From The Prostate Centre at Vancouver General Hospital, Vancouver, British Columbia V6H 3Z6, Canada
Received for publication, October 6, 2000
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ABSTRACT |
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Genes uniquely regulated by the androgen receptor
(AR) typically contain multiple androgen response elements (AREs) that
in isolation are of low DNA binding affinity and transcriptional activity. However, specific combinations of AREs in their native promoter context result in highly cooperative DNA binding by AR and
high levels of transcriptional activation. We demonstrate that the
natural androgen-regulated promoters of prostate specific antigen and probasin contain two classes of AREs dictated by
their primary nucleotide sequence that function to mediate
cooperativity. Class I AR-binding sites display conventional guanine
contacts. Class II AR-binding sites have distinctive atypical sequence
features and, upon binding to AR, the DNA structure is dramatically
altered through allosteric interactions with the receptor. Class II
sites stabilize AR binding to adjacent class I sites and result in
synergistic transcriptional activity and increased hormone sensitivity.
We have determined that the specific nucleotide variation within the AR
binding sites dictate differential functions to the receptor. We have
identified the role of individual nucleotides within class II sites and
predicted consensus sequences for class I and II sites. Our data
suggest that this may be a universal mechanism by which AR achieved
unique regulation of target genes through complex allosteric
interactions dictated by primary binding sequences.
Despite intensive efforts investigating how the androgen receptor
(AR)1 uniquely regulates
androgen-responsive promoters, the fundamental mechanisms governing
this specificity are still not completely understood. The specificity
of steroid receptor response can arise through at least five stratified
regulatory mechanisms, including: availability of constituents in the
tissue such as hormones, corresponding receptors, and transcriptional
coregulator proteins; influence of proximal transcription factors bound
to the promoter; cooperative binding to multiple hormone response
elements; and sequence-specific DNA target recognition of individual
binding sites (reviewed in Ref. 1). For the majority of nuclear
receptors, specificity of response is acquired largely by sequence
specificity of the primary DNA-binding site. This level of specificity
is commonplace with nuclear receptors within the thyroid, retinoid,
estrogen receptor (ER) subfamily that bind as homodimers and
heterodimers as well as orphan receptors that bind as monomers to
distinctive extended DNA sequences (2-7). Recently, evidence has
emerged that steroid receptors can also use sequence specificity to a degree to help discriminate their binding targets (1, 8, 9). However,
steroid receptors appear to often have overlapping sequence specificity
within the primary binding site and must employ additional mechanisms
to achieve exclusive regulation of their cognate regulated promoters
(10-12). In the case of AR, a number of studies have shown that AR
binds cooperatively to multiple androgen responsive elements (AREs)
within native promoters, and this is likely a fundamental mechanism for
AR-specific transcriptional activation (13-18).
By definition, cooperative DNA binding is the binding of a protein at
one DNA site that facilitates the binding of other protein molecules at
additional distant sites. This can occur by stabilizing the binding of
the resulting complex or by making the additional binding sites more
accessible (19-21). Stabilization of a complex typically involves
protein-protein interactions and results in distant DNA sites brought
together, looping out the intervening DNA and resulting in an
interactive cluster of transcription factors. If cooperative binding
involves creating access to DNA sites, then it is normally achieved by
allosteric interactions between the protein and DNA, which alters the
DNA in a manner that either restricts the interaction of a repressor
complex or chromatin structure or permits the interaction of a positive
acting factor (22-24). Examples of these allosteric cooperative
interactions have been well documented for the structures of the
NFAT1·Fos·Jun·ARRE2, MAT alpha 2·MCM1·STE6, and
E1·E2 transcription regulatory complexes, which reveal
dramatic changes in protein conformation and DNA bending upon complex
formation (19-21). Typically, cooperativity is studied between one
species of protein that facilitates another protein species binding.
However, in the case of AR it is apparent that AR cooperates with other
AR molecules to bind to specific arrangements of multiple AREs within a
given promoter (9, 14, 15, 18).
The AR binds as a homodimer to individual inverted hexameric DNA
half-sites that are spaced by 3 base pairs. Studies have shown that the
highest affinity binding for AR and glucocorticoid receptor (GR) occurs
on the imperfect palindrome, GGTACAnnnTGTTCT (1, 12). In nature,
however, androgen-regulated promoters do not contain this optimized
element and instead possess numerous variations of this consensus
sequence (reviewed in Ref. 1). In general, these variants are of lower
affinity compared with the idealized element, but are instrumental in
discriminating preferential steroid receptor binding and
transcriptional activation (1, 8, 9). Thus there is a wide range of
sequences that AR can bind, typically containing the core requirement
of three out of four guanines contacts at
GGTACAnnnTGTTCT.
Sequence variation of the binding site not only affects DNA binding
affinity, but also separately affects transcriptional activity in a
manner that is discordant with affinity (1).
By extrapolation of crystallographic studies of the GR DNA binding
domain (DBD) with DNA, it is assumed that AR uses the same amino acids
within its DNA recognition AR DNA-binding sites display an exceptional amount of variation in
sequence and as isolated binding sites are typically of relatively low
affinity and low transcriptional activity. However, it is clear from
studies with a number of promoters that physiological levels of
response by AR are achieved in conjunction with sets of AREs that are
organized into the appropriate spatial architecture (14, 17, 18). How
these AREs interact mechanistically in a cooperative manner for DNA
binding and in a synergistic manner for transactivation is not
understood. In this study we have identified and characterized the
nature and mechanism of cooperative binding of AR on androgen-regulated
regions of the probasin promoter and the PSA enhancer. We have
identified and characterized a subclass of AREs with distinctive
sequence variation that AR binds in an allosteric manner altering the
local DNA structure. These sites serve as a focal point that
facilitates binding to adjacent AREs of more conventional sequence
identity. This stabilized complex provides high levels of
transcriptional activation by AR and increased AR sensitivity to
androgen concentration. This phenomenon appears to be a universal
mechanism by which AR interacts cooperatively with DNA to enhance gene
expression levels. This furthers our understanding of how
androgen-responsive genes are regulated and how the precise nucleotide
sequences AREs dictate unique functions.
Recombinant AR DNA Binding Domain Fusion Protein--
The rat
AR-DBD (amino acids 524-648) was inserted into the EcoRI
and BamHI sites of pTrcHisC (Invitrogen, San Diego, CA) to construct the six-histidine residue N-terminal fusion protein AR-HisTag. The recombinant protein was expressed in Escherichia coli JM109 and purified on nickel-NTA-Sepharose according to the manufacturer's instructions (Qiagen Corp., Germany) except for the
following modifications: the column was washed once with 50 mM NaH2PO4, pH 8.0, 300 mM NaCl, 20 mM imidazole, and twice with 50 mM NaH2PO4 pH 8.0, 20 mM imidazole. Protein was eluted off the Ni-NTA column with
20 mM Hepes (pH 7.9), 100 mM KCl, 20%
glycerol, 1 mM DTT, and 250 mM imidazole.
