Departments of Internal Medicine and of Integrative Biology, Pharmacology, and Physiology, The University of Texas Medical School at Houston, Houston, Texas 77030
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ABSTRACT |
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The H+-K+-ATPase
2 (HK
2) gene plays a central role in
potassium homeostasis, yet little is known about its transcriptional control. We recently demonstrated that the proximal promoter confers basal transcriptional activity in mouse inner medullary collecting duct
3 cells. We sought to determine whether the
B DNA binding element at
104 to
94 influences basal HK
2 gene transcription in
these cells. Recombinant NF-
B p50 footprinted the region
116/
94 in vitro. Gel shift and supershift analysis revealed NF-
B p50- and
p65-containing DNA-protein complexes in nuclear extracts of mouse inner
medullary collecting duct 3 cells. A promoter-luciferase construct with
a mutated
104/
94 NF-
B element exhibited higher activity than the
wild-type promoter in transfection assays. Overexpression of NF-
B
p50, p65, or their combination trans-repressed the
HK
2 promoter. The histone deacetylase (HDAC) inhibitor
trichostatin A partially reversed NF-
B-mediated
trans-repression of the HK
2 promoter. HDAC6
overexpression inhibited HK
2 promoter activity, and
HDAC6 coimmunoprecipitated with NF-
B p50 and p65. These results suggest that HDAC6, recruited to the DNA protein complex, acts with
NF-
B to suppress HK
2 transcription and identify
NF-
B p50 and p65 as novel binding partners for HDAC6.
kidney; colon; trichostatin A; promoter
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INTRODUCTION |
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THE
H+-K+-ATPASE
2-SUBUNIT (HK
2)
is a member of the X+-K+-ATPase multigene
family, which includes the gastric H+-K+-ATPase
and the Na+-K+-ATPase isoforms. The
X+-K+-ATPases share homologous structures,
common catalytic mechanisms, and a requirement for heterodimeric
(
/
) assembly (22). HK
2 is principally
expressed in distal colon and the renal collecting duct, in which it
plays a critical role in potassium and acid-base homeostasis. Mice with
targeted ablation of the HK
2 gene develop fecal
K+ wasting and profound hypokalemia during potassium
(27) and sodium restriction (34).
HK
2 also appears to contribute to bicarbonate absorption
by the kidney (29) and distal colon (27) and
increased ammonium secretion in the inner medullary collecting duct
(IMCD) during chronic hypokalemia (37). Recent studies also suggest that HK
2, possibly functioning as an
Na+-K+ exchanger, participates in the chronic
adaptation to altered sodium and aldosterone balance (32).
The HK2 gene is differentially expressed in kidney and
distal colon under basal conditions and in response to chronic
potassium or sodium deprivation. Under basal conditions, the gene is
robustly expressed in distal colon, but only weakly expressed in the
renal medulla. After chronic K+ deprivation, however,
HK
2 gene expression is upregulated in the rat and mouse
kidney outer medulla but not in distal colon (1, 32). In
contrast, chronic sodium deprivation upregulates HK
2
expression in distal colon but not in the renal collecting duct
(32).
We recently isolated and completely sequenced the cDNA and structural
gene encoding the mouse HK2 gene and localized it to mouse chromosome 14C3 (41). Using deletion analysis of
promoter-reporter gene constructs, we demonstrated functional activity
of the HK
2 promoter in cultured renal collecting duct
cells and found that the proximal 177 bp of the promoter appear to be
essential for collecting duct-selective expression. This proximal
promoter region contains several consensus sequences for transcription
factors. Among these is an NF-
B site
104 GGGGCGTCCCC
94. The
mammalian NF-
B/Rel family comprises five known members: p50, p52,
p65, c-Rel, and RelB. NF-
B subunits form homo- or heterodimers
through the Rel homology domain, forming transcription factor complexes that exert a broad range of DNA-binding and -activation potentials (4). The protein is bound in the cytoplasm with members of the inhibitor of
B (I
B) family, which prevents phosphorylation of
the active unit and its translocation to the nucleus. A variety of
stimuli and signaling events leads to activation of NF-
B, and there
is evidence that NF-
B can transport between cytoplasm and nucleus
even in unstimulated cells. In several tissues, including kidney,
NF-
B DNA-binding activity is evident under basal conditions. It has
been suggested that NF-
B in these settings suppresses or activates
basal gene expression in these cells (10, 18). In this
context, it has been recently shown that NF-
B can suppress target
gene transcription by means of interactions of p65 with histone
deacetylase (HDAC)1 and HDAC2 corepressor proteins (3). Indeed, interaction with other transcription factors and accessory proteins appears to lend NF-
B versatility and specificity in mediating transcriptional responses.
