Extreme Position Dependence of a Canonical Hormone Response Element
Steven K. Nordeen,
Carol A. Ogden,
Laima Taraseviciene and
Benjamin A. Lieberman
Department of Pathology and Program in Molecular Biology
University of Colorado Health Sciences Center Denver, Colorado
80262
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ABSTRACT
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Hormone response elements (HREs) are
considered enhancers, activating transcription in a relatively
position- and orientation-independent fashion. Upon binding to an HRE,
steroid receptors presumably contact coactivators and/or proteins
associated with the transcription initiation complex. As a receptor
target site is moved further from a fixed position such as the TATA
box, not only will the spatial separation of the receptor with respect
to its interaction partners change, so will the orientation due to the
rotation of the DNA helix. Additional constraints may be imposed by the
assembly of DNA into chromatin. Therefore, we have endeavored to test
rigorously the assertion that HRE action is position independent. We
have constructed a series of 42 chloramphenicol acetyltransferase
expression vectors that contain a single progesterone/glucocorticoid
receptor-binding site separated from a TATA box by 4 to 286 bp. The
enhancer activity of the HRE was assessed after transient transfection
of progesterone receptor-expressing fibroblasts. We find that the
position of the HRE has a dramatic influence on induction by
progestins. When closely juxtaposed to the TATA box, the HRE was unable
to support a hormone response, perhaps due to direct steric hindrance
with the transcription initiation complex. Full activity was gained by
moving the HRE 10 bp further from the TATA sequence. As the HRE was
moved incrementally further, activity remained near maximal over the
next 26 bp. HRE activity then declined over the subsequent 26 bp and
remained low for another 2.5 helical turns. Surprisingly, a narrow
window of HRE activity occurred at an HRE-TATA box separation of
90100 bp. Little or no hormone-induced transcriptional activity was
observed when the HRE was positioned further from the TATA box. The
addition of a second HRE or a basal (nuclear factor-1) element failed
to relieve this constraint. A similar series of experiments was carried
out in a mammary carcinoma cell line that expressed high levels of both
glucocorticoid and progestin receptors. Data in these cells indicate
that glucocorticoids and progestins supported a similar HRE
position-activity profile, but this pattern of HRE activity was quite
distinct from that seen in fibroblasts. This may be indicative of cell
type-specific interactions between steroid receptors and
adapter/coactivator proteins or cell type-specific activities such as
acetylases or deacetylases participating in the steroid response.
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INTRODUCTION
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Enhancers are defined operationally as transcriptional control
elements that increase gene expression in a heterologous context and
independently of orientation and position. The hormone response element
(HRE) of the mouse mammary tumor virus (MMTV) promoter was among the
first transcriptional control regions shown to meet these operational
criteria (1). While it is clear that activity of enhancers may decrease
over some distance, activity is generally retained when an enhancer is
positioned at sites ranging over several hundreds of bases from the
promoter. Indeed, glucocorticoid response elements of the tyrosine
amino transferase and rat uteroglobin genes are found 23 kb from the
start of transcription of the natural gene (2, 3).
There are a number of constraints that have the potential to favor or
proscribe the activity of a transcriptional control element at
different positions. Models often depict the DNA or chromatin template
folding back so that transcription factors binding the enhancer can
interact with proteins at the initiation complex. Such cartoons fail to
consider the inherent stiffness of DNA. The helical nature of DNA also
compounds the difficulty of devising models that account for
position-independent interaction of transcription factors especially
between separation distances of less than the persistance length of DNA
(4) (
150 bp). Packaging of DNA into chromatin can deform the path of
the DNA template to juxtapose factors but also introduces its own set
of constraints with regard to position independence.
