Structural Requirements of the Glucocorticoid and Retinoic Acid Response Units in the Phosphoenolpyruvate Carboxykinase Gene Promoter
Takashi Sugiyama,
Donald K. Scott,
Jen-Chywan Wang and
Daryl K. Granner
Department of Molecular Physiology and Biophysics Vanderbilt
University School of Medicine Nashville, Tennessee 37232-0615
 |
ABSTRACT
|
---|
The phosphoenolpyruvate carboxykinase (PEPCK) gene
promoter contains a glucocorticoid response unit (GRU) that includes
three accessory factor-binding sites (AF1, AF2, and AF3), two
glucocorticoid receptor-binding sites (GR1 and GR2), and a cAMP
response element. All of these elements, and the proteins that
bind to them, are required for a complete glucocorticoid response. The
PEPCK promoter also contains a retinoic acid response unit (RARU) that
consists of two retinoic acid response elements (RARE1 and RARE2) that
bind retinoic acid receptor/9-cis-retinoic acid
receptor heterodimers. The sequences of RARE1 and RARE2 coincide
with those for AF1 and AF3, respectively. Thus, the PEPCK promoter can
mediate different hormone responses through hormone response units that
utilize common elements, but that bind different sets of proteins. We
reasoned that each response might require a unique structural assembly
and therefore tested how various arrangements of the PEPCK promoter
affect the actions of either glucocorticoids or retinoic acid.
The activation of the PEPCK gene in response to glucocorticoids
requires a specific set of cis-acting elements that must be
precisely positioned within the GRU. The distance between AF2 and GR1
is critical for the glucocorticoid response, and since the AF2 factor,
HNF3, interacts with GR in vitro, this protein-protein
interaction may be important for the glucocorticoid response. By
contrast, the distance and orientation requirements of AF1 and AF3 with
respect to GR1 are more flexible. In the case of the RARU, although the
relative positions of RARE1 and RARE2 are important for the retinoic
acid response, more tolerance for distance and stereospecific alignment
between RARE1 and RARE2 is allowed. In addition, we show that the GRU
and the RARU can act as a module, within a restricted region, in the
context of the PEPCK promoter and in limited contexts of a heterologous
promoter. These observations suggest that the structural requirements
of the GRU and the RARU are different, and this may have important
implications for how multiple hormonal signals are integrated through
this promoter.
 |
INTRODUCTION
|
---|
Phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) catalyzes
the conversion of oxaloacetate to phosphoenolpyruvate and is an
important rate-controlling step in hepatic gluconeogenesis. PEPCK is
not allosterically or posttranslationally modified. Rather, the
activity of PEPCK is modulated by altering the abundance of the
protein, i.e. it is regulated at the transcriptional level
by several hormones (1, 2, 3). PEPCK gene transcription is positively
regulated by glucagon (through cAMP), glucocorticoids, and retinoic
acid (RA), while insulin inhibits the transcription of the PEPCK gene
(2, 4, 5).
Induction of transcription of the PEPCK gene by glucocorticoids is
achieved through a complex glucocorticoid response unit (GRU) (6). The
GRU includes, as a linear array from 5' to 3', two accessory
factor-binding sites, AF1 and AF2, two glucocorticoid receptor
(GR)-binding sites, GR1 and GR2, and a third accessory factor-binding
site, AF3 (7, 8). These five elements are positioned between -455 to
-321 relative to the transcription initiation site (see Fig. 1
). An intact cAMP response element (CRE,
located between -93/-86) is also required for a full glucocorticoid
response, and thus is part of the GRU (Ref. 9 and Fig. 1A
). The CRE is
more than 200 bp from the rest of the GRU and is also required for
basal transcription of the PEPCK gene (10). Thus, for simplicity, the
GRU can be split into the distal component of the GRU (dGRU; AF1, AF2,
GR1, GR2, and AF3, Fig. 1A
) and the proximal component (the CRE, Fig. 1
). The proteins that mediate the accessory activities through AF1,
AF2, and AF3 have been identified. Both hepatic nuclear factor 4 (HNF4)
and chicken ovalbumin upstream promoter transcription factor (COUP-TF)
bind the AF1 element and act as accessory factors to the glucocorticoid
response (11). Members of the hepatic nuclear factor 3 (HNF3) family
and the CCAAT-enhancer binding protein (C/EBP) family bind to AF2,
although only binding of HNF3 specifically correlates with the ability
of the AF2 element to potentiate the glucocorticoid response (12).
COUP-TF also binds to AF3 and acts as the accessory factor through this
element (8). The CRE is important for basal transcription, the cAMP
response, and the glucocorticoid response (9, 10, 13). A number of
proteins bind the CRE, including CREB, C/EBP family members, and AP1.
CREB and GR interact in vitro (9), but this interaction has
not been proven to be required for the glucocorticoid response. In
fact, recent experiments suggest that C/EBPß is the accessory factor
acting through the CRE (K. Yamada and D. K. Granner,
unpublished observation).

View larger version (23K):
[in this window]
[in a new window]
|
Figure 1. Schematic Diagram of the PEPCK Gene Promoter GRU,
the Distal GRU, and the RARU
A, The cis elements and associated
trans-acting factors required for the glucocorticoid and
RA responses of the PEPCK gene are shown. The location of each element
with respect to the transcription start site is indicated above
the schematic of the PEPCK promoter. The sequence that contains
AF1, AF2, GR1, GR2, and AF3 is designated the distal component of the
GRU (dGRU) to distinguish it from the GRU, which contains the dGRU and
the CRE. B, The sequences of the accessory factor elements, AF1, AF2,
and AF3, as well as GR1 and GR2 are shown. The sequence of RARE1
contains a direct repeat of imperfect nuclear hormone receptor
half-sites separated by 1 bp. The sequence of RARE2 contains a direct
repeat of a degenerate RAR/RXR half-site ( ) and a consensus
half-site ( ) separated by 5 bp. AF3 is coincident with the -site.
The location of each cis element with respect to the
transcription start site is shown above these
sequences.
|
|
AF1, AF2, AF3, and the CRE are all pleiotropic elements. In addition to
their role in the glucocorticoid response, AF1 and AF3 also bind
heterodimers of retinoic acid receptor (RAR) and
9-cis-retinoic acid receptor (RXR) and mediate the RA
response of the PEPCK gene (14, 15). In this context, AF1 and AF3 are
referred to as retinoic acid response elements 1 and 2 (RARE1 and
RARE2), respectively. RARE1 contains imperfect direct repeat half-sites
separated by 1 bp (designated DR1), and RARE2 is a degenerate DR5-type
RARE (Fig. 1B
). Mutations in RARE1 and RARE2 each reduce the RA
response by about 50%, and RARE1 and RARE2 each confer a RA response
in the context of a heterologous promoter (4, 15). Therefore, both
RARE1 and RARE2 are required for the RA response of the PEPCK promoter
and, together, they constitute the PEPCK RA response unit (RARU, Ref.