Histone acetylation is a dynamic process regulated by the activities of
two histone-modifying enzymes, histone acetyltransferase(s) and
HDACs. Present models indicate that HDACs are recruited to target
sequences through protein-protein interactions (23). HDAC
activity results in histone hypoacetylation, chromatin condensation, and, generally, transcriptional repression (23).
HDACs are typically found as components of large corepressor complexes.
They affect transcriptional activity not only by modifying chromatin
structure but also by deacetylating transcription factors and altering
their transcriptional competency. For example, HDAC1 and HDAC2
have been shown to repress transcription through direct interaction with transcription factors. HDAC1 directly interacts with MyoD to
silence MyoD-dependent transcription of p21 (25). HDAC2
interacts with the YY1 transcription factors, converting them from
activators to repressors (39). Deacetylation of RelA by
HDAC3 serves as a molecular switch within the nucleus that controls the
NF-B transcriptional response (11).
In this report, we examined the role of basal NF-B expression on
HK
2 promoter activity in renal medullary collecting duct cells. We demonstrate that NF-
B acting through the
104/
94
element significantly suppresses transcriptional activity of the
HK
2 gene and that this inhibition involves recruitment
of HDAC6 to the NF-
B activation complex through protein-protein
interactions. These results provide the first evidence for regulatory
components of HK
2 gene transcription and identify novel
interactions of NF-
B p65 and p50 with specific HDAC proteins.
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MATERIALS AND METHODS |
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Cell culture and reagents.
Mouse IMCD-3 (mIMCD-3) cells, an immortalized cell line derived from
mIMCD (31), were cultured in DMEM supplemented with 10%
FBS at 37°C in a 5% CO2 environment. Polyclonal
antibodies recognizing NF-B p65, p52, p50, c-Rel, and STAT3 were
from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-FLAG M2 antibody
was from Sigma. Oligonucleotides were custom synthesized by Genosys (The Woodlands, TX). Lipofectamine 2000 reagent was from Invitrogen (Carlsbad, CA). The Dual-Luciferase Reporter Assay System and the
luciferase vectors pGL3-Basic and pRL-SV40, recombinant human NF-
B
p50 (rhNF-
B), and activator protein-2 (AP-2) nuclear extract were
from Promega (Madison, WI). The BCA protein estimation kit was from
Pierce Chemical. Enhanced chemiluminescence reagents were from Amersham
Pharmacia Biotech (Piscataway, NJ).
Plasmids and constructs.
pGL3-0.48MHK2, which contains a fragment spanning
from
477 to +253 of the murine HK
2 gene upstream of
the luciferase gene, has been previously characterized
(41). pGL3-0.48MHK
2mut, which harbors
a mutated NF-
B site at the
104 to
94 position
(TAGCCGTCCCC; mutated bases underlined), was
created by PCR overlap extension. pCMV500, a mammalian expression
vector containing the cytomegalovirus promoter, NF-
B expression
plasmids pRSV-p65 and dominant-negative pCMV-I
B
1-36
(8), and the NF-
B reporter construct
p36B(
)(NF-
B)3-luc, which contains three tandem copies
of the
B binding element (GGGGACTCTCCC) upstream of the prolactin
promoter sequence and fused to the coding sequence for the luciferase
gene (12), were gifts from Dr. Bharrat Agarwal (The
University of Texas M. D. Anderson Cancer Center). pRSV-p50, which encodes NF-
B p50, was kindly provided by Dr. Warren
S.-L. Liao (The University of Texas M. D. Anderson Cancer Center).
