From the Department of Biochemistry, Stockholm University, S-106 91 Stockholm, Sweden
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ABSTRACT |
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Expression of adenine nucleotide translocator
isoform 2 (ANT2) is growth regulated. In the present study, we report
the presence of a silencer region in the human ANT2 promoter and the
purification of a two-component factor that recognizes a specific
hexanucleotide element, GTCCTG, of the silencer. Transfection of
deletion constructs shows that ANT2 silencer activity extends over a
region of at least 310 nts. However, mutating the GTCCTG element
completely relieves silencing activity in the context of the human ANT2
promoter. The data suggest that the GTCCTG element might be required
for maintaining silencer activity of the extended silencer region. The
ANT2 silencer region cloned in front of the herpes simplex virus
thymidine kinase promoter confers nearly complete inhibition to the
heterologous promoter. However, unlike the ANT2 promoter, mutating the
GTCCTG element restores only partial activity to the herpes simplex
virus thymidine kinase promoter. A protein complex consisting of two
major polypeptides of 37 and 49 kDa was isolated from HeLa nuclear
extracts by affinity chromatography using the GTCCTG element as the
affinity resin. Cross-linking studies and Southwestern analysis
indicate that p37 binds DNA. p49 appears to be loosely associated with
the p37/DNA complex but is necessary for strong binding of p37. Our
data implicating the GTCCTG element directly in silencing of the ANT2
promoter, together with data from the literature reporting the presence of this element within the silencer region of several additional promoters, suggest a general role of the GTCCTG element in
transcriptional silencing.
The adenine nucleotide translocator
(ANT)1 proteins exchange
cytosolic ADP for mitochondrial ATP, thereby playing an essential role
in maintaining cell metabolism and growth. Recent findings also
implicate ANT in the initiation of events leading to apoptosis (1).
Mammalian ANT is encoded in three genes, ANT1, ANT2, and ANT3 (2-5),
that are expressed in a tissue-specific manner (6). ANT1 mRNA is
expressed predominantly in heart and skeletal muscle (7, 8) whereas
ANT2 mRNA is expressed in a broad range of tissues (6, 7, 9), but
predominantly those tissues that undergo rapid proliferation (6, 10).
However, the ANT2 isoform is unique in that it is also expressed in a
growth-dependent manner (11) in a wide variety of cell
lines. ANT2 expression is low in quiescent cells and is substantially
increased by factors that induce entrance into G1 and
subsequent cell growth (11). ANT2 expression in serum-activated NIH3T3
cells is inhibited by actinomycin D2 but not cycloheximide
(12), indicating that expression is regulated at the level of
transcription but does not require new protein synthesis.
The physiological significance of expression of just the ANT2 isoform
in a growth-dependent manner has not been delineated, although it is possible that slight kinetic differences in ADP/ATP exchange catalyzed by the isoforms (13) might provide the
growth-activated cell with an energetic advantage. To understand the
complex regulation of ANT2 expression, we have analyzed its promoter
(14). Our findings show that it is a complex array of positive and
negative activating regions, with the major activating sites residing
in a tandem Sp1 AB element (14) in the proximal promoter. We also identified a unique Sp1 site adjacent to transcription start that suppresses promoter activity when occupied by Sp1 in mammalian (14) and
Drosophila3 cells.
A second negative acting region was also identified in the distal
region of the ANT2 promoter (14). In the present study, we have
characterized this region. We show that the negative acting element is
a unique silencer region that is distributed over a region of about 300 nts, but whose silencer function is directed from an element containing
a GTCCTG core sequence. We also report the purification of a
two-component factor that binds to this element and appears to be
responsible for maintaining silencer function.