Part of the reason that enhancers are relatively position independent
may be that they are generally aggregates of individual elements
(enhansons, Ref.5) that together determine the overall properties of
an enhancer. The multipartite structure may disguise position
constraints if, at a given position, some enhansons are in a favorable
location even though others may not be. The functional synergism
between enhansons and between enhansons and elements of the basal
promoter may also serve to circumvent positional constraints. The
mechanistic basis of synergism between transcriptional control elements
is poorly understood. In this study we have sought to evaluate whether
a single steroid receptor-binding site, in the context of a promoter
lacking control elements other than a TATA box, can activate
transcription in a position-independent manner. Our data indicate that
the activation potential of an HRE with a simple promoter exhibits
remarkable position dependence. The pattern of this activity is,
however, not readily reconciled in terms of known structural
constraints of DNA or its packaging as chromatin.
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RESULTS
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Vectors for Assessing Position Effects on the Activity of an
HRE
To assess the position dependence of an HRE in a systematic
fashion, we engineered a series of reporter vectors with a promoter
comprised simply of a TATA box and an HRE (Fig. 1A
). The HRE is comprised of an optimized
15-bp sequence binding a glucocorticoid or progesterone receptor (PR)
dimer. This straightforward constitution obviates complicating factors
introduced due to the interplay and synergistic behavior between
multiple receptor recognition sites or between receptor-binding sites
and other transcription control elements. A polylinker sequence
separates the TATA box and HRE so that vectors with different spacing
between the two elements could be readily generated.

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Figure 1. HRE-TATA Spacing Vectors
A, Schematic of the series of HRE-TATA vectors. Different spacings of
the HRE and the TATA box were generated by digesting and religating the
polylinker sequence as described in Table 1 . B, HRE-44 is the vector
from which all other vectors of this series are derived. The number
indicates the number of base pairs separating the 5'-T of the TATA box
and the 3'-most position of the HRE (the first C of the
XhoI site). C, HRE 165 is derived from HRE 44 by the
insertion of a 121-bp polylinker sequence. Vectors with numbers greater
than 56 are derived directly or indirectly from HRE 165 (Table 1 ).
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The progenitors of all the vectors in this series are shown in Fig. 1
.
An optimal glucocorticoid/progestin response element sequence was
cloned into the XhoI site of the vector pE1bcat that
contains a 14-bp sequence encompassing the TATA box of the adenovirus
E1b gene upstream of the chloramphenicol acetyltransferase (CAT)
reporter gene (6) (Fig. 1A
). This created HRE-44 (Fig. 1B
), a
hormone-responsive vector in which 44 bp separates the 3'-most base of
the HRE and the 5'T of the TATA sequence. A polylinker sequence was
removed from the vector pBend2 (7) with XhoI and ligated
into the SalI site of HRE-44, creating HRE-165 (Fig. 1C
) and
HRE-286 (insertion of two copies of the 121-bp polylinker). All other
vectors were derived from manipulation of HRE-44 and HRE-165 as
outlined in Table 1
. In all, a series of
42 vectors was constructed with HRE-TATA spacing from 4286 bp.
HRE Activity Assay
Each HRE vector of the entire series was transfected into 4F
cells. 4F cells are Ltk - fibroblasts engineered to express the B
isoform of human PR. 4F cells express about 0.8 pmol receptors per mg
protein (8). These cells were chosen because they transfect well and
because the high expression of PR gave a robust hormone response with
HRE-44, the progenitor of the entire vector series. Although these
cells express GR, they do so at low levels and support only a small
induction of CAT activity in response to glucocorticoid with HRE-44.
