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INTRODUCTION |
Tissue remodeling in physiological and pathological conditions
such as trophoblast implantation, bone development, angiogenesis (1-3), and the spread of cancer (invasion/metastases) (4, 5)
requires proteolytic action to degrade the surrounding extracellular matrix (6) and to activate cytokines such as transforming growth factor-
and interleukin-1
(3, 7). There is now compelling evidence implicating the collagen-degrading 92-kDa type IV collagenase (MMP-9) in these processes. Thus, mice null for the MMP-9
gene exhibit an abnormal pattern of skeletal growth plate
vascularization (2). Additionally, MMP-9 is required for human
bronchial epithelial cell migration and spreading following injury (8).
In cancer, there is strong evidence implicating this type IV
collagenase in the spread of the disease. Thus, Bernhard et
al. (9) reported that the overexpression of this metalloproteinase
in rat embryo cells conferred a metastatic phenotype, whereas the
inhibition of MMP-9 expression by a ribozyme blocked metastasis of rat
sarcoma cells (10).
The MMP-9 gene located on chromosome 20 (11) covers
13 exons spanning 7.7 kilobases, (12) and its transcription yields a 2.5-kilobase mRNA (13). The regulation of MMP-9 protein levels has
been ascribed to transcriptional activation of the gene (14, 15),
reduced mRNA turnover (16), and altered translational efficiency
(17). The 5'-flanking sequence contains binding sites for AP-1, NF
B,
Sp1, and Ets transcription factors within the first 670 base pairs, and
these have been implicated in the regulation of MMP-9
gene expression by a variety of cytokines and contact inhibition (14,
18, 19). In addition, studies with transgenic mice have demonstrated
the requirement of regions
522/+19 and
2722/
7745 for
developmental regulation in mice and for tissue-specific expression in
osteoclasts and migrating keratinocytes, respectively (20, 21).
Although MMP-9 has been implicated in both physiological and
pathological processes, how its expression is regulated is still not
fully understood. In the last 5 years, a great deal of interest has
accrued in a group of genes collectively referred to as
metastases-associated genes, which modulate cancer metastases but not
tumorigenesis (22-24). One of these is the MTA1 gene, which is
overexpressed in metastatic mammary adenocarcinoma and prostate cancers
(25, 26) and subsequently determined to enhance the migration and invasion of immortalized human keratinocytes (27). The MTA1 gene
resides on chromosome 14q32.3 (28) and encodes a protein, which binds
histone deacetylases 1 and 2 (HDAC1 and
HDAC2)1 (29). Furthermore,
the MTA1 protein is one of the subunits of the human
nucleosome-remodeling and deacetylation complex shown previously
to regulate in vitro transcription of at least artificial reporter constructs via ATP-dependent nucleosome disruption
and changes in histone acetylation (30).
Because MMP-9 has a well established role in tumor cell invasion and
metastases and considering that MTA1 expression is associated with the
metastatic phenotype, we undertook the present study to determine
whether the expression of this collagenase is targeted by this
metastases-associated gene.
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EXPERIMENTAL PROCEDURES |
Plasmids and Antibodies--
A plasmid construct
expressing a myc-tagged human MTA1 (pBJ-myc/MTA1)
and its vector pBJ-Myc were as described previously (31). To construct
the MTA1-expressing vector in the Flp-In System (Invitrogen), the
pBJ-myc/MTA1 was digested with XbaI and BamHI. A 2.2-kilobase fragment corresponding to the MTA1
cDNA was purified and ligated into the pcDNA5/FRT vector. The
resulting MTA1-expressing plasmid was designated as
pcDNA5/FRT/MTA1.
Antibodies to acetylated histones 3 and 4 were purchased from Upstate
Biotechnology (Lake Placid, NY), whereas the rabbit antibody directed
against HDAC2 (H54) was obtained from Santa Cruz Biotechnology (Santa
Cruz, CA). Monoclonal antibodies to HDAC1 (H11), HDAC2 (C-8), and a
rabbit antibody to Mi2 (H-242) were purchased from Santa Cruz Biotechnology.
Cell Culture and Transfections--
Human fibrosarcoma cells
HT1080 were routinely maintained in McCoy 5A medium supplemented
with 10% fetal bovine serum and antibiotics. For transfections, cells
were transfected with poly-L-ornithine as described
previously (15, 32). Clones were selected with the appropriate
antibiotic: 600 µg/ml G418, 300 µg/ml zeocin, or 300 µg/ml
hygromycin B.
Establishment of Flp-In MTA1-expressing Cells--
This was
performed as described in the two-step protocol provided by the
manufacturer (Invitrogen). First, a Flp-In host cell line containing a
single copy integrated FRT sequence was established from HT1080 cells.
For this purpose, HT1080 cells were transfected with
pFRT/lacZeo and selected in zeocin-containing medium. The resistant clones were expanded and analyzed by Southern blotting (33).