Cell Culture and Transfection--
HeLa cells were grown to
subconfluence in Dulbecco's modified Eagle's medium containing 10%
(v/v) heat-inactivated fetal calf serum supplemented with 2 mM L-glutamine, 50 units of penicillin, and 50 µg/ml streptomycin. The cells (4 × 105) were plated
on 60-mm Petri dishes and transfected by the calcium phosphate method
(15) using 5-8 µg of reporter plasmid DNA containing the
chloramphenicol acetyltransferase (CAT) gene and 2-5 µg of control
plasmid DNA (RSV-LacZ or pLuc-ANT2( Plasmid Preparation--
Preparation of pCAT-ANT2(
Heterologous promoter constructs of the ANT2 silencer region and the
HSVtk promoter were prepared by cloning ANT2 promoter restriction
fragments into the pBLCAT2 (18) polylinker. The clones were checked for
correct orientation by restriction enzyme digestion, and the presence
of mutation within the Preparation of Nuclear Extracts--
HeLa and NIH3T3 cells were
grown to subconfluence in 175-cm2 flasks. Cells were
harvested, and nuclear extracts were prepared as described previously
(19). Rat liver nuclei purified as described previously (20) were
generously provided by Madeleine Kihlmark (Stockholm University,
Stockholm, Sweden), and nuclear proteins were extracted in high salt C
buffer as described previously (19). The protein was measured using the
Bio-Rad Protein Assay (Bio-Rad). Nuclear extracts were stored in
20-µl aliquots at Electrophoretic Mobility Shift Assay (EMSA)--
EMSA reaction
mixture (20 µl) containing 20 mM K+-Hepes (pH
7.9), 70 mM KCl, 10% glycerol, 5 mM
MgCl2, 2 mM dithiothreitol, 8 µM
ZnCl2, 1.5 µg of nuclear extract, 250 ng of
poly(dI·dC)·poly(dI·dC) (Pharmacia), and competitor DNA (where
indicated) was incubated on ice for 10 min. Radioactively labeled
oligonucleotide probe (10,000 cpm) was added, and the reaction was
incubated for 20 min at room temperature. Bound complexes were
separated on 5% native acrylamide gels in 0. 5× TBE (1× TBE = 90 mM Tris, 90 mM boric acid, and 2 mM EDTA, pH 8.0). The gels were dried and autoradiographed.
DNase I Protection Assay--
DNase I protection was performed
as described previously (16). Radioactive probes were prepared by
polymerase chain reaction using 5' 32P-labeled CAT primer,
M13 primer, and pCAT-ANT2( UV Cross-Linking and Southwestern Analysis--
Radioactive
oligonucleotide probes (ANT2
For Southwestern analysis, increasing amounts of HeLa nuclear extract
(10, 25, and 50 µg) were separated by 10% SDS-PAGE and electroblotted onto a nitrocellulose membrane. Membranes were treated
with guanidinium hydrochloride and incubated with radioactively labeled
oligonucleotides as described previously (21).
Protein Purification--
Nuclear extracts from 1.5 × 109 HeLa cells were purified (22) and separated on Sephadex
G-100 SF column (Pharmacia) in a buffer containing 20 mM
Hepes (pH 7.9) and 400 mM KCl. Eluted fractions were tested
for protein binding to the ANT2 Characterization of a Negative Regulatory Region in the ANT2
Promoter--
We previously identified a region of the human ANT2
promoter between nts Identification of DNA Binding Sites within the
Strong protection of region 2 suggests that the binding protein is
present in high amounts in HeLa nuclear extracts, or that it binds with
high affinity. Consistent with this conclusion, weak protection of the
major Sp1-activating sites within the proximal promoter requires three
times more HeLa nuclear extract (data not shown) than used here for the
protection of silencer region 2.
Identification of a Specific GTCCTG Element Binding
Activity--
To further study protein binding to region 2, a wild
type oligonucleotide covering the central part of region 2 (nts
The region 2-binding protein is also present in mouse NIH3T3 cells and
rat liver. Nuclear extracts from both sources exhibit specific binding
to the wild type probe (Fig. 3B). Thus, the silencer protein
appears to be widely expressed in mammalian cells.
As shown in Fig. 2A, three sites on the B silencer region
are contacted by nuclear extract components. To determine whether region 2 influences binding to the weaker sites, the mutated region 2 sequence was placed in the context of the long promoter fragment ( The GTCCTG Element Is Required for Silencing ANT2 Promoter
Activity--
To determine whether the strongly protected region 2 could influence silencer activity, deletion clones bearing the mutated GTCCTG element were constructed and fused with the CAT gene. Promoter activities of these clones and the corresponding wild type clones were
measured in transient transfection experiments (Fig.