With such a large series of vectors, it is not possible to assess all
in a single experiment. To normalize between experiments HRE-44 was
assessed in all experiments. The induction obtained with HRE-44 was set
at 100%, and induction data for other plasmids were calculated
relative to that standard within each experiment. The HRE activity data
for the entire set of plasmids are plotted in Fig. 2
relative to HRE-44. Each point
represents the average of data from 3 to 11 independent experiments
(median 5) where that spacing was assayed. It is predominantly the
poorly inducible to uninducible HRE-plasmids at the larger HRE-TATA
separations that were assayed only three times. Some plasmids
consistently exhibit greater inducibility than HRE-44 and many exhibit
less. When the HRE is closely juxtaposed to the TATA box (HRE-4), the
promoter is uninducible. It is likely that steric interference prevents
the simultaneous occupancy of the HRE and TATA box by receptor and the
basal transcription complex. However, moving the HRE an additional 8 bp
further from the TATA box permits recovery of some activity, while
increasing the separation another 2 bp (HRE-14) results in a plasmid
that supports an induction consistently greater than for HRE-44. The
HRE activity remains consistently greater than the HRE-44 standard for
all plasmids from HRE-14 to HRE-40. From HRE-40 through HRE-66 the HRE
inducibility declines steadily to baseline. Inducibility remains
negligible as the HRE-TATA separation is increased to about 88 bp.
There is a sharp peak of inducibility between HRE-88 and HRE-100.
Little or no HRE activity is exhibited by plasmids HRE-103 through
HRE-169. One even larger spacing tested, HRE-286, also exhibited no
inducibility.

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Figure 2. Position Dependence of the HRE in Response to the
Progestin R5020 in 4F Cells
In a large series of experiments each of 42 HRE vectors representing 38
different HRE-TATA box separations of 4286 bp was transfected into 4F
cells, and the inducibility was assessed by activity of the CAT
reporter. Inducibility of HRE 44 was assessed in each experiment, and
all inductions were normalized to HRE 44, which was assigned an
induction of 100%. HRE-44 is indicated by the solid
circle. The average CAT activity in 4F cells transfected with
HRE-44 was 4 pmol product/mg protein/min without hormone, a level
indistinguishable from assay background, and 1300 pmol product/mg
protein/min with hormone treatment. Thus, the average magnitude of
induction of HRE-44 by R5020 is more than 300-fold above background.
The other plasmids were assessed in 3 to 11 independent experiments
(median 5) and tested in duplicate transfections within an experiment.
It is generally the plasmids with the larger HRE-TATA separations where
little or no induction is observed that were repeated in only three
experiments. Several vectors, HRE-24, HRE-40, HRE-94, and HRE-165, were
constructed in two ways to test whether flanking polylinker sequences
were influencing inducibility. In each case, both vectors gave similar
results and data points represent the average of the two. Data points
at or below the horizontal dashed line are effectively
uninducible as compared with the activity of HRE-less pE1bCAT in
hormone-treated cells.
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It is clear from these data that the activity of a single HRE is highly
position sensitive. There is, however, no hint of a helical
periodicity. One can operationally define three domains each defined by
six or seven HRE plasmids and each spanning about two and one-half
helical turns. The closest to the TATA box is a region where HRE
activity is high. HRE activity declines steadily in the second domain
and is negligible in the third. Adjacent to this domain, there is a
narrow window where significant HRE function is recovered before again
falling to baseline at all greater HRE-TATA separations. This pattern
is not readily explicable in terms of the structure of a nucleosome or
the helical structure of DNA, as will be discussed.
HRE Position-Activity Profile: Progestins vs.
Glucocorticoids
We wanted to compare the activity profile of the same series of
vectors in response to glucocorticoids to test whether the two
receptors may exhibit distinct position preference for their shared
target element. 4F cells are glucocorticoid responsive. However, they
contain low levels of glucocorticoid receptors (GRs) and show only a
barely detectable response with HRE 44 and several of the other vectors
that respond well to progestin treatment in 4F cells. Therefore, we
have performed another large series of transfection experiments with
T47DA/12 cells, a derivative of T47D mammary carcinoma cells that
have been engineered to express high levels of GR along with the high
expression of endogenous PR. We have used these cells extensively to
compare glucocorticoid and progestin action (9, 10). Here we have
compared the induction of the series of HRE-spacing vectors by the
synthetic glucocorticoid, dexamethasone, and the synthetic progestin,
R5020. For every inducible vector, the absolute value of CAT activity
is higher with dexamethasone than R5020 despite the fact that there is
somewhat less GR than PR in these cells. We previously observed that
transiently transfected mouse mammary tumor virus promoter also
exhibits a greater response to glucocorticoids in these cells (9).
