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INTRODUCTION |
Epidermal growth factor receptor
(EGFR)1 is a
membrane-spanning 170-kDa glycoprotein that stimulates cell growth
after binding of specific ligands (1, 2). It has been found to be
expressed in many normal and malignant cell types (3). Overexpression of EGFR alone is sufficient to transform NIH 3T3 cells in an
EGF-dependent manner (4, 5). The level of EGFR expression
is primarily regulated by the abundance of its mRNA (6, 7).
Transcription of the EGFR gene starts at multiple initiation sites
within the GC-rich promoter that lacks a TATA or CAAT box (8). It has been shown that the first intron of several genes including EGFR has
important regulatory function (9-12). Two enhancer elements with
cooperative function in the receptor gene have been identified, upstream of the promoter and downstream in intron 1 (13), and EGFR
transcription is in part prematurely terminated in intron 1 (14). A
polymorphic simple sequence repeat (SSR) with 14-21 CA dinucleotides
and a heterozygosity of 72% in a Caucasian reference pedigree (15) was
revealed close to the downstream enhancer element. It was already
demonstrated for the acetyl-CoA carboxylase gene, that a CA repeat in
one of the two promoter regions can repress promoter activity (16).
This repression can be released by a tissue-specific factor or by
removal of the dinucleotide repeat. Therefore, we raised the question
whether the CA-SSR could also play a role in epidermal growth factor
receptor expression. The close proximity of the EGFR downstream
enhancer to the CA repeat in intron 1 led us to the hypothesis that
variations in the number of these dinucleotides could be partially
responsible for individual differences of the EGFR proto-oncogene
expression found in humans. To probe a potential regulatory function of
this highly polymorphic region, we investigated the influence of the CA-SSR on transcription activity in vitro and characterized
RNA synthesis in the EGFR 5'-region in relation to the number of
dinucleotide repeats. For this purpose, we applied an in
vitro method for quantitation of RNA synthesis from different DNA
templates in a reproducible, homogenous, and cell-free transcription
factor matrix. Nuclear extract from A431, a cell line with high EGFR
expression capacity, was used as a model for studying the regulation of
EGFR transcription. By using this approach, it was also possible to
detect all occurring RNA species simultaneously, including prematurely
terminated pre-mRNAs.
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EXPERIMENTAL PROCEDURES |
Cell Lines and DNA Preparation--
A431 (epidermoid carcinoma
cell line), MDA-MB-231, MDA-MB-468, BT-474 (cultured in RPMI 1640, 10%
FCS), HBL-100 (mammary cell line, cultured in RPMI 1640, 5% FCS), and
MCF-7 were purchased from the American Type Culture Collection (ATCC).
MKN7 (gastric carcinoma cell line, cultured in Dulbecco's modified
Eagle's medium/Ham's F-12, 1:1, 10% FCS) was a generous gift of
Prof. C. C. Benz, University of California San Francisco. If not
otherwise specified, the cell lines were derived from breast carcinomas
and cultured in Dulbecco's modified Eagle's medium with 10% FCS at
5% CO2. Genomic DNA of cell lines was prepared with the
QiaAmp tissue kit (Qiagen), according to the manufacturer's protocol.
The DNA concentration was determined with a UV photometer.
Determination of the Number of CA Repeats in Intron 1--
A
114-128-bp PCR fragment containing the polymorphic region was
amplified with 50 pmol of previously described primers (15). One of the
primers was labeled with fluorescein at the 5'-end. The 50-µl PCR
reaction mixture contained 200 ng of DNA of cultured cells, 1.5 mM MgCl2, 7.5% dimethyl sulfoxide (Sigma), 100 µM dNTP each (Perkin Elmer), 1× PCR amplification
buffer, 1.5 units of Taq polymerase (Promega), and light
white mineral oil (Sigma). After PCR, 1 µl of the products plus 0.3 µl of Genescan 500 TAMRA molecular weight standard (Perkin
Elmer-Applied Biosystems) were denatured in 12 µl of formamide,
separated in an Applied Biosystems Prism 310 genetic analyzer with POP4
polymer, and fragment lengths were determined.
