The Prostate Centre (C.C.N., S.C.H., R.J.S., H.C., N.B.,
P.S.R.) Jack Bell Research Centre Vancouver, British Columbia
V6H 3Z6, Canada
Department of Biology (B.F.K.) University
of Victoria Victoria, British Columbia V8W 3P6, Canada
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ABSTRACT |
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INTRODUCTION |
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Receptor-selective response on a promoter can arise through at least five stratified regulatory mechanisms, including availability of constituents, coregulators, proximal transcription factors, cooperative binding, and DNA target recognition. First, the most simplistic mechanisms of hormonal selectivity are the availability of types of steroid receptors or their cognate ligands in the cell (35). However, in many cell types, two or more steroid receptor types coexist with access to their respective ligands (30, 36, 37, 38). Second, cell type-specific coregulators may recognize a particular type of steroid receptor and, in turn, participate in directing the appropriate response (32). However, most coregulator proteins isolated at present have not shown an influence of DNA target sequence preference or discrimination between different receptor types (39, 40, 41, 42) with the possible exception of the recently described ARA70 (43) in some contexts, but not in others (44). Third, other transcription factors bound proximal to the receptor-binding site in a complex promoter may potentiate the relative responsiveness of one receptor type over another (33). This has been demonstrated for AR, where several AR-regulated promoters have been shown to have octamer transcription factor-1 (OCT-1) and/or nuclear factor-1 (NF1) binding sites adjacent to the AR-binding region that are involved in the establishment of full AR-specific response in this context (33, 45). However, NF1 is also instrumental in the activation of the MMTV promoter by the GR and thus, in itself, is not receptor type specific (46). Fourth, discrimination of steroid response may be derived from the biophysical architecture of receptor-binding sites within the promoter to promote receptor-preferential responses, such that particular configurations of sets of binding sites may act cooperatively for one receptor type specifically. This has been demonstrated for AR binding to the two sites separated by 80 bp on the probasin promoter (47, 48) and the multiple tandem AR binding sites on the mouse sex-limited protein (Slp) promoter (33, 49). However, in the case of the MMTV promoter, which is activated by GR, PR, and AR, the multiplicity of response elements in itself fails to discriminate between receptor types. Fifth, the natural genomic promoters that are regulated specifically by one steroid receptor type contain sequence variants of the idealized SRE, rather than displaying the canonical SRE sequence verbatim. These often less-than-subtle sequence variants of DNA-binding sites may provide preferential binding by a particular receptor type due to the unique deviation of nucleotide sequence of the response element (26, 27, 30, 31, 33, 47). This has recently been investigated for the selective binding of AR to the ARE2 found in probasin promoter in which the amino acid differences in second zinc finger and C-terminal extension of the AR-DBD in comparison to the GR-DBD provides receptor selectivity of response (21, 50). While all of these mechanisms may contribute collectively to receptor-specific response as a composite function, we have focused in the present study on the potential contribution of nucleotide deviations within the receptor binding site as partial discriminates of steroid receptor action.
During evolution, steroid receptors (AR, PR, GR, MR, and ER) likely
diverged in a coordinate manner with their DNA-binding targets to
provide nonoverlapping functions. Most dramatically, ER evolved amino
acids in the DNA recognition -helix that provided new discriminating
contacts to the central nucleotide of the half-site sequence (3, 5, 6, 51). However, due to the confinement of the highly conserved structural
features of the DBD for functional integrity and the relatively recent
common ancestry (1), it is possible that steroid receptors with an
identical DNA recognition
-helix (AR, GR, PR, MR) are capable of
binding both to a common variety of sequence (possibly a vestigial
sequence), as well as divergent sequences that display more receptor
selectivity. The development of steroid receptor-specific binding sites
from a common binding site may arise through two mechanisms. One
mechanism would be that a particular steroid receptor type could evolve
to have additional sequence-specific DNA contact potential that would
allow the receptor to bind with higher affinity to a particular
nucleotide sequence in comparison to other steroid receptors. This
strategy would result in the gain of affinity from a common ancestral
element, such as the canonical SRE. Alternatively, a second mechanism
is one in which a particular receptor type could secure a niche of
sequence specificity among related receptors through tolerance of a
nucleotide substitution in the DNA-binding target in comparison to
another receptor type. The overall affinity of this type of binding
site for the receptor may be lower than the canonical element but would
provide specificity by discouraging the binding of inappropriate
receptors, as shown recently for AR specificity of the ARE2 found in
the probasin promoter (21, 50). In nature the evolutionary pressure for
binding site selection of a steroid receptor likely weighs heavily on
receptor specificity of binding within a given range of DNA-binding
affinities. These mechanisms are not mutually exclusive, and it is
probable that most receptor-specific binding sites may have evolved
some divergent nucleotides for gain in energy contribution, while
selection of other nucleotide preferences are directed by differential
tolerance to certain nucleotides by competing receptor types (12).
In natural promoters, SREs display a great diversity in nucleotide sequence, some of which may contribute to a degree of receptor specificity, whereas other nucleotide substitutions may be incidental. Because so few natural androgen- or progesterone-regulated promoters have been isolated and characterized, it is difficult to determine the functionality of subtle nucleotide differences in the binding sites for providing receptor type discrimination by simple alignment of identified binding sites to derive a consensus.
In previous in vitro studies, attempts to identify androgen-, progesterone-, and glucocorticoid-specific response elements based on highest affinity selection strategies resulted in the confirmation of the proposed canonical SRE (26, 27, 28). These studies presumed that receptor specificity was synonymous to highest affinity binding. However, if nature derives receptor specificity from a balance of energetic contribution and nucleotide substitution tolerance, it is likely that receptor-specific binding sites may have a lower affinity than the idealized canonical element and therefore would be overlooked in the laboratory selection procedure because of the lack of consideration of this parameter. In fact, all receptor-specific elements characterized from natural promoters are of lower affinity than the canonical binding site, which is nondiscriminating for receptor-type responsiveness.
