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(Received for publication, July 13,
1995; and in revised form, September 14, 1995) From the
Diminished cellular responsiveness to transforming growth
factor-
Transforming growth factor- Much work
has recently been directed toward characterizing the different types of
TGF- Several tumor
cell lines, including retinoblastoma, pheochromocytoma, neuroblastoma,
and breast carcinoma, which are resistant to the growth inhibitory
effects of TGF- In order to study the
transcriptional regulation of human TGF-
Figure 1:
Nucleotide
sequence of the 5`-flanking region of the human TGF-
Figure 2:
Determination of the 5` ends of the
TGF-
Although S1 nuclease mapping of RNA ends is frequently a good
indicator of transcriptional start sites, the multiple bands revealed
by this assay prompted examination of the 5` ends of the TGF-
Figure 3:
In vitro transcription of the
deletion mutants of the TGF-
To further define the first positive regulatory element (-274
to -137), an additional series of CAT deletion constructs was
created from nucleotide -274 to -47, each ending at +2 (Fig. 4). Deletion of the sequences from -274 to
-219 led to no significant change in the level of activity.
However, removal of sequences from -219 to -200 decreased
activity 20-fold, and further deletion to -172 abolished nearly
all activity. This localized the positive regulatory element to within
this 48-base pair sequence where there is an AP1-like binding site
(-196; TTAGTCA; Fig. 1). Levels of transcription remained
minimal with sequential deletion of nucleotides -172 through
-100. However, when the region -100 to -67 was
deleted, activity returned to previous levels, indicating the presence
of a strong negative regulatory element in this region. Finally, the
promoter fragment -47/+2 displayed a relatively high level
of activity, which was significantly diminished by a substitution
mutation of the Sp1 site, implicating a role for Sp1 in transcriptional
activation from this region.
Figure 4:
Transcription of deletion mutants of the
-274 to -47 region of the TGF-
Figure 5:
Detection of nuclear proteins that bind to
the first positive regulatory element of the TGF-
To determine whether the
observed AP1-like consensus sequences present in the first positive
regulatory element are operative or whether other previously identified
transcription factors might be responsible for the strong enhancer
activity, a second mobility shift assay was performed. This time, the
radiolabeled first positive regulatory sequence was incubated with
HepG2 nuclear protein in the presence of a 100-fold molar excess of the
consensus sequences for AP1, AP2, and CRE. As shown in Fig. 5B (lanes 3 and 5), both the AP1
and the CRE recognition sequences were successful in competing with the
first positive regulatory element (-219/+172) for binding
with complex a but not complex b or the proteins
represented by the lower bands. AP2 failed to compete with the first
positive regulatory element for any protein. The target sequences for
AP1 and CRE are very similar. Complex a may therefore
represent an AP1 or CRE-like factor. The data further suggest that a
novel transcription factor complex or an uncommon consensus sequence is
responsible for the specific protein-DNA binding represented by complex b.
Figure 6:
Identification of first positive
regulatory element target sequences. A, sequences for the
sense strand of the mutant synthetic oligonucleotides. WT shows the wild type sequence. M2-M10 possess the
same sequence except for the 5-nucleotide substitutions shown. The
complementary antisense strand for each sequence was synthesized as
well to create a double-stranded oligonucleotide. B, EMSA
performed with labeled -219/-172 double-stranded
oligonucleotide incubated with HepG2 nuclear extract in competition
with mutant oligonucleotides from A. C, wild type
sequence of first positive regulatory element showing the target
sequences for complexes a and b.
Figure 7:
Detection of nuclear proteins that
interact with the second positive regulatory element of the TGF-
To determine whether any of
these bands represented known transcription factors, the second
positive regulatory element probe was mixed with nuclear protein and
incubated with the oligonucleotide target sequences for AP1, AP2, CREB,
and Sp1 (Fig. 7B). There was no evidence of binding to
any of these target sequences by complexes c, d, or e (lanes 3-6).
Figure 8:
Identification of second positive
regulatory target sequences. A, sense strand sequence for
series of mutant oligonucleotides. WT gives the wild type
sequence. M1-M8 contain the 5-nucleotide base
substitutions as shown. B, EMSA performed with labeled
+1/+50 double-stranded oligonucleotide incubated with HepG2
nuclear extract in competition with mutant oligonucleotides. C, wild type sequence of second positive regulatory element
showing the target sequences for complex a2, b2, c2, and d2.
Figure 9:
Relative contribution of first and second
positive regulatory elements to overall promoter activity. A,
schematic representation of series of TGF-
Isolated mutations of sequences Y and X in the
first positive regulatory element (-219M7/+35 and
-219M5/+35, respectively) or of sequence Z in the second
positive regulatory element (-219/+35M3) caused only a small
decrease in activity. Among the three individual mutations, the largest
decrease in activity to 82% of baseline, occurred with the isolated
mutation of sequence Y, which contains the putative AP1/CRE site.