The FLAG-tagged HDAC2 expression plasmid pME18S-FLAG-HDAC2 (38) was from Dr. Edward Seto (University of South
Florida). FLAG-tagged mammalian expression plasmids pBJ-HDAC1,
pBJ-HDAC4, pBJ-HDAC5, and pBJ-HDAC6 (13) were from Dr.
S. L. Schreiber (Harvard University).
In vitro DNase I footprinting.
DNase I footprinting analyses were performed with the Core Footprinting
System (Promega), according to the manufacturer's instructions. A PCR
fragment corresponding to 476 to +82 of the native murine
HK
2 5'-flanking region was obtained by using two primers: WZ162 (5'-ATCGAGACGCGTATAGATTCCCCGCCCCACCCTCATTTACAC-3', sense, MluI site attached for cloning) and
32P-labeled or unlabeled oligo 3 (5'-GTCCGGGTCCCTGAGTGGTGA-3', antisense). This fragment was used as the
DNA template for footprinting, and the unlabeled fragment also was
sequenced with oligo 3 as a marker (T7 Sequenase, version 2.0, DNA
sequence kit, Amersham Pharmacia Biotech). The transcription factors
examined were NF-
B p50 and, as a control, AP-2. For the NF-
B
experiment, the same final binding buffer was used as described
(21). Briefly, the labeled template DNA was incubated in a
final volume of 50 µl with or without 2 µl of recombinant human
NF-
B in the final buffer containing 10 mM HEPES (pH 7.9), 0.2 mM
EDTA, 50 mM KCl, 2.5 mM dithiothreitol, 10% glycerol, and 0.05%
Nonidet P-40 for 10 min on ice. After addition of 50 µl
Ca2+/Mg2+ solution (5 mM CaCl2, 10 mM MgCl2), 3 µl of DNase I was used for digestion for 1 min at room temperature. The reaction was terminated by adding 90 µl
of stop solution (200 mM NaCl, 30 mM EDTA, 1% SDS, 100 µg/ml yeast
RNA). Samples were extracted with phenol/chloroform/isoamyl alcohol,
precipitated, resuspended in sequencing loading buffer, and separated
on 8% sequencing gels. Bands were visualized by autoradiography.
EMSAs and supershift assays.
Nuclear extracts were prepared from mIMCD-3 cells as detailed in
earlier work from our laboratory (15). Double-stranded oligonucleotides were generated corresponding to nucleotides 109 to
90 containing the
B element (sense strand: wild-type,
5'-CCCCAGGGGCGTCCCCAGTG -3',
B binding element
underlined) and a mutated
B element
(5'-CCCCA
2 promoter and were end-labeled with
[
-32P]ATP (3,000 Ci/mmol) using T4 polynucleotide
kinase. Nuclear extract proteins (12 µg) were preincubated with or
without 4 µg antibodies specific for NF-
B p65, p50, p52, c-Rel, or
HDAC6 in the final binding buffer [10 mM Tris, pH 7.5, 50 mM NaCl, 0.1 mM EDTA, 1 mM dithiothreitol, 4% glycerol, and 1 µg poly(dI-dC)] at
4°C for 1 h or overnight. Binding reactions were performed for
30 min at room temperature by adding 1.75 pmol of duplex DNA probe
(~2 × 105 cpm) in the presence or absence of a
10-fold molar excess of nonradiolabeled competitor oligonucleotides.
The final reaction volume was adjusted to 20 µl. Aliquots of the
reactions were resolved on 4% native polyacrylamide gels in 0.5× Tris
borate-EDTA buffer. The gels were dried and exposed to X-Ray film with
an enhancing screen at
70°C to detect the DNA-protein and
DNA-protein-antibody complexes. Each observation represents a binding
reaction performed on a new nuclear extract preparation. Experiments
were replicated a minimum of three times, as indicated in the figure legends.
