 |
INTRODUCTION |
There are at least two nuclear receptors for estrogen receptor,
ER
1 (1, 2) and ER
(3).
Most breast cancers that occur in post-menopausal women overexpress
ER
(4). Patients with breast cancers that express ER
are more
likely to respond to hormonal therapy (4, 5) and have an improved
prognosis compared with patients with ER
-negative tumors (4, 6, 7).
Studies of breast cancer cell lines (8) and primary tumors (9, 10) have
indicated that transcription of the ER
gene plays an important role
in regulating the expression of ER
. Thus, understanding transcriptional regulation of the ER
gene will likely provide critical insights into the pathogenesis of hormone-responsive breast cancers.
Transcription of the ER
gene is complex and involves activity of
several distinct promoters (11-14). Functional promoter studies have
concluded that ER
expression in breast cancer cell lines and various
tissues is likely to involve trans-acting factors that have a specific
cell or tissue distribution pattern (15-19). There appear to be a
variety of factors that interact with the ER
promoter with
trans-activating (15-17) or trans-repressing (20) functions. There is
also evidence that ER
can autoregulate its own transcription (21,
22). Other studies suggest that the lack of ER
expression in
ER
-negative breast cancer cell lines and tumors may be controlled by
methylation of CpG islands in the 5' end of the ER
gene (23,
24).
The main ER
promoter, P1, initiates transcription at a cap site
previously mapped at the start of exon 1 (1). Exon 1 has a 233-base
5'-untranslated region preceding the AUG codon that initiates
translation of the ER
protein. Studies in ER
-positive breast
cancer cell lines have shown that transcription initiated at exon 1 accounts for 50-90% of all ER
mRNAs (18, 25). A functional
analysis of the main ER
promoter identified a factor, ERF-1, that
binds to high affinity sites in the untranslated region of exon 1 and
can trans-activate the cloned ER
promoter (15, 26). ERF-1 was
found to be a member of the AP2 family of transcription factors and has
been renamed AP2
(27).
All other ER
transcripts initiate at cap sites upstream of exon 1 and splice into a splice acceptor site at +163 in exon 1 (14). Although
some of these upstream exons have open reading frames, there is no
evidence that these are translated, and it appears that all alternate
5' exons are non-coding. Exon 1' has been reported to have two main cap
sites giving rise to alternate 5' exons of 110 bases or 1206 bases
(12). The short and long forms of exon 1' both utilize the splice donor
site at
1884 (location relative to cap site of P1). We had previously
described two additional alternate ER
transcripts called E and H
(14). Both the E and H transcripts are expressed in ER
-positive
breast cancer cell lines, primary breast cancers, ER
-positive
endometrial carcinoma cell lines, and normal endometrium. The existence
of the E and H transcripts of ER
were subsequently confirmed by
other investigators, and these exons were reported to be expressed in a
variety of tissues (25). The splice donor site of exon E was found to
be at
169. The H transcript was found to utilize two upstream exons, Ha and Hb, separated by an intron of 9 kbp (14). The genomic location
of the H exons was not determined but was concluded to be at least 20 kbp 5' to exon 1 (14). An additional liver-specific exon, called exon
C, has also been described (13). Exon C was reported to be spliced to
an exon with sequence matching exon Hb, and it was concluded that exon
C is farther 5' than exon Ha (14).
In order to define the location of the ER
promoters active in breast
cancer, a detailed analysis of the genomic structure of the 5' end of
the ER
gene including the alternate ER
transcripts E and H was
performed. Overlapping BAC clones have been isolated that generated a
contig map that includes all known ER
exons. The exon Ha was found
to be 124 kbp upstream of exon 1, and exon C was located 30-40 kbp 5'
to exon Ha. Together with the previously known exons of the ER
gene
that span a region of over 160 kb, the ER
locus was found to
encompass a genomic region of ~300 kbp. By using S1 nuclease and
5'-RACE, the cap sites for exons E and H have been mapped. Each of the
alternate upstream exons was found to be associated with DNase
I-hypersensitive sites specific to cells expressing ER
. The DNase
I-hypersensitive site, HS1, was mapped to the binding sites for AP2
in the untranslated leader of exon 1 and was invariably found in the
chromatin structure of ER
-positive cells. In human mammary
epithelial cells (HMECs), AP2
expression induced the
HS1-hypersensitive site and trans-activated the ER
promoter, which
was dependent upon the region of the promoter containing the HS1 site.
These findings provide additional evidence for a critical role for
AP2
in the oncogenesis of hormone-responsive breast cancer.
 |
EXPERIMENTAL PROCEDURES |
Cell Lines--
All cell lines were obtained from American Type
Culture Collection, Manassas, VA. Cells were maintained in minimal
essential media (MEM, Life Technologies, Inc.) supplemented with 10%
fetal bovine sera (FCS, Gemini BioProducts, Calabasas, CA), 25 mM HEPES, 26 mM sodium bicarbonate, 5000 units/ml penicillin G (Life Technologies, Inc.), 5000 µg/ml
streptomycin (Life Technologies, Inc.), and 6 ng/ml bovine insulin
(Sigma). HMECs were obtained from reduction mammoplasty and were a gift
from Dr. J. Dirk Iglehart, Boston. HMECs were maintained in DFCI-1
media (28). All cells were incubated at 37 °C in 5%
CO2.
BAC Library Screening--
BAC clones containing genomic DNA
from the 5' region of the human ER
gene were identified by
hybridization of DNA arrays with probes corresponding to the human
ER
exons 1', Ha and Hb. Nylon membranes arrayed with DNA from a
human BAC library were purchased from Research Genetics (Huntsville,
AL). Gel-purified insert DNA from clones of ER
exons Ha (pHa2.5) and
Hb (pHb4.1) were used for hybridization. Exon 1' sequences between
3090 and
2670 were PCR-amplified from MCF-7 genomic DNA using
primers ERSEQ1 (TCTAGAGCATGGGTGGCCAT) and ERSEQ2 (GTGCTCCTAGAGTGCCCACG) and TAQ polymerase. The cycle profile included an initial denaturation step of 94 °C for 2 min followed by 25 cycles of 94 °C for
30 s, 56 °C for 15 s, and 72 °C for 2 min and
terminated with a final extension step of 72 °C for 5 min. All DNAs
used for hybridization were gel-purified and labeled by random priming
with [
-32P]dCTP to specific activities greater than
7 × 108 dpm/µg.