Guided by the above observations, we have devised methodology to investigate the existence of receptor-specific binding sites by recapitulating these dual selection pressures that arise in a receptor-competitive environment. To identify receptor-specific DNA targets, we modified the common binding site selection assay (52, 53) to select high-affinity binding sites for the receptor of interest in the presence of a related competing receptor. This novel methodology resulted in the identification of preferred binding sites of AR and PR when competing with GR, which possess similarities to the few previously identified receptor-selective response elements. Most strikingly, the selected DNA sites were highly asymmetrical in the spacer and flanking regions, indicating that the homodimers likely bind in an allosteric manner. When the elements were tested in transactivation studies, the level of response by a given receptor was not directly proportional to DNA binding affinity and was greatly influenced by orientation. This suggests that subtle nucleotide changes in the DNA-binding targets may be significant for physiological responses, adding credence to the hypothesis that receptor response elements have evolved to secure a niche of DNA-binding targets rather than in isolation of each other to select for the absolute highest DNA binding affinity. We believe that the success of this approach provides a mechanism to address the evolution of DNA-binding targets of related transcription factors within a conserved gene family by taking into account the complexity of the competitive nuclear environment.
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RESULTS |
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The DNA sequences bound to the receptor-DBD in the randomized template
pool (composed of 10 of each possible sequence) were separated by
gel mobility shift analysis (Fig. 2
). The
receptor-dimer bound fraction of DNA sequences was excised and eluted
from the gel and amplified by PCR using the terminal primers (as
depicted in Fig. 1A
). One third of the PCR amplification reaction of
bound sequences was radiolabeled and then subjected to further rounds
of selection in a band shift assay with lower concentrations of
recombinant receptor-DBD to enrich for higher affinity binding sites.
After four rounds of selection, the highest affinity sequences selected
at 12 nM protein were cloned (Fig. 1A
, pathway A). A
minimum of 50 clones each were sequenced for statistical analysis by
Pearson
2 test of the highest affinity sequences for the
DBDs of AR, GR, and PR.
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Flanking Sequence Preferences
There was significant selection for flanking nucleotides of the
hexameric binding sites by AR when selected on the basis of highest
affinity. Flanking the variable half-site, reading 5' to 3', positions
-13 to -8 were selected to be
(-13N-G/t-g/t-G/t-a-G/a-8) (Fig. 3A). In
three positions, the selection of guanines by AR in the 5'-flanking
region was nearly as strong as the selection observed within the
half-site for GR and PR. The flanking sequences of the fixed half-site
from position +13 to +8 demonstrated slightly less selection
(+13N-N-g/t-t-G/t-T/g+8), but more notably the
flanking sequences of the variable half-site and fixed half-sites did
not mirror each other (an axis of symmetry would predict the selection
of (+13N-C/a-c/a-C-t-C/t+8) in the 3'-flanking
sequences). Likewise, the spacer nucleotides between the AR-bound
half-sites were not symmetrical. In the AR selected sequences, guanine
was predominant at +1 position without reciprocal selection for
cytosine at -1 position (-1t N
G+1). This reinforces earlier observations that
the optimal binding of AR homodimers is not perfectly symmetrical (28).
The GR had a strong selection for guanine at position +1, but had a
nearly equal preference for adenine at this position, which appeared to
be strongly selected against by AR.
The PR-selected flanking sequences were strongly selected and most
notably G/T rich for both the variable and fixed half-sites on the
strand of reference (Fig. 3B). Even more pronounced for the PR selected
sequences than the AR selection is the observation that the flanking
sequence preference is nearly antisymmetrical due to the extensive G/T
bias for selection, as a symmetrical interaction of a PR dimer that
favored G/T for one half-site would select C/A flanking nucleotides for
the opposing inverted half-site. Analysis of individual sequences
revealed that guanines most commonly occurred as doublets of GG in the
flanking sequences with a high frequency of guanines within the
variable half-site (data not shown). Likewise, the nucleotides in the
spacer between the PR half-sites
(-1T/g-g/t-g/t+1) were significantly selected
(particularly T at -1) and also in an asymmetric manner (Fig. 3B
). It
was also noted that in some instances (<10%) it was apparent that the
PR dimers were not binding to the classical inverted repeat spaced by
three nucleotides (IR+3), and the consensus-like half-sites on the
variable side ranged from an IR+2 to an IR+5 in spacing. These sites
may represent the binding of two monomers independently.
Orientation of Half-Sites
In general, the nucleotide sequence selected by GR appeared to be less
strongly selected in comparison to AR- and PR-selected sequences (Fig. 3C). Upon alignment of the GR sequences, it is apparent that in a
proportion of cases GR likely bound to two half-sites independently of
orientation and spacing of the classical SRE as an inverted repeat
spaced by three bases. Approximately 15% of the sequences appeared to
be bound as nearly perfect half-sites independent of the IR+3
orientation, with the greatest proportion binding as direct repeat
motifs with spacing separations from 3 to 9 bases (data not shown).
Again, these nonconventional steroid receptor binding sites may
represent double occupancy of two monomers because of the lack of
consistency of the spacing between the binding sites or in concurrence
with the recent data demonstrating that GR may bind to direct repeats
(54).
In spite of the variable nature of GR binding to 5' the randomized region, the 3' flanking sequences of the fixed GR half-sites illustrated sequence selection as (+13A/g-N-G-g/t-G-a+8) reading from position +13 to +8 toward the fixed half-site. The spacer region between the GR half-sites (-1g/t-a-G/A+1) nearly has an axis of symmetry and, similar to AR and PR, demonstrated the strongest preference for guanine at the +1 position. In contrast to AR, the GR had a strong secondary preference for adenine at +1, whereas adenine was selected against by AR at this position.
