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Advances in Brief |
Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, California 91010
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ABSTRACT |
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Introduction |
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TopABSTRACTIntroductionMaterials and MethodsRT-PCR Analysis.ResultsDiscussionREFERENCES |
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Our laboratory has functionally characterized promoters I.3
(10)
and II (11)
. We have also found a
silencer element (S1) that is situated between promoters I.3 and
II and that down-regulates the action of these two promoters
(12)
. Several nuclear receptors including ERR
-1 and
COUP-TF1 have been shown to be expressed in breast tissue and to
bind to S1 (13)
. A cAMP-responsive element (CREaro)
in the region upstream from promoter I.3 was also identified
(14)
. We have proposed that in normal breast stromal
cells, aromatase expression is driven by promoter 1.4, which is
regulated mainly by glucocorticoid, and the action of promoters 1.3 and
II is suppressed by the silencer S1 (14)
. However, in
cancer tissue, cAMP production increases (15)
, and
aromatase promoters are switched to cAMP-dependent promoters,
i.e., 1.3 and II. The recently identified CREaro is thought
to play an important role enhancing the function of promoter I.3. To
better understand the role of CREaro, we applied the yeast one-hybrid
approach to screen a human breast tissue hybrid cDNA expression library
for proteins binding to the CREaro region. Although we identified
cAMP-responsive element binding protein 1 when the library was
screened in the presence of
cAMP5
, the major proteins identified when the library was screened in the
absence of cAMP were the Snail and Slug zinc-finger proteins.
The snail gene was identified through the genetic analysis of dorsoventral pattering in Drosophila. Snail zinc-finger protein was thought to be required zygotically for mesoderm formation (16 , 17) . Drosophila Snail protein is a transcriptional repressor that acts to maintain the proper germ layers by repressing the expression within the mesoderm of regulatory genes involved in ectodermal development (18) . The snail genes have also been cloned in mouse (19) , Xenopus laevis (20) , chicken (21) , and zebrafish (22) . However, Snail had never been reported in humans before our isolation of the cDNA clone. We have named the human snail SnaH. Slug is a member of the snail family of zinc-finger proteins. It is involved in epithelial to mesenchyme cell transition that leads to the maintenance of the progress zone (23) and plays a role in limb bud development (24) . The cloning of the human Slug gene has been reported previously (25) .
We are excited about the Snail and Slug findings for two reasons. First, this was the first time that a human form of Snail cDNA was isolated (the cDNA sequence was submitted to GenBank on February 2, 1999). Second, this was also the first time that SnaH and Slug were found to be expressed in human breast tissue. A series of experiments with our full-length SnaH cDNA clone have been carried out. We performed DNA mobility shift analysis and mutation analysis, which indicated that SnaH binds to a segment in the promoter I.3 region that overlaps with CREaro. cDNA transfection experiments have revealed that SnaH acts as a repressor that reduces promoter I.3 activity. Very importantly, RT-PCR experiments have demonstrated that SnaH (as well as Slug) is expressed at a high level in normal breast epithelial cells and stromal fibroblasts but at a very low level in breast cancer cell lines and cancer tissue specimens. Our results support a specific action of SnaH on the activity of promoter I.3 of the human aromatase gene. A hypothesis for possible roles of SnaH and Slug in human breast tissue is presented.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsRT-PCR Analysis.ResultsDiscussionREFERENCES |
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-35Slabeled ATP and
[
-32P]ATP were from New England Nuclear
(Boston, MA). [14C]chloramphenicol
(D-threo-[dichloroacetyl-l-14C]chloramphenicol;
specific activity, 55 mCi/mmol) was from Amersham Life Science, Inc.
(Arlington Heights, IL). The CAT expression vector, pUMSVOCAT, was a
gift from Dr. K. Kurachi at the University of Michigan (Ann Arbor, MI).
Cell Culture.
Heat-inactivated FCS (10%, v/v; Gemini, Calabasas, CA) and 1x
antibiotics (Gemini) including 100 units of penicillin/ml, 100 µg of
streptomycin sulfate/ml, and 0.25 µg of fungizone/ml were routinely
added to media for all of the cell lines examined in this study. Hep-G2
cells, human hepatoblastoma cells, were grown in DMEM (Life
Technologies, Inc., Gaithersburg, MD) containing 1
mM sodium pyruvate. SK-BR-3 cells, human breast
adenocarcinoma cells, were maintained in McCoy’s 5a medium containing
2 mM L-glutamine. WS3PF, WS3TF, GI33PF, GI33TF,
and W88TF, human breast fibroblast cell lines, were grown in
Weymouth’s MB 752/1 medium and were the gifts of Dr. R. J.