Library membranes were prehybridized in 50% formamide, 5× SSC, 7%
SDS, 1% polyethylene glycol, 25 mM sodium phosphate
buffer, pH 6.7, and 0.5% non-fat dried milk at 42 °C for 1 h.
Twenty ml of hybridization solution per membrane were used. For
hybridization, the volume of hybridization solution was reduced to 6 ml
per membrane, and each probe was added to 5 × 105
dpm/ml and hybridized for 12-18 h at 42 °C. Following hybridization the membranes were washed twice in 2× SSC, 1% SDS at 42 °C and then twice in 0.1× SSC, 0.1% SDS at 65 °C. Positive signals were identified by overnight exposure to film.
Cultures of Escherichia coli harboring the BACs identified
in the initial screen were obtained from Research Genetics. A small amount of DNA was obtained from each culture using the vendor's protocols for secondary screening by PCR. DNA from 19 BACs were PCR-amplified with primers oEXON0INT and oEXON0-3' (14) for the
presence of ER
exon Hb sequences and with primers ERSEQ1 and ERSEQ2
for the presence of ER
exon 1' sequences as described above.
Mapping BAC Clones--
DNA from BACs containing portions of the
ER
upstream region was prepared from 1-liter cultures using a
Maxiprep kit from Qiagen (Valencia, CA) as described by the
manufacturer. For restriction enzyme digest analysis, 1-2 µg of BAC
DNA was digested in 20 µl with the appropriate enzyme and then
subjected to pulse field gel electrophoresis in 0.5× TBE at 140 V with
field switches increasing from 1 to 12 s over 20 h. Bands
were visualized by ethidium bromide staining and photographed. Band
sizes were calculated from standard curves constructed from molecular
weight markers.
For Southern blot analysis, DNAs were transferred to positively charged
nylon membranes (Hybond N+, Amersham Pharmacia Biotech) using protocols
provided by the manufacturer. The blots were hybridized with various
probes to identify the locations of the ER
exons relative to the
restriction sites mapped in each BAC. Probes used were the ER
exon
1', Ha and Hb probes described above. In addition, oligonucleotides
corresponding to ER
exon C (TTCACAATCAAAAGGATTGG) (13) and to the
ends of the BAC genomic DNA inserts were used. The sequences of the
ends of the BACs were determined by direct sequencing of BAC DNA.
S1 Nuclease Analysis--
S1 nuclease analyses were performed
essentially as described (18) with modifications of the protocols for
probe synthesis. Messenger RNA was isolated from MCF-7 cells using a
Fast Track mRNA isolation System (Invitrogen, Carlsbad, CA)
according to the manufacturer's instructions. The probe for analysis
of the ER
transcripts originating at the exon Ha promoter was
synthesized by single-sided PCR from a template that encompassed exon
Ha plus ~850 bases of upstream sequences. The template was
PCR-amplified from MCF-7 genomic DNA using the primer H199.4
(GAGAAGATTATCACTCAGAGAC) as the 3' primer and H853.3
(CGCCCCATTCTACCATTTTC) as the 5' primer. PCR conditions were as
described above except that the annealing temperature was 50 °C. To
synthesize a single-stranded probe complementary to the expected
mRNA sequence, PCR was performed using only H199.4 in the presence
of [
-32P]dCTP. After PCR the probe DNA was desalted on
a spin column to remove unincorporated label.
The single-stranded probe for analysis of the ER
transcripts
originating from the promoter associated with exon E was synthesized by primer extension using Klenow enzyme. The primer used was ERPRO30 (GTGCAGACCGTGTCCCCGCA) and was annealed to the denatured ER724-210LUC plasmid (15) as a template. The 3' end of the probe was defined by
cleavage with PvuII, and the single-stranded probe was
isolated from the template by electrophoresis through a 1.5%
alkaline agarose gel. The purified probe was eluted from the gel
using a Millipore Ultrafree DA spin cartridge (Millipore Corp., New
Bedford, MA).
Either the Ha or E probe (5 × 105 cpm) was hybridized
to 2 µg of MCF-7 mRNA or yeast tRNA overnight at 45 °C.
Nuclease S1 (500-2000 units/ml; Amersham Pharmacia Biotech) was added,
and the samples were incubated at 37 °C for 1 h. The reactions
were stopped, and the samples were extracted once with
phenol/chloroform (1:1) before analysis on a 6% acrylamide sequencing
gel. Signals were detected by overnight autoradiography.
5'-RACE Analysis--
Messenger RNA from MCF-7 cells was
analyzed by 5'-RACE using the 5'-RACE System from Life Technologies,
Inc., as recommended by the manufacturer. Initial reverse transcription
was primed with ERPRO22 (CCCTTGGATCTGATGCAGTA), which is located in
exon 1 at approximately +300. The gene-specific primer for the first round of PCR was ERPRO30, which anneals to sequences in exon 1 upstream
of ERPRO22. A second round of PCR amplification was performed using the
gene-specific primers ERPRO94 (GCTGGATAGAGGCTGAGTTT) and H199.4 for
exons E and Ha, respectively. RACE PCR products were cloned into pCR2.1
using a TA Cloning Kit (Invitrogen) as recommended. The location of the
5' end of each clone was determined by sequencing.