Mutations in the first positive regulatory element were then paired
with mutation of the second positive regulatory element and, as
expected, led to much more dramatic decreases in transcriptional
activity. When both X and Z were mutated (-219M5/+35M3),
activity fell to 56% of baseline levels. Combined Y and Z mutations
(-219M7/+35M3) decreased activity to 14% of baseline. Again,
mutation of sequence Y led to a more significant decrease in
transcription than mutation of X. Deletion of the second positive
regulatory element decreased transcription to a greater degree than
simply mutating the target sequence Z, confirming that sequence Z is
essential to activity of the second positive regulatory element but
suggesting that the mutation was not sufficient to inactivate the
entire target sequence. Comparing all constructs, the lowest level of
activity occurred with mutation of both the target sequences for the
first and second positive regulatory elements. Thus, the two target
sequences in the first positive regulatory element and the single
target sequence in the second positive regulatory element are critical
to conferring enhancer activity, and both positive regulatory elements
interact to contribute significantly to basal promoter activity. In 1985, Sporn and Roberts first suggested that defects in
the TGF- In
this report we present an expanded sequence for the promoter region of
TGF
Figure 10:
Schematic representation of the multiple
regulatory elements within the TGF-
Two distinct protein complexes demonstrate specific
binding to the first positive regulatory element. Complex a,
which may be identical to AP1 or CREB, binds to sequence Y (-196
to -189), and complex b binds to sequence X (-207
to -197). The second positive regulatory element is also
recognized specifically by two different protein complexes. In this
case complexes c, d, and e all bind to the
same target sequence Z (+11 to +25), although complex c appears to bind to a more limited portion (+16 to +21). Mutational analysis reveals that the two positive regulatory
elements cooperate with the promoter region to sustain basal levels of
promoter activity. Maximum levels of transcription were achieved with
intact first and second positive regulatory elements. Mutation of
individual target sequences in either first or second positive
regulatory element impaired transcription only slightly; however,
mutation of both first and second positive regulatory sequences
together led to marked declines in transcriptional activity (Fig. 9, A and B). This study presents
sequencing data for the human TGF- This study has shown that the promoter region of the
human TGF-
Volume 270,
Number 49,
Issue of December 8, 1995 pp. 29460-29468
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Type II Receptor Gene (*)
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(TGF-) is frequently correlated with decreased
transcription of the type II receptor for TGF- (TGF- RII). We
have cloned and characterized the human TGF- RII promoter and,
using S1 nuclease mapping and 5` rapid amplification of cDNA ends
polymerase chain reaction, have identified five alternative
transcription start sites within the region -33 to +57. DNA
transfection experiments and electrophoretic mobility shift assays have
revealed the existence of five distinct regulatory regions including
two positive regulatory elements and two negative regulatory elements
in addition to the core promoter region. The first positive regulatory
element (-219 to -172) interacts with two distinct nuclear
protein complexes, at least one of which appears to be a previously
unidentified transcription factor. The second positive regulatory
element (+1 to +35) also interacts with two separate protein
complexes, both of which appear to be novel transcription factors.
Deletion of either positive regulatory element markedly decreased
expression of the target gene, suggesting that both positive regulatory
elements are necessary for basal expression levels of TGF- RII.
(TGF-) (
)is a
homodimeric, 25-kDa peptide that plays a critical role in many cellular
processes, including regulation of the cell cycle, cell
differentiation, extracellular matrix synthesis, and modulation of the
synthesis of other growth factors and their receptors
(Massagué, 1990; Roberts and Sporn, 1990).
