Multiple Pit-l-Binding
Sites Facilitate
Estrogen Responsiveness
of the
Prolactin Gene
Barbara
E. Nowakowski
and Richard
A. Maurer
Department of Cell Biology and Anatomy
Oregon Health Sciences University (B.E.N.,
Portland, Oregon 97201
R.A.M
Molecular Biology Ph.D. Program
University of Iowa
Iowa City, Iowa 52242
Previous studies have shown that estrogen responsiveness of the rat PRL gene requires the presence
of both the estrogen receptor and the tissue-specific
transcription
factor, Pit-l. To examine the contribution of individual Pit-l-binding
sites in permitting
an
estrogen response, we mutated specific sites in both
the proximal and distal regions of the rat PRL gene.
The studies reveal that mutation
of Pit-l-binding
sites in either the proximal or the distal region can
have an effect on estrogen
responsiveness.
The
most important Pit-l-binding
site appears to be the
site in the distal enhancer, which is adjacent to the
estrogen
receptor-binding
site. However, mutation
of combinations
of other Pit-l-binding
sites reveals
that these sites also contribute
to the estrogen
response of the PRL gene. The binding sequences
for
another transcription
factor cannot substitute for Pit1 sites in bringing
about a wild-type
estrogen
response, as shown by replacement
of Pit-l-binding
sites with a consensus
CAMP-responsive
element.
Conversion
of the imperfect
palindromic
estrogen
response
element of the PRL gene to a perfect
palindrome
eliminated
the positive effects of an intact 1D Pit-l-binding
site. To examine potential physical interactions
between the estrogen receptor and
Pit-l, a protein interaction
assay was performed.
The results
demonstrate
that labeled
estrogen
receptor can bind to Pit-l immobilized
on glutathione
agarose beads. However, most of the interaction
between Pit-l and the estrogen receptor appears to
be DNA dependent.
Overall, the results demonstrate
a distributed
role for multiple Pit-l sites in permitting
an estrogen response of the rat PRL gene. (Molecular Endocrinology
8:1742-1749,1994)
the transcription
of specific genes. Early studies demonstrated that estrogen increases PRL messenger RNA
through effects at the transcriptional
level (1). The finding that estrogenic induction of PRL gene transcription
is not dependent on protein synthesis suggested that
this regulation probably involves a direct effect of the
receptor (2). Subsequently,
it was shown that a region
located approximately
1.7-l 5 kilobases
upstream
from the transcription initiation site was required for an
estrogen response of the PRL gene (3, 4). This upstream region is designated the distal enhancer of the
PRL gene. The distal enhancer region was also found
to contain a high affinity binding site for the estrogen
receptor (ER). The sequence of the receptor-binding
site, TGTCACTATGTCC,
resembles the palindromic
consensus ER-binding site, GGTCANNNTGACC,
but is
not a perfect palindrome (3,4). A mutation that disrupts
this imperfect palindrome abolishes estrogen responsiveness of the PRL gene (5). The combination of these
findings suggested that the estrogen response of the
PRL gene was mediated by binding of the ER to a site
in the distal enhancer region of the gene. This receptorbinding site was thought to function as an estrogenresponsive DNA element.
Subsequent studies demonstrated
that the estrogen
response of the PRL gene is more complicated
and
requires DNA elements other than the ER-binding site.
These studies demonstrated
that the ability of estrogen
to activate transcription
of the PRL gene appears to
require the presence of both the ER and the tissuespecific transcription factor, Pit-l (6, 7). Pit-l is a member of a group of transcription
factors that are designated POU factors (8, 9). Pit-l binds to multiple sites in
both the proximal region and the distal enhancer of the
PRL gene and is crucial for pituitary-specific expression
of the PRL gene (6, 7, 1 O-l 6). One of the Pit-l -binding
sites in the rat PRL gene is located immediately adjacent to the ER-binding site (11, 17). Deletion studies
demonstrated
that Pit-l-binding
sites in the distal enhancer are required to permit a response to estrogen
(6). Furthermore,
in heterologous
cells, the distal enhancer of the PRL gene was not able to mediate a
INTRODUCTION
The rat PRL gene has provided a useful system
examine the requirements
for estrogenic regulation
0t?8aa810/94$03.00/0
Molecular Endowtnology
Copyright CD 191994 by The Endocrine
to
of
Sodety
1742
Pit-l
and Estrogen
Responsiveness
response to estrogen unless the cells were transfected
with an expression vector for Pit-l (6,7). These studies
suggest that the estrogen response of the PRL gene
requires a functional “unit” involving several factorbinding sites, similar to the glucocorticoid
response unit
of the phosphoenolpyruvate
carboxykinase
gene (18).