15 and see Fig. 1A
). It is noteworthy that RA is required for the
proper expression and regulation of the PEPCK gene in transgenic mice,
and that the RARU apparently mediates these effects (16, 17). AF2, in
addition to its glucocorticoid accessory factor activity, mediates a
substantial portion of the inhibitory response of insulin and is a
component of an insulin response unit [IRU (18)]. The CRE, along with
a C/EBP
-binding site in the P3I region of the PEPCK promoter
(located between -246/-238), mediates the cAMP response (10, 19).
Thus, each hormone response is mediated by a different set of multiple
elements, termed hormone response units (HRUs). Furthermore, many of
the individual elements participate in more than one HRU and are
capable of binding more than one set of protein complexes.
In the case of the glucocorticoid response of the PEPCK gene, the
substitution of the AF2 element with certain transcription
factor-binding sites replaces accessory activity, but other binding
sites are ineffective (12). This suggested to us that specific
transcription factors are required for accessory factor activity and
that a precise structure of the GRU might be required for the proper
induction of the PEPCK gene by glucocorticoids. The interaction between
nuclear factors and DNA is an essential event for transcriptional
regulation in eukaryotic cells. DNA-binding nuclear factors interact
with one another and also with other proteins, termed coactivators, to
form complex networks of DNA-protein-protein interactions (20, 21).
Thus, the 5'-regulatory regions of genes are often comprised of large
nucleoprotein complexes whose correctly assembled structures are
required for the proper regulation of the gene. For instance, Thanos
and Maniatis (22) have shown that the virus-mediated induction of the
human interferon-ß (IFNß) gene requires a specific set of
regulatory elements that must be precisely arranged in relation to the
helical phasing of the DNA template (22).
If specific DNA elements can bind different trans-acting
factors with similar affinity, and these factors are present in
approximately equal abundance in the nucleus, what determines the
specificity of the hormone response? A structure/function analysis of
the PEPCK promoter was initiated in an effort to explore this question.
Here we show that there are some similarities, but also some important
differences, in the structural requirements for function through the
distal component of the GRU and the RARU in the PEPCK promoter (Fig. 1
). The activation of the PEPCK gene in response to glucocorticoids
requires that a specific set of cis-acting elements be
positioned correctly within the GRU. The distance and spatial alignment
between AF2 and GR1 is critical for the glucocorticoid response, but
more flexibility is allowed for the distance and orientation of AF1 and
AF3 with respect to GR1. In contrast, the relative positions of RARE1
and RARE2 to one another are important for the RA response through the
RARU, but their specific alignment is less important. In addition, we
show that the dGRU and the RARU can act as modules within a restricted
region in the context of the PEPCK promoter and in limited contexts in
a heterologous promoter. These observations suggest that different sets
of promoter structures are required for different hormone responses and
have important implications for how multiple hormonal signals are
integrated through promoters.
 |
RESULTS
|
---|
A Complete Glucocorticoid Response Is Dependent on the Order and
Position of the Accessory Factor Elements
We tested whether the accessory factor (AF) elements of the GRU
are restricted to specific positions in the PEPCK promoter (Fig. 2
). Each AF element was switched with
another AF element, in a variety of combinations, in the context of the
intact PEPCK promoter. The exact spacing between the individual
elements was maintained in accordance with the native PEPCK promoter. A
60% reduction of the glucocorticoid response occurred when either AF1
or AF3 was put in the place of AF2 (designated 11G3 and 13G3,
respectively, see Fig. 2
, lines 2 and 3). The loss of function is
equivalent to a 5-bp block mutant in AF2 or a complete deletion of the
element (7, 12, 30). While it is possible that the alignment of the
substituting element is not optimal, the simplest explanation is that
there is no tolerance for a substitution of elements at AF2. By
contrast, the substitution of either AF1 for AF3, or AF3 for AF1 (12G1
and 32G3, respectively), resulted in a complete glucocorticoid response
(Fig. 2
, lines 4 and 5); thus, AF1 and AF3 are interchangeable. This
result is consistent with our previous observation that COUP-TF
functions as an accessory factor for the glucocorticoid response
through both AF1 and AF3 (8, 11). The insertion of AF2 in place of AF1
(22G3) or AF3 (12G2) resulted in a 60% and 40% reduction of the
glucocorticoid response, respectively (Fig. 2
, lines 6 and 7). This
substitution is the functional equivalent of the double-point mutations
that completely disable AF1 or AF3 as accessory elements (8, 11). In
addition, constructs were made wherein combinations of two of the AF
elements were changed. In these constructs (21G3 or 13G2), all three
individual AF elements are present in the dGRU, but they are in
the wrong sequence. Neither of these constructs supported a complete
glucocorticoid response (Fig. 2
, lines 8 and 9). These results suggest
that a complete glucocorticoid response requires the correct order and
position of the AFs.

View larger version (31K):
[in this window]
[in a new window]
|
Figure 2. The Effects of Substitutions of Accessory Factor
Elements in the dGRU or the RARU on the Function of the PEPCK Promoter
in Response to Glucocorticoids or RA
Substitution mutations of accessory factor elements were made in the
context of the full-length wild-type PEPCK promoter-CAT reporter
(pPL32). The ability of the constructs (lines 29) to confer a
glucocorticoid or an RA response was compared with that of pPL32. A
hatched oval represents an element that is substituted
by another accessory factor element. Ten micrograms of reporter plasmid
and 5 µg of pSV2GR or pRShRAR were cotransfected into H4IIE cells,
and CAT activity was measured in cell extracts 18 h after
treatment of the cells with or without 0.5 µM
dexamethasone (Dex) or 2 µM
all-trans-retinoic acid (RA). The data are expressed as
the mean (±SEM, n = 6) of the percentage of the
wild-type Dex or RA response. All of the reporter constructs provided
very similar basal activities in all of these experiments (data not
shown).
|
|
A Complete RA Response Requires Two Properly Positioned RAREs
We have previously shown that RARE1 and RARE2 are both required
for a complete RA response and together constitute the RARU in the
PEPCK promoter (Ref. 15 and see Fig. 1
). We tested whether the changes
made in the constructs described above affected the RA response. The
replacement of RARE1 (22G3) or RARE2 (12G2) with AF2 diminished the RA
response by about 60% and 40% from the fully induced wild-type value,
respectively (compare lines 1, 6, and 7 in Fig. 2
). This is the
functional equivalent of a deletion or substitution mutation of these
elements (4, 15). By contrast, the replacement of RARE1 with RARE2
(32G3), or vice versa (12G1), maintained the RA response (Fig. 2
, lines
4 and 5). Thus, RARE1 and RARE2 are interchangeable. The addition of a
third RARE at the AF2 site had no effect on the RA response (compare
lines 1, 2, and 3 in Fig. 2
). On the other hand, the inverted exchange
of RARE1 and AF2 (21G3) or AF2 and RARE2 (13G2) reduced the RA response
by 2550% (Fig. 2
, lines 8 and 9) despite the fact that the promoter
still contains two RAREs. Together, these data suggest that AF2
occupies a position within the PEPCK promoter that is incapable of
supporting an RARE or, alternatively, that the distance between RARE1
and RARE2 is important for the RA response (see below). Thus, while
each RARE contributes about 50% of the RA response, these elements may
not be completely independent.
