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(HNF4) Gene Impair Recruitment of p300, a Key Transcriptional Coactivator
Unité 459 INSERM Laboratoire de Biologie Cellulaire Université H. Warembourg Lille, France F 59045
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(HNF4) is a
nuclear receptor involved in glucose homeostasis and is required for
normal ß-cell function. Mutations in the HNF4 gene are associated
with maturity-onset diabetes of the young type 1. E276Q and
R154X mutations were previously shown to impair intrinsic
transcriptional activity (without exogenously supplied coactivators) of
HNF4. Given that transcriptional partners of HNF4 modulate its
intrinsic transcriptional activity and play crucial roles in HNF4
function, we investigated the effects of these mutations on
potentiation of HNF4 activity by p300, a key coactivator for
HNF4. We show here that loss of HNF4 function by both mutations
is increased through impaired physical interaction and
functional cooperation between HNF4 and p300. Impairment of
p300-mediated potentiation of HNF4 transcriptional activity is of
particular importance for the E276Q mutant since its intrinsic
transcriptional activity is moderately affected. Together with previous
results obtained with chicken ovalbumin upstream promoter-transcription
factor II, our results highlight that impairment of recruitment
of transcriptional partners represents an important mechanism leading
to abnormal HNF4 function resulting from the MODY1 E276Q mutation.
The impaired potentiations of HNF4 activity were observed on the
promoter of HNF1, a transcription factor involved in a
transcriptional network and required for ß-cell function. Given its
involvement in a regulatory signaling cascade, loss of HNF4 function
may cause reduced ß-cell function secondary to defective HNF1
expression. Our results also shed light on a better structure-function
relationship of HNF4 and on p300 sequences involved in the
interaction with HNF4. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(HNF4) is a transcription factor
required for normal early embryogenesis (1, 2). HNF4 plays a central
role in the coordination of a complex transcription factor network that
defines the hepatocyte phenotype (3, 4, 5) and in the regulation of normal
pancreatic endocrine function (6). It controls expression of the
transcription factors HNF1 and HNF6 (5, 6, 7, 8, 9); the former is required
for pancreatic endocrine cell function and differentiation (10, 11),
whereas the latter plays a central role in pancreatic endocrine
differentiation at precursor stage (12).
HNF4 is a member of the hormone nuclear receptor superfamily (13).
It has a modular structure comprising a DNA binding domain (DBD)
located in the C domain and two activation function modules AF-1
(residues 1–24, located in the A domain) and AF-2 (residues 360–366,
located in the E domain) (14, 15). The HNF4 AF-2 module does not
exhibit an autonomous transactivation activity and requires the
sequence 128–366 (located in D–E domains) for its full activity (15).
Protein-protein interactions and synergies with other transcription
factors such as chicken ovalbumin upstream promoter transcription
factors (COUP-TFs) (16) and nuclear transcription factor Y
(NPY) (17) or with coactivators of the p160 family (18, 19) and the
CREB binding protein (CBP) (19, 20, 21, 22) enhance the transcriptional
activity of HNF4. CBP has been recently shown to be required for
HNF4 function: through acetylation of HNF4, which is required for
its nuclear retention, CBP increases HNF4 DNA binding and
transcriptional activities (22). CBP and the closely related p300
protein (together referred to as CBP/p300) (23) serve as coactivators
for other hormone nuclear receptors. CBP/p300 contains three LXXLL
motifs located in the N-terminal receptor interaction domain (RID),
near the C/H1 domain and in the C-terminal sequence, respectively (24)
(scheme in Fig. 1C
). Requirement of the
RID in CBP/p300- mediated activation of nuclear receptors depends on
nuclear receptors. The RID, which was initially shown to interact with
thyroid hormone receptor (TR) and retinoic acid receptor (RAR)
homodimers (24), is required to enhance activity of the peroxisome
proliferator-activated receptor
(PPAR)-retinoid X receptor
(RXR) heterodimer (25) but is dispensable for enhanced transactivation
by TR-RXR and RAR-RXR heterodimers (26, 27). CBP/p300-mediated
transcriptional potentiation of the activities of these two latter
heterodimers requires steroid receptor coactivator-1 (SRC-1) (26). On
the other hand, CBP-mediated activation of HNF4, which acts as a
homodimer (28), does not require SRC-1 (21). The N-terminal third of
CBP (sequence 1–771) forms a much stronger complex with full-length
HNF4 than its C terminus (21). Regions of HNF4 interacting with
CBP have been mapped to the AF-1 and to the sequence required for full
activity of the AF-2 [amino acids (aa) 128–366 (21)]. For
convenience, this latter sequence will be named D–E domains
hereafter.