Amplification and Purification of the 4,050-bp EGFR PCR
Fragment--
250 ng of genomic DNA were used in a 50-µl PCR
reaction with 0.55 mM MgCl2, 7.5%
Me2SO, 50 µM dNTP each, 1× PCR amplification buffer (Promega), a blend of 2.5 units Taq polymerase
(Promega) with 1 unit Pwo polymerase (Roche Molecular Biochemicals),
and 35 pmol of the following primers: B1/1, CCT TCA GAG ACA GCA AAG GGC; B1/2, CCT GAA ACC AGA ACT CGG ACA AGG C (5'-3'). The polymerase mixture was added to the reaction after overlaying with mineral oil, 4 min of denaturation at 100 °C, and cooling to 95 °C. Cycling profile: 1st cycle, 95 °C for 30 s; 62 °C for 1 min 30 s; 72 °C for 5 min; 2nd to 32nd cycle: 95 °C for 2 min 30 s;
62 °C for 1 min 30 s; 72 °C for 5 min in a Robocycler
Gradient 40 (Stratagene). After amplification, several 50-µl aliquots
of reaction mixtures were combined, DNA was ethanol precipitated,
resolved in TE buffer and electrophoresed through a 0.7% agarose gel
in 1× TBE running buffer. The product band was cut from the gel and
purified using QiaEx (Qiagen). Finally, the UV-quantitated DNA was
again ethanol precipitated and resolved in diethyl pyrocarbonate
treated water. The products were stored at
20 °C until used for
further analysis.
Heterodimer Analysis--
Equal amounts of the purified EGFR
4,050-bp PCR products from A431 and each other cell line were combined
and denatured for 4 min at 100 °C under a mineral oil layer.
Following the addition of one-ninth volume 10× PCR amplification
buffer for efficient hybridization, the mixture was cooled for 10 min
in a 42 °C heating block and kept at room temperature until loaded
with Ficoll loading dye on a native 5% PAA, 1× TBE mini gel.
Electrophoresis was carried out at 15 V/cm for 3 h. Homo- and
heterodimers were detected by ethidium bromide staining.
In Vitro Run-off Assay--
Transcription in vitro
was carried out as described previously (17) with the following
modifications. Approximately 50 ng of PCR product was combined with 6 µl of 1× transcription buffer (20 mM HEPES (pH 7.9), 100 mM KCl, 0.5 mM dithiothreitol, 0.2 mM EDTA, 20% glycerol, in diethyl pyrocarbonate-treated
water), 1 µl of RNasin, 2 µl of 50 mM
MgCl2, 1 µl of 10 mM NTP mix (all Promega), 5 µl of A431 nuclear extract (25 µg of protein, Santa Cruz
Biotechnology), and diethyl pyrocarbonate-treated water to a final
volume of 25 µl. Following careful mixing, transcription took place
for 1 h at 30 °C, before 2.9 µl of 10× restriction buffer
and 10 units of the restriction enzyme PstI were added to
obtain smaller DNA fragments and improve efficiency of the later
transfer to a nylon membrane. The solution was incubated further for
1 h at 37 °C. Subsequently, 71 µl of stop solution (0.3 M Tris (pH 7.4), 0.3 M sodium acetate, 0.5%
SDS, 2 mM EDTA, 3 µg/ml tRNA) was added, and the nucleic
acids were extracted with 100 µl of phenol/chloroform/isoamyl alcohol
(25:24:1) before ethanol precipitation in the presence of 1 µl of
GlycoBlue coprecipitant (Ambion) to increase recovery.