The Precise Distance between AF2 and GR1 Is Critical for a
Complete Glucocorticoid Response
Since a specific combination and arrangement of AF
elements is required for a proper glucocorticoid response, we tested
whether the helical orientation or precise distance between each AF
element of the PEPCK gene promoter affects the glucocorticoid response.
Constructs were made wherein 5- or 10-bp insertions, which result in a
half- or full-helical turn of DNA, respectively, were placed at
strategic locations within the PEPCK promoter. The glucocorticoid
response was reduced by about 40% when a half-helical turn (5 bp) was
introduced between AF2 and GR1 (Fig. 3
, line 2). The glucocorticoid response was also reduced by 40% when an
additional 5-bp insertion was placed between AF1 and AF2, which
effectively rotates the AF2 element one half-helical turn with respect
to the rest of the promoter (Fig. 3
, line 3). In these circumstances
AF2 was essentially not involved in the glucocorticoid response (Fig. 3
, lines 2 and 3). However, insertion of a 5-bp sequence between AF1
and AF2 did not affect the glucocorticoid response (Fig. 3
, line 4),
and a 5-bp insertion placed on both sides of the AF3 element had no
effect on the glucocorticoid response (Fig. 3
, line 5). These
results suggest that, while the helical alignment of AF1 or AF3 and
GR1 is not important, the alignment of AF2 and GR1 is critical for
a full glucocorticoid response. In fact, 5-bp insertions on both sides
of GR1, which result in a half-helical turn of GR1 with respect to AF2
and AF3, markedly reduced the glucocorticoid response (Fig. 3
, line 6).
However, an insertion of 10 bp between AF2 and GR1, which maintains the
relative positions of the binding sites on the same face of the DNA
helix, did not completely restore the glucocorticoid response (Fig. 3
, line 7). These results, in the aggregate, suggest that the distance
between AF2 and GR1 is critical for a complete glucocorticoid
response.

View larger version (34K):
[in this window]
[in a new window]
|
Figure 3. The Effects of 5- or 10-bp Insertions on the
Function of the PEPCK Promoter in Response to Glucocorticoids or RA
A series of constructs were made wherein 5- or 10-bp insertions were
placed at strategic positions within the PEPCK promoter, as described
in Materials and Methods. Ten micrograms of the reporter
constructs and 5 µg of pSV2GR or pRShRAR were cotransfected into
H4IIE cells. After 18 h of treatment, with or without 0.5
µM Dex or 2 µM RA, cells were harvested and
CAT activity was measured in the cell lysates. The results are
expressed as the mean (±SEM) of the percentage of the
wild-type Dex or RA response from at least seven separate experiments.
The basal expression of CAT activity from the reporter constructs used
in these experiments was not significantly different (data not
shown).
|
|
We next inserted a 5- or 10-bp segment between the dGRU and the
proximal promoter to determine whether a certain alignment of the dGRU
and the proximal promoter, i.e. the NF1 element, the CRE,
and the TATA box (Ref. 10 and see Fig. 1
), is required for a maximal
response to glucocorticoids. As shown in Fig. 3
, there was no
difference between the wild-type promoter and these two constructs
(compare lines 1, 8, and 9 in Fig. 3
). These results suggest that the
glucocorticoid response is not sensitive to changes of the helical
phasing between the dGRU and the proximal promoter.
A Specific Alignment of RARE1 and RARE2 Is Not Critical for a
Complete RA Response
Since the GRU and the RARU share two elements (AF1/RARE1 and
AF3/RARE2), one might expect that any perturbations in the DNA template
that affect the function of the GRU should also affect the RARU.
Intriguingly, none of the constructs described in Fig. 3
affected the
RA response, so precise positioning of RARE1 and RARE2 is not required
within the RARU. In addition, the relative alignment of the RARU and
the proximal promoter does not affect the RA response. These
experiments demonstrate that the structural requirements of the GRU and
the RARU are different.
HNF3 Associates with GR in Vitro
The requirement for a precise distance between AF2 and GR1 for a
full glucocorticoid response suggested that a protein-protein
interaction between HNF3 and GR may be involved. Pull down experiments
were performed to test whether a glutathione S-transferase
(GST)-GR fusion protein and 35S-labeled HNF3ß interact
in vitro. As shown in Fig. 4
, left panel, appreciable binding of HNF3ß to GST-GR was
detected. The converse experiment was also performed wherein
35S-labeled GR coeluted with a His-tagged HNF3
(Fig. 4
, right). The presence of 0.5 µM dexamethasone
in the binding reactions did not significantly alter the extent of
binding of HNF3 to the GR (Fig. 4
).

View larger version (40K):
[in this window]
[in a new window]
|
Figure 4. The Interaction of HNF3 and GR in
Vitro
Experiments were performed to determine whether HNF3 and GR can
interact in vitro. In the experiment illustrated in the
left panel, GST-GR or GST bound to glutathione-agarose
beads was incubated with 35S-labeled HNF3ß in the
presence or absence of 0.5 µM dexamethasone. The bound
proteins were eluted from the beads, subjected to SDS-PAGE, and
visualized by autoradiography. The converse experiment was also
performed wherein 35S-labeled GR was incubated with
His-tagged HNF3 bound to a nickel-chelating Sepharose column. The
bound proteins were also separated on SDS-PAGE gel and visualized by
autoradiography (right panel). These results are
representative of three separate experiments.
|
|
The dGRU and RARU Function within a Restricted Region of the PEPCK
Promoter
We interpret the substitution and phasing experiments described
above to mean that there are specific requirements for the arrangements
of the transcription factors bound to the regulatory elements
comprising the dGRU or the RARU, and that this assemblage provides a
functional unit for specific hormone responses. A test of the dGRU and
the RARU as modular units in the PEPCK promoter was conducted using a
series of constructs wherein insertions or deletions were made between
the dGRU/RARU and the proximal promoter. All of the constructs were
designed so that the length of the insertion or deletion was a multiple
of 10.5; thus the dGRU/RARU and the TATA box maintain their relative
positions on the DNA helix. The insertion of 126 or 252 bp between the
dGRU/RARU and the proximal promoter decreased the glucocorticoid and
the RA responses by about 50% and 30%, respectively. This suggests
that insertions that increase the distance between the dGRU/RARU and
the proximal promoter compromise the ability of the promoter to confer
a complete glucocorticoid response, and to a lesser extent, a complete
RA response (compare lines 1, 2, and 3 in Fig. 5
). The deletion of 63 or 126 bp between
the dGRU and the proximal promoter did not reduce the glucocorticoid
response (Fig. 5
, lines 4 and 5). In fact, the glucocorticoid response
was increased by 2-fold when 126 bp was deleted, although basal
transcription was not affected in any of these constructs (data not
shown). However, a 189-bp deletion resulted in a reduction of the
glucocorticoid response by about 50% (Fig. 5
, line 6). By contrast,
neither of these deletions affected the RA response. These results
demonstrate that the dGRU functions within a restricted region in the
context of the PEPCK promoter and may be dependent on other promoter
elements not yet defined. By contrast, the RARU is relatively
unaffected by its position in the promoter.