Santen at the University of Virginia Health Science Center,
(Charlottesville, VA). MCF-7 cells, human breast adenocarcinoma cells,
were grown in Eagle’s MEM with nonessential amino acids and sodium
pyruvate. MDA-MB-231, a human breast adenocarcinoma cell line, was
maintained in Leibovitz’s L-15 medium. MCF-10A and HBL-100
are noncancerous breast epithelial cell lines. MCF-10A was grown in
DMEM and Ham’s nutrient mixture F-12 containing 5% equine serum, 2
µM insulin, 20 ng/ml epidermal growth factor, 0.5
µg/ml hydrocortisone, and 0.1 µg/ml chorela toxin. HBL-100 was
grown in McCoy’s 5a medium containing 2 mM
L-glutamine.
Library Screening.
A hybrid cDNA mammary gland library containing 3.5 x 106
independent clones was purchased from
Clontech Laboratories, Inc. (Palo Alto, CA). The library was titered
and amplified according to Appendices B and C of MATCHMAKER GAL4
Two-Hybrid User Manual (Clontech Laboratories, Inc.). The library
screening followed the MATCHMAKER One-Hybrid User Manual (Clontech
Laboratories, Inc.). Complementary strands of a 27-bp aromatase genomic
fragment (designated CREaroCF), which included a stretch of eight
nucleotides of CREaro sequences, were synthesized to contain three
tandem copies with an EcoRI site at the 5' end and
XhoI and XbaI sites at the 3' end. The sequence
for the sense strand is: 5'-CCCCCCGAATTCAACCTGCTGATGAAGTCACAAAATGA-CAACCTGCTGATGAAGTCACAAAATGACAACCTGCTGATGAAGTCACAAAATGACTCTAGACTCGAGCCCC-3'.
The complementary strands were annealed and cloned into pHISi and
pLacZi vectors. The resulting constructs were linearized with
XhoI and NcoI, respectively, to facilitate
integration into the yeast host YM4271 strain genome.
Transformation of yeast cells was carried out using the Yeast Maker kit
(Clontech Laboratories, Inc.). Integrated pHISi construct yeast strain
was used as a reporter to screen the mammary gland cDNA library.
Integrated pLacZi construct yeast strain was used to reconfirm positive
clones using a ß-galactosidase assay. Positive clones coding for
fusion proteins that interact with CREaroCF were isolated from yeast
hosts and retransformed into E. coli DH5. Sequences of
positive clones were then subjected to a database search
(BLAST6
) for homology.
DNA Sequencing and Analyses.
DNA sequencing for short stretches of DNA for checking orientation of
inserts was performed manually in the laboratory using the T7 Sequenase
Version 2.0 (Amersham Life Science, Cleveland, OH) kit and
-35S-labeled ATP for labeling. DNA analyses
were performed using GCG Wisconsin software packages. A human
Snail homologue (designated SnaH; H stands for Human or
Homo sapiens) has been identified, and a 1696-bp SnaH cDNA
sequence was submitted to GenBank (accession no. AF125377) on February
2, 1999.
Expression of SnaH in E. coli.
A 795-bp cDNA containing the coding region of SnaH was generated by PCR
using a set of primers containing an EcoRI restriction site.
The DNA fragment was subcloned into the expression vector pET-28a(+)
containing the T7 promoter (Novagen, Inc., Madison, WI). The correct
orientation, sequence, and in-frame of the fragment in the vector were
confirmed by direct DNA sequencing using T7 promoter primer
(5'-TAATACGACTCACTATAGGG-3'). The confirmed construct was designated
pET-SnaH. A host cell BL21DE3 was transformed with pET-SnaH, cultured
in LB containing 15 µg/ml of kanamycin, and induced by the
addition of 1.0 mM
isopropyl-1-thio-ß-D-galactopyranoside
at 37°C for 2 h. The induced cells were harvested by
centrifugation. The recombinant SnaH protein was solubilized and
refolded according to the pET System Manual and Protein Refolding Kit
(Novagen, Inc.). Briefly, the cells were resuspended in 0.1 culture
volume of 20 mM Tris-HCl (pH 7.5)/10
mM EDTA/1% Triton X-100 and then put on ice.
They were then treated with lysozyme (100 µg/µl) at 30°C for 15
min, sonicated five times (each time for 4 x 10 s)
on ice at 40% Output (Branson Sonifier 450), and centrifuged at
10,000 x g for 10 min. Most of the SnaH
protein was found to be in the resulting pellet, as demonstrated by
10% SDS-PAGE. The pellet was washed several times and then solubilized
in 50 mM
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
(pH 11.0) containing 0.3% N-lauroylsarcosine. The
solubilized sample was incubated at room temperature for 15 min and
centrifuged at 10,000 x g for 10 min at room
temperature. The detergent in the supernatant was slowly removed by two
dialyses using 50x volumes of 20 mM Tris HCl (pH
8.5) containing 0.1 mM DTT and then dialyzed two
more times in the same buffer without DTT (3 h each). The concentration
of the renatured protein was determined by the Bradford method
(26)
.
