From the
The androgen receptor (AR) is a nuclear transcription factor
that is essential for development of the male urogenital tract. In the
current work, we have characterized the mouse androgen receptor
suppressor (mARS). A single, 20-base pair, region
(TCCCCCCACCCACCCCCCCT) was sufficient for suppression in
chloramphenicol acetyltransferase assays. Northern analysis indicated
that translational regulation is not necessary for the suppression.
Analysis of the AR mRNA half-life indicated that the mARS does not
affect AR RNA degradation. Gel mobility assays showed that the mARS is
bound by multiple proteins that can recognize single-stranded DNA and
RNA. In addition, differing proteins are expressed in distinct tissues.
Purification of some of these proteins has shown that a doublet of 33
and 35 kDa binds to the G-rich strand and that a 52-kDa protein binds
to the C-rich strand. Southwestern blots have confirmed that these
proteins are indeed recognized by the mARS. The results of these
experiments indicate that the AR 5`-untranslated region contains a
suppressor element that can be bound by multiple proteins. The mARS
appears to be acting either by altering transcription initiation or
blocking transcription elongation. Characterization of this suppressor
may provide insight into the physiological means by which the AR is
regulated.
The androgen receptor (AR)
The AR cDNA has been
cloned and characterized from several different species
(11, 12, 13, 14) . In addition, the
5`-flanking region of the gene has been cloned and shown to contain two
promoters. Promoter-1 is the 5`-most promoter and is located
approximately 1000 bp upstream from the ATG translation start site. For
the purposes of this paper, the 5`-most site of transcription
initiation from promoter-1 of the mouse AR will be numbered +1
(13) . Promoter-2 is located approximately 160 bp downstream
from promoter-1
(15) . The function(s) of the large region from
promoter-2 to the translation start site remained unknown until a novel
regulatory element in this region was discovered by this laboratory
(16) . It was shown using CAT assays that the 5`-untranslated
region of the AR contains a cis-acting suppressor element that was
termed the mouse androgen receptor suppressor (mARS). The mARS is
capable of down-regulating AR or thymidine kinase promoters if it is
inserted 3` to the site of transcription initiation. It was also shown
that the mARS is capable of binding to proteins using both footprinting
and bandshift assays. In the current work, we further characterize the
specific sequences of the mARS, the mechanism of mARS action, and the
proteins that bind to the mARS.
The observation that the mARS was bound by
single-stranded DNA-binding proteins led us to question earlier
indications that it could be bound as double-stranded DNA
(16) .
Initial observations indicated that the double-stranded mARS could also
bind to protein, but it seemed possible that this was an artifact.
Shifts would be created if unannealed single-stranded DNA was present
with the double-stranded DNA probes. To confirm that single-stranded
DNA was present with the double-stranded DNA probe, we electrophoresed
the single-stranded C-rich sense strand, the single-stranded G-rich
antisense strand, and the double-stranded mARS on the same gel, in the
absence of protein. Fig. 4 C shows that the three
different probes have different electrophoretic mobilities and that the
double-stranded probe does contain unligated single-stranded DNA.
Additional experiments indicated that all double-stranded probes
created in this way contained unligated single-stranded DNA (data not
shown).
In order to test to see if the mARS could be bound as
double-stranded DNA, increasing quantities of nuclear extracts were
added to a constant amount of double-stranded mARS. If the mARS is
bound as double-stranded DNA, then increasing amounts of shifted probe
should be seen. However, the increasing amounts of nuclear extracts did
not result in increasing amounts of shifted probe
(Fig. 4 D). This indicates that only residual amounts of
single-stranded mARS are binding in these experiments. This residual
single-stranded sense and single-stranded antisense mARS is again seen
below the major band of labeled, double-stranded probe in
Fig. 4D, lane1. Based on these data,
proteins appear to be able to bind in a sequence-specific manner to
either the sense or antisense single-stranded mARS DNA, but the
proteins appear to have little or no ability to bind double-stranded
mARS DNA.
The 5`-flanking region of the mouse androgen receptor has
been shown to contain a region that is capable of suppressing activity
from both the AR gene promoter and the heterologous thymidine kinase
promoter. The region appears to function by suppression of AR
transcription, since it is not dependent on translational effects and
does not facilitate RNA degradation. We have termed this region the
mARS.