Aberrant TGF- function has been implicated in the pathogenesis of
many diseases including arthritis (Lafyatis et al., 1989),
hepatitis (Castilla et al., 1991), atherosclerosis (Chen et al., 1987; Grainger et al., 1993), and
glomerulonephritis (Border et al., 1990). It has also been
suggested that in some cases, diminished responsiveness to TGF-
may underlie the process of malignant transformation (Wakefield and
Sporn, 1990). This decreased responsiveness to TGF- could be
caused by defects not only in TGF- expression or activation but
also by defects in the regulation of TGF- receptors. receptors and their intracellular signaling pathways as well
as identifying their role in cell regulation and pathology (Miyazono et al., 1994; Kingsley, 1994; Massagué,
1992). Three distinct cell surface receptors, types I, II, and III,
have been cloned and characterized (Wang et al., 1991;
Lopez-Casillas et al., 1991; Lin et al., 1992; Moren et al., 1992; Franzen et al., 1993; He et al., 1993; Attisano et al., 1993). Type I and type II
receptors are transmembrane serine/threonine kinases that together are
sufficient for signal transduction. The type III receptor is a
transmembrane proteoglycan without intrinsic signaling ability but that
may facilitate the binding of TGF- to the type II receptor (Wrana et al., 1992). The most commonly held model for receptor
action proposes that the type I and type II receptors form a
heteromeric complex that is essential for signaling responses (Wrana et al. 1994). It is therefore likely that a mutation in either
receptor could result in a loss of responsiveness to TGF- (Wrana et al., 1992; Bassing et al., 1994;
Cárcamo et al., 1994)., also fail to express the type II receptor (Park et al., 1994; Kimchi et al., 1988; Sun et
al., 1994). In a previous study, our laboratory described a series
of gastric cancer cell lines in which resistance to TGF-
correlated with gross structural mutations in the type II receptor
gene. There were two notable exceptions in which Southern analysis
yielded a gene without gross deletions or rearrangements, but no type
II receptor protein or mRNA was produced. This suggested that
abnormalities in transcriptional regulation of the type II receptor may
also be involved in the escape from TGF- growth control frequently
observed in the process of carcinogenesis. RII, we cloned and
sequenced 1.9 kilobase pairs of the 5`-flanking region and used S1
nuclease mapping and 5`RACE PCR studies to identify five alternative
transcription start sites within a region from residue -33 to
+57. The human hepatoma HepG2 cell line was selected for this
study because of its high level of TGF- RII expression. Using a
series of promoter-CAT deletion constructs transfected into HepG2
cells, we identified two distinct positive regulatory elements at
-219 to -172 and +1 to +35. Electrophoretic
mobility shift assays (EMSAs) and mutational analysis were then
utilized to define two target sequences in the first positive
regulatory element and one target sequence in the second positive
regulatory element. One protein interacting with the first positive
regulatory element may be an AP1 or CREB-like transcription factor. The
other two target sequences do not share homology with any previously
reported consensus sequences and may be recognized by novel
transcription factor complexes.
Cloning the Promoter Region of the TGF-
A human genomic library was obtained (Clontech)
and screened by standard methods using the 5` end fragment of the human
TGF- Type
II Receptor RII cDNA. Four overlapping clones of the promoter region were
isolated, and subfragments were cloned into the pTZ18 vector
(Pharmacia) and sequenced in both directions by the Sanger
dideoxynucleotide method (U. S. Biochemical Corp. sequence kit).S1 Nuclease Determination of Transcription Start
Sites
A P-labeled DNA probe was generated
spanning the putative transcription start site(s). A plasmid containing
the 2.7-kilobase pair XbaI-HindIII genomic DNA
fragment (10 µg) was digested by the EagI restriction
enzyme, which cut 138 base pairs 3` of the published cDNA end (Lin et al., 1992) and left a 5` overhang. The end was then labeled
as described (Geiser et al., 1991), and the 5` end of the
probe was released by digestion with XbaI. The probe (100,000
cpm) was then hybridized to 80 µg of total RNA from human
adenocarcinoma A549 and DU145 human prostatic adenocarcinoma cell lines
in hybridization buffer (80% formamide, 400 mM NaCl, 0.1% SDS,
20 mM Tris, pH 7.4, and 1 mM EDTA) overnight at 55
°C. S1 nuclease digestion was then done with 150 units of enzyme
(Boehringer Mannheim) for 1 h at 37 °C. Samples were extracted with
phenol/chloroform and ethanol precipitated and then loaded (in 50%
formamide dye) onto a 6% denaturing acrylamide gel.