Electrophoresis, Nucleic Acid Transfer, Hybridization, and
Detection--
Nucleic acid pellets were resolved and denatured at
95 °C in formamide loading dye and electrophoresed for 2.5 h at
25 V/cm through an 8-cm, 4% PAA, 8 M urea gel in 1× TBE.
For nucleic acid transfer to a Hybond N+ nylon membrane (Amersham
Pharmacia Biotech), the gel was semi-dry blotted with 0.5× TBE for 45 min at 200 mA. After the transfer, the damp filter was UV irradiated at
302 nm on a transilluminator for 1 min and treated three times with
boiling 1× SSC, 0.1% SDS solution to omit unspecific background in
nonradioactive detection. For hybridization, the filter was
pre-incubated for 1 h at 68 °C in hybridization buffer (5×
SSC, 0.1% SDS, 5% dextran sulfate, 5% liquid block, Amersham
Pharmacia Biotech) before 5 ng/ml fluorescein-UTP (RNA labeling mix,
Roche Molecular Biochemicals) labeled RNA probe was added. The EGFR
5'-region-specific probe was generated by T7 RNA polymerase from a
4,050-bp PCR product with a corresponding promoter site incorporated in
the downstream primer. Hybridization was carried out at 68 °C for
16 h. Stringency washes were 10 min at room temperature in 1×
SSC, 0.1% SDS, and 2× 10 min at 68 °C in 0.5× SSC, 0.1% SDS.
Subsequently, the bands were detected with the Gene Images detection
module (Amersham Pharmacia Biotech), involving an anti-fluorescein
monoclonal antibody coupled to alkaline phosphatase and CDP-Star
chemiluminescence substrate.
Quantification of Transcription Activity in Vitro--
Digital
chemiluminescence imaging was carried out with a LumiImager (Roche
Molecular Biochemicals), and band intensities were integrated after
60-min exposures with the LumiAnalyst (Roche Molecular Biochemicals)
software. For quantification, intensities of the two RNA bands were
normalized to the 1,051-bp DNA template band in the same lane.
Ribonuclease Protection Assay--
In vitro run-off
transcription was performed as described but without PstI
treatment. DNA was instead digested with 10 units of RNase-free DNase I
(Roche Molecular Biochemicals), phenol/chloroform/isoamyl alcohol
extracted and precipitated in the presence of 50 µg of yeast RNA and
a fluoresceinated antisense RNA, transcribed from a 572-bp PCR product
plus T7 RNA polymerase promoter at the 3'-end. The probe spanned a
region from nucleotide +892 to +1463 in the EGFR intron 1. RNase
protection analysis was performed with the HybSpeed ribonuclease
protection assay kit (Ambion) according to the included protocol. After
final precipitation of protected fragments, the RNA was denatured and
electrophoresed through a 6% PAA, 8 M urea mini gel. RNA
transfer and nonradioactive detection was performed as described.
Competitive RT-PCR--
A specific competitor with an internal
deletion of 20 nt of a 143-bp fragment specific for the exon 1-intron 1 boundary of the EGFR gene was constructed according to the method of
Celi et al. (24). The resulting PCR product contained a T7
RNA polymerase promoter and was transcribed in vitro. The
gel-purified competitor RNA was quantitated by UV absorption
measurement. 500 ng of total RNA from cell lines and 10
18
or 10
19 mol of the competitor was subjected to reverse
transcription and PCR (Titan One Tube RT-PCR system, Roche Molecular
Biochemicals) with 20 pmol each of the following specific primers:
primer A, GAG AGC CGG AGC GAG CTC TTC GG; primer B: GAG CCG CGA GAC ACG CCC TTA CC. The two RT-PCR products were separated, and fluorescence was detected with a Prism 310 genetic analyzer. Peaks were integrated with the Genescan software (Perkin Elmer-Applied Biosystems). The molar
amounts of the pre-mRNA template was calculated by the method
reported by Roetger et al. (19). To eliminate the influence of gene amplification or loss of heterozygosity on protein expression, protein concentrations were divided by the EGFR gene dosage.