Recently, it has been shown that the estrogen response of the PRL gene involves communication
between the distal enhancer and proximal promoter elements. One line of studies demonstrated
that estrogen
treatment resulted in an increase in nuclease hypersensitivity of the proximal promoter region (19). Subsequently, a nuclear ligation assay was used to demonstrate that the distal enhancer of the rat PRL gene is
located physically close to the proximal region (20). This
finding raises the strong possibility that the estrogen
response involves functional and physical interactions
between the distal and proximal promoter elements. As
Pit-l binds to multiple sites in both the distal enhancer
and proximal regions of the PRL gene, this tissuespecific transcription
factor may be a candidate for
mediating interactions between these two regions. In
the present study, we examined the role of individual
Pit-l-binding
sites in both the proximal and distal enhancer regions in mediating an estrogen response of
the rat PRL gene. The results demonstrate
that the
estrogen response requires multiple Pit-l -binding sites
in both the distal and proximal regions of the PRL gene.
The requirement for the presence of Pit-l to activate
multiple sites is probably a substantial part of the mechanism that prevents estrogen from activating transcription of the PRL gene in nonpituitary tissues that contain
ER.
RESULTS
Estrogen Responsiveness
of the PRL Gene
Can Be Abolished
by Mutation of Proximal Pit-lBinding Sites
To test the possibility that Pit-l-binding
sites in the
proximal region of the PRL gene may contribute to the
estrogen response, we prepared reporter genes in
which varying combinations
of proximal Pit-l -binding
sites were mutated. For these studies, a reporter gene
was prepared in which restriction sites were introduced
into the PRL 5’-flanking region so that cassettes containing the proximal or distal regions could be easily
replaced. The sites were introduced in a manner so that
the normal spacing between the distal enhancer and
proximal region was maintained. We then tested reporter genes in which various combinations
of Pit-lbinding sites in the proximal region of the PRL gene
were disrupted by clustered point mutations (Fig. 1).
The four Pit-l-binding
sites in the proximal region are
designated the 1 P, 2P, 3P, and 4P sites, with the 1 P
site located closest to the start of transcription
(17).
Mutation of individual lP, 2P, 3P, or 4P sites or a
combination
of 2P, 3P, and 4P sites reduced basal
1743
Fig. 1. Mutation
of Multiple Pit-l -Binding Sites in the Proximal
Region of the Rat PRL Gene Affects Estrogen Responsiveness
GH3 cells were transfected
with wild-type
or mutant reporter
genes containing
1.9 kilobasepairs
of the PRL gene linked to
luciferase.
The mutant
reporter
genes contained
clustered
point mutations
that disrupted
specific Pit-l-binding
sites in
the proximal
region of the PRL gene (41). A schematic
map is
shown, which indicates
the relative positions
of Pit-l -binding
sites and the specific sites that were disrupted
by mutations.
Pit-l -binding sites are numbered
as described
by Nelson et al.
(17) with sites in the proximal
region designated
1 P to 4P and
sites in the distal region designated
1 D to 4D. Also indicated
is the position of the ER-binding
site (ER) in the distal enhancer
(3). After transfection,
the cells were treated with no addition
(basal) or 10 nrv estradiol (E2) and then collected 24 h later for
analysis of reporter
gene activity. Results are from two separate experiments,
which each contained
three separate
transfections
for each DNA construct.
Data are presented
as a
percentage
of the wild type values and fold inductions in
response
to estradiol
over basal for each mutation.
