| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
by Tumor Suppressor p53: Involvement of the Ligand-Binding Domain and Histone Deacetylase Activity
Environmental Toxicology Graduate Program (Y.M.), Departments of Cell Biology and Neuroscience (S.D.S., F.M.S.) and Biochemistry (G.W., X.L.), University of California, Riverside, California 92521
Address all correspondence and requests for reprints to: Frances M. Sladek, Ph.D., Department of Cell Biology and Neuroscience, 5429 Boyce Hall, University of California, Riverside, Riverside, California 92521.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
1 (HNF41), an orphan receptor required for liver
differentiation. Our results show that HNF41-mediated
transactivation is repressed by p53 but that the mechanism of
repression is not due to inhibition of HNF41 DNA binding. Rather,
transfections with Gal4 fusion constructs indicate that the repression
is via the ligand-binding domain of HNF41. Furthermore, we found
that p53 in human embryonic kidney whole-cell extracts preferentially
bound the ligand-binding domain of HNF41 and that the
activation function 2 region was required for the binding. Competition
for coactivator CREB binding protein could not entirely account for the
repression but trichostatin A, an inhibitor of histone deacetylase
activity, could reverse p53-mediated repression of
HNF41. In contrast, p53-mediated repression of
transcriptional activation of the same promoter by another
transcriptional activator, CCAAT/enhancer-binding
protein-, could not be reversed by the addition of
trichostatin A. These results suggest that p53, like other
transcriptional repressors, inhibits transcription by multiple
mechanisms, one of which involves interaction with the ligand-binding
domain and recruitment of histone deacetylase activity. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
There are several reports of p53 blocking transcription by nuclear receptors. Nuclear receptors are a superfamily of ligand-dependent transcription factors that play a critical role in growth, development, and differentiation of nearly every cell type in the body. Members of the superfamily include steroid receptors, vitamins A and D receptors, TR, and a large number of orphan receptors, some of which have recently been found to respond to fatty acids (PPARs), bile acids (farnesoid X receptor), cholesterol derivatives (liver X receptor), and xenobiotics (steroid and xenobiotic receptor, benzoate X receptor, and pregnane X receptor) (reviewed in Refs. 9, 10, 11, 12, 13). Like other transcriptional activators, nuclear receptors are known to activate transcription by binding specific DNA response elements in promoter/enhancer regions of genes and subsequently recruiting a variety of different coactivator complexes which, by some poorly defined mechanism, activate transcription by RNA polymerase II (reviewed in Ref. 14). p53 has been reported to block the activation of several different nuclear receptors by binding the DNA binding domain (DBD) of the receptor and, in at least some cases, subsequently inhibiting DNA binding [TR (15), GR (16), ER (17)].
Since overexpressed p53 is known to correlate with poor liver
differentiation in liver cancer (18, 19, 20), we wanted to
determine whether p53 might also affect hepatocyte nuclear factor 41
(HNF41), an orphan nuclear receptor (NR2A1) known to be important
for liver differentiation. In addition to liver, HNF41 is also
expressed in adult kidney, intestine, and pancreas but not
other tissues (reviewed in Ref. 21). HNF41 is
considered to be an orphan receptor in that a ligand has not yet been
definitively identified for it (22, 23). However, HNF41
is known to be essential for development in organisms ranging from
insects to mammals and to regulate many essential genes related to
nutrient transport and metabolism. For example, HNF41 positively
regulates genes involved in the transport of lipids and vitamins as
well as genes involved in lipid, amino acid, and glucose metabolism. It
also regulates genes involved in the regulation of several serum
proteins such as blood coagulation factors, erythropoietin, and
antithrombin III. HNF41 is directly linked to several human
diseases: the HNF4 gene has been found to be mutated in
maturity-onset diabetes of the young type 1, and HNF41 DNA binding
sites have been found to be mutated in patients with hemophilia B
Leyden and maturity onset diabetes of the young type 1
(21). Therefore, since a tissue-specific knockout
indicates that HNF41 is essential for proper functioning of the
adult liver (24), repression of HNF41-mediated
transcription is anticipated to impede the function of the liver and
possibly other organs as well.