Characterization of the mARS has shown that it is bound by
multiple, sequence-specific, proteins. Some of these proteins are
capable of binding the single-stranded C-rich sense strand of the mARS
DNA or RNA, and a different protein(s) binds to the single-stranded
G-rich antisense mARS DNA. Proteins capable of specifically binding to
the mARS are found in all tissues tested to date. However, these
tissues differ in quantity of binding and appear to contain different
proteins or protein complexes capable of binding to the mARS. Thus, it
is possible that the mARS may have tissue-specific functions.
Southwestern analysis and protein purification have revealed a 52-kDa
band that binds to the C-rich sense strand of the mARS and a doublet of
33 and 35 kDa that binds to the G-rich antisense strand. The complexes
are identical in size to those seen using unpurified nuclear extracts.
Interestingly, increasing amounts of purified liver extract caused the
lower band of the complex formed with the C-rich sense strand
(Fig. 7, lane3) to disappear, suggesting that
the two complexes may represent monomer and dimer complexes (data not
shown).
Information about the specific proteins that bind to the
mARS is of great interest. It is exciting to note that a second
suppressor with some sequence similarity has been shown to exist
upstream of the promoter-1 in the AR gene
(19) . Additional
experiments may determine the interactions of these two suppressors.
Reports exist in the literature of other single-stranded DNA binding
proteins. A very large family of evolutionarily conserved Y-box
proteins have been well characterized
(20) . Analysis of the
literature has revealed three potential proteins that bind to sequences
similar to the mARS. The SW2 protein is a negative regulator of
catalase gene expression
(21) , and the heterogeneous
ribonucleoprotein particle proteins K and A1 serve as positive
transcriptional elements for the c- myc gene
(22) . All
of these proteins function by influencing transcription from a location
that is 5` to transcription initiation. Protein sequence or antibodies
should help determine whether any of these proteins are involved in the
AR gene suppression.
The mechanism by which these proteins regulate
AR transcription is open to speculation. Two potential models of
transcriptional suppression are possible, based on the current data.
One model involves inhibition of transcription initiation by binding of
the protein(s) to the mARS. The DNA must then bend back so that the
mARS protein(s) could interact with other members of the transcription
complex to prevent transcription from initiating. Another model
(Fig. 9) involves blockage of transcription elongation by the
protein(s) binding to both the C-rich strand of the mARS and the G-rich
strand of the mARS. This model is favored by us for two reasons. First,
it accounts for the fact that in previous experiments
(16) the
mARS is capable of suppressing transcription only if it is located 3`
to transcription initiation. Second, it explains the need for two
distinct sets of single-stranded DNA binding proteins. It is possible
that as the DNA is transformed from a double-stranded state to a
single-stranded state during the process of transcription, the mARS
binding proteins may be able to interact with the DNA and form a
roadblock to transcription elongation.
In conclusion,
these studies further support the novel observation that the mouse
androgen receptor has a suppressor region located in the
5`-untranslated region of the gene. We have termed this region the mARS
and have shown that the mechanism of suppression is transcriptional.
The proteins involved in the suppression exhibit several interesting
properties. Further study of these proteins should yield additional
information about regulation of the AR specifically and about
transcription in general.
We thank Marceil Blexrud for excellent technical
assistance in conducting the experiments for this manuscript. We also
thank Dr. Leen Blok for discussions about and critical review of this
manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
is a nuclear
phosphoprotein that serves as a transcription factor whose function is
modulated by testosterone and dihydrotestosterone. The AR is essential
during development and maintenance of the male urogenital organs and
has been implicated in a number of diseases. It is expressed at a
relatively low level and has been shown to be regulated in a very
complex manner that is dependent on both tissue and developmental
effects
(1) . Recent studies have shown that the AR can be
regulated by androgens
(2, 3) , follicle-stimulating
hormone
(4) , epidermal growth factor
(5) , and the cAMP
pathway
(6, 7) . Tissue-specific factors regulating the
AR have not yet been defined, although an age-dependent factor has been
implicated in the expression of AR during development of the rat liver
(8) . The regulation of the AR occurs at several levels,
including translation, mRNA stability
(9) , and transcription
(10) . Further definition of the specific factors involved in AR
regulation should allow a better understanding of how the AR is
controlled during normal and diseased states.