Protein concentrations were determined by Bradford Assays.
Full-length Androgen Receptor--
Nuclear extracts were
prepared from HeLa cells containing the stable transfection FLAG tag
full-length androgen receptor (HeLa-fAR), a kind gift from Michael
Carey, UCLA (18). Cells were incubated 24 h in Dulbecco's
modified Eagle's medium containing 10% fetal bovine serum and 1 nM R1881 (PerkinElmer Life Sciences) prior to extraction by
a method modified from a previous study (18). Briefly, cells were
washed twice with phosphate-buffered saline, harvested, and centrifuged
10 min at 1000 rpm. The cell pellet was washed once with ice-cold
phosphate-buffered saline and resuspended in 5 volumes of buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT), centrifuged 5 min at
1850 × g and resuspended in 3 volumes of buffer A. After 10 min on ice the cells were homogenized with 10 strokes in a
Dounce homogenizer, and the nuclei were pelleted by centrifugation for
15 min at 3300 × g. Nuclei were resuspended in 1 volume of buffer C (20 mM HEPES, pH 7.9, 25% v/v glycerol, 20 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride,
0.5 mM DTT) and incubated at 4 °C for 30 min with gentle mixing. The nuclear extract was obtained by centrifugation at 21,000 × g for 30 min.
Oligonucleotides--
Fragments of the rat probasin promoter
were generated by PCR amplification using the DMS Methylation Protection and Methylation
Interference--
Methylation protection of either the rat probasin
promoter and the human PSA enhancer regions was performed by a modified protocol.2 In brief,
histidine-tagged AR-DBD (7.2 µg, 13.6 µM) was incubated at room temperature with 2.0 µg of poly(dI-dC) (Amersham Pharmacia Biotech) and DNA binding buffer (DBB: 20 mM HEPES, pH 7.9, 100 mM KCl, 10% glycerol, 1 mM DTT). To each
reaction 350,000 dpm (26.5 fmol) of
32P-single-end-labeled DNA probe was added, and the binding
reaction was brought to equilibrium at room temperature. To methylate
the protein-bound DNA probe, dimethyl sulfate (DMS) was added to a final concentration of 19 mM and incubated at room
temperature for exactly 2 min. The methylation process was stopped by
loading the reaction onto a 5% (29:1) polyacrylamide gel containing
0.5× TBE while it was running at 16 V/cm at room temperature. DNA
treated in the same manner, but without AR-DBD added, was used as a control.
After separation by polyacrylamide gel electrophoresis, bands
indicating protein-bound and protein-free probes were excised and the
DNA was eluted. The methylated DNA was cleaved using 1 M
piperidine, and the denatured fragments were separated on a 6% (29:1)
denaturing polyacrylamide gel containing 1× TBE and 8.3 M
urea. Gels were dried and autoradiographed, and the developed images
were scanned using a Hewlett-Packard 6300-dpi resolution scanner. Bands
were quantitated and compared using ImageQuaNT (5.0). Methylation
interference of the G-1 and ARE2 fragments of the probasin promoter
involved premethylating the DNA fragments before carrying out the
binding reaction. The 32P-single-end-labeled DNA probe (200 fmol) in DMS buffer (50 mM sodium cacodylate, pH
8.0, 10 mM MgCl2, and 1.0 µg of calf thymus DNA) was incubated with 45 mM DMS for exactly 2 min at room
temperature. The reaction was stopped with DMS Stop Solution (1.5 mM sodium acetate, pH 7.0, and 1.0 M
Gel Mobility Shift Assays--
For gel shift analysis of
relative binding affinities, radiolabeled fragments of the probasin
promoter were obtained by PCR amplification in the presence of Transcriptional Activation--
Luciferase reporter plasmids
were created by introducing BamHI/HindIII ends to
each fragment of the probasin promoter by PCR and cloning into the
corresponding restriction sites of pTK-luc (ATCC). LNCaP cells were
then transfected with 0.2 µg of reporter plasmid, 1.2 µg of rat
androgen receptor in pRcCMV (Invitrogen), and 8 ng of the renilla
expression plasmid pRLTK (Promega) using Lipofectin (Life
Technologies). Cells were incubated for 22 h in 24-well plates
containing either 5% charcoal-stripped medium alone or with R1881
(PerkinElmer Life Sciences), ranging in concentration from 0.001 to 5 nM. Cells were washed and harvested with passive lysis
buffer (Promega). Luciferase activity of 20-µl aliquots of lysate was
determined using the Dual-Luciferase Reporter assay system (Promega) on
a luminometer (Berthold, Germany). Luciferase activity was normalized
for transfection efficiency using renilla activity. Experiments were
done in triplicate, averaged, and expressed both in relative luciferase
units (RLU) and as -fold induction.
DMS Protection Reveals Atypical Androgen Receptor Binding
Sites--
The AR has been shown to bind to the probasin promoter in a
highly cooperative manner (14). Two androgen response elements (AREs)
have been previously characterized in the proximal promoter (15). These
AREs, referred to as ARE1 and ARE2, are separated by 80 base pairs. If
either ARE1 or ARE2 is mutated to a nonbinding form, then binding to
the other ARE is impaired and transcriptional activity is greatly
compromised (15). In other data, in which the intervening sequence has
been deleted, transcription activation by AR is greatly impaired
(9). Therefore these data suggest that AR binds cooperatively to
the probasin promoter in a functionally significant manner and that
this cooperative binding is reliant on the integrity of the intervening
sequence (9). To investigate the nature of AR's interaction with the
probasin promoter, we employed chemical probe methodologies to
investigate the base-specific contacts of AR. To initiate these
studies, we used recombinant his-tagged purified AR-DBD extending from
amino acids 524 to 648 of the rat AR and DMS-based methylation
interference (MeI) to examine the contacts made to guanines by AR. In
this methodology the DNA is premethylated prior to binding to the
protein of interest. If the methylation of a specific guanine
interferes with DNA binding, then the DNA molecule segregates into the
unbound or free fraction following gel shift separation and is missing
in the corresponding bound fraction. On the other hand, if a methylated
guanine does not interfere with binding of the protein, then the DNA
molecule appears at equal concentrations in the bound and free
fractions. This methodology has been instrumental in indicating
DNA-protein contacts to guanines of many bimolecular interactions.
However, due to the cooperativity between AR-DBDs bound to the multiple AREs on the probasin promoter, this approach did not produce an interference pattern on the intact promoter, whereas isolated AREs do
show MeI patterns (data not shown). This result is most likely, because
the prevention of one guanine contact was compensated for by contacts
at additional sites and stabilized by protein-protein interactions, a
characteristic result we have found of MeI assays throughout these
studies when applied to highly cooperative binding sites.