RACE PCR to Determine 5` RNA Ends
Total
RNA from A549 (4 µg) was reverse-transcribed (Perkin-Elmer RT-PCR
kit) at 42 °C for 1 h using random primers. The resulting cDNA was
then tailed with dGTP using terminal transferase (Lif Technologies
Inc.) to create a 5` end with an oligo(dG) stretch. This product was
then amplified by PCR using the oligonucleotide 5`-GGCCGAGGGAAGCTGCACAG
(+137 to +119 relative to the published cDNA end) and an
oligo(dC) (Geiser et al., 1991). The amplified product was run
on an agarose gel, blotted to nytran, and hybridized to a labeled
upstream oligonucleotide (+137 to +119) for evidence of
amplified receptor product. The positive product was then amplified a
second time using the oligonucleotide 5`-GAGTCCGGCTCCTGT CCCGAG
(+118 to +98) and oligo(dC). The product was cloned into the
AT cloning vector (Invitrogen), and individual clones were sequenced to
determine the 5` ends.Nuclear Extracts
Nuclear extracts of
HepG2 cells were prepared as described (Kim et al., 1994) with
minor variation. Monolayers of HepG2 cells (3 
10
to
5 10) were harvested by scraping, washed in cold
phosphate-buffered saline, and incubated in 2 packed cell volumes of
buffer A (10 mM HEPES, pH 8.0, 1.5 mM MgCl
, 10 mM KCl, 0.5 mM
dithiothreitol, 200 mM sucrose, 0.5 mM phenylmethanesulfonyl fluoride, 1 µg of both leupeptin and
aprotinin/ml, and 0.5% Nonidet P-40) for 5 min at 4 °C. The crude
nuclei released by lysis were collected by microcentrifugation, rinsed
once in buffer A, and resuspended in 2/3 packed cell volume of buffer C
(20 mM HEPES, pH 7.9, 1.5 mM MgCl, 420
mM NaCl, 0.2 mM EDTA, 0.5 mM phenylmethanesulfonyl fluoride, 1.0 MM dithiothreitol, and 1.0
µg of both leupeptin and aprotinin/ml). Nuclei were incubated on a
rocking platform at 4 °C for 30 min and clarified by
microcentrifugation for 5 min. The resulting supernatants were diluted
1:1 with buffer D (20 mM HEPES, pH 7.9, 100 mM KCl,
0.2 mM EDTA, 20% glycerol, 1 mM dithiothreitol, 0.5
mM phenylmethanesulfonyl fluoride, and 1 µg of both
leupeptin and aprotinin/ml).Cell Culture, DNA Transfection, and CAT
Assays
A549 human lung adenocarcinoma cells were grown in
Dulbecco's modified Eagle's medium supplemented with 5%
fetal bovine serum and DU145 human prostatic adenocarcinoma cells were
grown in Dulbecco's modified Eagle's medium/Ham's
F-12 medium supplemented with 10% fetal bovine serum. HepG2 cells were
grown in minimal essential medium supplemented with 10% fetal bovine
serum. For transient expression assays, cells were plated at 1.2
10/100-cm dish and cultured for 24 h before
transfection by the calcium phosphate coprecipitation method with
5-10 µg of the appropriate plasmids purified by banding in
CsCl. Cells were harvested 48 h after the addition of DNA. The extracts
were then assayed for CAT activity. All transfections were repeated a
minimum of three times. For normalization of transfection efficiencies
in HepG2 cells, a growth hormone expression plasmid (pSVGH) was
included in cotransfections. Growth hormone expression was quantified
using a growth hormone detection kit (Nichols Institute).CAT Plasmids and Expression
Constructs
DNA constructs were generated by polymerase
chain amplification using genomic DNA containing the 5`-untranslated
region of the TGF- type II receptor gene as a template. Amplified
DNA fragments were cloned into the promoterless CAT expression plasmid
(pGEM4-SV0CAT) (Kim et al., 1989) using HindIII and KpnI or XbaI restriction sites built into the
oligonucleotides used for amplification. The sequences of the
PCR-generated portions of all constructs were verified by DNA
sequencing. The constructs were named pTRII-n, where n is the
distance in nucleotides from the transcription initiation site. The
plasmid containing the CAT gene alone was used as the control. All CAT
construct plasmids were purified by two sequential CsCl banding steps.EMSA
Double-stranded oligonucleotides
representing the first and second enhancer regions as well as a series
of mutant oligonucleotides for each region were generated using an
oligonucleotide synthesizer. Two oligonucleotides,
TRII(-219/-172) and TRII(+1/+50), were
labeled using a fill in reaction with
[
-P]dCTP (50 µCi at 3,000 Ci/mmol) and
the Klenow fragment of Escherichia coli DNA polymerase I. The
fragments were then gel purified using a 6% polyacrylamide gel and
autoradiography to locate the specific fragment. Binding reactions
contained 10 µg of nuclear extract protein, buffer (10 mM Tris, pH 7.5, 50 mM NaCl, 1 mM dithiothreitol, 1
mM EDTA, and 5% glycerol), 2 µg of poly(dI-dC), and 30,000
cpm of
P-labeled DNA in a volume of 10 µl. Reactions
were incubated at room temperature for 20 min. Competition reactions
were performed by adding an unlabeled double-stranded oligonucleotide
to the reaction mixture. Reactions were electrophoresed on a 6% NOVEX
precasted nondenaturing polyacrylamide gel at 100 V for 1 h in a 100
mM Tris borate-EDTA buffer. Gels were vacuum dried and
analyzed by autoradiography.
Isolation of 5`-specific Human TGF-
A 5` segment of the TGF- RII
Genomic Clones RII cDNA was
used to screen a human lambda phage genomic DNA library for clones
containing the promoter region. Four independent clones were isolated
that overlapped in the 5`-untranslated and promoter regions.