To identify single integrants of the FRT sequence, DNA from the clones
digested with HindIII was resolved by agarose gel
electrophoresis and, after transfer to a nylon membrane, was hybridized
with a 290-base pair 32P-labeled LacZ cDNA fragment
derived by digesting pFRT/lacZeo with EcoRV and
ClaI. DNA containing a single integrated
pFRT/lacZeo copy is identified by a single hybridizing band.
A clone designated as F8 was thus obtained, expanded, and transfected
with 1.8 µg of the Flp-In recombinase-encoding pOG44 and 0.2 µg of
pcDNA5/FRT/MTA1 or pcDNA5/FRT. Transfected cells were selected,
pooled, and cultured in hygromycin B-containing media.
Western Blotting--
Cells were washed with cold
phosphate-buffered saline and lysed in modified radioimmune
precipitation assay buffer (50 mM Tris-HCl, pH 7.4, 1%
Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, 1 mM NaF,
and 1× proteinase inhibitor (Roche Molecular Biochemicals)) on ice for
30 min. Cell lysates were clarified by centrifugation, and 60 µg of
protein were resolved by SDS-polyacrylamide gel electrophoresis.
Proteins were transferred to a nitrocellulose membrane, blocked with
5% milk, and incubated with a Myc monoclonal antibody (9E10) or a goat
anti-MTA1 antibody (A-18) (Santa Cruz Biotechnology) at 4 °C
overnight. After extensive washing, the membrane was incubated with a
horseradish peroxidase-conjugated secondary antibody and visualized by ECL.
Quantitation of MMP-9 and MMP-2 mRNA--
Preparation of
total RNA and reverse transcription were as described previously
(15). cDNA was then subjected to multiplex PCR with 5 nM MMP-9 primers (5'-GAGGTTCGACGTGAAGGCGCAGATG-3' and 5'-CATAGGTCACGTAGCCCACTTGGTC-3') and 0.5 nM
-actin
primers (5'-ACACTGTGCCCATCTACGAGG-3' and
5'-AGGGGCCGGACTCGTCATACT-3'). To ensure that amplification was in
the linear range, PCR was terminated at cycles 18, 20, and 22, respectively. PCR products were resolved by agarose gel electrophoresis, DNA was transferred to a nylon membrane, and the
membrane was hybridized with a 32P-labeled MMP-9 cDNA.
Linearity of the PCR amplification was confirmed with serial dilutions
of MMP-9 cDNA.
Quantitation of MMP-2 mRNA was accomplished by Northern blotting as
described previously (34) using stringency washes performed with 0.1×
SSC, 0.1% SDS at 65 °C.
Zymography--
Condition medium was collected, and
aliquots normalized for varying cell numbers were subjected to
zymography as described by us previously (34).
Co-immunoprecipitation--
Cells were lysed in IPH
buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, 1 mM
phenylmethylsulfonyl fluoride, 1× proteinase inhibitor) on ice for 30 min. After clarification by centrifugation, cell lysate (3 mg of
protein) was incubated with 40 µl of an anti-c-Myc-agarose conjugate
(Sigma) at 4 °C overnight. Agarose beads were then washed three
times in 1 ml of IPH buffer and boiled in 25 µl of loading
buffer, and eluted proteins were resolved by SDS-PAGE. Blots were
probed with the indicated antibody, and immunoreactive proteins
were visualized by ECL.
Real-time PCR--
This method was performed with an ABI Prism
7000 sequence detection system (Applied Biosystems, Foster City, CA)
according to the manufacturer's instructions. DNA samples were mixed
with 1× SYBR Green PCR Master Mix (Applied Biosystems) and 1 µM of each primer and loaded into the ABI Detection
System. After incubating at 95 °C for 10 min to activate the
AmpliTaq Gold enzyme, the mixtures were subjected to 40 amplification
cycles (15 s at 95 °C for denaturation and 1 min for annealing and
extension at 60 °C). After PCR, dissociation curves were generated
with one peak, indicating the specificity of the amplification. A
threshold cycle (Ct value) was obtained from each amplification curve
using the software provided by the manufacturer.
Chromatin Immunoprecipitation Assays--
ChIp assays were
performed essentially as described previously but with modifications
(15). Cells (1 × 107) were treated with 1%
formaldehyde and lysed in SDS lysis buffer (1% SDS, 10 mM
EDTA, and 50 mM Tris-HCl, pH 8.1). The chromatin samples
were sonicated to reduce DNA length to 200-500 base pairs and
precleared with protein A-agarose beads. Antibodies (2 µg) were added
to the chromatin samples and incubated at 4 °C overnight. To
minimize nonspecific binding, single-stranded DNA/protein A-agarose beads (Upstate Biotechnology) were incubated with 1% bovine serum albumin at room temperature for 30 min. 40 µl of treated beads was
then added to the chromatin samples and incubated at 4 °C for 1 h. The beads were then washed five times as described previously. Immunoprecipitates were then eluted, cross-links were reversed, and
after proteinase K treatment, DNA was recovered by phenol/chloroform extraction and co-precipitation with glycogen. DNA was dissolved in 20 µl of TE buffer (10 mM Tris, 1 mM, pH
8.0). For immunoprecipitating MTA1-bound chromatin, the same
protocol was employed with the exception that anti-c-Myc-agarose
conjugate (A7470, Sigma) was used after pretreatment with 1% SDS and 1 mg/ml single-stranded DNA at room temperature for 30 min.
