The Phosphoenolpyruvate Carboxykinase Gene Glucocorticoid Response Unit: Identification of the Functional Domains of Accessory Factors HNF3ß (Hepatic Nuclear Factor-3ß) and HNF4 and the Necessity of Proper Alignment of Their Cognate Binding Sites
Jen-Chywan Wang,
Per-Erik Strömstedt1,
Takashi Sugiyama and
Daryl K. Granner
Department of Molecular Physiology and Biophysics Vanderbilt
University Medical School Nashville, Tennessee, 37232-0615
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ABSTRACT
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Complete induction of hepatic
phosphoenolpyruvate carboxykinase (PEPCK) gene transcription by
glucocorticoids requires a complex glucocorticoid response unit (GRU).
The GRU is comprised of two glucocorticoid receptor (GR)-binding sites
(GR1 and GR2) and four accessory factor-binding sites [AF1, AF2, AF3,
and cAMP response element (CRE)] that bind distinct transcription
factors. Hepatic nuclear factor 4 (HNF4) and chicken ovalbumin upstream
promoter transcription factor (COUP-TF) bind to the AF1 element and
account for AF1 activity. Members of the hepatic nuclear factor 3
(HNF3) family bind to the AF2 element and provide AF2 activity. In this
report, we show that the functions of AF1 and AF2 are dependent on
their positions in the promoter, since they cannot substitute for each
other nor can they be exchanged without a reduction in the response to
glucocorticoids. We also identified the domains of HNF4 and HNF3ß
that are required for the AF1 and AF2 activities, respectively. The
carboxy-terminal transactivation domain of HNF4 (amino acids 128374)
confers most of the AF1 activity, while the carboxy-terminal
transactivation domain of HNF3ß (amino acids 361458) mediates AF2
activity. These domains of HNF4 and HNF3ß appear to have distinct
roles in the response to glucocorticoids, as there are unique
structural requirements for each, as judged by the failure of most
other classes of transactivation domains to serve as accessory factors.
These results suggest that the regulation of the PEPCK gene by
glucocorticoids requires specific interactions between GR, accessory
factors, and coactivators, and that the transactivation domains of AF1
and AF2 are of fundamental importance in the assembly of this
multiprotein complex.
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INTRODUCTION
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The hepatic phosphoenolpyruvate carboxykinase (PEPCK) gene, which
encodes a rate-controlling enzyme of hepatic gluconeogenesis, is under
multihormonal control at the transcriptional level (1, 2).
Transcription of the PEPCK gene is stimulated by glucocorticoids,
retinoic acids, and glucagon (via cAMP), while insulin and glucose
repress PEPCK gene transcription (1, 2, 3, 4, 5). A complex glucocorticoid
response unit (GRU) in the PEPCK gene promoter is required for a
complete glucocorticoid response. The GRU consists of two adjacent
glucocorticoid receptor (GR)-binding sites (GR1 and GR2) and four
accessory elements: AF1, AF2, AF3, and the cAMP response element (CRE)
(see Fig. 1
) (6, 7, 8). GR1 and GR2, alone
or in combination, are unable to confer a glucocorticoid response when
placed in the context of a heterologous promoter (9). Although none of
the four accessory elements mediates a glucocorticoid response itself,
deletion or mutation of any accessory element in the PEPCK promoter
causes about a 5070% reduction in the glucocorticoid response (6, 8, 10). Furthermore, any combination of two mutations of AF1, AF2, or AF3
essentially abolishes the glucocorticoid response (6). Thus, the PEPCK
GR1 and GR2, unlike simple glucocorticoid response elements (GREs) that
can mediate a glucocorticoid response, are inactive in the absence of
at least two accessory elements.

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Figure 1. Schematic Diagram of the PEPCK GRU
The PEPCK GRU includes two GR-binding sites (GR1 and GR2) and four
accessory elements (AF1, AF2, AF3, and the CRE) and their associated
trans-acting factors (lower panel). The
position of each cis-element, relative to the
transcription initiation site, is indicated above the
figure.
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AF1 activity is provided by either of two members of the nuclear
receptor superfamily, hepatic nuclear factor 4 (HNF4), or chicken
ovalbumin upstream promoter transcription factor (COUP-TF) (11).
COUP-TF also binds to the AF3 element and serves as an accessory factor
for the glucocorticoid response (6). Members of the hepatic nuclear
factor 3 (HNF3) and CCAAT enhancer binding protein (C/EBP) families
bind to the AF2 element (12, 13). The binding of HNF3, but not C/EBP,
to this element correlates with AF2 activity (14). Hence, HNF3 has been
identified as an accessory factor for the glucocorticoid response.
Finally, the CRE binding protein (CREB) and C/EBP family members bind
to the CRE (12, 15, 16). Although a physical interaction between GR and
CREB has been identified in vitro (8), it appears that
C/EBPß functions as the accessory factor for the glucocorticoid
response through the CRE (16A ).
In experiments reported in this paper, we show that AF1 cannot
substitute for AF2, and vice versa, as the exchange of these elements
in the PEPCK GRU results in about a 50% reduction of the
glucocorticoid response. Thus, AF1 and AF2 both function in a
position-dependent manner. We also identify the domains within HNF4
(the AF1 factor) and HNF3ß (the AF2 factor) required for accessory
factor activity. In both cases, previously identified transactivation
domains are critical for accessory factor activity. Several distinct
transactivation domains identified in other proteins cannot replace the
HNF3 and HNF4 activities in the context of the PEPCK GRU. These results
indicate that glucocorticoid-stimulated PEPCK gene transcription
requires functional interactions between GR and the transactivation
domains of at least two accessory factors, or between these two
components and a third, coregulatory protein, and that these
interactions require a specific spatial organization of the components
of the GRU.
