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Departments of Obstetrics and Gynecology and Molecular Genetics (S.S., S.E.B.), University of Illinois at Chicago, Chicago, Illinois 60612; Department of Obstetrics and Gynecology (K.T.), Tohoku University School of Medicine, Sendai 980-8574, Japan; and Department of Obstetrics and Gynecology (M.S.), School of Medicine, Kanazawa University, 13-1 Takara-machi, Kanazawa, Japan 920-0934
Address all correspondence and requests for reprints to: Serdar E. Bulun, M.D. Departments of Obstetrics and Gynecology and Molecular Genetics, The University of Illinois at Chicago, 820 South Wood Street, M/C 808, Chicago, Illinois 60612. E-mail: sbulun{at}uic.edu.
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INTRODUCTION |
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Several studies have demonstrated concentration gradients of P450arom expression within the breast bearing a tumor (1, 2, 3). In breast cancer patients, total P450arom transcript levels were significantly higher in adipose tissue proximal to a tumor in comparison with adipose tissue distal to a tumor (1, 2, 3, 11, 12). Previous studies from this laboratory and elsewhere have demonstrated that a switch in the use of CYP19 gene promoters in the breast adipose fibroblasts surrounding malignant epithelial cells is, at least in part, responsible for the excessive P450arom expression noted in breast cancer (3, 12, 13, 14). The current report describes identification of a novel 101-bp first exon that comprises the 5'-UTR of a significant portion of P450arom transcripts using 5'-rapid amplification of cDNA ends (RACE) generated libraries of human breast cancer tissues. 2 Subsequent cloning and functional characterization of the genomic region upstream of exon I.7 revealed a novel promoter regulated by transcription factors (e.g. GATA-2) typically found in endothelial cells. The activity of promoter I.7 located about 36 kb upstream of the human CYP19 (P450arom) gene coding region gives rise to splicing of this 101-bp exon onto a common splice site 38 bp upstream of the translation start codon (ATG) of the P450arom mRNA.
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). A basic local alignment search tool (BLAST) search analysis mapped this 101-bp sequence onto a region 36,343 bp 5'-upstream of the exon II of the human CYP19 gene (Fig. 1). Following the traditional nomenclature adopted to identify various untranslated first exons of human CYP19 gene, we named this 101-bp novel exon as exon I.7. In these two cancer RNA samples, sequencing analysis also revealed previously identified 5'-UTRs, namely promoter II-, I.3-, and I.4-specific sequences (see below).
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). Four of 14 clones (
29%) contained exon I.7. The other P450arom clones contained promoter II, exon I.3, and exon I.4, which was consistent with previously published data (11, 12, 13, 14). We found only exon I.4-containing P450arom transcripts in a cancer-free breast tissue, used as negative control (Table 1). Next we prepared replica plates of bacterial colonies representing the library of 5'-RACE clones of human breast cancer RNA samples from four additional patients. As summarized in Table 2, the colony hybridization experiments demonstrated that of a total of 270 breast cancer tissue P450arom transcripts, 114 (or 40%) transcripts carried exon I.7. The ratio of I.7-specific transcripts to total transcripts varied from 29%–54% in samples represented in Table 1.
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). We should note that the minor RT-PCR products represented as faint bands larger than the expected size for exon I.7-specific product probably represent minor splice variants. We have not come across any unexpected sequences although we sequenced a total of 22 exon I.7-specific 5'-RACE clones generated from three separate breast cancer tissues and one male buttock adipose tissue (data not shown; Table 1
and Fig. 1). Because these faint bands could be detected by Southern hybridization but not on agarose gels, we did not have a chance to isolate and sequence them.
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Identification of the Transcription Initiation Site
Based on the initial sequence information obtained from the longest exon I.7 clone found in the 5'-RACE-generated library, the most distal 5'-nucleotide was an adenine, defining the total length of exon I.7 as 101 bp. To ascertain whether additional transcription start sites were present, we generated an exon I.7-specific 5'-RACE sublibrary of P450arom mRNA transcripts expressed in male buttock adipose tissue. All but one of the eight randomly selected clones of PCR products subjected to direct sequencing had the previously identified 5'-boundary, i.e. an adenine nucleotide positioned 101 bp upstream of the guanine of the splice acceptor site of coding exon II (Fig. 1). These results are indeed in agreement with a potential transcription initiator (Inr) noted in the promoter I.7 sequence (Fig. 3 and see below).