Transient transfection and reporter gene assays.
mIMCD-3 cells grown in 24-well plates were transiently transfected
using the LipofectAMINE 2000 Reagent (Life Technologies) as detailed in
our laboratory's previous work (41). For comparative purposes, the cells were cotransfected with the Renilla
luciferase expression plasmid pRL-SV40 (10 ng/well) to control for
transfection efficiency and other assay-to-assay variability.
Trans-repression/trans-activation experiments
used 0.8 µg of pGL3-0.48MHK2,
pGL3-0.48MHK
2mut, or
p36B(
)(NF-
B)3-luc and 0.2 µg of pRSV-p65, pRSV-p50,
dominant-negative pCMV-I
B
1-36 or insertless expression
vector, or the FLAG-tagged expression vectors pBJ-HDAC1,
pME18S-FLAG-HDAC2, pBJ-HDAC4, pBJ-HDAC5, or pBJ-HDAC6. In the
cotransfection experiments with pRSV-p65 and pRSV-p50 together, 0.1 µg of each plasmid was used. Twenty-four hours later, firefly and
Renilla luciferase activities in 5- to 10-µl lysate
samples were measured in a Turner Systems 20/20 luminometer using the
Dual-Luciferase Reporter Assay System (Promega) following the
manufacturer's protocol. Firefly luciferase activity was normalized for Renilla luciferase activity in the lysates. The results
were recorded as "normalized MHK
2 promoter
activity." In some experiments, the cells were treated after
transfection with vehicle or trichostatin A (TSA; 100 nM) for 16 h, after which cell lysates were prepared and luciferase assays
performed. As TSA treatment also dramatically affects expression of
Renilla luciferase expression plasmid pRL-SV40, firefly
luciferase activities in these experiments were normalized to the total
lysate protein used in the assay. Each experimental observation
represents the mean of results from at least three independent
transfections. HDAC isoform overexpression was confirmed by analyzing
immunoblots of cell lysates prepared from the transfected cells with
antibodies against the specific HDAC isoform and with the anti-FLAG M2
antibody. Roughly comparable levels of overexpression were observed for
HDAC1, HDAC2, HDAC4, HDAC5, and HDAC6 (not shown).
Coimmunoprecipitation.
mIMCD-3 were harvested, lysed in JLB buffer (50 mM
Tris · HCl, pH 8, 150 mM NaCl, 10% glycerol,
0.5% Triton X-100) containing 1 mM PMSF, and 3% protease inhibitor
cocktail, and precleared by incubating with 20 µl/ml protein
A/G-agarose beads (Santa Cruz) for 1 h at 4°C. After briefly
spinning down, the supernatant was added with anti-FLAG M2,
anti-NF-B p65, or anti-NF-
B p50 antibodies overnight at 4°C,
followed by the addition of 20 µl of protein A/G-agarose beads.
Immunoprecipitates were washed four times in JLB buffer, resuspended in
SDS sample buffer, boiled for 5 min, and analyzed on 4-15% linear
gradient Tris · HCl gels (Bio-Rad). Proteins were
electrophoretically transferred to polyvinylidene difluoride membranes
and incubated with anti-FLAG M2 overnight, followed by extensive washes
and incubation with secondary antibody conjugated to horseradish
peroxidase. Antigen-antibody complexes were detected with enhanced
chemiluminescence reagent (ECL, Amersham).
Data analysis.
Potential regulatory motifs in the HK2 gene were
identified with TESS software (http://www.cbil.upenn.edu/tess).
Quantitative data were analyzed for significance using
t-test, two-sample assuming equal variances, with
significance assigned at P < 0.05.
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RESULTS |
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NF-B p50 footprints the HK
2 gene in vitro at
116 to
94.
The proximal promoter of the HK
2 gene was initially
analyzed by TESS software. A putative NF-
B and an overlapping AP-2
binding site were identified at
104 to
93, with the NF-
B site
ranging from
104 to
94 and the AP-2 site from
99 to
93. To
determine the significance of these sites, DNase I footprinting assay
with recombinant NF-
B p50 (Fig. 1) and
AP-2 was performed. Protected or hypersensitive sites in the region
116/
94 were detected in the binding reactions that included NF-
B
p50, when compared with the reaction in which p50 was omitted.