Construction of CAT Expression Plasmids
mARS
mutants were constructed by annealing oligonucleotides that contained
SalI sites and ligating the double-stranded nucleotides into
the XhoI site in the thymidine kinase promoter containing
pBLCAT2 vector. Clones were sequenced using the fmol sequencing system
(Promega).
Cell Culture and Transfection
Procedure
GT1-7 cells are a mouse hypothalamic cell line
(kindly provided by Dr. P. Mellon see Ref. 17) and express low levels
of AR transcripts as shown by analysis of steady-state levels of RNA on
a Northern blot (data not shown). The cells were grown to confluency in
T-175 flasks using Dulbecco's modified Eagle's medium (with
pen/strep) with 5% charcoal-stripped fetal calf serum, 5%
charcoal-stripped horse serum. The cells were harvested using
trypsin-EDTA, washed (2 times), and counted. Cells (20
10
) were pelleted, resuspended in 400 µl of 0.1%
glucose/phosphate-buffered saline containing 15 µg of CAT vector
and 5 µg of RSV-
-GAL vector, and electroporated at 960
microfarads and 0.35 kV. The cells were incubated on ice for 7 min,
resuspended in 1 ml of phosphate-buffered saline with 4%
charcoal-stripped fetal calf serum, incubated at room temperature for 7
min, and resuspended in 42 ml of Dulbecco's modified
Eagle's medium with 5% charcoal-stripped fetal calf serum, 5%
charcoal-stripped horse serum. Ten ml of cell suspension were plated
into each of four 100 mM culture dishes and harvested 48 h
later with phosphate-buffered saline, 2 mM EDTA. Cells were
lysed in 100 mM Tris (pH 7.8), 0.1% Triton X-100, and the
resulting supernatant was assayed for CAT and RSV-
-GAL activity.
In brief, the CAT assay consisted of 50 µl of nuclear extract and
100 µl of Tris/Triton X-100 buffer placed in a scintillation vial
and incubated at 70 °C for 10 min. The mixture was cooled to room
temperature and overlaid with 100 µl of acetyl-CoA mixture (2.5
mM chloramphenicol, 0.1 M Tris (pH 7.8), 2 µl of
[
H]acetyl-CoA (5 Ci/mM)) added, and 2 ml
of Ecoscint O scintillation fluid. After acetylation, the
H
entered the organic phase and was counted a minimum of 5 times over
2-6 h. CAT activity was calculated by linear regression and
normalized to RSV-
-GAL values. Cells that were used in the RNA
half-life experiments were treated with actinomycin D at a final
concentration of 5 µg/ml and harvested 48 h after transfection.
RNA Preparations
Total RNA was isolated from
tissues or cells by lysing the cells with 3 M LiCl, 6
M urea. The samples were homogenized on ice for 1 min with a
Polytron and incubated on ice for 3-18 h. The samples were
centrifuged 20 min at 25,000 rpm. The pellet was resuspended in ES
(0.1% SDS, 0.2 mM EDTA), extracted with phenol once and
chloroform/isoamyl alcohol twice, and precipitated by adding one-tenth
volume of 3 M NaAc/HAc (pH 5.2) and 2.5 volumes of EtOH. The
RNA pellet was resuspended in water and quantitated. RNA prepared from
transfected cells was treated with 0.3 units of DNase I/20 µg of
RNA for 10 min at 30 °C.
RNA Separation and Blotting
RNA was separated by
electrophoresis on a 1% agarose gel that contained 2% formaldehyde
using 1 MOPS buffer (0.02 M MOPS, 0.005 M
NaAc, 0.001 M EDTA). The RNA was transferred to a Hybond-N
membrane (Amersham Corp.) and UV cross-linked to the membrane using a
Stratalinker (Stratagene). The probes were labeled by denaturing 125 ng
of DNA in 12 µl of water by heating to 100 °C for 3 min and
adding 4.5 µl of oligonucleotide labeling buffer (0.2 mM
dCTP, 0.2 mM dTTP, 0.2 mM dGTP, 50 mM
Tris-HCl (pH 7.8), 5 mM MgCl
, 10 mM
2-mercaptoethanol), 0.5 µl Klenow (1 unit), and 3 µl of
[
-
P]dATP. The probes were incubated for 45
min at 25 °C and purified over a Sephadex G-50 column. Blots were
prehybridized for 1 h at 42 °C with hybmix (45% formamide, 0.5%
SDS, 10% Denhardt's solution, 10 mM phosphate buffer,
15% dextran sulfate, and 150 µg/ml salmon sperm DNA). Probes (1
10
) were denatured by heating at 100 °C
for 3 min, added to the hybmix, and incubated at 42 °C for 18 h.