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is involved in glucose homeostasis, and loss of HNF4
function results in the decreased expression of genes involved in
glucose transport and metabolism (6, 29) and in insulin secretion (6).
Mutations in the HNF4 gene are associated with type 1
maturity-onset diabetes of the young (MODY1), a monogenic form of
diabetes characterized by abnormal pancreatic ß-cell function (30).
Studies of functional properties of several MODY1-associated mutations
of HNF4 have shown that mutations can have variable effects on the
ability of HNF4 to transactivate a reporter gene (29, 31, 32, 33, 34, 35, 36, 37). We
have previously shown that mutations E276Q and R154X impair HNF4
functions. Mutation E276Q affects the interaction and synergy with
COUP-TFII, which enhances HNF4 activity on the HNF1 promoter
(33). Mutation R154X decreases the transcriptional activity of HNF4,
the decrease being more pronounced in pancreatic ß-cells compared
with non-ß-cells. In addition, in the former cells, R154X exhibits a
slight dominant-negative behavior (36). HNF4 R154X has intact AF-1
and DNA binding domains but lacks the E domain, which is involved in
interaction with CBP/p300. The E276Q mutation is located in this E
domain. Taking into account the crucial role of CBP/p300, we
investigated the effects of these two mutations on the physical
interaction and functional cooperation between HNF4 and p300. |
RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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transcriptional activity on several
promoters in various cell lines. Interestingly, the transcriptional
activity of R154X remained equal to one-third that of wild-type HNF4
on the HNF1 promoter in HEK 293T cells (Ref. 36 and Fig. 1A
). We
investigated whether this low transcriptional activity could be
enhanced by the coactivator p300. Cotransfection of p300 and HNF42
increased HNF1 promoter activity 3.8-fold over the activation by
HNF42 alone (Fig. 1B). Coexpression of p300 and R154X stimulated
HNF1 promoter activity only 2.3-fold over the activation by R154X
alone (Fig. 1B). Transfection of p300 in the absence of transfected
HNF4 did not increase the activity of the HNF1 promoter in HEK
293T cells (data not shown), indicating that the effect of p300 is
mediated through HNF4.
These results led us to compare interactions of wild-type and mutated
HNF4 with p300 by pull-down assays using the three
glutathione-S-transferase (GST)-p300(1–595),
GST-p300(1–340), and GST-p300(340–528) chimeras. GST-p300(1–595)
contains two LXXLL motifs located in the receptor interaction domain
(RID) and near the C/H1 domain, respectively, whereas GST-p300(1–340)
and GST-p300(340–528) contain only one of these LXXLL motifs (Fig. 1C)
(24). HNF42 and R154X were retained on both p300(>NOREF>1–595) and
p30040–528) fragments (Fig. 1D, lanes 5, 6, 9, and 10).
Nevertheless, quantification of intensities of bands indicated that,
compared with wild-type HNF42, R154X bound three times less
efficiently to these two p300 fragments (Fig. 1D, graph).
The specificity of these complexes was ascertained by the inability of
GST alone to bind HNF42 and R154X (Fig. 1D, lanes 3 and 4). HNF42
and R154X were not bound by the p300(1–340) fragment (Fig. 1D, lanes 7
and 8) although GST-p300(1–340) was readily expressed (Fig. 1D, inset).