Figure 3
compares the glucocorticoid and
progestin induction for the entire series of HRE-TATA-spacing vectors.
The activity of each spacing vector is normalized to the induction of
HRE 44 with that hormone. The overall HRE activity-position profile is
similar for both receptors showing only some minor quantitative
differences that are less remarkable than the extent of similarity.
Indeed, the two patterns are, for the most part, superimposable,
suggesting that the two receptors act or are acted upon by similar
mechanisms to regulate transcription in T47D/A12 cells.

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Figure 3. Position Dependence of the HRE in Response to
Progestin or Glucocorticoid in T47DA/12 Cells
Plasmids from the HRE library were transfected into T47D/A12 cells,
and CAT activity was assessed after treatment with R5020,
dexamethasone, or vehicle. HRE-44 was assessed in every experiment. All
R5020 inductions were normalized to the R5020 induction of HRE-44, and
all dexamethasone inductions were normalized to the dexamethasone
induction of HRE-44. Hormone inductions of HRE-44 were assigned an
induction value of 100% (solid symbols). The average
CAT activity in T47D/A12 cells transfected with HRE-44 was 3 pmol
product/mg protein/min without hormone, a level indistinguishable from
assay background. Activity after R5020 treatment averaged 27 pmol/mg
protein/min and, after dexamethasone treatment, 66 pmol/mg protein/min.
These activities represent a magnitude of induction 9-fold and 22-fold
above background. Data points for the remaining plasmids represent the
average of two to six independent experiments with each transfection
done in duplicate in an experiment. In general, it is the uninducible
plasmids at the larger HRE-TATA separations for which only two
independent experiments have been done.
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HRE Position-Activity Profile: Cell Type Dependence
It is readily apparent that the HRE position-activity profile for
progestin induction differs between 4F fibroblasts and T47D/A12
mammary carcinoma cells. R5020 induction data from Figs. 2
and 3
have
been replotted on the same graph for direct comparison (Fig. 4
). Both cell types exhibit a narrow
activity peak at about HRE-94 but from there the profiles are quite
different. In T47D/A12 cells there is a very narrow peak at HRE-82
that is completely absent in 4F cells. Note that the same plasmid
preparation of HRE-82 was used in both series of experiments. In
T47D/A12 cells there is a peak of activity at HRE-48 and modest
inducibility from HRE-14 through HRE-30 in contrast to the broad
pattern of activity of HRE-14 through HRE-40 in 4F cells. The
differences in the HRE position-activity profile in the two cell lines
suggest that protein-protein interactions between receptor and other
components of the transcription apparatus differ between cell
types.

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Figure 4. HRE Position Dependence: 4F vs.
T47D/A12 Cells
R5020 induction data from Figs. 2 and 3 have been replotted to compare
directly the HRE position-activity profile in the two cell lines.
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Multiple Elements and HRE Action at a Distance
The data we have presented indicate that an isolated HRE in
the context of a simple promoter composed of only a TATA box does not
act in a position- independent manner. Indeed, HRE activity is
extremely position dependent for both GR and PR action. Thus, in this
circumstance, an HRE does not fulfill the operational criteria of an
enhancer. One possibility suggested by these data is that position
independence may be conferred by the interaction of multiple elements
acting in a synergistic fashion just as position-dependent SV40
enhansons interact to create a position-independent enhancer (5). We
tested whether the responsiveness of a poorly inducible HRE vector
could be restored by introduction of a second HRE or another
transcription control element at a position where the second element
cannot itself promote transcription. An 18-bp oligonucleotide with an
HRE sequence was cloned in both orientations at the SpeI
site of HRE-165 (Fig. 1C
). This HRE is separated from the TATA box by
127 bp in orientation 1 or 130 bp in orientation 2 and from the distal
HRE by 41 and 38 bp, respectively. The two HRE constructs were
transfected into 4F fibroblasts, and the induction of CAT activity by
R5020 was compared with HRE-165, HRE127, or HRE-44. As before,
induction of the latter averaged more than 300-fold and was assigned a
value of 100%. Consistent with the data of Fig. 2
, little induction of
HRE-165 and HRE-127 was seen (3.4% ± 2.7 and 10.8% ± 8.5, Fig. 5
). The two HRE constructs were somewhat
inducible (25.0% ± 10.8 and 27.8% ± 8.8 compared with the HRE-44
single HRE construct). Thus, a pair of HREs can support more
induction than each does alone, but the modest synergism seen suggests
the existence of additional constraints and/or the need for additional
mechanisms to relieve spatial limitations of HRE action. It should be
noted that the two HRE constructs, although less inducible than HRE44,
still exhibit 80-fold inductions in these circumstances. This suggests
that an alternative way to approach these data is to ask not why the
more distant elements give small inductions but rather why HRE-44 and
closer elements are so effective.