DNase I-hypersensitive Site Assay--
DNase I-hypersensitivity
was analyzed in a variety of ER
-positive and ER
-negative cell
lines. Cells were harvested during exponential growth, and then washed
once with cold phosphate-buffered saline (PBS). The cells were then
washed once with cold buffer A (15 mM Tris, pH 7.4, 60 mM KCl, 15 mM NaCl, 0.2 mM EGTA,
0.2 mM EDTA, 0.25 M sucrose, 1 mM
dithiothreitol, 0.15 mM spermine, 0.5 mM
spermidine) and then resuspended in 5 ml of cold buffer A with 0.2%
Nonidet P-40. The cells were lysed in a Dounce homogenizer using 10 strokes of a B pestle on ice; lysis was checked by trypan blue
exclusion. Nuclei were pelleted and resuspended in 2.5 ml of buffer B
(buffer A minus sucrose). To 0.5-ml aliquots of nuclei varying
quantities of DNase I (0, 200, 400, 600, 800, 1200, 1600 units/ml;
Roche Molecular Biochemicals) were added, and then MgCl2 was added to 5 mM, and the samples were incubated on ice
for 15 min. The reactions were stopped by addition of EDTA to 50 mM. Following addition of SDS to 0.5% and proteinase K to
1 mg/ml, the samples were incubated overnight at 37 °C. Residual
protein was removed by extraction once each with phenol,
phenol/chloroform (1:1), and chloroform. Following ethanol
precipitation the nucleic acid pellets were dissolved in 10 mM Tris, pH 8.0, 0.5 mM EDTA, and the
absorbance at A260 was measured.
Ten micrograms of each sample were cleaved with the appropriate
restriction enzyme overnight at 37 °C. For analysis of
hypersensitive sites near exon 1, the samples were then electrophoresed
through 1% agarose (SeaKem GTG, BioWhittaker Molecular Applications,
Rockland, ME) using 0.5× Tris/acetate/EDTA buffer and transferred to
nylon membranes for hybridization as described above. The resulting blots were hybridized with the exon 1' probe. For analysis of hypersensitive sites near exons Ha/Hb, samples were subjected to pulse
field gel electrophoresis following restriction enzyme cleavage. DNA
was immobilized on nylon membranes and hybridized with the same exon Ha
probe used to screen the BAC library.
AP2
Antibody Production--
Antigen for the production of an
AP2
-specific polyclonal antibody (AP) was generated by cloning a
fragment of AP2
in frame with glutathione S-transferase
(GST) to create a fusion protein. A fragment corresponding to
nucleotides 474-607, which encodes amino acids 150-187, was generated
by PCR and cloned in frame in the pGEX-4T3 vector (Amersham Pharmacia
Biotech). The identity of the clone was confirmed by sequencing the
entire insert from both directions. The clone was transformed into XL-1
Blue cells (Stratagene), and the production of a fusion protein of the
proper size was confirmed by SDS-polyacrylamide gel electrophoresis. Large scale production of the fusion protein was induced with growth of
the transformed cells in the presence of
isopropyl-1-thio-
-D-galactopyranoside. Bacterial lysates
were incubated with glutathione-agarose beads, washed with PBS, and the
fusion protein eluted with the addition of 5 mM
glutathione. The fusion protein was injected into rabbits, and antisera
were generated by CalTag Laboratory (Healdsburg, CA). Following
production of a polyclonal antisera, affinity purification was
performed by passing antisera first over a GST affinity column to bind
selectively antibodies directed to the GST portion of the fusion
protein, and then over a GST-AP2
affinity column with subsequent
elution of the affinity-purified antibody. Affinity columns were
prepared using Affi-Gel 10 supports (Bio-Rad). Gel shift assays were
performed as described previously (15). In supershift assays, 2 µl of
AP antisera was used. The rabbit polyclonal antibody to AP2, SC-184,
was obtained from Santa Cruz Biotechnology, Santa Cruz, CA.
Construction of AP2
/pAdTrack-CMV and AP2
/pAdTrack-CMV
Shuttle Vector--
In order to generate AP2
and AP2
adenoviral
constructs, the AP2
and AP2
cDNAs were first cloned into a
shuttle vector. By using a previously described AP2
clone retrieved
from a MCF7 expression library (27) as template, AP2
cDNA was
PCR-amplified from the translation start site at +167 to the stop codon
at +1519 using a 5' primer (GACGCAGATCTCCATGTTGTGGAAAATAAC)
containing a BglII site and a 3' primer
(TCGTTCTCGAGTTATTTCCTGTGTTTCTCC) containing an XhoI
site. AP2
cDNA was PCR-amplified from the translational start
site to the stop codon using an AP2
cDNA clone (26) as template.
PCR was performed using a 5' primer AP2
5' SalI
(CGATCCGTCGACATGCTTTGGAAATTGACG) containing a SalI site and a 3' primer AP2
3' XbaI
(GGGAGGTCTAGATCACTTTCTGTGCTTCTC) containing an XbaI site.
The AP2
and AP2
cDNA fragments were ligated into the
pAdTrack-CMV shuttle vector (gift of Dr. Burt Vogelstein, The Johns
Hopkins University) (29) after digestion with appropriate
restriction enzymes to create AP2
/pAdTrack-CMV and
AP2
/pAdTrack-CMV. pAdTrack-CMV also encodes the GFP protein so that
viral production can be monitored by fluorescence.
Generation of Recombinant AdAP2
, AdAP2
, and AdWT Adenoviral
Plasmids--
To generate AdAP2
, AdAP2
, and AdWT adenoviral
recombinants, 1 µg of AP2
/pAdTrack-CMV, AP2
/pAdTrack-CMV, or
pAdTrack-CMV plasmid DNA was linearized overnight at 37 °C with
PmeI, extracted two times with phenol/chloroform and once
with chloroform, and followed by ethanol precipitation.
Cotransformation of linearized DNA and 100 ng of pAdEasy-1 adenoviral
backbone vector (gift of Dr. Vogelstein) (29) into electrocompetent
E. coli BJ5183 cells was performed in 2.0-mm cuvettes at 2.5 kV, 200 ohms, and 25 microfarads using a Bio-Rad Gene Pulser
electroporator (Bio-Rad). Recombinants were selected for kanamycin
resistance, and recombination was confirmed by restriction digestion
with PacI, BamHI, BstXI, and NdeI. True recombinants were retransformed into
electrocompetent E. coli DH10B (Life Technologies, Inc.) as
described above, and DNA was purified using the Plasmid Maxi Kit
(Qiagen) according to the manufacturer's protocol.
