelberg, Germany)
provided pBLCAT2 (pTKCAT; Ref. 33). Herbert H. Samuels (New York
University) provided the cDNA for chicken TR
(6). Plasmid
Bluescript KS+ was obtained from Stratagene Cloning Systems. Fragments
of chicken malic enzyme DNA containing putative regulatory sequences
were inserted into the multiple cloning site 5
to the virus thymidine kinase (TK) promoter in pBLCAT2.
A double-stranded herpes simplex polymerase chain reaction-generated
DNA fragment (ME
3903/
3703) was subcloned into the SphI and BamHI sites of pTKCAT to generate
p[ME
3903/
3703]TKCAT (pT3RU-TKCAT) (32). Polymerase chain
reaction-generated fragments of DNA were subcloned into the
HindIII and BamHI sites of pTKCAT to make
p[ME
3474/
2715]TKCAT. Most of the TKCAT plasmids containing
individual T3REs were constructed by inserting annealed complementary
36-bp oligonucleotides into the corresponding restriction sites of
pTKCAT. T3RE 7 was subcloned into the HindIII and
BamHII sites of pTKCAT, so that the insertion sites were the
same as those in p[ME
3474/
2715]TKCAT. Synthetic oligonucleotides
were treated with T4 polynucleotide kinase prior to annealing and
ligation. Structures of the resulting plasmid DNAs were confirmed by
restriction mapping and partial sequence analyses.
Cell Culture and Transient Transfection--
Hepatocytes were
isolated from livers of 19-day-old chick embryos; suspended in Waymouth
medium MD 705/1 supplemented with penicillin (60 µg/ml), streptomycin
(100 µg/ml), insulin (50 nM), and corticosterone (1 µM); and incubated in 35-mm tissue culture dishes at
40 °C in 5% CO2 in air (34). Cells were transfected 20 h after plating, using 40 µg of
LipofectACETM/plate. Each plate contained 5 µg of plasmid
DNA: pTKCAT (1 µg) or the molar equivalent of other test constructs,
pRSV-LUC (0.5 µg), and pBluescript KS+ (balance). Transfection medium
was removed at 24 h and replaced with the medium described above,
with or without 1.6 µM T3.
Analysis of Cell Extracts and Statistics--
For each
treatment, cells from two plates were combined and harvested at 48 h after adding T3, lysed by three cycles of freezing and thawing, and
analyzed for protein content (35), luciferase (36), and CAT (37)
activities. Cell lysates to be used for CAT assays were heat-treated
for 30 min at 60 °C; denatured protein was removed by
centrifugation. CAT activity was normalized to protein in the unheated
extracts (CAT activity/µg of protein/15 h) and then corrected for
transfection efficiency. Statistical significances of differences were
determined by the Wilcoxon signed rank test (38). S.E. values are
provided to indicate the degree of variability.
Gel Electrophoretic Mobility Shift Assay--
Each
oligonucleotide probe contained a 5
-extension and was labeled by a
fill-in reaction catalyzed by the Klenow fragment of E. coli
DNA polymerase I. Probes were purified over BioSpin-6 columns.
Blunt-ended, double-stranded competitor oligonucleotides were the same
lengths as the corresponding labeled probes. Nuclear extracts were
prepared from chick embryo hepatocytes incubated for 48 h with
insulin and corticosterone, plus or minus T3 for 24 h. Extracts
were prepared in 20 mM Hepes, pH 7.9, 1.5 mM
MgCl2, 25% glycerol, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.42 M NaCl, and the
protease inhibitors leupeptin (10 mM), benzamidine (2 µg/ml), aprotinin (0.7 µg/ml), and phenylmethylsulfonyl fluoride (0.5 mM) (37). Chicken TR
protein was expressed in
E. coli and purified by heparin-agarose column
chromatography (6). Binding reactions contained 7 µg of nuclear
protein, 0.3 ng of [
-32P]dCTP-labeled double-stranded
DNA (30 nt), 2 µg of poly(dI/dC), 0.01% Nonidet P-40, 0.8 µg of
bovine serum albumin, and 5% glycerol (v/v) plus or minus competitor
DNA in a total volume of 20 µl. Reactions were incubated at room
temperature for 15 min, after which antibody, if any, was added, and
the reaction was incubated for another 15 min. DNA-protein complexes
were subjected to electrophoresis in 6% nondenaturing polyacrylamide
gels at 18 mA in 110 mM Tris base, 110 mM boric
acid, and 2.4 mM EDTA at 4 °C. Gels were dried and
subjected to autoradiography at
70 °C for 16 h. In one
experiment, 25 mM Tris base, 0.19 M glycine, 1 mM EDTA buffer was used instead of the Tris borate buffer
(37) (noted in figure legend).
 |
RESULTS |
T3REs of the Malic Enzyme Gene Are Functionally
Distinct--
Individual putative T3REs were subcloned into pTKCAT and
transfected into hepatocytes to determine how much T3 responsiveness each T3RE would confer when it lacked the context of the entire T3RU
(Fig. 1). T3RE 2 (32) and putative T3RE 5 bestowed large inductions by T3 and conferred basal levels that were 90 and 50% lower, respectively, than that of the entire T3RU. Cells
transfected with putative T3REs 3, 4, and 6 showed small but
statistically significant responses to T3, but they did not show
significant reductions in basal activity vis à vis the
entire T3RU. Putative T3RE 7 conferred an induction by T3 that was
similar in magnitude to that conferred by the
3474 to
2715 bp
region. Thus, in addition to T3RE 2, putative T3REs 3-7 are authentic
T3REs with varying abilities to confer T3 responsiveness to linked
genes. Interestingly, T3 responsiveness for T3RE 5 was higher than that
for a larger DNA fragment that contained T3REs 4, 5, and 6 (results not
shown), suggesting that factors other than TR are involved in T3
responsiveness of the entire T3RU.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 1.