Estimation of Cognate Contacts to the Variable Half-Site
For binding sites selected by GR in the classical IR+3 orientation,
there was also a less defined consensus in the variable half-site. This
less stringent selection of the variable half-site may be related to
the ability of GR to bind to one half-site, and through cooperative
interactions between the GR proteins tether the dimeric partner
molecule over a noncognate site, as demonstrated in the crystal
structure of a GR dimer bound to an IR+4 (2, 55). To address this
issue, the data were analyzed to assess the flexibility of the binding
site recognition in the variable half-site by determining a cognate
score for the selected variable half-site for each receptor. The
cognate score was determined by assigning one point for each nucleotide
match to the consensus G/A-G-T/G-A-C-A (06, for each selected
sequence) divided by the number of sequences analyzed. Thus a score of
6 indicates that all sequences in the variable half-site were a perfect
match to the consensus, a score of 5 meant that, on average, five of
the six nucleotides conformed to the consensus and so on. A low cognate
score for a dimer indicates lack of sequence recognition possibly due
to protein-protein interactions resulting in tethering from the fixed
half-site. AR demonstrated the highest cognate score with 4.98, PR
scored 4.06, and GR scored 3.83. The selection of noncognate (<2 of 6
matches to the consensus half-site at the expected IR+3 location was
seen for 22.6% of the GR-, 17.3% of the PR-, and 5.8% of the
AR-selected sequences. We did not find evidence of AR-DBD binding to
direct repeat-like elements as reported earlier for a recombinant
AR-DBD-glutathione-S-transferase (GST) fusion protein tested
on a random pool of DNA containing a fixed half-site of TGTTCT (13). In
contrast, this study did not detect any conventional IR+3 type of
binding site for the AR-DBD (13). These effects may be due to GST
dimerization of the fusion proteins, which has been reported to affect
the DNA binding characteristics of GST-DBD fusion proteins (69, 70). In
our study, the DBDs of GR and PR have less restriction of binding to
the variable half-site in the presence of a canonical half-site,
whereas AR is quite restricted in its sequence recognition, both in
terms of orientation and sequence deviation.
Intolerance of Cytosines
One notable feature of all three steroid receptor selections of
sequence is that, on the reported reference strand (Fig. 1B), there is
an extreme bias against cytosines except at the known contacting base
pair at position -3 in the variable half-site (Fig. 3
). To test
whether this was an artifact of the random parental probe preparation,
the random oligonucleotide population was sequenced directly by
PCR-based sequencing using one of the terminal primers. Each nucleotide
in the randomized region appeared to be evenly represented (data not
shown). Since this guanine-rich strand is paired to the cytosine-rich
bottom strand, we would have to assume that this is not a PCR or
nucleotide bias of the selection procedure. This selection against
cytosines is also apparent in the AR consensus sequence and in
site-directed mutagenesis studies of binding sites of GR and PR
(26, 27, 28). From these data we conclude that steroid receptors may have a
strand bias for guanines and/or against cytosines particularly in the
regions flanking the half-sites. In particular, both AR- and
PR-selected populations show a very strong preference for guanines
three nucleotides upstream from the variable half-site at position -10
(Fig. 3
, A and B), which is also in concurrence with earlier data of AR
specificity (28).
Receptor-Selective Binding Site Enrichment
The methodology developed for selection of receptor type-specific
binding sites was designed to mimic the physiological abundance of
steroid receptors in a given cell type. While GR is ubiquitous in
tissue distribution, AR and PR are more limited to particular tissue
types and are often at similar levels to GR (30, 56). It follows that
in many tissues the sex steroid receptors could be in competition with
GR for potential DNA-binding sites. The CAAB methodology was designed
to isolate AR-specific sequences from a pool of templates that was
preenriched for medium-to-high affinity AR DNA-binding sites in the
Round II selection (schematically illustrated in Fig. 1A, pathway B).
The AR-preferential elements were isolated by titration of GR into the
binding reaction resulting from the radiolabeled Round II selection to
competitively remove common binding elements or SREs. The AR- and
GR-bound fractions of DNA could be segregated by the characteristic
faster mobility of the 87-amino acid DBD fragment of GR relative to the
124-amino acid DBD of AR used in the gel shift assay in this study
(Fig. 2B
). The AR-dimer-bound DNA sequences that were still apparent on
an autoradiogram at the highest concentration of competing GR (a 3-fold
excess) in the first competitive round were excised and eluted from the
gel and amplified by PCR for enrichment of AR-specific sequences. The
amplified ARE-enriched sequences were radiolabeled and enriched further
for AR-selective sequences in a gel mobility shift assay, in which AR
was present at 50 nM with titration of GR (50
nM, 150 nM, 300 nM). In this second
competitive round of ARE selection, GR levels could be increased to a
6-fold excess of GR to AR, indicating that ARE enrichment had occurred.
The AR-preferential sequences were then cloned and sequenced. The same
protocol was used for selection of PR-selective sequences in the
presence of GR. However, in the case of PR, GR could only be titrated
to a 3-fold excess before the PR-specific band was undetectable. This
difference in AR and PR resilience to GR competition may be due to the
greater level of phylogenetic divergence of AR from GR in comparison to
PR. The AR and PR populations of DNA-binding sites that withstood the
competitive binding pressure of GR were considered to be enriched for
AR-selective and PR-selective sequences, respectively, and were
compared with sequences that were selected solely on the basis of
optimizing affinity for AR, PR, or GR.