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2.4 kb) was amplified by PCR using a positive phage (EMBL3) clone containing a genomic sequence (15 kb) as the template. Sequence analysis confirmed the presence of exon I.7 in this clone. Incidentally, a BLAST search analysis aligned this genomic sequence to a region about 36 kb upstream of the first coding exon (exon II) of the human CYP19 (P450arom) gene. A number of smaller fragments of this clone encompassing the 5'-flanking region of exon I.7 were derived by restriction digestion and/or PCR to generate various deletion mutants fused to luciferase reporter constructs described below.
Structural Features of Promoter I.7
Figure 3 depicts the DNA sequence of the 719-bp 5'-flanking region of exon I.7. A computational analysis (using the TFSEARCH database available at http://www.cbrc.jp/research/db/TFSEARCH.html) of the 800-bp fragment spanning the -719/+81-bp region of exon I.7 revealed the presence of several consensus binding sequences for a number of trans-activating regulatory proteins. The proximal 5'-flanking region was devoid of a canonical TATAA or CAAT element. Notably, an initiator (Inr) or cap signal for transcription initiation at -4/+6 bp and two consensus GATA binding motifs at positions -146/-141 bp and -196/-191 bp were predicted (15, 16). Some other cis-acting motifs predicted for promoter I.7 include two consensus c-Ets sites (GGAA) at -166/-163 and -285/-282 bp, two reverse c-Ets sites (TTCC) at -126/-123 bp and -182/-179 bp, an E47 site at -247/-243 bp, a sterol response element binding protein-1 site at -454/-443 bp, a hepatocyte nuclear factor-3ß site at -601/-590 bp, and an Oct-1 site at -637/-624 bp.
Functional Analysis of Promoter I.7 in Various Cell Types
The transcriptional activity of promoter I.7 was assayed by ligating selected segments of the 5'-flanking region of exon I.7 upstream of the luciferase reporter gene in pGL3-Basic vector. These deletion constructs were then transiently transfected into various cell types, as described in Materials and Methods. Because we observed elevated levels of exon I.7-specific P450arom transcripts in breast cancer tissues, we first examined the basal activity of promoter I.7 in the breast cancer cell line MCF-7. Interestingly, transfection of MCF-7 cells with equal molar amounts of various reporter constructs of promoter I.7 resulted only in a marginal increase (2-fold) of maximal promoter activity (-299/+81 bp) compared with the minimal promoter construct containing a -35/+81-bp sequence (Fig. 4). On the other hand, transfection of normal female breast adipose fibroblasts did not show any promoter activity at all (data not shown). We should note that we routinely transfect primary breast adipose fibroblasts with P450arom promoter II luciferase constructs to study regulation of this promoter and observe up to about a 6-fold increase in basal activity in serial deletion constructs (17).
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As shown in Fig. 5A, transient transfection of equal molar amounts of various deletion constructs of the putative promoter I.7 into the human microvascular endothelial cells (HMEC-1) resulted in substantial increases (up to 4-fold) in the luciferase activity compared with a promoterless pGL3 basic vector or the construct containing a minimal fragment (-35/+81 bp) of promoter I.7. The highest promoter activity was observed in the reporter construct containing the -299/+81-bp region. Deletion up to the -35-bp position resulted in a complete loss of promoter activity, indicating that the sequence from -299 to -35 bp (which included two GATA sites) is critical for the basal promoter activity. On the other hand, addition of 5'-upstream sequences up to position -2344 bp gradually reduced the promoter activity (Fig. 5A).
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ctcg did not affect the promoter activity significantly (Fig. 5A). In contrast, mutation of the -196/-191-bp GATA site (GATA ctcg) resulted in 55% reduction in luciferase activity (Fig. 5A). Mutations of both of these GATA sites abolished promoter I.7 activity in HMEC-1 cells. Taken together, these results demonstrated that even though both GATA motifs are required for optimal promoter activity, the GATA site at -196/-191 bp is more critical for sustaining the basal activity of promoter I.7 in endothelial cells.