Two different approaches were used to analyze the MMP-9 promoter
fragments in the immunoprecipitated samples. For conventional PCR, a pair of primers (P1) spanning
634/
484
(5'-ATTCAGCCTGCGGAAGACAG-3' and 5'-ACTCCAGGCTCTGTCCTCTT-3') was used
with the following PCR cycle parameters: denaturation at 94 °C for
30 s; annealings at 56 °C for 30 s, and extension at
72 °C for 30 s. The number of PCR cycles are indicated in the
figure legends. Alternatively, real-time PCR was done as described
above to quantify the immunoprecipitated DNA. Two primer sets were used
for amplifying the
657/
484 and
86/+34 regions, respectively: PN1,
5'-TGTCCCCTTTACTGCCCTGA-3' and 5'-ACTCCAGGCTCTGTCCTCTT-3'; and P3,
5'-TGACCCCTGAGTCAGCACTT-3' and 5'-CTGCCAGAGGCTCATGGTGA-3'. A
Ct value was calculated by subtracting the Ct value for the 2%
input sample from the Ct value for the immunoprecipated (IP) sample,
i.e.
Ct = Ctinput
CtIP. The percentage of the total input amount for
the IP sample then was calculated by raising 2 to the
Ct power,
i.e. % total of IP sample = 2
Ct × 2 as
described previously (35).
Restriction Enzyme Accessibility Assays--
Restriction enzyme
accessibility assays were performed essentially as described elsewhere
(36, 37). 5 × 10 6 cells were resuspended in
hypotonic buffer (10 mM Tris-HCl, pH 7.4, 10 mM
NaCl, 3 mM MgCl2, 0,15 mM spermine,
and 0.5 mM spermidine) and incubated on ice for 5 min.
Nonidet P-40 was then added (final concentration = 0.5%), and the
cells were vortexed and incubated on ice for another 5 min. Nuclei were
harvested by centrifugation, washed twice with RE buffer (10 mM Tris-HCl, pH 7.4, 50 mM NaCl, 10 mM MgCl2, 0.2 mM EDTA, 0.2 mM EGTA, 1 mM dithiothreitol, 0.15 mM spermine, and 0.5 mM spermidine), and then
resuspended in 90 µl of buffer H (Roche Applied Science). Restriction
digestions were performed with 100 units of PstI for 15 min
at 37 °C. The reactions were terminated with 2× proteinase K buffer
(100 mM Tris-HCl, pH 7.5, 200 mM NaCl, 2 mM EDTA, and 1% SDS). Reaction mixtures were then
supplemented with 50 µl of 2× proteinase K buffer, 50 µl of RE
buffer, 80 µg of proteinase K, and 50 µg of RNase A and incubated
at 37 °C overnight. Genomic DNA was harvested by phenol/chloroform
extractions and ethanol precipitation and then dissolved in TE
buffer. To determine the amount of uncut DNA, 150 ng of genomic DNA was
used for real-time PCR with primer set P3. The amount of uncut DNA was
determined by plotting the Ct value of the samples in a standard Ct
curve made by co-amplifying varying amounts of undigested genomic DNA
(60-180 ng) (37).
DNase I Hypersensitivity Assays--
Nuclei were purified from
3 × 107 cells as described above, washed, and
resuspended in 500 µl of DNase I digestion buffer (15 mM
Hepes, pH 7.6, 60 mM KCl, 15 mM NaCl, 0.2 mM EDTA, 0.2 mM EGTA, 320 mM
sucrose, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol, 0.5 mM spermidine, and 0.15 mM spermine) containing varying amounts of DNase I
(Worthington Biochemicals, Lakewood, NJ). Digestions were carried out
on ice for 30 min following the addition of 2.5 µl of 1 M
MgCl2. The reaction was terminated by the addition of 25 µl of 0.5 mM EDTA solution followed by sequential
incubations with 100 µg of RNase A at 37 °C for 1 h and with
400 µg of proteinase K, 0.6% SDS at 37 °C overnight. DNA was
purified by with phenol, phenol/chloroform, and ether; precipitated
with ethanol; and dissolved in TE buffer. DNA (30 µg) was
digested with 150 units of ApaLI, and fragments were
resolved by gel electrophoresis and transferred to a nylon membrane.