AR-Selective Sequences
In comparison to the sequences selected for AR based solely on
increasing affinity, the sequences selected to be preferentially bound
by AR when competing with GR illustrated several changes in the
distribution of nucleotides (compare Fig. 3A with Fig. 4A
). Within the variable half-site
selected by AR, there was an increased prominence of adenine in the -5
position (-7GGT/AACA-2) and in
the -2 position an increase in guanine and, to a much lesser
extent, thymidine, in correspondence with a decrease in adenine
(-7GGTACG/t-2). This suggests
that the half-site
(-7GGAACG/t-2) may
contain sequence determinants that either favor binding by AR and/or
are less tolerated by GR. When examining the individual sequences
selected in context, the degenerate consensus sequence
(-7G/A-G-A/T-A-C-G-2) was represented 15% of
the time in the AR population selected on the basis of affinity alone,
whereas in the AR-selective population this degenerate hexamer was
enriched to 40% of the population. This degenerate sequence was not
found in the GR-selected sequences (data not shown). The sequence
(-7AGTACT-2) found in the ARE2 of
the probasin promoter (21, 50) was also enriched in the AR-selective
population in comparison to the AR and GR sequences selected solely on
the basis of affinity.
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The AR-selective sequences also demonstrated differences in the
flanking nucleotide preferences in comparison to AR affinity-based
sequences. In general, the distribution of selected nucleotides in the
5'-flanking region of the AR-selected population was less strongly
selected adjacent to the variable half-site with the exception of the
guanine at -12, but more highly selected adjacent to the 3' fixed
half-site for guanine at position +12 in comparison to the high
affinity AR sequences (compare Figs. 4A and 3A
). This suggests that the
selection of nucleotides in the flanking sequences may add to affinity,
while only those at -12 and +12 play a more specific role in
receptor-type discrimination.
PR-Selective Sequences
The sequences that were selected by PR in the presence of GR
competition showed a different pattern of nucleotide preferences (Fig. 4B). Within the variable half-site, nucleotide frequency distribution
was altered at each position. In position -7, there was a 50%
increase in the adenine selection. In position -6, there was a notable
enrichment in the occurrence of thymidine, which is unexpected because
the guanine in the canonical sequence at this position is presumably a
conserved arginine contact throughout the receptor superfamily (2, 3, 9). However, a thymidine at this position has been demonstrated to be a
major contributor to PR-specific induction of a distal element of MMTV
in comparison to GR (26). At position -5 there is an increased
occurrence of cytosine, with a complementary decrease in guanine. The
emergence of cytosine may be due to the apparent intolerance to
cytosine by GR at this location as only 2 cytosines out of a possible
53 at this location were seen during GR affinity-based selection (Fig. 3C
). In position -2, there was a significant increase in thymidine
concomitant with the loss of adenine. The similarities of the PR- and
AR-selective sequences in the presence of competing GR may reflect the
relative tolerance levels of these steroid receptors for these
nucleotide variations.
One of the most striking features of the changes in flanking nucleotide
selection between the PR sequence selected by affinity and those
selected by competition with GR is the predominance of the selection of
guanines on the reference strand; in 8 of the 12 given random flanking
positions, guanines were selected more than 37% of the time (Fig. 4B).
The bias for guanines in the flanking regions of the PR binding sites
was apparent with sequences selected by PR for affinity alone, but was
further enriched in the PR-selective population at nearly every
location. In this PR-selective population, nearly all putative binding
sites conformed to the classical orientation and spacing of IR+3 by the
steroid receptors, so we do not believe that this enrichment in
guanines is an artifact of misaligned half-sites.
Amino Acid Regions That Dictate Receptor-Selective Nucleotides
The data described above indicated that AR, GR, and PR had
individual preferences for nucleotides within and flanking their DNA
binding sites. To determine the amino acid regions of these receptor
DBDs that were responsible for receptor-specific nucleotide
discrimination, we created two chimeric DBDs of the AR and GR utilizing
the conserved HindIII restriction site within the sequence
encoding the first zinc finger-like module (Fig. 2). The first chimeric
DBD, AHG, consisted of the first 24 amino acids of the
AR-DBD and the corresponding following 63 amino acids of GR ending at
the cryptic Factor Xa cleavage site. The complementary chimeric
GHA consisted of the first 24 amino acids of GR-DBD
followed by the corresponding 100 amino acids of the AR-DBD. These
chimeric GHA- and AHG-DBDs were then used for
selection of the highest affinity DNA sequences and compared with the
sequences selected by the native AR- and GR-DBDs. This analysis
demonstrated that the selection of the guanines in the 5'-flanking
region at positions -12 and -10 of the variable half-site was
associated with the amino-terminal 24 amino acids of the AR-DBD (Fig. 5
, A and B). Furthermore, these data
demonstrated that the AR-derived amino acids 548648 were associated
with the AR-specific selection of G/A at position -8 and thymidine at
position +8 on the reference strand immediately flanking the half-site
(Fig. 5
, A and D). The preference of the GR-DBD for a guanine at
position +11 segregated with the amino acid region of the GR-derived
amino acids 426490 (Fig. 5
, B and C). Interestingly, the preference
for an adenine by GR at position 1 in the spacer was primarily dictated
by the GR region from 426 to 490 (Fig. 5
, B and C). Only the intact
AR-DBD possessed a discrimination against adenine at this position,
which may suggest a restrictive function that deters binding AR from a
site with an adenine at this location.
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Although there were slight differences in the methylation interference patterns, the results do not provide substantial evidence that AR or PR is able to make novel guanine hydrogen bond contacts on these DNA elements which would provide a receptor-specific energy contribution that could elicit receptor specificity. It is feasible that these elements do not provide additional base pair contacts for their respective receptors, but they may instead possess determinants that deter the binding of inappropriate receptors, or through subtle DNA conformations, including bending and nearest neighbor effects, that may accommodate one receptor type more so than another.