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RESULTS
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AF1 and AF2 Function in a Position-Dependent Manner
Constructs were made with various combinations of AF1 and
AF2 substitutions in the context of the wild-type PEPCK promoter
ligated to the chloramphenicol acetyltransferase (CAT) reporter gene
(pAF12, also referred to as pPL32 in this and previous publications)
to compare the roles of AF1 and AF2 in the PEPCK GRU (Fig. 2
). When the AF2 element was replaced by
an AF1 element (pAF11) or, conversely, when the AF2 element was put
at the AF1 site (pAF22), glucocorticoid responses were 6070% lower
than those obtained from the wild-type pAF12 reporter (Fig. 2
). These
responses are equivalent to the glucocorticoid response obtained when
either AF1 or AF2 is deleted or mutated (7, 10). Thus, these results
show that the AF1 element is inert at the position of the AF2 element,
and the AF2 element is inactive at the AF1 site in the PEPCK GRU, and
suggest that AF1 and AF2 have distinct functions. These results are
consistent with our previous observations (17). Next, the AF1 element
and the AF2 element were switched with each other (pAF21, Fig. 2
) to
determine whether their relative positions within the GRU are critical.
If a specific spatial alignment between AF1, AF2, and the other members
of the GRU is not important for a complete glucocorticoid response, and
only the presence of these transcription factors is required, this
construct should confer a glucocorticoid response similar to that of
the wild-type pAF12 reporter. However, the pAF21 reporter construct
conferred a much weaker glucocorticoid response than did pAF12. Thus,
the positional alignment of AF1 and AF2 in the PEPCK gene promoter
appears to be critical for mediating optimal accessory factor
activity.

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Figure 2. The Alignment of AF1 and AF2 Is Critical
H4IIE cells were cotransfected with 10 µg of various reporter
constructs [either pAF12 (pPL32), pAF11, pAF22, or pAF21] and
5 µg of an expression plasmid that encodes the GR. Cells were treated
with or without 500 nM dexamethasone (DEX) for 1824 h,
and CAT activity was measured. Results are presented relative to the
wild-type glucocorticoid response (pPL32) and represent the mean
± SE of the number of experiments indicated.
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The Carboxy-Terminal Transactivation Domain of HNF3ß Confers AF2
Activity
The distinct and specific roles of AF1 and AF2 in the GRU
suggested by the results obtained in the previous experiment prompted
us to localize the domains required for the accessory factor activities
in HNF4 and HNF3. We first focused our attention on HNF3. A reporter
construct was made wherein the AF2 element was replaced by a yeast
GAL4-binding site (pGAL4AF2, Fig. 3A
) in the context of the wild-type
promoter construct. The response to dexamethasone in cells transfected
with pGAL4AF2 was about 30% that of the wild-type pPL32
(Fig. 3A
) and is similar to that obtained when the AF2 element is
deleted or mutated (a 5070% reduction) (7, 10). Cotransfection of
the pGAL4AF2 construct with expression vectors that encode
the GAL4 DNA-binding domain (DBD) ligated to various domains of HNF3ß
was performed to identify the region(s) within HNF3ß required for AF2
activity (Fig. 3
). HNF3ß was used in this study because it represents
the major constituent that binds to the AF2 element in H4IIE hepatoma
cells (14).

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Figure 3. The C-Terminal Transactivation Domain of HNF3ß Is
Sufficient for AF2 Activity
A, H4IIE cells were transfected with pPL32, the wild-type PEPCK
promoter reporter construct or with pGAL4AF2, wherein the
AF2 element is replaced with a GAL4-binding site. In this and
subsequent experiments, the amount of DNA transfected is described in
Materials and Methods. All cells were cotransfected with
the GR expression vector. Cells were treated with or without 500
nM dexamethasone (DEX) for 1824 h, and CAT activity was
measured. Results are presented as the fold induction and represent the
average ± SE. of the number of experiments indicated
in parenthesis. B, Expression plasmids encoding one of the various
GAL4·HNF3ß fusion proteins and the GR expression vector were
cotransfected with pGAL4AF2, and the cells were treated
with or without 500 nM dexamethasone. The results are
presented as described above. The schematic structure of HNF3ß
protein is also shown. The shaded areas represent the
domains critical for the transactivation activity of HNF3ß
(20 ). The conserved regions of HNF3 , -ß, and - (I-IV)
are also shown.
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Cotransfection of H4IIE cells with the pGAL4AF2 reporter
construct and an expression plasmid that encodes full-length HNF3ß
fused to GAL4 DBD had no effect on the glucocorticoid response, as CAT
expression was equivalent to that detected in cells transfected with a
simple GAL4 DBD construct (data not shown). This result is perhaps not
surprising since the GAL4·HNF3ß chimeric protein contains two
separate DBDs. A similar phenomenon, very low transcriptional activity,
was observed when chimeric proteins that contain the GAL4 DBD and
either full-length C/EBP
, C/EBPß, and ATF-2 are tested (18, 19).
Strong transcriptional activity is observed only when the DBDs of
C/EBP
, C/EBPß, and ATF-2 are deleted (18, 19). We therefore
constructed a series of expression plasmids that encode proteins in
which the GAL4 DBD is ligated to various segments of HNF3ß, all of
which lack the DBD of the latter. GAL4·HNF3ß (1157), like the
GAL4 DBD, did not influence the glucocorticoid response (Fig. 3B
, lines
1 and 2). In contrast, cotransfection of cells with the
pGAL4AF2 construct and an expression plasmid encoding
GAL4·HNF3ß(257458) restored the glucocorticoid response to a
level near that found in cells transfected with pPL32 (Fig. 3B
, line
3). Thus, the C-terminal domain of HNF3ß mediates most of the AF2
activity. Two additional plasmids, wherein smaller segments of the
C-terminal portion of HNF3ß were fused to the GAL4 DBD, were
cotransfected with pGAL4AF2 to further localize the minimal
domain required for AF2 activity. While the expression of
GAL4·HNF3ß(361458) provided a robust glucocorticoid response in
H4IIE cells, nearly the equal of that obtained from pPL32,
GAL4·HNF3ß(257360) expression failed to enhance the
glucocorticoid response (Fig. 3B
, lines 4 and 5). Hence, the accessory
activity of AF2 is conferred by the region located between amino acids
361458 (a.a. 361458) of HNF3ß.