4). Normalized CAT activity was increased
up to 3.7-fold in the mutated clones, and, in the case of mutated
fragments Silencing Activity Can Be Conferred to a Heterologous
Promoter--
Fig. 5A shows
that silencing could be conferred to the heterologous HSVtk promoter in
transfected HeLa cells. Silencing function was retained in the
Characterization of a Specific Protein that Binds Region 2 of the
Silencer--
A computer search of a vertebrate transfactor database
(24) failed to reveal a transfactor that specifically recognizes the
region 2 sequence. To estimate the size of the protein binding to this
DNA sequence, both cross-linking and Southwestern blot analysis were
performed. Using HeLa nuclear extracts, a single polypeptide with an
apparent mass of 33 kDa is cross-linked to the wild type ANT2
The presence of a single cross-linked band on SDS gels (Fig.
6A) suggests that the DNA-binding protein binds as a
monomer, although the symmetric organization of the GTCCTG element is
suggestive of a dimer binding site. However, we could not eliminate the
possibility that the DNA-binding protein interacts with additional
factors, because protein cross-linkers were not used in our experiments.
The DNA sequence of region 2 (noncoding strand,
5'-CAGGACCAGGAC-3') resembles a general binding
site for the family of Ets proteins (5'-C/AGGAA/T-3'; Ref.
25). However, antibodies against the conserved region of human Ets-1
protein (amino acids 362-374) failed to recognize the DNA-binding
protein in an EMSA supershift assay (data not shown). This antibody
(catalogue number sc-112X; Santa Cruz Biotechnology, Inc.) cross-reacts
with several members of the Ets family.
DNA Affinity Purification of the Region 2-binding Protein from HeLa
Nuclear Extracts--
A protein that binds specifically to the GTCCTG
element was purified from HeLa nuclear extracts by DNA affinity
chromatography using a biotin-labeled region 2 oligonucleotide (ANT2
The affinity-purified fraction contained two major polypeptides with
molecular masses of 49 and 37 kDa that co-eluted from the affinity
resin between 0.2 and 0.4 M NaCl (Fig. 7A). Only residual binding of these polypeptides was observed to columns containing the mutated oligonucleotide ligand (data not shown). The
apparent mass of the smaller polypeptide is identical to that of the
DNA-binding protein identified with Southwestern blot analysis and
agrees well with that observed by cross-linking. Based on these
criteria, we conclude that p37 most probably binds DNA.
Although p49 appears to co-elute with p37 in Fig. 7A, in
other experiments, proportionately more p49 is eluted at lower salt concentrations (0.2 M), suggesting that the p49 association
with the p37/DNA complex is weaker than the p37/DNA contact itself.
The functional integrity of the DNA affinity-purified protein was
tested by EMSA using the ANT2 In the present study, we describe a silencer region within the
human ANT2 promoter and report the purification of factors that are
essential to silencing. The silencer region extends over 310 nts (nts
Although both the A and B regions are required for maximal silencing of
transcription, the B region, and more specifically, one element within
the B region, is required for the expression of the A region's
silencing capacity. This element, encompassing 29 nts with a central,
directly repeated hexanucleotide, 5'-GTCCTG-3', is the most strongly
protected DNase I site within the B region. The central role of the
hexanucleotide repeat is clearly shown by the finding that the mutated
form (GTCCTG to GTAATG) abolishes silencer function. The GTCCTG element
thus appears to organize the entire silencer region in a manner that is
not yet understood. Indeed, the role of the A region remains an enigma.
It is possible that specific suppressor proteins that are not detected
by our methods bind to region A. If present, however, their association with DNA would probably be controlled by protein binding to the GTCCTG
element. A second explanation might be that the silencer region
includes a nucleosome assembly site. The A region contains putative AT
tracts that are important in nucleosome assembly (27, 28). Nucleosome
assembly is strongly influenced by specific DNA-binding proteins,
including hormone receptors. Thyroid hormone and
9-cis-retinoic acid receptor were reported to disrupt
chromatin structure and facilitate transcriptional repression in the
absence of hormone by a mechanism that does not involve deacetylases
(29). Region B of the ANT2 silencer is footprinted over a conserved hormone response element half site (AGGTCA), but this footprint is not
altered by mutating the GTCCTG element that prevents silencing. Whatever the mechanisms of suppression through the A + B silencer region, it is clear that the factor binding to the GTCCTG element is of
central importance.