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Figure 5. An Additional HRE or Basal Element Fails to Relieve
Spatial Constraints on HRE Action
4F cells were transfected with plasmids containing a second HRE or a
basal transcription element (NF-1). In the 2HRE vectors, both
individual HREs are at positions that permit little or no inducibility.
The activity of the HRE-NF-1 vector, like the HRE vectors, was at or
below background in the absence of hormone (R5020). The results are
representative of three or four experiments. The mean induction
relative to HRE-44 is plotted ± 1 SE.
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We also tested whether the presence of a target sequence for a basal
transcription factor could functionally synergize with a single HRE to
relieve spatial constraints on hormone inducibility. We chose to
introduce a nuclear factor-1 site into the HindIII site of
HRE-165 since a nuclear factor-1 site is present in the strongly
inducible mouse mammary tumor virus promoter. However, the nuclear
factor-1 site did not promote R5020 inducibility in the context of the
HRE-165 promoter (Fig. 5
). The presence of the nuclear factor-1 site
also failed to stimulate a detectable glucocorticoid induction in
response to dexamethasone in 4F cells (data not shown). These results
highlight how little we really understand about the spatial constraints
on factor-factor interaction and the mechanisms that account for the
relative lack of such constraints on HRE action in the context of
natural control elements.
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DISCUSSION
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HREs have been classified as enhancers based on data indicating
steroid-dependent transcriptional activation to be relatively
insensitive to position, orientation, or the basal promoter employed.
This is consistent with observations that the HREs of natural promoters
are found at varying distances from the promoter. Glucocorticoid
response elements have been identified more than 2 kb distant from the
transcription initiation site (2, 3). However, natural steroid response
elements are generally composed of multiple receptor binding sites that
may be located over some distance and that may act synergistically.
Also, steroid receptors may work in conjunction with other factors such
that these additional promoter elements may have significant influence
on the hormone response. Such assemblages in the tyrosine amino
transferase gene and the phosphoenolpyruvate carboxykinase gene have
been termed glucocorticoid-respons(iv)e units (11, 12, 13). Thus, natural
response elements and response units may achieve position
independence as the result of the contribution of multiple elements to
activity.
