(Received for publication, July 13, 1995; and in revised form, September 14, 1995)
From the
Diminished cellular responsiveness to transforming growth
factor- (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.
Transforming growth factor- (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.
Much work
has recently been directed toward characterizing the different types of
TGF- 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).
Several tumor
cell lines, including retinoblastoma, pheochromocytoma, neuroblastoma,
and breast carcinoma, which are resistant to the growth inhibitory
effects of TGF-, 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.
In order to study the
transcriptional regulation of human TGF- 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.
Figure 1:
Nucleotide
sequence of the 5`-flanking region of the human TGF- 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.
Figure 2:
Determination of the 5` ends of the
TGF- 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.
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- 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).
Figure 3:
In vitro transcription of the
deletion mutants of the TGF- 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.
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- 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.
Figure 5:
Detection of nuclear proteins that bind to
the first positive regulatory element of the TGF- 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.
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-
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.
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- 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.
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- 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.
In
this report we present an expanded sequence for the promoter region of
TGF 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).
Figure 10:
Schematic representation of the multiple
regulatory elements within the TGF- 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.
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- 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. (
)
This study has shown that the promoter region of the
human TGF- 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.