T3 responsiveness conferred by putative T3REs
of the chicken malic enzyme gene. A, T3REs of the T3RU. The
T3RU spans bp 3894 to 3764 of the malic enzyme gene and is
contained within p[ME 3903/ 3703]TKCAT; putative T3REs 2, 3, 4, 5, and 6 contain 26 bp of malic enzyme DNA fused to pTKCAT. Chick embryo
hepatocytes were transiently transfected using LipofectACE (40 µg/plate) and p[ME 3903/ 3703]TKCAT (1 µg/plate or an equimolar
amount of the other constructs), pRSV-LUC (0.5 µg/plate), and
pBluescript DNA (sufficient to bring DNA to 5.0 µg/plate) and treated
with or without T3 for 48 h. Activities of the promoter constructs
were expressed as CAT activity; CAT activity of cells transfected with p[ME 3903/ 3703]TKCAT treated with T3 was set equal to 100, and those transfected with other constructs were normalized thereto. Each
point represents the mean ± S.E. of five or six independent sets
of hepatocytes, using at least two independently prepared batches of
each plasmid. Basal CAT activities were corrected for differences in
transfection efficiency by dividing by luciferase activity of the same
extract (light units/µg of protein). Relative basal CAT activities
were calculated by setting the corrected CAT activities for T3-treated
hepatocytes transfected with p[ME 3903/ 3703]TKCAT equal to 1 and
adjusting all other activities proportionately. For each construct,
-fold response to T3 was calculated by dividing the relative CAT
activity for hepatocytes treated with T3 (+T3) by that for
hepatocytes not treated with T3 ( T3). The -fold responses were calculated for individual experiments and then averaged; they are
not the same as the quotients of the averaged relative CAT activities.
Statistical significance between means within a column
(p < 0.05) is as follows. a,
versus p[ME 3903/ 3703]TKCAT. CAT and luciferase
activities of extracts from T3-treated hepatocytes transfected with
pMET3RU2TKCAT were 25.6 ± 6.1 (mean ± S.E.,
n = 6) percentage of conversion/15 h/µg of protein
and 13 ± 3 × 103 (mean ± S.E.,
n = 6) light units/µg of protein, respectively. CAT
and luciferase activities of extracts from non-T3-treated hepatocytes
transfected with pT3RUTKCAT were 0.09 ± 0.02 (mean ± S.E., n = 6) percentage of conversion/15 h/µg of
protein and 8.0 ± 1.8 × 103 (mean ± S.E.,
n = 6) light units/µg of protein, respectively. B, T3RE 7 of DNase I-hypersensitive site III. Relative CAT
activities were calculated by setting the CAT activities for T3-treated
hepatocytes transfected with p[ME 3474/ 2715]TKCAT DNA to 100 and
adjusting all other activities proportionately. Basal CAT activities
were corrected for differences in transfection efficiency by dividing by luciferase activity of the same extract (LUC; light
units/µg protein). Relative basal CAT activities were calculated by
setting the corrected CAT activities for T3-treated hepatocytes
transfected with p[ME 3474/ 2715]TKCAT equal to 1 and adjusting all
other activities proportionately. Statistical significance between
means within a column (p < 0.05) is as follows.
a, versus p[ME 3474/ 2715]TKCAT; b, versus pTKCAT. CAT and luciferase activities
of extracts from T3-treated hepatocytes transfected with
pME[ 3474/ 2715]TKCAT were 4.1 ± 0.8 (mean ± S.E.,
n = 6) percentage of conversion/15 h/µg of protein
and 7.4 ± 0.4 × 103 (mean ± S.E.,
n = 6) light units/µg of protein, respectively. CAT
and luciferase activities of extracts from non-T3-treated hepatocytes
transfected with pME[ 3474/ 2715]TKCAT were 0.6 ± 0.1 (mean ± S.E., n = 6) percentage of conversion/15
h/µg of protein and 4.3 ± 0.2 × 103
(mean ± S.E., n = 6) light units/µg of protein,
respectively.
|
|
Several mechanisms could explain the differences in T3 responsiveness
bestowed by the various T3REs. First, sequence-dependent differences in binding affinities of the different T3REs for TR/TR homodimers or RXR/TR heterodimers could influence responsiveness. Second, some T3REs may bind TR/TR homodimers and others RXR/TR; the
latter should elicit greater responsiveness. Third, the binding of
heterodimers to T3REs may induce sequence-dependent
structural changes in TR/TR or RXR/TR complexes which, in turn, alter
affinity for a bridging factor. Fourth, non-TR proteins may compete for the site that binds TR/TR or RXR/TR dimers;
sequence-dependent affinity of the T3REs for such proteins
may control the extent of binding of TR to the T3RE. Fifth, non-TR
proteins may bind near or adjacent to TR/TR or RXR/TR dimers and
regulate the transactivation activity of bound TR. These hypotheses
were tested by assessing binding of nuclear proteins to wild-type and
mutant T3REs and promoter function of constructs containing the same
wild-type and mutant sequences.