Human GATA-2 Transactivates Promoter I.7 in a Concentration-Dependent Manner in HMEC-1 Cells
To determine whether GATA proteins could modulate promoter I.7 activity, we introduced the -299/+81 promoter I.7 reporter plasmid construct along with the mammalian expression vector pMT2-hGATA-2 (18), which contained a full-length human GATA-2 cDNA into HMEC-1 cells. As shown in Fig. 5B, cotransfection of pMT2-hGATA-2 (100 ng) resulted in approximately 5-fold stimulation in the luciferase activity of the reporter plasmid -299/+81 in HMEC-1 cells compared with the empty expression vector used as a control. Moreover, addition of increasing amounts (10–100 ng/well) of pMT2-hGATA-2 expression vector elicited a concentration-dependent increase in promoter I.7 activity in HMEC-1 cells. Taken together, these results demonstrate the ability of GATA-2 to transactivate P450arom promoter I.7 in a concentration-dependent fashion in endothelial cells.
The GATA Binding Site at -196/-191 bp Is Protected by HMEC-1 Nuclear Protein(s) in Deoxyribonuclease I (DNase I) Footprinting
In vitro footprinting revealed that a nuclear extract from HMEC-1 cells protected a sequence flanking the -196/-191-bp GATA site from digestion by DNase I (Fig. 6). Two additional footprints at an E47 and Ets binding sites positioned at -247/-243 bp and -166/-163 bp, respectively, were also detected (Fig. 6).
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, incubation of nuclear extracts from HMEC-1 cells with the 32P-labeled oligonucleotide probe resulted in the formation of one specific band (lane 2), which was abolished by 50-fold molar excess of unlabeled probe (lane 3). In contrast, preincubation with an oligonucleotide, which also represents the sequence at -209/-180 bp but with a mutation in the -196/-191-bp GATA site (GATA ctcg), did not compete with the wild-type probe (lane 4), demonstrating that the -196/-191-bp GATA sequence is a critical requirement for binding activity.
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, lanes 5 and 6, respectively). In contrast, the addition of a control antibody, anti-E47, had no effect (lane 7), confirming the specific binding of GATA-2 to the -196/-191-bp GATA site in promoter I.7. |
DISCUSSION |
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The GATA transcription factor family consists of six homologous members, and, by virtue of its highly conserved zinc finger DNA binding domains, they all potentially recognize a (G/A) GAT (T/A)(A/G) motif (25, 26). Even though the GATA proteins display distinct but overlapping patterns of expression in a variety of tissues, gene disruption experiments revealed critical requirements for GATA-1 and GATA-2 in hematopoiesis and megakaryopoiesis (25, 26). In addition to a high level of expression in hematopoietic stem cells, mast cells, and megakaryocytes, abundant expression of GATA-2 has also been noted in vascular endothelial cells (26).
We demonstrated a positive concentration gradient of promoter I.7-specific P450arom mRNA levels in breast tissues bearing carcinomas, where mRNA levels were strikingly higher in cancer tissue and tissue proximal to carcinomas compared with adipose tissue biopsies distal to the tumor or cancer-free breast adipose tissues. We hypothesize that increased vascularity and, thus, numbers of endothelial cells with promoter I.7 activity in carcinoma tissue or breast tissues proximal to carcinomas form the basis for these observations. In fact, we and others consistently observed that P450arom protein is present in endothelial cells of the capillaries in breast cancer and surrounding adipose tissue (Sasano, H., personal communication, and our unpublished observations). A corollary to this observation is the body of literature showing P450arom expression in endothelial cells of human and murine vena cava, coronary artery, and aorta (22, 23, 24).
We do not know the functional implications of the high levels of promoter I.7-specific P450arom transcripts found in pregnant abdominal adipose tissue and normal male buttock adipose tissue. Increased vascularity in adipose tissue during pregnancy may be a potential explanation. Additionally, it is tempting to speculate that P450arom promoter I.7 activity levels exhibit a sexually dimorphic pattern in human adipose tissue. Experiments are under way to test these hypotheses.