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RESULTS |
Sequence of the EGFR Promoter/Enhancer Region Reveals No Mutations
in Cell Lines but Contains a Polymorphism in Intron 1--
To
characterize sequence and function of the EGFR promoter/enhancer
region, we amplified a 4,050-bp PCR product from several cell lines. It
contained most of the upstream enhancer (nucleotides
1439 to
1109;
+1 corresponds to the start of exon 1), the promoter region, exon 1, a
polymorphic CA-SSR, and a downstream enhancer site in intron 1 (+1788
to +2318) of the EGFR gene (Fig. 1).
Because of the locally very high GC content and the ability of this
region to form stem loops and triplex structures (8, 9), especially stringent conditions with unusually low Mg2+ concentration
under 1 mM, an organic solvent that reduces secondary structure formation (18) and a special blend of Taq DNA
polymerase with a proof reading enzyme had to be applied to obtain this
long amplicon. The exact number of CA repeats in the PCR products was determined by microsatellite PCR and fragment analysis with an internal
DNA size standard. We used heterodimer analysis to check for further
differences in the sequence of the EGFR promoter/enhancer region in the
cell lines used for this study. A431, an epidermoid carcinoma cell line
with an amplified EGFR gene, was used as a reference, because reported
EGFR sequence data were obtained from this cell line (14). After
hybridization of the A431 4,050-bp PCR product with the fragment from
each other cell line, we found homodimer bands of identical products
and heterodimers bands formed by nonidentical fragments (Fig.
2). Heterodimers run more slowly in
native PAA gels due to the presence of unpaired bases in DNA double
strands. The PCR product of A431 formed no detectable heterodimer when
denatured and rehybridized alone. MDA-MB-231 with the same CA-SSR
(Table I) also provided no detectable
heterodimers with A431. All other cell lines with higher numbers of CA
repeats formed a single heterodimer band running more slowly with
increasing differences in their polymorphic regions. Even the 2-bp
difference in A431/MDA-MB-468 heterodimers was clearly detectable.
Because we were able to detect differences with an at least 2-bp
resolution and heterodimer bands corresponded exactly to the previously
determined allelic pattern of cell lines, we conclude that detected
heterodimer bands were induced only by known polymorphic differences,
and there was no evidence for mutations in these cell lines.

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Fig. 1.
Structure of the 4,050-bp EGFR 5'-region PCR
product. The product was PCR amplified using the indicated primers
B1/1 and B1/2 (small arrows). The sequence contains the
downstream enhancer, a highly polymorphic CA-dinucleotide repeat
(waved line), exon 1 (solid box), the promoter
region, and most of the upstream enhancer. The antisense RNA probe used
for ribonuclease protection assays is shown as an arrow
below the structure (RPA probe).
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Fig. 2.
Heterodimer formation. EGFR 4,050-bp PCR
products from cell lines with the previously determined number of CA
repeats on top of each lane were denatured and hybridized before
electrophoresis through a native 5% polyacrylamide, 1× TBE mini gel
at 15 V/cm for 3 h. Bands were stained with ethidium bromide. A
DNA size standard with fragment lengths in kilobase pairs is shown in
the left lane (M). The arrow on the
right side indicates the position of homodimers with regular
electrophoretic mobilities. Heterodimers were detected above the
homodimer band when two fragments with different numbers of CA repeats
were involved. Retention of heterodimers increases with growing
differences in the polymorphic region. No additional bands are
detected.
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Table I
Characteristics of the cell lines used
The number of CA repeats in intron 1 of the EGFR gene was determined by
microsatellite PCR, EGFR gene dosages, previously published (19), or
unpublished data from our laboratory. EGFR pre-mRNA concentrations
were measured by competitive RT-PCR.