The present studies were designed to systematically test whether a
single HRE in the context of a basal promoter composed only of a TATA
box is capable of functioning in a position-independent manner. In PR
expressing 4F fibroblasts, the HRE exhibited a complex pattern of
distance dependence as shown in Fig. 2
. There was no evidence of a
10-bp periodicity that would implicate a constraint imposed by the
helical nature of DNA. However, we find that the distance-activity
pattern depicted in Fig. 2
is also not easily accommodated in terms of
nucleosome structure. It should be noted that the beginning of the
broad plateau of high HRE activity (HRE-14 to HRE-40) is positioned
about 80 bp (or one wrap of the DNA around a nucleosome) from the sharp
spike of activity exhibited by HRE 94a, HRE-94b, and HRE-98. However,
arguing against a role for nucleosomes in this pattern are the reports
that although transiently transfected DNA is packaged into chromatin,
the nucleosomes are not arrayed in the same specific pattern as seen
with stable transfections (15). Yet, it is conceivable that binding of
a steroid receptor could determine the local positioning of nucleosomes
around the promoter. This could favorably position the TATA box so as
to permit access by the TFIID complex. Arguing against such simple
models accounting for the position dependence of the HRE are the
progestin induction data in T47D/A12 cells. The HRE position-activity
profile obtained in this line is quite different than that of 4F cells
(Fig. 4
). In both cases, however, a peak of activity is seen at an
HRE-TATA spacing of 94 bp, and no induction is seen at spacings greater
than 100 bp. It is not obvious what determines this limit nor the
cell-specific pattern of induction at lesser spacing of the HRE from
the TATA box. Nonetheless, these data would argue that it is the
interaction of the receptor with factors expressed in a cell
type-specific manner rather than general factors that determine hormone
responsiveness of this simple promoter. Such cell-specific factors
could be involved in the tissue-specific expression of agonist activity
by steroid antagonists.
We had anticipated that glucocorticoid response might show a different
HRE position-activity profile than the progesterone response. Counter
to these expectations, GR and PR mediated a similar pattern of response
throughout the library of HRE position vectors. Thus, with this
template the activation domains of the two receptors may interact in a
similar manner with other components of the transcription apparatus.
There clearly must exist other mechanisms to distinguish glucocorticoid
and progestin action since the two hormones may have very different
physiological activities in tissues such as the mammary epithelium that
express both receptors. In other work we have shown that a stably
integrated mouse mammary tumor virus promoter is differentially induced
by glucocorticoids in a mammary carcinoma cell line (10) in a
locus-specific fashion (J. R. Lambert and S. K. Nordeen,
submitted), implying that the packaging of promoters as stable
chromatin and the influence of the surrounding chromatin may play a
commanding role in governing hormone inducibility.
In summary, we have used a strategy that employs simplified promoter
constructs to make interpretation of the results as straightforward as
possible. Of course, there are caveats to any reductionist approach. As
with natural promoters, it is not possible to guarantee that any DNA
sequence is completely inert. Thus, we cannot rule out that the
polylinker sequence is structured in some unusual fashion that could
affect hormone action or that factors, possibly cell type specific, may
bind to the polylinker DNA, thereby enhancing or suppressing hormone
action. We have considered this where possible. For example, certain
constructs, such as the active HRE-94 spacing, were made in more than
one way to minimize the possibility that a chance juxtaposition
fortuitously created a factor-binding site or an unusual DNA structure.
Over the entire set of HRE-TATA vectors, various portions of the
polylinker sequence are present in different vectors, suggesting that
no single sequence could easily account for the complex
spacing-activity patterns observed or the cell type differences of this
pattern.
Introduction of a second HRE or a nuclear factor-1 site into the poorly
inducible HRE-165 construct results at best in limited relief of
spatial constraints on hormone induction. Again, in this reductionist
approach it is difficult to guarantee that functional synergism would
not have been seen if the constructs were formulated differently. A
GRE2tkCAT construct exhibited considerably more
inducibility than a single GRE construct (16), but this is a more
complicated circumstance since the basal thymidine kinase promoter
contains at least three elements, two GC boxes and a CCAAT box. Sathya
et al. (17), in a paper published after submission of our
work, analyzed the functional consequences of multiple estrogen
response elements (EREs) and indicated that a single ERE 52 bp upstream
of the TATA box of a minimal promoter gave a borderline induction
(1.2-fold). This was improved only marginally by the addition of a
second ERE (1.9-fold induction). Addition of a third or fourth ERE did
lead to a synergistic increase in inducibility (16- and 38-fold,
respectively). These authors went on to show that the presence of other
basal elements in the promoter, spacing between the EREs, and position
of the EREs with respect to the promoter all influenced the functional
synergism of EREs (17). Together, this work and ours suggest that the
synergistic interactions between multiple HREs or between HREs and
other transcription control elements must not be completely promiscuous
and are, therefore, subject to constraints of their own. These data
highlight the paucity of our understanding of how steroid receptors and
other transcription factors can communicate with the basal
transcription apparatus in many different promoter contexts and over a
remarkable variety of configurations. No understanding of mechanisms of
steroid receptor action will be complete without a better
conception of how receptors deal with these cir-cumstances.