The blots were washed and exposed to x-ray film.
Bandshift Analysis
Ribonucleotides were
synthesized using the RNA phosphoramidites from Applied Biosystems
(Foster City, CA). Oligonucleotides and oligoribonucleotides
corresponding to the sequences illustrated were either used as
single-stranded DNA or single-stranded RNA or annealed to form a
double-stranded DNA product, P-end-labeled with T
polynucleotide kinase and purified on Biogel P-60 columns
(Bio-Rad). Binding reactions were carried out in 25-µl volumes
containing 2 µg of protein from the nuclear extract (unless
otherwise stated in the figure legends), 1 µg of poly(dI-dC),
varying amounts of unlabeled competitor, and 5
binding buffer
(4% glycerol, 1.0 mM MgCl
, 50 mM NaCl, 10
mM Tris (pH 7.5), 0.5 mM dithiothreitol). After
incubation for 15 min on ice, 4
10
cpm of probe was
added and incubated for an additional 30 min. Samples were
electrophoresed on a 6 or 8% acrylamide gel (38:1 cross-linking) at 125
V at room temperature with circulating cold water using 0.5
TBE
buffer (0.45 mM Tris base, 0.45 mM boric acid, 1
mM EDTA (pH 8.0)). Gels were dried and exposed to x-ray film
at -70 °C, except for where the gel was imaged on a
PhosphorImager (see Fig. 4 C) (Molecular Dynamics,
Sunnyvale, CA).
Figure 4:
The
mARS is bound by proteins as single-stranded DNA. A, bandshift
analysis of the single-stranded, C-rich, sense strand of the mARS. The
C-rich strand of the mARS (+861/+880) shown in Fig. 3 was
labeled and used as a probe. The molar excess is shown above the competitors. Single-stranded cold competitor is shown where
numbers represent starting and ending bp, and Ts are mutant
competitors. Addition of 2 µg of GT1-7 protein is shown as a
+ above the lanenumbers. B,
bandshift analysis of the single-stranded, G-rich, antisense strand of
the mARS. The G-rich strand of the mARS underlined in Fig. 1
was labeled and used as a probe. The molar excess is shown above the
competitors. Single-stranded cold competitor is shown where numbers
represent starting and ending bp and As are mutant
competitors. Addition of 2 µg of GT1-7 protein is shown as a
+ above the lanenumbers. C,
single-stranded and double-stranded bandshift probes have different
mobilities in the absence of protein. The C-rich strand, G-rich strand
and the double-stranded mARS were labeled for use as bandshift probes
( lanes1-3, respectively). The probes were
separated on a 6% acrylamide gel in the absence of protein. The exact
bases used are shown above the lanes. D,
bandshift analysis of double-stranded mARS. The double-stranded mARS
was labeled, and 5 10
counts were incubated with
the amounts of GT1-7 nuclear extracts shown above each
lane. The complexes were then separated on a 6% acrylamide
gel.
Protein Separation and Southwestern
Analysis
Protein was mixed with an equal volume of 2
Laemmli buffer (125 mM Tris-HCl (pH 6.8), 20% glycerol, 4%
SDS, 0.005% bromphenol blue, 5%
-mercaptoethanol) and incubated
for 5 min at 100 °C. Samples were loaded on a 14% Tris-glycine gel
(Novex), and run at 150 V for 1.5 h in 1
Tris-glycine SDS
running buffer (2.5 mM Tris-Base, 19.2 mM glycine,
0.01% SDS). Protein size was determined using Rainbow
high
molecular weight range markers (Amersham Corp.). Proteins were allowed
to renature by incubation in 50 mM Tris-HCl (pH 7.4) with 20%
glycerol for 1 h and transferred to nitrocellulose membrane in 12
M Tris, 96 mM glycine with 20% methanol using 275 mA
for 2 h. The membranes were incubated with TBST blocking buffer (10
mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.05% Tween 20, 2%
skim milk) for 1 h at 25 °C. Single-stranded end-labeled probe (1
10
cpm/ml, prepared as described above) was added
and allowed to incubate for 1 h at 25 °C. The blots were washed 2
times with TBST (10 mM Tris-HCl (pH 7.4), 150 mM
NaCl, 0.05% Tween 20) for 5 min each time and exposed to
autoradiography film.