DNA was indirectly labeled by hybridizing with a
32P-labeled probe (PCR derived with the
5'-CAGTCCACCCTTGTGCTCTT-3' and 5'-CTTCAGATACGCCCATCACC-3' primer
set using the genomic clone RP11.465L10) spanning +92/+284 of the
MMP-9 gene.
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RESULTS |
Generation of MTA1-expressing HT1080 Cells--
To answer the
question as to whether MTA1 expression regulates metalloproteinase
expression, we first generated HT1080 cells expressing the MTA1 coding
sequence. To eliminate the confounding issue of clonal variation due to
a heterogeneous cell population and random integration of the
expression construct, we used the Flp-In system (Invitrogen). First, a
host clone was generated by stably transfecting HT1080 cells with an
expression construct (pFRT/lacZeo) bearing a
zeocin-resistant coding sequence and the Flp-In system recombination
target site (FRT). Zeocin-resistant clones were subjected to
Southern blotting (Fig. 1A) to
select a clone bearing a single copy of the integrated plasmid. Clone F8 was determined to bear a single copy by virtue of the single band
generated when the genomic DNA was digested with HindIII and
probed with the lacZ cDNA sequence. In contrast, clone
F9 contained multiple FRT copies as indicated by the several bands hybridizing with the probe. Subsequently, clone F8 was co-transfected with a Flp recombinase-encoding plasmid (pOG44) and the pcDNA5/FRT expression vector bearing nothing or the full-length MTA1 coding sequence (pcDNA5/FRT/MTA1). The Flp recombinase catalyzes a
homologous recombination event between the FRT sites in clone F8 and
the pcDNA5/FRT-expressing vector (empty or bearing the MTA1 coding sequence). Because the pcDNA5/FRT expression vector encodes a hygromycin B-resistant coding sequence, resulting transfectants were
selected in hygromycin B and pooled. Western blotting demonstrated MTA1
protein in the cells (designated MTA1) transfected with the pcDNA5/FRT/MTA1 plasmid when compared with the cells bearing the empty vector (designated FRT) or the F8 host clone (Fig.
1B).

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Fig. 1.
Establishment of Flp-In MTA1-expressing
cells. A, HT1080 cells were transfected with a vector
(pFRT/lacZeo) bearing a FRT site followed by selection of
resistant clones in zeocin-containing medium. DNA from resistant clones
(F8, F9) and parental cells was resolved by gel electrophoresis,
transferred to a nylon membrane, and hybridized with a
32P-labeled lacZ fragment. B, clone
F8 was transfected with pcDNA5/FRT/MTA1 or the empty vector and
subsequently selected in hygromycin B-containing medium. Equal protein
from pooled resistant clones (MTA1) or clones bearing the empty vector
(FRT) were subjected to Western blotting for MTA1 protein. Data are
representative of at least duplicate experiments.
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Diminished MMP-9 Activity/mRNA Levels in
MTA1-expressing HT1080 Cells--
To determine whether MTA1 expression
altered metalloproteinase expression, conditioned medium was analyzed
by zymography. An enzymatic activity indistinguishable in size (92 kDa)
from MMP-9 was detected in the control (FRT) cells. The intensity of this band was greatly induced by PMA (Fig.
2A), a known stimulant of
MMP-9 expression (38). In contrast, the MTA1-expressing cells showed
diminished basal MMP-9 activity and demonstrated an attenuated response
to the phorbol ester. Equally important, a gelatinolytic activity
migrating at the 72-kDa position, which is encoded by a separate type
IV collagenase (MMP-2) (11, 12, 39), was unaffected by MTA1
expression arguing against a generalized repressive effect of this
metastases-associated gene. To corroborate these data, total RNA was
extracted from cells treated under identical conditions and analyzed
for steady-state MMP-9 transcript (Fig. 2B). Again, when
compared with the FRT cells, the MTA1-expressing cells contained a
lower basal level of MMP-9 mRNA and was induced to a lesser extent
by the phorbol ester. In contrast, both basal and PMA-induced MMP-2
mRNA levels were unaffected by MTA1 expression (Fig.
2C). Thus, attenuated MMP-9 activity is due to a less
abundant MMP-9 mRNA in the MTA1-expressing cells.

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Fig. 2.
MTA1 represses constitutive and PMA-induced
MMP-9 expression. A, aliquots of conditioned medium from FRT
and MTA1 cells treated with or without PMA treatment (100 nM, 24 h) were normalized for cell number differences
and subjected to zymography using an acrylamide gel impregnated with 1 mg/ml gelatin. B, total RNA (2 µg) extracted from the
indicated cells was reverse-transcribed, and cDNAs were subjected
to multiplex PCR using primers for MMP-9 and -actin. PCR products
were resolved by agarose gel electrophoresis and indirectly labeled
using a 32P-labeled MMP-9 probe. Band intensity was
determined by densitometry using Quantity One software. C,
total RNA was subjected to Northern blotting for MMP-2 transcript.