View larger version (22K):
[in this window]
[in a new window]
|
Figure 5. The Effects of Insertions or Deletions between the
dGRU and the Proximal Promoter on the Glucocorticoid or RA Response
A series of constructs were made that contained insertions or deletions
between the dGRU and the NF1-binding site in the context of the PEPCK
promoter fused to CAT, as described in Materials and
Methods. These reporter plasmids (10 µg) were cotransfected
with pSV2GR or pRShRAR (5 µg) into H4IIE cells. Cells were
incubated for 18 h with or without Dex (0.5 µM) or
RA (2 µM), and CAT activity was measured in cell
extracts. The data are expressed as the mean (±SEM) of the
percentage of the wild-type Dex or RA response from at least six
independent experiments. The basal expression of CAT activity from
these reporter constructs was not significantly different (data not
shown).
|
|
The dGRU and RARU Can Function within the Context of a Heterologous
Promoter
We tested whether the dGRU/RARU functions as a modular unit in the
context of the minimal thymidine kinase (tk) promoter ligated to the
chloramphenicol acetyltransferase (CAT) reporter gene (tkCAT). Neither
tkCAT alone, nor a construct wherein GR1 is ligated to tkCAT (GR1tk),
conferred a glucocorticoid response (Fig. 6A
, lines 1 and 2). This result confirms
previous observations that GR1 or GR2, either alone or in combination,
cannot confer a glucocorticoid response in a heterologous promoter
context (27). A construct was made wherein the dGRU/RARU was placed
upstream of the tk promoter [dGRU/RARU(-467/-314)tk] to test
whether the addition of the three accessory elements help provide a
glucocorticoid response in this context. This construct gave a modest,
4.5-fold glucocorticoid response when transfected into H4IIE cells
(Fig. 6A
, line 3). These results are in contrast to those of Imai
et al. who found that the sequences from -467 to -200 of
the PEPCK promoter, placed upstream of the tk promoter, failed to
confer a glucocorticoid response (9). These results, along with the
results presented in Fig. 5
, suggest that the distance between the dGRU
and the proximal promoter is a critical parameter for the proper
functioning of the dGRU. Alternatively, there may be undefined
repressor elements in the -467/-200 construct that are absent in the
-467/-321 construct. To test this, a construct was made
[dGRU/RARU(-467/-113)tk] wherein the dGRU was placed upstream of
the tk promoter so that the distance between the dGRU and the proximal
promoter was the same as that in the PEPCK promoter. This construct
conferred the full 15-fold glucocorticoid response (Fig. 6A
, line 4), a
somewhat surprising result since the tk promoter does not contain a CRE
(located at -93/-86 in the PEPCK promoter) but it could contain a
cis-acting element(s) that substitutes for the CRE in this
context. Another possibility is that one or more elements between -200
and -113 contribute to the glucocorticoid response. Alternatively, the
distance between the dGRU and the start site, or the position the dGRU
occupies in this context, is optimal.

View larger version (22K):
[in this window]
[in a new window]
|
Figure 6. The dGRU/RARU Can Function in the Context of a
Heterologous Promoter
Constructs were made wherein the sequence of GR1, RARE1, RARE2,
dGRU/RARU(-467/-314), or dGRU/RARU(-467/-113) was ligated upstream
of the minimal tkCAT reporter gene (tkCAT). H4IIE cells were
transiently transfected with 10 µg of reporter plasmids and 5 µg of
pSV2GR (panel A) or pRShRAR (panel B). After transfection, cells
were cultured for 18 h with or without 0.5 µM Dex
(panel A) or 2 µM RA (panel B), the cells were harvested,
and CAT activity was measured in cell extracts. The data are expressed
as the mean of the fold induction (±SEM) in response to
each hormone from at least three experiments.
|
|
We also compared the ability of the RARU, and the individual RAREs, to
confer a RA response in the context of a heterologous promoter. The RA
response conferred by tkCAT was compared with two constructs wherein
RARE1 or RARE2 was ligated to tkCAT (compare lines 1, 2, and 3 in Fig. 6B
). While tkCAT did not provide a response to RA, the AF1(RARE1)
construct conferred a 2-fold greater RA response than did the
AF3(RARE2) construct. These results are consistent with previous
observations, including a 5'-deletion analysis (4, 15). When the entire
RARU was placed upstream of the tk promoter, the RA response was not
any greater than that conferred by RARE1 alone in the same context
(compare lines 2 and 4 in Fig. 6B
). However, when the RARU was placed
in a position wherein the distance between the RARU and the start site
of the tk promoter was identical to that of the PEPCK promoter, there
was a 13-fold RA response, which is about 2-fold greater than when the
RARU was positioned 200 bp closer to the proximal promoter (compare
lines 4 and 5 in Fig. 6B
). This result suggests that the RARU is either
in an optimal position for transactivation or that there are accessory
elements in the sequence between -321 and -113. The data presented in
this figure suggest that the dGRU/RARU functions as a modular enhancer
unit, and it responds to two different hormones in the context of a
minimal heterologous promoter. In addition, these data, along with
those displayed in Fig. 5
, suggest that the distance between the
dGRU/RARU and the proximal promoter of the PEPCK gene is optimal for
these two hormonal responses.
 |
DISCUSSION
|
---|
Transcription-regulatory regions of eukaryotic cell promoters
generally consist of clusters of transcription factor-binding sites
(31) that can provide a coordinated, specialized response to an
environmental challenge. For example, the virus-induced enhancer
complex of the IFNß gene promoter is comprised of a CRE, an NF-
B
binding site, and an IFNß response element (22). These binding sites
all act as simple elements when placed in heterologous contexts. In
contrast, the IFNß promoter is exclusively activated by virus
infection and not by any of the effectors that act on the isolated
elements. Viral induction is achieved by the assembly of a higher-order
enhancer complex, termed an enhanceosome, that requires a precise
helical phasing relationship between the individual transcription
factor- binding sites. The substitution of any transcription
factor-binding sites, or a change of the helical phasing between
different transcription factor-binding sites, decreases the magnitude
of viral induction (22, 32).