Therefore, we turned to methylation protection (MeP)-based assays in
which the DNA·protein complex is formed first and then exposed to DMS
to methylate guanines that are not involved in hydrogen bonds with the
protein.2 Protein·DNA complexes are then isolated by
electrophoretic mobility shift assay, the bound fraction is isolated
and eluted, and methylation patterns are analyzed on a denaturing gel
in comparison to control DNA that has not been bound by protein to
improve the sensitivity and specificity of DMS protection-based
assays.2 DMS is an ideal chemical modifier probe, because
it methylates all guanines equally on free DNA and, therefore, the
cleavage patterns produce a uniform ladder of guanines within the
sequence. Typically, this approach of MeP provides parallel information to MeI with respect to specific guanine contacts by DNA binding proteins, because they are uniquely protected from DMS attack when the
protein is bound and noncontacted guanines are methylated with the same
efficiency as in the unbound DNA control. However, if the DNA is
locally distorted by binding of a transcription factor, guanines can be
hypersensitive to DMS attack (40, 41).
The results from MeP assays of the intact probasin promoter confirmed
the previously identified ARE1 and ARE2 and illustrated the expected
protection pattern of conserved guanine contacts of the nuclear
receptor superfamily with no change in DMS sensitivity to the
noncontacted guanine residues within the binding sites (
Because of the unusual nature of these AR-binding sites containing
hypersensitive guanines in the third position of the half-site, we have
referred to these individual sites within the probasin promoter as G-1
and G-2. The unusual binding patterns of G-1 and G-2 with respect to
DMS hypersensitivity have lead us to classify these AR interactions as
class II type binding in contrast to the typical binding pattern
hereafter called class I type binding as demonstrated on ARE1 and ARE2.
Binding of Class II Sites Is Independent of Adjacent Class I
Sites--
The apparent binding of the AR-DBD to the class II G-1 and
G-2 sequences could be a function of weak interactions that are buttressed by the two class I AR-DBD homodimers bound to ARE1 and ARE2,
respectively. If these AR dimers physically interact in a cooperative
manner, the intervening DNA must assume a loop structure. Therefore, it
is possible that the interactions of class I AR dimers on ARE1 and ARE2
could alter the DNA conformation such that further interactions of the
class I dimers to distant sites are permitted. This could result in DNA
torsional strain causing the guanine hypersensitivity seen on the G-1
and G-2 regions. Alternatively, G-1 and G-2 could be bound
independently by two additional AR homodimers and the hypersensitivity
of these sequences may be intrinsic to the nature of AR-DBD binding to
this complex promoter with four interacting sites. To distinguish
between these two possibilities the four individual binding elements
were tested in isolation for their ability to bind to AR. These four
individual fragments all bound AR-DBD and were each further
characterized for DNA binding affinity by Scatchard analyses. These
analyses were performed using a constant amount of purified AR-DBD and increasing concentrations of radiolabeled DNA in an electrophoretic mobility shift-based assay to avoid inconsistencies caused by protein
dilution over large ranges of concentration. These data demonstrated
that the relative DNA binding affinities of the AR-DBD were 6.3 nM for ARE2, 11 nM for ARE1, 14 nM
for G-1, and 18 nM for G-2. Although the isolated class I
sites have slightly higher affinity than the class II sites of the
probasin promoter, all elements displayed appreciable binding activity.
Because it was demonstrated that G-1 and G-2 were independent binding
sites of AR-DBD homodimers, we next wanted to determine whether the DMS
hypersensitivity seen on the G-1 and G-2 elements was an intrinsic
property of the individual G-1 and G-2 primary DNA sequence or if
hypersensitivity was a result of the multiple complex interactions in
the context of the promoter containing AREs interacting in a highly
cooperative manner. Using the MeP assay as described above, it was
found that the relative pattern of protection and hypersensitivity of
AR binding to the isolated G-1 and G-2 elements was highly similar to
the pattern observed when this technique was applied to the entire
probasin promoter or its fragments (Fig.
3). Full-length flag-tagged AR prepared from a stably transfected HeLa cell line also demonstrated the hypersensitive pattern seen with the AR-DBD (data not shown) (18). To
investigate further how the hypersensitive guanines were involved in
binding to the class II sites, we performed MeI in which the DNA is
premethylated as discussed above. In the MeI analysis we found that the
premethylation of the guanines prevented binding by AR to both class I
and class II binding sites (Fig. 3). We interpret this intriguing set
of findings to mean that the guanines initially involved in hydrogen
bonds with AR in the recognition of the class II binding sites, as they
are in class I sites. However, after this initial recognition
interaction, there is an allosteric conformational change to the class
II binding sites in which the interaction with guanines is altered and
becomes hypersensitive to methylation. In summary, these results
demonstrate that G-1 and G-2 can bind AR independently, likely in an
allosteric manner and that the specific nucleotide sequence of these
class II elements dictates the hypersensitivity to guanine methylation
within their half-sites.
Class II Type Binding Sites Nucleate the Cooperative Binding to
Adjacent Class I Sites--
To investigate the functional role of
these class II binding sites in terms of cooperative binding by AR to
the probasin promoter, we created a series of DNA fragments of various
regions of the promoter containing combinations of ARE1, G-1, ARE2, and
G-2 in their native context. We analyzed combinations of the class I and class II elements to determine the relative contribution of individual binding sites to cooperativity as judged by complex formation as a function of increasing DNA concentration as was performed above for singular elements (Fig.
4) (39). These analyses revealed that the
combination of ARE1 and G-1 (with Kd values of 11 and 14 nM in isolation, respectively) resulted in a
cooperative interaction that half-saturated the DNA at more than
a 10-fold lower concentration (0.9 nM). Similarly ARE2 and G-2 interacted in a cooperative manner to shift the rate of complex formation approximately an order of magnitude lower in concentration. The promoter fragment containing G-1, ARE2, and G-2 interacted with the
highest degree of DNA binding cooperativity resulting in half-saturated
binding at 0.11 nM, which is over 50 times stronger binding
than ARE2, the individual element with the highest DNA binding
affinity. Interestingly, the addition of ARE1 to this extremely
cooperative DNA fragment of G-1, ARE2, and G-2 resulted in weakening
the overall strength of the DNA binding complex with all four sites
occupied to ~1.2 nM to achieve half saturation. Comparable binding results were observed with increasing AR-DBD protein
concentration in the presence of a constant amount of DNA. In thorough
examination of AR binding to this promoter containing all four binding
sites, it was apparent that the binding curve was biphasic. The order
of complex formation appears to be occupation of G1-ARE2-G2 at low
concentrations of protein, followed by additional binding to ARE1 as
protein concentration increases. These results indicate that class II
elements are instrumental in providing the dramatic level of DNA
binding cooperativity seen on the probasin promoter. It is also clear
that the four individual elements interact in a complex manner in which
G-1 can interact individually with ARE1 or ARE2-G2. However, if both
ARE1 and ARE2-G2 are present on the same DNA fragment, then the
interaction between G-1 and ARE2-G2 is weakened, presumably, through an
interaction with ARE1, thereby affecting the overall stability of the
complex.