A search of the eucaryotic promoter data base (GenBank) shows that
hexanucleotide repeat (GTCCTGGTCCTG) is rarely found in mammalian
promoters. By contrast, the hexanucleotide (GTCCTG) is present in a
large number of promoters. In most cases, it appears to be part of an
enhancing or activating element (30-33). However, in glutathione
transferase P (34), apolipoprotein AI (35), and the major
histocompatibility complex class II HLA-DRA (36) promoters, this
element is present within a silencer region. It is not clear, however,
if the same proteins are responsible for silencing these and the ANT2
promoters. Thus, whether the GTCCTG element recognizes a specific
silencing factor remains an open question.
The ANT2 GTCCTG element-binding protein isolated by us is composed of
two major polypeptides of 37 and 49 kDa. Cross-linking studies show
that only p37 binds DNA. p49 appears to be loosely associated with p37,
and the two can be partially separated during chromatographic
separation. However, p49 appears to strengthen the binding of p37,
because chromatographic fractions containing predominantly p37 bind the
DNA probe weakly. Thus, p49 appears to function as an ancillary protein
to p37. This behavior is reminiscent of that displayed by members of
the Ets family. For example, GABP Multiple forms of the NF1 family were recently reported to bind to the
glutathione transferase P promoter silencer (43). Four major
polypeptides were isolated in a complex that bound to the dominant GPS4
silencer element containing the GTCCTG element (43). These polypeptides
ranged from 33 to 41 kDa, close to the mass of the ANT2 p37-binding
protein. However, a conserved NF1 binding element could not be detected
by a computer search of the ANT2 silencer. Thus, it seems unlikely that
we have isolated members of the NF1 family, although this remains to be
rigidly excluded. There are, however, several interesting similarities between the glutathione transferase P and ANT2 silencer regions: (a) both contain multiple protein binding sites,
(b) mutation in single sites results in a significant
increase in promoter activity, and (c) despite the fact that
expression of the glutathione transferase P gene is restricted to tumor
cells derived from the liver, silencer activity was observed with no
tissue restriction (34). This correlates well with previous results
from ANT2 transfection experiments (14) and with the observation that
ANT2 GTCCTG binding activity is widely distributed in mammalian tissues
(Fig. 3B).
A 36-kDa DNA-binding protein was isolated (44) that most probably binds
to the human HLA-DRA Vbox silencer (36) that contains a GTCCTG element.
This polypeptide has not been identified, and its relationship to the
ANT2 p37 DNA-binding proteins is unknown. As discussed above, there are
reasonably strong arguments that p37 might belong to a class of
silencer proteins that have not previously been identified. In view of
the apparent broad distribution of GTCCTG binding activity, it is
important to identify p37 and establish its role in promoter silencing.
The role of the silencer region in expression of the ANT2 gene also
remains to be delineated. The ANT2 gene was isolated as one of the
early immediate genes during the growth stimulation of quiescent cells
(11). Several laboratories demonstrated regulation of the ANT2 gene
under different physiological conditions. ANT2 transcripts are
up-regulated in activated T cells and various growth-activated cells
(45-47) and are down-regulated in differentiating HL-60 cells (48),
myoblasts (7, 8), quiescent cells (11), and cells approaching
confluence (48). We recently identified a far upstream promoter region
(
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
235/+46)). Enzymatic activities
(CAT,
-galactosidase, and luciferase) were measured according to the
manufacturer's instructions (16).
647/+46) and
pCAT-ANT2(
235/+46) was described previously (14). To construct
pCAT-ANT2(
546/+46) and pCAT-ANT2(
413/+46), corresponding ANT2
promoter fragments prepared by restriction enzyme digestion
(BamHI/PstI and DraI/PstI, respectively) were cloned into pCATbasic (Promega).
pCAT-ANT2(
647/+46)mut was constructed by the overlap-extension
polymerase chain reaction (17). A mutated (CC to AA) oligonucleotide
covering nts
338 to
318
(5'-CCTAGTAATGGTAATGCTCC-3') of the ANT2
promoter was synthesized and used as a primer for introducing the
mutation into the
647/+46 fragment. The M13 (nts 2230-2208) and CAT
(nts 2307-2285) primers of pCATbasic were also used. All other
deletion clones containing mutated oligonucleotide
338/
318 were
constructed by restriction deletion of pCAT-ANT2(
647/+46)mut.