The 361458 domain includes the primary transactivation domain of
HNF3ß (20). Two regions within this domain are highly conserved among
HNF3 family members: a.a. 369387 (conserved box II) and a.a. 445458
(conserved box III) (see Fig. 4B
) (20).
The transactivation capacity of HNF3ß is severely reduced when either
of these two conserved boxes is deleted or mutated (20). We therefore
made the expression plasmid GAL4·HNF3ß(388458), wherein conserved
box II is deleted, and GAL4·HNF3ß(361442), wherein conserved box
III is deleted (Fig. 4B
). Cotransfection of GAL4·HNF3ß(388458)
with pGAL4AF2 into H4IIE cells provided a partial
glucocorticoid response, but cotransfection of GAL4·HNF3ß(361442)
did not provide any accessory factor activity (Fig. 4
, lines 3 and 4).
These data suggest that, although box III contributes most of the
activity of AF2, boxes II and III are both required for full AF2
activity (compare lines 2 and 4, Fig. 4B
).

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Figure 4. Conserved Regions II and III in HNF3 Are Required
for AF2 Activity
A, H4IIE cells were transfected with pPL32, the wild-type PEPCK
promoter reporter construct, or with pGAL4AF2, wherein the
AF2 element is replaced with a GAL4-binding site. All cells were
cotransfected with the GR expression vector. Cells were treated with or
without 500 nM dexamethasone (DEX) for 1824 h, and CAT
activity was measured. Results are presented relative to the wild-type
glucocorticoid response (pPL32; top panel) and represent
the average ± SE of the number of experiments
indicated. B, An expression plasmid encoding one of the various
GAL4·HNF3ß fusion proteins was cotransfected with the GR expression
vector and with pGAL4AF2. Cells were treated and results
are presented, with or without 500 nM dexamethasone, as
described above. The schematic structure of HNF3ß protein is shown,
as described in Fig. 3 .
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The inherent capacity for the activation of basal transcription by each
of the GAL4·HNF3ß fusion protein expression vectors was monitored
by cotransfecting them with a CAT reporter plasmid that contains five
copies of a GAL4-binding site positioned upstream of a E1B TATA box
[(GAL4)5E1bCAT, Fig. 5
].
HepG2 cells, previously used to map the transactivation domain of
HNF3ß (20), were also used in this study since transfection of large
amounts of DNA were required to detect basal activity in H4IIE cells.
The relative activity of each of the GAL4·HNF3ß chimeric proteins
is presented in Fig. 5
. For comparison, a qualitative indication of AF2
activity of the same constructs is also presented. The activity of
GAL4·HNF3ß(361458) is 2-fold higher than that of
GAL4·HNF3ß(388458) and 15-fold higher than that of
GAL4·HNF3ß(361442) (Fig. 5
, lines 5, 6, and 7). Thus, the
transactivation capacity of these three GAL4·HNF3ß fusion proteins
correlates well with their ability to support AF2 activity.
Interestingly, expression of GAL4·HNF3ß(257458), which mediates
AF2 activity, provided little basal activity (Fig. 5
, line 3); perhaps
there is a repression domain for basal activity located between a.a.
257360. The N-terminal portion of HNF3ß contains a region that is
conserved among HNF3 family members (box IV, a.a. 1493), and this may
be important for transactivation activity (34, 37). However,
GAL4·HNF3ß(1157) did not mediate AF2 activity nor did it activate
the (GAL4)5E1bCAT reporter gene (Fig. 5
, line 2). This is
consistent with previous results, which also suggest that this
conserved region cannot function as an independent activation domain
(20). The expression of the GAL4·HNF3ß chimeric proteins was
confirmed in an independent gel mobility shift experiment using nuclear
extracts prepared from GAL4·HNF3ß fusion protein-expressing cells.
COS cells were used for this experiment because of the very low
transfection efficiency of H4IIE hepatoma cells. All of the chimeric
proteins were expressed efficiently, albeit at somewhat different
levels (Fig. 6
). In fact,
GAL4·HNF3ß(361458), which provides the strongest AF2 activity,
was expressed at a low level relative to the other GAL4·HNF3ß
chimeric proteins (Fig. 6
).

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Figure 5. Comparison of the Basal Transactivation and AF2
Activities of Various GAL4·HNF3ß Fusion Proteins
Expression plasmids (7.5 µg) that encode the GAL4·HNF3ß fusion
constructs were cotransfected into HepG2 cells with a reporter
construct (2.5 µg) that contains five tandem copies of the
GAL4-binding site inserted into the E1b promoter
[(GAL4)5E1bCAT)]. Results are presented relative to the
activity of GAL4 DBD protein and are the average ± SE
of at least five experiments. The AF2 activity of each GAL4·HNF3ß
fusion protein is also shown. The schematic structure of the HNF3ß
protein is shown, as described in Fig. 3 .
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Figure 6. Expression and DNA Binding of Various GAL4-HNF3ß
Fusion Proteins
COS cells were transfected with expression plasmids (40 µg) that
encode various GAL4-HNF4 fusion constructs [1: GAL4DBD; 2:
GAL4·HNF3ß(1157); 3: GAL4·HNF3ß(257458); 4:
GAL4·HNF3ß(257360); 5: GAL4·HNF3ß(361458); 6:
GAL4·HNF3ß(361442); and 7: GAL4·HNF3ß(388458)]. Whole-cell
extracts were prepared, and the gel mobility shift assay was used to
analyze expression levels (as described in Materials and
Methods). The arrow indicates the various
GAL4·HNF3ß fusion proteins. Each protein was supershifted by prior
incubation with an antibody directed against the GAL4 DBD.