Production of AdAP2
, AdAP2
, and AdWT Adenoviruses in 293 Cells--
AdAP2
, AdAP2
, and AdWT adenoviruses were produced as
described previously (29) with several modifications. Briefly, 2 × 106 293 cells were plated in 25-cm2 flasks
24 h before transfection in MEM with 10% FCS so that cells had
reached 70-80% confluence by 24 h. On the day of transfection, 4 µg each of AdAP2
, AdAP2
, and AdWT DNA were linearized by
PacI digestion. Each linearized DNA was mixed with 20 µl
of LipofectAMINE (Life Technologies, Inc.) in 500 µl of Opti-MEM
(Life Technologies, Inc.) and incubated at room temperature for 15-30
min according to the manufacturer's protocol. Meanwhile, the cells
were washed once with 3 ml of Opti-MEM. After incubation, the lipid/DNA
mixes were brought up to 2 ml with Opti-MEM and overlaid onto the 293 cells. After incubation at 37 °C, 5% CO2 for 4 h,
the transfection mix was removed, replaced with 6 ml of MEM + 10% FCS,
and returned to the incubator. Cells were monitored by fluorescence
microscopy for GFP expression over 7-9 days at which time most of the
cells were fluorescent and had detached from the flask. Cells were
collected, pelleted, resuspended in 2 ml of 1× PBS buffer, and
subjected to 4 cycles of freeze/thaw/vortex (dry ice/37 °C).
AdAP2
, AdAP2
, and AdWT adenoviruses were then plaque-purified by
infecting 5 × 105 293 cells in 35-mm plates with 100 µl of serial dilutions of viral supernatants from
10
1 to 10
4 made in
Opti-MEM. After a 1-h incubation at 37 °C, 5% CO2,
cells were overlaid with 3 ml of 0.8% agarose in MEM + 10% FCS and
returned to the incubator. Plates were monitored for plaque formation
and GFP expression over 9 days at which time plaques were isolated as
agarose plugs into 200 µl of MEM + 10% FCS and subjected to 3 freeze/thaw (dry ice/37 °C) cycles. Fifty µl of viral lysate was
used to infect 2 × 106 293 cells in a
25-cm2 flask, and the cells were harvested as described
above at 3-4 days when the cells were at least 50% detached. Virus
was then titered by GFP expression, and 3 more rounds of infection were performed at an m.o.i. of 0.1 to generate higher titer viral stocks. A
final round of infection was performed at an m.o.i. of 5 using 5 × 108 293 cells in six 175-cm2 flasks. Cells
were harvested at 60 h post-infection and after 4 cycles of
freeze/thaw in 8 ml of 1× PBS, the virus was purified by CsCl banding
using a density of 1.35 g/ml CsCl in a SW41Ti rotor at 32,000 rpm,
10 °C, 18-24 h. Virus was collected in ~1 ml with an 18-gauge
needle and dialyzed against 1 liter of Storage Buffer (5 mM
Tris, pH 8.0, 50 mM sodium chloride, 0.05% bovine serum
albumin, and 25% glycerol) at 4 °C for 6 h. Viruses were titered by GFP expression in both 293 cells, and HMECs generally resulted in titers of 1011 plaque-forming units/ml in 293 cells and 108 plaque-forming units/ml in HMECs.
Confirmation of AdAP2
, AdAP2
, and AdWT
Identity--
In order to confirm the identities of AdAP2
,
AdAP2
, and AdWT viruses, DNA was isolated from the viruses using the
DNeasy Tissue Kit (Qiagen) according to the manufacturer's protocol
for non-nucleated blood. Viral DNA was subjected to PCR amplification using 3 sets of primer pairs for AP2
and AP2
and one set for AdWT. Primer pairs for AdAP2
were composed either of one primer derived from the pAdTrackCMV vector sequence and one primer from AP2
(primer pair 1, GFP-AdTrack/ APseq6; primer pair 2, Rt.
Arm-AdTrack/Apseq2) or of two primers from the pAdTrack-CMV sequence
(primer pair 3, GFP-AdTrack/Rt. Arm-AdTrack). For AdAP2
, primer pair
1 was composed of GFP-AdTrack and AP2
5' SalI, and primer
pair 2 was composed of Rt. Arm-AdTrack and AP2
3' XbaI.
Primer pair 3 remained the same. Viral DNA for AdWT was amplified using
only primer pair 3. Primer sequences are as follows: GFP-AdTrack
(GCCGTCCTCGATGTTGTGGCGGATC); APseq6 (CATCAAAGAAGCCCTGATT G); Rt.
Arm-AdTrack (CATCAAACGAGTTGGTGCTCATGGC); and Apseq2 (GTGCTGCCCGGCGGAGGAGA).
Infection of HMEC with Adenoviruses--
The day before
infection, a total of 3 × 107 HMEC cells for each
virus was plated in three 175-cm2 flasks. The next day,
media were removed from the flasks, and the cells were washed with 5 ml
of Opti-MEM. Cells were infected at an m.o.i. of 10 for 24 h at
which time they were analyzed for DNase I hypersensitivity as described above.
Trans-activation of the ER
Promoter--
The ER
promoter
constructs used have been described previously (15). The constructs
ER3794-230LUC and ER3794-0LUC were previously called ER3500-230LUC
and ER3500-0LUC, respectively. However, sequence analysis has shown
that the 5' end of the constructs are at
3794 bp relative to the P1
cap site. Transfections in HMECs were performed in triplicate using
FuGene 6, and luciferase expression was normalized using
-galactosidase expression as described previously (26).
 |
RESULTS |
Genomic Mapping of the Alternate ER
Exons--
We had
previously identified alternate 5' exons of the human ER
gene that
were expressed in breast cancer cell lines and primary breast tumors
(14). However, genomic mapping with genomic lambda clones failed to
provide a contig spanning the entire region. It remained to be
determined how large the intron was between exon Hb and the splice
acceptor site in exon 1. The location of the liver-specific exon C (13)
also had not previously been determined. In order to locate the 5'
alternative exons Ha, Hb, and C, a BAC library was screened using
probes representing exons 1', Ha and Hb. Of the BAC clones identified
in the initial screen only BAC 542K7 encompassed sequences from both
exon 1' and exon Hb and therefore was likely to span the intron
separating these two exons. This BAC clone also hybridized to a probe
specific for exon Ha. Three other BACs including BAC 295K8 were
isolated that contained sequences from exons Ha and Hb but not exon 1'. BAC 295K8 also hybridized to a probe for the liver-specific exon C.