pCAT-ANT2(
546/
235)wt and pCAT-ANT2(
546/
235)mut, which were used
as templates for the polymerase chain reaction to synthesize DNase I
footprint probes, were created by removing the
SmaI/PstI
235/+46 fragment from
pCAT-ANT2(
546/+46)wt or pCAT-ANT2(
546/+46)mut, respectively, followed by T4 DNA polymerase blunting and plasmid re-ligation. All
clones were checked by restriction enzyme digestion and sequencing.
338/
318 element was confirmed by sequencing.
70 °C.
546/
235)wt or pCAT-ANT2(
546/
235)mut
as the templates. The probe (20,000 cpm) was partially digested by
RNase-free DNase I (Boehringer Mannheim) in the presence or
absence of HeLa nuclear extract (20 µg) and separated on a 6%
acrylamide sequencing gel.
338/
318wt or ANT2
338/
318mut) were
incubated with HeLa nuclear extracts under the exact conditions
described above for the EMSA. Samples were placed 5 cm from a UV
Transluminator (Ultra-Violet Products, Inc.) and exposed to UV light
for 10, 20, or 30 min. Subsequent DNase I/micrococcal nuclease
treatment (17) was omitted due to the 5' end-labeling of the probes.
Cross-linked complexes were separated by 10% SDS-PAGE, dried, and autoradiographed.
338/
318 oligonucleotide by EMSA.
Active fractions were pooled and adjusted to 20 mM Hepes
(pH 7.9), 100 mM KCl, 5 mM MgCl2,
and 10% glycerol. To remove nonspecific DNA binding proteins, the
protein sample was repeatedly incubated (1 h, 4 °C) with mutated
ANT2
338/
318 oligonucleotide coupled to CNBr Sepharose 4B
(Pharmacia) (23). The flow-through was collected and incubated (1 h,
4 °C) with 100 µl of immobilized streptavidin (Boehringer
Mannheim) to remove streptavidin-binding proteins. Biotinylated ANT2
338/
318 oligonucleotide probe (850 pmol) was added, and the samples
were incubated for 1 h at 4 °C. Fresh streptavidin resin (100 µl) was added, and the incubation was repeated. The resins with bound
DNA and proteins were collected, washed in buffer containing 20 mM Hepes (pH 7.9), 5 mM MgCl2, 10%
glycerol, 0.1% Triton X-100, and 0.1 M NaCl, and eluted
stepwise with the same buffer containing increasing concentrations (0.1-1.0 M) of NaCl. Finally, streptavidin resins were
washed with 100 µl of 0.1 M glycin (pH 2.5).
RESULTS
647 and
235 (14) that exhibited negative
regulation in three different transfected cell lines (JEG3, NIH3T3, and
HeLa), suggesting the involvement of a factor(s) common to all of the cell lines tested. To further define this region of the ANT2 promoter, additional 5' deletion clones were constructed and tested in transient transfection experiments using HeLa cells (Fig.
1). Deletion of nts
647 to
546 had no
significant influence on the promoter activity. By contrast, the
stepwise deletion of nts
546 to
413 and nts
413 to
235 each
resulted in an approximately 2-fold increase in ANT2 promoter activity.
Thus, negative regulation is spread over an extended area of about 310 nts (nts
546 to
235). To emphasize the bipartite distribution of
the negative acting region (Fig. 1), we shall refer to fragment
546/
413 and fragment
412/
235 as silencer regions A and B,
respectively. Together, the A and B regions decrease ANT2 promoter
activity by 70-75%.
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Fig. 1.
Analysis of the ANT2 silencer region in
transient transfection experiments. Deletion constructs of the
human ANT2 promoter were transfected into HeLa cells. Numbering is
relative to the transcription start site (+1). Mean values ± S.E.
of four independent experiments are shown. The overall structure of the
ANT2 promoter is also shown. The five represent Sp1 elements that
determine proximal promoter activity (14). The A and B negative
silencer regions defined in this study are indicated. The three DNase
I-protected sites in silencer region B (see Fig. 2) are shown as
.