Contribution of Class I and Class II Sites to Transcriptional
Activation on the Probasin Promoter--
To determine whether class II
AR-binding sites contribute to the transcriptional activity of the
probasin promoter in response to hormone induction, we tested
combinations of ARE1, G-1, ARE2, and G-2 as well as the individual
class I and class II elements for transactivation potential in the
presence of increasing concentrations of the synthetic androgen R1881
in LNCaP cells. All individual class I and class II sites displayed low
levels of transcriptional activity (less than 2000 RLUs) that were
maximal at 0.5 nM R1881corresponding to levels of 12-, 23-, 25-, and 33-fold, for G-1, ARE1, G-2, and ARE2, respectively (Fig.
5a). Combining class I and II
sites resulted in an increase in the sensitivity to hormone
concentration maximal at ~0.05 nM R1881, but the
magnitude of transcriptional activation of ARE1-G1 and ARE2-G2 was at
most additive of the activity of the individual elements (Fig.
5b). The addition of G-1 to the ARE2-G2 binding element
resulted in dramatic synergistic transcriptional activation to a
maximum of 169-fold induction (~20,000 RLUs), consistent with the
increase in DNA binding cooperativity when G-1 is combined with ARE2-G2
(Fig. 5c). The addition of ARE1 to the highly cooperative
DNA binding region containing G-1, ARE2, and G-2 resulted in a biphasic
curve from 0.01 to 0.05 nM followed by a sharp increase in
level of activation to culminate in a 5-fold enhanced transcriptional
response (~100,000 RLUs) compared with G-1-ARE2-G2 alone and more
than 30-fold greater than any individual element (Fig.
5c).
The higher relative transcriptional activity of ARE1-G1-ARE2-G2 is in
contrast to the cooperative binding data, which demonstrated that the
addition of ARE1 decreased the overall binding strength of the complex.
Together these data suggest that a biphasic curve of activity arises
from the highly cooperative DNA binding complex at low concentrations
of R1881, presumably corresponding to concentration of activated
AR in the nucleus, whereas, at high levels of R1881 (activated AR), the
inclusion of ARE1 is able to provide highly synergistic levels of
transcriptional activation. In summary, these data suggest that unique
combinations of class I and class II sites are required for maximal
transcriptional activation by AR and that each class I and class II
element plays a unique functional role in transcriptional activation,
DNA affinity, and complex stability that in a composite fashion
culminates in a high level of transcriptional induction. Furthermore,
these results illustrate that androgen-regulated promoters can respond
in a rheostat fashion to hormone concentration generally reflecting the
degree of cooperative DNA binding to the given promoter.
Characterization of class II Binding Sites of AR on the PSA
Enhancer--
The AR has been reported to bind cooperatively to a
number of androgen-regulated promoters (9, 17, 18, 38). To determine whether the principles of class I and class II AR binding sites that we
have documented for the probasin promoter also applied to other
cooperative promoters, we investigated the characteristics of AR
binding to the PSA enhancer. This enhancer has recently been
characterized in detail, identifying four AR binding sites, referred to
in the earlier study as V, IV, III, and IIIa, which interact in a
cooperative manner, and each site contributes to full androgen
induction (18). To determine whether this cooperatively bound enhancer
of the PSA gene contained class II binding sites for
the AR, we used DMS protection assays as described for the probasin
promoter. This analysis demonstrated that two of the previously
characterized AREs, V, and IIIa, within the PSA enhancer had the same
distinctive features of hypersensitivity reported above for the
probasin promoter (Fig. 6). The high
affinity PSA enhancer site III was similar to a class I element. The
PSA element, IV, was similar to the class II sites but does not possess
enough guanines to confidently classify this element. From this
analysis of the PSA enhancer it was evident that additional guanines
immediately flanking the half-sites present only in the PSA enhancer
sequence showed hypersensitivity to DMS. Therefore, this analysis
provides additional information that the structural distortion induced by AR binding to these class II sites may extend over an area of at
least 17 base pairs.
Consensus Sequence for Hypersensitive class II Sites of AR--
To
determine the consensus features of class II binding sites of AR, the
elements that displayed the characteristic hypersensitive guanines
within the probasin promoter and PSA enhancer were aligned to identify
similarities. The numbering scheme for this analysis refers to the
reference strand with the base location indicated in reference to the
central nucleotide of the palindrome as 0 and ascending toward the
3'-half-site and descending toward the 5'-half-site (Fig.
7). This comparison of AR-binding sites
within the PSA enhancer and probasin promoter derived a unique
consensus of distinctive features that distinguish class I and class II sites. Both class I and II sites have a Cys at Sequence Determinants for DMS-hypersensitive AR-binding
Sites--
Because class II sites appeared to have distinctive
consensus nucleotides and demonstrated atypical AR binding patterns in isolation, we next investigated the nucleotide determinants of the
class II sites that result in the allosteric structural changes upon AR
binding. To do so, we focused on the G-1 binding site from the probasin
promoter using site-directed mutagenesis followed by analysis of the
DMS protection patterns after binding of AR. The distinctive features
of the class II type binding sites in comparison to class I sites,
other than the aforementioned guanines in the third position of the
half-site, is that the spacer region was relatively AT-rich in
comparison to class I type AREs. In addition it was noted that class II
sites had a Thr 6 base pairs 5' of each half-site and that in the
3'-half-site the sixth nucleotide was a purine in contrast to the
consensus pyrimidine in this location of class I sites. These
individual mutations were tested for their potential contribution to
hypersensitivity as before (Fig. 8). The
results from DMS protection assays demonstrated that conversion of the
Thr at position
In summary, these analyses clearly demonstrate that AR binds to two
structurally and functionally distinct classes of AREs that are
directed by allosteric interactions of the binding complex. AR binding
to class I sites have been previously recognized and use conventional
nucleotide contacts used by most nuclear receptors. These are of low
DNA binding affinity and transcriptional activity in isolation. In
contrast, class II binding sites result in DNA structural alterations
that display DMS hypersensitivity to particular guanines expected to be
contacted, but are also of low affinity and transcriptional activation
as individual elements. Unique combinations of class I and class
II sites result in dramatic cooperative DNA binding affinity and a
highly synergistic effect upon transcriptional activity. This complex,
composite function that facilitates cooperative DNA binding and
achieves a higher level of transcriptional response is dictated by the
primary nucleotide sequence to which the AR binds.
The AR can bind to a large repertoire of sequence variants that
resemble an inverted palindromic repeat spaced by 3 base pairs. The
sequence identity of the individual AR binding sites within natural
promoters may be incidental or may impart function to the receptor
response. In a previous analysis of AR binding sites, we demonstrated
that some of the nucleotide variation within AR binding sites aid in
discrimination of binding by GR and PR (1). Likewise, some nucleotide
substitutions within the binding site affected transcriptional activity
in a manner that was discordant with their effects on DNA binding
affinity (1, 34-36). Thus, the nucleotide sequence of an ARE can
separately function to help discriminate both preferential steroid
receptor binding and transcriptional activation. Our previous study
also provided evidence that AR binds to a nearly palindromic DNA
binding site in an asymmetrical manner that extends 5 base pairs away
from the previously recognized target. Together this suggests that AR
binds to the DNA in an allosteric manner and that the nucleotide
sequence of the response element imparts functional information to the
AR-protein to which it is bound.