Specific Binding of Proteins to T3REs--
Each T3RE bound
monomeric and dimeric TR
that had been partially purified from
E. coli transformed with an expression vector for chicken
TR
(results not shown). Nuclear proteins from T3-treated hepatocytes
also bound to each T3RE (Fig. 2).
Antibodies (IgG) to either cTR
or cRXR caused the same two closely
spaced complexes to be supershifted, indicating that they contained
both TR and RXR (bracketed in Fig. 2). Proteins that bind to
TR or RXR also should be supershifted by TR or RXR antibodies. Thus,
binding of TR accessory proteins, TR-interacting proteins, or
integrator proteins to RXR/TR heterodimers may account for the two
closely spaced bands (24-26, 39-41). Alternatively, the two complexes
may contain RXR and/or TR of different sizes. These results suggest that binding of TR/TR homodimers, as opposed to RXR/TR heterodimers, is
not responsible for the weak functional activities of T3REs 3, 4, 6, and 7.

View larger version (83K):
[in this window]
[in a new window]
|
Fig. 2.
Binding of RXR/TR heterodimers to T3REs of
the chicken malic enzyme gene. Gel electrophoretic mobility shift
analyses were conducted using [ -32P]dATP-labeled
double-stranded probes of 30 nucleotides as indicated below
each gel. Nuclear extracts were prepared from hepatocytes incubated
with T3 for 24 h as described under "Experimental Procedures." Each reaction contained 7 µg of nuclear protein. Antibody to cTR (1 µl) or cRXR (0.25 µl) was added to the binding reaction as indicated in the figure and allowed to incubate at room
temperature for 15 min. DNA-protein complexes were visualized on 6%
nondenaturing acrylamide gels. The position of the RXR/TR heterodimers
is indicated by a bracket to the left of each set
of gels.
|
|
Unlabeled fragments containing each of the T3REs and DR4 competed for
RXR/TR complexes when tested against each labeled T3RE (Fig.
3). When T3RE 3 was the probe, unlabeled
T3REs 2, 4, and 5 competed less effectively for RXR/TR than unlabeled
T3RE 3, suggesting that affinity of the weak T3RE 3 for RXR/TR may be higher than those of the strong T3REs 2 and 5. Thus, differing affinities of the T3REs for RXR/TR may not cause the differences in
responsiveness to T3.

View larger version (89K):
[in this window]
[in a new window]
|
Fig. 3.
Non-RXR/TR proteins bind specifically to
T3REs. Nuclear extracts were prepared from hepatocytes incubated
with or without T3 for 24 h as described under "Experimental
Procedures" and indicated for each panel. Gel
electrophoretic mobility shift analyses were conducted using
[ -32P]dATP-labeled double-stranded probes (30 nt).
Each reaction contained 7 µg of nuclear protein. DNA-protein
complexes were visualized on 6% nondenaturing acrylamide gels. The
positions of RXR/TR heterodimers are denoted by a bracket to
the left of each set of gels. A, T3RE 2 was the
labeled probe. Double-stranded competitor T3REs were added to the
binding reactions at either a 100- or 500-fold molar excess over probe
as indicated. DR4 is an idealized T3RE with direct repeat orientation
and a 4-bp spacer; half-sites match the consensus sequence AGGTCA.
B, T3RE 4 was the probe. Double-stranded competitor T3REs
were added to the binding reactions at 100-fold molar excess over
probe. C, T3RE 3 was the probe. Other conditions were as
described for T3RE 4. D, T3RE 5 was the probe. Other
conditions were as described for T3RE 4. E, T3RE 7 was the
probe. Other conditions were as described for T3RE 4. IC,
insulin plus corticosterone; ICT3, insulin plus
corticosterone plus T3.
|
|
Antibodies to TR or RXR did not supershift or inhibit binding of all
complexes bound to the T3REs; additional factors bound to each T3RE but
did not form stable complexes with RXR/TR (Figs. 2 and 3,
A-E). Migration patterns of the non-RXR/TR complexes formed
with T3RE 2 were similar to those formed with T3RE 3 or 4 but different
from those with T3REs 5 or 7, and that with T3RE 5 was different from
that with T3RE 7. The non-RXR/TR complex(es) bound to each T3RE
appeared to be specific for that T3RE. Competitions of DNA-protein
complexes with a 100-500-fold molar excesses of the other T3REs failed
to displace, or displaced poorly, the non-RXR/TR complexes on
nonidentical T3REs. These results suggest that different non-RXR/TR
proteins bind to each of the T3REs.
The pattern of migration of the non-RXR/TR proteins and competition for
binding of non-RXR/TR proteins by the various T3REs were similar for
T3REs 5 and 6. Furthermore, T3REs 5 and 6 contain 21 bp of overlap.
T3RE 5, however, bestowed a much higher T3 responsiveness to
transfected cells than T3RE 6. A series of binding and transfection experiments suggested that only one of these two DR4 elements is
functional in the natural gene (results not shown). No additional analyses of T3RE 6 are reported in this study.