Restriction fragments of the lambda phage inserts were subcloned and
sequenced to derive the promoter sequence shown in Fig. 1. The
sequence obtained includes 1883 nucleotides upstream and 35 nucleotides
downstream of the 5`-most residue of the human TGF- RII precursor
cDNA (Lin et al., 1992).
type II
receptor gene. The 1.883-kilobase pair fragment was subcloned into the
pTZ18 vector, and the sequence extending upstream of the TGF- type
II receptor 5`-cDNA was obtained by the dideoxy chain termination
method. Potential Sp1 binding sites are indicated by a double
line. The single lines indicate potential AP1 binding
sites.
Analysis of the 5`-flanking Region of the Human
TGF-
Examination of the human
TGF- Type II Receptor Gene RII gene sequence 5` to the first major transcription
initiation site designated as -1 reveals several notable
features. No consensus CAAT or TATA boxes exist near the published cDNA
end. The sequence GGGCGG is found at two positions -25 and
-143 (Fig. 1) and corresponds to the sequence identified
as the binding site for the transcription factor Sp1, commonly seen in
the promoters of viral and cellular housekeeping genes. The consensus
sequence of the transcription factor AP1 is also identified at two
positions, -1213 (TGACTCA) and -195 (TTAGTCA).Transcription Initiation Sites of the 5`-flanking
Region of the Human TGF-
To
identify the true start site(s) of transcription, S1 nuclease mapping
was used on RNA isolated from two cell lines known to express the type
II receptor, A549 human lung adenocarcinoma and DU145 human prostatic
adenocarcinoma. A DNA probe was generated that would hybridize to the
first 138 nucleotides of the mRNA (from the published cDNA sequence)
and any RNA upstream (5`) of the published sequence. As shown in Fig. 2A, several putative start sites were repeatedly
seen that represent RNA ends both longer and shorter than the published
cDNA 5` end. These heterologous start sites appear to span 90
nucleotides, from -33 to +57 relative to the cDNA 5` end.
The same probe hybridized to tRNA did not result in any protected
bands, thus demonstrating the completion of the S1 nuclease digestion. Type II Receptor Gene
RII mRNA by S1 nuclease assay and RACE PCR. A, A549
and DU145 cell mRNAs were studied by S1 nuclease protection assay
utilizing an XbaI-EagI fragment labeled at the
5`-end of the EagI site. tRNA was used as control, and the
size of the protected fragments was measured with a sequencing ladder. B, RACE PCR was performed to determine the 5` ends of the RNA
as described under ``Materials and Methods.'' The results are
as shown. The asterisk indicates the published transcription
start site.
RII
mRNA through 5` RACE PCR. Fig. 2B shows that
heterogeneous clones representing heterologous start sites were
observed. Of the six clones sequenced, two were longer than the
published cDNA (by 4 and 35 nucleotides) and four were shorter (by 30,
36, and 38 nucleotides). These results indicate a range of transcripts
spanning from -35 to +38, confirming the heterogeneous
nature of transcriptional start sites observed in the S1 assays.
Whereas the cloned 5` ends approximated the S1 nuclease band sizes,
some differences are evident that probably reflect deficiencies
inherent in the assays (i.e. RNA secondary structure
inhibiting reverse transcriptase in the 5` RACE PCR or S1-sensitive
sequence sites).Cellular Expression Directed by the 5`-flanking
Region of the Human TGF-
To study in vitro transcriptional
regulation of TGF- Type II Receptor Gene Reveals Two
Distinct Positive Regulatory Elements and Two Negative Regulatory
Elements RII, we selected the human hepatoma HepG2 cell
line, which has been shown to have the highest basal level of
expression of TGF- RII of any line studied. In order to identify
the sequences essential for transcription of the TGF- RII gene,
progressively shorter fragments of the 5`-flanking region fused with
the coding region of the bacterial CAT gene in the plasmid pGEM-SV0CAT
were transfected into HepG2 cells. As seen in Fig. 3, construct
pTRIIP(-1240/+50) generated a similar level of CAT
activity as the longer constructs, -1430/+50,
-1670/+50, and -1883/+50. Transcription doubled
upon deletion of the sequence between -1240 and -504
(pTRIIP(-504/+50)), suggesting the presence of a weak
negative regulatory element in this region. Eliminating the sequence
-274 to -137 (pTRIIP(-137/+50)) resulted in
a dramatic drop in transcriptional activity pointing to a very strong
positive regulatory element localized to this region. Deletion of the
sequence -137 to -47 (pTRIIP(-47/+50)) led
to a 10-fold increase in activity pointing to the presence of a second
strong negative regulatory element within this region. Finally, the
shortest construct, pTRIIP(+2/+50), demonstrated a
significant level of activity compared with the control SVoCAT
construct, indicating the presence of a second functional positive
regulatory element. Of all constructs evaluated, pTRIIP-274 and
pTRIIP-504 displayed the highest level of activity. The most
dramatic change in activity was seen with deletion of the region
-274 to -137 containing the putative first positive
regulatory element. Examination of this region reveals at least two
potential recognition sequences for transcription factors AP1
(-195; TTAGTCA) and Sp1 (-143; GGGCGG, Fig. 1).