In the present study we have investigated the nature in which AR binds
cooperatively to multiple AREs within androgen-regulated promoters.
This study has led to the identification of two distinct classes of AR
binding sites. Class I sites display conventional binding patterns in
terms of guanine contacts. Class II are distinguished by their unusual
patterns of hypersensitivity seen by DMS protection assays in response
to interaction with AR. Class II sites function to facilitate
cooperative binding to adjacent class I sites, presumably through local
DNA structural alterations. Both class I and class II sites in
isolation are of low binding affinity and low transcriptional activity,
requiring over 0.5 nM R1881 for full induction in transient transfection assays. Whereas, specific combinations of class I and
class II sites found in androgen-regulated promoters synergistically increase DNA binding affinity, hormone sensitivity, and levels of
transcription in comparison to singular AREs. The degree of DNA binding
cooperativity most strongly correlates to the high levels of
transcriptional induction at very low levels of hormone.
Summarizing the data obtained in this study and other studies of the
probasin promoter, it is most likely that G-1, ARE2, and G-2 interact
cooperatively to increase the stability of the DNA-binding complex and
correspondingly are activated at relatively low levels of hormone
concentration. At higher levels of androgens, this most highly
cooperative complex for DNA binding may be poised to stabilize binding
of an additional AR dimer to ARE1. The additional interaction with ARE1
decreases the physical stability of the overall complex, but provides
maximal transcriptional activation.
Our working hypothesis is that the AR dimer bound to ARE1 may interact
most efficiently with coregulator proteins to induce androgen-dependent transcription from this promoter,
whereas ARE2 in this context is more integrally involved in
protein-protein interactions with AR molecules bound to G-1 and G-2. In
the highly stable G-1·ARE2·G-2 complex, the interaction site
for AR coregulators of the three homodimers may be less accessible.
Therefore, when ARE1 is mutated, transcriptional activity is lost
possibly due to a lack of efficient coactivator interactions with the
remaining complex, whereas when ARE2 is mutated, transcriptional
activity is greatly decreased due to impairment of DNA binding
cooperativity (14). Thus each of the four elements imparts a different
function that cumulates in a high level of transcriptional activity. In contrast, when these elements are bound by AR in isolation of other AR
sites, they exhibit a low level of transcriptional activity, and only
at high levels of hormone, compared with the highly cooperative DNA-binding complexes, which are activated at much lower concentrations of hormone. We interpret this shift in the concentration of hormone induction to be a function of titration of activated AR to the nucleus.
Highly cooperative AR complexes would require a correspondingly lesser
amount of activated AR for activation of transcription, as paralleled
by the cooperative DNA binding data. This study demonstrates that the
DNA-binding sites respond in a sequence- and
concentration-dependent manner to the binding of AR and
that AR reacts differentially, in terms of function, to binding DNA dependent upon the nucleotide sequence. This evidence strongly suggests
that the AR interaction with DNA is not an inert binding process or
docking station but is in fact mutually allosteric in a functionally
significant manner. This model gives credence to the hypothesis that
sequence variation of AREs is not incidental in natural promoters, but
imparts function to the response to hormone induction in a complex
composite manner.
In examination of other androgen-regulated promoters that interact with
the AR in a cooperative manner, we have identified putative class II
binding sites, which suggests that this is a universal mechanism by
which the AR binds to multiple AREs in a cooperative manner. In
building on previous work and the studies reported here we hypothesize
that DNA sequence dictates function to the AR that is mediated by
sequence-specific allosteric interactions. These mechanisms, when used
in combination, may provide for a rheostat of androgenic response both
in terms of amplitude and duration, thereby allowing a specific gene to
customize the transcriptional response to a hormone stimulus in a
promoter/gene-specific fashion.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix (CGSCKVFFKRAAE) to form similar
hydrogen bonds to nucleotides within the half-sites for
sequence-specific recognition (25, 26). The most fundamental contacts
obligatorily conserved throughout the nuclear receptor family are
between the first lysine, which bonds to the second guanine
GGTACA of the half-site and the arginine, which binds to a
conserved G base paired to cytosine GGTACA (26-28). These anchoring base contacts are universal within the nuclear receptor family and have been demonstrated for a large number of receptors by a
variety of techniques (4, 29, 30). Further discrimination between
half-sites is achieved by a specific van der Waals contact between the
valine and the thymidine base paired to A, GGTACA for AR,
GR, and progesterone receptor (PR) (26-28). The ER binds to an
inverted repeat spaced by 3 base pairs with the sequence, AGGTCAnnnTGACCT, by discrimination with its DNA recognition
-helix (CEGCKAFFKRTIQ) using its unique glutamate in a bond to
cytosine base paired to the characteristic guanine of an estrogen
response element AGGTCA (31). The alanine in the DNA
recognition
-helix restricts ER from binding to a sequence
containing an AGGACA (7, 32, 33). Although the core
consensus half-sites extend for 6 base pairs, other nucleotides are not
known to engage in base-specific bonds, but the conservation of these
nucleotides imply functional significance. Of interest is the
observation that alteration of the sixth nucleotide to a T or G
increases transcriptional activity despite lowering DNA binding
affinity for AR and PR (1, 34-36). In other studies, it has been shown
that particular nucleotides are discriminated against due to
incompatibility of a receptor DBD structure and a given sequence, which
provides another level of discrimination (1, 7, 25, 37). Therefore,
there are general core nucleotide requirements of a steroid response
element, but there is also nucleotide variation within the binding site that provides function apart from the receptor binding affinity to the DNA.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
286 to + 28 portion of
the probasin promoter inserted BamHI/HindIII into
pBluescript (Stratagene, La Jolla, CA) as a template. The fragments
studied are as follows: ARE1 G-1 ARE2 G-2 (
269 to
77), G-1 ARE2 G-2
(
229 to
77), ARE1 G-1 (
269 to
164), ARE2 G-2 (
150 to
77),
ARE1(
269 to
210), ARE2 (
150 to
105), G-1(
229 to
164), and
G-2 (
121 to
77). In addition, six sets of complementary 29-mers
corresponding to the wild-type G-1 binding site and five mutant forms
were synthesized: wild-type G-1: 5'-CTTATTAGGGACATACCCACAAAT-3';
A195C: 5'-CTTAATAGGGACATAAAGCCCCCAAATAA-3'; C197T:
5'-CTTAATAGGGACATAAAGCTCACAAATAA-3'; G-207A:
5'-CTTAATAGGAACATAAAGCCCACAAATAA-3'; T215G:
5'-CGTAATAGGGACATAAAGCCCACAAATAA-3'; Spacer mutant:
5'-CTTAATAGGGACACGGAGCCCACAAATAA-3'. Oligonucleotides were
synthesized by the Nucleic Acid and Protein Services, University of
British Columbia, Vancouver, Canada.