A chimera comprising two copies of the HNF4 AF-1 module fused to
GAL4 DBD has been shown to interact with fragment 1–771 of CBP (14).
R154X binding to p300 is likely mediated through the AF-1 module
located in the sequence 1–24 since it has been shown that a HNF4
fragment spanning residues 45–142 does not interact with CBP (21). To
ascertain this hypothesis, the mutation F19D, which abolishes the
transcriptional activity of the AF-1 module (14), was introduced in
R154X. This amino acid residue is located within the sequence (aa
13–24) involved in the interaction between HNF4 AF-1 and CBP (14).
Results presented in Fig. 2A show that
mutation F19D strongly impaired the transactivation activity of R154X.
The drop in R154X-F19D activity is not due to its decreased expression
(Fig. 2A, inset). In addition, R154X-F19D activity was
poorly enhanced by p300: the activity was enhanced 1.17-fold whereas
that of R154X was enhanced 2.30-fold (Fig. 2B). By pull-down
experiments, we observed that the F19D mutation nearly abolished
interaction of R154X with p300 (Fig. 2C). These results confirm that
AF-1 is involved in R154X transcriptional activity and in potentiation
of this latter by p300.
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to
investigate the AF-1 contribution toward the potentiation of
full-length HNF4 activity by p300. F19D mutation resulted in a drop
of 50% in the transactivation activity of HNF42 (Fig. 3A). Conversely, the F19D mutation did
not alter the p300-mediated enhancement of HNF42 transcriptional
activity (Fig. 3B) despite a decrease in interaction with
p300(340–528) (Fig. 3C). These results show that the AF-1 is required
for the full intrinsic activity of HNF4 (without exogenously
supplied coactivators) but exhibits a secondary role in the
p300-mediated potentiation of this transcription factor.
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2 and HNF42-E276Q, which were
expressed at the same levels (Fig. 4A, inset), exhibited similar transactivation activities on the
HNF1 promoter (Fig. 4A). On the other hand, the p300-mediated
enhancement of these activities was markedly different: it was much
higher for HNF42 than for its E276Q mutant (Fig. 4B), thus
demonstrating that the E276Q mutation impairs the cooperation between
HNF4 and p300. We then investigated whether this impairment could be
correlated with a decrease in physical interaction between HNF4 and
its coactivator by pull-down experiments. In these experiments we used
GST-p300(1–595) and GST-p300(340–528) but not GST-p300(1–340), which
could not bind HNF4 (see Fig. 1D). We have previously shown that
wild-type and E276Q HNF42 do not bind GST (33). The E276Q mutation
significantly impaired the HNF4-p300 interaction as evidenced with
both GST-p300(1–595) and GST-p300(340–528) proteins (Fig. 4C).
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AF-2
Activation Function AF-2 activation function does not exhibit an
autonomous transcriptional activity and requires the sequence spanning
residues 128–366 for its full activity (15). The E276Q mutation is
located within this sequence, which encompasses the HNF4 D–E
domains. We therefore investigated the effect of the E276Q mutation on
the activity of the D–E domains. To this end, wild-type or mutated
HNF4 fragments spanning the sequence 128–369 fused to the GAL4 DBD
were expressed. These chimeras correspond to GAL4-HNF4(128–369) and
GAL4-HNF4(128–369)-E276Q and were denoted WT and E276Q,
respectively, in Fig. 5. In HEK 293T
cells, p300 alone elicited a 3-fold activation of the
(UAS)2xtk promoter containing two GAL4 response
elements (UAS) (Fig. 5A, lane b). GAL4-HNF4(128–369) and
GAL4-HNF4(128–369)-E276Q exhibited indistinguishable activities on
this promoter when p300 was not cotransfected (Fig. 5A, lanes c
and e). On the other hand, cotransfection of p300 resulted in a
greater enhancement of activity for GAL4-HNF4(128–369) than for
GAL4-HNF4(128–369)-E276Q (Fig. 5A, compare lanes d and f).