Like other nuclear receptors, HNF41 recruits coactivators
of the p160 and p300/CBP families via an interaction that depends
primarily on the activation function 2 (AF-2) region (putative helix
12) in the ligand-binding domain (LBD) (25, 26, 27, 28, 29). These
coactivator complexes contain or are associated with histone acetylase
activity, which serves to decondense the chromatin, thereby
facilitating transcription initiation (8, 14, 30).
However, unlike most other receptors, HNF41 interacts with
coactivators and activates transcription in a constitutive fashion,
i.e. in the absence of an exogenously added ligand.
In the current work, using transient transfection analysis, we
show that p53 represses HNF41-mediated activation on a
HNF41-responsive promoter (derived from the apolipoprotein B (apoB)
gene) in several different cell lines. We also provide evidence
indicating that the repression is via a mechanism not previously
described for p53 but one that is reminiscent of corepressors. Namely,
repression occurs via interaction of p53 with the LBD of HNF41.
Furthermore, the interaction appears to be dependent on the presence of
the AF-2 and to result in recruitment of HDAC activity. Finally, we
present evidence suggesting that this mechanism of repression may not
be applicable to basic leucine zipper protein CCAAT/enhancer-binding
protein- (C/EBP), indicating that p53, like other corepressors,
inhibits transcription by multiple mechanisms.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
1-Mediated Transactivation in
Vivo1-mediated
transactivation, transient transfection assays were performed in three
different cell lines: Saos-2, a human osteosarcoma cell line that is
deficient in both endogenous p53 and endogenous HNF41 protein (our
unpublished results and this study); HepG2, a human hepatocellular
carcinoma cell line that is at least partially differentiated for the
liver phenotype and expresses endogenous HNF41 (Ref. 31
and our unpublished data) and low levels of wild-type p53 protein
(32); and 293T, a human embryonic kidney cell line that
transfects very well and displays no endogenous HNF41 protein but
contains high levels of wild-type p53 protein (see Fig. 1B
). The results indicate that in all
three cell lines, wild-type p53 repressed transactivation by
transfected HNF41 from a reporter construct containing four HNF41
sites from the human apoB promoter (Fig. 1A, compare lanes 4, 8, and 11
to lanes 3, 7, and 10, respectively). In HepG2 cells, p53 also
repressed transcription of the reporter construct alone, presumably by
interference with the endogenous HNF41 protein (compare lane 6 to
lane 5). The repression was relatively greater in the Saos-2 cells than
in the HepG2 and 293T cells, which may or may not be related to the
fact that the Saos-2 cells do not express endogenous p53 protein. To
verify that repression was not due to a loss of expression of HNF41
protein, immunoblot analysis was performed on the same whole-cell
extracts from 293T cells as those used to determine the luciferase
activity in Fig. 1A. The results indicate that the level of HNF41
protein was not affected by cotransfection of the p53 vector (Fig. 1B, top panel; compare lane 3 to lane 2), indicating that the
repression is via blocking of HNF41 function.
|
1 DNA Binding1-mediated
transactivation via inhibition of DNA binding, as has been reported for
other nuclear receptors (15, 16, 17), EMSA was performed with
a 32P-labeled oligonucleotide containing the
HNF41 element from the human apoB promoter and HNF41 and p53
proteins expressed in mammalian cells (Fig. 2A). We used two different immunoaffinity
methods to purify p53: one was via an antisera (12CA5) that recognizes
the hemagglutinin (HA) tag on the N terminus, and the other was via an
antisera (421) that recognizes the C terminus of p53 (Fig. 2B). (The
421 antisera has been shown previously to activate p53 DNA binding,
suggesting that it may change the conformation of the protein
(33).) The results indicate that, even in the presence of
excess amounts of p53 protein (up to 200 ng vs.
approximately 20 ng of HNF41 protein, as determined by immunoblot
analysis, data not shown), regardless of the p53 purification method,
the ability of HNF41 to bind DNA was not altered (Fig. 2A, compare
lanes 2, 3, and 4 to lane 1 and lanes 6, 7, and 8 to lane 5). This
suggests that the repression observed in vivo was not due to
an inhibition of HNF41 DNA binding but to an alternative
mechanism.
|
LBD LBD is known to be essential for transactivation
in vivo, we hypothesized that p53 might interfere with the
function of the LBD. We therefore constructed a Gal4 fusion with the
HNF4 LBD (Gal4.HNF4.LBD) (Fig. 3A) and
cotransfected it into 293T cells along with the p53 expression vector.