Extensive angiogenesis, a hallmark of aggressive tumor growth, consists of endothelial cell proliferation, migration, and tube formation, yielding increased vascular density (27). We predict that endothelial cell-specific promoter I.7 activity may be the key to excessive expression of exon I.7 in breast tumors. Additionally, it is possible that paracrine factors from adjacent neoplastic or nonneoplastic cells may also contribute to the aberrant expression of exon I.7 in the vascular endothelium in breast cancer tissues. We propose that excessive levels of promoter I.7-specific P450arom mRNA in breast tumors contributing to the elevated in situ aromatase activity and local estrogen biosynthesis leading to the growth of breast cancer. In addition to its well recognized role as a potent mitogen to malignant breast epithelial cells, the ability of estrogen to stimulate angiogenesis, at least in part, by increasing vascular endothelial growth factor production leading to neovascularization, have also been demonstrated (28, 29, 30, 31). Estrogen was also shown to preserve the actin cytoarchitecture during metabolic stress, rescue endothelial cells from hypoxia-induced apoptosis, and induce the migration of endothelial cells leading to tube formation (32). Moreover, recent literature is suggestive of important physiological and pathological roles of estrogen in coronary artery disease and rheumatoid arthritis (33, 34, 35, 36). In summary, estrogen appears to play important roles in endothelial cell physiology and angiogenesis. In this regard, an endothelial cell-specific P450arom promoter giving rise to local estrogen formation with potential intracrine and paracrine effects may have significant impacts on human physiology and pathology.
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MATERIALS AND METHODS |
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Fetal calf serum, media for cell cultures, LipofectAMINE PLUS reagent, Platinum Taq Polymerase, Superscript preamplification system, and oligonucleotides used as PCR primers or probes were obtained from Life Technologies, Inc. (Gaithersburg, MD). Sources of kits and reagents used in various experiments were indicated in appropriate contexts. Antibodies against GATA-1, GATA-2, and E47 used in supershift experiments were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Dual-Luciferase Reporter Assay System was purchased from Promega Corp. (Madison, WI). Other standard biochemicals and restriction enzymes used in the experiments were products of Sigma (St. Louis, MO), Life Technologies, Inc., or Roche Molecular Biochemicals (Indianapolis, IN). A human genomic library in EMBL3-SP6/T7 vector was procured from CLONTECH Laboratories, Inc. (Palo Alto, CA). A mammalian expression plasmid of human GATA-2 cDNA (pMT2 hGATA-2) was a gift from Dr. Stuart H. Orkin (18).
RNA Isolation, RT-PCR, and Southern Blot Analysis
Total RNA was isolated by the guanidinium thiocyanate-cesium chloride method (38). In the reverse transcription (RT) reaction 2 µg total RNA were reverse transcribed employing Superscript II enzyme (Life Technologies, Inc.) using random hexamers as the primer. For the amplification of exon I.7-specific P450arom mRNA species, the sense primer was designed as the +48/+67-bp sequence in exon I.7, whereas antisense primer was a 20-bp oligonucleotide (5'-ATTCCCATGCAGTAGCCAGG-3') complementary to the +272/+ 291-bp sequence in coding exon III of the P450arom cDNA. PCR was carried out employing the Ampli-Taq PCR system (Perkin-Elmer Cetus, Norwalk, CT) in a 20-µl reaction containing 8 pmol of each primer and 1 µl of the reverse transcription reaction as the PCR template. The PCR conditions were as the following: one cycle of 94 C for 1 min, followed by 30 cycles of 94 C for 30 sec, 55 C for 30 sec, and 72 C for 1 min with a final extension at 72 C for 5 min. The expected size of the PCR product was 259 bp. The PCR products were fractionated on a 2% agarose gel, transferred to a Zeta-Probe membrane (Bio-Rad Laboratories, Inc., Hercules, CA), and hybridized with a 18-bp radiolabeled oligonucleotide (5'-TCTGAGGTCAAGGAACAC-3') complementary to a sequence in the middle of the amplified PCR product. Additionally, we confirmed the fidelity of three amplified fragments by sequencing. As an internal control, the coding region of the housekeeping gene human glyceraldehyde-3-phosphate dehydrogenase was amplified from the reverse transcription mix and analyzed in an agarose gel containing ethidium bromide.