Protein Purification
CNBr-activated Sepharose 4B
(Pharmacia Biotech Inc.) was coupled to single-stranded DNA using
multimers of the mARS C-rich strand (TCCCCCCACCCACCCCCCCT),
G-rich strand (AGGGGGGGTGGGTGGGGGGA)
, mutated C-rich strand
(TCTCCTCACTCACCTCTCCT)
or mutated G-rich strand
(AGGAGAGGTGAGTGAGGAGA)
according to manufacturer's
specifications. Protein was mixed with dI-dC in a 4:1 ratio in binding
buffer with no dithiothreitol (see ``Bandshift Analysis'')
and incubated at 4 °C for 10 min. The protein was applied to either
the C-rich or the G-rich column and eluted in 1-ml fractions with
increasing amounts of NaCl. Fractions were assayed for DNA binding
activity, and active fractions were applied to columns with the mutated
DNA to remove nonspecific single-stranded DNA binding proteins. The
flow-through was then applied to the C-rich or G-rich column again and
eluted with 1-ml fractions using increasing NaCl. The fractions were
assayed for DNA binding and protein content. Protein visualization was
achieved by silver staining
(18) after separation on a
Tris-glycine SDS gel as described above.
Mutating the mARS Results in Increased CAT
Activity
In order to better define the mARS and demonstrate that
the mARS is capable of suppressing a heterologous promoter, a set of
constructs was created in which a single copy of the mARS or a portion
of the mARS was inserted in the correct orientation in the pBLCAT2
vector. In addition, a set of control constructs was created that
contained a mutated mARS or portion of the mARS
(Fig. 1 A). The mARS was inserted 3` to the transcription
start site of the pBLCAT2 vector, which contains the thymidine kinase
promoter, since previous work in this laboratory had shown that the
mARS was not functional if placed 5` to the transcription start site
(16) . Fig. 1 B shows that the mARS-containing
construct +861/+880 was suppressed by 51% as compared with
its mutant partner +861/+880T. The two constructs that
contained only a portion of the mARS (+872/+880 and
+861/+868) had 39 and 11% suppression as compared with their
mutants. Previously
(16) , a construct that contained the
homologous AR promoter had shown a 58% reduction in CAT activity when
compared with a mutant construct in which three cytosines had been
changed to thymines in the mARS. This indicates that the mARS region
from +861 to +880 is capable of suppressing a heterologous
promoter in much the same manner as the homologous promoter is
suppressed.
Figure 1:
The mARS suppresses CAT activity from a
heterologous promoter. A, sequences of mARS that were inserted
into the pBLCAT2 vector. Construct names ending with a T were
mutated. Specific bases that were mutated are shown in
boldface. B, the bar graph shows percent activity of
each construct. Bars represent the average activity from two
separate experiments. The filleddiamonds are the
values from experiment one. The opencircles show the
values from experiment two. Mutated mARS constructs were assigned a
value of 100% as compared with the same unmutated construct. Duplicate
plates were used in each experiment. GT1-7 cells were transfected
by electroporation and extracts normalized to
RSV--GAL.
The mARS Reduces the Amount of AR-initiated RNA
We
next investigated the mechanism by which the mARS causes suppression.
Because the mARS is located in the 5`-untranslated region of the AR
gene, we asked the question, are the mARS effects prior to the level of
RNA translation? In order to investigate this, we transiently
transfected GT1-7 cells, harvested total RNA, and analyzed the
effect of the mARS. Using this method, changes in the amount of CAT RNA
should reflect mARS effects. Fig. 2, lane1,
shows RNA from GT1-7 cells transfected with vector alone, which
does not contain a promoter. The faint band indicates that some
nonspecific transcription initiation is occurring or that a small
amount of residual plasmid DNA remains. Lane2 shows
the amount of total RNA that an mARS-containing construct produced.
Lanes3- 5 show RNA produced by
constructs from which the mARS has been mutated ( lane3) or deleted ( lanes4 and 5).
The increased amounts of transcripts detected in the deletion
constructs clearly illustrate that these bands do represent the proper
gene products and that the mARS is capable of suppressing the amount of
transcripts. As a control, a construct that contained an RSV-driven
- gal gene was cotransfected with the CAT vectors, and the
blot was reprobed for
-Gal RNA. These data illustrate that the
mARS is exerting its influence prior to translation.