type II receptor promoter. A, the structure of human TGF- type II receptor
promoter-CAT chimeric constructs. Progressively shorter fragments of
the 5`-flanking region of the type II receptor gene were ligated to the
bacterial chloramphenicol acetyltransferase gene. The first number
gives the first nucleotide of the promoter sequence, e.g. -1883 is position -1883 relative to the published cDNA
start site. All promoter fragments ended at +50. Constructs were
transfected into HepG2 cells, and the cells were harvested after 48 h.
CAT assays were performed a minimum of three times. The right hand
column gives representative CAT activities obtained. B,
results from a representative CAT assay.
type II receptor
promoter. A, the structure of additional human TGF- type
II receptor promoter-CAT chimeric constructs. A series of deletion
constructs from the region -274 to -47 of the type II
receptor gene were ligated to the bacterial CAT gene and assayed in
HepG2 cells a minimum of three times as before. The right hand
column gives representative CAT activities. B, results
from a representative CAT assay.
Identification of Nuclear Proteins Interacting with
the First Positive Regulatory Element (-219 to
-172)
To identify any nuclear proteins associating
with the first positive regulatory element (-219 to -172),
EMSA was performed as described above using a double-stranded P-labeled oligonucleotide containing the sequence for the
first positive regulatory element. The reaction mixture was then
electrophoresed on a polyacrylamide gel and viewed by autoradiography.
The results are shown in Fig. 5A. In the absence of an
unlabeled competitor oligonucleotide (lane 1), two strong
upper bands (complex a and complex b) and multiple
weak lower bands are apparent. It is clear that these bands represent
specific binding of protein to the target oligonucleotide sequence,
because binding to the labeled probe diminishes with increasing
concentrations of unlabeled competitor (lanes 2-6).
Complex a was competed out more readily than complex b, suggesting that complex b binds with greater
affinity or to a longer target sequence.
type II receptor
promoter. A, electrophoretic mobility shift assay. A 5`
end-labeled oligonucleotide representing the first positive regulatory
element (-219 to -172) was incubated with 10 µg of
purified HepG2 nuclear extract, and the resulting DNA-protein complexes
were resolved by native polyacrylamide gel electrophoresis and
autoradiography. Two bands are visualized (a and b),
as well as multiple fainter bands of higher mobility. Specific binding
is demonstrated by progressive disappearance of the bands with
increasing concentrations of the unlabeled competitor oligonucleotide
(-219 to -172). B, the same labeled
oligonucleotide and nuclear protein in competition with various
synthetic double-stranded oligonucleotides corresponding to the
consensus sequences for AP1, AP2, CRE, and Sp1. Lane 2 shows
competition with unlabeled -219/-172 oligonucleotide.
Reactions were carried out with 100-fold molar excess of
competitors.
Identification of Nuclear Protein Recognition
Sequences within the First Positive Regulatory Element
To
determine which sequences within the first positive regulatory element
of the TGF RII promoter are required for specific binding to
complexes a and b, we synthesized a series of mutant
oligonucleotides derived from the first positive regulatory element (Fig. 6A). Each mutant oligonucleotide contained a
4-base pair substitution in which pyrimidine pairs were converted to
purine pairs and vice versa. A mobility shift assay was then performed
using a radiolabeled first positive regulatory element probe incubated
with HepG2 nuclear protein in competition with the series of mutant
oligonucleotides. As shown in Fig. 6B, substitution of
nucleotides -207 to -192 (lanes 5-8, M4-M7, ACTGTGTGCACTTAGT) led to decreased competition
for binding to complex b with the most marked reduction
resulting from mutation of the central nucleotides -203 to
-199 (lane 6, M5). The target sequence for
complex b must therefore reside within this 16-nucleotide
segment. Mutation of nucleotides -195 to -188 (lanes 8 and 9, M7 and M8, TAGTCATT) led to
decreased competition for binding to complex a. This region
shares homology with AP1 and CREB consensus sequences. The first
positive regulatory element therefore contains at least two distinct
sequences demonstrating specific binding to different nuclear proteins (Fig. 6C).