5'-RACE
5'-RACE was performed using the Smart RACE cDNA Amplification Kit of CLONTECH Laboratories, Inc. following the instructions provided by the manufacturer with minor modifications. Briefly, adaptor-ligated, double-strand cDNA was synthesized using ribonuclease H-reverse transcriptase, Superscript II (Life Technologies, Inc.), and a 33-bp antisense primer corresponding to the +485/+517-bp coding region (exon IV) of the human P450arom cDNA as the primer (5'-GCCTTTCTCATGCATACCGATGCACTGCAGGCC-3'). The first round of PCR was conducted using 1 µl of the adaptor-ligated 5'-RACE-ready cDNA as the template and universal primer, which is a forward primer complementary to the adaptor (component of the kit provided by CLONTECH Laboratories, Inc.) and a 21-bp antisense primer (5'-CAGGAATCTGCCGTGGGAGAT-3') complementary to the P450arom cDNA at +302/+323 bp (exon III). To increase the specificity of the reaction, a nested PCR amplification of the 1:100 diluted primary PCR reaction was performed using the nested universal primer complementary to the adaptor sequence and a 20-bp antisense primer (5'-CAGGCACGATGCTGGTGATG-3') representing the +160/+179-bp sequence (exon II) of the human P450arom mRNA. The resulting PCR products were fractionated using a 2% agarose gel. All bands within the range of 150 to 300 bp were excised and cloned into pGEM-T-Easy plasmid vector for sequence analysis. The novel exon I.7 was discovered as the 5'-end in certain portions of these clones. To map the transcription initiation site of exon I.7, exon I.7-specific PCR was used employing the RACE-ready cDNA as templates and the nested universal primer and a 32-bp antisense primer (5'-AGAGTCCCTTGCTGATTTCACCCCTTTCTCCG-3') complementary to the +76 to +101-bp sequence in exon I.7 plus the first 6 bp of the coding Exon II. The PlatinumTaq Polymerase enzyme system supplied by Life Technologies, Inc. was employed in all PCR experiments.
Colony Hybridization
Bacterial colonies (a total of 270) representing a library of 5'-RACE clones of P450arom transcripts expressed in breast cancer tissue specimens were replica-plated. Each replica plate was hybridized with an 18-bp radiolabeled oligonucleotide (5'-TCTGAGGTCAAGGAACAC-3') complementary to the sequence within the coding exon II of the P450arom cDNA or a 21-bp oligonucleotide complementary to the +27/+47-bp sequence (5'-GATGAAGTGTTGTATCTTAGG-3') of exon I.7.
Mapping of Exon I.7 to the Human Genome
Nucleotide sequence information of the exon I.7 (101 bp) and the human full-length aromatase cDNA (GenBank accession no.: M22246) were subjected to the BLAST homology search (39) against the Human Genome Resource database of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/genome/seq/HsBlast.html).