A schematic map of the PRL 5’-flanking
region indicates the position
of wild-type
Pit-l -binding sites (M), mutant Pit-l -binding sites
(W), and the ER-binding
site
activity significantly, but had little effect on the ability of
estrogen to stimulate reporter gene expression. However, a mutation encompassing
all of the proximal Pit1 -binding sites (1 P, 2P, 3P, and 4P mutant) produced
a very large decrease in basal expression (lOO-fold),
which was near the level observed with promoterless
controls. Considering
the construct containing mutations in all of the proximal Pit-l-binding
sites appears
to be essentially inactive, it is probably not appropriate
to consider the estrogen responsiveness of this reporter
gene.
The finding that mutation of all of the Pit-l-binding
sites in the proximal region substantially
reduced the
estrogen response may simply indicate a requirement
for the presence of proximal binding sites for transcription factors, leading to the assembly of a competent
transcription
complex. Alternatively,
there may be a
specific requirement
for Pit-l. To test this possibility,
two different approaches
were employed. In the first
approach, the proximal region and other sequences
were deleted so that the distal enhancer was moved
adjacent to the TATA box of the PRL gene (Fig. 2A). In
this case, an estrogen response was increased somewhat. Thus, the proximal Pit-l sites are not required if
the distal region is moved from its usual, far up-stream
position so that it is adjacent to the start of transcription.
Vol8No.12
MOL ENDO. 1994
1744
B.
Fig. 2. Effects of Deletion or Substitution of Proximal Pit-lBinding Sites on Estrogen Responsiveness of the PRL Gene
Reporter genes containing varying portions of the rat PRL
gene, as indicated, were transfected into GH3 cells. The distal
plus TATA construct contains the -1769 to -1495 region of
the PRL gene ligated to the -29 to 38 region of the PRL gene.
For the distal plus TK construct, the -208 to 38 proximal
region of PRL gene was replaced by the -105 to 58 region of
the herpes simplex TK promoter (Distal PRL+TK). After transfection, the cells were treated with no addition (basal) or 10
nM estradiol (E2) and collected 24 h later for analysis of reporter
gene activity. Results are from two separate experiments,
which each contained three separate transfections for each
DNA construct. Values are the mean + SEM, with data expressed as a percentage of wild type values or fold inductions
in response to estradiol.
In the secondapproach, the proximal regionof the PRL
gene was replacedwith the thymidine kinasepromoter
(Fig. 28). Again, the estrogen responsewas similarto
that obtained with the wild-type construct. These findings suggest that when the distal enhancer is located
in its normal position, a transcriptionally competent
proximal region is required. However, this functional
proximal region does not necessarilyneed to contain
Pit-l -bindingsites.
Distal Enhancer Pit-l-Binding
for Estrogen Responsiveness
Sites Are Required
A previous study found that deletion of the sequences
in the -1769 to -1665 regionof the PRL gene reduced
responsesto estrogen, even though this region does
not includethe ER-bindingsite (6). There are four Pit1-bindingsitesin the distal enhancer(designated1D to
4D), and the deletion that diminishedthe estrogen
responseremoved the 4D and 3D Pit-l-binding sites.
To directly examine the contributions of specific distal
Pit-l bindingsites to the estrogen response,individual
sites were disrupted by clustered point mutations(Fig.
3). Mutation of the 1D Pit-l-binding site substantially
reduced basal expression and also reproducibly decreasedthe responseto estrogen. Mutations of the 2D
or 3D sites also reduced basal activity, but did not
diminishthe estrogen response.Although mutation of
the individual2D or 30 sites did not affect the estrogen
response,simultaneousdisruptionof both sites slightly
diminishedthe estrogen response in a reproducible
23 * 3
28*02
2D. 30
Rl”M”,
15*5
3.3 * 0.3
10. 2D. 3D
mutant
11*3
,.a*o.,
Fig. 3.
ltiple Pit-l -Binding
Sites in the Distal Enhancer on Estrogen Responsiveness
Individual Pit-l-binding sites in the distal enhancer were
mutated by oligonucleotidedirected
mutagenesis, as described in Materials and Methods.