DNA Mobility Shift Analysis.
The oligonucleotide with one copy of CREaroCF
(5'AACCTGCTGATGAAGTCACAAAATGAC-3') and its antisense strand
oligonucleotide were synthesized by the DNA/RNA synthesis facility at
the City of Hope National Medical Center/Beckman Research Institute.
These two oligonucleotides were annealed together at 95°C for 10 min
and then cooled down slowly to room temperature. The double-stranded
oligonucleotides were end-labeled with
[-32P]ATP using T4 kinase and used as probes
in DNA mobility shift analysis. DNA mobility shift analysis was
conducted as described by Singh et al. (27)
.
Briefly, 10 µl of recombinant SnaH protein was incubated with 60,000
cpm of 32P-labeled probe at room temperature for
30 min in a total volume of 20 µl of the DNA mobility shift analysis
buffer containing 12 mM HEPES (pH 7.9), 100
mM KCl, 10 µM
ZnCl2, 1 mM DTT, 12% (v/v)
glycerol, 0.05% NP40, 20 mg/ml BSA, and 0.1 mg/ml of poly(dI-dC). The
mixture was then electrophoresed on a 4% acrylamide gel with
0.5 x Tris-borate EDTA. Gels were dried and
autoradiographed.
For the competition assays, the conditions for binding of SnaH protein to each probe were the same as those described above except that the appropriate amounts of the unlabeled oligonucleotides were supplemented in the binding reaction mixture 10 min before adding the labeled probe. To evaluate the interaction of SnaH protein with CREaroCF, five nonradioactive mutated double-stranded oligonucleotides were used as competitors in the DNA mobility shift analysis. The sequence of these oligo-mutants and the wildtype CREaroCF are as follows (only sense strands are shown): wild-type CREaroCF, 5'-AAC CTG CTG ATG AAG TCA CAA AAT GAC-3'; Mu 1, 5'-TTG CTG CTG ATG AAG TCA CAA AAT GAC-3'; Mu 2, 5'-AAC GAC CTG ATG AAG TCA CAA AAT GAC-3'; Mu 3, 5'-AAC CTG CAC TTG AAG TCA CAA AAT GAC-3'; Mu 4, 5'-AAC CTG CTG ATG TTG TCA CAA AAT GAC-3'; Mu 5, 5'-AAC CTG CTG ATG AAG TTG CAA AAT GAC-3'. The CREaro sequence is in bold, Drosophila Snail-binding site-like sequence is underlined, and the mutated bases are in italic.
CAT Expression Plasmids.
A low background promotorless CAT expression vector, pUMSVOCAT
(28)
, with modification at its cloning sites
(29)
was used for preparation of the CAT expression
constructs. The 1.2-kb aromatase genomic fragment containing 836 bp of
5' flanking sequences of promoter I.3, promoter I.3 region, and the
entire sequence of exon I.3 was subcloned into pBluescript vector,
termed pBS-1.2-kb aro. This plasmid was used as a template DNA in the
PCR reactions to generate a set of 5' or 3' deletion constructs with
sets of 5' and 3' primers flanking the designated regions with an
artificial HindIII site at the 5' end and a XbaI
site at the 3' end. The fragments, -144/+5 bp, -76/+5 bp, and -32/+5
bp, were restricted with HindII/XbaI, purified on
the agarose gel, and subcloned into the
HindII/XbaI site of the modified pUMSVOCAT
vector. These constructs were designated pUMS-144/+5CAT,
pUMS-76/+5CAT, and pUMS-32/+5CAT, respectively.
Transfection and CAT Assays.
Hepatoblastoma cell line Hep-G2 cells and breast adenocarcinoma
SK-BR-3 cells were cotransfected with pSG5-SnaH, pSV-ß-Gal,
and pUMS-144/+5CAT, pUMS-76/+5CAT, or pUMS-32/+5CAT. Transfection
was carried out using lipofectin according to the provided protocol
(Life Technologies, Inc.). Briefly, 1 µg of each of the
aforementioned constructs in the lipofectin reagent was added to
4 x 105
cells seeded in 60-mm
tissue culture dishes. After an overnight incubation, the transfected
cells were washed twice with appropriate volume of the regular growth
medium and cultured for 24 h. The cells were then harvested by
removal from the plates with a scraper, pelleted by centrifugation, and
resuspended in 200 µl of 0.25 M Tris-HCl (pH 8.0). The
cells were disrupted to release proteins by three quick freeze and thaw
cycles. Aliquots of the lysates were used to determine protein
concentration in each sample. An equal amount of protein from each
sample was used for the CAT assays. Briefly, the appropriate amounts of
proteins were incubated overnight at 37°C in a mixture containing
[14C]chloramphenicol and
n-Butyryl-CoA. 14C-labeled CoA was
extracted with xylene and subjected to scintillation readings. An equal
amount of total protein was also used in the ß-galactosidase assay
for transfection efficiency normalization.