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EGFR Transcription Activity in Vitro Declines with Increasing
Numbers of CA Repeats in Intron 1--
To examine the influence of the
CA-SSR in intron 1 on EGFR gene transcription we used seven PCR
products from cell lines that contained no detectable mutations within
the 5'-region but different numbers of CA repeats from 16 to 26. Rare
alleles with 14, 15, or 19 CA repeats with frequencies below 3% could
not be found in any for us accessible cell line. Transcription in
vitro of these PCR products with A431 nuclear extracts resulted in
RNA double bands of approximately 1,550 and 1,650 nt from all
templates. Fig. 3A shows
representative results from one of three experiments with accordant
findings. The DNA template is detected as multiple fragments due to
PstI restriction digestion. Unspecific origin of the
observed RNA bands can be ruled out because of the stringent hybridization and washing conditions and the negative control experiments that displayed no transcription-derived bands (Fig. 3A, +RNase, no DNA). In an experiment with template amounts
from 10 to 100 ng, we showed that the RNA product yield divided by the
DNA template amount is approximately constant within these limits (Fig.
3B). When band intensities of the 1,650-nt nuclear transcription in vitro products are divided by the amount of
template DNA (represented by the 1,051-bp PstI fragment),
significant differences between PCR products with different numbers of
CA repeats become obvious (Fig. 5A). Relative transcript
amounts vary 5-fold from fragments with 16-21 CA. In triple
experiments, a coefficient of variation of 33% was found. A
correlation of transcription activity in vitro with the
number of CA repeats in intron 1 is manifested by declining relative
transcription activities with increasing counts of dinucleotides.
Approximately the same course is revealed when using the 1,550-nt
instead of the 1,650-nt transcript band for quantitation. The ratio of
the two run-off products (1,650:1,550 nt) fluctuates around 0.8 (data
not shown), a value that reflects the use of two major in
vitro transcription start sites under our conditions.

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Fig. 3.
In vitro run-off transcription of
the EGFR gene. A, EGFR 4,050-bp PCR products from the
indicated cell lines were used as templates for run-off transcription
with A431 nuclear extracts. After transcription, DNA was digested with
PstI, electrophoresed through a denaturing 4%
polyacrylamide, 1× TBE gel, and transferred to a nylon membrane. RNA
products and DNA fragments were detected after hybridization with a
fluoresceinated RNA probe. The DNA template is detected in fragments of
1,051, 848, 706, and 412 nt, and run-off transcripts are sized
approximately 1,650 and 1,550 nt. The sizes of an RNA standard are
shown on the left side. Controls without DNA template and
with RNase treatment in the right lanes revealed no RNA
products. B, determination of the ratio of RNA product
intensity to the DNA 1,051-bp band intensity dependent on the DNA
template amount. The ratio is approximately constant between 10 and 100 ng DNA.
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Nuclear Transcription in Vitro Produces EGFR-specific
Pre-mRNA--
We further characterized and confirmed the identity
of the two in vitro transcribed pre-mRNA species by a
ribonuclease protection assay. We hybridized nuclear transcription
in vitro products in solution with an RNA probe that spanned
the approximate region of transcription termination estimated from the
length of nuclear run-off transcription products (Fig.
4). The probe included the CA repeat with
5'- and 3'-flanking regions plus a 20-nt T7 RNA polymerase promoter at
the 5'-end. Controls of the RNase protection assay demonstrated
integrity of the RNA probe (
RNase) and the absence of self-protecting
structures or unspecific hybridization (
Transcr.). Hybridization of
the probe with nuclear run-off products and subsequent digestion of
single-stranded RNA (+RNase) provided a single product that was
slightly shorter than the undigested probe. Consequently, the point of
termination can be mapped to a site immediately before the 3'-end of
the probe at nucleotide +1,463. Since there is no second termination
site detectable within the region of the elongation block in our
assays, we deduce that the double band is due to two major start sites
between nucleotides
107 and
257 (8) in transcription in
vitro under our conditions.

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Fig. 4.
Ribonuclease protection assay of the nuclear
in vitro transcribed EGFR RNA species. Run-off
transcription products were hybridized in solution with a 592-nt RNA
probe that spanned the region of transcription termination in intron 1 (nucleotides +892 to +1463, see also Fig. 1) before single-stranded RNA
was digested by RNase treatment. The observed band (+RNase)
runs slightly faster than the undigested probe ( RNase). A
negative control that was not allowed for run-off transcription
( Transcr.) revealed no bands. The left lane
contains an RNA size marker with the indicated fragment lengths.
