©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of the Promoter Region of the Human Transforming Growth Factor- Type II Receptor Gene (*)

(Received for publication, July 13, 1995; and in revised form, September 14, 1995)

Hyun W. Bae (§) Andrew G. Geiser(§)(¶) David H. Kim (§) Michelle T. Chung James K. Burmester (**) Michael B. Sporn (§§) Anita B. Roberts Seong-Jin Kim (¶¶)

From the Laboratory of Chemoprevention, NCI, National Institutes of Health, Bethesda, Maryland 20892-5055

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Diminished cellular responsiveness to transforming growth factor-beta (TGF-beta) is frequently correlated with decreased transcription of the type II receptor for TGF-beta (TGF-beta RII). We have cloned and characterized the human TGF-beta 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-beta RII.


INTRODUCTION

Transforming growth factor-beta (TGF-beta) (^1)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-beta 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-beta may underlie the process of malignant transformation (Wakefield and Sporn, 1990). This decreased responsiveness to TGF-beta could be caused by defects not only in TGF-beta expression or activation but also by defects in the regulation of TGF-beta receptors.

Much work has recently been directed toward characterizing the different types of TGF-beta 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-beta 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-beta (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-beta, 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-beta 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-beta growth control frequently observed in the process of carcinogenesis.

In order to study the transcriptional regulation of human TGF-beta 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-beta 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.


MATERIALS AND METHODS

Cloning the Promoter Region of the TGF-beta Type II Receptor

A human genomic library was obtained (Clontech) and screened by standard methods using the 5` end fragment of the human TGF-beta 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 times 10^6 to 5 times 10^6) 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(2), 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(2), 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 times 10^6/100-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-beta 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 pTbetaRII-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, TbetaRII(-219/-172) and TbetaRII(+1/+50), were labeled using a fill in reaction with [alpha-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.


RESULTS

Isolation of 5`-specific Human TGF-beta RII Genomic Clones

A 5` segment of the TGF-beta 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-beta RII precursor cDNA (Lin et al., 1992).


Figure 1: Nucleotide sequence of the 5`-flanking region of the human TGF-beta type II receptor gene. The 1.883-kilobase pair fragment was subcloned into the pTZ18 vector, and the sequence extending upstream of the TGF-beta 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-beta Type II Receptor Gene

Examination of the human TGF-beta 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-beta Type II Receptor Gene

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.


Figure 2: Determination of the 5` ends of the TGF-beta 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-beta 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-beta Type II Receptor Gene Reveals Two Distinct Positive Regulatory Elements and Two Negative Regulatory Elements

To study in vitro transcriptional regulation of TGF-beta RII, we selected the human hepatoma HepG2 cell line, which has been shown to have the highest basal level of expression of TGF-beta RII of any line studied. In order to identify the sequences essential for transcription of the TGF-beta 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 pTbetaRIIP(-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 (pTbetaRIIP(-504/+50)), suggesting the presence of a weak negative regulatory element in this region. Eliminating the sequence -274 to -137 (pTbetaRIIP(-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 (pTbetaRIIP(-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, pTbetaRIIP(+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, pTbetaRIIP-274 and pTbetaRIIP-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).


Figure 3: In vitro transcription of the deletion mutants of the TGF-beta type II receptor promoter. A, the structure of human TGF-beta 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-beta type II receptor promoter. A, the structure of additional human TGF-beta 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.


Figure 5: Detection of nuclear proteins that bind to the first positive regulatory element of the TGF-beta 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.

Identification of Nuclear Protein Recognition Sequences within the First Positive Regulatory Element

To determine which sequences within the first positive regulatory element of the TGFbeta 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).


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.



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.


Figure 7: Detection of nuclear proteins that interact with the second positive regulatory element of the TGF-beta 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).

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).


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.



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-beta 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).


Figure 9: Relative contribution of first and second positive regulatory elements to overall promoter activity. A, schematic representation of series of TGF-beta 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.


DISCUSSION

In 1985, Sporn and Roberts first suggested that defects in the TGF-beta 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-beta1; 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-beta1 (Birchenall-Roberts et al., 1991). Transfecting human breast carcinoma and hepatoma cells lacking type II receptor with wild type TGF-beta RII restores sensitivity to TGF-beta 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-beta and possessed structural mutations in TGF-beta 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-beta 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-beta RII gene causing resistance to growth inhibition by TGF-beta. Inactivation of TGF-beta RII may be a common occurrence in epithelial malignancies. By permitting escape from regulation by TGF-beta, 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 TGFbeta 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-beta 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-beta 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-beta RII promoter region. The human TGF-beta 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. (^2)

This study has shown that the promoter region of the human TGF-beta 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-beta RII promoter in different cell lines as well as to purify and characterize the involved binding proteins.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
These authors contributed equally to this work.

Present address: Lilly Research Labs, Lilly Corporate Center, Indianapolis, IN 46285.

**
Present address: Marshfield Medical Research and Education Foundation, Marshfield, WI 54449.

§§
Present address: Dept. of Pharmacology, Dartmouth Medical School, Remsen 524, Hanover, NH 03755.

¶¶
To whom correspondence should be addressed: Bldg. 41, Rm. B1106, National Cancer Institute, NIH, Bethesda, MD 20892-5055. Tel.: 301-496-5391.

(^1)
The abbreviations used are: TGF-beta, transforming growth factor-beta; TGF-beta RII, TGF-beta type II receptor; RACE, rapid amplification of cDNA ends; PCR, polymerase chain reaction; CAT, chloramphenicol acetyltransferase; EMSA, electrophoretic mobility shift assays.