The indicated wild-type or
mutant PRL-luciferase fusion genes were transfected into GH3
cells maintained in estrogen-depleted medium by electroporation. The cells were treated with no addition (basal) or 10 nM
estradiol (E2) and collected after 24 h for analysis of reporter
gene activity. Results are from two separate experiments,
which each contained three separate transfections for each
DNA construct. Data are presented as percentage of wild type
values and fold inductions in response to estradiol. A schematic map of the PRL Y-flanking region indicates the positions
of wild-type Pit-l-binding sites (RI), mutant sites disrupted by
a clustered point replacement 0, and the wild-type ER-
fashion. Combinedmutation of the 1D, 2D, and 3D Pitl-binding sites had a substantialeffect to diminishthe
estrogen response.
Distal Enhancer Pit-l-Binding
Sites Cannot Be
Replaced with Binding Sites for CAMP response
element (CRE)-binding
protein (CREB)
The finding that mutations that disrupted the lD, 2D,
and 3D sites substantially reduced estrogen responsiveness demonstrates a role for these distal Pit-lbinding sites in facilitating the estrogen response.This
might reflect either a particular requirementfor Pit-l or
a more general requirement,which could be fulfilled by
the binding of any transcription factor to the distal
enhancer.To addressthis question, we converted Pit1-binding sequencesin the distal enhancerto consensus CAMP responseelements(Fig. 4). In an attempt to
compensatefor possibleinterference between binding
of ER and CREB, we preparedthree different mutations
in which the 1D site was replacedwith a CRE. The first
of these mutations was centered in the Pit-l-binding
site. The other two 1D to CRE replacements were
placed either 3 or 6 basepairsup-stream of the first
CRE. Each of the mutations that placed a CRE within
the 1D site region reduced estrogen responsiveness.
The reduction in responsivenessto estrogen was comparable to the results obtained with simply disrupting
the 1D site (the 1D to CRE mutations reducing estrogenie induction to about 60% of wild type, similarto
the effects of the 1D mutation, as shown in Fig. 3).
Mutation of both the 2D and the 3D Pit-l -bindingsites
to CREs alsoreduced the ability of the gene to respond
Pit-l and Estrogen Responsiveness
1745
1 D Pit-l
Binding Site
wild type
CRE-1
CRE-2
CRE-3
Estrogen Receptor
Binding Site
AGTGCA
TGTCACTATGTCC
TGACGTCA
TG ACGTCA
Distal Region
TAG
I
Proximal
Region
wild type
Basal
(percent)
100
E2 Induction
(fold)
CAMP Induction
(fold)
2.7 i 0.2
11 +l
1D to CRE-1
1 D to CREQ
lDtoCRE-3
6.5 i 0.6
[-,I//-
2D&3D
to CRE
111 *28
1.6 zt 0.2
12kO.5
6022
1.9 f 0.2
15*1
24*11
1.9 50.2
22zt4
Fig. 4. Replacement of Distal Pit-l-Binding Sites with CAMP Response Elements Does not Permit a Wild-Type Estrogen Response
Individual Pit-l-binding sites in the distal enhancer were mutated by oligonucleotidedirected
mutagenesis, so that 8 basepairs
within the binding site were replaced with the sequence TGACGTCA, a consensus CRE. For the 1 D Pit-l -binding site, three
different CRE replacements were prepared, as indicated. The wild-type or mutant PRL-luciferase fusion genes were transfected
into GHa cells maintained in estrogen-depleted medium. The cells were treated with no addition (basal), 10 nM estradiol (E2), or 0.5
mM chlorophenylthiocAMP and collected 24 h later for analysis of reporter gene activity. Results are from two separate experiments,
which each contained three separate transfections for each DNA construct. Data are presented as a percentage of wild type values
and fold inductions in response to estradiol or the CAMP analog. A schematic map of the PRL 5’-flanking region indicates the
positions of wild-type Pit-l-binding sites @), mutant sites disrupted by a clustered point replacement m, and the wild-type ER-
to estrogen, and again, the effects were similar to
simply inactivating these sites. As the 2D and 3D sites
are located more than 30 basepairs away from the ER-
bindingsite, it seemsunlikely that bindingof transcription factors at these sites reducesthe ability of the ER
to bind to DNA. It shouldalso be noted that each of the
CRE substitutionssignificantly enhancedthe response
to CAMP, indicating
that CREB or related factors prob-
ably occupy these sites in vivo.