Loading equality was checked by photography of the ethidium-stained gel
(lower panels) and densitometry. The data are typical of
three separate experiments.
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Reduced DNase I/Endonuclease Hypersensitivity at the in
Vivo MMP-9 Promoter in the MTA1-expressing Cells--
The ability of
MTA1 to repress MMP-9 expression might be direct or indirect via the
modulated expression of other genes. If the effect of MTA1 is direct,
we predict that MTA1 would be bound to the endogenous MMP-9
promoter. As a first step in this direction, we identified the regions
of the in vivo MMP-9 promoter targeted by MTA1.
Nuclei from the MTA1 or FRT cells were incubated with varying amounts
of DNase I, DNA purified and digested to completion with
ApaLI. Three hypersensitive regions were identified (Fig. 3, parentheses) with
one located approximately at
650/
450 and the other two closely
spaced regions located at
120/+1 relative to the transcriptional
start site. Both distal (
650/
450) and proximal (
120/+1) regions
were of particular interest because they contain several transcription
factor binding sites (distal region AP-1, PEA3, Sp1, and NF
B;
proximal region AP-1) reported previously by our group and others (14,
18, 40) to regulate MMP-9 expression. Importantly, the
intensity of these hypersensitive regions was diminished in the
MTA1-expressing clone indicating a more compact chromatin structure in
these cells. Densitometric analysis (Quantity One software, Hercules,
CA) indicated a 35-60% reduction in the intensity of the
hypersensitive regions in the MTA1-expressing clone.

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Fig. 3.
Reduced DNase I hypersensitivity of the MMP-9
promoter in MTA1-expressing cells. Nuclei from FRT and MTA1 cells
were digested with increasing amounts of DNase I followed by DNA
extraction. Equal DNA (30 µg) was digested overnight with
ApaLI and subjected to Southern blotting using a
32P-labeled MMP-9 probe as indicated.
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To corroborate these results, we subjected the more
proximal region of the MMP-9 promoter to endonuclease
accessibility assays. Nuclei from the FRT and MTA1 cells were isolated
and incubated with PstI. DNA was then extracted and
subjected to real-time PCR using primers (Fig.
4A), which span the
PstI site in the proximal DNase I hypersensitive region. In
this method, PCR amplification can only occur with the uncut DNA
because the primers span the PstI restriction site. This
assay revealed a dramatic reduction in the percent of
PstI-cut DNA (Fig. 4B) in the MTA1-expressing cells, further indicating a more compact chromatin structure of the
proximal MMP-9 promoter region in vivo.

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Fig. 4.
Reduced restriction enzyme accessibility
within the proximal MMP-9 promoter in MTA1-expressing cells.
A, schematic description of the proximal MMP-9 promoter
indicating the PstI cleavage site and the region amplified
by PCR. B, nuclei from FRT and MTA1 cells were digested with
100 units PstI (37 °C, 15 min) followed by DNA extraction
and purification. DNA (150 ng) was subjected to real-time PCR using the
primer set P3, and the absolute amount of uncut DNA was determined by
plotting Ct values against a standard Ct/DNA curve. Data are expressed
as mean values ± S.D. of three separate experiments.
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MTA1 Is Bound to the MMP-9 Promoter in Vivo--
We then
determined whether the distal DNase I hypersensitive region, which
contains multiple regulatory cis elements, was bound with
MTA1. Because the cloning strategy to generate the cells bearing the
MTA1 sequence integrated at the FRT site had removed the myc
tag, we stably transfected parental HT1080 cells with a
myc-tagged MTA1 sequence. After selection of a clone (M10) with G418, MTA1 protein expression was confirmed by Western blotting using an anti-myc antibody (Fig.
5A). In contrast, MTA1 protein was undetectable in clone P derived from HT1080 cells transfected with
the empty vector. Subsequently DNA proteins were cross-linked in the
myc-tagged MTA1-expressing clone M10 and the control clone P. Chromatin was prepared and sheared by sonication, and protein-bound DNA was immunoprecipitated with the anti-myc tag antibody.
Precipitated DNA spanning the
634/
484 MMP-9 promoter
region was amplified by PCR. The anti-myc antibody
efficiently precipitated (Fig. 5C) the endogenous
MMP-9 promoter fragment from the MTA1-expressing clone M10,
whereas no detectable signal was apparent with control clone P. Furthermore, the MMP-9 promoter sequence was not detected when the anti-myc antibody was deleted from the reaction.
These data indicate that MTA1 is bound to the MMP-9 promoter
in vivo.

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Fig. 5.