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Level of EGFR pre-mRNA Expression in Vivo Roughly Reflects the
Transcription Activity in Vitro--
After we have shown that
transcription activity in vitro depends on the number of CA
repeats in intron 1, we wanted to know if the data in vitro
correspond to the effective EGFR RNA expression in vivo. We
measured the amount of EGFR pre-mRNA specific for the end of exon 1 and start of intron 1 from the indicated cell lines by competitive
RT-PCR (Table I). Fig. 5B
shows the results with respect to the CA-SSR and after normalization to
an EGFR gene dosage of 1. A431 and MDA-MB-468 have EGFR genes amplified 33-fold or 15.5-fold, respectively and MCF-7 is hemizygous for EGFR as
determined by competitive differential PCR (19). It is obvious that the
fragments with 16 and 20 CA repeats exhibit disproportionately elevated
pre-mRNA expression levels when compared with the other fragments.
But in general, molar amounts of pre-mRNA normalized for gene
dosages also show a tendency of declining expression with increasing
numbers of CA repeats.

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Fig. 5.
Transcription activity in vitro
and in vivo. A, run-off
transcription product bands were quantitated in relation to the DNA
content of each lane. Relative RNA quantity therefore indicates the
run-off transcription activity in vitro. Results were
combined and arranged in order of the number of CA repeats in DNA
templates. Activities are given in percent, relative to the fragments
with 16 CA dinucleotides. B, in vivo levels of
EGFR exon 1/intron 1-specific pre-mRNA in cell lines determined by
competitive RT-PCR. Amounts per 500 ng of total RNA are given in
attomoles. Individual values for each cell line (see Table I) were
arranged in order of the number of CA repeats in the EGFR intron 1 and
divided by the EGFR gene dosage to avoid effects of gene amplification
and loss of heterozygosity.
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DISCUSSION |
Transcription of the EGFR gene and the structure of the 5'-region
of the gene has been extensively investigated. The promoter region as
well as parts of intron 1 are exceptionally GC-rich and potential
secondary structures like stem loops and triple helices were described
(8, 9). A short palindrome sequence 2 kilobase pairs upstream from exon
1 with predicted cruciform structure has been linked to termination of
transcription in nuclear run-on experiments with isolated A431 nuclei
(14). To characterize the action of promoter and enhancers relative to
the length of a polymorphic region in intron 1, we decided to use an
approach with transcription in vitro of PCR products that
contained all known regulatory elements of the EGFR gene. In seven
tumor cell lines, we found no signs of mutation in the 5'-region by
heterodimer analysis at a resolution of at least 2 bp (Fig. 2).
Therefore we used PCR products from these cell lines as templates in
our assay and determined the number of CA repeats in intron 1.
Transcription in vitro mimics the initiation of
transcription in vivo in a constant transcription factor
matrix. Influences of differentially expressed transcription factors or
gene copy numbers are excluded by use of cell free extracts in excess
and DNA transcription templates in any concentration. A431 nuclear extract provides elevated transcription activity. Hence, products are
easy to analyze and the situation in cells with high amounts of
positive transcription factors but also with transcriptional repressors
(14, 20) active on the EGFR gene promoter and enhancers can be
examined. Quantification of transcripts appropriately reflects the
activity of promoters and enhancers in the DNA applied as long as it is
normalized to the DNA content of the in vitro run-off mixture, all other components are present in the same concentrations and activities and nucleic acid transfer efficacy is identical. Therefore, transcription activities may be compared only on the same
blot and if the same batch of nuclear extract has been used. In
contrast to reporter gene assays, the necessity of cloning DNA
fragments into expression vectors and limitations due to unknown plasmid transfection efficiencies and post-transcriptional regulation are omitted. Moreover, additional information can be obtained, for
example about different RNA species and initiation or termination sites
within the examined DNA segment. Problems connected with quantitation
of signals on x-ray films like low linearity of signal/response ratios
and the requirement for multiple exponations were resolved by use of a
chemiluminescence imager with a high dynamic quantification range.