Southern blot analysis was used to generate a restriction map, to
locate the positions of the ER
exons, and to determine the overlap
in the two BAC clones (data summarized in Fig.
1). Previous results using lambda genomic
clones had demonstrated that exon Hb was at least 20 kb upstream of
exon 1' (14). The results from mapping BAC 542K7 and 295K8 revealed
that exon Hb was more than 110 kb upstream of exon 1' and that the
distance between the cap site for exon Ha and the cap site for exon 1 was ~124 kb (Fig. 1). Southern blot analysis revealed that the
liver-specific exon C was located on a SmaI/PacI
fragment between 30 and 40 kb upstream of exon Ha.

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Fig. 1.
Genomic map of 5' region of the
ER gene. A genomic map of the 5' end of
the ER gene is shown with restriction sites. Sites in
parentheses indicate that not all sites of that enzyme are
shown. The location of the ER exons C, Ha, Hb, 1', E, and
1 are shown. The positions of BAC clones BAC 542K7 and BAC
295K8 are shown below the map.
|
|
Mapping the Cap Sites for the Alternate ER
Transcripts--
The
location of the alternate 5' exons of the human ER
gene suggests
that there are separate ER
promoters that may be independently regulated. Functional promoter studies have been performed for promoters initiating transcription at exons 1 (15) and 1' (17), but no
studies have been performed on promoters that may be involved in
expression of exons E or Ha. In order to further localize possible promoters controlling expression of these two alternate exons, experiments to locate the cap sites of the E and H transcripts were
performed using S1 nuclease and 5'-RACE. The results of these findings
are shown in Fig. 2. For the S1 analysis,
MCF-7 mRNA was hybridized to a probe extending several hundred
bases upstream of the end of the longest cDNA previously identified
for ER
transcripts initiating with exon Ha (14). The analysis
revealed multiple species of protected fragments with 5' ends between
15 bases (Fig. 2A, fragment J) and 80 bases (Fig. 2A,
fragment A) upstream of the 5' end of the longest cDNA clone
previously obtained for Ha.

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Fig. 2.
S1 nuclease and 5'-RACE for exons Ha and
E. A, mapping for exon Ha. Results of S1 nuclease are
shown that demonstrate 10 start sites over a 66-base region. The main
S1 fragments are labeled A-J. RNA used in the experiments
is from MCF-7 cells (lanes 1 and 2) or tRNA
(lanes 3 and 4). Increasing concentration of S1
nuclease was used as indicated. To the right of the gel is
the sequence locating the approximate position of the S1 nuclease
products. Closed circles represent the 5'-terminal
nucleotide from the 5'-RACE clones. Arrow indicates the
position of the longest cDNA previously reported for ER H
transcripts. B, mapping for exon E. S1 nuclease results
shown for exon E presented in a manner similar to exon Ha. Five main S1
nuclease fragments are labeled A-E. Fragment A is ~120 bp
upstream of the cluster represented by B-E. Closed
circles represent the 5'-terminal nucleotide of 5'-RACE clones.
Arrow indicates the position of the longest cDNA
previously reported for ER E transcripts.
|
|
To validate that the cap sites of Ha had been correctly identified by
S1 nuclease analysis, 5'-RACE was performed on MCF-7 mRNA. A clear
single PCR product was seen after the second round of PCR using a
gene-specific primer in exon Ha (data not shown). Twelve clones with
inserts were sequenced to determine the 5'-most base, and the results
were correlated with the S1 nuclease analysis data (see Fig.
2A). The circles plotted on the right
side of the vertical sequence represent the locations of 5'-most
bases from these 12 clones. Similar to the S1 analysis, RACE clones
displayed a 66-base range in length, which coincided with the mRNA
ends identified by S1 analysis. Two clusters of clones were observed that terminated ~45 bases and 57 bases upstream of the previous end
noted from ER cDNA clones. Two clones mapped to within 2 bases of
the longest S1 product (fragment A), and one
clone corresponded to the smallest S1 product (fragment
J). These results demonstrate that exon Ha represents a
genuine ER
cap site with multiple start sites scattered over ~60
bp.
Exon E was previously identified as an alternative 5' exon from
screening a cDNA library and was localized between exon 1' and exon
1 (14). The 3' end of exon E was identified 169 bp upstream of the main
transcriptional start site for the ER
gene. In order to confirm that
ER
transcripts have a cap site associated with transcription
initiated at exon E, a similar analysis to that for exon Ha was
undertaken. A single strand DNA probe extending from the 3' end of exon
E sequences to several hundred bases upstream was hybridized to MCF-7
mRNA. S1 nuclease analysis of the hybrids revealed several
protected species as was observed for exon Ha (Fig. 2B). A
prominent protected band was observed that corresponded to an mRNA
end ~120 bases upstream of the end previously identified from the
longest cDNA clone for exon E (Fig. 2B, fragment A). Four additional protected species were identified, the 5'-most of which
(fragment B) was 8 bp from the previously identified cDNA 5' end. The other three protected species (fragments
C-E) corresponded to shorter mRNAs.
The 5'-RACE analysis of ER mRNA from MCF-7 cells using exon
E-specific primers produced two prominent bands of ~200 and 300 bp
(data not shown). Fourteen of the RACE clones were sequenced, and the
5'-most base correlated with the S1 analysis data (Fig. 2B).
One clone extended several bases beyond the length of S1 fragment A,
and the rest extended over an 80-base range with lengths similar to
fragment B-E identified by S1 analysis. A cluster of 7 clones had 5'
ends within an 11-base range just 3' to the fragment corresponding to
S1 product B. Since the S1 nuclease fragment sizes are estimated from
the mobility in gel electrophoreses, the S1 nuclease results and
5'-RACE results are in excellent agreement.