The antisense of this protected region reads as
5'-GGGGACGCCCCTGGGGTAGGAAA-3', with the NF-
B site
underlined. In addition, no footprint was observed with recombinant AP-2 under the condition tested (data not shown).
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109/
90 Sequence binds
NF-
B p65 and p50.
Binding of NF-
B to the
104/
94 element was further investigated
by gel shift assays with mIMCD-3 cell nuclear extracts and the
radiolabeled
109/
90 sequence containing the
B element as probes.
Sequence-specific DNA-protein complexes were detected (Fig.
2A). Supershift assays
demonstrated that antibodies to NF-
B p65 and p50 supershifted the
DNA-protein complex (Fig. 2B). In contrast, the mobility of
the DNA-protein complex in the presence of antibodies to p52, c-Rel, or
STAT3 (as a negative control) was not altered. Furthermore, mutation of
these sites dramatically decreased, although did not completely
prevent, NF-
B binding to this site (data not shown). These data are
consistent with the conclusion that NF-
B p50/p65 heterodimers are
basally expressed in the nuclei of mIMCD-3 cells.
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NF-B p65 and p50 trans-repress the HK
2 promoter.
To determine the functional effect of NF-
B on HK
2
promoter activity,
trans-activation/trans-repression assays were
performed in mIMCD-3 cells. Overexpression of NF-
B p65 or p50
resulted in ~22 and ~30%, respectively, lower rates of
HK
2 promoter activity compared with vector-transfected
controls (Fig. 3A).
Overexpression of NF-
B p65 and p50 together resulted in a dramatic,
synergistic inhibition (~87%) of HK
2 promoter
activity (Fig. 3A). In concert with these findings,
overexpression of an I
B
mutant designed to limit NF-
B
translocation to the nucleus resulted in a 40% increase in
HK
2 promoter activity (Fig. 3A). Importantly,
overexpression of NF-
B p65 or p50 in these same cells strongly
activated activity of the NF-
B reporter plasmid
p36B(
)(NF-
B)3-luc (Fig. 3B), indicating that the classic gene activation by NF-
B signaling under these conditions was intact and that the observed inhibitory effects on
HK
2 promoter activity likely reflected the influence of
the promoter context and/or coregulatory molecules.
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TSA augments and HDAC6 overexpression inhibits HK2
promoter activity.
NF-
B is known to interact with HDAC1, HDAC2, and HDAC3 (3, 11,
19) in other cell types and promoter contexts (3), and HDACs are known to act as corepressors in some instances. To
determine whether the state of histone acetylation influences the
ability of NF-
B to trans-repress the HK
2
promoter in mIMCD-3 cells, HK
2 promoter-luciferase
activity was measured in the presence and absence of the potent and
specific HDAC inhibitor TSA. TSA treatment promoted a 2.5-fold
increase in basal HK
2 promoter activity (Fig.
4A). As shown in Table
1, TSA treatment resulted in
significantly greater activity of the NF-
B consensus element reporter in the vector-transfected controls c-transfected with either
the wild-type HK
2 promoter
(pGL3-0.48HK
2) or the HK
2 promoter
harboring the mutation in the NF-
B site
(pGL3-0.48HK
2mut). However, p65, p50, and
p65+p50 had less ability to inhibit
pGL3-0.48HK
2mut promoter activity compared with the
wild-type pGL3-0.48HK
2 construct in the presence of
TSA, indicating that TSA partially reversed the NF-
B-dependent
trans-repression of the HK
2 promoter (Table 1). These findings are consistent with the involvement of an HDAC in
the process. In accordance with these findings,
overexpression of HDAC6 inhibited HK
2 promoter activity
(Fig. 5A). Interestingly, the
inhibitory effect appeared to be specific for HDAC6, because overexpression of HDAC1, HDAC3, HDAC4, and HDAC5 had no appreciable effect on HK
2 promoter activity (Fig. 5A),
despite roughly comparable levels of expression (not shown).