In a study of the acetyl-CoA carboxylase gene, it was demonstrated
(16), that a nonpolymorphic sequence of 28 CA repeats within the
promoter region can repress the activity of one of the two promoters by
70%. However, in this special case mediation of promoter inhibition
seems to require a CAAT box. In the case of EGFR, a similar effect is
observed although there is no CAAT box in the promoter region, and the
CA-SSR is located more than 1,000-bp downstream of the promoter.
Interestingly, differences in the number of CA repeats in the EGFR
intron 1 show different levels of transcription modulation. Fig.
5A shows the association of decreasing transcriptional
activity with increasing numbers of CA repeats. In addition, it could
be demonstrated by ribonuclease protection assays that transcription
in vitro with A431 nuclear extracts terminates at a site
near the polymorphic region in intron 1 (Fig. 4). As already mentioned
in a previous study, EGFR transcription was found to be in part
prematurely terminated approximately 2-kilobase pairs downstream of
exon 1, a site about 540-bp further downstream than our observed
termination site. This discordance may be explained by the use of a
1-kilobase pair probe in the earlier reported run-on hybridization
experiments, which covers our termination sites as well as the more
downstream site. Therefore multiple termination sites spanned by this
probe would appear as a single block. On the other hand, presence of a
low abundant longer transcript that was not detected in our experiments
is possible, because transfer efficiency of lengthy nucleic acids from
PAA gels is significantly reduced. We conclude that our results suggest
a dual function of the polymorphic region. First, an indirect effect that enhances or represses transcription in vitro up to
5-fold depending on the number of CA repeats, and second, a block of RNA elongation unaffected by the length of the SSR.
To examine whether the observed effect in vitro also has
importance in vivo, we measured EGFR pre-mRNA expression
levels of the cell lines used and normalized the results to the EGFR
gene dosages. Fig. 5B also shows declining transcription
levels with increasing numbers of CA repeats except for the 20 CA
allele. The corresponding cell line MKN7 obviously uses other
mechanisms to up-regulate EGFR transcription despite the CA-SSR effect.
The cell lines with 16 CA fragments also appear to further enhance EGFR
transcription. As seen in the in vitro experiments, the
number of CA repeats can mediate an up to 5-fold transcription
repression or activation in our A431 model. Up-regulation by the action
of transcription factors could easily overcome this more basal effect. Taken together, these data demonstrate that
allele-dependent modulation of EGFR transcription can be
observed in carcinoma cell lines in vivo, but not
surprisingly, there are other regulation mechanisms that can outweigh it.
To elucidate the function of the CA stretch in transcription
modulation, we considered properties affecting the bendability of the
EGFR downstream enhancer region. Bending of DNA in a
sequence-dependent manner has an important function in many
biological events like DNA replication, site-specific recombination,
and transcription (21). Helical conformation analysis (22, 23) on the
basis of the CA-SSR and flanking sequences indicate that the
dinucleotide repeat is highly flexible. The intrinsic DNA curvature
propensity of the poly-CA stretch, a measure for helical asymmetry
frequently associated with a rigid conformation, is remarkably low,
whereas the bendability is prominently elevated. The longer the CA
stretch, the longer the highly bendable section becomes, too. This
could favor a DNA secondary structure that supports or hinders binding of a factor to the neighboring enhancer element if the polymorphic segment is prolonged. In this way, a transcriptionally active protein
that binds downstream of the CA-SSR could serve as a mediator of allele
dependent modulation of EGFR transcription.
This report describes the association of decreased transcription
activity with a prolonged polymorphic sequence in the EGFR intron 1 close to an enhancer region. The results are important for the
understanding of EGFR proto-oncogene expression and probably other
genes with comparable constellations of CA repeats and
transcriptionally regulative elements. The knowledge of microsatellite
function in relation to negative or positive enhancers provides new
insights in individually different gene expression and the linkage of
inherited polymorphisms to serious diseases like cancer.