Medium containing T3 affected binding and migration of the non-RXR/TR
complexes with some of the T3REs. For T3REs 2 (results not shown) and 4 (Fig. 3B), the migration patterns and extents of binding of
non-RXR/TR complexes to each T3RE were similar in extracts prepared
from cells incubated with or without T3. In contrast, for T3REs 3, 5, and 7 (Fig. 3, C-E), major non-RXR/TR binding complexes
were formed when the nuclear extracts were from hepatocytes not treated
with T3; these complexes were absent in extracts prepared from
T3-treated cells. Antibodies to TR and RXR did not supershift
non-RXR/TR complexes in extracts prepared from cells that were not
treated with T3, confirming that these complexes did not bind to TR or
RXR under these conditions. T3RE 7 also bound non-RXR/TR complexes that
formed in extracts prepared from T3-treated cells, but not, or less so,
in those from cells not treated with T3. In sum, the non-RXR/TR
complexes appear to contain factors that are specific for the elements
to which they bind; binding of these factors may contribute to the
differences in T3 responsiveness and basal activities of cells
transfected with TKCAT linked to the different T3REs.
T3 in the medium caused a slight increase in the mobility of complexes
formed between most of the T3REs and RXR/TR (Fig. 3). A similar
phenomenon has been reported previously (42). For most of the T3REs,
the extent of binding of RXR/TR was unaffected by T3. For T3RE 7, however, increased binding of RXR/TR in extracts from T3-treated cells
was correlated with decreased binding of one of the non-RXR/TR
complexes. At this T3RE, RXR/TR and one of the non-RXR/TR proteins may
bind to the same site and compete with one another for binding to the
site in a T3-dependent manner.
T3RE 5--
A gel electrophoretic mobility shift assay that used a
high ionic strength buffer permitted resolution of the slowly migrating complex containing T3RE 5 into two distinct bands (Fig.
4C). The mutations in mD
reduced binding of RXR/TR to T3RE 5 because they destroyed the
downstream half-site of T3RE 5 (Fig. 4B). In addition, however, they reduced binding of protein (NP5) in the more slowly migrating of the two non-RXR/TR complexes. Formation of this complex may be dependent on binding of RXR/TR to the T3RE. The mutations in m3
also reduced binding of NP5 (Fig. 4C), but they had no effect on binding of RXR/TR (Fig. 4B).

View larger version (42K):
[in this window]
[in a new window]
|
Fig. 4.
T3RE 5 and its mutants: binding of
nuclear proteins and T3 responsiveness of transfected cells.
A, nucleotide sequences of T3RE 5 (half-sites
underlined) and its mutant forms (changes shaded). B, double-stranded competitor DNAs were
added to the binding reactions at a 100-fold molar excess with respect
to labeled probe. Gel electrophoretic mobility shift analyses were
conducted as described in the legend to Fig. 3 and used
[ -32P]dATP-labeled double-stranded probes (30 nt). The
position of the RXR/TR heterodimers is denoted by a bracket
to the left of the set of gels. C, localization
of a high molecular weight protein that binds specifically to the
3 -flanking region of T3RE 5. The position of the high molecular weight
protein is indicated by an arrowhead. This gel
electrophoretic mobility shift analysis was conducted using
Tris-glycine buffer rather than Tris borate as in B.
D, hepatocytes were transfected as described in the legend to Fig. 1. The results are expressed as described in the legend to Fig.
1 and represent the means ± S.E. of five or six independent experiments using at least two independently prepared batches of each
plasmid. Relative CAT activities were calculated by setting the
corrected CAT activities for T3-treated hepatocytes transfected with
pT3RE 5[ME 3794/ 3769]TKCAT equal to 100 and adjusting all other
activities proportionately. Basal CAT activities were corrected for
differences in transfection efficiency by dividing by luciferase activity of the same extract (light units/µg of protein). Relative basal CAT activities were calculated by setting the corrected CAT
activities for T3-treated hepatocytes transfected with pTKCAT equal to 1 and adjusting all other activities proportionately. Statistical significance between means within a column
(p < 0.05) is as follows. a,
versus pT3RE 5[ME 3794/ 3769]TKCAT; b,
versus pTKCAT. CAT and luciferase activities of extracts
from T3-treated hepatocytes transfected with pT3RE
5[ME 3494/ 3769]TKCAT were 0.4 ± 0.1 (mean ± S.E.,
n = 6) percentage of conversion/15 h/µg of protein
and 6.3 ± 1.0 × 103 (mean ± S.E.,
n = 6) light units/µg of protein, respectively. CAT
and luciferase activities of extracts from non-T3-treated hepatocytes
transfected with pTKCAT were 0.08 ± 0.01 (mean ± S.E., n = 6) percentage of conversion/15 h/µg of protein
and 3.8 ± 0.6 × 103 (mean ± S.E.,
n = 6) light units/µg of protein, respectively.
|
|
Mutant m3
of T3RE 5 was introduced into the TKCAT vector (p5
m3
[ME
3794/
3769]TKCAT) to characterize function of NP5 (Fig. 4D). This mutation caused a significant decrease in T3
responsiveness. The decrease was due entirely to elevated basal
activity. The NP5 binding activity was present only in extracts made
from hepatocytes that were not treated with T3. This suggests that the
binding of NP5 to T3RE 5 may contribute to the silencing function of
TR.