The results indicate that the Gal4.HNF4.LBD fusion protein activated
transcription well (Fig. 3B, compare lane 4 to lane 3), as expected
from previous results (34). However, cotransfection with
the p53 expression vector repressed the Gal4.HNF4.LBD-mediated
transcription to approximately 50% (compare lane 6 to lane 4) but had
no significant effect on the basal activation by the Gal4 DBD (compare
lane 5 to lane 3), which, in any case, was only slightly above the
reporter alone (compare lane 3 to lane 1). Similar p53-mediated
repression of activation by Gal4.HNF4.LBD was observed in Saos-2,
HepG2, and 293 cells (data not shown). These results suggest that p53
inhibits transactivation by HNF4 by interfering with the function of
the LBD and are consistent with the notion that p53 does not inhibit
HNF41 DNA binding.
|
LBD, a
glutathione-S-transferase (GST) pull-down assay was
performed with whole-cell extracts from 293T cells, and the presence of
p53 protein was verified by immunoblot analysis. The results indicate
that the endogenous p53 protein bound very well (well over 10%) to the
GST.HNF4.LBD but only marginally to the GST.HNF4.DBD and not at all to
the GST control (Fig. 3C
, top panel; compare lane 4 to lanes
3 and 2). Since 293T cells express the SV40 large T antigen, and since
large T antigen is known to interact with p53 (33), we
performed a similar experiment with whole-cell extracts from 293 cells
that lack the large T antigen and observed the same result, namely that
the endogenous p53 protein bound the HNF4.LBD but not the GST control
or the HNF4.DBD (Fig. 3C, bottom panel; compare lane 4 to
lanes 2 and 3). Since p53 also repressed transactivation by
Gal4.HNF4.LBD in 293 cells (data not shown), these results suggest that
repression of and interaction with the HNF4 LBD by p53 is not
mediated via the SV40 large T antigen.
Since the AF-2 region of the LBD is essential for interaction of
nuclear receptors with coactivators and corepressors (14),
we next determined whether the AF-2 of HNF41 was also required for
interaction with p53. The results indicate that the LBD construct
lacking the AF-2 (GST.HNF4.127.360) (see Fig. 3A) did not bind p53
protein in 293T whole-cell extracts (Fig. 3D, compare lane 4 to lane
5), indicating that the AF-2 is required for interaction with p53 and
suggesting that inhibition is via the AF-2 of HNF41. (The construct
lacking the AF-2 did, however, bind in vitro translated
HNF41, indicating that the lack of binding to p53 was not due to a
gross malfolding of the bacterially expressed GST protein; data not
shown).
Repression by p53 Is Only Partially Recovered by Overexpression of
CBP
The AF-2 of nuclear receptors is known to interact with
coactivators CBP/p300 (reviewed in Ref. 35), and p53 is
known to interact with p300 (36) and repress activation by
other transcriptional activators via competition for p300 (4, 5). Therefore, to determine whether repression of
HNF41-mediated transactivation is due to sequestration of limiting
amounts of an endogenous coactivator such as CBP/p300, we overexpressed
CBP in the transient transfection assay. The results indicate that CBP
enhanced the ability of HNF41 to activate its reporter construct
both in the absence and presence of cotransfected p53 (Fig. 4, compare lane 6 to lane 4 and lane 7 to
lane 5). However, they also indicate that even in the presence of the
exogenously added CBP, the cotransfected p53 still reduced the
luciferase activity to approximately 60% compared with 46% in the
absence of added CBP (lanes 7 and 5). These results indicate that there
is only limited reversal of the p53-mediated repression by
cotransfection with CBP, indicating that a mechanism of repression
other than sequestration of CBP by p53 is involved.