Binding-Affinity Discrimination Between GR, PR, and AR on Selected
Sequences
Since the representative ARE and PRE elements did not demonstrate
significant differences in guanine interactions as analyzed by
methylation interference, we investigated the DNA-binding kinetics of
AR, PR, and GR to these DNA elements in comparison to a control
canonical SRE element (Table 1, panel B). The binding affinity
of each receptor on each idealized element was determined by gel
mobility shift using a constant amount of recombinant protein and
increasing concentration of radiolabeled DNA target element followed by
quantitation by phosphorimaging followed by Scatchard analysis. The
binding affinity for each element is expressed as a dissociation
constant (Kd) in nanomolar concentration in Table 1
,
panel B. This analysis showed that for the canonical element, GR binds
with approximately 20-fold greater affinity than AR, whereas analysis
of the AR-selective elements demonstrated that GR binds only 2- to
3-fold better than AR to ARE-hasp and ARE-sp, respectively. Thus GRs
avidity for these different elements is drastically reduced 20- to
50-fold on the AREs relative to the SRE. Similarly, PR binds 35-fold
less well than GR to the canonical element yet approximately equally
well as the GR on the two idealized PREs, whereas AR is severely
compromised for binding to the PREs. The above data are similar to
previous evidence that natural AREs show approximately this
differential binding affinity for AR and GR (30).
Transactivation of Representative DNA Elements by the AR, GR, and
PR
To determine whether the representative DNA elements acted
preferentially in terms of transactivation by the AR, GR, or PR, single
copies of the response elements were cloned into the luciferase
reporter plasmid pMLuc containing a minimal promoter of MMTV and
cotransfected into PC3 cells. Receptor-selective elements in both
orientations were compared with the canonical SRE expressed as fold
induction in the absence or presence of the cognate hormone (Fig. 6). The AR activated the SRE in either
orientation approximately 7-fold in the presence of androgens. In spite
of its lower DNA binding affinity, the ARE-sp-selective element gave an
11-fold induction in the reverse orientation and a 6.6-fold induction
in the forward direction averaged in three independent experiments. The
PR most efficiently activated the PREhasp in the reverse orientation
(5.3-fold) and 3.4-fold in the forward orientation in comparison to
3.3-fold activation on the higher affinity SRE. In contrast, the GR
most efficiently activated the SRE to approximately 14-fold and to a
lesser degree activated the AREsp approximately 9-fold and the PREhasp
approximately 7.5-fold regardless of orientation in all cases. These
preliminary results suggest that there is not a direct linear
relationship between DNA binding affinity and transcriptional
activation and furthermore that minor changes of nucleotide sequence
within the half-site can effect the transcriptional activity of the
response element by a given receptor in an orientation-dependent
manner.
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DISCUSSION |
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The specificity of response by the steroid receptors most likely arises
at many levels of promoter regulation as discussed previously. In this
study we have limited our examination to the nucleotide determinants
within the receptor DNA-binding site that are fundamental to
receptor-type DNA binding specificity. To regulate the correct subset
of genes, these highly conserved steroid receptors must be able to
discriminate between closely related DNA sequences. The selection of
the appropriate receptor binding sites is a balance of energy
contribution from positive nucleotide contacts and negative factors
that inhibit the binding of a receptor to noncognate sites. Within the
greater context of the nuclear receptor superfamily, this principle has
been well studied (11, 12). In these studies, it has been demonstrated
that the derivation of the P-box amino acids in the DNA recognition
-helix between the ER/T3R and GR/AR/PR families results in
discrimination of the central nucleotides of the half-site,
AGGTCA and AGAACA, respectively (5, 6, 7). The
discrimination of the fourth base pair (AGAACA) is due to a
base-specific contact, whereas discrimination of the third base pair by
GR (AGAACA) is likely due to a restrictive function rather
than a positive base-specific contact (2, 11, 12). Similarly, the
fourth base pair of the ER/T3R family half-site (AGGTCA)
provides a positive contact as well as a restriction to binding to a
noncognate site by the P-box amino acids, whereas the third base pair
(AGGTCA) provides binding energy (3, 10, 57). Thus, the
discrimination between binding sites for the ER/T3R and GR/AR/PR
subfamilies of receptors can be attributed to the obvious functional
differences in the characteristic P-box amino acid residues within the
DNA recognition
-helix that make nucleotide contacts in the
half-site. Further discrimination of binding sites between receptors
within the ER/T3R family occurs by other features of the binding site
architecture by virtue of spacing, orientation, and flanking sequence
differences of the half-sites. However, the steroid receptors under
investigation in the present paper have a nearly identical DNA
recognition
-helix, and all bind primarily to nearly identical
inverted half-sites spaced by 3 bp (2). The conservation of these
parameters results in an enigma of how a steroid receptor can
discriminate its respective DNA-binding target. Indisputably, the
majority of DNA sequence discrimination resides in the DNA recognition
-helix of the minimal DBD (2, 3, 9), but subtle differences in amino
acid content here and in other regions outside of the zinc finger
modules may directly or indirectly influence DNA sequence selection as
shown for T3R, vitamin D3 receptor (VD3R), and AR (21, 58, 59). Within the minimal 75-amino acid DBD used for crystallography
studies of GR, the corresponding regions of AR and PR are 76% and 84%
conserved, respectively. Comparison of the 124-amino acid DBD regions
used in this study show more divergence, in that AR is only 49% and PR
is only 56% homologous to GR. Thus, using larger DBD fragments of
these steroid receptors may accentuate receptor-specific contributions
to DNA target selection as recently seen in studies of AR specificity
(21, 49). Other more distal regions of the receptors may influence the
stability of DNA-protein interaction; however, the intent of our study
was to use extended DBDs of the steroid receptors to investigate
whether the nonconserved amino acids within the DBD had the potential
to discriminate between subtle nucleotide derivations within DNA
binding sites.