T3RE 2--
Mutant forms of T3RE 2 were linked to TKCAT (Fig.
5A), and their promoter
activities were tested. Mutation of the 5
-flanking region did not
reduce T3 responsiveness significantly compared with that of wild-type
T3RE 2 (Fig. 5B). Mutation of the spacer region (2mS)
reduced (by 93%) but did not eliminate T3 responsiveness. Mutation of
the 3
-flanking region (2m3
) increased T3 responsiveness by almost
5-fold. Mutations that caused a change in response to T3 did not change
basal activity. Thus, the sequences flanking the downstream half-site
are important for the characteristic response of T3RE 2 to T3.

View larger version (49K):
[in this window]
[in a new window]
|
Fig. 5.
T3RE 2 and its mutant forms: T3
responsiveness of transfected cells and binding and relative affinity
of nuclear proteins. A, sequences of wild-type and mutant
forms of T3RE 2. Changed nucleotides are shaded; boxed
regions denote T3RE half-sites. B, hepatocytes were
transfected as described in the legend to Fig. 1. The results are
expressed as described in the legend to Fig. 1 and represent the
means ± S.E. of six independent experiments using at least two
independently prepared batches of each plasmid. Relative CAT activities
were calculated by setting the corrected CAT activities for T3-treated
hepatocytes transfected with pT3RE 2[ME 3883/ 3858]TKCAT equal
to 100 and adjusting all other activities proportionately. Basal CAT
activities were corrected for differences in transfection efficiency by
dividing by luciferase activity of the same extract (light units/µg
of protein). Relative basal CAT activities were calculated by setting
the corrected CAT activities for T3-treated hepatocytes transfected
with pTKCAT equal to 1 and adjusting all other activities
proportionately. Statistical significance between means within a column
(p < 0.05) is as follows. a,
versus pT3RE 2[ME 3883/ 3858]TKCAT; b,
versus pTKCAT. CAT and luciferase activities of extracts
from T3-treated hepatocytes transfected with pT3RE
2[ME 3883/ 3858]TKCAT were 0.7 ± 0.2 (mean ± S.E.,
n = 6) percentage of conversion/15 h/µg of protein
and 4.0 ± 0.9 × 103 (mean ± S.E.,
n = 6) light units/µg of protein, respectively. CAT
and luciferase activities of extracts from non-T3-treated hepatocytes
transfected with pTKCAT were 0.05 ± 0.01 (mean ± S.E., n = 6) percentage of conversion/15 h/µg of protein
and 2.5 ± 0.7 × 103 (mean ± S.E.,
n = 6) light units/µg of protein, respectively. C, double-stranded competitor DNAs were added to the binding
reactions with nuclear extract at either 100- or 500-fold molar excess
over probe as indicated. Gel electrophoretic mobility shift analyses were conducted using an [ -32P]dATP-labeled
double-stranded probe (30 nt). Nuclear extracts were prepared from
hepatocytes in culture incubated in the absence of T3; each reaction
contained 7 µg of nuclear protein. DNA-protein complexes were
visualized on a 6% nondenaturing acrylamide gel. The position of the
RXR/TR heterodimers is denoted by a bracket. Binding of a
novel low molecular weight protein specific to T3RE 2 is indicated by
an arrowhead.
|
|
Mutation of the upstream half-site (2mU) had no effect on binding of
the novel protein specific for T3RE2 (NP2). Mutation of any other
region of T3RE 2, including the 3
-flanking region, inhibited binding
of NP2 (Fig. 5C). The 2mD competitor DNA did not compete for
RXR/TR at 100- and 500-fold molar excesses, consistent with this region
of T3RE 2 being part of a functional TR binding site. Despite having
opposing effects on T3 responsiveness, mutants 2mS and 2m3
had similar
protein binding profiles; both bound RXR/TR but not NP2 (Fig.
5C). These results suggest that the binding of RXR/TR and
NP2 may be coupled; NP2 may have TR-dependent and TR-independent modes of binding, or the NP2 complex may be composed of
two or more proteins that bind differentially.
Conversion of Weaker T3REs into Stronger Ones--
The binding
site for a novel protein may not be easily localized within a T3RE
because sequence and orientation of the T3RE may affect binding of both
RXR/TR and novel proteins as well as function. For example, adenosine
and thymidine in positions III and IV of the spacer, respectively, are
unfavorable for high affinity binding of RXR/TR to a DR4 T3RE and
reduce T3 responsiveness. Guanosines in positions II and III are
critical for TR binding in the downstream half-site (3) but are less
critical for RXR binding to the upstream half-site (5). A cytosine in
position VI of the downstream half-site inhibits binding of TR/TR (16). The dinucleotides TG or TA immediately upstream of either half-site combined with the appropriate hexameric sequence improves T3
responsiveness. The stronger T3REs of the chicken malic enzyme gene (2 and 5) contain only favorable nucleotides, and weaker T3REs 3, 4, and 7 contain unfavorable nucleotides (Fig. 6).
We next determined whether changing unfavorable nucleotides to
favorable nucleotides would improve T3 responsiveness of weak T3REs
and, if so, whether it increased or decreased binding of novel
proteins.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 6.