Site-directed Mutagenesis.
The mutants were generated by using a PCR-based mutagenesis method
described by Nelson and Long (30)
. Briefly, the PCR
mutagenesis used three forward primers, the nucleotide sequences of
which were as follows with the mutated bases and amino acids in bold:
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PCR was carried out with a DNA Thermal Cycler 480 (Perkin-Elmer Corp., Norwalk, CT). The reaction mixture (in 100 µl) contained five units of Taq DNA polymerase, 20 ng of pSG5-SnaH as template, and 200 nmol each of forward and reverse primers. The PCR cycles were 1 min at 94°C to denature the template DNA, 1 min at 50°C to allow the primers to anneal, and 1 min at 72°C for DNA extension, and they were cycled 30 times. The PCR products were resolved over 1.5% agarose gel, purified by using QIAGEN Gel Extraction Kit (Qiagen, Inc., Chatsworth, CA), and cloned by using TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA). After blue-white selection, transformed competent cells were grown in LB media, and plasmids were extracted by using QIAprep Spin Miniprep Kit (Qiagen, Inc.) followed by EcoRI digestion. SnaH fragments were purified from agarose gel and were inserted into pSG5 expression vector (Stratagene, La Jolla, CA) digested with EcoRI. The orientation of SnaH-fragments was confirmed by restriction enzyme digestion and sequencing.
Constructs of wild-type SnaH or its mutants (SnaH-P2A, -F5A, and -V7A) were transiently cotransfected with pUMS-76/+5CAT and pSV-ß-Gal into HepG2 cells growing on 6-well plates as described above. After washing cells with PBS, cells were lysed in Reporter Lysis Buffer (Promega, Madison, WI), and cell extracts were collected by scraping. The lysates were centrifuged, and the supernatants were used for ß-galactosidase assay to define transfection efficiency. Remaining extracts were heated for 10 min at 60°C followed by centrifugation, and the supernatants were used for CAT assay as described above.
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RT-PCR Analysis. |
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TopABSTRACTIntroductionMaterials and MethodsRT-PCR Analysis.ResultsDiscussionREFERENCES |
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The semiquantitative analyses were performed using the Titan one tube RT-PCR system (Roche Molecular Biochemical). All of the samples were first analyzed using ß-actin primers, and the RNA levels for the analyses of both SnaH and human Slug were normalized based on the results from analyses with a set of ß-actin primers. RT-PCR conditions were as indicated below. After 1-h incubation at 50°C, 25 cycles of PCR were run at 94°C for 30 s, 50°C for 1 min, and 68°C for 2 min. The reaction was ended with a 7-min elongation period at 68°C. We performed PCR at a nonsaturating condition, i.e., 25 cycles. In this way, at least we could compare the relative expression levels of nuclear receptors in our samples. It has been shown that with 25 cycles, product accumulation is exponential (7) . Furthermore, the quantity of PCR products generated with 25 cycles of PCR increases in a mRNA concentration-dependent manner (7) . In addition, a proper control such as PCR analysis with RNA, without treating with reverse transcriptase, was performed to assure us that the PCR products were derived from mRNA. The sequences for the primers and hybridization probes are shown below. SnaH: forward, 5'-AAT CGG AAG CCT AAC TAC AGC GAG-3'; reverse, 5'-CCT TGG CCT CAG AGA GCT GG-3'; hybridization probe, 5'-TGG GCC TCC CTT CGG CTC CAG GAG AGT CCC AGG GTG G-3'; Slug: forward, 5'-GCG CTC CTT CCT GGT CAA GAA GCA TTT CAA CG-3'; reverse, 5'-CTC ACA TAT TCC TTG TCA CAG TAT TTA CAG-3'; hybridization probe, 5'-CCC AGC TAC CCA ATG GCC TCT CTC CTC TTT CCG-3'; Aromatase: forward primer, 5'-GAC TCT AAA TTG CCC CCT CTG; reverse primer, 5'-GTG CCC TCA TAA TTC CAC AC; hybridization probe, 5' ATG GTT TTG GAA ATG CTG AA; ß-actin: forward, 5'-AGG AGC ACC CCG TGC TGC TGA-3'; reverse, 5'-CTA GAA GCA TTT GCG GTG GAC-3'; hybridization probe, 5'-CAT CAC CAT TGG CAA TGA GCG GTT CCG CTG -3'.
Hybridization and Chemiluminescent Detection.