546/
235 Region
of the ANT2 Promoter--
Protein binding sites on the silencer region
were determined by DNase I protection using ANT2
546/
235 as the
probe and HeLa nuclear extracts (Fig.
2A). This probe includes both
the A and B silencer regions. However, DNase I-protected sites were
found only in the B region (nts
413/
235). The most strongly
protected area covers nucleotides
339 to
310 (region 2, Fig.
2A). The core sequence of this site consists of a
hexanucleotide direct repeat (GTCCTG) with no spacing (Fig.
2B). Two additional weakly protected regions are located
upstream and downstream from region 2. Region 1 is weakly protected
over a 14-nt sequence that contains a nuclear receptor half-site
(AGGTCA) on the noncoding strand. Region 3, which is located upstream
from region 2, covers a 27-nt-long sequence and includes strong DNase
I-hypersensitive sites. With the exception of a very weak correlation
to the NF1 element, no conserved transfactor binding elements could be
identified in region 3 by computer analysis (24).
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Fig. 2.
DNase I protection analysis of the ANT2 A and
B silencer regions. A, a DNA probe corresponding to the ANT2
A and B silencer regions (nts 546/
235, Fig. 1) was used in the
DNase I protection assay. Protection by HeLa nuclear extracts was
observed only in the B region. Protected regions are
numbered. B, the DNA sequence (coding strand) of
the ANT2 promoter B silencer region (nts
413/
235) is shown.
Protected regions are indicated by lines and
numbers above the sequence. The arrow in the
region 1 indicates a nuclear receptor half site. Arrows in
region 2 indicate the two hexanucleotide direct repeats that
characterize the strong protein-binding GTCCTG element. The mutated
GTCCTG element (CC
AA) used in additional experiments is shown. The
asterisks in region 3 denote a DNase I-hypersensitive
site.
338 to
318) and a mutated oligonucleotide (CC to AA; Fig. 2B)
of the same region were used in EMSA. Competition experiments showed that binding to the wild type probe was nearly eliminated by a 5-fold
molar excess of the unlabeled probe but was unaffected by a 50-fold
excess of the mutated probe (Fig.
3A). In addition, no binding
was observed when the mutated oligonucleotide served as the EMSA probe
(data not shown).
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Fig. 3.
Protein binding to the ANT2 region 2 silencer
element is specific. A, EMSA was performed with a
32P-labeled ANT2 338/
318 wt probe and HeLa nuclear
extract. The same amount of HeLa nuclear extract (1.5 µg) was used in
all lanes except lane P (probe only). Increasing amounts of
wt or mut competitor oligonucleotide were added to the binding
reactions. Mutations within probe
338/
318 are shown in Fig.
2B. A specific bandshift is indicated by an
arrow. B, EMSA was performed with
32P-labeled ANT2
338/
318 wt probe and nuclear extracts
(1.5 µg) from HeLa and NIH3T3 cells or rat liver. ANT2
338/
318 wt
or mut competitor oligonucleotides were added in 20-fold molar
excess. C, a DNase I footprint assay was carried out on the
546/
235 ANT2 probe bearing mutations in the GTCCTG element. wt
or mut probes were digested by DNase I in the presence or absence
of HeLa nuclear extract. Only the sequence of the B silencer region is
shown, because no protection of region A was observed. The changes in
the DNase I digestion pattern within region 2 are due to the
introduction of the mutation (Fig. 2). The numbering of the protected
regions corresponds to that in Fig. 2.
546/
235). This construct was subjected to DNase I protection analysis with HeLa nuclear extract (Fig. 3C). As expected,
protection of the mutated region 2 site is abolished, and, in addition,
the hypersensitive bands that surround it are lost. However, protection of regions 1 and 3 is not significantly affected by a mutation in
region 2, suggesting that protein binds to these sites independent of
region 2.
546/+46 and
413/+46, promoter activity was restored to
levels comparable to those obtained with clones in which the A and B
silencer regions were deleted (clone
235/+46, Fig. 1). This
observation indicates that the mutation in the GTCCTG element region 2 is able to eliminate silencing from the distal A suppressor region (nts
546/
413). Thus, the silencer protein appears to play a central role
in organizing or maintaining the entire silencer region.
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Fig. 4.