|
1 Is Reversed by HDAC Inhibitor
Trichostatin A (TSA)1-mediated transcription by recruiting HDAC
activity. To test this hypothesis, transient transfection assays into
293T cells with the Gal4 fusion constructs were performed in the
presence of TSA, a specific inhibitor of HDAC activity (37, 38). The results indicate that in the presence of TSA, the
repression by p53 of the Gal4.HNF4.LBD-mediated transactivation was
completely reversed (Fig. 5A; compare
lane 8 to lane 6 vs. lane 4 to lane 2), indicating that
recruitment of HDAC activity to the HNF4 LBD may be involved in
p53-mediated repression.
|
1 by using the HNF41 reporter
construct ApoB.-85 -47.E4.Luc. The results indicate that TSA
partially reversed the repression of HNF41 by p53 (Fig. 5B; compare
lane 4 to lane 2). The significance, if any, of the fact that 100%
reversal was achieved with the Gal4.HNF4.LBD, but not the full-length
HNF41, is not known but could be due to the fact that other domains,
such as the DBD, the AF-1, and the F domain, are present in the
full-length HNF41 construct but not the Gal4.HNF4.LBD construct.
Since we had previously reported that the -85 to -47 region of
the apoB promoter also responds to the basic leucine zipper protein
C/EBP (39), and since C/EBP has been shown
previously to be repressed by p53 (40), we tested whether
that repression might also be reversed by TSA. The results indicate
that treatment with TSA did not reverse the repression of C/EBP by
p53 (Fig. 5B; compare lane 8 to lane 6), suggesting that repression of
C/EBP by p53 may act via a mechanism other than recruitment of HDAC
activity. These and the previous results suggest that the effect of the
reversal of p53-mediated repression by TSA is a specific one and might
be related to interaction with the LBD.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
1 in vivo (Fig. 1). We
also show that the repression is not due to an inhibition of HNF41
DNA binding by p53 but rather to an inhibition of LBD function (Figs. 2 and 3). We show that endogenous p53 in 293T and 293 whole-cell extracts
can interact in vitro with the HNF4 LBD in an
AF-2-dependent fashion (Fig. 3). Repression could not be completely
accounted for by competition for limiting amounts of coactivators such
as CBP (Fig. 4). Rather, it appeared to be due, at least partially, to
recruitment of HDAC activity, as it was reversed by addition of TSA, a
specific inhibitor of HDAC activity (Fig. 5). In contrast, repression
of basic leucine zipper protein C/EBP by p53 could not be reversed
by TSA (Fig. 5), suggesting that there may be multiple mechanisms of
repression. Taken together, our results support the notion that p53
represses transactivation of at least certain nuclear receptors by a
mechanism other than inhibition of DNA binding and one that involves
the LBD, the AF-2, and recruitment of HDAC activity.
New Mechanism of p53-Mediated Repression
Previous work by others showed that p53 inhibited transactivation
of certain nuclear receptors by interaction with the DBD and inhibition
of DNA binding (15, 16, 17, 41). Our results indicate that p53
also inhibits transactivation by HNF41 albeit via a different
mechanism, one that is more reminiscent of a corepressor than an
inhibitor of DNA binding. Hallmarks of corepressors include repression
of nuclear receptors only in the absence of ligand, interaction with
the receptor LBD, and recruitment of HDAC activity (14),
conditions all found in this study. What is not known at this point is
whether binding of the endogenous p53 in the cell extracts to the
HNF4 LBD is direct or indirect. Whereas we ruled out involvement of
the SV40 T antigen (Fig. 3), it is still possible that some cellular
protein or another viral protein is required for the interaction.