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The Transactivation Domain of HNF4 Provides AF1 Activity
The approach described above was also used to localize the
functional domain(s) of HNF4 required for AF1 activity. The
glucocorticoid response in H4IIE cells transfected with a
pPL32-derived reporter construct in which the AF1 element is replaced
with a GAL4-binding site (pGAL4AF1) was 6070% that of
wild type (Fig. 7A
), an effect similar to
that seen when AF1 is deleted or mutated (7, 10). The glucocorticoid
response did not change when cells were cotransfected with
pGAL4AF1 and a plasmid that encodes the GAL4 DBD (Fig. 7B
, line 1). However, the glucocorticoid response was strongly enhanced
when a plasmid that encodes a chimeric protein consisting of the
full-length HNF4 and the GAL4 DBD [GAL4·HNF4(1455)] was
cotransfected into cells with pGAL4AF1 (Fig. 7B
, line 2).
Cotransfection of a GAL4DBD·HNF4 chimeric protein that lacks the
C-terminal 81 amino acids [GAL4·HNF4(1374)] with the
pGAL4AF1 reporter construct into cells provided a slightly
greater glucocorticoid response than did GAL4·HNF4(1455) (Fig. 7B
, line 3). Thus, the 374455 a.a. segment of HNF4 is dispensable for AF1
activity. Since the entire C-terminal transactivation domain of HNF3ß
is required for AF2 activity, it was of interest to determine whether
the two transactivation domains of HNF4 are critical for AF1 activity.
One of these domains is located within the first 24 amino acids of the
N-terminal region of the protein, and the second resides between amino
acids 128370 (21). Therefore, GAL4·HNF4(145), which contains the
N-terminal transactivation domain, or GAL4·HNF4(128374), which
contains the C-terminal transactivation domain, was independently
cotransfected with pGAL4AF1 into H4IIE cells to determine
whether either transactivation domain harbors AF1 activity. Expression
of GAL4·HNF4(145) in cells provided no significant AF1 activity
(Fig. 7B
, line 6). In contrast, the glucocorticoid response in cells
transfected with GAL4·HNF4(128374) was similar to that observed in
cells that express GAL4·HNF4(1455) (Fig. 7B
, line 5). Thus, the
C-terminal transactivation domain of HNF4 provides most of the AF1
activity. The region from a.a. 361- 366 is a highly conserved motif
among members of the nuclear receptor superfamily, and it is essential
for activity mediated through the C-terminal transactivation domain
(21). Cotransfection of GAL4·HNF4(1360), an expression plasmid that
lacks this region, with the pGAL4AF1 reporter construct did
not enhance the glucocorticoid response (Fig. 7B
, line 4). A
single-point mutation, L366E, causes a severe reduction in the
transactivation activity of HNF4 (21). The same mutation, in the fusion
construct GAL4·HNF4(1374/L366E), also abolishes AF1 activity (Fig. 7B
, line 7). In conclusion, these data indicate that the C-terminal
transactivation domain of HNF4 is indeed critical for its ability to
serve as accessory factor 1 in the glucocorticoid response.

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Figure 7. The C-Terminal Transactivation Domain of HNF4 Is
Sufficient for AF1 Activity
A, H4IIE cells were transfected with pPL32, the wild-type PEPCK
promoter reporter construct, or pGAL4AF1, wherein the AF1
element is replaced with a GAL4-binding site. All cells were
cotransfected with the GR expression vector and were treated with or
without 500 nM dexamethasone (DEX) for 1824 h. CAT
activity was then measured as described above. Results are presented
relative to the wild-type glucocorticoid response (pPL32; top
panel) and represent the average ± SE of the
number of experiments indicated. B, An expression plasmid encoding one
of the various GAL4·HNF4 fusion proteins and the GR expression vector
were cotransfected with pGAL4AF1. A schematic
representation of the structure of the HNF4 protein is shown. The
shaded areas represent the transactivation domain of
HNF4 (21 ). The asterisk (*) indicates the mutation site
(L366E) in one of the GAL4·HNF4fusion proteins.
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A comparison of basal transactivation with AF1 activity was performed
by cotransfecting the various GAL4·HNF4 fusion protein plasmids with
the (GAL4)5E1bCAT reporter construct into H4IIE cells. In
general, the transactivation capacities of GAL4·HNF4(1374),
(1360), and (1374/L366E) correlate with their ability to provide
AF1 activity (Fig. 8
, lines 57).
Interestingly, GAL4·HNF4(145) was the strongest activator of the
(GAL4)5E1bCAT reporter gene, but it demonstrated very low
AF1 activity (Fig. 8
, line 3). In contrast, GAL4·HNF4(128374)
mediates AF1 activity, but its transactivation activity is 8-fold lower
than that of GAL4·HNF4(145) (Fig. 8
, line 4). These results suggest
that a specific domain, rather than general transactivation capacity,
is the determinant of AF1 activity. Although GAL4·HNF4(1455),
(1360), and (1374/L366E) provided similar basal transcriptional
activity, only GAL4·HNF4(1455) mediates AF1 activity, and it is the
only one of these three constructs that contains the C-terminal
transactivation domain. The basal transcriptional activity of
GAL4·HNF4(1455) is 4-fold lower than GAL4·HNF4(1374) (Fig. 8
, lines 2 and 5). This result is consistent with the presence of a
repression domain for basal transcriptional activity located between
a.a. 374 and 455 (21). However, this repression domain has only a
slight effect on AF1 activity (Figs. 7
and 8
). The expression of the
GAL4·HNF4 chimeric proteins was confirmed by an independent
assessment in which gel mobility shift experiments were performed using
nuclear extracts from COS cells that expressed distinct GAL4·HNF4
fusion proteins (Fig. 9
).
GAL4·HNF4(1374), which provides the strongest AF1 activity, was
expressed at a level similar to that of GAL4·HNF4(1374/L366E) and
GAL4·HNF4(1360) and was expressesed at a level lower than
GAL4·HNF4(1455), GAL4·HNF(145), and GAL4·HNF4(128374) (Fig. 9
). These results indicate that the inability of GAL4·HNF(145),
GAL4·HNF4(1374/L366E), and GAL4·HNF4(1360) to confer AF1
activity is not due to inadequate expression in the cells.