Some years ago we showed that the GR-binding sites (GR1 and GR2) in the
PEPCK gene promoter are associated with other, functionally essential
transcription factor binding sites (7). It now appears that, in their
natural context, most (if not all) hormone response elements, including
GREs, confer their cognate responses when in association with
DNA-binding accessory factors. These accessory factor-binding sites are
often located in the proximity of the HRE and together constitute what
has been referred to as HRUs (6). In the case of the PEPCK gene,
several HRUs have been described in some detail. A common theme is that
each of these HRUs contains multifunctional elements that can bind to
more than one set of proteins and thereby mediate responses to
different signals. Thus, the RARU is composed of RARE1 and RARE2 (that
bind RAR/RXR heterodimers), and these are also components of the GRU,
which consists of at least six DNA elements. This overlapping set of
HRUs, inclusively termed the PEPCK gene metabolic control domain,
provides an integrated response of multiple hormonal and metabolic
stimuli. For example, regulation of the transcription of the PEPCK
gene, which encodes the enzyme critical for gluconeogenesis, is
controlled by a metabolic control domain that consists of
glucocorticoid, RA, cAMP, and insulin HRUs (6).
We have now demonstrated that the RARU and GRU of the PEPCK gene
promoter operate properly only when in an ordered structure. Despite
having common elements, the structural requirements for the
glucocorticoid and the RA response units are different. The proper
positions of AF1, AF2, and AF3 are required for a complete
glucocorticoid response (Fig. 2
). AF1 and AF3 are interchangeable,
which is consistent with the fact that both elements bind COUP-TF, and
this orphan nuclear hormone receptor can confer accessory factor
activity from both AF elements (8, 11). AF2 cannot substitute for
either AF1 or AF3, and, conversely, neither AF1 nor AF3 can substitute
for AF2 (Fig. 2
and Ref. 12). A previous study also provided evidence
that AF2 is functionally different from AF1 and AF3. Scott et
al. (27) replaced GR1 with a high-affinity palindromic GRE
in the context of the PEPCK promoter and found that, while AF1 and AF3
are no longer required for a complete glucocorticoid response, AF2 is
still required. In addition, Wang et al. (12) found
that AF2 activity has a relatively specific requirement for HNF3. Thus,
if AF2 is replaced with a binding site for SP1, or is mutated so that
C/EBP isoforms (but not HNF3) can bind, there is a reduction in the
glucocorticoid response equivalent to the removal of AF2. There is also
a relatively specific requirement for the factors that bind to AF1 and
AF3 (Fig. 2
, and J.-C. Wang and D. K. Granner, unpublished
data). This is in contrast to earlier studies which noted that
essentially any transcription factor placed in close proximity to a
canonical GRE potentiates the glucocorticoid response (33). The
requirement for specific trans-acting factors that bind in
the proper spatial alignment is reminiscent of the IFNß example cited
above and implies that multiple protein-protein interactions are made
in the context of a large nucleoprotein complex whose integrity is
essential for a complete glucocorticoid response.
The precise distance between AF2 and GR1 is critical for the
glucocorticoid response, since insertions of 5 or 10 bp between the two
elements diminish the response. Thus, it is possible that an
interaction between HNF3 and GR, which we demonstrate herein, may be
important for the glucocorticoid response. A similar situation exists
in a number of gene promoters (34, 35, 36, 37). The major histocompatibility
class (MHC) II human leukocyte antigen-DR
gene (HLA-DRA) provides
one example. Vilen et al. demonstrated that a number of
upstream regulatory elements, including the Y box, the X box, and the S
element, are important for constitutive and IFN
-inducible expression
of the MHC II HLA-DRA gene (35). The precise spacing between the S
element and the X box is required for the proper expression of this
gene. The authors conclude that two different proteins may interact
with the S and X sites and that rigid protein-protein interaction
requirements are highly dependent on the interelement distance. With
this in mind, it is noteworthy that the spacing between AF2 and GR1 is
exactly conserved at 18 bp in the PEPCK gene promoters of rat, mouse,
and human. By contrast, the spacing between AF1 or AF3 and GR1 is
variable in these species (Ref. 38 and unpublished data by C. Williams,
D. K. Granner, and R. Chalkley). In this context, it is
noteworthy that 5- and 10-bp insertions between AF1 and GR1, or GR2 and
AF3, had no effect on the glucocorticoid response (Fig. 3
).
The RARU and the distal component of the GRU are both contained within
the same 140-bp segment of the PEPCK promoter (-460 to -320) and
share two elements (AF1/RARE1 and AF3/RARE2). Thus, it is interesting
that, in contrast to the glucocorticoid response, the RA response is
not sensitive to changes in the helical alignment of elements within
this segment of DNA (Fig. 3
). One possible explanation is that RARE1
and RARE2 both act as independent RA-stimulated enhancers and are not
part of a higher ordered structure that requires the proper placement
of these elements for a complete RA response. However, an arrangement
where RARE1 and RARE2 are placed relatively close together, so that one
or the other element occupies the AF2 position in the PEPCK promoter,
results in an incomplete RA response (Fig. 2
). In addition, the RARU
confers a
7-fold RA response in the context of the tkCAT promoter,
but a 13-fold RA response when an additional 200 bp of the PEPCK
promoter are added, suggesting that this sequence contains accessory
elements that are necessary for the RA response (Fig. 5
). Thus, while
the RAREs are not completely autonomous and require a higher ordered
structure to function properly, the actual structural requirements of
the RARU differ from those of the GRU.
The protein-DNA interactions between hormone receptor and cognate DNA
element, and between accessory factors and their DNA elements,
described here and elsewhere, are probably not sufficient to explain
how discrimination between glucocorticoid and RA signals is provided at
the PEPCK gene promoter. One or more of the several coactivators known
to be involved in the action of members of the steroid hormone receptor
superfamily could provide the required selectivity. This class of
proteins includes SRC-1 and family members, TIF1 and TIF2/GRIP1, as
well as CREB-binding protein (CBP) and its homolog, p300 (39, 40, 41, 42, 43).
Accordingly, the ligand, either glucocorticoid or RA, by binding to and
activating its cognate receptor, could initiate a series of protein-DNA
and protein-protein interactions that result in the assembly of
different, complex nucleoprotein structures that activate transcription
of the PEPCK gene. Korzus et al. demonstrated that
RAR-mediated transcription requires CBP/p300, SRC-1, p/CAF, and p/CIP
(44, 45, 46), while signal transducer and activator of transcription-1
(STAT-1)-mediated transcription requires CBP/p300 and p/CIP. In the
PEPCK promoter, there may also be different sets of cofactors assembled
in a ligand-specific manner that may thus specify either the
glucocorticoid or RA response.