-mercaptoethanol), and the DNA was ethanol-precipitated. The
methylated probe was then used in a protein-binding reaction with
AR-DBD as described in the methylation protection protocol. The bands
indicating bound and free (in the presence of protein) and unbound (no
protein) probes were excised, and the DNA was eluted. The DNA was
cleaved using 90 mM NaOH and examined as described in the
methylation protection protocol.
-dCTP
(Amersham Pharmacia Biotech) at 50 mCi/mmol specific activity. Probes
were gel-purified as described above. Binding assays were
carried out by preincubating AR-DBD-histidine tag protein (10 pmol)
with 1 µg of poly(dI-dC) and DBB in a 10-µl volume for 10 min. Increasing amounts of the respective radiolabeled probe were then
added in 2 µl of DBB and incubated at room temperature for 10 min.
Samples were then loaded onto a 5% polyacrylamide gel and
electrophoresed for 90 min at 20 V/cm. The gels were dried and
autoradiographed on Biomax MR film (Eastman Kodak Co.). For DNA binding
analysis, bands corresponding to the bound protein·DNA complex and
free DNA were excised from the dried gel, and the activity of each band
was determined by scintillation counting. Relative binding affinity for
the probes containing more than one binding site (ARE1 G-1 ARE2 G-2,
ARE1 G-1, and G-1 ARE2 G-2) were compared by plotting bound
versus free values averaged from three independent
repetitions for each probe. Binding constants for probes containing one
(dimeric) binding site (ARE1, G-1, ARE2, and G-2) were determined by
Scatchard analysis as described previously (39).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
241ATAGCAtctTGTTCT
227)
and
(
136AG- TACTccaAGAACC
122)
(Fig. 1). However, in addition to these
previously recognized binding sites of AR to the probasin promoter, the
DMS protection assays revealed two additional potential AR binding
sites with atypical half-site sequences located at (
209 to
196) and
(
107 to
93). Most intriguingly, these AR binding sites were unusual with respect to their pattern of protected guanines and DMS
hypersensitivity. To resolve these complexes of AR-DBD binding to the
probasin promoter in more detail, we repeated the MeP assay using
smaller fragments of the probasin promoter extending from (
269 to
164) and (
229 to
77) (Fig. 2,
a and b). These higher resolution analyses
demonstrated that the guanines in the fifth position of the atypical
half-sites were protected as to be expected for nuclear receptor
interaction with DNA
(
209GGGACAtaaAGCCCA
196)
and
(
107ATGACAcaaTGTCAA
93).
However, both of the atypical sites had guanines in the third position
of the presumed 5'-half-sites that showed dramatic DMS hypersensitivity
and even more pronounced DMS hypersensitivity in the 3'-half-sites
(
209GGGACAtaaAGCCCA
196)
and
(
107ATGACAcaaTGTCAA
93).
Hypersensitivity to DMS methylation is most commonly attributed to
structural alteration of the DNA, including strand displacement, kinking, and other forms of local DNA structure deformation (42, 43).
According to our understanding of the binding of nuclear receptors to
their response elements, the DNA is in B-form and the guanine in the
second location of the half-site AGCCCA should be
protected, not overtly hypersensitive (26, 28, 31, 44). The most
striking consistent feature of these newly identified AR-binding sites
is that the hypersensitive guanines are located in the position of the
half-site that is a characteristic discriminatory nucleotide within
estrogen receptor (ER)-responsive binding sites (
209GGGACAtaaAGCCCA
196)
and
(
107ATGACAcaaTGTCAA
93)
(27, 31, 45, 46). However, the conservation of the adenine adjacent to
the guanine on these binding sites prohibits interaction with ER (7,
32) (data not shown). Interestingly, in a previous study of binding
site selection, we have shown that a guanine at this location is
preferred for PR and equally preferable to the consensus thymidine for
AR (1).
View larger version (65K):
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Fig. 1.
The probasin reveals four AR-DBD-binding
sites using methylation protection assay. The AR-DBD was bound to
the probasin promoter ( 269 to
77) and analyzed by DMS-based
methylation protection. Class I binding sites, ARE1 and ARE2, and class
II binding sites, G-1 and G-2, are illustrated with their sequences.
Arrows indicate half-site location and orientation.
Open circles represent protected guanines residues, whereas
solid circles represent guanines hypersensitive to
DMS.
View larger version (66K):
[in a new window]
Fig. 2.
Pronounced DMS hypersensitivity in the 5'-
and 3'-half-sites of class II AR-binding sites on the probasin
promoter. The probasin promoter fragments from ( 269 to
164)
and (
181 to
77) were bound by AR-DBD and assayed by methylation
protection. Arrows indicate half-site orientation and
location. Open circles represent protected guanines, and
solid circles represent hypersensitive guanines.
A, class I binding element, ARE1, and class II binding
element, G-1, are illustrated with their nucleotide sequence.
B, class I binding element, ARE2, and class II binding
element, G-2, are illustrated with their nucleotide sequence.
View larger version (45K):
[in a new window]
Fig. 3.
Class II binding elements demonstrate
intrinsic DMS hypersensitivity upon AR-DBD binding. Methylation
protection assays on individual class II AR-binding sites.
Arrows denote half-site orientation and location for each
binding element sequence. Open circles represent protected
guanines, whereas solid circles represent hypersensitive
guanines. A, methylation protection of class II AR-binding
element G-1 contained within 231 to
164. B, methylation
protection of class II AR-binding element G-2 contained within
128 to
56. C, methylation interference assay demonstrates that
conventional guanine contacts are necessary for AR binding to G-1 and
ARE2.
View larger version (13K):
[in a new window]
Fig. 4.
Class II AR-binding sites facilitate DNA
cooperative binding on the probasin promoter. Relative binding
affinities of probasin promoter elements were determined by gel shift
assay using 5 nM purified His-tagged AR-DBD with increasing
concentrations of radiolabeled DNA as indicated. Bound and free
fractions were isolated using a gel shift assay, excised, and counted.
The change of the bound over free ratio is plotted as a function of
increasing input DNA. Only values corresponding to the linear portion
of the binding curve are given. ARE1, open circles; ARE2,
filled circles; G1, open squares; G2,
filled squares; A1-G1, open triangles; A2-G2,
filled triangles; G1-A2-G2, open diamonds;
A1-G1-A2-G2, filled diamonds.
View larger version (19K):
[in a new window]
Fig. 5.
Class I and class II AR-binding sites display
low levels of transcriptional activity in isolation and interact
synergistically in combination. a, transactivation
activity of individual elements was tested in the presence of
increasing concentrations of R1881. ARE1 (open circles),
ARE2 (filled circles), G-1 (open squares), and
G-2 (filled squares). The boundaries of each site within the
promoter are indicated in Ref. 43. LNCaP cells transfected with pTK-luc
reporter plasmids containing the fragments were incubated 22 h
with the indicated concentration of R1881 then assayed for luciferase
activity. Transcriptional activity is reported as relative luciferase
units (RLU) corrected for background. b, effects on
transactivation activity of combining class I and class II AR binding
sites of the probasin promoter as assayed in A. Levels for
ARE1-G1 (open triangles), ARE2-G2 (filled
triangles) are shown. c, transcriptional activity of
multiple class I and class II AR binding sites of the probasin
promoter, G-1 ARE2 G-2 (closed diamonds) and ARE1 G-1 ARE2
G-2 (open diamonds) were tested as described above.