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Figure 8. Comparison of the Basal Transactivation and AF1
Activities of Various GAL4·HNF4 Fusion Proteins
Expression plasmids (2 µg) that encode various GAL4·HNF4 fusion
constructs were cotransfected into H4IIE cells with a reporter
construct (5 µg) that contains five tandem copies of the GAL4-binding
site inserted into the E1b promoter [(GAL4)5E1bCAT].
Results are presented relative to the activity of GAL4 DBD protein and
represent the average ± SE of at least five
experiments. The AF1 activity of each GAL4·HNF4fusion protein is also
shown. The asterisk (*) indicates the mutation site (L366E) of the
GAL4·HNF4fusion protein.
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Figure 9. Expression and DNA Binding of Various GAL4-HNF4
Fusion Proteins
COS cells were transfected with expression plasmids (40 µg) that
encode various GAL4·HNF4 fusion constructs [1: GAL4 DBD; 2:
GAL4·HNF4(1455); 3: GAL4·HNF4(145); 4: GAL4·HNF4(128374);
5: GAL4·HNF4(1374); 6: GAL4·HNF4(1374/L366E); and 7:
GAL4·HNF4(1360)]. Whole-cell extracts were prepared, and the gel
mobility shift assay was used to analyze expression levels (as
described in Materials and Methods). The result using
the GAL4 DBD was also shown in Fig. 6 . The arrow
indicates the various GAL4·HNF4 fusion proteins. Each protein was
supershifted by prior incubation with an antibody directed against the
GAL4 DBD.
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The Activity of Other Transactivation Domains at AF1 and AF2,
Respectively
Plasmids that encode fusion proteins of the GAL4 DBD and
various transactivation domains were cotransfected with either
pGAL4AF1 or pGAL4AF2 reporter genes (Figs. 10
and 11
), to determine whether other classes
of transactivation domains can substitute for the accessory factor
activities of HNF3 and HNF4. Cotransfection of H4IIE cells with the
pGAL4AF1 construct and an expression plasmid that encodes
GAL4·E1A, which consists of the GAL4 DBD and the CR3 region of
adenovirus E1A protein, provided a 2- to 3-fold induction of the basal
promoter activity (data not shown), and the glucocorticoid response was
similar to that obtained from GAL4·HNF4(1374) (Fig. 10B
, lines 2
and 3). GAL4·E1A also provided a 3- to 5-fold increase of basal
promoter activity and conferred some AF2 activity when cotransfected
with pGAL4AF2 into the cells (data not shown and Fig. 11B
, lines 2 and 3). GAL4·VP16, which contains the GAL4 DBD and the
C-terminal acidic transactivation domain of herpes simplex virus VP16
protein, also resulted in a 2- to 3-fold increase of basal promoter
activity from pGAL4AF1 and a 3- to 5-fold increase from
pGAL4AF2 (data not shown). However, GAL4·VP16 conferred
only partial AF1 activity and failed to provide AF2 activity (Fig. 10B
, line 4, and Fig. 11B
, line 4). GAL4·SP1, which consists of the
glutamine-rich activation domain of SP1 fused to the GAL4 DBD, and
GAL4·CTF, which consists of the proline-rich activation domain of
NF1/CTF fused to the GAL4 DBD, did not increase the basal promoter
activity (data not shown) or provide accessory factor activity from
either element (Fig. 10B
, lines 5 and 6, and Fig. 11B
, lines 5 and 6).
Hence, among these four distinct classes of transactivation domains,
only the E1A CR3 region provided strong AF1 activity, and none afforded
complete AF2 activity. These results suggest there is a high degree of
specificity associated with the transactivation domains of HNF4 and
HNF3ß in their roles as accessory factors in the PEPCK GRU.

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Figure 10. Domain Specificity of AF1 Activity
A, H4IIE cells were transfected with pPL32, the wild-type PEPCK
promoter reporter construct or with pGAL4AF1, wherein the
AF1 element is replaced with a GAL4-binding site. All cells were
cotransfected with the GR expression vector. Cells were treated with or
without 500 nM dexamethasone (DEX) for 1824 h, and CAT
activity was measured. Results are presented relative to the wild-type
glucocorticoid response (pPL32) and represent the average ±
SE of the number of experiments indicated. B, An expression
plasmid, encoding one of the various GAL4 DBD and transactivation
domain fusion proteins, and the GR expression vector were cotransfected
with pGAL4AF1. Other details are as described in panel A.
|
|

View larger version (18K):
[in this window]
[in a new window]
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Figure 11. Domain Specificity of AF2 Activity
A, H4IIE cells were transfected with pPL32, the wild-type PEPCK
promoter reporter construct, or with pGAL4AF2 wherein the
AF2 element is replaced with a GAL4-binding site. All cells were
cotransfected with the GR expression vector. Cells were treated with or
without 500 nM dexamethasone (DEX) for 1824 h and CAT
activity was measured. Results are presented relative to the wild-type
glucocorticoid response (pPL32) and represent the average ±
SE of the number of experiments indicated. B, An expression
plasmid, encoding one of the various GAL4 DBD and transactivation
domain fusion proteins, and the GR expression vector were cotransfected
with pGAL4AF2. Other details are as described in panel A.
|
|
 |
DISCUSSION
|
---|
Much has been learned from the analysis of simple hormone response
elements (HREs) placed, as single or multimerized copies, in the
context of heterologous promoter reporter gene constructs. It is not
clear, however, whether such receptor-binding elements are sufficient
for function in native promoters. Multicomponent assemblies, including
the HRE and cis-elements with associated DNA-binding
proteins (so-called accessory factors), in an arrangement referred to
as a hormone response unit (HRU), appears to be much more common
(22). Additional complexity is provided by a number of coregulators,
which can bind to the hormone receptor and/or the accessory factors to
modulate the rate of transcription of a given gene. A specific HRU thus
consists of a number of protein-DNA and protein-protein interactions
involving the hormone receptor, accessory factors (>30 have been
identified) and coregulators.