The RT-PCR products (10 µl each) were run on 1.5% agarose gel,
transferred to the positively charged Zeta membrane (Bio-Rad
Laboratories, Hercules, CA) in 20 x SSC, and UV
cross-linked. All of the probes were labeled with DIG-11-ddUTP
using the 3'-end oligonucleotide labeling kit (Roche Molecular
Biochemical). The blots were prehybridized in DIG-Easy-Hyb (Roche
Molecular Biochemical) for 1 h and hybridized overnight at each
hybridization temperature with each probe. The hybridization
temperature was generally 5 to 10°C below the melting temperature of
the probe. After hybridization, the blots were washed two times for 1
min in 2 x SSC, 0.1% SDS, and two times for 15 min in
0.1 x SSC, 0.1% SDS.
The RT-PCR products were detected according to the manufacturer’s instructions. After hybridization and washes, membranes were rinsed briefly in washing buffer (maleic acid buffer). The membranes were incubated for 30 min in blocking solution, then in anti-DIG-AP (1:10,000) solution for 30 min, and then two times for 15 min in washing buffer and equilibrated for 2 min in detection buffer. CSPD read-to-use solution (Roche Molecular Biochemical) was applied on the membranes that were incubated for 5 min at room temperature. After incubation for 15 min at 37°C, the membranes were exposed to Lumi-film Chemiluminescent detection film (Roche Molecular Biochemical) for 15–30 min at room temperature. Image density was quantified with an Imaging Densitometer (Model GS-670; Bio-Rad Laboratories).
Overexpression of SnaH in MDA-MB-231 Breast Cancer Cell Line by
Stable cDNA Transfection.
The SnaH cDNA was generated by PCR with the EcoRI
restriction site at both ends and then ligated into the mammalian
expression vector, pHß-Apr-1-Neo, after a published procedure
(32)
. The orientation and sequence of the cDNA was
confirmed by dideoxy sequencing.
MDA-MB-231 cells were cultured in RPMI medium 1640, containing 10% fetal bovine serum (Omega Scientific, Inc., Tarzana, CA), 5 ml of (10,000 units/ml) antibiotic-antimyotic (Life Technologies, Inc., Grand Island, NY), 2 mM L-glutamine, and 1 mM sodium pyruvate (IS; Irvine Scientific, Santa Ana, CA) in 500 ml medium for each cell culture. When cells had grown about 70% confluent, Phß-Apr-1-Neo vector and plasmid pHß-SnaH were introduced into the cells with lipofectin. The transfection experiments were performed using lipofectin, following the manufacturer’s protocol (Bethesda Research Laboratories). For the selection of transfected cells, the beginning concentration of G418 was 0.6 mg/ml in 5-ml culture volume. The G418 concentration was increased at an increment of 0.1 mg/ml G418/week, and the final concentration of G418 was 1 mg/ml.
Semi-quantitative Analysis of Aromatase and SnaH in Transfected
MDA-MB-231 Cell Lines.
The SnaH-stable-transfected MDA-MB-231 and the vector-transfected cells
were grown in culture media containing 100 µg/ml G418. For RT-PCR,
total RNAs were extracted from cultured cells using TRIzol (Life
Technologies, Inc.). PCR was carried out under the conditions indicated
below. ß-actin (15)
, SnaH (25)
, aromatase (35)
cycles of PCR
were run at 94°C for 1 min, 50°C for 1 min, and 68°C for 2 min.
The reaction was ended with a 10-min elongation period at 68°C. The
PCR products were run on 1.5% agarose gel, transferred to the
positively charged Zeta membrane GT (Bio-Rad) in 20 x SSC, and UV cross-linked. All of the probes were labeled with
32P using T4 DNA kinase. The blots were
prehybridized in 20 mM sodium phosphate buffer (pH 7.2)
containing 7% SDS for 1 h and hybridized overnight at 41°C
(aromatase) or 65°C (SnaH and ß-actin). After hybridization, the
blots were washed at hybridization temperature two times for 30 min in
20 mM sodium phosphate buffer (pH 7.2) containing 5% SDS
and two times for 30 min in 20 mM sodium phosphate buffer
(pH 7.2) containing 1% SDS. The membranes were exposed to X-ray films
for 6 h-3 days at -80°C. Image density was quantified with an
Imaging Densitometer (Model GS-710; Bio-Rad).
Statistical Analysis.
The results were analyzed by the Student t test. Results
were considered significant when P < 0.05.
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Results |
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TopABSTRACTIntroductionMaterials and MethodsRT-PCR Analysis.ResultsDiscussionREFERENCES |
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Sequence Analysis and Characterization of Human Snail Homologue
(SnaH).