Mutation of the ER-Binding Site to a Fully
Palindromic, Consensus Estrogen Response
Element (ERE) Reduces the Effect of Mutations in
the 1D Pit-l-Binding Site
The ER-bindingsite of the distal enhancer, TGTCACTATGTCC, resemblesa consensus ERE, but is an
imperfect
palindrome.
It seemed possible that the pres-
ence of this nonconsensusreceptor-bindingsite might
contribute to the requirementfor a functional interaction
between the ER and Pit-l. Therefore, two point mutations were introduced by site-directed mutagenesisto
generate the palindromic sequence, GGTCACTATGACC. The palindromic ER-binding site mutant was
tested in the presence
of a wild-type
1 D Pit-l -binding
site or a disrupted 1D site (Fig. 5). Interestingly, in the
presence of a wild-type 1D site, the palindromicERbindingsite increasedbasal activity, but did not result
in increasedresponsivenessto estrogen. However, in
the context
of a disrupted
1 D Pit-l-binding
site, the
palindromicER-bindingsite yielded a modest increase
in estrogen responsiveness.Thus, disruptionof the 1D
bindingsite has very different effects dependingon the
nature of the ER-bindingsite, either wild type or palindromic.
Analysis of the Possible Association of the ER with
Pit-l in Solution
One mechanismthat might permit Pit-l to facilitate the
effects of ER on transcriptionwould involve interaction
of the two moleculesin solution. The finding that the
glucocorticoid receptor can interact with either AP-1
(21-23) or CREB (24) and that vitamin D receptors can
also interact with AP-1 (25) offers support for the possibility that membersof the steroid receptor family can
interact with other transcription factors in solution.
Therefore, we used a protein:protein interaction assay
to assessthe ability of the ER to interact with Pit-l in
solution. For this assay, a glutathion&-transferase
MOL ENDO. 1994
1746
Fig. 5. Replacement of the ER-Binding Site of the PRL Gene
with a Fully Palindromic Binding Site Results in an Altered
Functional Interaction with the 1 D Pit-l -Binding Site
The ER-binding site (ER) of the rat PRL gene was altered
by in vitro mutagenesis to the palindromic sequence GGTCACTATGACC (nuclaotide substitutions used to create the perfect
palindrome are italicized).
A reporter plasmid containing the
palindromic ER-binding site as well as a mutation in the 1D
Pit-l-binding site was also prepared. The indicated wild-type
or mutant PRL-luciferase fusion genes were transfected into
GH3 cells maintained in estrogendepleted medium by electroporation. The cells were treated with no addition (basal) or 10
nM estradiol (E2) and collected after 24 h for analysis of
reporter gene activity. Results are from two separate experiments, which each contained three separate transfections for
each DNA construct. Values are the mean f SEM, with data
expressed as a percentage of wild type values or fold inductions in response to estradiol. A schematic map of the PRL
5’-flanking region indicates the positions of wild-type Pit-lbinding sites (f@),the mutant Pit-l-binding site 0, and the ER-
(GST)-Pit-l fusion protein was synthesized in bacteria,
and this protein was immobilizedon a glutathione agarose column. We then tested for retention of radiolabeledER on a columncontainingthe immobilizedGSTPit-l fusion protein (Fig. 6). Our initial findings
suggested that there might be an easily detectable
interactionbetween the ER and Pit-l, as more receptor
was retainedon a columncontaining GST-Pit-1 (Fig. 6,
lane 3) than on control columnscontaining either GST
alone (Fig. 6, lane 2) or the glutathione agarose beads
(Fig. 6, lane 1). However, it has been suggested that
contaminating DNA can stabilize DNA-dependent associations that appear to be DNA independent (26).
This DNA-dependentbindingcan be inhibitedby several
treatments, includingethidium bromide or micrococcal
nuclease,which do not affect DNA-independentassociations. We, therefore, tested for interaction between
ER and Pit-l in the presence of ethidium bromide or
micrococcalnuclease,treatments that should be effective in minimizing DNA-dependent interactions (26).