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MATERIALS AND METHODS
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Plasmids
A hormone-responsive reporter gene vector bearing a single HRE
was constructed by introducing a synthetic, optimized HRE into the
XhoI site of plasmid pE1bCAT (6). The promoter of the E1bCAT
consists of a 14-bp DNA sequence spanning the TATA box of the
adenovirus E1b gene embedded in a polylinker sequence upstream of the
CAT reporter gene. The 3'-most base of the HRE is separated by 44 bp
from the first T of the TATA box. This vector is termed HRE-44 (Fig. 1
). A 121-bp XhoI fragment containing a polylinker sequence
from pBend2 was cloned at the SalI site of HRE-44 between
the HRE and the TATA box. The separation of the HRE and TATA box was
increased to 165 bp (HRE-165). A clone that contains two copies of the
polylinker was also isolated (HRE-286). All of the remaining plasmids
in the HRE series were derived from HRE-44 or HRE-165 and their
products by cutting with the indicated restriction enzyme(s), blunting
the ends where necessary with Klenow polymerase or Mung Bean nuclease,
and religation (Table 1
). The sequence of all HRE plasmids was
verified, and the instances of deviation from the predicted joint are
indicated in Table 1
.
The 2HRE-1 and 2HRE-2 plasmids were constructed by introducing the
oligonucleotide; 5'-CTAGTGGTACAAACTGTT-3'; 3'-ACCATGTTTGACAAGATC-5'
into the SpeI site of HRE-165. The plasmid 2HRE-1 has one
copy of the oligonucleotide in the orientation shown. 2HRE-2 has one
copy of the oligonucleotide in the opposite orientation. HRE-nuclear
factor-1 (NF-1) was constructed by introducing the oligonucleotide
5'-AGCTTGGCTTGAAGCCA-3'; 3'-ACCGAACTTCGGTTCGA-5' into the
HindIII site of HRE-165.
Transfection and Reporter Assays
The development of PR-expressing 4F fibroblasts and
GR-expressing T47D/A12 mammary carcinoma cells has been described (8, 9, 18). Both lines are cultured in modified Eagles medium supplemented
with 5% FBS, glutamine, and penicillin + streptomycin. Additionally,
4F medium contains HAT (hypoxanthine-aminopterin-thymidine), and
T47D/A12 medium contains Geneticin. Geneticin is not included when
stock cultures are split for transfection experiments as GR expression
has been found to be stable for some time in the absence of continued
selection. Both cell lines were transiently transfected using a
diethylaminoethyl (DEAE)-dextran protocol.
4F cells were plated on 60-mm culture dishes at 1.4 x
106 cells per dish the day before transfection. For
transfection, cells were exposed to 1 ml growth medium containing 200
µg/ml DEAE-dextran, 2 µg/ml test plasmid, 0.5 µg/ml of internal
transfection control plasmid (pSV2luc), and 100
µM chloroquine for 2 h. Transfection medium was
replaced by shock buffer (137 mM NaCl, 5 mM
KCl, 0.7 mM Na2HPO4, 6
mM glucose, 21 mM HEPES, pH 7.1) with 15%
dimethylsulfoxide for 6 min before cultures were returned to growth
medium. Two days after the start of transfection, R5020 (10
nM) or vehicle was added and the incubation was continued
for 24 h before harvest. For both 4F cells and T47D/A12 cells
all plasmids were transfected in duplicate dishes for each
treatment.