Sequence analysis of the 1696-bp EcoRI/XhoI
insert from positive clone 6A, which contains a sequence that is
homologous with known Snail sequences from other species, revealed an
ORF. The 795-bp ORF shares highest homology with mouse Snail
zinc-finger protein Sna (83% identity; Fig. 1
). The encoding protein was named SnaH, where H stands for
human or Homo sapiens. There are 61-bp upstream and 840-bp,
including a poly(A) sequence, downstream from the ORF region (Fig. 2
). The translated SnaH contains 264 amino acids and four
zinc fingers (Fig. 2)
. The length of the SnaH is identical to mouse Sna
but significantly shorter than Drosophila Snail, which is
390 amino acids in length. We have performed DNA mobility shift
analyses and transcriptional function studies using expression
constructs containing the coding region of SnaH cDNA.
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. We generated a series of five double-stranded oligonucleotide
competitors that contain di- or tri-nucleotide mutations in the
CREaroCF (as indicated in "Materials and Methods"). These
oligomutants were independently tested in a DNA mobility shift analysis
to determine whether they could compete with a radiolabeled wild-type
CREaroCF probe for the binding of SnaH protein. If the mutated
competitors are able to compete with the wild-type probe for SnaH
protein binding, then the nucleotides that were mutated are not
involved in the binding. Conversely, if the oligomutants cannot compete
with the wild-type probe for binding, then the regions containing
mutated nucleotides are probably involved in the interaction with the
DNA-binding proteins. We have demonstrated that the recombinant SnaH
interacts with CREaroCF in a dose-dependent manner and that nonspecific
DNA, poly(dI-dC) cannot compete with the binding in DNA mobility shift
analysis (data not shown). We added 30-, 60-, 120-fold molar excess of
the unlabeled wild-type CREaroCF or five oligomutants in the reaction
mixture in the competition experiments. Whereas Mu 1, Mu 2, and Mu 5
competed as well as the wild type, Mu 3 and Mu 4 oligonucleotides did
not compete as well as the wild type (Fig. 4)
. The results of the DNA mobility shift analysis lead us to propose
that the region at -70 bp to -61 bp, -CTG ATG AAG T-, is involved in
the interaction with SnaH.
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. As a control experiment, SnaH did not
have an effect when the analysis was performed using a reporter
construct that did not contain the SnaH-binding site (i.e.,
-32/+5; see Fig. 5B
). In addition, the inhibitory activity
of SnaH was not modified by incubating cells in the presence of
forskolin (data not shown), indicating that cAMP is not needed for the
interaction of SnaH with its binding element. Our results indicate that
SnaH protein binds to the region upstream from promoter I.3 in the
human aromatase gene and reduces the promoter I.3 activity.
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. These results indicate that
the NH2-terminal SNAG domain of SnaH is important
for its repressor activity.
Expression of SnaH and Slug in Breast Cell Lines and Tissue
Specimens.
We examined the levels of SnaH and Slug mRNAs through RT-PCR
Southern analysis with RNA isolated from 55 breast cancer tissues, 5
breast fibroblast primary cell lines, 3 breast cancer cell lines, 1
hepatoblastoma Hep-G2 cell line, and 2 noncancer breast epithelial cell
lines. As can be seen in Fig. 6
, the two breast normal epithelial cell lines (i.e., MCF-10A
and HBL-100) and five breast stromal fibroblast cell lines have
relatively high levels of SnaH and Slug RNA messages, but aromatase
mRNA was not detected in these cell lines. On the other hand, aromatase
mRNA can be detected in breast cancer cell lines, and the levels of
SnaH and Slug mRNAs in these cell lines are low. SnaH expression was
not detected in MCF-7 and MDA-MB-231 cell lines, and Slug expression
was not detected in the MCF-7 cell line. Most interestingly, SnaH mRNA
was detected in only 16 of 55 breast cancer specimens, and Slug
mRNA was absent in all of the 55 specimens. On the other hand,
aromatase mRNA was detected in 54 of the 55 specimens. Although
additional analyses on normal breast tissues are needed, our results
indicate that these two zinc-finger proteins are regularly expressed in
normal breast tissue, and that their expressions are reduced in breast
cancer tissue.
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. These results indicate that overexpression of SnaH in MDA-MB-231
cells suppresses aromatase expression.