DNase I-hypersensitive Sites--
The location of DNase
I-hypersensitive sites has been shown to correspond to the position of
binding by transcription factors involved in transcriptional regulation
of a gene (30). Therefore, mapping DNase I-hypersensitive sites can
provide important information about the location of the transcriptional
regulatory regions. Previously published data (31) had identified three
DNase I-hypersensitive sites near exon 1 that were specific to
ER
-positive cells, HS1 located near the cap site for exon 1, HS2 at
approximately
350 bp, and HS3 at approximately
2000 bp (positions
relative to the cap site for exon 1). A repeat of this experiment
confirmed these results and localized the sites with more precision
(Fig. 3A). The first
hypersensitive site (HS1) was observed within the 5'-untranslated region of exon 1 at approximately +200 bp. A second hypersensitive site
was observed at
800 bp (HS2), and a third hypersensitive site (HS3)
was seen at
2000 bp. In agreement with previous work, each of these
three hypersensitive sites was specific to ER
-positive cells, and we
have adopted the earlier nomenclature. The location of HS1 coincides
with binding sites for AP2
, which were previously identified in a
functional promoter analysis of the main ER
promoter (15). HS3
appears to be associated with exon 1' and may be functionally related
to the P0 promoter (also known as the B promoter) (17) for exon 1'.
Exon 1' may be transcribed as a 110-base or 1206-base exon both of
which end at a splice donor site at
1884 (12) indicating that there
may be two alternate transcriptional start sites. If the smaller exon
is expressed, HS3 would be mapped just upstream of the start of exon
1'. HS2 is located adjacent to exon E and may relate to the promoter
controlling transcription starting at this cap site.

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Fig. 3.
DNase I-hypersensitive sites in region of
ER cap sites. A,
hypersensitive sites in region of exons 1, E, and 1'. Hypersensitive
sites were determined using chromatin extracted from MCF-7
(ER -positive) and MDA-MB-231 (ER -negative) cells. Chromatin was
untreated (0) or treated with increasing amounts of DNase I
as indicated. Hypersensitive sites HS1, HS2, and HS3 correspond to
sites specific to ER -positive cells as reported previously (30). Map
below shows schematic location of exons 1, E, and
1' relative to location of hypersensitive sites and probe
used in the hybridization. Arrows indicate major cap sites
identified. B, hypersensitive sites in region of exons Ha
and Hb. Results for mapping DNase I-hypersensitive sites are presented
in a manner similar to exons 1, E, and 1'. Data demonstrate
hypersensitive site HS4, which was specific to ER -positive cells.
Location of site was confirmed in two experiments with digestion with
SwaI or XhoI. Hypersensitive sites HS5 and HS6
were identified in both cell lines.
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DNase I-hypersensitive site analysis was performed using an exon Ha
probe on Southern blots of DNA from MCF-7 and MDA-MB-231 cells to
locate potential regulatory elements involved in transcription of the H
transcript (Fig. 3B). Southern analysis of DNA digested with
SwaI revealed a 32-kb fragment in both MCF-7 and MDA-MB-231 DNA, which is consistent with the size of the SwaI fragment
determined from the BAC mapping experiments. An additional band
migrating at 15 kb was observed in MCF-7 DNA treated with DNase I (Fig. 3B, top panel, HS4). This band mapped a hypersensitive site
that is specific to ER
-positive cells to a location ~5 kb
downstream of exon Hb. Southern analysis of DNA digested with
XhoI demonstrated a band at 48 kb in both cell lines. A
prominent band of lower mobility (bottom panel, HS4) was
observed in MCF-7 DNA treated with DNase I but not in similarly treated
MDA-MB-231 DNA. This band maps to the same location as HS4 in the
top panel. Additional faster migrating bands were observed
in both sets of DNA (lower panel, HS5 and HS6)
and are, therefore, not specific to ER
-positive cells. These bands
mapped hypersensitive sites to the locations indicated on the diagram
below the autoradiographs. HS5 mapped to a location ~2 kb
upstream of exon Hb, and HS6 mapped to a location near or within exon Ha.
The HS1-hypersensitive Site Is Present in ER
-positive
Cells--
The location of the HS1-hypersensitive site suggests that
this alteration of chromatin structure is required for transcription from the main ER
promoter. If this hypothesis were correct, the HS1
site should be found invariably in cells that express ER
. The
chromatin structure of a panel of ER
-positive and ER
-negative cell lines was previously examined for the presence of the HS1 site
(31). Fig. 4 shows the results of an
analysis of the HS1 site in an additional panel of cell lines. The
ER
-positive breast carcinoma cell lines T47-D, ZR75-1, BT20, and
MDA-MB-361 all demonstrated the HS1 site. The ER
-positive
endometrial cancer cell line ECC-1 also demonstrated the
HS1-hypersensitive site. In distinct contrast, the HS1 site was not
found to be present in an analysis of the chromatin structure of the
ER
-negative cell lines HEC1A, HBL-100, or HeLa (see Fig. 4).
Together with the results in Fig. 3A, this analysis of the
HS1 site in 10 cell lines demonstrated a striking correlation between
the expression of ER
and the presence of the HS1 site. Previous
studies in these cell lines have shown that each of the cell lines that
demonstrate the HS1 site (MCF7, T47-D, ZR75-1, BT20, MDA-MB-361, and
ECC-1) had been found to overexpress AP2
, whereas those cell lines
that lack the HS1 site (MDA-MB-231, HEC1A, HBL-100, and HeLa) express
negligible amounts of the AP2 factors (15, 32). This result suggests
that the HS1 site may be generated by AP2 binding to chromatin in the
ER
promoter.

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Fig. 4.
Analysis of HS1 in a panel of cell
lines. A panel of ER -positive breast carcinoma (T47-D, ZR75-1,
BT20, MDA-MB-361), ER -positive endometrial carcinoma (ECC-1),
ER -negative endometrial carcinoma (HEC1A), ER -negative breast
carcinoma (HBL-100), and an ER -negative cervical carcinoma cell line
(HeLa) were analyzed for the presence of the HS1-hypersensitive site.
All ER -positive lines demonstrated the presence of HS1, whereas none
of the ER -negative lines had the site.