The PEPCK gene GRU is an example of the HRU described above. A complete
response of the PEPCK gene to glucocorticoids requires the presence of
four accessory factor elements and associated proteins (AF1, AF2, AF3,
CRE) and two GREs (GR1 and GR2) with associated ligand-receptor
complexes (see Fig. 1
). The loss of function of any one of these
accessory element factor complexes results as a
50% reduction of
the glucocorticoid response (6, 8, 10). Loss of function of any
combination of two results in a complete loss of the glucocorticoid
response. Similarly, a partial response is obtained if either of the
GREs is deleted, but GR1 is quantitatively more important than GR2 (9).
It is evident that considerable redundancy has been built into this
system. Having defined the components of the GRU, we can now begin to
address several key questions. For example, are there unique structural
requirements with respect to the orientation, positioning, and spacing
of the accessory factors with respect to each other and the GREs? What
domains are responsible for the accessory factor action of these
proteins? Are coregulatory proteins involved? And finally, how does all
of this come together to control the rate of transcription of the PEPCK
gene? The work in this paper addresses the first two of these
questions.
In the studies described above, it is apparent that AF1 cannot
substitute for AF2, and vice versa. Also, the two elements do not work
well when inverted with respect to the rest of the GRU (Fig. 2
).
Therefore, it appears that a specific spatial organization of AF1 and
AF2 within the PEPCK gene GRU is critical for the complete
glucocorticoid response. These results also suggest that AF1 and AF2
have distinct functions. If so, specific domains that either activate
transcription directly, or through a coregulatory intermediate, should
be present in the accessory factors that bind to these elements, namely
HNF4 and HNF3.
AF1 activity is conferred by the carboxy-terminal transactivation
domain of HNF4 (a.a. 128374) (Fig. 8
). It is not known whether this
domain functions directly or indirectly. The fact that this accessory
function can be assumed by the activation domains of E1A or VP16, which
have no obvious similarity to the HNF4 domain (Fig. 9A
), suggests that
a protein-protein interaction is involved. HNF4 binds to TFIIB and
facilitates formation of the preinitiation complex (23); however, an
HNF4 mutant that lacks the C-terminal activation domain still binds to
TFIIB (23), so the effect may be mediated through the N-terminal
transactivation domain, a region not necessary for accessory factor
action in our studies (Fig. 8
). Several coactivators, such as steroid
receptor coactivator-1 (SRC-1) (24), CBP/p300 (25, 26, 27), glucocorticoid
receptor interacting protein 1 (GRIP1)/TIF2 (28, 29, 30), and
RAC3/ACTR (31, 32, 33), associate with the C-terminal activation domain of
various members of the nuclear receptor superfamily. These proteins,
including HNF4, have very similar C-terminal transactivation domains
(21). Indeed, we and others have recently shown that SRC-1, GRIP1, and
CBP/p300 function as coactivators for HNF4 (34, 35). The CR3 region of
E1A exhibits AF1 activity, so this domain and the C-terminal region of
HNF4 may interact with a common coactivator. The CR3 domain associates
with a wide variety of transcription factors and components of the
basal transcription machinery, including the TATA-box binding
protein (TBP), certain TBP-associated factors (TAF) such as human TAF
135 (36), Drosophila TAF 110, and TAF 250 (37), and several
other DNA binding proteins, including ATF2, USF, and SP1 (38). However,
the interaction between the CR3 region of E1A and SRC-1, GRIP1, and
CBP/p300 has not been reported. Therefore, it is also possible that
distinct coactivators provide AF1 activity in the context of the PEPCK
GRU; thus, HNF4 and E1A may each interact with different coactivators.
COUP-TF also serves as accessory factor 1 for the PEPCK glucocorticoid
response through both the AF1 and AF3 elements (6, 11). COUP-TF
functions as both a positive and negative regulator of gene
transcription (39), and it is not known whether COUP-TF uses the same
mechanism as HNF4 to provide AF1 activity. We are currently identifying
the domain(s) that provide AF1 activity in COUP-TF, and this analysis
should clarify the issue of whether unique or common coactivators are
involved.
AF2 activity is provided by the C-terminal transactivation domain of
HNF3ß, which contains two regions that are conserved in the various
members of the HNF3 family of transcription factors (20). Region III
(a.a. 445458) contributes most of the AF2 activity, although region
II (a.a. 369387) is required for complete AF2 activity (Fig. 4
).
These regions do not have amino acid sequence motifs common to known
transactivation domains (20). However, region III is a potent
transactivator when fused to the GAL 4 DBD (Fig. 5
). No coactivator has
been identified to date that specifically interacts with the C-terminal
transactivation domain of HNF3ß. The CR3 domain of E1A provides
substantial AF2 activity (Fig. 11
), but VP16, in contrast to the case
with AF1, is inactive. Future experiments should clarify whether this
distinction is important. Furthermore, we have recently found that
HNF3ß and GR interact in vitro (17). However, the
C-terminal transactivation domain of HNF3ß does not interact with GR
in glutathione-S-transferase pull-down experiments
(J.-C. Wang and D. K. Granner, unpublished observation). More
studies will be required to determine whether the previously noted
interaction between HNF3ß and GR is physiologically important.
The identification of the domains of HNF4 and HNF3ß necessary for
accessory factor activity brings us a step closer to understanding how
the GRU works. In a previous paper we suggested that AF1 might
stabilize the binding of GR to GR1, and that AF2 could be required for
achieving the maximal transactivation potential of the GRU (9). This
hypothesis is based on the observation that the AF1 element is no
longer required for the PEPCK gene glucocorticoid response when GR1, a
low-affinity GR-binding site, is replaced by a strong GR-binding site
(9). In contrast, the AF2 element is still required for a complete
glucocorticoid response in this situation. The observation that AF1 and
AF2 cannot substitute for one another, and that these elements must be
oriented in a certain way with respect to each other and with the
GR-binding sites, coupled with the identification of the
transactivation domains of HNF4 and HNF3ß, should allow us to test
the hypothesis that the assembly of a stereospecific nucleoprotein
complex results in the stabilization of GR binding to the promoter and
allows for efficient activation of the basal transcription machinery.
GR may act as a signal transducer that triggers the assembly of this
nucleoprotein complex rather than as a transactivator, since neither
GR1 nor GR2 can mediate a glucocorticoid response by themselves.