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Discussion |
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TopABSTRACTIntroductionMaterials and MethodsRT-PCR Analysis.ResultsDiscussionREFERENCES |
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The nucleotide and amino acid sequence analyses revealed that the
similarity of human, mouse, Xenopus, and
Drosophila Snail extends throughout the coding region, and
the human and mouse Snail proteins are shorter than the others (Fig. 2)
. Both the coding region and amino acid sequences of SnaH share the
highest homology with those of mouse Snail (Sna, 83%; xsna, 54.5%;
snail, 39.0% homology, respectively). The zinc-finger regions are
highly conserved among them. Structural and functional conservation
suggests that these proteins may have originated in the extremely
ancient common ancestors of insects and vertebrates. However, SnaH has
only four zinc fingers and does not encode for the first zinc finger
found in Xenopus and Drosophila. Mouse and
zebrafish Snail proteins also have only four zinc fingers. These
observations suggest that the first zinc finger in
Drosophila and Xenopus snail may not be critical
for the protein functions or may have a different function. In
addition, the translated SnaH contains 264 amino acids (Fig. 2)
. The
length of the SnaH is identical to mouse Sna but significantly shorter
than Drosophila Snail, which is 390 amino acids in length.
By DNase I footprinting analysis, Kasai et al. (38) and Ip et al. (39) identified Drosophila Snail-binding sites in the promoter regions of the gene single-minded (sim), ANCACCTGTTNNCA, and rhomboid (rho), C(A/C)ACTTGC, respectively. Mauhin et al. (40) defined six Snail-binding sequences (CAGGTG, CAAGTG, CAGGTT, CAGATG, CACGTG, and CATGTG) using a pool of oligonucleotides with random sequences, and the base frequency analysis suggested that each motif has its own binding capacity. On the basis of our DNA mobility shift analysis, it was found that SnaH protein binds to the CREaroCF region and probably interacts with the region between -70 bp to -61 bp, CTGATGAAGT, relative to the human aromatase transcriptional start site of promoter I.3. Because SnaH is a four-finger protein instead of a five-finger protein, as is the Drosophila Snail, it is not surprising that, of the two species, Snail recognizes different DNA sequences. Furthermore, the findings from our laboratories and others suggest that SnaH binds to DNA with a broad specificity.
The functional analysis revealed that SnaH is a repressor that reduces
the activity of promoter I.3 of the human aromatase gene. We used two
reporter constructs containing CREaro (i.e., -144/+5 and
-76/+5) and performed transfection experiments in two mammalian cell
lines, SK-BR-3 and HepG2. SK-BR-3 is a breast cancer cell line. The
HepG2 cell line (human hepatoblastoma cell line) was chosen because
aromatase is expressed in this cell line. We have also performed the
control experiment using a reporter construct that does not contain the
SnaH-binding site (i.e., -32/+5). SnaH did not have an
effect when the analysis was performed using the latter construct,
supporting the hypothesis that SnaH suppresses the promoter activity by
interacting with the proposed SnaH-binding site. As indicated in
"Results," the SnaH expression was not detected in the human breast
cancer MDA-MB-231 and MCF-7 cell lines. To further analyze the effect
of SnaH on aromatase expression, we have generated a
SnaH-overexpressing MDA-MB-231 cell line. The aromatase mRNA level in
the SnaH-expressing cell line was found to be one third that of the
vector-transfected cell line, confirming that SnaH suppresses aromatase
expression (Fig. 7)
. Attempts were also made to generate a
SnaH-expressing MCF-7 cell line. However, we have not yet been able to
obtain any live cells after the transfection process.
As shown in Fig. 2
, SnaH, mSna, and Xenopus snail have the
identical NH2-terminal seven-amino acid SNAG
domain. Our results from site-directed mutagenesis experiments confirm
that this domain is important for the repressor activity of SnaH (see
Fig. 5C
).
The proposed SnaH-binding element overlaps six nucleotides with CREaro,
an enhancer element situated upstream from promoter I.3 of the human
aromatase gene (see Fig. 3
). It is thought that SnaH can quench the
CREaro activity. "Quenching" is a form of gene regulation whereby
activators and repressors cooccupy neighboring sites in a target
promoter, but the repressor blocks the ability of the activator to
contact the transcription complex (41)
. Gray et
al. (41)
have reported that Snail is not a dedicated
repressor but, instead, appears to block disparate activators. We
identified SnaH (and Slug) in a human mammary cDNA library in the
absence of cAMP. In addition, these zinc-finger proteins are expressed
at a relatively high level in normal breast epithelial cells and
fibroblasts, but their expression levels in breast cancer tissue are
very low. The present observation and our previous work lead us to
propose that in normal breast epithelial cells and stromal fibroblasts,
SnaH is expressed and prevents proteins from binding to CREaro. This
results in a suppression of promoter I.3 activity. On the other hand,
in cancer tissue, human aromatase promoter usage switches to promoter
I.3, which is in response to cAMP activation of cAMP-responsive
element-binding proteins and to a decrease in SnaH (and Slug)
expression.