Nucleotide sequences of functional
T3REs. The 5 - and 3 -end points are listed for each T3RE;
half-sites are boxed. Unfavorable nucleotides are indicated
by a shaded circle, favorable nucleotides by a shaded
square.
|
|
T3RE 3 contains unfavorable nucleotides in positions IV of the spacer
and VI of the downstream half-site. Favorable nucleotides were inserted
at these positions either simultaneously (3mS-mD) or separately (3mS
and 3mD) (Fig. 7A). Compared
with cells transfected with wild-type T3RE 3, T3 responsiveness of
hepatocytes transfected with p3mS-mD[ME
3833/
3808]TKCAT or
p3mD[ME
3833/
3808]TKCAT were increased only about 2-fold (Fig.
7B). Mutation of the spacer had no effect on T3
responsiveness (3mS). Basal activity conferred by the double mutant was
reduced compared with that for the wild type and was significantly
different from that conferred by either pT3RE 2[ME
3883/
3858]TKCAT
or pTKCAT. Thus, a large increase in T3 responsiveness of T3RE 3 may
require more than high affinity binding of RXR/TR.

View larger version (44K):
[in this window]
[in a new window]
|
Fig. 7.
T3RE 2, T3RE 3 and mutant forms of T3RE 3: T3
responsiveness of transfected cells and binding of nuclear proteins.
A, sequences of wild-type T3RE 2 and wild-type and mutant
forms of T3RE 3. Changed nucleotides are shaded; boxed
regions denote T3RE half-sites. B, hepatocytes were
transfected as described in the legend to Fig. 1. The results are
expressed as described in the legend to Fig. 1 and represent the
means ± S.E. of 6-10 independent experiments using at least two
independently prepared batches of each plasmid. Relative CAT activities
were calculated by setting the corrected CAT activities for T3-treated
hepatocytes transfected with pT3RE2TKCAT equal to 100 and adjusting all
other activities proportionately. Basal CAT activities were corrected
for differences in transfection efficiency by dividing by luciferase
activity of the same extract (light units/µg of protein). Relative
basal CAT activities were calculated by setting the corrected CAT
activities for T3-treated hepatocytes transfected with pTKCAT equal to
1 and adjusting all other activities proportionately. Statistical significance between means within a column is as follows. a,
versus pT3RE2TKCAT (p < 0.01);
b, versus pT3RE3TKCAT (p < 0.05); c, versus pTKCAT (p < 0.01);
d, versus pTKCAT (p < 0.02). CAT
and luciferase activities of extracts from T3-treated hepatocytes
transfected with pT3RE2TKCAT were 2.9 ± 0.6 (mean ± S.E.,
n = 6) percentage of conversion/15 h/µg of protein
and 13 ± 2 × 103 (mean ± S.E.,
n = 6) light units/µg of protein, respectively. CAT
and luciferase activities of extracts from non-T3-treated hepatocytes
transfected with pTKCAT were 0.09 ± 0.01 (mean ± S.E., n = 6) percentage of conversion/15 h/µg of protein
and 5.8 ± 0.6 × 103 (mean ± S.E.,
n = 6) light units/µg of protein, respectively. C, double-stranded competitor DNAs were added to the binding
reactions with nuclear extract at a 100-fold molar excess over probe as indicated. Gel electrophoretic mobility shift analyses were conducted using an [ -32P]dATP-labeled double-stranded probe (30 nt). Nuclear extracts were prepared from hepatocytes in culture
incubated with or without T3 for 24 h; each reaction contained 7 µg of nuclear protein. DNA-protein complexes were visualized on a 6%
nondenaturing acrylamide gel. The position of the RXR/TR heterodimers
is denoted by a bracket. Binding of a novel high molecular
weight protein specific to T3RE 3 is indicated by an
arrowhead.
|
|
Competition with wild-type or mutated versions of T3RE 3 was used to
localize binding of the high molecular weight complex (Fig.
7C) found in cells incubated without T3. Only 3mD DNA failed to compete with wild-type T3RE 3 for binding of HNP3 (high molecular weight novel protein of T3RE 3). This result suggests that in extracts
from cells not treated with T3, the unfavorable nucleotide in the
downstream half-site may contact HNP3. Decreased binding of HNP3 to 3mD
correlated with increased T3 responsiveness of cells transfected with
p3mD[ME
3833/
3808]TKCAT. Extracts from cells incubated with or
without T3 also contain a low molecular weight protein that binds to
T3RE 3 and potentially could inhibit function of T3RE 3.
T3RE 4 contains one unfavorable nucleotide in position II of the
downstream half-site; this substitution should exert a strong negative
effect on function. This nucleotide was changed to a more favorable one
in p4mD[ME
3809/
3784]TKCAT and in a double mutant, p4
m5
-mD[ME
3809/
3784]TKCAT (Fig.
8A). The latter construct contains a second mutation in the 5
-flanking region that also should
improve function. The constructs that contained the 4mD substitutions
conferred increased responses to T3 (Fig. 8B). Although basal activities were increased significantly relative to that bestowed
by wild-type T3RE 4, relative CAT activities in the presence of T3 were
increased much more, resulting in T3 responsiveness similar to that
conferred by T3RE 2. Insertion of a more favorable nucleotide in the
5
-flanking region increased basal but not T3-induced activity and
reduced the "-fold" T3 response to that for the vector alone. These
results suggest that the unfavorable nucleotide in the downstream
half-site of T3RE 4 suppresses T3 responsiveness of a potentially
strong T3RE.