Alternatively, the transactivation domain of GR may contribute to the
overall transcriptional capacity of the PEPCK GRU, but only in the
presence of the interaction and/or transactivation domains of the
accessory factors.
The PEPCK gene GRU is conceptually similar to the interferon ß
(IFNß) enhanceosome. Viral induction of the IFNß gene requires the
functional interaction of several regulatory elements, designated PRD
I, II, III, and IV. PRD II, III, and IV bind the transcription factors
NF
B, IRF1, and an ATF/c-Jun heterodimer, respectively. Substitution
of PRD IV with PRD II markedly reduces the viral induction (40) just as
substitution of the AF1 and AF2 elements reduces the glucocorticoid
response of the PEPCK gene. Interestingly, when the transactivation
domains of either NF
B or IRF1 are replaced by the transactivation
domain of VP16, viral induction of IFNß enhanceosome is markedly
decreased even though these constructs provide much stronger basal
transcription activity (41). Furthermore, replacement of the
transactivation domain of p65 by IRF1 reduces viral induction of IFNß
enhanceosome activity (41), a result also similar to those described in
this paper. The assembly of a high-order nucleoprotein complex is
required for the highly specific activation of IFNß enhanceosome, and
CBP/p300 serves as an integrator in this complex (41, 42, 43). It is
conceivable that the activation of PEPCK gene transcription by
glucocorticoids may use a similar mechanism. The next challenge will be
the identification of coactivators that interact with the functional
domains of HNF3ß and HNF4 and mediate AF1 and AF2 activities.
The multicomponent GRU may have evolved to provide both tissue-specific
expression and versatile hormonal regulation of hepatic PEPCK gene
transcription in response to a wide variety of environmental cues. For
example, PEPCK gene transcription is positively regulated by
glucocorticoids in liver and negatively regulated in adipose tissue (1, 44). The liver-enriched transcription factors, HNF3 and HNF4, serve as
AF2 and AF1, respectively, but these factors are not present in adipose
tissue. Thus, tissue-specific accessory factors may allow for
differential regulation of PEPCK gene transcription by the same
effector in different tissues.
A simple GRE represents either an "all or none" or "on and off"
switch, whereas the GRU allows for a graded response, and the multiple
GR-binding sites and accessory factors provide inherent intraunit
redundancy. Interestingly, the PEPCK GRU is part of a much more complex
assembly of elements and factors, which we have termed a metabolic
control domain (MCD) (45). The MCD consists of the response units for
several hormones and metabolites, and it provides an integrated
response to the demands of the organism for tightly regulated
gluconeogenesis. In addition to their role in the glucocorticoid
response, the AF1 and AF3 elements also function as retinoic acid
response elements (RARE 1 and 2) by binding a heterodimeric complex
that consists of the retinoic acid receptor and
9-cis-retinoic acid receptor (RAR/RXR) (5, 46, 47). RARE1
and RARE2 collectively constitute an RARU and mediate the retinoic acid
response that is necessary for gluconeogenesis (48). The CRE is an
accessory element in the glucocorticoid response, and is part of a cAMP
response unit that mediates the stimulatory effect of glucagon (15, 49, 50), which is the primary hormonal stimulator of gluconeogenesis.
Finally, the AF2 element, as part of a multicomponent IRU, also
mediates the repressive effect of insulin by binding an, as yet
unidentified, protein (51) and thus participates in the down-regulation
of gluconeogenesis. Thus, the different sets of proteins that bind to
the PEPCK MCD, of which the GRU is an integral part, determine the
direction and magnitude of PEPCK gene transcription and provide for
additivity, synergism, intra- and inter-unit redundancy, and the
dominance of one effect over another. This complex system has
presumably evolved to provide the array of responses necessary to
maintain a rate of gluconeogenesis compatible with the adaptive needs
of the organism.
 |
MATERIALS AND METHODS
|
---|
Plasmid Constructions
Oligonucleotides used in plasmid constructions (listed in Table 1
) were synthesized by an Expedite 8909
oligonucleotide synthesizer (Perceptive Biosystems, Framingham, MA).
The plasmids pGAL4AF1, pGAL4AF2, pAF11, and
pAF22 were derived from pPL32 by PCR-based mutagenesis using the
corresponding oligonucleotides and a 3'-chloramphenicol
acetyltransferase (CAT) primer. pPL32 is a fusion gene construct that
contains the PEPCK gene promoter sequence from nucleotides -467 to +69
(relative to the transcription start site) ligated to the CAT reporter
gene (52). pPL32 was cleaved with HindIII and
BglII, and the wild-type PEPCK sequence was replaced with
the various PCR-derived sequences. The entire sequence of the subcloned
DNA fragment was confirmed by sequencing. The plasmid pAF21 was
generated by the same method, using the pAF11 reporter plasmid as a
template and oligonucleotide AF22 as a primer. The reporter plasmid
(GAL4)5E1bCAT has been previously described (53).
The expression plasmids for GAL4·HNF4 chimeras were generated in two
steps. First, the nucleotide sequences of rat HNF4 corresponding to
a.a. 145, 1455, 1374, 1360, and 128374 were generated by PCR
using primers H4/N/1 and H4/C/45, H4/N/1 and H4/C/455, H4/N/1 and
H4/C/374, H4/N/1 and H4/C/360, and H4/N/128 and H4/C/374, respectively.