To identify sequence-specific binding determinants, investigators have used exponential amplification-based evolution assays, such as the SAAB assay. This has been extremely useful in defining the highest affinity binding sites for nuclear hormone receptors such as the canonical SRE (28, 59, 60). However, since evolution does not take place in a simple bimolecular environment, we believe that it is important to consider the potential competing pressures of closely related transcription factors. To reproduce these dual pressures, we modified the SAAB assay to select high-affinity sequences with one receptor in the presence of a related receptor to segregate common binding sequences of related receptors from receptor type-specific sequences. Our analysis compared the selection of DNA sequences that were bound by AR, PR, and GR in isolation to sequences bound by these receptors in the presence of a competing GR population in an effort to recreate the natural setting of AR and PR. These data demonstrate that the DBDs of AR, PR, and GR have different DNA binding site preferences that extend 5 bp away from the half-site dictated by amino acids N-terminal to the first zinc finger as well as within the IR+3. Furthermore, our data provide evidence that the AR and PR bind to an IR+3 in an asymmetric fashion that ultimately effects transcriptional activity.
To determine whether this theoretical approach predicts for
biologically relevant sites, we have correlated the selected sequence
content of our theoretical receptor-specific binding sites to AREs and
PREs found in natural promoters. Considering the constraints of our
assay and the diversity of nucleotide deviation of natural AREs, it is
remarkable that our analysis correlated with features of many naturally
occurring AREs (Fig. 7). A distinctive
feature of naturally occurring AREs is the variant guanine or
thymidine in the -2 or +2 position of the binding site that is present
in 15 of 18 aligned natural AREs. The C3 gene contains a well
characterized ARE in the first intron, which has a guanine in the -2
position (AGTACG) that has been shown to contribute to
AR-selective binding (34). The presence of thymidine in this position
is perhaps the most common nonconsensus feature of AREs, as illustrated
in the kallikrien family of androgen-responsive genes that includes the
prostate-specific antigen (PSA) gene. The human kallikrien gene, hKLK2
GGAACA GCA AGTGCT is highly androgen inducible and is one
nucleotide different than the well studied ARE in the PSA promoter
located at -160 (61). Notably, the PSA promoter has two characterized
AREs that act synergistically, both of which have a thymidine at +2
position of the binding site, AGAACA GCA AGTGCT and GGATCA
GGG AGTCTC (62). However, both of these elements in
isolation demonstrate activity with GR and AR (62). The primary ARE of
the probasin gene also has a thymidine at the indicated position in
both half-sites, AGTACTccaAGAACC (47). The
probasin element displays strong androgenicity in the context of its
native promoter and has recently been shown to be preferentially bound
by AR in comparison to GR (21, 30, 50, 63). The androgen regulation of
aldose reductase-like protein in the mouse vas deferens occurs
similarly through a proximal ARE with a thymidine in the sixth position
of the half-site TGAAGT tcc TGTTCT (64) as does the
junctional regulatory element of human glycoprotein hormone
-subunit gene GGTACT TGG TGTAAT (65).
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Strikingly, the flanking nucleotides of natural AREs have a remarkable
conservation of the selected nucleotide preferences shown in our
analysis. In particular, of the collated nucleotides of 18 natural
AREs, nine of the sequences have a guanine flanking the 5' half-site at
position -8, and six of those have a thymidine flanking the
corresponding 3'-half-site at position +8. A secondary predominance at
these positions was seen in 8/18 natural AREs with a thymidine or
adenine as a 5'-flanking nucleotide and with a guanine as a 3'-flanking
nucleotide. Our data suggest that these flanking nucleotides may
be selected by amino acids within the core of the DBD, consistent with
results of methylation interference analysis. Remarkably, there was a
predominance of T/G present in 14 of 18 AREs at position -11 flanking
the 5'-half-site in the collation of natural AREs (Fig. 7).
While some consistencies can be drawn between our nucleotide selection data and natural AREs, this analysis is limited by the scarcity of AREs presently known and confounded by the fact that many AREs are complex sites that overlap with other transcription factor-binding sites (Factor IX with HNF4 and ARR with AP1) or AR with itself (Slp promoter). Additionally, many genes regulated by AR require two binding sites acting cooperatively (probasin, PSA, hKLK, Slp, 6-PF-2K, and the AR gene). Furthermore, it should be noted that not all AREs listed are primarily regulated by AR (e.g. MMTV sites, 6-PF-2K). Thus if particular nucleotides in these natural AREs have a specific function, they may have evolved under the influence of multiple selection pressures, not just simply competition with GR.
The sequence specificity of PR has been less thoroughly characterized than that for AR and GR. However, it has been shown that a thymidine at position -6 (GTTACA) is partially responsible for inducing PR- preferential activation of this element in MMTV (26). Mutation of the spacer region of a PRE, particularly at position 0 also effected the relative affinity of PR for binding to this site (26), demonstrating that nucleotides not thought to be contacted directly by the receptor can effect DNA binding characteristics.
It is interesting to note in our data that for both AR and PR the DNA binding affinity is not necessarily proportional to the magnitude of transcriptional activation from the response element similar to that observed earlier (26). For instance, some nucleotides selected in our assay by virtue of receptor-DNA binding specificity, have reduced DNA binding affinity in comparison to the canonical element, but enhance the DNA transcriptional potency of the DNA element. This discordance of binding affinity with transactivation was demonstrated earlier for PR using site-directed mutagenesis of the PRE in the context of MMTV (26). This observation is most pronounced for the selected guanine at position -2, which dramatically decreased binding affinity to 1% of that seen with the consensus adenine, but was 3 times more transcriptionally active than the canonical element (26), similar to our observations. To a lesser extent, the nucleotide substitution of thymidine for adenine at -1 in the spacer region selected in our assay for PR specificity decreased binding 50%, but increased transcriptional activation 3.6-fold (26). Conversely, some nucleotides that increase DNA binding affinity, in both our affinity assay and in the earlier study of PR specificity, occur at a lower frequency in our receptor selectivity assay and impede transcriptional potency (26). This is illustrated by both thymidine to guanine substitutions at -5 and -6, which increase DNA binding affinity 1.5- and 3-fold respectively; whereas they are only 50% as transcriptionally active as would be expected from affinity alone.