View larger version (52K):
[in this window]
[in a new window]
|
Fig. 8.
T3RE 2, T3RE 4, and mutant forms of T3RE 4:
T3 responsiveness of transfected cells and binding and relative
affinity of nuclear proteins. A, sequences of wild-type T3RE
2 and wild-type and mutant forms of T3RE 4. Changed nucleotides are
shaded; boxed regions denote T3RE half-sites.
B, hepatocytes were transfected as described in the legend
to Fig. 1. The results are expressed as described in the legend to Fig.
1 and represent the means ± S.E. of 6-10 independent experiments
using at least two independently prepared batches of each plasmid.
Relative CAT activities were calculated by setting the corrected CAT
activities for T3-treated hepatocytes transfected with pT3RE2TKCAT
equal to 100 and adjusting all other activities proportionately. Basal
CAT activities were corrected for differences in transfection
efficiency by dividing by luciferase activity of the same extract
(light units/µg of protein). Relative basal CAT activities were
calculated by setting the corrected CAT activities for T3-treated
hepatocytes transfected with pTKCAT equal to 1 and adjusting all other
activities proportionately. Statistical significance between means
within a column is as follows. a, versus
pT3RE2TKCAT (p < 0.05); b,
versus pTKCAT (p < 0.01); c,
versus pT3RE4TKCAT (p < 0.05). CAT and
luciferase activities of extracts from T3-treated hepatocytes
transfected with pT3RE2TKCAT were 2.9 ± 0.6 (mean ± S.E.,
n = 10) percentage of conversion/15 h/µg of protein
and 13 ± 2 × 103 (mean ± S.E.,
n = 10) light units/µg of protein, respectively. CAT
and luciferase activities of extracts from non-T3-treated hepatocytes
transfected with pTKCAT were 0.09 ± 0.01 (mean ± S.E., n = 10) percentage of conversion/15 h/µg of protein
and 5.8 ± 0.6 × 103 (mean ± S.E.,
n = 10) light units/µg of protein, respectively. C, double-stranded competitor DNAs were added to the binding
reactions at a 100-fold molar excess over probe as indicated. Gel
electrophoretic mobility shift analyses were conducted using
[ -32P]dATP-labeled double-stranded probe (30 nt).
Nuclear extracts were prepared from hepatocytes in culture incubated
with or without T3 for 24 h; each reaction contained 7 µg of
nuclear protein. DNA-protein complexes were visualized on a 6%
nondenaturing acrylamide gel. The position of the RXR/TR heterodimers is denoted by a
bracket. Binding of a novel low molecular weight protein
specific for T3RE 4 is indicated by an arrowhead.
D, double-stranded competitor DNAs were added to the binding
reactions at a molar excess of 1-500-fold over probe as indicated. Gel
electrophoretic mobility shift analyses were conducted using
[ -32P]dATP-labeled double-stranded probe (30 nt).
Nuclear extracts were prepared from hepatocytes that were treated with
T3; each reaction contained 7 µg of nuclear protein. DNA-protein
complexes were visualized on a 6% nondenaturing acrylamide gel.
Radioactivity in the individual complexes of the dried acrylamide gel
was quantitated using an InstantImager (Packard); radioactivity present
in the lane without nuclear extract was subtracted as background.
|
|
Except for the single substitution at position II of the downstream
half-site, all mutations of T3RE 4 decreased the ability of competitor
oligonucleotides to bind to NP4 (novel protein of T3RE 4) (Fig.
8C). Mutant 4mD competed better than the wild-type T3RE 4 for binding of both RXR/TR and NP4 to the wild-type probe (Fig.
8D). The increased binding affinity of 4mD is correlated with a dramatic increase in T3 responsiveness of cells transfected with
p4mD[ME
3809/
3784]TKCAT. Thus, high affinity RXR/TR binding and
the ability to bind NP4 may be characteristic of a very strong T3RE.
T3RE 7 contains five unfavorable nucleotide contacts. One construct was
designed with favorable substitutions at all sites (p7
m5
-mS-mD[ME
3081/
3056]TKCAT) to determine if T3 responsiveness could be improved. Unexpectedly, these favorable mutations resulted in
a loss of T3 responsiveness (results not shown). Basal activity of the
mutated T3RE 7 was not significantly different from that for wild-type,
so that decreased activation by T3 was the affected step. This confirms
the unusual nature of T3RE 7.
 |
DISCUSSION |
These studies were designed to examine T3RE function and
modulation of TR action using physiologically relevant natural T3REs. The natural genes for human and rat growth hormone were used in earlier
transfection studies, but rat liver TR that was partially purified and
devoid of other DNA-binding proteins was used with radiolabeled T3 in
the corresponding T3RE binding analyses. Thus, only TR and proteins
that interacted directly with TR were detected (14, 43). Our binding
analyses used endogenous proteins rather than purified or in
vitro translated receptors. We wanted to know if non-RXR/TR
proteins bound to these T3REs. Differential functional activity of
T3REs could be due to the binding of different proteins and/or to
differences in T3RE structure. We report here that novel proteins bind
to and alter the function of T3REs; for at least one T3RE, they do so
without interacting directly with TR or RXR/TR. For some T3REs, an
alternative explanation is that nucleotide sequence influences
conformation of RXR/TR and alters interactions with coactivators and/or
corepressors.