Figure 2:
Northern blot analysis of total RNA
harvested from mARS lacking and mARS containing cells. Total RNA was
prepared from GT1-7 cells harvested 40 h after they were
transiently transfected with mARS-containing constructs ( lane2) and mARS-lacking constructs ( lanes3-5). Constructs transfected are shown above the lanes. Lane1 ( CAT3) RNA
from the vector lacking any promoter insert. Lanes2-5 are RNA from pBLCAT3 vector with fragments of
mAR promoter inserted into the vector. The exact bases inserted are
shown above the lanes where +1 represents the 5`-most site of transcription initiation (13). The
-146/+971T ( lane3) contains three point
mutations in the mARS that have been previously described (18). Probe
was prepared as described using a 300-bp fragment of the pBLCAT3 vector
that extended downstream from the multiple cloning region to the
EcoRI site. CAT RNA shows amount of RNA produced by
constructs. -Gal RNA is from an RSV-
-Gal vector that
was simultaneously transfected and is shown as a control for
transfection and loading efficiency.
The mARS Does Not Affect RNA Turnover
In order to
further investigate the mechanism of mARS action, we determined the
potential effects of the mARS on the half-life of CAT RNA from
transiently transfected GT1-7 cells (Fig. 3). A CAT
construct that contained the native mARS (-146/+971) or a
mutated mARS (-146/+971T) was transfected, and RNA synthesis
was halted by the addition of actinomycin D for varying amounts of time
(Fig. 3 A). The autoradiogram was analyzed by
densitometry and normalized to the 28 S ribosomal band. As had
previously been shown, mutation of the mARS caused a marked increase in
total RNA. However, analysis of the rate of RNA degradation
(Fig. 3 B) indicates that native mARS had a very similar
rate of turnover as compared with the mutated mARS. This indicates that
the mARS is not functioning by facilitating RNA degradation.
Figure 3:
Northern blot analysis of RNA half-life
from suppressor containing (-146/+971) and suppressor
lacking (-146/+971T) constructs. A, Twenty-four h
after transient transfection into GT1-7 cells, actinomycin D was
used to block synthesis of new RNA. Time of treatment with actinomycin
D ( ActD) is shown above the lanes in hours. The lanes that contain RNA from mARS-containing
constructs are designated with a +, and the lanes that contain RNA
from mutated mARS constructs are designated with a -. CAT RNA
shows amount of construct RNA remaining after varying times of
actinomycin D treatment. The 28 S ribosomal band was used for
normalization. B, graph of RNA half-life from mARS-containing
and mARS-lacking constructs. The graph shows percent of mRNA on a log
scale verses time of actinomycin D treatment in hours. Opensquares represent the mARS containing points. Closedsquares represent the mARS-lacking
points.
Single-stranded mARS Is Specifically Bound by
Proteins
In order to characterize the proteins that had been
shown previously
(16) to interact with the mARS, we performed
bandshift analyses on the mARS DNA. Fig. 4 A shows that
when a single-stranded, C-rich, sense strand mARS DNA probe (+861
to +880) is incubated with nuclear extracts from the GT1-7
cells, a specific protein-DNA interaction occurs ( lanes2-5). In addition, an unlabeled, mutated,
single-stranded oligonucleotide (shown in Fig. 1) was not able to
compete ( lanes6-8), indicating that the
protein-DNA interaction is a sequence-specific interaction.
Fig. 4B shows that the single-stranded, G-rich,
antisense strand is also able to bind specifically to protein
( lanes2-5), although the bound complexes
appear to be different from those seen in Fig. 4 A.
Lanes6-8 indicate that an unlabeled, mutated,
single-stranded oligonucleotide is unable to compete for binding,
indicating that this complex is also dependent on specific DNA
sequences.
The mARS Binds Multiple Proteins
In order to
investigate the possibility that more than one protein-DNA complex is
forming, we performed an additional bandshift experiment with several
unlabeled competitors. Fig. 5shows bandshifts with two different
labeled probes. In lanes1-3, a single-stranded
probe consisting of the C-rich sense strand of the mARS was used. The
protein-DNA complex that was formed with this probe was competed away
by the C-rich sense strand of the mARS ( lane2).