T47D/A12 cells were plated on 60-mm culture dishes at 1.4 x
106 cells per dish the afternoon before transfection. For
transfection, cells were exposed to 1 ml of growth medium containing
500 µg/ml DEAE-dextran, 2 µg/ml test plasmid, and 0.1 µg/ml of
internal transfection control plasmid [cytomegalovirus(CMV)-ßgal).
The transfection solution was replaced after 4 h with 1 ml shock
buffer with 15% dimethylsulfoxide for 6 min. This was replaced by
growth medium containing 100 µM chloroquine for 2 h
before returning to growth medium. Two days after the start of
transfection, hormone (R5020 10 nM or dexamethasone 100
nM) or vehicle was added and the incubation was continued
for 24 h before harvest.
To harvest, cells were rinsed twice with wash buffer (40 mM
Tris-HCl, 140 mM NaCl, 1 mM EDTA, pH 7.4) and
then lysed with 500 µl lysis buffer (20 mM
K2HPO4, 5 mM MgCl2,
0.5% Triton X-100, pH 7.8). Lysate was transferred to a microfuge
tube, and insoluble debris was pelleted by centrifugation. Reporter
gene assays were performed on the cleared lysate.
CAT activity was assessed by an enzymatic/organic extraction method
described previously (19). Acetylation cocktail (200 µl) was
incubated for 5 min at 37 C. To this was added 50 µl of lysate
supernatant, and the incubation was continued for 4 h. The
components of the cocktail were at the following concentrations after
addition of lysate: Tris-Cl, pH 7.8, 100 mM;
MgCl2, 6 mM; KCl, 100 mM; sodium
acetate, 0.4 mM; ATP, 3 mM; Coenzyme A, 0.4
mM; chloramphenicol, 1 mM. Each reaction also
contained 7.8 µCi [3H]acetate and 0.015 U acetyl CoA
synthetase (Sigma, St. Louis, MO). The CAT assays were stopped by
pipetting an aliquot (100 µl) into 1 ml benzene and immediately
vortexed. The phases were separated by centrifugation. An aliquot (750
µl) of the upper phase was removed to a scintillation vial. The
benzene was evaporated overnight in a fume hood to lower background
(19), and scintillation fluor (Beckman Ready Safe, Beckman Instruments,
Fullerton, CA) was added to the dry scintillation vials. Duplicate
aliquots were assayed from each reaction.
For luciferase assays, lysate (1550 µl) was added to 350 µl
luciferase buffer (100 mM K2HPO4, 1
mM dithiothreitol, 5 mM ATP, 15 mM
MgSO4, pH 7.8) The reaction was initiated by the injection
of 100 µl 1 mM luciferin and light output integrated for
10 sec after a 2-sec delay using a Monolight 2001 luminometer
(Analytical Luminescence Laboratories, Ann Arbor, MI). For
ß-galactosidase, 25 µl of lysate were added to 100 µl of
Galactolight reaction buffer containing the Galacton-Plus substrate
(Tropix, Bedford, MA), and the mixture was incubated at room
temperature for 1 h. Using the Monolight 2001 luminometer,
readings were taken after the injection of 100 µl Galacto-light
accelerator into the reaction mixture. CAT induction data calculated by
normalizing for the internal transfection control were compared with
induction data normalized to protein content of the lysate or to
unnormalized induction data. Similar results were obtained by all three
methods.
 |
ACKNOWLEDGMENTS
|
---|
Cell culture medium was obtained through the Tissue Culture and
Monoclonal Antibody Core of the University of Colorado Cancer Center.
Some of the vector sequence confirmation was done by the Sequencing
Core of the Cancer Center.
 |
FOOTNOTES
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Address requests for reprints to: Steven K. Nordeen, Department of Pathology B216, University of Colorado Health Sciences Center, 4200 East 9th Avenue, Denver, Colorado 80262.
This work was supported by NIH Grant DK-37061.
Received for publication June 11, 1997.
Revision received December 9, 1997. Revision received February 10, 1998.
Accepted for publication February 16, 1998.
 |
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