Treatment with ethidium bromide resulted in a major
decreasein the amount of ER bound to the GST-Pit-l
column(Fig. 6, lane 6) and micrococcalnucleasealso
decreasedbindingof ER to the GST-Pit-1 column(Fig.
6, lane 9) although not to the same extent as the
ethidium bromide treatment. Similar results were obtained with propidium iodide (data not shown). Thus,
much of the interaction between the ER and Pit-l
appearsto be DNA dependent.
DISCUSSION
We examined the role of specific DNA elements in
permittingestrogenic regulationof PRL gene transcrip-
Vol8No.12
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6. Analysis of the Interaction of Pit-l and Rat ER in Vitro
Either GST or a GST-Pit-1 fusion protein was immobilized
on glutathione agarose beads and incubated with [“S]ER in
the presence of 10 nM estradiol. Some samples were also
incubated with 50 fig/ml ethidium bromide (lanes 4, 5, and 6)
or with 0.8 U micrococcal nuclease (lanes 7, 8, and 9). After
washing, the beads were boiled in buffer containing sodium
dodecyl sulfate and &mercaptoethanol, and the proteins were
resolved by denaturing polyacrylamide gel electrophoresis. As
a standard, a sample of the cell-free translation reaction was
also electrophoresed on the gel (lane 10); an arrow indicates
the migration of the [35S]ER.
Fig.
tion. Thesestudiesextend previousobservationswhich
demonstratedthat the estrogen response of the PRL
gene requiresmore than the simplepresenceof an ERbinding site (6, 7). The present findings demonstrate
that Pit-l-binding sites in both the proximal and distal
regions of the PRL gene are important for estrogen
responsiveness.Furthermore, Pit-l makes a relatively
specializedcontribution to the estrogen response,as
replacement of distal Pit-l-binding sites with binding
sites for CREB did not permit the full estrogen
response.
Of the eight known Pit-l-binding sites within the 5’flanking region of the PRL gene (11, 17) the 1D site,
which is immediately adjacent to the ER-bindingsite,
appears to be most important for the estrogen response. Disruption of the 1D site led to a detectable
decreasein estrogen responsivenessof the PRL gene.
In contrast, disruption of any other singlePit-l-binding
site had little if any effect on estrogen responsiveness.
On the other hand, sites other than the 1D site do
appear to contribute to estrogen responsiveness,as
demonstrated by the fact that combined mutation of
the 1D, 2D, and 3D sites resultedin decreasedresponsivenessto estrogen compared to the effect of the 1D
mutation alone. Thus, the mechanismthat permits Pit1 to facilitate estrogen responsivenessmust accom-
Pit-l
and Estrogen
Responsiveness
modate a distributed role involving multiple Pit-l sites.
It also appears that functional interaction between Pitl-binding sites and the ER is at least partially due to
the nonconsensus
nature of the ER-binding site in the
PRL gene. Conversion
of the site to a consensus
palindromic site eliminated the positive functional interaction between the ID Pit-l-binding
site and the ERbinding site. Previous studies have demonstrated
that
weak nonconsensus
steroid response elements may
require cooperativity
with other transcription
factors
(27). However, these previous findings suggested that
many different transcription factors can functionally synergize with steroid receptors. In contrast, the present
findings suggest a relatively stringent requirement
for
Pit-l-binding
sites in the distal enhancer of the PRL
gene. It is interesting that conversion of the weak
nonconsensus
ER-binding site of the PRL gene to a
fully palindromic site did not have the expected effects.
Surprisingly, the consensus palindromic site led to an
increase in basal expression without an increase in the
fold stimulation by estradiol. Interestingly, disruption of
the 1 D Pit-l-binding
site in the context of the palindromic ER-binding site led to decreased basal expression and an increased response to estrogen. The mechanisms that mediate these responses are not clear.
Perhaps the presence of a palindromic ER-binding site
adjacent to a Pit-l site permits the ER to stimulate
transcription in the absence of estradiol.