These experiments were also carried out in Hela cells in which the
p300-mediated enhancement of HNF4 activity was previously documented
(21). Again in these cells, the transactivation activities of
GAL4-HNF4(128–369) and GAL4-HNF4(128–369)-E276Q were similar
when p300 was not cotransfected (Fig. 5B, lanes c and e), and
cotransfection of p300 resulted in a higher enhancement of activity for
GAL4-HNF4(128–369) than for GAL4-HNF4(128–369)-E276Q (Fig. 5B, compare lanes d and f). Therefore, in both cell lines, the E276Q
mutation markedly impaired the cooperation between p300 and the HNF4
D–E domains. This impairment was correlated with a decrease in
physical interaction between p300 and HNF4 D–E domains, as
evidenced by pull-down assays (Fig. 5C).
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bound
efficiently to GST-p300(1–595) and GST-p300(340–528) but not to
GST-p300(1–340) (Fig. 1D). To determine more precisely the sequence of
p300 required for interaction with HNF4, we compared the abilities
of GST-p300(340–528) and GST-p300(355–528) to interact with wild-type
HNF4. In this experiment, we also analyzed binding of R154X and
GAL4-HNF4(128–369), which contain only one of the activation
function modules: AF-1 and AF-2, respectively. Removal of 15 amino acid
residues at the N terminus of the p300(340–528) fragment resulted in a
dramatic decrease in its interaction with full-length HNF4 and
GAL4-HNF4(128–369) (Fig. 6A, lanes
3 and 9 compared with lanes 2 and 8, respectively) but did not affect
binding of R154X (lanes 5 and 6 in Fig. 6A). The faint bands of
full-length HNF4 and GAL4-HNF4(128–369) retained on
GST-p300(355–528) were not due to a low expression of this latter
protein (Fig. 6A, inset). These results indicate that the
p300 sequence 340–354 is required for efficient binding of full-length
HNF4 and HNF4(128–369) but is dispensable for binding of R154X.
To confirm the role of this sequence in p300 recruitment by HNF4, in
a functional context ex vivo, we tested whether the
p300(302–443) fragment (Fig. 6B), containing this sequence 340–354
but lacking the domain involved in histone acetyltransferase
(HAT) activity, has a dominant negative effect on the
cooperation between p300 and HNF4. In this assay, the fragment
p300(355–443) (Fig. 6B) was used as a control. When cotransfected with
HNF4 and full-length p300, the fragment p300(302–443) exhibited a
dominant negative effect that was not observed with the fragment
p300(355–443) (Fig. 6 C). Taken together, results of in
vitro protein-protein interaction and ex vivo
transactivation experiments obviously demonstrated the involvement of
the sequence 340–354 of p300 in the potentiation of HNF4
activity.
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is AF-2 independent (21). Nevertheless,
results in Fig. 6A indicated that p300 (340–354) efficiently
interacted with HNF4 constructs containing the AF-2 module
[full-length HNF4 and HNF4(128–369)] but did not interact with
R154X lacking the AF-2. The presence of a LXXLL motif within the p300
sequence 340–354 (at position 342–346) and involvement of LXXLL
motifs in the interaction between coactivators and AF-2 modules of
nuclear receptors (27) led us to reevaluate the role of the HNF4
AF-2 module in binding to p300(340–528). To this aim, we compared
binding to GST-p300(340–528) of HNF4-
F and HNF4-AF-2,
which only differ by the lack of AF-2 in the latter (Fig.