However, it should be noted that p53 repressed activation by HNF41
in cell lines lacking known viral genes (Fig. 1; Saos-2 and HepG2) and
that repression was via the LBD in those cell lines as well (data not
shown). Finally, it appears that repression via interaction with the
LBD may be applicable to other nuclear receptors as we have evidence
that repression of RAR/RXR activity by p53 is reversed by the addition
of ligand, suggesting involvement of the LBD (data not shown).
Multiple Mechanisms of p53-Mediated Repression
In addition to inhibition of DNA binding and recruitment of HDAC
activity, there appears to be additional mechanisms of repression
mediated by p53 since TSA did not completely reverse the repression of
full-length HNF41 (Fig. 5B). One such mechanism could be competition
for coactivators such as CBP (Fig. 4). p53 also repressed C/EBP by a
mechanism other than recruitment of HDAC activity (Fig. 5B), which may
or may not involve inhibition of C/EBP DNA binding (40, 42). Others have also found multiple mechanisms of repression by
p53 of another liver-specific gene, although both of those mechanisms
required binding of p53 to a p53 response element (43).
This mechanism, however, is apparently not involved here as we saw no
evidence of p53 binding the ApoB element in the gel shift assay (Fig. 2A), and p53 did not repress the ApoB reporter construct in the absence
of HNF41 in Saos-2 cells (Fig. 1A). The existence of
multiple mechanisms of repression is evidently not uncommon for
corepressors. Two well characterized transcriptional repressors,
silencing mediator of retinoid and thyroid hormone receptors (SMRT) and
retinoblastoma protein (Rb), have also been found to repress
transcription by multiple mechanisms, one of which involves recruitment
of HDAC activity under certain conditions and another that involves
interaction with the basal transcription machinery
(44, 45, 46).
Consequences of p53-Mediated Repression
HNF4 and C/EBP are both critical for liver function and
differentiation (24, 47, 48). Therefore, repression of
their transcriptional activity by p53 could explain why overexpression
of p53 has been shown to correlate with poor liver differentiation in
liver cancer (18, 19, 20). Furthermore, p53-mediated
repression of C/EBP and HNF41 could play a role in liver toxicity
of adenoviral vectors, such as those used in gene therapy, as an
increase in p53 protein levels due to adenovirus infection correlated
with a decrease in albumin gene expression, presumably via inhibition
of C/EBP/ß transcriptional activity (40). Under
normal conditions in vivo, however, wild-type p53 protein
levels are very low and are increased only in response to cellular
stress such as DNA damage, withdrawal of growth factors, and hypoxia
(reviewed in Ref. 49). It has been assumed, with good
reason, that tight regulation of p53 protein levels is required to
prevent blockage of the cell cycle and induction of apoptosis. Perhaps
another reason to keep p53 protein levels low is to avoid interference
with normal cellular processes such as differentiation. Finally, an
increase in p53 protein levels may also serve as a mechanism to prevent
commitment of cellular resources for processes such as differentiation
during times of stress.
In conclusion, this report of p53-mediated repression of an additional nuclear receptor suggests that, in addition to cell cycle control, DNA repair, and apoptosis, repression of transcriptional activators involved in cellular differentiation might be a general property of p53. And, like those other processes, it appears that there are multiple mechanisms by which p53 carries out this repression, one of which is to interact with the LBD and recruit HDAC activity.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
1
(pMT7.HNF41) (50), wild-type human p53 in pcDNA1
(pcDNA-p53) (51), and rat basic-leucine zipper protein
C/EBP (pMT2.C/EBP) (52) have been described
previously. The HNF4 GST fusion constructs were made by inserting
PCR products from the original rat HNF41 cDNA (53)
corresponding to fragments containing amino acids (aa) 127–374
(GST.HNF4.LBD), aa 45–125 (GST.HNF4.DBD), and aa 127–360
(GST.HNF4.127.360) into the EcoRI/XhoI sites of
the pGEX6P-1 vector (Pharmacia Biotech, Uppsala, Sweden),
using appropriate primers containing EcoRI (sense) or
XhoI (antisense) sites. Gal4.HNF4.LBD (pFA-HNF4.LBD) was
constructed by first inserting the appropriate
BamHI/NotI fragment from the GST.HNF4.LBD vector
containing aa 127–374 of rat HNF41 into the pcDNA3.1 vector
(Invitrogen, Carlsbad, CA). Subsequently, the appropriate
BamHI/XbaI fragment from pcDNA3.1 was inserted in
frame with Gal4.DBD in the pFA-CMV vector (Stratagene, La
Jolla, CA). The reporter construct ApoB.-85-47.E4.Luc was constructed
by ligating four tandem copies of a double-stranded oligonucleotide
(ApoB.-85-47) corresponding to nucleotides -85 to -47
relative to the transcriptional start site of the human apoB gene and
containing a four-base overhang on the 5'-end (underlined)
(5'-GATCCGGGAGGCGCCCTTTGGACCTTTTGCAATCCTGGCGCTC-3',
top strand) into the BamHI site of pZLuc.E4, which contains
the adenovirus E4 TATA box driving the firefly luciferase gene.