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HDAC6 and NF-B interact in mIMCD-3 cells in vivo.
EMSA and supershift studies using the
104/
94
B element as
probe, nuclear extracts from mIMCD-3 cells, and anti-HDAC6 antibodies or IgG (as a negative control) demonstrated that anti-HDAC6
partially supershifted the
B-specific DNA-protein complex
(Fig. 6). In agreement with these results
and those of the HK
2 promoter assays, coimmunoprecipitation experiments demonstrated interaction of HDAC6 and
NF-
B proteins (Fig. 7). HDAC6 was
overexpressed as a FLAG-tagged construct in mIMCD-3 cells, and the
cells were then subjected to coimmunoprecipitation with antibodies to
NF-
B p50, NF-
B p65, the FLAG epitope (as a positive control), or
IgG (as a negative control), followed by blotting with the anti-FLAG M2 antibody. As seen in Fig. 7, both NF-
B p50 and p65 coprecipitated with HDAC6.
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DISCUSSION |
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In this study, we demonstrated that NF-B p65 and p50 DNA
binding activities are basally expressed in cultured renal collecting duct cells (Fig. 2) and that these transcription factors serve to
trans-repress the HK
2 gene by binding to a
B element in the proximal promoter region of this gene (Fig.
3A). The fact that blockade of HDAC activity with TSA
relieves NF-
B-mediated trans-repression of the
HK
2 gene (Fig. 4) and that HDAC6 overexpression inhibits HK
2 promoter activity (Fig. 5) indicates that the state
of histone or NF-
B acetylation strongly influences
HK
2 promoter activity. We further demonstrate that
NF-
B p50 and p65 interact with HDAC6 in supershift experiments with
the
104/
94
B element (Fig. 6) and in coimmunoprecipitation
assays (Fig. 7). These results provide the first data concerning
transcriptional regulation of the HK
2 gene, give a novel
example of the ability of NF-
B to suppress basal gene expression,
and identify for the first time HDAC6 as a binding partner with NF-
B
p50 and p65. However, it remains to be defined whether p50 and p65 are
subject to acetylation/deacetylation and whether HDAC6 is directly
involved in this process.
The fact that the 104/
94 region was footprinted in vitro by NF-
B
p50 (Fig. 1) and that mutation of the
B element partially relieved
repression of HK
2 promoter activity (Fig. 3A)
suggests that much of the effect of NF-
B on the promoter was direct
and mediated by DNA binding. However, because the effect was only partial, and because overexpression of NF-
B p65 or p65/p50, but not
p50 alone, still inhibited the mutant HK
2 promoter (Fig. 3A), other undefined mechanisms are apparently involved. The
B element rests 53-bp upstream of the TATA box and is flanked by an
AP-2 site. However, AP-2 did not footprint the region in vitro (Fig.
1). Because the combination of p50 and p65 overexpression synergistically suppressed HK
2 promoter activity (Fig.
3A) and because these NF-
B proteins contributed to the
B-specific DNA-protein complexes in supershift assays (Fig.
2B), we conclude that p50/p65 heterodimers are likely the
dominant NF-
B species binding the HK
2 promoter and
the most effective in trans-repressing the
HK
2 gene.
NF-B is subject to complex control. The principal mode of regulation
has long been considered to be the retention of NF-
B in the
cytoplasm by association with I
B proteins and its release for
translocation to the nucleus on I
B phosphorylation and degradation (4). This regulatory pathway appears to be operative at
least to a degree in our study, because overexpression of an I
B
dominant-negative mutant augmented basal HK
2 promoter
activity (Fig. 3A). In addition, NF-
B has been shown to
shuttle between cytoplasm and nucleus under basal conditions. Chen et
al. (11) showed that deacetylation of RelA by interaction
with HDAC3 promotes effective binding to I
B and results in
I
B
-dependent nuclear export of the complex. Ashburner et
al. (3) established that NF-
B p65 in the
nucleus of unstimulated cells interacts with HDAC1 and HDAC2 to
suppress basal gene expression. The glucocorticoid receptor has also
been shown to recruit HDAC2 to the p65-cAMP response element binding protein (CBP) histone acetyltransferase complex to inhibit
IL-1
-induced gene expression (19). NF-
B has been
found in the nucleus of unstimulated cells, in which it is theorized to
repress and/or activate basal gene expression (10). The
differential association of NF-
B with coregulatory proteins may
regulate this function. CBP and p300 coactivators interact with p65 to
enhance its ability to activate transcription (4). The
histone acetyltransferase function of the p300/CBP-associated factor
coactivator (9) and the steroid receptor coactivator-1
(28) were shown to interact with p50 to potentiate
NF-
B-mediated trans-activation.