7A). Deletion of the AF-2 resulted in a
significant decrease in binding to p300(340–528) (Fig. 7B), thus
demonstrating the crucial role of the HNF4 AF-2 in binding to this
p300 fragment.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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gene impair the functional
cooperation and physical interaction between HNF4 and p300. In the
case of the R154X mutant, this impairment can be ascribed to the lack
of AF-2, which plays a crucial role in the recruitment of p300 as
discussed below. In the case of the E276Q mutant, this impairment is
ascribed to an alteration of the interaction and cooperation between
p300 and the D–E domains, which are required for full
activity of the HNF4 AF-2 activation function. Impairment
of the p300-mediated potentiation of HNF4 transcriptional activity
is of particular importance for the E276Q mutant of which the intrinsic
transcriptional activity (without exogenously supplied coactivators) is
moderately affected (33, 35) by comparison with that of the R154X
mutant (35, 36). Another MODY1-associated HNF4 mutant, R127W, was
shown recently to have no effect on the p300-mediated enhancement of
HNF4 activity (37). The localization of this mutation in the HNF4
DBD (aa 50–142), which does not interact with CBP/p300 (21), may
account for the lack of change in CBP/p300 recruitment.
Many authors who study the interaction and cooperation between nuclear
receptors and their coactivators assume that they act synergistically
when their combined effects on enhancement of transcription of the
target promoter are higher than the sum of their individual effects.
Nevertheless, we cannot rule out that other models of interaction may
be involved in p300 recruitment by HNF4. Whatever the mechanism
involved in this interaction, our results clearly showed that
recruitment of p300, which has been shown to be essential for
transcription by HNF4, was impaired by the studied HNF4
mutations.
The ability of CBP/p300 to interact with the HNF4 AF-1 in the
context of the chimera GAL4 (AF-1)2x has been
shown previously (14). CBP/p300 interacts with the HNF4 fragments
1–128 (21) and 1–153 (present work), which contain the AF-1. Our
results showing significant decreases in interaction and cooperation
between R154X and p300 by the mutation F19D indicate unambiguously that
the AF-1 is actually involved in p300 recruitment to this truncated
HNF4. In addition, our results extend these findings by
demonstrating for the first time that the HNF4 AF-1 activity can be
enhanced by p300.
Mutation F19D resulted in a 50% decrease of full-length HNF42
activity despite the presence of the AF-2, a result consistent with
that of Green et al. (14) and which confirms the crucial
role of the AF-1 in the transcriptional activity of full-length
HNF4. The AF-1 role in full-length HNF4 activity does not depend
on p300 recruitment since the F19D mutation did not alter the
p300-mediated enhancement of full-length HNF4 transcriptional
activity. This result fits with those obtained by Dell and
Hadzopoulou-Cladaras (21) using a complete deletion of the AF-1. It
appears therefore that, even if p300-mediated potentiation of truncated
HNF4 lacking the AF-2, i.e. R154X, is exerted through the
AF-1, p300-mediated potentiation of HNF4 proteins containing the
AF-2 is exerted mainly through this latter activation function. This
suggests that, in the context of full-length HNF4, the p300-mediated
enhancement of AF-2 activity is strong enough to compensate for the
decrease in cooperation between p300 and the AF-1 by the F19D mutation.
This hypothesis is supported by the drop of 60% in cooperation between
p300 and full-length HNF4 due to the E276Q mutation located in the
D–E domains required for full activity of the AF-2. The role of the
AF-1 in the CBP/p300 action is much more crucial for other nuclear
receptors than for HNF4. Indeed, CBP/p300-mediated potentiation of
AF-1 plays a major role in the transcriptional activities of androgen
receptor, estrogen receptor-, and estrogen receptor-ß since
mutations in AF-1 cause reductions in ligand-induced functional
synergism between their AF-1 and AF-2 mediated by CBP/p300 (38, 39).
Previous mapping of CBP sequences interacting with full-length or
truncated HNF4 used either a CBP fragment spanning the sequence
271–451 (20) and corresponding to sequence 251–436 of p300 (23) or a
CBP fragment spanning the sequence 1–771 (14, 21). We have shown that
the p300 sequence 340–528 is sufficient for binding full-length
HNF4 as well as HNF4 fragments encompassing sequences 1–153 and
128–369 containing the AF-1 and AF-2, respectively. Interestingly, we
have observed that within the p300 fragment spanning residues 340–528,
the sequence 340–354, which contains a LXXLL motif, is required for
efficient interaction with the full-length HNF4 and the
HNF4(128–369) fragment containing the AF-2. Our results thus allow
a more precise mapping of sequences of p300 involved in its interaction
with HNF4.