Discordant binding affinity with transcriptional activation has also been documented for the C3 element with a variant guanine at -2 (AGTACG TGA TGTTCT), which lowers the DNA binding affinity for AR in comparison to a canonical site (GGTACA TGA TGTTCT), but simultaneously increases the transactivation potential by AR over the canonical element from 4- to 5.9-fold (34, 67). Thus it appears that this nucleotide substitution may have dual functions by influencing DNA binding specificity to promote preferential binding by AR and PR, and increase transactivation potential by an unknown mechanism. Further investigation of the influence of these nucleotide substitutions on transcriptional activity by steroid receptors is currently underway.
Another marked feature of our present analysis and those of previous investigators is that the preferred binding sites for all steroid receptors, including natural response elements, is, paradoxically, asymmetrical, and reorientation of the binding sites dramatically alters transcriptional activation (B. Matusik, personal communication and Refs. 26, 27, 28). Previous data have demonstrated asymmetry for the core half-sites, and our data have extended that observation to the spacer region and flanking sequences of both half-sites. This suggests that features of the DNA-binding region not directly contacted by amino acids of the DBD, possibly through DNA bending or other conformational changes, may differentially effect DNA binding specificity in a receptor-specific manner. The occurrence of extended influence of the DNA target adds to the complexity of the recognition site in perhaps an unrecognized manner.
One general feature of receptor-specific elements shown by in vitro DNA binding analysis is that they are of lower affinity in comparison to the canonical binding site. Conversely, the high-affinity canonical site is nondiscriminating for receptor-type responsiveness. Our data suggest that the optimal DNA binding affinity potential has not been realized by the receptor in the evolution of natural target elements to accommodate the needs of specificity. We hypothesize that suboptimal receptor binding sites (in terms of DNA binding affinity) may evolve to: 1) promote receptor specificity through discouraging the binding of inappropriate receptors by mechanisms of differential tolerance; 2) optimize promoter function through asymmetrical directionality of interactions; and 3) promote transcriptional activation by allosteric interactions through particular nucleotide deviations. Our data provide circumstantial evidence that these sequence variants in the half-sites contribute to DNA binding specificity of androgen or progesterone responses. In other studies, it is apparent that receptor specificity of the elements is often lost as the response elements are dissected and removed from their native promoters. This indicates that numerous characteristic features of the promoter and nuclear environment are integral parts of a composite function that collectively culminates in receptor specificity.
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MATERIALS AND METHODS |
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Oligonucleotides
All oligonucleotides used in this study were synthesized by
Dalton Chemicals (Toronto, Ontario, Canada) using trityl-on
synthesis followed by purification by TOPC (trityl-on purification
cartridge) chromatography. Selected oligonucleotides were
digested with BglII and cloned into the BamHI
site of pBluescript SK+/- (pBS) and propagated in JM109. All plasmids
were sequenced by PCR-based dideoxy termination (Fmole Sequencing Kit,
Promega Corp., Madison, WI) and analyzed on a gradient
sequencing gel. The synthetic, idealized binding elements were cloned
into the SmaI site of pBS in both the forward and reverse
orientations as determined by sequence analysis. The fragments were
excised with XhoI and SmaI for methylation
interference studies and binding affinity analysis.
Gel Mobility Shift Analysis
Purified DBD proteins were preincubated in DNA-binding buffer
and 0.2 mg poly dIdC for 10 min at room temperature in a volume of 10
µl. The radiolabeled probe was then added in a volume of 2 µl and
incubated at room temperature for 10 min. Samples were electrophoresed
at 15 V/cm on a 5% (29:1/acrylamide-bisacrylamide) gel in
0.5xTris-borate-EDTA that had been prerun for 10 min before
loading. Gels were either dried and autoradiographed on BioMax film
(Eastman Kodak Co., Rochester, NY) or selected DNA
fragments were isolated from the desired bands of gels after exposure
to BioMax film. DNA was eluted from gel fragments in Maxam and Gilbert
elution buffer (0.5 M NH4Ac, 0.1% SDS, 1
mM EDTA) after rotation overnight at room temperature.
Eluates were ethanol precipitated and resuspended in DNA binding
buffer.
The DNA sequences bound to the receptor-DBD in the randomized template
pool were separated by gel mobility shift analysis. The receptor
dimer-bound fraction of DNA sequences was excised and eluted from the
gel and amplified by PCR using the terminal primers (as depicted in
Fig. 1A). One third of the PCR amplification reaction of bound
sequences was radiolabeled by direct incorporation and subjected to
further selection in a band shift assay with a titration (50
nM, 300 nM, 600 nM) of lower
concentrations of recombinant receptor-DBD (to 50 nM
protein) to enrich for higher affinity binding sites. After four rounds
of sequential selection with a titration of 12 nM, 20
nM, and 50 nM in the final round, the
highest affinity sequences selected at 12 nM were cloned
(Fig. 1A
, pathway A). A minimum of 50 clones each were sequenced for
analysis of the highest affinity sequences for the DBDs of AR, GR, and
PR.
Selected Amplification and Binding Assays
The randomized oligonucleotide population (73 pmol) was
radiolabeled by primer extension with 584 pmol RUP (reverse
universal primer), 40 µCi in the presence of
-dAT32P, using the Klenow fragment of DNA
polymerase I in each initial round of selection to ensure that copies
of the 4.4 x 1012 possible sequences were present in
each initial binding reaction. The gel-purified radiolabeled probe was
incubated with either 100 nM AR-DBD, GR-DBD, or PR-DBD that
was purified from Factor Xa-cleaved GST fusion proteins to liberate the
respective 124-amino acid DBD of AR or PR from the GST protein moiety.