Mutating the region 2 GTCCTG element relieves
silencing imparted by the entire ANT2 Ab silencer region. ANT2
promoter fragments bearing the wt or mut GTCCTG elements were
transfected into HeLa cells. The fragments contained either the A and B
silencer regions or only the B region, as indicated. CAT activity was
normalized for transfection efficiency. All values were set relative to
the wild type 647/+46 promoter fragment. The data represent the mean
value ± S.E. of three independent experiments. Each experimental
point was done in triplicate.
413/
235 ANT2 promoter fragment that includes the GTCCTG element.
Interestingly, silencing of the thymidine kinase promoter reached
87-95%, which was higher than that observed in 5' deletion
experiments with the native ANT2 promoter. As a control, the region
immediately upstream of the ANT2 silencer (nts
647/
546) that lacks
the GTCCTG element was also cloned in front of the thymidine kinase
promoter and transfected into HeLa cells. This fragment exhibited no
silencer properties (Fig. 5A). Mutated GTCCTG elements
partially relieved the silencing of the heterologous ANT2-thymidine
kinase promoter (Fig. 5B), but unlike the native ANT2
promoter, full restoration was not achieved.**
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Fig. 5.
The ANT2 silencer confers inhibition to a
heterologous promoter. A, ANT2 promoter fragments were
cloned in front of the HSVtk promoter and transfected into HeLa cells.
CAT activity was normalized for transfection efficiency. All values
were set relative to the activity of pBLCAT2 plasmid containing only
the HSVtk promoter. ANT2 647/
546 fragment was used as a negative
control. The location of the wild type GTCCTG element is indicated by
. B, ANT2 promoter fragments containing the mutated
GTCCTG element (indicated by
) were cloned in front of the HSVtk
promoter. Wild type and mutated clones were transfected into HeLa
cells. CAT activity was normalized on transfection efficiency control.
The values of the activities of mutated clones were set relative to the
corresponding wild type clones. All data represent the mean value ± S.E. of three independent experiments. Each experimental point was
done in triplicate.
338/
318 probe, but not to the mutated probe (Fig.
6A). No other specific signals
were detected, even under prolonged UV light exposure (data not shown).
Southwestern blot analysis with wild type ANT2
338/
318
oligonucleotide as the probe also identified a single band that did not
hybridize with the mutated probe (Fig. 6B). The apparent
mass of this polypeptide was 37.9 kDa. Because the mobility of
cross-linked polypeptides can be affected by a covalently attached DNA
probe, we conclude that the molecular mass of the DNA-binding protein
is most likely around 38 kDa. This is confirmed by an analysis of the
purified factor (see below).
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Fig. 6.
UV cross-linking and Southwestern analysis of
proteins binding to the GTCCTG element. A,
32P-labeled wt or mut ANT2 338/
318 probes were
incubated with HeLa nuclear extract under EMSA conditions. Samples were
exposed to UV light for the period of time indicated, separated by 10%
SDS-PAGE, and autoradiographed. A specific signal is indicated by an
arrow. B, Southwestern analysis. Increasing
amounts of HeLa nuclear extract (as indicated) were separated by 10%
SDS-PAGE and blotted on nitrocellulose membrane. Proteins were
renatured as described under "Experimental Procedures." The
membranes were incubated with 32P-labeled wt or mut ANT2
338/
318 oligonucleotides. A signal specific for the wild type probe
is indicated by an arrow.
338/
318) as the affinity ligand (Fig.
7A). The fraction from which
protein was purified by the affinity chromatography step (Fig.
7A) had previously been treated with a DNA resin containing
the region 2 oligonucleotide bearing a mutated GTCCTG element. Thus,
the protein purified in Fig. 7A binds specifically to the
wild type GTCCTG element. Fig. 7A also shows that the
polypeptides were not retained on immobilized streptavidin in the
absence of the ANT2
338/
318 oligonucleotide ligand. Together, the
above experiments show that the purified polypeptides associate
specifically with the region 2 GTCCTG element.
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Fig. 7.