A single point mutation of amino acid 366 (L366E) in the nucleotide
sequence corresponding to a.a. 1374 was generated by PCR using
primers H4/N/1 and H4/C/374/M366. These PCR fragments were then
digested with BamHI and KpnI, or BamHI
and XbaI for the 145 fragment, and subcloned into the
simian virus 40 (SV40) enhancer-driven GAL4 expression plasmid pSG424
(54). The expression plasmids for GAL4·HNF3ß were generated by the
same method. The nucleotide sequences of rat HNF3ß that correspond to
a.a. 1157, 257458, 257360, 361458, 361442, and 388458 were
generated by PCR using primers H3/N/1 and H3/C/157, H3/N/257 and
H3/C/458/X, H3/N/257 and H3/C/360, H3/N/361 and H3/C/458, H3/N/361 and
H3/C/442, and H3/N/388 and H3/C/458/K, respectively. These PCR
fragments were then digested with BamHI and KpnI,
or BamHI and XbaI for the 1157 and 257458
fragments, and subcloned into pSG424. The sequence of all subcloned DNA
fragments was verified by DNA sequencing. The expression plasmids,
GAL4·VP16, GAL4·E1A, GAL4·SP1, and GAL4·CTF/NF1, have been
described previously (53, 55, 56). pSV2GR, which expresses the rat GR
under control of the SV40 promoter, was provided by K. R. Yamamoto
(University of California at San Francisco). RSV (Rous sarcoma
virus)-ß-galactosidase was provided by M. A. Magnuson
(Vanderbilt University, Nashville, TN).
Cell Culture, Transient Transfection, and CAT Assays
The methods of transfection and maintenance of H4IIE and HepG2
hepatoma cells have been described previously (7, 13). In the
experiments performed with H4IIE cells, the dexamethasone response
varied from 7- to 20-fold (with dexamethasone/without dexamethasone).
The variation was passage and batch dependent. For this reason, we
expressed the data as a percentage of the wild-type dexamethasone
response, which gave much more consistent results. For experiments that
map the accessory domains of HNF3ß and HNF4, 10 µg of the reporter
construct (pGAL4AF2 or pGAL4AF1) were
cotransfected with 5 µg of GR expression vector, and 1, 2.5, 5, or 10
µg of expression plasmid encoding the various GAL4·HNF3ß or
GAL4·HNF4 fusion proteins. The optimal hormonal responses from each
titration experiment were pooled. The optimal concentration for the
expression plasmids encoding the GAL4·HNF4 fusion proteins was
consistently 1 µg. However, the concentration required for an optimal
glucocorticoid response using the GAL4·HNF3ß expression plasmids
encoding the various GAL4DBD and transactivation domain fusion proteins
ranged from 5 to 10 µg. In the experiments using HepG2 cells, CAT
activity was normalized to ß-galactosidase activity.
ß-Galactosidase activity was measured by adding 25 µl cell extract
to 175 µl assay of buffer (60 mM
Na2HPO4, 40 mM
NaH2PO4, 10 mM KCl, 1
mM MgSO4, and 50 mM
ß-mercaptoethanol), and 80 µl
o-nitrophenol-ß-D-galactopyranoside (ONPG)
solution (4 mg/ml ONPG, 72 mM
Na2HPO4, 200 mM
NaH2PO4). ß-Galactosidase activity was
monitored by absorbance at 420 nm. COS-1 cells were grown in DMEM
containing 10% FCS (GIBCO/BRL, Gaithersburg, MD). The calcium
phosphate method was used to transfect COS cells. Briefly, the calcium
chloride-plasmid DNA precipitate was added to 11.5 x
106 cells. After incubation at 37 C for 1820 h, cells
were washed with PBS and then placed in fresh culture medium and
incubated for an additional 24 h. Whole-cell extracts of COS cells
were prepared as described previously (57). Briefly, the cells were
washed with PBS, collected by centrifugation, and resuspended in 5060
µl of lysis buffer [20 mM HEPES (pH 7.9), 0.4
M NaCl, glycerol 25%, 1 mM EDTA, 2.5
mM dithiothreitol, and 1 mM
phenylmethylsulfonylfluoride]. Cells were kept on ice for
1520 min, frozen at -70 C, and thawed on ice. The cell suspension
was then centifuged for 10 min in a microfuge at 4 C. The supernatant
was used in the gel mobility shift experiments.
Gel Mobility Shift Assays
The gel mobility shift assays were performed as described
previously (58) with certain modifications. Briefly, whole-cell
extracts (5 µg) were incubated in the presence or absence of a GAL4
DBD antibody (Santa Cruz Biotechnology, Santa Cruz, CA) in a 10-µl
reaction for 1015 min and then mixed with 10 µl of a reaction
buffer containing 30,000 cpm 32P-labeled DNA probe
(5'-CACACGGAGGACTGTCCTCCGACCA-3'), 20 mM HEPES, pH 7.7, 100
mM NaCl, 5 mM MgCl2, 10
µM ZnCl2, 6% glycerol, 0.6 µg
poly(dI·dC) (Pharmacia, Piscataway, NJ), and 2 µg salmon sperm DNA,
for another 1015 min at room temperature. The reaction mixtures were
then loaded onto a 4.8% polyacrylamide gel in 0.5x Tris-borate/EDTA
electrophoresis buffer (TBE). After electrophoresis at 20 mA for 150
min at room temperature, the gels were dried and exposed to an x-ray
film (Eastman Kodak, Rochester, NY).
 |
ACKNOWLEDGMENTS
|
---|
We thank Drs. Frances Sladek and Rob Costa for providing HNF4
and HNF3ß cDNA, respectively. We thank Dr. James Manley for providing
GAL4·CTF expression plasmid and Dr. Yoel Sadovsky for providing
GAL4·SP1 expression plasmid. We also thank Drs. Roland Stein, Mina
Peshavaria, and Rob Hall for providing reagents and helpful
discussions; Drs. Calum Sutherland and Don Scott, for a critical
reading of this manuscript; Cathy Caldwell for her excellent technical
assistance; and Deborah C. Brown for helping to prepare this
manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Daryl K. Granner, Molecular Physiology and Biophysics, Vanderbilt School of Medicine, 707 Light Hall, Nashville, Tennessee 37232-0615. E-mail:
Daryl.Granner{at}mcmail.vanderbilt.edu
This work was supported by NIH Grant DK-35107 and by the Vanderbilt
Diabetes Research and Training Center (Grant DK-20593).
1 Present address: Astra Draco AB, Cell and Molecular Biology, 221 00
Lund, Sweden. 
Received for publication August 24, 1998.
Revision received December 22, 1998.
Accepted for publication January 8, 1999.
 |
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