However, the unlabeled single-stranded oligonucleotide, which
corresponds to the G-rich antisense strand, was not able to compete for
the complex ( lane3). In lanes4-6, a single-stranded probe consisting of the
G-rich antisense strand of the mARS was used. However, the protein-DNA
complexes formed using this probe were not able to be competed for by
the single-stranded C-rich sense strand of the mARS ( lane5). The single-stranded G-rich antisense strand was able
to compete for binding with the complexes ( lane6).
The difference in the shifted complex seen when lanes4 and 5 of Fig. 5are compared with
lane2 of Fig. 4 B appears to be due to
the increased amount of protein that was bound and slightly better
resolution of the gel in Fig. 4 B. The difference in the
mobility of the probe in lane5 of Fig. 5as
compared with lanes4 and 6 appears to be
due to the single-stranded antisense probe annealing to the unlabeled
single-stranded sense competitor. These results confirm that one
protein-DNA complex is able to bind to the single-stranded C-rich sense
strand of the mARS. A distinctly different protein-DNA complex binds to
the single-stranded G-rich antisense strand of the mARS.
Figure 5:
Bandshift analysis showing multiple
proteins bind to the mARS. Bandshifts were performed using 0.5 µg
of GT1-7 nuclear extract for lanes1-3 and 2.0 µg of GT1-7 nuclear extract for lanes4-6. Extracts were incubated with 5
10
counts of the mARS oligonucleotides and separated on a
6% acrylamide gel. Two different probes were radioactively labeled and
are shown above the arrows. Single-stranded
sense mARS ( mARS (C)) was used for lanes1-3, and single-stranded antisense ( mARS
(G)) was used for lanes4-6. Cold
competitors were used in 50-fold molar
excess.
The mARS Binds Proteins from Multiple Tissues
In
order to further characterize the suppressor proteins, we performed
bandshifts using nuclear extracts from multiple tissues.
Fig. 6A shows that the single-stranded C-rich sense
strand of the mARS interacts with proteins from all tissues examined.
The mARS proteins from most tissues formed similar complexes. However,
the complex formed with the nuclear extract from the submandibular
gland was smaller. The single-stranded G-rich antisense strand of the
mARS also interacted with proteins from all tissues examined
(Fig. 6 B) but with different complexes from those seen
with the single-stranded C-rich sense strand of the mARS. These data
demonstrate that the mARS proteins are found in many different tissues
and suggest that the two strands of DNA may interact with different
proteins.
Figure 6:
Proteins from multiple tissues bind to the
mARS. A, bandshifts of the single-stranded, C-rich, sense
strand of the mARS bound by proteins from multiple tissues. Bandshift
analysis was performed as described above using the C-rich sense strand
of the mARS as the labeled probe. Specific tissues that the nuclear
extracts were made from are shown above the gel.
B, bandshifts of the single-stranded, G-rich, antisense strand
of the mARS. Bandshift analysis was performed as described above except
that the labeled probe was produced using only the G-rich antisense
strand of the mARS. Specific tissues that the nuclear extracts were
made from are shown above the
gel.
Determination of the Size of the Proteins That the mARS
Binds to
To determine the size of the different mARS proteins,
Southwestern analysis and protein purification were performed.
Southwestern analysis allows one to analyze proteins that have been
size-fractionated on an SDS-polyacrylamide gel for their ability to
bind to a radioactive DNA probe. The proteins that bind to the
single-stranded C-rich sense strand of the mARS and the proteins that
bind to the single-stranded G-rich antisense strand of the mARS were
examined, and the results are shown in Fig. 7. Southwestern
analysis of unpurified protein from the liver demonstrated that the
sense strand binds two different sized proteins ( lane1). The major protein bound is 52 kDa, and the minor
protein bound is 90 kDa. A purified fraction from the liver was
obtained using a DNA affinity column specific for the sense strand of
the mARS. On a silver-stained gel, the purified fraction exhibited a
single protein band of approximately 52 kDa ( lane2).
This purified fraction retained the ability to form a complex with the
single-stranded C-rich sense strand of the mARS as shown by bandshift
analysis ( lane3). A Southwestern blot of unpurified
protein from the spleen using the antisense mARS as a probe is shown in
lane4. Two bands were observed at approximately 35
and 28 kDa. These proteins were then purified using a DNA affinity
column specific for the antisense strand of the mARS. Silver staining
revealed a doublet of 33 and 35 kDa ( lane5). The
purified proteins retained the ability to bind to the antisense DNA as
shown by a bandshift assay ( lane6). These results
indicate that at least one protein of approximately 52 kDa is capable
of binding to the sense strand of the mARS and that a doublet of 33 and
35 kDa is capable of binding to the antisense strand of the mARS.