Some hormone response elements are functional when present in tandem or
in the context of other elements but fail to bestow large responses
when utilized individually (43-46). Plasmid T3RE2TKCAT bestowed T3
responsiveness equivalent to that of the idealized T3REs, pTREpalTKCAT
and pDR4TKCAT, each of which contain two or more copies of the T3RE. A
version of T3RE 4, p4mD[ME
3809/
3784]TKCAT, that contains only
favorable nucleotides conferred T3 responsiveness equivalent to those
of the strong idealized T3REs and T3RE2. Thus, weak T3REs can become
strong T3REs if appropriate bases are changed.
High affinity RXR/TR binding is a critical factor in the T3
responsiveness conferred by strong T3REs (5, 14, 16, 47). Given their
variable responsiveness, we were surprised that all of the chicken
malic enzyme T3REs bound RXR/TR; binding of TR/TR was not detected
using nuclear extracts from hepatocytes. Binding of TR/TR dimers or TR
monomers was observed only with partially purified cTR
. RXR/TR bound
to 4mD with higher affinity than it bound to T3RE 4, suggesting that
differences in RXR/TR binding affinity may account for the observed
functional differences. However, the affinity of a non RXR/TR protein
also was increased.
Binding sites for the novel proteins that bound to T3RE 3 and 5 were
localized to specific sequences in each T3RE. Decreased binding of HNP3
was correlated with a small but significant increase in T3
responsiveness. In contrast, decreased binding of NP5 resulted in a
large decrease in T3 responsiveness. Both HNP3 and NP5 bound to T3REs
only in the absence of T3, suggesting that the changes in
responsiveness may have been due to changes in basal activities rather
than decreased levels of induced activities. HNP3 was tentatively characterized as a inhibitor of TR's repressor function; mutation of
the putative binding site for HNP3 caused a decrease in basal activity
and a comparable increase in T3 responsiveness. The action of NP5
resembled that of a group of TR cofactors known as co-repressors because it augmented the silencing function of TR. Interestingly, binding of NP5 required nucleotides just 3
of the downstream T3RE 5 half-site; to our knowledge this is the first report of 3
-flanking
sequences playing a role in the function of T3REs.
NP5 differs from other co-repressors in that it binds specifically to
the DNA and fails to supershift upon the addition of TR antibody. Other
TR co-repressors interact directly with TR to enhance the silencing
function when T3 is absent (18, 19, 48-50). NCoR may be an exception
because it binds to both DNA and TR. The region to which NP5 bound was
necessary for its binding to T3RE 5 but was not sufficient to confer
binding of NP5 in the context of T3RE 3 (results not shown); additional
specific sequences may be required for NP5 binding.
NP4 may be a co-activator of T3RE function; increased binding is
correlated with increased T3 responsiveness. In addition, NP4 bound to
DR4, and DR4 shows a high degree of T3 responsiveness in transfected
hepatocytes (results not shown). The binding site for this protein was
not localized, because most mutations used to localize the site
disrupted its binding. Binding of NP4 may be dependent on the binding
of RXR/TR and/or nucleotide sequence of the T3RE. RXR/TR forms in
solution through an interface in the ligand binding domain of each
receptor and forms a second interface in the DNA binding domains; the
second interface restricts the dimer to specific direct repeat
sequences separated by four nucleotides (13). The orientation of the
dimer on the element is critical for high affinity binding and
subsequent transactivation by ligand; changes in conformation of the
element upon binding of RXR/TR dimers may be critical to effective
transactivation. DNA bending may be the way by which the element
acquires the required conformation (5, 14, 49).
When bound by RXR/TR, T3RE 2, T3RE 4, and DR4 may have similar
conformations that are recognized by NP4 and possibly NP2. Interestingly, DR4 competed with T3RE2 for the binding of NP2 and
competed with T3RE 4 for the binding of NP4. By contrast, T3RE 2 and
T3RE 4 (or 4mD) were unable to compete for the binding of NP4 and NP2,
respectively. This suggests that the structural conformation of the
T3RE may be important for the binding of NP2 and NP4 to DR4 and
that additional features may be involved in conferring the specificity
of NP2 and NP4 binding to T3RE 2 and T3RE 4, respectively.
The T3 responsiveness of T3RE 7 was decreased when "favorable"
nucleotide substitutions were made, whereas that for other T3REs
increased. This T3RE has an unusual nucleotide in position VI of each
half-site as well as an unusual C in position I of the downstream
half-site. Other T3REs also contain such unusual nucleotides; the Sp1
and NF-
B motifs in the human immunodeficiency virus type-1 long
terminal repeat can bind to and be transactivated by cTR
(51). The
viral protein Tat requires specific DNA sequences to convert a
functionally inactive TR binding site in the Sp1 element to an
active site, confirming the importance of other factors in T3RE
function.
We thank Dr. Sung Soo Chung for helpful
discussions and technical expertise. We are indebted to Drs. Herbert H. Samuels and Pierre Chambon for providing the cDNA for chicken TR
cloned into the pET8c expression vector and the monoclonal antibody to
cRXR, respectively.