MTA1 binds the MMP-9 promoter in
vivo. A, HT1080 cells were transfected with
an expression vector encoding a c-myc-tagged MTA1 protein or
the empty vector. Positive clones were selected in G418-containing
medium. Equal protein from a resistant clone (M10) or an empty vector
clone (P) was subjected to Western blotting for MTA1 protein using an
anti-c-Myc antibody. B, schematic description of the distal
MMP-9 promoter indicating the region spanned by the PCR primers.
C, the indicated clones were treated with 1% formaldehyde
followed by shearing of the chromatin by sonication. c-Myc-tagged MTA1
protein-DNA was precipitated with anti-c-Myc-agarose beads. DNA was
eluted from the agarose beads, cross-links were reversed, and the DNA
was purified and subjected to PCR using primer set P1. PCR was
performed for 30 cycles for the input sample, whereas one-tenth of the
PCR products from the other two groups were subjected to a second PCR
with an additional 12 cycles. The experiment was performed twice.
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HDAC2 Interacts with MTA1 and Is Bound to the
657/
484 MMP-9 Promoter Region in MTA1-expressing HT1080
Cells--
Since MTA1 has previously been shown to bind HDAC1 and
HDAC2, we considered the possibility that the more compact
MMP-9 promoter regions as shown above was due to the
recruitment of these deacetylases. To address this possibility, M10 and
P cell lysates were immunoprecipitated with the anti-myc
antibody, and the immunoprecipitate was subjected to Western blotting
for the HDACs. HDAC2 was co-immunoprecipitated from lysates of the MTA1
clone (Fig. 6), whereas this deacetylase was undetectable with control clone P. In contrast, HDAC1 protein was
undetectable in these co-immunoprecipitation assays using either clone
(data not shown). These data reveal MTA1-HDAC2 protein-protein interactions in MTA1-expressing cells.

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Fig. 6.
HDAC2-MTA1 protein-protein interactions.
Proteins from the indicated clones were immunoprecipitated with the
anti-c-Myc-agarose beads and subjected to Western blotting for either
the HDAC1 or MTA1 protein. The data are representative of two separate
experiments.
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Using ChIp assays, we then determined whether MTA1 targeted HDAC2 to
the regulatory regions of the MMP-9 promoter identified by
DNase I hypersensitivity assays. Primer sets PN1 and P3 (Fig. 7A) corresponding to the
proximal and distal hypersensitive regions were employed. Chromatin was
immunoprecipitated with the anti-HDAC2 antibody, and the
immunoprecipitated DNA was quantitated by real-time PCR (Fig. 7,
B and C). For the MTA1-expressing clone M10, the anti-HDAC2 antibody precipitated the distal MMP-9 promoter
fragment as evident using the PN1 primer set (Fig. 7B). In
contrast, the amount of MMP-9 promoter precipitated by the
HDAC2 antibody in the MTA1-deficient control clone P was barely above
background (No Ab). The difference between the M10
and P clones in the amount of MMP-9 promoter fragment
precipitated was statistically significant (p = 0.003).
Although the anti-HDAC2 antibody precipitated the proximal
MMP-9 promoter region as evident using primer set P3, the
amount of DNA precipitated from the MTA1 clone M10 and the control
clone P was essentially the same (p > 0.05) (Fig.
7C). These data suggest that MTA1 targets HDAC2 to the more
distal regulatory region of the MMP-9 promoter.

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Fig. 7.
Binding of HDAC2 to the distal MMP-9 promoter
region is MTA1-dependent. A, schematic of
the MMP-9 promoter indicating the distal and proximal regions that the
primer sets (PN1, P3) amplify. B and C, the
MTA1-expressing clones (M10) and empty vector clone (P) were subjected
to chromatin immunoprecipitation assays using a rabbit antibody against
HDAC2. Precipitated DNA was subjected to real-time PCR for quantifying
DNA within the distal (B) or proximal (C) MMP-9
promoter region using the primer sets PN1 and P3, respectively. Data
are shown as mean values ± S.D. of six independent
determinations.
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Reduced Histone Acetylation at the
657/
484 MMP-9
Promoter Region in the MTA1-expressing Cells--
Since the data above
indicated that targeting of HDAC2 to the
657/
484 MMP-9
promoter region was MTA1-dependent, reduced DNase I
hypersensitivity might very well reflect diminished histone acetylation
at this region, thereby creating a more compact structure resistant to
digestion. To address this possibility, ChIp assays were repeated using
antibodies directed at acetylated histones H3 and H4. Using the primer
pair PN1 spanning the
657/
484 MMP-9 promoter region, we
found that either one of these antibodies precipitated diminished the
amounts of the MMP-9 promoter from the MTA1 clones
(p = 0.0011 and 0.0035 for acetylated
histone H3 and acetylated histone H4, respectively) (Fig.