One mechanism that might account for the requirement for both Pit-l and the ER would involve physical
interactions between these two proteins. Indeed, the
specialized nature of the functional interaction between
Pit-l and the ER is consistent with a possible physical
interaction between the ER and Pit-l. Although we have
been able to detect interactions between immobilized
Pit-l and the ER, most of this interaction appears to be
DNA dependent. However, there is a small amount of
residual binding of ER to GST-Pit-1 in the presence of
treatments
that inhibit DNA-dependent
interactions.
Thus, it is possible that there is a weak physical interaction between the ER and Pit-l in solution. This may
at least partially account for the role of Pit-l in facilitating
the estrogen response of the PRL gene. An alternative
mechanism that might permit Pit-l to facilitate the estrogen response would involve effects to alter the interaction between the distal enhancer and the proximal
promoter of the PRL gene. Analysis of nuclease sensitivity has shown that estrogen treatment induces hypersensitivity in both the distal enhancer and the proximal region of the PRL gene, but not in the DNA
between these two regions (19). This finding suggests
that binding of an activated ER to the distal enhancer
in some way communicates
with the proximal region.
Recently, Cullen et al. (20) used a ligation assay to
provide evidence that in chromatin, the distal enhancer
is in close association with the proximal promoter region. It is possible that the multiple Pit-l-binding
sites
in both the proximal and distal regions are important
for this physical interaction between the distal enhancer
and the proximal region. Such an interaction would
1747
probably involve additional factors. Proteins related to
the yeast factors, SWIl, SWl2, and SWl3, would be
reasonable candidates for such factors. These proteins
have been found to be important for transcriptional
responses to steroid receptors (28). The SW11 , SWl2,
and SW13 proteins are known to play a role in the
transcription of a number of regulated yeast genes (2932) and may act through altering chromatin structure
(33, 34). The involvement of SWll, SWl2, and SW13 in
regulating
chromatin
structure
and permitting
responses to steroid receptor suggests that similar mammalian proteins might be involved in permitting communication
between
the distal enhancer
and the
proximal region of the PRL gene as well as facilitating
a response to estrogen.
There are now a number of examples of steroidresponsive
genes that contain multicomponent
response elements. These complex regulatory units are
quite different from simple response elements, in which
binding of the receptor to a specific site is sufficient to
permit a response to a hormone. The present finding
that Pit-l-binding
sites in both the proximal and distal
regions are important for the estrogen response suggests that the combination
of these regions can be
considered an estrogen-responsive
unit. The glucocorticoid response unit of the phosphoenolpyruvate
carboxykinase gene is also a rather complex structure.
The phosphoenolpyruvate
carboxykinase
glucocorticoid response unit includes two binding sites for the
glucocorticoid
receptor as well as binding sites for
nonreceptor factors, designated the AFl and AF2 sites
(18). The AFl site also functions as a retinoic acid
response element (35, 36) whereas the AF2 site functions as an insulin- and phorbol ester-responsive
element (37, 38). The proliferin gene contains a composite
glucocorticoid
response element that binds multiple
factors and permits both positive and negative regulation from a single element (21). The binding of multiple
factors to these complex structures presumably permits tissue-specific expression and particular patterns
of regulation that could not be achieved with simple
response elements.
MATERIALS
Reporter
AND
Gene
METHODS
and Expression
Vector
Construction
A reporter
gene containing
1.9 kilobase
pairs of 5’-flanking
sequence
and the promoter
from the rat PRL gene linked to
luciferase was prepared
using previously
described
constructs
(3,39,40).
To facilitate the replacement
of the proximal
region
of the PRL gene with fragments
containing
previously
prepared
clustered
point mutations
(41) a Sac1 site at position
-617
was removed,
and a new Sacl site was introduced
at position
-219
by oligonucleotide-directed
mutagenesis.
Specific sites
in the distal enhancer
region were altered by oligonucleotidedirected mutagenesis,
so that the core of each Pit-l-binding
site was disrupted.
The specific oligonucleotides
used to create the 1 D, 2D, and 3D mutations
were ACTTTGGAGTGCATGCCCCCCGTACGllTTGTCACTATGT,
GACATCAT-TTAGTCCCGAGAGCCAACATGAGTGGAACT,
and AAGAAAGTCATCAGCAACGGACGCGGCCGCACAACGAATGACATCAfi-
MOL
1748
ENDO.