Identification of Nuclear Proteins Interacting with
the Second Positive Regulatory Element
To identify specific
binding of proteins to the second positive regulatory element (+1
to +50), we employed the same strategy. An oligonucleotide
representing the second positive regulatory element was synthesized and
radiolabeled with P. HepG2 nuclear protein was combined
with the labeled second positive regulatory element probe and incubated
with increasing concentrations of unlabeled oligonucleotide. This assay
was repeated several times, and representative results are shown in Fig. 7A. Two strong upper bands consistently appeared
(complexes c and d) along with at least one weaker
lower band (complex e) and represented specific binding
because these bands progressively disappeared with increasing
concentrations of unlabeled competitor.
type II receptor promoter. A, EMSA. Labeled double-stranded
oligonucleotide +1/+50 was incubated with HepG2 nuclear
extract, and the resulting DNA-protein complexes were resolved by
native polyacrylamide gel electrophoresis and autoradiography. Four
bands are visualized. The two upper bands were consistently present
with multiple repetitions of the assay. Lower bands of higher mobility
were variably present at variable intensities. Specific binding is
demonstrated by progressive disappearance of the bands with increasing
concentrations of unlabeled competitor oligonucleotide. B, the
same labeled oligonucleotide and nuclear extract in competition with
consensus sequences for AP1, AP2, CRE, and Sp1. Lane 2 shows
competition with unlabeled +1/+50
oligonucleotide.
Identification of Nuclear Protein Recognition
Sequences within the Second Positive Regulatory
Element
Another set of oligonucleotides was synthesized in
which the wild type second positive regulatory element was serially
mutated with sequential 5-base pair substitution mutations (Fig. 8A). EMSA was performed using the second positive
regulatory element probe, nuclear protein, and the mutant
oligonucleotides. The results are shown in Fig. 8B.
Competition for binding to complex c was abolished by mutation
of nucleotides +16 to +20 (AAGTG, M4), whereas competition
for binding to complexes c, d, and e was
abolished by mutations through a longer sequence from +11 to
+29 (Fig. 8B, M3-M6). Therefore,
the second positive regulatory element appears to contain at least one
nuclear protein recognition sequence from +11 to +29 and
possibly a second nested within the first (Fig. 8C).
This sequence does not match any published binding site for previously
described transcription factors, suggesting that the second positive
regulatory element, as well as the first positive regulatory element,
may be regulated by an unidentified transcription factor(s).
Comparing Transcriptional Activity Directed by the
First and Second Positive Regulatory Elements
To evaluate
the relative contributions of the first and second positive regulatory
elements to the overall promoter activity levels, another series of CAT
contructs was created containing various combinations of mutations and
deletions in the two target sequences of the first positive regulatory
element, designated X and Y, and the single target sequence of the
second positive regulatory element, designated Z. Fig. 9A presents a schematic of the construct series. Construct
-219/+35 contained the wild type human TGF- RII
promoter sequence from nucleotide -219 to +35. The presence
of a bar represents the intact wild type target sequence, and
absence of the bar indicates that the sequence has been
mutated. Thus, construct -219/+35M3 carried the promoter
sequence with a 5-base pair substitution mutation in Z (+11 to
+16, AGTTT-CTGGG). Similarly, -219M7/+35 carried
a substitution mutation in Y (-195 to -192,
TAGT-GCTG), whereas -219M5/+35 carried a mutation in X
(-203 to -200, TGTG-GTGT). -219M5/+35M3
combined mutations in both X and Z. -219M7/+35M3 combined
mutations in Y and Z. Constructs -219M5/+2,
-219M7/+2, and -219/+2 contained a truncated
promoter sequence from -219 to +2 in which the second
positive regulatory element was deleted. These CAT constructs were
transfected into HepG2 cells, and the transcriptional activity was
assayed. As anticipated, the highest level of transcription occurred
with both intact first and second positive regulatory elements
(-219/+35).
type II receptor
promoter-CAT constructs. X and Y mark the positions
of the two target sequences within the first positive regulatory
element, and Z marks the position of the second positive
regulatory target sequence. The presence of the shaded bar signifies the wild type sequence, and its absence indicates that
the sequence has been mutated. The arrow marks the
transcriptional start site +1. B, CAT assay results after
transfection of constructs into HepG2 cells and 72 h of incubation. The bottom row shows unacetylated forms, the middle row shows monoacetylated forms, and the top row shows
diacetylated forms.