Purification of a protein complex that
exhibits specific binding to the GTCCTG element. A,
silver-stained SDS-PAGE. The last two purification steps
(Streptavidin-control column followed by a
Streptavidin-affinity column) are shown. The applied protein
fractions had been prepurified by gel filtration and by passage through
a resin containing the mutated ANT2 338/
318 oligonucleotide (see
"Experimental Procedures"). Left panel, a negative
control showing the proteins that bound to the streptavidin-control
resin lacking an oligonucleotide ligand. Right panel, the
flow-through fraction from the streptavidin-control column was
separated on the streptavidin-DNA affinity column using a salt gradient
(as indicated), followed by wash at low pH (W). The two
major affinity-purified polypeptides of 49 and 37 kDa are
indicated. B, EMSA of eluates from the streptavidin-DNA
affinity column. Salt concentrations of the eluates are indicated as in
A. Radioactively labeled ANT2
338/
318 wt oligonucleotide
was used as a probe (lane P, probe only). A protein sample
taken before the addition of biotinylated oligonucleotide was used as a
positive control (lane C). The specific band is indicated by
an arrow.
338/
318 wt oligonucleotide (see
above) as the probe (Fig. 7B). Fractions eluted from the affinity resin with 0.2 or 0.4 M salt containing both p37
and p49 gave a single, strongly shifted band identical to that obtained with crude nuclear extracts. Thus, the isolated fraction appears to be
fully active. However, the formation of a strong DNA complex appears to
require both p37 and p49, because a bandshift was obtained with
fractions eluting at 0.2 and 0.4 M NaCl that contain
roughly similar amounts of p37 and p49, but not with the 0.6 M fraction that includes little p49.
DISCUSSION
546 to
235) and was divided by the transfection of deletion
constructs into two regions, which were designated A and B. DNase
I-protected sites are restricted to the B region, but both regions are
required for complete silencing. Silencing by the A and B regions
appears to be additive rather than synergistic. The combined A and B
regions completely suppress the activity of the heterologous HSVtk
promoter, showing that it functions as a general, position-independent
silencer (26).
binding to DNA is greatly enhanced
by GABP
(37, 38), and Elk-1 (39) and Sap-1 (40) require contact with
the serum response factor before contacting the serum response element.
Nuclear respiratory factor-2 (41), a GABP family factor that functions
as a transcriptional activator in the promoters of several OXPHOS genes
(see Ref. 42 for a review), was recently described. Although the ANT2
silencer element contains repeated CCT motifs, which could form the
core binding element of the GABP proteins, GABP
has a larger mass (58 kDa) (41) than the ANT2-binding protein (37 kDa). Furthermore, to
our knowledge, there is no report that the GABP proteins function in
any capacity than activating. Because of this, and because of our
inability to induce supershifts with antibodies against general Ets
proteins, we conclude that the ANT2 binding factors most probably do
not belong to the Ets family.
1237/
546) that appears to be responsible for the down-regulation
of ANT2 transcript during the growth of NIH3T3 cells to
confluence.2 However, the nature of this suppression and
its mechanism are not known; therefore, we cannot exclude the
interaction of this region with other elements within the ANT2 promoter
including the silencer described in this work.
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ACKNOWLEDGEMENTS |
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We thank Madeleine Kihlmark for providing rat liver nuclei and Zdenek Hodny for help with protein purification procedures.
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FOOTNOTES |
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* This study was supported by a grant from the Swedish Natural Sciences Research Council (to B. D. N.) and by a visiting scientist fellowship (to K. L.) from the Wenner-Grenska Samfundet.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.
Permanent address: Cancer Research Institute, Slovak Academy of
Sciences, SK-833 91 Bratislava, Slovakia.
§ To whom all correspondence should be addressed: Dept. of Biochemistry, Arrhenius Laboratories, Stockholm University, S-106 91, Sweden. Tel.: 46-8-162488; Fax: 46-8-153679; E-mail: buck{at}mail.biokemi.su.se.
The abbreviations used are: ANT, adenine nucleotide translocator; HSVtk, herpes simplex virus thymidine kinase; CAT, chloramphenicol acetyltransferase; wt, wild type; mut, mutant; nt, nucleotide; EMSA, electrophoretic mobility shift assay; PAGE, polyacrylamide gel electrophoresis; GABP, GA-binding protein; NF1, nuclear factor-1.
2 Unpublished observations.
3 A. Zaid, R. Li, K. Luciakova, P. Barath, S. Nery, and B. D. Nelson, unpublished observations.
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REFERENCES |
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