The snail gene was originally identified in
Drosophila (16)
. It acts to restrict
neuroectoderm and neural fate in the invaginating mesoderm
(17)
as a regulator of neurogenesis (42)
and
is necessary for the maintenance of vestigial expression in the wing
disc (43)
. Xsna, a Xenopus gene, is
expressed zygotically in all of early mesoderm (20)
. A
mouse gene (Sna) encodes a 264-amino acid protein that
contains four zinc fingers, like the SnaH. Sna transcripts
are expressed throughout postimplantation development
(44)
. CSnR is expressed in the right-hand
lateral mesoderm during normal chick development (21)
. Our
study represents the first direct functional analysis of human Snail,
SnaH. Although we do not yet know the role of this protein in early
development, we have demonstrated a specific role for this protein in
modulating estrogen formation in breast tissue. A recent report by
Batlle et al. (45)
has suggested that Snail is
a repressor of E-cadherin gene expression in epithelial tumor cells.
The study was mainly performed using mouse Snail, i.e., Sna.
As shown in our RT-PCR studies and in Northern analysis by Batlle
et al. (45)
, SnaH is expressed at a higher
level in noncancer cell lines and fibroblasts than in breast cancer
cell lines and breast tumor specimens. The role of SnaH may not be as
simple as that proposed by Batlle et al. (45)
.
The Drosophila Snail contains a P-DLS-K sequence and
was thought to interfere with the interaction between CtBP and
adenovirus E1A protein, which is involved in transcriptional activation
and tumorigenesis (46)
. It is important to point out that
SnaH is shorter than the Drosophila Snail and that the
P-DLS-K sequence is not present in SnaH (see Fig. 1
).
The Slug gene is a second member of the snail family. Slug expression is correlated with areas of undifferentiated mesenchyme at various stages of tissue differentiation, suggesting a role in maintaining the mesenchymal phenotype and repressing the differentiation processes in early development (24) . The mouse slug (Slugh) is not required for mesoderm formation or for neural crest generation, migration, or development in mice (47) . These results indicate that neither the expression pattern nor the biological function of the Slug gene is conserved among all of vertebrates. The findings that Slug is expressed in noncancer cell lines and fibroblasts and expressed at very low levels in breast cancer cell lines and tumor tissue are very interesting. Because the Slug cDNA clones that we isolated from yeast one-hybrid screening were not full-length clones, we did not include the Slug functional studies in this investigation. We have recently generated the full-length Slug clone by RT-PCR, and experiments are being designed to study the role of this protein in breast cancer development.
The Escargot is a third member of the Snail family. The Drosophila Escargot has a 72% nucleotide identity with Snail and a 76% nucleotide identity with xsnail (48) . The protein has a similar zinc-finger domain. Escargot protein regulates tracheal branch fusion in Drosophila. During development of tubular networks, epithelial tubes must fuse to each other to form a continuous network. Little is known about the cellular mechanisms or molecular control of epithelial tube fusion. Escargot is an early fusion marker. Its ectopic expression activates the fusion process and suppresses branching throughout the tracheal system, leading to ectopic tracheal connections that resemble certain arteriovenous malformations in humans (49) .
In summary, using the yeast one-hybrid approach to screen a human breast tissue hybrid cDNA expression library, we found that a zinc-finger transcriptional factor Snail (SnaH) and another member of the Snail family, Slug, interacted with a regulatory region near promoter I.3 of the human aromatase gene. In addition, these proteins are expressed in noncancerous cell lines and stromal fibroblasts, but they are expressed at significantly lower levels in breast cancer cell lines and tumor tissue. Our functional analysis revealed that SnaH suppresses the activity of promoter I.3 of the human aromatase gene. These results explain, in part, our previous findings that promoter I.3 is down-regulated in normal breast tissue by SnaH and a previously characterized silencer element (12) . On the other hand, promoter I.3 activity is up-regulated in breast cancer tissue, possibly by a decrease of the expression of SnaH and Slug. It is not unexpected that these zinc-finger proteins have additional functions in human breast tissues. Considering the regulatory action of Escargot, a member of the Snail family, on tracheal branch fusion in Drosophila, we propose that SnaH/Slug may be involved in the development of ductal networks in the breast. This hypothesis is presently being examined in our laboratory.
|
FOOTNOTES |
|---|
1 Supported by the NIH Grants CA44735, ES08258,
and CA65767. 
2 These authors equally contributed to this
work.
3 To whom requests for reprints should be
addressed, at City of Hope, Beckman Research Institute, Division of
Immunology, 1450 East Duarte Road, Duarte, CA 91010-0269. Phone:
(626) 359-8111, extension 2601; Fax: (626) 301-8186;
4 The abbreviations used are: RT-PCR, reverse
transcription-PCR; cAMP, cyclic AMP; CAT, chloramphenicol
acetyltransferase; ORF, open reading frame.
5 K. Wu, D. Zhou, and S. Chen, unpublished
observations.
6 Internet address:
http://www.ncbi.nlm.nih.gov/.
Received 3/23/00. Accepted 12/28/00.
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