We have observed that the HNF4 AF-2 plays a crucial role in
interaction with the p300(340–528) fragment, whereas Dell and
Hadzopoulou-Cladaras have shown that interaction of the CBP(1–771)
fragment with HNF4 is AF-2 independent (21) (referred to as AF-2 AD
in this reference). This discrepancy is not due to the presence of the
RID in the CBP(1–771) fragment since both our results and those of
Yoshida et al. (20) showed that the RID is dispensable for
interaction of CBP/p300 with HNF4. Nevertheless, we cannot exclude a
slight difference of behavior between CBP and p300 despite their very
close structures (23).
Cooperation between HNF4 and other transcription factors or
coactivators is crucial in HNF4-mediated transcriptional activation
(16, 17, 18, 40, 41, 42, 43, 44). Some of these transcriptional partners interact
directly with HNF4 (16, 17, 18, 44). In addition to alteration in p300
recruitment by R154X and E276Q mutations, we have previously shown that
alteration in synergy with the transcription factor COUP-TFII by the
E276Q mutation strongly impairs HNF4 function (33). Alteration
in recruitments probably occurs with other HNF4 partners since
the R154X and E276Q mutations also impair the in vitro
interaction with the GRIP-1 (glucocorticoid receptor interacting
protein-1) coactivator (J. Eeckhoute, unpublished results).
Therefore, our results emphasize that impairment of recruitment
of transcriptional partners by MODY1-associated HNF4 mutations most
likely represents a key mechanism leading to HNF4 loss of
function.
The impairment of p300-mediated enhancement of HNF4 activity
by MODY1 mutations has been observed on the HNF1 promoter. HNF1
is essential for insulin gene transcription and also regulates
expression of genes involved in glucose transport and
metabolism and in insulin secretion (11). In addition, loss of HNF1
function results in defective insulin secretion responses to glucose
(10, 11), and mutations in the HNF1 gene are associated with another
form of MODY, MODY3 (45). Interestingly, patients with MODY1 and MODY3
exhibit similar clinical profiles (46). Also, recent results
showed that the phenotype and gene expression patterns of INS-1
ß-cells expressing a dominant negative HNF4 are strikingly similar
to those of INS-1 ß-cells expressing a dominant negative HNF1 (6),
which led the authors to suggest that loss of HNF4 function may
cause abnormal ß-cell function secondary to defective HNF1
function. Therefore, impairment by R154X and E276Q mutations in the
p300 recruitment leading to a reduced activity of the HNF1 promoter
is of major interest. Nevertheless, as for other MODY1-associated
HNF4 mutations, further studies are needed to link our mechanistic
information to comprehension of ß-cell dysfunction and MODY1
physiopathology. Indeed, whatever the mechanisms underlying HNF4
loss of function (either deletion of the AF-2 activation function
for the Q268X and R154X mutations or impaired recruitment of HNF4
partners such as COUP-TF and p300 for the E276Q mutation), the
scientific community is still unable to explain clearly how these
HNF4 mutations lead to ß-cell abnormal function and to development
of diabetes. These mutations, as well as mutations in the HNF1 gene,
are supposed to cause haploinsufficiency or reduced gene dosage (35, 47) since they do not result in proteins exhibiting dominant negative
activities on the wild-type protein expressed from the unaffected
allele [except for a slight dominant negative behavior of R154X in
HIT-T15 cells (36)]. Strikingly, although the Q268X exhibits a
complete lack of transcriptional activity, its expression in ß-cells
did not result in deficient insulin secretion (6). Furthermore, the
huge variability in the clinical characteristics of patients with MODY1
diabetes suggests the existence of other factors (environmental,
genetic) influencing the phenotype of MODY1 patients (46).