pZLuc.E4 was generated by removal of an approximately 100-bp
BamHI fragment containing five RGC response elements from
5RGC.E4.Luc (54). The reporter construct pFR-luc contains
a synthetic promoter with five Gal4 binding elements driving the
expression of the luciferase gene (Stratagene). All PCR
constructs were verified by dideoxy sequencing. pRc/RSV-mCBP.HA.RK
containing full-length mouse CBP was kindly provided by R. Goodman.
Transient Transfection Assays
Cell lines—human embryonic kidney cells (293 and 293T), human
hepatocarcinoma cells (HepG2), and human osteogenic sarcoma cells
(Saos-2)—were routinely maintained in DMEM supplemented with
penicillin-streptomycin and 10% FBS (5% for 293 and 293T cells) at 37
C under 5% CO2. One day before transfection,
cells were seeded in six-well plates (Falcon 3046, Becton Dickinson and Co., Franklin Lakes, NJ) at 0.5 x
106 cells per well. DNA mixtures containing 2
µg luciferase reporter construct, 0.1–1.0 µg of HNF4 expression
vector, and 0.1–1.0 µg p53 expression vector (pcDNA-p53), as
indicated, were added to each well using Lipofectin according to the
manufacturer’s protocol (Life Technologies, Inc.,
Gaithersburg, MD). Total DNA was brought up to 10 µg with either the
appropriate amount of empty vector (e.g. pcDNA1 for samples
lacking p53) or salmon sperm DNA. After approximately 24 h,
transfected cells were harvested and luciferase activity was determined
as previously described (55). For cell treatment, cells
were treated with 100 ng/ml TSA (Sigma, St. Louis, MO) for
8 h before harvest. All assays were performed in triplicate in at
least two independent experiments, unless indicated otherwise. Results
are given in relative light units (RLU, an arbitrary unit) or fold
induction (RLU relative to transfections lacking expression vectors) as
indicated.
GST Pull-Down Assay
In vitro protein-protein interaction assays were
performed using GST fusion proteins expressed in E. coli
strain BL21(DE3)(pLysS) and bound to glutathione-agarose
(Sigma) using the manufacturer’s protocol
(Pharmacia Biotech). In general, 20 µl of a 1:1
GST-protein/glutathione agarose bead slurry (i.e. 10 µl
packed beads, 
10–20 µg protein) in 100 µl NETN
equilibration buffer (20 mM Tris-HCl, pH 8.0, 100
mM NaCl, 1 mM EDTA, 0.01%
Nonidet P-40) were incubated with 100 µl of 10 mg/ml whole-cell
extract (see below) for 12 h at 4 C with gentle agitation. Protein
complexes were isolated by pelleting the beads at 3,000 rpm for 30 sec
in a Sorvall MC 12-V Microfuge (E.I. Dupont de Nemours & Co.,
Inc., Newtow, CT) and washing three times with 400 µl of
NETN equilibration buffer followed by resuspension in SDS-sample
loading buffer. After fractionation by 10% SDS-PAGE, proteins were
transferred to polyvinylidene difluoride membrane (Immobilon,
Millipore Corp., Bedford, MA) for immunoblot analysis (see
below). GST.HNF4.DBD beads were prewashed in buffer containing 1
M NaCl to remove contaminating DNA. Whole-cell
extracts were prepared by harvesting approximately
107 293 or 293T cells by gentle scraping in 1 ml
ice-cold PBS and resuspending in 0.2 ml ice-cold lysis buffer [50
mM Tris-HCl (pH.7.4), 150
mM NaCl, 5 mM EDTA, 0.5%
Nonidet P-40, 0.5 mM phenylmethylsulfonyl
fluoride, 1 mM dithiothreitol]. The lysis
mixture was incubated for 1 h at 4 C and then cleared by
centrifugation at 12,000 rpm for 15 min at 4 C.