8A). In contrast, primer set
P3, which amplifies the
86/+34 MMP-9 promoter region, indicated little change (p = 0.4062 and 0.0595, respectively) between the MTA1 and the FRT clones in either histone H3
or H4 acetylation (Fig. 8B). These data corroborate the
previous experiments showing MTA1-dependent targeting of
HDAC2 to the more distal MMP-9 promoter region.

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Fig. 8.
MTA1-dependent hypoacetylation of
histones H3 and H4 at the distal but not proximal MMP-9 promoter
region. Protein-DNA complexes in FRT and MTA1 cells were
cross-linked and subjected to chromatin immunoprecipitation assays with
antibodies against acetylated histones H3 and H4. Precipitated DNA was
subjected to real-time PCR assays for quantifying DNA within the distal
and proximal MMP-9 region using the primer sets PN1 (A) and
primer set P3 (B), respectively. Data are shown as average
values ± S.D. of six separate determinations.
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Trichostatin A Only Partially Relieves MTA1-repressed MMP-9
Expression--
The observations that HDAC2 binding (and histone
deacetylation) to the proximal MMP-9 promoter region was unaffected by
MTA1 could not explain the reduced DNase I hypersensitivity of this region evident in the MTA1-expressing clones (Fig. 3). Therefore, we
speculated that this metastases-associated gene might regulate MMP-9
expression via a mechanism that was both dependent on and independent
of histone deacylation. To answer this question, MTA1 and FRT clones
were treated with trichostatin A (TSA), a broad HDAC inhibitor, and
analyzed for MMP-9 activity. TSA increased constitutive and
PMA-inducible MMP-9 mRNA levels in both FRT and MTA1-expressing
clones (Fig. 9A). However, TSA
was unable to restore either the basal or the PMA-induced MMP-9
mRNA levels in the MTA1-expressing cells to that achieved in the
FRT control cells. Furthermore, TSA failed to increase chromatin
accessibility at the proximal MMP-9 promoter region in the
MTA1-expressing cells over and above that achieved with the FRT cells
(Fig. 9B). These data suggest that a more compact chromatin
conformation at the proximal MMP-9 promoter region occurs
independently of changes in histone deacetylation.

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Fig. 9.
Repression of MMP-9 expression by MTA1 occurs
via a histone acetylation-dependent and independent
mechanism. A, the indicated cells were treated with
100 nM TSA for 2 days. Constitutive or PMA-induced MMP-9
expression was determined by reverse transcriptase-PCR as described in
Fig. 2B. B, nuclei from the indicated cells
treated with or without TSA was digested with PstI followed
by real-time PCR assays as described in Fig. 4. Data are representative
of duplicate experiments (A) and are shown as average
values ± S.D. for six separate determinations
(B).
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Since changes in histone acetylation could not entirely
account for the MTA1-repressed MMP-9 expression, we considered the possibility that chromatin remodeling of the proximal MMP-9
promoter might occur via an additional mechanism. Previous studies have demonstrated that the Mi2 protein, which confers
ATP-dependent chromatin-remodeling activity (30), is
present in the nucleosome remodeling and deacetylase complex.
Thus, we determined whether Mi2 was bound by the MMP-9
promoter in vivo. As a first step in this direction, we
immunoprecipitated the M10 and P clones with the anti-Myc antibody and
Western blotted for Mi2 (Fig.
10A). Mi2 protein was
co-immunoprecipitated with MTA1 in the M10 clone but not in control
clone P. These data indicate MTA1-Mi2 protein-protein interactions at
least in the MTA1-expressing cells. We then performed ChIp assays to
determine whether Mi2 was bound by the MMP-9 promoter. Interestingly, although the proximal primer set yielded negative data
(data not shown), the distal primer set amplifying the
657/
484 sequence revealed that this chromatin-remodeling protein was indeed bound to the MMP-9 promoter in the M10 clone. In contrast,
the MMP-9 promoter was not immunoprecipitated with this
antibody in the MTA1-deficient control clone P. Thus, the ability of
MTA1 to repress MMP-9 expression may reflect, in part, the recruitment of Mi2 to the promoter. Presumably, like the p300/CREB-binding protein-associated factor (41), Mi2 acts at long range on the proximal
region to achieve chromatin remodeling.

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Fig. 10.
MTA1 recruits the chromatin-remodeling
enzyme Mi2 to the MMP-9 promoter. A, protein from the M10
and empty vector P clones was immunoprecipitated with
anti-c-Myc-agarose beads. Immunocomplexes were analyzed by Western
blotting for the Mi2 and MTA1 proteins. B, DNA-protein
complexes from the indicated cells were cross-linked with formaldehyde
and subjected to chromatin immunoprecipitation assays with a rabbit
anti-Mi2 antibody. The MMP-9 promoter fragment was amplified by PCR (35 cycles) using the primer set P1. The experiment was performed
twice.