Cloning of the 5'-Flanking Region of Exon I.7
The 5'-upstream region of the newly identified untranslated first exon I.7 in the human CYP19 gene was cloned from a EMBL3-SP6/T7 human genomic library constructed from human placental DNA partially digested with Sau3AI restriction enzyme (CLONTECH Laboratories, Inc.). For library screening employing oligonucleotides as probes, the protocol recommended by the manufacturer was followed (Lambda Library User Manual, PT1010-1, CLONTECH Laboratories, Inc.). Briefly, the human EMBL3 genomic library was sequentially screened by plaque lift hybridization using two oligonucleotide probes. For the first screening, we used a 21-bp oligonucleotide complementary to the +27/+47-bp sequence in exon I.7. The positive plaques were then rescreened with a second 20-bp oligonucleotide complementary to the +48/+67-bp sequence in exon I.7. The sequences of these two oligonucleotides were 5'-GATGAAGTGTTGTATCTTAGG-3' and 5'-GGCTCCATCTACAAGGATGA-3', respectively. Using purified DNA of a positive phage clone as the template, we amplified a region (2.4 kb) by PCR using a primer pair complementary to +51/+81-bp sequence of exon I.7 and the T7 primer flanking the EMBL-SP6/T7 cloning site. For the PCR we employed the Expand Long Template PCR System (Roche Molecular Biochemicals) and 200 ng purified phage DNA (as template). The PCR conditions were as follows: one cycle of denaturation at 95 C for 3 min, followed by 25 cycles of 15 sec at 94 C, 1 min at 42 C, 12 min at 68 C, followed by a final extension at 68 C for 10 min. The gel-purified genomic fragment (2.4 kb) was subsequently cloned into the pGEM-Teasy vector (Promega Corp.) employing the T-tailing method. The cloned fragment was sequenced to its entirety by the primer walking method using ABI PRISM BigDye Terminator Cycle sequencing kit and ABI PRISM 377 automated sequencer (PE Applied Biosystems, Foster City, CA).
Generation of Luciferase Reporter Constructs
Deletion of specific regions of the 2.4-kb cloned DNA fragment was accomplished through the use of available restriction sites and subcloning into compatible sites of the luciferase reporter plasmid pGL3-basic. In some cases, PCR was used to create flanking restriction sites for cloning purposes.
Mutations were performed using the QuikChange Site-Directed Mutagenesis Kit from Stratagene (La Jolla, CA), following the protocol provided by the manufacturer. A 380-bp fragment corresponding to the -299- to +81-bp region cloned into the pGL3-Basic vector was used as a template for PCR amplification. Mutations were introduced into putative GATA binding sites in this -299/+81-bp construct. For the -146/-141-bp GATA site, the sequence GAGATAAT was changed to GActcgAT, and for the -196/-191 GATA site, GTGATAGT was changed to GTctcgGT. The mutated bases are shown in lowercase. The mutations were confirmed by direct sequencing, and the 380-bp fragment (-299/+81 bp) containing the desired mutations was isolated from the original pGL3 construct and cloned into a separate MluI-BglII-digested empty pGL3-Basic vector.
Plasmids used in transfection experiments were purified using an EndoFree Plasmid Isolation Kit (QIAGEN, Valencia, CA), and purity was verified by A260/A280 absorbance and agarose gel electrophoresis. In all constructs, orientation of inserts was verified by direct sequencing.
Transfection of HMEC-1 and MCF-7 Cells with Luciferase Reporter Constructs
HMEC-1 cells were grown in endothelial basal medium MCDB-131 (Life Technologies, Inc.) containing appropriate supplements as described previously (16). MCF-7 cells were grown as recommended by ATCC. The day before transfection, cells were plated into six-well tissue culture dishes at a density such that the cells reached 70–80% confluence by the time of transfection. Transfections were performed using LipofectAMINE PLUS reagent (Life Technologies, Inc.) following the protocol provided by the manufacturer. One microgram of the -299/+81-bp luciferase reporter construct or equimolar amounts of various deletion constructs were used. As an internal control, 25 ng of pRL (Renilla luciferase)-Null plasmid (Promega Corp.) was cotransfected. Three hours after transfection, the transfection medium was removed by aspiration, 3 ml of complete medium (containing serum and antibiotics) were added, and the plates were returned to the incubator. In the case of HMEC-1 cells, transfections were carried out using Lipofectin reagent (Life Technologies, Inc.) for 5 h. At 48 h post transfection, medium was removed and wells were rinsed with PBS to remove detached cells and residual growth medium. Next, 250 µl of 1x passive lysis buffer provided in the Dual-Luciferase Reporter Assay System (Promega Corp.) were added to each well. Cells were dispersed by scraping with a disposable plastic cell lifter and subjected to one cycle of freeze (-80 C)/thaw (room temperature) to ensure complete cell lysis. Ten microliters of the supernatant were used to assay luciferase activities. Firefly and Renilla luciferase activities were sequentially measured using the Dual-Luciferase Reporter Assay System (Promega Corp.) following the manufacturer’s instructions. Luciferase activities were determined using a Lumat LB 9507 Luminometer equipped with a variable auto dual injector (Berthold Technologies, Bad Wildbad, Germany) The protocol included a 1-sec predelay followed by a 20-sec measuring period. The Renilla luciferase activity, expressed from the pRL-Null vector, provided an internal control to monitor transfection efficiency. Firefly luciferase activities were normalized based on the Renilla luciferase activity in each well. Statistical analysis and graphic representation of the results were accomplished using the SigmaPlot 5 Program (SPSS, Inc., Chicago, IL).