Immunoblot Analysis
Immunoblot analysis was performed as previously described
(55) except that the blot was preincubated in blocking
buffer [5% nonfat dried milk, 0.01% sodium azide, 1x Tris-buffered
saline with Tween 20 (TBST)] and then incubated for 16 h
with a 1:5,000 dilution of 445 antiserum for HNF41
(53) or 0.5 µg/ml final concentration of DO-1 antibody
for p53 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA)
in blotting buffer (1% nonfat dried milk, 0.002% sodium azide, 1x
TBST). The blot was washed twice with 1x TBST and incubated for 2
h in the same blotting buffer with a 1:5,000 dilution of goat
antirabbit (for 445) or goat antimouse (for DO-1) IgG Fab'
conjugated to alkaline phosphatase (The Jackson Laboratory, West Grove, PA). The blot was washed three times in
1x TBST and developed with nitroblue tetrazolium and
5-bromo-4-chloro-3-indolylphosphate toluidinium using a standard
protocol (56).
EMSA
Shift reactions and gel analysis were carried out as previously
described, with minor modifications (55). A standard
mobility shift reaction mixture (15 µl) contained 400 ng of COS-7
cell crude nuclear extract with overexpressed HNF41 incubated at 4 C
for 1 h with purified p53. The incubations were continued for
another 30 min at room temperature with 3 ng of
32P-labeled double-stranded oligonucleotide probe
(ApoB.-85-47) and nonspecific DNA [1 µg of poly(dIdC), 1 µg of
sonicated denatured salmon sperm DNA] before 5 µl were loaded onto a
6% native polyacrylamide gel in 0.25x Tris-borate-EDTA. After
electrophoresis, gels were subjected to autoradiography for 16 h
at room temperature. Crude nuclear extracts of pMT7.
HNF41-transfected COS-7 cells were prepared as previously described
(55). HA epitope-tagged wild-type human p53 was
immunoaffinity purified with either the 421 antibody (recognizes the C
terminus of p53) or the 12CA5 antibody (recognizes the HA tag at the N
terminus) antibody from HeLa cells infected with a recombinant vaccinia
virus as previously described (54, 57).
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
Abbreviations: aa, Amino acid; AF-2, activation function 2;
apoB, apolipoprotein B; CBP, CREB binding protein; C/EBP,
CCAAT/enhancer-binding protein-; CREB, cAMP response element binding
protein; DBD, DNA-binding domain; GST,
glutathione-S-transferase; HA, hemagglutinin; HDAC,
histone deacetylase; HNF, hepatocyte nuclear factor; LBD,
ligand-binding domain; RLU, relative light units; TBST,
Tris-buffered saline with Tween 20; TSA, trichostatin A.