Figure 7:
Protein purification and size
determination of the mARS proteins. Southwestern analysis ( lanes1 and 4) was performed using 2 µg of total
nuclear extract from liver ( 1) or spleen ( 4).
Single-stranded probes corresponding to the sense ( 1) and
antisense ( 4) mARS were labeled and used as probes. Purified
protein is shown as silverstained fractions from DNA affinity columns
( lanes2 and 5), which were specific for the
single-stranded sense ( 2) and single-stranded antisense
( 5) mARS. The fractions were generated by using increasing
amounts of NaCl to elute bound protein. Nonspecific DNA binding
proteins were removed by applying the extracts to DNA affinity columns
that contained single-stranded mARS with point mutations inserted.
Bandshifts ( lanes3 and 6) were performed as
described previously using the same fractions as shown in lanes2 and 5.
RNA Corresponding to the mARS Can Be Bound by
Protein
Because the mARS is found in the 5`-untranslated region
of the AR gene, we asked if RNA from the mARS could be bound by
protein. Fig. 8shows a bandshift experiment in which a
radioactively labeled RNA probe that corresponds to the mARS sense
strand from +861 to +880 is bound by protein(s) from the
GT1-7 cells ( lane2). One-hundred-fold cold
+861/+880 RNA competitor was able to compete for RNA binding
( lane3), but the mutated +861/+880 RNAU was not able to compete for binding ( lane4), indicating that the protein-RNA interaction is a
sequence-specific interaction. In order to determine if the same
proteins that bound the mARS RNA could bind to the mARS DNA, we used
the single-stranded, C-rich, sense strand of DNA and the
single-stranded, G-rich, antisense strand of DNA as cold competitors
for the RNA binding. The data indicate that the C-rich, sense strand of
DNA is able to compete for binding ( lane5) but that
the G-rich, antisense strand of DNA is not able to compete for binding
( lane6). This experiment indicates that the mARS RNA
can be bound by protein and that the protein may be identical to that
bound to the C-rich DNA strand of the mARS.
Figure 8:
Bandshift analysis of proteins binding to
the mARS RNA Bandshifts were performed using 0.4 µg of GT1-7
nuclear extract. Extracts were incubated with 5 10
counts of the labeled mARS oligoribonucleotide and separated on a
8% acrylamide gel. The sequence of the unmutated mARS RNA
( +861/+880 RNA) was UCCCCCCACCCACCCCCCCU and the
mutated mARS RNA ( +861/+880 RNA U) was
UCUCCUCACCCACUCUCUCU. The sequences of the cold competitor DNA is shown
in Fig. 3. Specific cold competitors used are shown above each
lane.
Figure 9:
Potential model of mARS action. Stylized
model of transcription attenuation by the mARS. The mARS bpC (mouse androgen receptor suppressor binding protein C) represents
protein(s) capable of binding to the C-rich sense strand of the mARS
DNA ( double- strandeddarkgrayribbon) or the mARS RNA ( single- strandedlightgrayribbon). The mARS bpG (mouse androgen receptor suppressor binding protein G) represents
protein(s) capable of binding to the G-rich antisense strand of the
mARS. Dashed, two- headedarrows indicate the potential for mARSbpG protein sequestration by mARS
RNA and subsequent transcription elongation. Single- headedsolidarrow with X represents
elongation blockage. Single- headedsolidarrow without X represents transcription elongation. The
largeoval represents the general transcription
apparatus.
Additional information about
the potential mechanism of mARS suppression is gained by the knowledge
that both the C-rich sense strand of the mARS DNA and the mARS RNA can
compete for the same protein(s). In this way, the formation of the
roadblock may be directly affected by the amount of AR RNA present
(Fig. 9). Other laboratories have shown that some single-stranded
DNA binding proteins are capable of binding to RNA and sequestering
proteins that negatively regulate transcription
(23) . This
could be a method for ensuring that transcription is maintained in a
situation where the protein is a transcriptional suppressor. Further
study of the mARS proteins should yield interesting details about this
newly emerging area of transcriptional regulation.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.