Specificity of the hormone response in the PEPCK gene can be explained
by assuming that specific enhanceosomes are assembled in response to
each ligand, but the fact remains that the GRU and RARU share DNA
elements that have the capacity to bind different transcription
factors. With the exception of the RAR, these factors (COUP-TF, HNF4,
RXR) are in abundance in H4IIE cell nuclei, and all bind with
approximately the same affinity to the same contact points in the
AF1/RARE1 and AF3/RARE2 elements. Hall et al. (11)
demonstrated that RAR overexpression, in the absence of RA, had no
effect on the glucocorticoid response and suggested that RAR/RXR
heterodimers, while capable of binding AF1 and AF3, are not accessory
factors for this response. However, the possibility that ligand-bound
RAR/RXR may act as an accessory factor for the glucocorticoid response
has not been excluded. In this regard, it is interesting to speculate
that the integration of multiple hormone responses may proceed through
hybrid HRUs that contain portions of each individual HRU. Accordingly,
the overall regulatory structure, the metabolic control domain, would
consist of a number of DNA elements, DNA-binding proteins, and
protein-binding coregulatory proteins from which various combinations
are assembled in response to different signals. This general mechanism
would provide the selectivity and specificity, the different degrees of
repression, additivity, or synergism, and for the dominance of one
response over another, which are required of genes that encode proteins
involved in complex processes such as gluconeogenesis.
 |
MATERIALS AND METHODS
|
---|
Plasmid Construction
The construction of the plasmid pPL32, which contains the
PEPCK promoter from -467 to +69 relative to the transcription start
site ligated to the CAT reporter gene, has been described (23).
Site-directed mutations of pPL32, including AF1(+5)AF2, AF2(+5)GR1,
(+5)AF2(+5), 11G3, 22G3, and 21G3 were made using the PCR megaprimer
method. Other plasmids, including 32G3, 12G1, 12G2, 13G3, 13G2,
AF2(+5)GR1, AF3(+5)P4, AF3(+10)P4, (+5)AF3(+5), AF2(+10)GR1,
GR1(+5)GR2, dGRU/RARU(-63)NF1, dGRU/RARU(-126)NF1, and dGRU/RARU
(-189)NF1 that were used for substitution, spacing, and deletion
experiments, were constructed using the Quick Change site-directed
mutagenesis kit (Stratagene, La Jolla, CA) according to the
manufacturers instructions. Some of the constructs were made by
inserting a 5-bp (5'-TGCAG-3') or a 10-bp (5'-TGCAGTGCAG-3') sequence
(24, 25) into various sites within the PEPCK promoter and are
designated with a (+5) or (+10). The sequences of the oligonucleotides
used in this study are shown in Table 1
.
The mutations of pPL32 that had DNA fragments inserted between the dGRU
and the distal boundary of the basal promoter, demarcated as 5'-end of
the NF1 element, termed dGRU/RARU(+126)NF1 and dGRU/RARU(+252)NF1, were
created in three steps. First, DNA fragments of 126 or 252 bp were
generated by PCR using the plasmid pBR322 as a template and the primers
(+126/+252)a and (+126)b, or the primers (+126/+252)a and (+252)b,
respectively. The primer (+126/+252)a contains a sequence that
generates HindIII and XbaI sites after
amplification, and the primers (+126)b and (+252)b each contain
sequences that generate a single SalI site. Second, the PCR
products were digested with HindIII and SalI,
and the resulting fragments of 126 or 252 bp were then subcloned into
the HindIII-SalI sites of pPL33, which contains
the PEPCK promoter from -310 to +69 ligated to CAT, to generate the
plasmids pPL33(+126) and pPL33(+252), respectively. Finally, an
XbaI fragment that contains the dGRU was amplified from
pPL32 by PCR using the primer dGRUa, which generates an XbaI
site, and the primer dGRUb. This fragment was subcloned into the
XbaI sites of pPL33(+126) and pPL33(+252).
The plasmid dGRU/RARU(-467/-113)tk was constructed in two steps.
First, the nucleotide sequence of pPL32 from -467 to -113 was
amplified by PCR using the primers dGRU (-467/-113)a and
dGRU(-467/-113)b that generate a BamHI site and a
BglII site, respectively. Second, the
BamHI-BglII fragment was then subcloned into the
BamHI site of tkCAT. The plasmid tkCAT contains the tk
promoter from -105 to +51 ligated to the CAT reporter gene (26). The
plasmid dGRU/RARU(-467/-314)tk was constructed in a similar manner by
amplifying the sequences of pPL32 between -467 and -314 by PCR using
the primers dGRU (-467/-314)a and dGRU (-467/-314)b that generate a
HindIII and a BamHI site, respectively. The
HindIII and BamHI fragments were then subcloned
into the HindIII and BamHI sites of tkCAT. The
plasmids GR1tk, AF1(RARE1)tk, and AF3(RARE2)tk have been previously
described (4, 15, 27). The sequences for AF1, GR1, and AF3 are included
in Fig. 1
.
The cDNA that encodes rat HNF3ß was a gift from Dr. Robert Costa
(University of Illinois, Chicago). The plasmid was digested with
EcoRI, and the resulting fragment was subcloned into the
pSG5 vector (Stratagene) for the in vitro synthesis of
labeled HNF3ß protein. To generate constructs for the overexpression
of a GST-fusion protein in Escherichia coli, the full-length
cDNA that encodes GR was amplified by PCR using the primers GRa and
GRb, which generate an EcoRI and a SalI site,
respectively. The PCR product was digested with EcoRI and
SalI and subcloned into the pGEX-5X-1 plasmid (Pharmacia,
Piscataway, NJ). The resulting plasmid was named GST-GR. The expression
vector that encodes rat HNF3
, with an amino-terminal histidine tag,
was a gift from Dr. Kenneth Zaret (Brown University, Providence,
RI).
The DNA sequence of all constructs was verified by dideoxy sequencing.
All oligonucleotides were produced on a Perceptive Biosystems Expedite
8909 DNA synthesizer located in the Vanderbilt University Diabetes
Research and Training Center.
Transient Transfection
The maintenance and transfection of H4IIE cells and the
measurement of CAT activity have been described previously (14, 23, 28). The mammalian expression vectors, pSV2GR and pRShRAR, were
provided by Keith Yamamoto (University of California, San Francisco)
and Ronald Evans (Salk Institute, San Diego, CA), respectively.