View larger version (62K):
[in a new window]
Fig. 6.
The PSA enhancer ( 4366 to
3874 bp) has
three class II AR-binding sites as revealed by methylation protection
analysis. In all panels, open circles represent
protected guanines, whereas solid circles represent
hypersensitive guanines. Arrows denote half-site orientation
within each ARE. a, the AR-binding site III has a class I
protection pattern, whereas AR-binding site V has similarity to class
II type protection/hypersensitive patterns. b, AR-binding
site IIIA has a class II protection/hypersensitive pattern.
c, the locations of the class I and class II sites within
the PSA enhancer sequence are indicated.
3 and a Gly at +3.
Class II sites uniquely have a Gly at
5 and a Cys at +4 and/or +5. The most prominent feature of class II sites is a purine at +7,
which displays a highly conserved pyrimidine in class I sites. Interestingly, class II sites also contain a Tht at position
13 in
all cases and a corresponding Ala at +13 in all but one case. Additionally, there is a tendency to have a higher AT-rich flanking region and spacer in class II sites in comparison to class I sites.
View larger version (43K):
[in a new window]
Fig. 7.
Alignment of AREs into proposed class II and
class I AR-binding sites. ARE sequences from the probasin
promoter, PSA enhancer, PSA promoter, and Slp gene.
Consensus features are highlighted by solid circles for
class II and open circles for class I, with conserved guanine contacts
underlined. Italicized elements are proposed by
sequence similarity to the class designation.
13, six bases 5' of the half-site, decreased the
guanine hypersensitivity in the 5'-half-site but did not affect the
binding pattern of the 3'-half-site. Likewise, conversion of the
half-site spacer nucleotides from TAA to CGG also decreased the
hypersensitivity in the 5'-half-site alone. When the Ala at position +7
of the 3'-half-site was converted to a Cys, the DMS protection binding
pattern of the 3'-half-site reverted to the pattern observed with a
class I type binding site but did not alter the hypersensitivity in the
5'-half-site. Therefore, these results suggest that class II type
binding by AR is a composite of influences, primarily distinguished by
the nonconsensus purine nucleotide at the +7 position of the
3'-half-site, but is also influenced by the identity of other
nucleotides in the flanking and spacer regions.
View larger version (21K):
[in a new window]
Fig. 8.
Nucleotide determinants of class II
AR-binding sites as determined by site-directed mutagenesis. To
quantitate the relative protection or hypersensitivity of each guanine
band the band intensity of bound AR-DBD to the probasin G-1 wild-type,
A195C, T215G, or SPACER oligonucleotides indicated was compared
with the unbound oligonucleotide using ImageQuaNT 5.0 densitometry
analysis. Hypersensitive guanines are shown with positive intensity
relative to the unbound band and are illustrated using black
bars. Protected guanines are shown with negative intensity
relative to the unbound band and are illustrated using gray
bars. ARE half-site orientation and location are indicated by
arrows. a, the wild-type G-1 oligo shows the
typical class II methylation pattern. b, changing the
upstream conserved thymidine at position-13 to a guanine decreased the
hypersensitivity on the 5'-half-site at position 5. c,
mutating the 3'-half-site such that it more resembles a class I
half-site by changing the adenine at position +7 to a cytosine resulted
in an overall decrease in hypersensitivity in the 3'-half-site and a
newly protected cytosine at position +6. d, changing the ATT
spacer region to a CGG decreased the hypersensitivity on the
5'-half-site at position
5.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We are appreciative of many helpful discussions with Drs. Paul Rennie, Robert Matusik, and Susan Kasper. C. Nelson gratefully acknowledges her support as a Medical Research Council of Canada Scholar.
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FOOTNOTES |
---|
* This work was supported in part by Medical Research Council of Canada (MRC) Operating Grant 14099.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.
An MRC Scholar. To whom correspondence should be addressed: The
Prostate Centre at Vancouver General Hospital, 2660 Oak St., Vancouver,
British Columbia V6H 3Z6, Canada. Tel.: 604-875-4282; Fax:
604-875-5654; E-mail: ccnelson@interchange.ubc.ca.
Published, JBC Papers in Press, October 30, 2000, DOI 10.1074/jbc.M009170200
2 Reid, K. J., and Nelson, C. C. (2001) Biotechniques 30, in press.
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ABBREVIATIONS |
---|
The abbreviations used are: AR, androgen receptor; ER, estrogen receptor; ARE, androgen response element; GR, glucocorticoid receptor; DBD, DNA binding domain; PR, progesterone receptor; DTT, dithiothreitol; PCR, polymerase chain reaction; DBB, DNA binding buffer; DMS, dimethyl sulfate; RLU, relative luciferase units; MeI, methylation interference; MeP, methylation protection; R1881, synthetic androgen methyl-trienolone.
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---|
1. |
Nelson, C. C.,
Hendy, S. C.,
Shukin, R. J.,
Cheng, H.,
Bruchovsky, N.,
Koop, B. F.,
and Rennie, P. S.
(1999)
Mol. Endocrinol.
13,
2090-2107 |
2. | Yu, V. C., Delsert, C., Andersen, B., Holloway, J. M., Devary, O. V., Naar, A. M., Kim, S. Y., Boutin, J. M., Glass, C. K., and Rosenfeld, M. G. (1991) Cell 67, 1251-1266[Medline] [Order article via Infotrieve] |
3. | Zhang, X. K., Hoffmann, B., Tran, P. B. V., Graupner, G., and Pfahl, M. (1992) Nature 355, 441-446[CrossRef][Medline] [Order article via Infotrieve] |
4. | Kliewer, S. A., Umesono, K., Mangelsdorf, D. J., and Evans, R. M. (1992) Nature 355, 446-449[CrossRef][Medline] [Order article via Infotrieve] |
5. | Wahlstrom, G. M., Sjoberg, M., Andersson, M., Nordstrom, K., and Vennstrom, B. (1992) Mol. Endocrinol. 6, 1013-1022[Abstract] |
6. | Zhao, Q., Khorasanizadeh, S., Miyoshi, Y., Lazar, M. A., and Rastinejad, F. (1998) Mol. Cell 1, 849-861[Medline] [Order article via Infotrieve] |
7. | Zilliacus, J., Carlstedtduke, J., Gustafsson, J. A., and Wright, A. P. H. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 4175-4179[Abstract] |
8. |
Claessens, F.,
Alen, P.,
Devos, A.,
Peeters, B.,
Verhoeven, G.,
and Rombauts, W.