Received for publication June 1, 2001. Accepted for publication October 1, 2001.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
. In:
Burris TP, McCabe ERB, eds. Nuclear receptors and genetic
diseases. London: Academic Press; 309–361
. J Mol Biol 302:831–851[CrossRef][Medline]
(nuclear receptor 2A1) is essential for
maintenance of hepatic gene expression and lipid homeostasis. Mol Cell
Biol 21:1393–14031. Mol Cell Biol 19:6509–6522 bind to overlapping regions of
the apolipoprotein B gene promoter and synergistically activate
transcription. J Biol Chem 268:16831–16838-fetoprotein gene expression by specific DNA binding. Mol Cell Biol 19:1279–1288 knockout mice. Science 269:1108–1112 gene in adult mouse liver. Mol Cell Biol 17:6014–6022[Abstract]
. J Biol Chem 272:1218–1225This article has been cited by other articles:
 |
|
 |
|
  J. Y. L. Chiang Bile acids: regulation of synthesis J. Lipid Res., October 1, 2009; 50(10): 1955 - 1966. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Hannemann, K. Rosenke, J. M. O'Dowd, and E. A. Fortunato The Presence of p53 Influences the Expression of Multiple Human Cytomegalovirus Genes at Early Times Postinfection J. Virol., May 1, 2009; 83(9): 4316 - 4325. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. W. Hwang-Verslues and F. M. Sladek Nuclear Receptor Hepatocyte Nuclear Factor 4{alpha}1 Competes with Oncoprotein c-Myc for Control of the p21/WAF1 Promoter Mol. Endocrinol., January 1, 2008; 22(1): 78 - 90. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Sun, V. Montana, K. Chellappa, Y. Brelivet, D. Moras, Y. Maeda, V. Parpura, B. M. Paschal, and F. M. Sladek Phosphorylation of a Conserved Serine in the Deoxyribonucleic Acid Binding Domain of Nuclear Receptors Alters Intracellular Localization Mol. Endocrinol., June 1, 2007; 21(6): 1297 - 1311. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Benoit, A. Cooney, V. Giguere, H. Ingraham, M. Lazar, G. Muscat, T. Perlmann, J.-P. Renaud, J. Schwabe, F. Sladek, et al. International Union of Pharmacology. LXVI. Orphan Nuclear Receptors Pharmacol. Rev., December 1, 2006; 58(4): 798 - 836. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Maeda, T. C. Hunter, D. E. Loudy, V. Dave, V. Schreiber, and J. A. Whitsett PARP-2 Interacts with TTF-1 and Regulates Expression of Surfactant Protein-B J. Biol. Chem., April 7, 2006; 281(14): 9600 - 9606. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Briancon, A. Bailly, F. Clotman, P. Jacquemin, F. P. Lemaigre, and M. C. Weiss Expression of the {alpha}7 Isoform of Hepatocyte Nuclear Factor (HNF) 4 Is Activated by HNF6/OC-2 and HNF1 and Repressed by HNF4{alpha}1 in the Liver J. Biol. Chem., August 6, 2004; 279(32): 33398 - 33408. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Fukazawa, Y. Maeda, F. M. Sladek, and L. B. Owen-Schaub Development of a Cancer-Targeted Tissue-Specific Promoter System Cancer Res., January 1, 2004; 64(1): 363 - 369. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Eeckhoute, B. Oxombre, P. Formstecher, P. Lefebvre, and B. Laine Critical role of charged residues in helix 7 of the ligand binding domain in Hepatocyte Nuclear Factor 4{alpha} dimerisation and transcriptional activity Nucleic Acids Res., November 15, 2003; 31(22): 6640 - 6650. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. E. Torres-Padilla, F. M. Sladek, and M. C. Weiss Developmentally Regulated N-terminal Variants of the Nuclear Receptor Hepatocyte Nuclear Factor 4alpha Mediate Multiple Interactions through Coactivator and Corepressor-Histone Deacetylase Complexes J. Biol. Chem., November 15, 2002; 277(47): 44677 - 44687. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Maeda, C. Rachez, L. Hawel III, C. V. Byus, L. P. Freedman, and F. M. Sladek Polyamines Modulate the Interaction between Nuclear Receptors and Vitamin D Receptor-Interacting Protein 205 Mol. Endocrinol., July 1, 2002; 16(7): 1502 - 1510. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. D. Ruse Jr., M. L. Privalsky, and F. M. Sladek Competitive Cofactor Recruitment by Orphan Receptor Hepatocyte Nuclear Factor 4{alpha}1: Modulation by the F Domain Mol. Cell. Biol., March 15, 2002; 22(6): 1626 - 1638. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