receptor system might, in some situations, account for
resistance to its effects on growth in some situations. There is now
substantial evidence to support this early speculation. For example,
human esophageal epithelial cells stably transfected with cyclin D1 are
resistant to the growth inhibitory effects of TGF-1; these cells
express normal levels of the type I receptor but markedly reduced
levels of the type II receptor (Okamoto et al., 1994). Murine
myeloid cells infected with the src oncogene express
significantly higher levels of the type II receptor and show increased
sensitivity to the growth inhibitory effects of TGF-1
(Birchenall-Roberts et al., 1991). Transfecting human breast
carcinoma and hepatoma cells lacking type II receptor with wild type
TGF- RII restores sensitivity to TGF- and decreases
tumorigenicity in transplanted breast cancer cells (Sun et
al., 1994; Inagaki et al., 1993). Recently, we have
reported that a majority of human gastric carcinoma cell lines acquired
resistance to growth inhibition by TGF- and possessed structural
mutations in TGF- RII (Park et al. 1994). Instances in
which cells failed to express RII mRNA despite the absence of apparent
structural deletions or rearrangements of the gene introduced the
possibility of a promoter defect and first suggested that
transcriptional regulation may play an important role in controlling
TGF- RII expression. Most recently, Markowitz et al.,
(1995) have identified a subset of colon cancer cell lines in which
defective DNA repair mechanisms consistently lead to characteristic
mutations in the TGF- RII gene causing resistance to growth
inhibition by TGF-. Inactivation of TGF- RII may be a common
occurrence in epithelial malignancies. By permitting escape from
regulation by TGF-, such mutations confer a strong growth
advantage to affected cell populations. Decreased transcription of RII
mRNA can have the same effect as mutation of the structural gene. RII and describe the existence of at least five distinct
regulatory regions including two positive regulatory elements
(-219 to -172 and +1 to +35) and two negative
regulatory elements (-1240 to -504 and -100 to
-67; Fig. 10) in addition to the core promoter region
(-47 to -1; Fig. 10). One negative element located
between 0.5 and 1.2 kilobase pairs upstream from the transcriptional
start site(s) was not extensively examined in this study. Deletion of
this region increased transcription approximately 2-fold. The first
positive regulatory element (-219 to -172) is required for
basal transcriptional activity because its deletion allows the powerful
second negative regulatory element (-100 to -67) to repress
transcription completely regardless of the presence of the core
promoter and second positive regulatory element (see
-137/+50 in Fig. 3and -172/+2 in Fig. 5). Transcription directed by the core promoter region is
dependent on an Sp1 consensus sequence at -25. Mutation of this
sequence reduces transcription by 70% (-47Sp1 mt/+2 in Fig. 4).
type II receptor promoter.
Relative positions of the four identified regulatory elements have been
mapped to the regions shown. E1 and E2 indicate the
first and second positive regulatory elements, respectively, P signifies the core promoter, and N indicates a negative
regulatory element. Positions of putative Sp1 sites and a potential AP1
or CRE/ATF site are labeled. The arrow marks the +1
transcriptional start site.
RII promoter region that agrees
well with a previously published report (Humphries et al.,
1994) and also extends the known sequence an additional 930 base pairs
upstream. However, unlike the earlier report, this study shows the
heterogeneous nature of the transcriptional start sites and presents
functional data regarding the regulation of transcription from the
human TGF- RII promoter region. The human TGF- RII promoter
is similar to other promoters lacking TATA and CAAT boxes in that
transcription is initiated from multiple start sites separated by as
much as 90 nucleotides surrounding the previously published cDNA 5` end
(Lin et al., 1992). The identification of start sites at
+30 and further downstream complements the recognition of a
positive regulatory element at +11 to +25. Sequence analysis
reveals multiple sites homologous to known transcription factor
consensus sequences. Two putative Sp1 sites are located at -143
and -25. The -25 site is responsible for at least 70% of
basal activity from the -47/-1 core promoter region. Two
putative AP1/CREB binding sites have also been recognized at -669
and -196. The -669 site is located in a region that
contains a weak negative regulatory element, but further analysis is
required to determine if this site is functional. The -196 site
is located in the first positive regulatory element and corresponds to
the binding site for complex a (Fig. 6C). EMSA
performed with the labeled first positive regulatory element and HepG2
nuclear extract in competition with unlabeled AP1 and CRE consensus
sequences confirmed that complex a specifically bound to
AP1/CRE-like sequences. Purified AP1 and CRE/ATF protein also
demonstrated specific binding to the first positive regulatory element. () RII gene contains multiple components including two
positive regulatory elements and two negative regulatory elements in
addition to the core promoter. Such a high level of structural
complexity suggests a correspondingly high level of functional
intricacy. Multiple nuclear proteins have been shown to bind
specifically to the two positive regulatory elements, and it is likely
that these proteins include previously unidentified transcription
factors. Studies are currently underway to define the activity of the
TGF- RII promoter in different cell lines as well as to purify and
characterize the involved binding proteins.
), transforming growth factor-;
TGF- RII, TGF- type II receptor; RACE, rapid amplification of
cDNA ends; PCR, polymerase chain reaction; CAT, chloramphenicol
acetyltransferase; EMSA, electrophoretic mobility shift assays.)
We thank Herbert Y. Lin and Harvey F. Lodish for the
TGF- type II receptor cDNA as well as Erwin
Böttinger, David Danielpour, and Robert Lechleider
for helpful suggestions.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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