GST-Fusion Protein Pull-Down Assay
The GST-GR fusion proteins were expressed in TOPP3 cells
(Stratagene) by induction with 0.4 mM
isopropyl-ß-D-thiogalactopyranoside at 30 C for
4 h. Cell pellets were lysed and sonicated, after which an extract
of soluble protein was prepared by centrifugation. An extract
containing the GST fusion protein was mixed with glutathione-Sepharose
(Sigma Chemical Co., St. Louis, MO) and incubated at 4 C. One microgram
of various supercoiled DNA plasmids was transcribed in vitro
then translated in the presence of [35S]methionine (ICN
Pharmaceuticals, Inc., Costa Mesa, CA) in the TNT-coupled
reticulocyte lysate system (Promega, Madison, WI). GST-GR fusion
proteins were bound to glutathione-agarose beads in GST binding/washing
buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl,
100 mM EDTA, 1% Triton X-100/1 mM
phenylmethylsulfonyl fluoride, and 10 mM dithiothreitol)
and were mixed with 10 µl of a reticulocyte lysate that contained
in vitro-translated proteins. This was incubated at 25 C for
1 h in the presence or absence of 0.5 µM
dexamethasone. The formed complex was washed three times with 500 µl
of the binding/washing buffer and then once with a buffer that contains
10 mM Tris-HCl, pH 8.0, 50 mM NaCl, and 5
mM EDTA. Bound proteins were eluted by boiling in 50 µl
of 2x SDS loading buffer and analyzed by 10% SDS-PAGE.
His-Tagged Fusion Protein Pull-Down Assay
The expression and purification of rat His-tagged HNF3
have
been described previously (29). The His-tagged protein was incubated
with a nickel-chelating Sepharose slurry (Novagen, Madison, WI)
at 4 C. The formed His-tagged HNF3
/Sepharose complex was then washed
twice with binding buffer (20 mM Tris-HCl pH7.9, 5
mM imidazole, 0.5 M NaCl) and once with washing
buffer (20 mM Tris-HCl, pH 7.9, 60 mM
imidazole, 0.5 M NaCl). Ten microliters of
[35S]methionine-labeled protein and 200 µl of binding
buffer were then added to the complex, and the protein-binding reaction
was carried out for 1 h at 25 C in the presence or absence of 0.5
µM dexamethasone. The mixture was washed three times with
binding buffer and then once with washing buffer. The resin was
suspended in 2x SDS loading buffer and analyzed by 10% SDS-PAGE.
 |
ACKNOWLEDGMENTS
|
---|
We would like to thank Cathy Caldwell for her excellent
technical assistance, Deborah Caplenor Brown for preparation of the
manuscript, Robert K. Hall and Mary E. Waltner-Law for their careful
review of the manuscript, Leena George for preparing purified
His-tagged HNF3
, and Kazuya Yamada for his invaluable advice.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Daryl K. Granner, Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615.
This work was supported by NIH Grant DK-35107 and the Vanderbilt
Diabetes Research and Training Center (DK-20593).
Received for publication January 7, 1998.
Revision received July 6, 1998.
Accepted for publication July 11, 1998.
 |
REFERENCES
|
---|
-
Granner DK, Pilkis S 1990 The genes of hepatic glucose
metabolism. J Biol Chem 265:1017310176[Free Full Text]
-
OBrien RM, Bonovich MT, Forest CD, Granner DK 1991 Signal
transduction convergence: phorbol esters and insulin inhibit
phosphoenolpyruvate carboxykinase gene transcription through the same
10-base-pair sequence. Proc Natl Acad Sci USA 88:65806584[Abstract]
-
Hanson RW, Reshef L 1997 Regulation of phosphoenolpyruvate
carboxykinase (GTP) gene expression. Annu Rev Biochem 66:581611[CrossRef][Medline]
-
Lucas PC, OBrien RM, Mitchell JA, Davis CM, Imai E, Forman
BM, Samuels HH, Granner DK 1991 A retinoic acid response element is
part of a pleiotropic domain in the phosphoenolpyruvate carboxykinase
gene. Proc Natl Acad Sci USA 88:21842188[Abstract]
-
Sasaki K, Cripe TP, Koch SR, Andreone T, Peterson DD, Beale
EG, Granner DK 1984 Multihormonal regulation of phosphoenolpyruvate
carboxykinase gene transcription: dominant role of insulin. J Biol
Chem 259:1524215251[Abstract/Free Full Text]
-
Lucas PC, Granner DK 1992 Hormone response domains in gene
transcription. Annu Rev Biochem 61:11311173[CrossRef][Medline]
-
Imai E, Stromstedt P, Quinn PG, Carlstedt-Duke J, Gustafsson
J, Granner DK 1990 Characterization of a complex glucocorticoid
response unit in the phosphoenolpyruvate carboxykinase gene. Mol Cell
Biol 10:47124719[Medline]
-
Scott DK, Mitchell JA, Granner DK 1996 The orphan receptor
COUP-TF binds to a third glucocorticoid accessory factor element within
the phosphoenolpyruvate carboxykinase gene promoter. J Biol
Chem 271:3190931914[Abstract/Free Full Text]
-
Imai E, Miner JN, Mitchell JA, Yamamoto KR, Granner DK 1993 Glucocorticoid receptor-cAMP response element-binding protein
interaction and the response of the phosphoenolpyruvate carboxykinase
gene to glucocorticoids. J Biol Chem 268:53535356[Abstract/Free Full Text]
-
Quinn PG, Wong TW, Magnuson MM, Shabb JB, Granner DK 1988 Identification of basal and cyclic AMP regulatory elements in the
promoter of the phosphoenolpyruvate carboxykinase gene. Mol Cell Biol 8:34673475[Medline]
-
Hall RK, Sladek FM, Granner DK 1995 The orphan receptors
COUP-TF and HNF-4 serve as accessory factors required for induction of
phosphoenolpyruvate carboxykinase gene transcription by
glucocorticoids. Proc Natl Acad Sci USA 92:412416[Abstract]
-
Wang J-C, Stromstedt P-E, OBrien RM, Granner DK 1996 Hepatic
nuclear factor 3 is an accessory factor required for the stimulation of
phosphoenolpyruvate carboxykinase gene transcription by
glucocorticoids. Mol Endocrinol 10:794800[Abstract]
-
Liu J, Park EA, Gurney AL, Roesler WJ, Hanson RW 1991 Cyclic
AMP induction of phosphoenolpyruvate carboxykinase (GTP) gene
transcription is mediated by multiple promoter elements. J Biol
Chem 266:1909519102[Abstract/Free Full Text]
-
Hall RK, Scott DK, Noisin EL, Granner DK 1992 Activation of
the PEPCK retinoic acid response element is dependent on a retinoic
acid receptor/coregulator complex. Mol Cell Biol 12:55275535[Abstract]
-
Scott DK, Mitchell JA, Granner DK 1996 Identification and
characterization of a second retinoic acid response element in the
phosphoenolpyruvate carboxykinase gene promoter. J Biol Chem 271:62606264[Abstract/Free Full Text]