(1996)
J. Biol. Chem.
271,
19013-19016 |
9. |
Kasper, S.,
Rennie, P. S.,
Bruchovsky, N.,
Lin, L.,
Cheng, H.,
Snoek, R.,
Dahlman-Wright, K.,
Gustafsson, J.-A.,
Shiu, R. P. C.,
Sheppard, P. C.,
and Matusik, R. J.
(1999)
J. Mol. Endocrinol.
22,
313-325 |
10. | Cato, A., Henderson, D., and Ponta, H. (1987) EMBO J. 6, 363-368[Abstract] |
11. | Rundlett, S., and Miesfeld, R. (1995) Mol. Cell. Endocrinol. 109, 1-10[CrossRef][Medline] [Order article via Infotrieve] |
12. | Roche, P. J., Hoare, S. A., and Parker, M. G. (1992) Mol. Endocrinol. 6, 2229-2235[Abstract] |
13. |
Grad, J. M.,
Dai, J. L.,
Wu, S.,
and Burnstein, K. L.
(1999)
Mol. Endocrinol.
13,
1896-1911 |
14. |
Kasper, S.,
Rennie, P.,
Bruchovsky, N.,
Sheppard, P.,
Cheng, H.,
Lin, L.,
Shui, R.,
Snoek, R.,
and Matusik, R.
(1994)
J. Biol. Chem.
269,
31763-31769 |
15. | Rennie, P., Bruchovsky, N., Leco, K., Sheppard, P., McQueen, S., Cheng, H., Snoek, R., Hamel, A., Bock, M., MacDonald, B., Nickel, B., Chang, C., Liao, S., Cattini, P., and Matusik, R. (1993) Mol. Endocrinol. 7, 23-36[Abstract] |
16. | Adler, A. J., Danielsen, M., and Robins, D. M. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 11660-11663[Abstract] |
17. | Adler, A., Scheller, A., and Robins, D. (1993) Mole. Cell. Biol. 13, 6326-6335[Abstract] |
18. |
Huang, W.,
Shostak, Y.,
Tarr, P.,
Sawyers, C.,
and Carey, M.
(1999)
J. Biol. Chem.
274,
25756-25768 |
19. | Travers, A. (1998) Curr. Biol. 8, 616-618 |
20. | Kerppola, T. (1998) Structure 6, 549-554[Medline] [Order article via Infotrieve] |
21. | Senear, D., Ross, J., and Laue, T. (1998) Methods 16, 3-20[CrossRef][Medline] [Order article via Infotrieve] |
22. | Hager, G., Archer, T., Fragoso, G., Bresnick, E., Tsukagoshi, Y., John, S., and Smith, C. (1993) Cold Spring Harbor Symp. Quant. Biol. 58, 63-71[Medline] [Order article via Infotrieve] |
23. | List, H. J., Lozano, C., Lu, J., Danielsen, M., Wellstein, A., and Riegel, A. T. (1999) Exp. Cell Res. 250, 414-422[CrossRef][Medline] [Order article via Infotrieve] |
24. |
Pham, T. A.,
Hwung, Y. P.,
Mcdonnell, D. P.,
and Omalley, B. W.
(1991)
J. Biol. Chem.
266,
18179-18187 |
25. | Gewirth, D., and Sigler, P. (1995) Nature Struct. Biol. 2, 386-394[Medline] [Order article via Infotrieve] |
26. | Luisi, B. F., Xu, W. X., Otwinowski, Z., Freedman, L. P., Yamamoto, K. R., and Sigler, P. B. (1991) Nature 352, 497-505[CrossRef][Medline] [Order article via Infotrieve] |
27. | Schwabe, J. W. R., Chapman, L., Finch, J. T., Rhodes, D., and Neuhaus, D. (1993) Structure 1, 187-204 |
28. | Rastinejad, F., Perlmann, T., Evans, R., and Sigler, P. (1995) Nature 375, 203-211[CrossRef][Medline] [Order article via Infotrieve] |
29. | Manglesdorf, D., Thummel, C., Beato, M., Herrlich, P., Schutz, G., Umesono, K., Mark, M., Chambon, P., and Evans, R. (1995) Cell 83, 835-839[Medline] [Order article via Infotrieve] |
30. | Glass, C. K. (1994) Endocrine Rev. 15, 391-407[Medline] [Order article via Infotrieve] |
31. | Schwabe, J. W. R., Chapman, L., Finch, J. T., and Rhodes, D. (1993) Cell 75, 567-578[Medline] [Order article via Infotrieve] |
32. | Nelson, C. C., Hendy, S. C., Faris, J. S., and Romaniuk, P. J. (1994) Mol. Endocrinol. 8, 829-840[Abstract] |
33. | Zolfaghari, A., and Djakiew, D. (1996) Prostate 28, 232-238[CrossRef][Medline] [Order article via Infotrieve] |
34. | Lieberman, B., Bona, B., Edwards, D., and Nordeen, S. (1993) Mol. Endocrinol. 7, 515-527[Abstract] |
35. |
Tan, J.,
Marschke, K.,
Ho, K.,
Perry, S.,
Wilson, E.,
and French, F.
(1992)
J. Biol. Chem.
267,
4456-4466 |
36. | Claessens, F., Celis, L., Devos, P., Peeters, B., Heyns, W., Verhoeven, G., and Rombauts, W. (1993) Biochem. Biophys. Res. Commun. 191, 688-694[CrossRef][Medline] [Order article via Infotrieve] |
37. |
Nelson, C. C.,
Hendy, S. C.,
and Romaniuk, P. J.
(1995)
J. Biol. Chem.
270,
16981-16987 |
38. | Dai, J. L., and Burnstein, K. L. (1996) Mol. Endocrinol. 10, 1582-1594[Abstract] |
39. |
Nelson, C.,
Hendy, S.,
Faris, J.,
and Romaniuk, P.
(1996)
J. Biol. Chem.
271,
19464-19474 |
40. | Espinas, M., Roux, J., Pictet, R., and Grange, T. (1995) Mol. Cell. Biol. 15, 5346-54[Abstract] |
41. | Ramesh, V., and Nagaraja, V. (1996) J. Mol. Biol. 260, 22-33[CrossRef][Medline] [Order article via Infotrieve] |
42. | Werner, M. H., Gronenborn, A. M., and Marius Clore, G. (1996) Science 271, 778-784[Abstract] |
43. |
Dickerson, R. E.
(1998)
Nucleic Acids Res.
26,
1906-1926 |
44. |
Rastinejad, F.,
Wagner, T.,
Zhao, Q.,
and Khorasanizadeh, S.
(2000)
EMBO J.
19,
1045-1054 |
45. | Mader, S., Kumar, V., de Verneuil, H., and Chambon, P. (1989) Nature 338, 271-274[CrossRef][Medline] [Order article via Infotrieve] |
46. | Danielsen, M., Hinck, L., and Ringold, G. M. (1989) Cell 57, 1131-1138[Medline] [Order article via Infotrieve] |