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AP2
Induced the HS1-hypersensitive Site in HMECs--
The
location of HS1 mapped to the untranslated region of exon 1 and
coincides with the location of high affinity binding sites for the AP2
transcription factors (15). Functional analysis of the main ER
promoter indicated that AP2
as well as AP2
are able to induce
transcription from the main ER
promoter by binding to the AP2 sites
found in the untranslated leader of exon 1 (15, 26). To define better
which AP2 proteins may be involved in regulation of the ER
gene in
MCF7 cells, an AP2
-specific antisera was generated. As seen in Fig.
5, the commercially available polyclonal antibody, SC-184, is able to supershift purified AP2
and AP2
. The
antisera, AP, is specific for AP2
and does not supershift purified
AP2
(Fig. 5). An analysis of MCF-7 nuclear extracts indicates that
the majority of AP2 activity in MCF-7 is supershifted with the AP
antisera. Densitometer analysis indicated that ~75% of the AP2
activity in MCF-7 nuclear extract is supershifted by the
AP2
-specific antisera. Assuming that homo- and heterodimers of
AP2
will supershift, it is estimated that ~50% of the AP2 activity in MCF-7 cells is AP2
.

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Fig. 5.
Gel shift with
AP2 -specific antisera. Gel shift using
AP2-binding site probe and purified AP2 , AP2 -GST fusion protein
(26), or MCF-7 nuclear extract. Antibody used in supershift is either
SC-184, which shifts both AP2 or AP2 , and antisera AP, which is
AP2 -specific. Free probe is not shown. The panel on the
right shows that the majority of AP2 activity in MCF-7 cells
is supershifted with AP antisera.
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Functional promoter analysis established an important role for AP2
in regulating the expression of the main ER
promoter in
ER
-positive cells (15). HS1 was specific to the chromatin of
ER
-positive cells, and the location of HS1 coincided with the
location of the AP2-binding sites. These two findings led us to
hypothesize that binding of AP2
to high affinity sites in the ER
promoter induces the HS1 site. HMECs express minimal amounts of ER
mRNA or protein and are generally considered to be ER
-negative.
An adenovirus was engineered that expressed AP2
(AdAP2
) and was
used to induce overexpression of AP2
in HMECs. Infection of HMECs
with AdAP2
induced high levels of AP2 expression, which was
supershifted with AP2 polyclonal antibody, SC-184 (Fig. 6A). However, no AP2 activity
was detected in HMECs infected with wild-type adenovirus. AP2
expression was able to induce the HS1-hypersensitive site in ER
exon
1 (Fig. 6B). However, HS2 and HS3 were not induced by AP2
expression. Infection of HMECs with AdWT did not induce formation of
any hypersensitive sites in the ER
exon 1 region (Fig.
6B). These experiments were repeated using an adenoviral construct that expressed the AP2
protein. Identical results were obtained (data not shown) indicating that binding of either AP2
or
AP2
is capable of altering the chromatin structure of the ER
promoter and inducing the HS1 site which is a characteristic of the
chromatin in ER
-positive cells.

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Fig. 6.
HS1 DNase I-hypersensitive site is induced in
HMECs by AP2 . A, AP2 activity.
A demonstrates that infection of HMECs with AdAP2 virus
generated AP2 activity that co-migrates with activity in MCF-7 nuclear
extract and is supershifted with the AP2 antibody SC-184. B,
hypersensitive site in HMECs. B, demonstrates that the
AdAP2 virus induced the HS1-hypersensitive site in HMECs. Infection
of HMECs with AdWT had no effect.
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AP2
Induces Transcription of the ER
Promoter in
HMECs--
Expression of the endogenous ER
gene was examined in
HMECs in which AP2 expression was induced by viral infection. After viral infection with AdAP2
, AdAP2
, or AdWT, no changes in the level of endogenous ER
expression were detected in HMECs (data not
shown). This result suggests that either additional factors or
additional changes in chromatin structure are required to induce endogenous ER
expression in mammary epithelial cells. To investigate these possibilities further, HMECs were transfected with a reporter construct in which the ER
promoter was inserted upstream of
luciferase. Fig. 7 shows the results of
experiments in which an AP2 expression construct was co-transfected
into HMECs with ER
promoter reporter plasmids. As seen in Fig. 7,
the pGL2-Basic reporter has relatively low basal activity in HMECs. The
activity of pGL2-Basic was not altered by co-transfection of an AP2
expression construct. An ER
promoter reporter containing the
untranslated leader to +230 (ER3794-230LUC) had low levels of basal
expression in HMECs that was identical to the promoterless pGL2-Basic
construct. However, AP2
was able to trans-activate the ER
promoter in HMECs and induced expression from the promoter by
approximately 10-fold. An ER
promoter truncation that deletes the
untranslated leader (ER3794-0LUC) lacks the region of the promoter
containing the HS1-hypersensitive site. ER3794-0LUC has identical
basal expression in HMECs as the larger construct containing the
untranslated leader. However, this construct is profoundly reduced in
its ability to be trans-activated by AP2
. These results clearly
demonstrate that AP2
can trans-activate the ER
promoter in HMECs
by interacting with the region of the promoter encompassing the
HS1-hypersensitive site.

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Fig. 7.
ER promoter analysis
in HMECs. HMECs were transfected with the reporter plasmids
pGL2-Basic, ER3794-230LUC, or ER3794-0LUC. The AP2 expression
plasmid, AP2 /pcDNA3.1 (26), or vector alone, pcDNA3.1, was
co-transfected. Luciferase expression was normalized with a
-galactosidase control vector. Results are the average and standard
deviation from three transfections.
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DISCUSSION |
Breast cancers that express ER
are more likely to occur in
postmenopausal women (4), are associated with a better prognosis (6,
7), and are more likely to respond to hormonal therapy (4, 5) than
tumors that do not express the receptor. ER
-positive breast cancers
overexpress ER
protein and have 10-100-fold more ER
than normal
mammary epithelial cells (33). Studies of breast cancer cell lines (8)
and primary carcinomas (9, 10) indicate that transcriptional regulation
is a critical level of control of ER
expression. The genomic map of
the 5' region of the ER
locus described in this study provides a
physical map of the ER
cap sites and the location of regulatory
regions involved in transcriptional control. Thus, these data provide
the basis for a functional analysis of the alternate ER
promoters in
breast cancer. The hypersensitive site, HS1, is invariably found to be
a feature of chromatin in cell lines that express ER
. The
location of the HS1 site corresponds to binding sites for the AP2
transcription factors in the ER
promoter, and the HS1 site can be
generated in HMECs by expression of the AP2
transcription factor. We
have further shown that AP2
can trans-activate the ER
promoter in
HMECs by interacting with the region of the promoter encompassing the
HS1 site. Since ER
expression is necessary for hormone response,
these results provide further evidence that overexpression of AP2
is
a critical mechanism in the oncogenesis of hormone-responsive breast cancer.