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DISCUSSION |
We report herein the repressive effect of the
metastases-associated gene MTA1, on MMP-9 expression mediated via its
direct interaction with the collagenase promoter. These findings are the first to demonstrate regulation of a protease by a
chromatin-remodeling activity. Furthermore, with the exception of the
estrogen-regulated genes c-myc and pS2 (29), this
is the first endocrine-independent gene shown to be targeted by MTA1.
Our findings that a chromatin-remodeling activity regulates MMP-9
expression indicates an additional level of control of expression for
this gene previously only alluded to by transgenic approaches in which
MMP-9 promoter constructs were stably integrated into the genome (20,
21).
It does not appear that MTA1 has a pleiotropic effect on gene
expression because the expression of the 72-kDa type IV collagenase encoded by a separate gene (MMP-2) was unaffected.
Additionally, preliminary expression array studies indicate few other
genes regulated by MTA1. How the specificity for the MMP-9
promoter is conferred remains to be determined. In this regard, it is
noteworthy that another chromatin-remodeling activity (Swi/Snf complex)
in NIH3T3 cells and yeast also demonstrates selectivity toward subsets of genes (42, 43), although like MTA1, the mechanism by which this
specificity is conferred is presently unknown.
Our study bears an important difference to that of Mazumdar et
al. (29) who investigated the regulation of estrogen-responsive genes by MTA1. Thus, although the repressive effect of MTA1 on an
estrogen response element-driven promoter was entirely abrogated by the
HDAC inhibitor TSA, this was not the case for MMP-9 expression. This
difference might suggest that estrogen-responsive genes are repressed
entirely via changes in histone acetylation, whereas MTA1 regulates
MMP-9 expression through a mechanism that is both dependent and
independent upon histone deacetylation. Indeed, our observation that
the Mi2 protein, which confers chromatin-remodeling activity
independent of histone deacetylation (30), co-immunoprecipitated with
MTA1 and was targeted to the MMP-9 promoter in
vivo might explain the inability of TSA to fully restore MMP-9
expression in the MTA1-expressing cells.
How might MTA1 repress MMP-9 expression? We initially hypothesized that
a more compact chromatin structure achieved by increased histone
deacetylation (44) might reduce the binding of transcription factors to
previously characterized cis elements (14, 18, 40) as we
reported for the KiSS-1 metastases suppressor gene (15).
However, our analysis using ChIp (data not shown) failed to show
altered occupancy of the AP-1, NF
B, and Sp1 motifs in the
MMP-9 promoter, which we and others (14, 18, 40) had previously reported to be regulatory for the expression of this collagenase. Furthermore, our DNase I hypersensitivity experiments did
not reveal novel MMP-9 regulatory elements in the MTA1-expressing cells. These data, although not exhaustive, would argue against the
possibility that MTA1 suppresses MMP-9 synthesis by way of reducing the
access of transcription factor(s) to their cognate sequences in the
MMP-9 promoter. A second possibility is that the
trans-acting activity of the MMP-9 promoter-bound
transcription factors is modulated by MTA1 or the chromatin structure.
Such a control mechanism has been demonstrated for the Gcn4 and Hap4 transcription factors in yeast (45) and for p53 and Fos/Jun dimers in
mammalian cells (46, 47), albeit in response to the Swi/Snf
chromatin-remodeling complex. A third possibility is that a
MTA1-mediated change in chromatin conformation culminates in decreased
accessibility to the basal transcriptional machinery. Indeed, our data
showing reduced PstI endonuclease accessibility proximal to
the TATA box would be consistent with this latter scenario.
We were initially surprised by the observation that MTA1 repressed
MMP-9 synthesis since expression of either gene correlates with the
metastatic phenotype (25, 48, 49). Several reasons could explain this
unexpected finding. First, it is now well accepted that MMP-9
expression in many human malignancies occurs in the neighboring stromal
cells rather than the tumor cells themselves (50-54). Certainly,
in situ hybridization experiments support this contention in
breast cancer (55), a tissue in which increased MTA1 expression was
first described (25). It is tempting to speculate that the absence of
MMP-9 expression in these tumor cells reflects the increased expression
of MTA1 (25). Alternatively, studies reporting that MTA1 expression
correlates positively with tumor metastases are based on
immunohistochemical and in situ hybridization analyses, and
it is unclear whether the antibodies or RNA/cDNA probes employed in
those investigations were specific for MTA1 or recognized other related
proteins such as MTA-L1 (56) or the recently identified naturally
occurring short form of MTA1 (57).
In conclusion, we have provided bona fide evidence for a
direct role of the MTA1 co-repressor in the regulation of MMP-9
expression through a mechanism that is both dependent on and
independent of histone deacetylation. MMP-9 is the first protease to be
shown to be regulated by a chromatin-remodeling activity and adds to a
short list of MTA1-regulated genes, which at present only includes the
estrogen-responsive genes pS2 and c-myc (29).