1994
Vol8No.
TAGGAA,
respectively.
Mutation
of the ER-binding
site in the
distal enhancer
to a fullv Dalindromic
seouence
was accomplished
by in vitro mutagenesis
using the oligonucleotide
seouence
AAAATGCAlllTGGTCACTATGACCTAGAGTGCTT’. Conversion
of Pit-l-binding
sequences
to consensus
CREs was accomplished
so that a portion of each Pit-l -binding
site was replaced
with a consensus
CREB-binding
site, 5’TGACGTCA3’.
All mutated
sequences
were confirmed
by
dideoxy
chain termination
sequence
analysis (42).
Cell Culture
and Transfections
GH3 pituitary tumor cells were maintained
in monolayer
culture
in Dulbecco’s
Minimum
Essential
Medium
containing
15%
horse serum and 2.5% fetal calf serum. Before transfection,
the medium was changed
to phenol-red
free Dulbecco’s
Minimum Essential
Medium
supplemented
with 10% gelding
serum, which was treated to remove
residual estrogen
(43).
and the cells were maintained
in this medium for 48 h. The
cells were transfected
by electroporation,
as described
previously (44). and harvested
24 h after transfection.
Luciferase
activity was determined
as previously
described
(45).
Generation
of GST-Pit-1
Fusion
Proteins
The complete coding sequence
of Pit-l was ligated into pGEXKG (48)..Escherichk
cob harboring
the expression
vector for
GST-Pit-1
or GST alone was induced with 0.5 mM isooropvlfi-o-thiogalactopyranoside;
harvested;
resuspended
in 20 ‘n&
HEPES (pH 7.6) 4 mM EDTA, 6 mM dithiothreitol,
and 0.4 mM
phenylmethylsulfonylfluoride;
and lysed by passing the mixture
twice through a French Press (SLM Instruments,
Urbana, IL).
The homogenate
was centrifuged
for 20 min at 20,000 rpm in
a Beckman
Ti45 rotor (Beckman,
Palo Alto, CA). The supernatant was incubated
with glutathione
agarose
beads at 4 C
and washed in the homogenization
buffer.
In Vitro Binding
of Rat ER to Pit-l
For synthesis
of radiolabeled
ER, the complete
coding sequence of the rat ER (47) was subcloned
into the Bluescript
vector (Stratagene,
La Jolla, CA). Radiolabeled
rat ER was
produced
using the T7coupled
transcription
translation
system (Promega,
Madison,
WI) in the presence
of [35S]methionine. The radiolabeled
ER was incubated
with 10 nM 17@estradiol on ice. GST-Pit-l
or GST bound to glutathione
agarose beads was washed
twice in a buffer containing
50 I?IM
KCI. 20 mM HEPES (DH 7.9). 2 mM EDTA. 0.1% Nonidet P40, ‘5 mM dithiothreitol,
10%. glycerol,
and 0.5% nonfat dry
milk. The beads were then incubated
with labeled ER for 60
min with gentle mixing at 4 C, washed four times, boiled in
buffer containing
1% sodium dodecyl
sulfate and @-mercapmethanol,
and analyzed
by denaturing
polyacrylamide
gel
electrophoresis.
Some of the samples
were treated
with 50
fig/ml ethidium bromide,
which was present in all of the incubation and wash buffers.
Other
samples
were treated
by
incubation
of the beads with micrococcal
nuclease
[0.8 U in
50 ~1 50 mM NaCI, 10 mrv Tris (pH 7.0) and 4 mM CaCI, for 1
h at 37 C] before the beads were incubated
with the ER.
We thank
B. Maurer
for aid in preparing
this manuscript.
Received April 14,1994.
Revision received August 5,1994.
Accepted August 9, 1994.
Address
requests
for reprints
to: Richard
A. Maurer,
Department
of Cell Biology and Anatomy,
L215, Oregon
Health
Sciences
University,
3181 S.W. Sam Jackson
Park Road,
Portland, Oregon 97201.
This research
(to R.A.M.).
was
supported
by USPHS
Grant
12
DK-40339
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