Factor Xa digestion of the GST-GR-DBD fusion protein produced an
87-amino acid fragment of the GR-DBD due to an additional cryptic
Factor Xa cleavage site in the hinge region, which removed the unique
receptor C-terminal region (depicted in Fig. 2A
) resulting in a
comparatively faster mobility of GR in gel shift assay (Fig. 2B
). The
DNA sequences bound to the receptor in the randomized template pool
were separated by gel mobility shift analysis. The receptor-bound
fraction of DNA sequences was excised and eluted from the gel, ethanol
precipitated, and resuspended in 10 µl of dH2O. Five
microliters of the isolated bound DNA were amplified by PCR using 50
pmol of RUP and FUP (forward universal primer) primers in PCR
reaction buffer containing 20 mM Tris-HCl (pH 8.4), 50
mM KCl, 0.2 mM deoxynucleoside
triphosphates, 1.5 mM MgCl2, and
Taq DNA polymerase (Life Technologies, Inc.,
Gaithersburg, MD). All PCR reactions were cycled as follows:
denaturation at 95 C for 1 min, annealed at 55 C for 1 min, and
extended at 72 C for 1.5 min. One third of the purified PCR
amplification reaction of bound sequences was radiolabeled by direct
incorporation and subjected to further selection in a band shift assay
with a titration (50 nM, 300 nM, 600
nM) of lowering concentrations of recombinant receptor-DBD
(to 50 nM protein) to enrich for higher affinity binding
sites. After four sequential rounds of selection with a titration of 12
nM, 20 nM, and 50 nM in the final
round, the highest affinity sequences selected at 12 nM
were cloned. A minimum of 50 clones were sequenced for analysis of the
highest affinity sequences for AR, GR, or PR. Compiled sequences were
statistically analyzed for significance of frequency distribution of
nucleotides by Pearson
2 test for goodness of fit in
which a P value of 1.0 indicates a random distribution of
nucleotides. The nucleotides in a given position were subjected to the
Pearson
2 test with 3 degrees of freedom to generate P1.
If P1 was less than 0.4, then P2 was generated on the remaining
nucleotide population by Pearson
2 test with 2 degrees
of freedom. Strongly selected nucleotides (P < 0.08)
were denoted in uppercase while nucleotide selected for to a
significant but lesser degree (P between 0.4 and 0.08) were
shown in lowercase.
After the second round of affinity enrichment of binding sites, selection of receptor type-specific sequences was performed by incubation of the medium-high affinity targets with 50 nM AR or PR concurrent with GR at a molar excess of 1-, 2-, or 3-fold. The bands of bound AR or PR at the highest concentration of GR still detectable after autoradiography were selected for another round of enrichment at a 1-, 3-, or 6-fold molar excess of GR in the binding reaction. AR-bound sequences isolated in the presence of a 6-fold excess of GR- and PR-bound sequences isolated in the presence of a 3-fold excess of GR were cloned and sequenced as described above.
Binding Affinity
For binding affinity analysis a constant amount of recombinant
protein (4 nM) was incubated with increasing concentrations
of radiolabeled DNA, as determined by the specific activity of
incorporation, ranging from 0.1 nM to 25 nM.
Bound and free fractions were separated by gel mobility shift assays,
and the dried gels were exposed on a phosphorimage screen.
Quantification of bound and free fractions were determined using a
Storm 860 phosphorimager (Molecular Dynamics, Inc.,
Sunnyvale, CA). Binding constants were determined by Scatchard analysis
as previously described (68).
Methylation Interference
The indicated binding sites were excised from pBluescript in
both orientations and uniquely end labeled by fill-in with the Klenow
fragment of DNA PolI in the presence of
-dCT32P. DNA was methylated by treatment with
dimethylsulfate. Methylation interference was performed using
approximately 30 nM protein incubated with 500,000 cpm of
single end-labeled methylated probe for 10 min at room temperature.
Bound and free fractions were separated by gel mobility shift assay and
excised and eluted from the gel in elution buffer with rotation
overnight. DNA was ethanol precipitated, resuspended in 10%
piperidine, incubated at 95 C for 30 min, and dried in a Savant speed
vac. The pellet was resuspended in water twice and dried in the speed
vac. The dried pellet was quantitated by Cherinkov counting and
resuspended to 2500 cpm/ml. Cleaved DNA products (5000 cpm) were
separated on a 10% acrylamide gel/8.3 M
urea/1xTris-borate-EDTA by electrophoresis at 80 watts for
2 h. Gels were dried and exposed to autoradiographic film or the
phosphorimage screen for quantification using the Storm 680 and
Imagequant software (Molecular Dynamics, Inc.).
Transcriptional Activation
The indicated response elements were cloned into the
EcoRV site of pBS (Stratagene, La Jolla, CA)
and excised by either BamHI and HindIII digestion
or BamHI and SacI digestion and cloned into the
corresponding restriction sites in the pMLUC reporter plasmid
(ATCC, Manassas, VA). PC3 cells were transfected with 0.2
µg of either rAR, hGR, or hPR cloned into
pRcCMV(Invitrogen, San Diego, CA), with 0.2 µg of
reporter plasmid and 5 ng of renilla expression plasmid, pRLTK
(Promega Corp.) using Lipofectin (Life Technologies, Inc.) in 24-well plates. Cells were either incubated in 5%
charcoal-stripped serum alone or with the addition of 10 nM
DHT, 10 nM dexamethasone, or 10 nM R5020 for
22 h. Cells were harvested with passive lysis buffer
(Promega Corp.), and 20 µl of lysates were analyzed
using Dual-Luciferase Reporter Assay System (Promega Corp.) on a luminometer (Berthold, Germany). Luciferase
activity was corrected using renilla activity and experiments done in
triplicate were averaged and expressed as fold induction.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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C.N. was a recipient of a National Cancer Institute of Canada Senior Research Fellowship and an Medical Research Council (MRC) Centennial Fellowship, supported by the funds of the Canadian Cancer Society and the MRC, respectively. This research was supported by an operating grant from the Medical Research Council of Canada.
Received for publication November 6, 1998. Revision received August 13, 1999. Accepted for publication September 8, 1999.
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REFERENCES |
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