The exons 1-8 of the ER
gene encode the ER
protein and span a
region of ~160 kbp of genomic DNA (34). We have developed a contig
map that includes all of the known 5' non-coding exons of the ER
gene that adds an additional 160-170 kbp to the ER
gene locus,
which brings the total size of the ER
locus to ~300 kbp. We were
surprised to find that exon Ha is ~124 kbp upstream of the coding
region of the ER
gene and that the intron between exon Hb and the
splice acceptor site in exon 1 is over 110 kb. Exon C has been
described as a liver-specific ER
exon (13). Exon C has been reported
to splice to exon Hb (14), and it was fortuitous that this exon
was also in the contig. Exon C is located ~30-40 kbp upstream of
exon Ha, contrary to the genomic location for exon C in a previous
report (25).
The two alternate ER
transcripts, E and H, are both initiated with
genuine cap sites indicating that there are separate ER
promoters
controlling expression of the ER
gene. In addition, there are
multiple cap sites for exons Ha and exon E, as demonstrated by the S1
nuclease analysis and 5'-RACE results (Fig. 2). In the case of exon Ha,
these cap sites are clustered over ~70 bp, whereas for exon E, there
appears to be two separate clusters of cap sites separated by 120 bp.
Multiple cap sites are a common feature of TATA-less promoters, which
is consistent with the lack of clear TATA elements associated with
either the exon Ha or E 5'-flanking sequence.
It is interesting that the ER
gene is controlled by several
different promoters. The biologic basis for a gene having multiple promoters that encode the same protein is not entirely clear. A recent
paper (35) described the expression of an isoform of ER
that lacked
the amino terminus of the protein. The truncated ER
isoform
repressed trans-activation of the full-length ER
protein. This
isoform was encoded by an H-type transcript that skipped exon 1 and
spliced directly to exon 2. It seems likely that one purpose of these
alternate cap sites may be to regulate expression of ER
proteins
with alternate function. There may be additional factors involving
tissue-specific expression that requires the existence of alternate cap
sites. Defining the genomic organization, cap sites, and hypersensitive
sites associated with these exons is an important step toward
determining the regulation of these alternate ER
promoters.
DNase I-hypersensitive sites are regions of chromatin that are open and
accessible to DNase digestion (30). These sites correspond to the
location of important regulatory regions of eukaryotic gene promoters.
Indeed, hypersensitive sites specific to active transcription of ER
are located near each of the ER
cap sites. However, eukaryotic
regulatory signals can be far removed from a cap site, and often a
hypersensitive site may identify the location of a regulatory sequence
relevant to regulation of one or more promoters. Our results are in
agreement with other studies that have described hypersensitive sites
of the ER
gene near exon 1 (31). In addition, a new hypersensitive
site, HS4, has been identified that is specific to ER
-positive
cells. The HS4 site is just downstream of exon Hb and may represent a
regulatory element controlling expression of ER
transcription
initiated at exon Ha.
The HS1 site has been localized to the untranslated leader of the main
ER
promoter in exon 1 and is found in all ER
-positive breast and
endometrial carcinoma cell lines examined ((31) Figs. 3 and 4) but is
not a characteristic of the chromatin structure of HMECs (Fig. 6) or
ER
-negative cell lines ((31) Figs. 3 and 4). The location of the HS1
site corresponds to the region of the ER
promoter that was defined
in a functional assay to be responsible for ER
-specific
transcription (15, 26). This region of DNA bound a factor, initially
called ERF-1 (15), that was found to be overexpressed in ER
-positive
breast and ER
-positive endometrial cancer cell lines. The ERF-1
factor was cloned and found to be identical to AP2
(27). ERF-1 in
MCF-7 nuclear extract is composed of AP2
but contains other AP2
factors as well, presumably AP2
(Fig. 5). These two AP2 factors have
identical binding specificity and both are able to induce expression
from the cloned ER
promoter (26). The data herein show that AP2
and AP2
are able to generate the HS1-hypersensitive site in HMECs,
indicating that these factors are able to induce changes in the
chromatin structure that is known to be associated with transcription
of the ER
gene. We have further shown that AP2
can trans-activate
the ER
promoter in HMECs and that trans-activation requires the
region of the promoter that contains the HS1 site. These results
provide compelling evidence that AP2
has an important role in
regulating transcription of the ER
gene by altering chromatin
structure of the promoter.
Comparing the genomic structure and expression of the ER
gene in
HMECs to hormone-responsive breast cancer can be a useful cell culture
model to dissect the oncogenic processes that leads to a
hormone-responsive tumor. Overexpression of AP2 factors appears to be
one important step. This conclusion is supported by data that
demonstrated overexpression of AP2
in breast cancer compared with
normal mammary epithelial cells (36). This report also demonstrated a
significant correlation between AP2
expression and the
ER
-positive breast cancer phenotype (36). However, we were not able
to detect an increase in ER
mRNA in HMECs infected with the
AdAP2
or AdAP2
viruses (data not shown). This result is not
surprising since other investigators (20) have reported the existence
of factors in ER
-negative cells that repress expression of ER
. In
addition, other HS1 sites are present within the promoter region of the
ER
gene that were not generated by expression of AP2 factors alone
(Fig. 6) indicating that other trans-activating factors may also be
necessary to induce overexpression of the ER
gene in HMECs.
Identifying other factors involved in the regulation of ER
gene
transcription will be an area for further investigation. In addition,
there may be other changes of chromatin structure that are needed to
induce overexpression of ER
. Determining what other changes, in
addition to AP2, are required to induce overexpression of ER
will
help to define the mechanisms of oncogenesis of hormone-responsive breast cancer.