Advances in clinical studies and cell biology of Langerhans
islets, together with functional studies of HNF4 mutants, are
clearly required for a better understanding of the physiopathology of
MODY1 diabetes. The present work, exploring the mechanisms by which the
mutations E276Q and R154X could affect the function of HNF4,
represents one contribution to achieve this goal. This work also sheds
new light on the crucial roles played by the glutamic acid residue at
position 276 and by the AF-2 activation function module in recruitment
of the p300 coactivator and therefore in the structure-function
relationship of this nuclear receptor.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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2 and its mutants E276Q, -F, and
-AF-2 cloned in the expression vector pcDNA3 were described
previously (33, 36, 48). The wild-type and R154X human HNF42 cloned
in the vector pcDNA3.1/HisB were described in Ref. 36 . Plasmids pcDNA3
HNF42-F19D and pcDNA3.1HisB HNF4-R154X-F19D were cloned using the
QuikChange site-directed mutagenesis kit from Stratagene
(La Jolla, CA) and primers spanning nucleotides 42–78 (taking A
of ATG of initiation methionine at position 1) with mutations T
G and
TA at positions 55 and 56, respectively. The plasmids pCMVß-NHA
p300 and its pCMVß control vector, pCMVmycNLS p300(302–443),
pCMVmycNLS p300(355–443), and pGEX2TK-p300(1–595), -p300(1–340),
-p300(340–528), and -p300(355–528) were generous gifts from Dr.
S. R. Grossman. The pGAP HNF4(128–369) vector expressing
wild-type HNF4(128–369) fused to the GAL4 DBD (aa 1–147)
corresponds to plasmid GAL AF-2 described in Ref. 48 ; its name has been
modified to comply with the AF-2 terminology now restricted to the
highly conserved module in helix 12 of nuclear receptors. The plasmid
pGAP HNF4(128–369)-E276Q was prepared by a similar method using
pcDNA3 HNF42-E276Q as template. The mouse HNF1
promoter-luciferase reporter construct was described in Ref. 33 . The
(UAS)2xtkLUC containing two copies of the
GAL4-binding element upstream of the thymidine kinase (TK) promoter was
a gift from V.K.K. Chatterjee. All constructs were verified by DNA
sequencing.
Cell Culture and Transient Transfection Assays
HEK 293T and Hela cells (2 x 105
cells and 1.5 x 105 cells per 12-well
dishes, respectively) were grown and transfected as in Ref. 33 except
that 1 µg of reporter plasmid, 50 ng of HNF4 expression plasmid,
and 500 ng of p300 expression vector were used unless otherwise
specified in the figure legends. Luciferase activities were measured
using the SteadyGlo buffer (Promega Corp., Madison, WI)
and the Lumicount apparatus (Packard Instruments, Meriden,
CT).
Western Blot Assays
Western blot assays were carried out as described previously
(33).
In Vitro Protein-Protein Interaction
Pull-down assays were performed as described previously (33)
using immobilized GST-p300 proteins and [35S]
labeled wild-type or mutated HNF42 which were synthesized in
vitro using reticulocyte lysates (Promega Corp.).
Binding of proteins was quantified by the PhosphorImager
apparatus using ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA).
Data Analysis
Statistical analysis was performed by Student’s t
test for unpaired data using the Prism software. The significance has
been considered at *** P < 0.001; ** P
< 0.01; * P < 0.05.
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ACKNOWLEDGMENTS |
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The authors are indebted to S. Grossman and M. P. Calos for providing plasmids expressing the p300 coactivator and HEK 293T cells, respectively. They acknowledge P. Lefebvre for critically reading the manuscript and I. Briche for her skillful technical assistance.
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FOOTNOTES |
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Received for publication November 13, 2000. Revision received March 26, 2001. Accepted for publication March 29, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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in Xenopus: rapid activation by HNF4 and delayed
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