Histone acetylation and deacetylation play essential roles in modifying
chromatin structure and regulating expression of eukaryotic genes.
HDACs are part of transcriptional corepressor complexes. HDAC interacts
with NcoR (nuclear corepressor) and SMRT (silencing mediator of
receptor transcription) to mediate nuclear receptor repression, as well
as with the Mad-Max complex to confer transcriptional repression
(2, 16, 17). In our study, TSA treatment increased HK2 promoter activity, consistent with the model that
TSA blocks activity of HDAC, resulting in hyperacetylation of histones
and, consequently, a higher level of gene expression (40).
Consistent with this, overexpression of HDAC6 inhibited
HK
2 promoter activity (Fig. 6). We do not know whether
TSA promotes dissociation of HDAC from p65 or p50, facilitating the
binding of CBP or other coactivators.
Acetylation regulates transcription factors other than NF-B,
including p53 (6), GATA-1 (7), and MyoD
(25), and it alters transcription factor function in
several ways, including altering protein-protein interactions
(5), affecting conformation (33), and
altering half-life (26). Direct interaction with HDACs has
only been demonstrated for a few transcription factors. Retinoblastoma
protein (24) and DNA topoisomerase II (36) interact directly with HDAC1, SP1 (20) and YY1
(35) interact with HDAC2, GATA-2 couples with HDAC3 and
HDAC5 (30), and myocyte enhancer factor 2 was shown to
interact directly with HDAC4 and HDAC5 (14). Our finding
of an interaction between HDAC6 and NF-
B p50 and p65 adds to this
list. Why specific HDAC isoforms are active in some cell types but not
others likely relates to the specific promoter context and other
coregulatory molecules. Nonetheless, this protein-protein interaction
could lend cell specificity and versatility to the regulatory response.
In addition, the ability of NF-
B to function as both activator and
repressor in mIMCD-3 cells indicates that this transcription factor may exist in different complexes with different acetylation/deacetylation patterns, resulting in differential DNA binding specificities and
regulation of expression of its target genes.
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ACKNOWLEDGEMENTS |
---|
The authors thank Dr. Bharrat Agarwal, The University of Texas M.D.
Anderson Cancer Center, for the gift of the NF-B and mutant I
B
expression plasmids, Dr. Warren S.-L. Liao, The University of Texas
M.D. Anderson Cancer Center, for the gift of the NF-
B expression
plasmid, Dr. Edward Seto, University of South Florida, for the
FLAG-tagged HDAC2 expression plasmid pME18S-FLAG-HDAC2, and Dr. S. L. Schreiber, Harvard University, for the FLAG-tagged expression
plasmids pBJ-HDAC1, pBJ-HDAC4, pBJ-HDAC5, and pBJ-HDAC6. The authors
also thank Dr. Jun Chen and Sandra Higham for technical assistance.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-47981.
Address for reprint requests and other correspondence: B. C. Kone, Depts. of Internal Medicine and of Integrative Biology, Pharmacology, and Physiology, Univ. of Texas Medical School at Houston, 6431 Fannin, MSB 4.138, Houston, TX 77030 (E-mail: Bruce.C.Kone{at}uth.tmc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
July 9, 2002;10.1152/ajprenal.00156.2002
Received 22 April 2002; accepted in final form 19 June 2002.
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