Preparation of Nuclear Extracts from HMEC-1
Nuclear extracts were prepared from HMEC-1 cells using the NE-PER Nuclear and cytoplasmic extraction reagent kit supplied by Pierce Chemical Co. Protein content of the prepared nuclear extract was determined by the BCA-200 Protein Assay Kit supplied by Pierce Chemical Co.
DNase I Footprinting
For in vitro DNase I footprinting, a SacI–XbaI fragment containing the -287/-92-bp region of promoter I.7 was gel purified and was labeled by filling in with Klenow fragment using 
[32P]dCTP. Because SacI restriction digestion creates a 3'-protruding end, Klenow filling is restricted to the XbaI-digested end of the fragment. The labeled fragment was purified using a QIAquick PCR Purification Kit (QIAGEN), and radioactivity was quantitated by scintillation counting. The footprinting was performed using the SureTrack Footprinting Kit (Amersham Pharmacia Biotech, Arlington Heights, IL) and following the protocol provided by the manufacturer, except that the binding reaction was conducted in a 20-µl reaction volume, and the volume was adjusted to 50 µl by adding 30 µl of 1x binding buffer before proceeding with DNase I digestion. Samples were analyzed on a 6% sequencing gel. A sequencing ladder was obtained by loading a sample of the probe that had been treated with formic acid and piperidine to cleave at G and A residues.
EMSA
A 30-mer oligonucleotide (representing the -209- to -180-bp sequence), which harbors the -196/-191-bp GATA motif, was radiolabeled using 
[32P]ATP and T4 polynucleotide kinase. The labeled oligonucleotide was purified using a QIAquick Nucleotide purification Kit (QIAGEN), and radioactivity was quantified by scintillation counting. Approximately 30,000 cpm of labeled probe and 250 ng of HMEC-1 nuclear extract were mixed with indicated amounts of oligonucleotide competitors or antibodies (supershift) in a gel shift binding buffer supplied by Promega Corp. The reaction mix had been incubated on ice for 1 h before adding the labeled probe (30,000 cpm), after which the incubation was continued for 20 min at 30 C. Samples were then analyzed on a 5% nondenaturing acrylamide gel using 0.5x Tris-borate-EDTA as the running buffer. The dried gel was exposed to Biomax MR autoradiographic film (Eastman Kodak Co., Rochester, NY).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Present address: Department of Pathology, Duke University Medical Center 3712, Durham, North Carolina 27710. 
Abbreviations: BLAST, Basic local alignment search tool; DNase I, deoxyribonuclease I; HMEC-1, human microvascular endothelial cells; P450arom, aromatase cytochrome P450; RACE, rapid amplification of cDNA ends; UTR, untranslatory region.
2 The nucleotide sequences reported in this paper have been submitted to the GenBank/EBI data bank with accession nos. AF419337 and AF419338.
Received for publication March 28, 2002. Accepted for publication July 5, 2002.
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REFERENCES |
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and ß. Proc Natl Acad Sci USA 97:10972–10977NURSA Molecule Pages Link:
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 J. Brosens, H. Verhoeven, R. Campo, L. Gianaroli, S. Gordts, J. Hazekamp, L. Hagglund, T. Mardesic, E. Varila, J. Zech, et al. High endometrial aromatase P450 mRNA expression is associated with poor IVF outcome Hum. Reprod., February 1, 2004; 19(2): 352 - 356. [Abstract] [Full Text] [PDF]
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-146/-141, pGATA
X174 DNA/HinfI markers (Promega Corp.); G+A, Maxam-Gilbert sequencing ladder of the probe DNA.