-
Shin D-J, Tao A, McGrane MM 1995 Effects of vitamin A
deficiency and retinoic acid treatment on expression of a
phosphoenolpyruvate carboxykinase-bovine growth hormone gene in
transgenic mice. Biochem Biophys Res Commun 213:706714[CrossRef][Medline]
-
Shin DJ, McGrane MM 1997 Vitamin A regulates genes involved in
hepatic gluconeogenesis in mice: phosphoenolpyruvate carboxykinase,
fructose-2,6-bisphosphatase and
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. J Nutr 127:12741278[Abstract/Free Full Text]
-
OBrien RM, Lucas PC, Forest CD, Magnuson MA, Granner DK 1990 Identification of a sequence in the PEPCK gene that mediates a negative
effect of insulin on transcription. Science 249:533537[Medline]
-
Lamers WH, Hanson RW, Meisner HM 1982 Cyclic AMP stimulates
transcription of the gene for cytosolic phosphoenolpyruvate
carboxykinase gene in rat liver nuclei. Proc Natl Acad Sci USA 79:51375141[Abstract]
-
Pugh BF, Tjian R 1990 Mechanism of transcriptional activation
by Sp1: evidence for coactivators. Cell 61:11871197[Medline]
-
Tjian R, Maniatis T 1994 Transcriptional activation: a complex
puzzle with few easy pieces. Cell 77:58[Medline]
-
Thanos D, Maniatis T 1995 Virus induction of human IFNß gene
expression requires the assembly of an enhanceosome. Cell 83:10911100[Medline]
-
Peterson DD, Magnuson MA, Granner DK 1988 Location and
characterization of two widely separated glucocorticoid response
elements in the phosphoenolpyruvate carboxykinase gene. Mol Cell Biol 8:96104[Medline]
-
Vilen BJ, Cogswell JP, Ting JP-Y 1991 Stereospecific alignment
of the x and Y elements is required for major histocompatibility
complex class II DRA promoter function. Proc Natl Acad Sci USA 11:24062415
-
Osawa H, Robey RB, Printz RL, Granner DK 1996 Identification
and characterization of basal and cyclic AMP response elements in the
promoter of the rat hexokinase II gene. J Biol Chem 271:1729617303[Abstract/Free Full Text]
-
McKnight SL, Kingsbury R 1982 Transcriptional control signals
of a eukaryotic protein-coding gene. Science 217:316324[Medline]
-
Scott DK, Stromstedt P-E, Wang J-C, Granner DK 1998 Further
characterization of the glucocorticoid response unit in the
phosphoenolpyruvate carboxykinase gene. The role of the glucocorticoid
receptor binding sites. Mol Endocrinol 12:482491[Abstract/Free Full Text]
-
Nordeen SK, Green PPI, Fowlkes DM 1987 A rapid, sensitive, and
inexpensive assay for chloramphenicol acetyltransferase. DNA 6:173178[Medline]
-
Zaret KS, Stevens K 1995 Expression of a highly unstable and
insoluble transcription factor in Escherichia coli:
purification and characterization of the fork head homolog
HNF3
. Protein Expr Purif 6:821825[CrossRef][Medline]
-
Mitchell J, Noisin E, Hall R, OBrien R, Imai E, Granner DK 1994 Integration of multiple signals through a complex hormone response
unit in the phosphoenolpyruvate carboxykinase gene promoter. Mol
Endocrinol 8:585594[Abstract]
-
Dynan WS 1989 Modularity in promoters and enhancers. Cell 58:14[Medline]
-
Kim TK, Maniatis T 1998 The mechanism of transcriptional
synergy of an in vitro assembled interferon-ß
enhanceosome. Mol Cell 1:119129
-
Schule R, Muller M, Kaltschmidt C, Renkawitz R 1988 Many
transcription factors interact synergistically with steroid receptors.
Science 242:14181420[Medline]
-
Wu L, Berk A 1988 Constraints on spacing between transcription
factor binding sites in a simple adenovirus promoter. Genes Dev 2:403411[Abstract]
-
Vilen BJ, Penta JF, Ting P-Y 1992 Structural constraints
within a trimeric transcriptional regulatory region. J Biol Chem 267:2372823734[Abstract/Free Full Text]
-
Tansey WP, Schaufele F, Heslewood M, Handford C, Reudelhuber
TL, Catanzaro DF 1993 Distance-dependent interaction between basal,
cyclic AMP, and thyroid hormone response elements in the rat growth
hormone promoter. J Biol Chem 268:1490614911[Abstract/Free Full Text]
-
Ambrosetti D-C, Basilico C, Dailey L 1997 Synergistic
activation of the fibroblast growth factor 4 enhancer by Sox2 and Oct-3
depends on protein-protein interactions facilitated by a specific
spatial arrangement of factor binding sites. Mol Cell Biol 17:63216329[Abstract]
-
OBrien RM, Printz RL, Halmi N, Tiesinga JJ, Granner DK 1995 Structural and functional analysis of the human phosphoenolpyruvate
carboxykinase gene promoter. Biochim Biophys Acta 1264:284288[Medline]
-
Beato M, Sanchez-Pacheco A 1996 Interaction of steroid hormone
receptors with the transcription initiation complex. Endocr Rev 17:587609[Medline]
-
Kamei Y, Xu L, Heinzel T, Torchia J, Kurokawa R, Gloss B, Lin
S, Heyman RA, Rose DW, Glass CK, Rosenfeld MG 1996 A CBP integrator
complex mediates transcriptional activation and AP-1 inhibition by
nuclear receptors. Cell 85:403414[Medline]
-
Hong H, Kohli K, Trivedi A, Johnson DL, Stallcup MR 1996 GRIP1, a novel mouse protein that serves as a transcriptional
coactivator in yeast for the hormone binding domains of steroid
receptors. Proc Natl Acad Sci USA 93:49484952[Abstract/Free Full Text]
-
LeDouarin B, Zechel C, Garnier JM, Luts Y, Tora L, Pierrat B,
Heery D, Gronemeyer H, Chambon P, Losson R 1995 The N-terminal part of
TIF1, a putative mediator of the ligand-dependent activation function
(AF-2) of nuclear receptors, is fused to B-raf in the oncogenic protein
T18. EMBO J 14:20202033[Abstract]
-
Voegel JJ, Heine MJS, Zechel C, Chambon P, Gronmeyer H 1996 TIF2, a 160 kDa transcriptional mediator for the ligand-dependent
activation function AF-2 of nuclear receptors. EMBO J 15:36673675[Abstract]
-
Korzus E, Torchia J, Rose DW, Xu L, Kurokawa R, Mclnerney EM,
Mullen TM, Glass CK, Rosenfeld MG 1998 Transcription Factor-Specific
Requirements for Coactivators and Their Acetyltransferase Functions.
Science 279:703707[Abstract/Free Full Text]
-
Yang XJ, Ogryzko J, Nishikawa J, Howard BH, Nakatani Y 1996 A p300/CBP-associated factor that competes with the adenoviral
oncoprotein E1A. Nature 382:319324[CrossRef][Medline]
-
Torchia J, Rose DW, Inostroza J, Kamei Y, Westin Y, Glass CK,
Rosenfeld MG 1997 The transcriptional co-activator p/CIP binds CBP and
mediates nuclear-receptor function. Nature 387:677684[CrossRef][Medline]