(^2)
D. H. Kim, M. T. Chung, A. B. Roberts, and S.-J. Kim, unpublished data.


ACKNOWLEDGEMENTS

We thank Herbert Y. Lin and Harvey F. Lodish for the TGF-beta type II receptor cDNA as well as Erwin Böttinger, David Danielpour, and Robert Lechleider for helpful suggestions.


REFERENCES

  1. Attisano, L., Cárcamo, J., Ventura, F., Weis, F. M. B., Massagué, J., and Wrana, J. L. (1993) Cell 75, 671-680 [Medline] [Order article via Infotrieve]
  2. Bassing, C. H., Yingling, J. M., Howe, D. J., Wang, T., He, W. W., Gustafson, M. L., Shah, P., Donahoe, P. K., and Wang, X.-F. (1994) Science 263, 87-89 [Medline] [Order article via Infotrieve]
  3. Birchenall-Roberts, M. C., Falk, L. A., Kasper, J., Keller, J., Faltynek, C. R., and Ruscetti, F. W. (1991) J. Biol. Chem. 266, 9617-9621 [Abstract/Free Full Text]
  4. Border, W. A., Okuda, S., Languino, L. R., Sporn, M. B., and Ruoslahti, E. (1990) Nature 346, 371-374 [CrossRef][Medline] [Order article via Infotrieve]
  5. Cárcamo, J., Weis, F. M., Ventura, F., Wieser, R., Wrana, J. L., Attisano, L., and Massagué, J. (1994) Mol. Cell. Biol. 14, 3810-3821 [Abstract]
  6. Castilla, A., Prieto, J., and Fausto, N. (1991) N. Engl. J. Med. 324, 933-940 [Abstract]
  7. Chen, J. K., Hoshi, H., and McKeehan, W. L. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5287-5291 [Abstract]
  8. Franzen, P., ten Dijke, P., Ichijo, H., Yamashita, H., Schulz, P., Heldin, C.-H., and Miyazono, K. (1993) Cell 75, 681-692 [Medline] [Order article via Infotrieve]
  9. Geiser, A. G., Kim, S.-J., Roberts, A. B., and Sporn, M. B. (1991) Mol. Cell. Biol. 11, 84-92 [Medline] [Order article via Infotrieve]
  10. Grainger, D. J., Kirschenlohr, H. L., Metcalfe, J. C., Weissberg, P. C., Wade, D. P., and Lawn, R. M. (1993) Science 260, 1655-1658 [Medline] [Order article via Infotrieve]
  11. He, W. W., Gustafson, M., Hirose, S., and Donahoe, P. K. (1993) Dev. Dyn. 196, 133-142 [Medline] [Order article via Infotrieve]
  12. Humphries, D. E., Bloom, B. B., Fine, A., and Goldstein, R. H. (1994) Biochem. Biophy. Res. Commun. 203, 1020-1027 [CrossRef][Medline] [Order article via Infotrieve]
  13. Inagaki, M., Moustakas, A., Lin, H. Y., Lodish, H. F., and Carr, B. I. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5359-5363 [Abstract]
  14. Kim, S. J., Glick, A., Sporn, M. B., and Roberts, A. B. (1989) J. Biol. Chem. 264, 402-408 [Abstract/Free Full Text]
  15. Kim, S. J., Park, K., Rudkin, B. B., Dey, B. R., Sporn, M. B., and Roberts, A. B. (1994) J. Biol. Chem. 269, 3739-3744 [Abstract/Free Full Text]
  16. Kimchi, A., Wang, X.-F., Weinberg, R. A., Cheifetz, S., and Massagué, J. (1988) Science 240, 196-199 [Medline] [Order article via Infotrieve]
  17. Kingsley, D. M. (1994) Genes & Dev. 8, 133-146
  18. Lafyatis, R., Thompson, N. L., Remmers, E. F., Flanders, K. C., Roche, N. S., Kim, S.-J., Case, J. P., Sporn, M. B., Roberts, A. B., and Wilder, R. L. (1989) J. Immunol. 143, 1142-1148 [Abstract/Free Full Text]
  19. Lin, H. Y., Wang, X.-F., Ng-Eaton, E., Weinberg, R. A., and Lodish, H. F. (1992) Cell 68, 775-785 [Medline] [Order article via Infotrieve]
  20. Lopez-Casillas, F., Cheifetz, S., Doody, J., Andres, J. L., Lane, W. S., and Massagué, J. (1991) Cell 67, 785-795 [Medline] [Order article via Infotrieve]
  21. Markowitz, S., Wang, J., Myeroff, L., Parsons, R., Sun, L., Lutterbaugh, J., Fan, R. S., Zborowska, E., Kinzler, K. W., Vogelstein, B., Brattain, M., and Wilson, J. K. V. (1995) Science 268, 1336-1338 [Medline] [Order article via Infotrieve]
  22. Massagué, J. (1990) Annu. Rev. Cell Biol. 6, 597-641 [CrossRef]
  23. Massagué, J. (1992) Cell 69, 1067-1070 [Medline] [Order article via Infotrieve]
  24. Miyazono, K., ten Dijke, P., Ichijo, H., and Heldin, C.-H. (1994) Adv. Immunol. 55, 181-220 [Medline] [Order article via Infotrieve]
  25. Moren, A., Ichijo, H., and Miyazono, K. (1992) Biochem. Biophys. Res. Commun. 189, 356-362 [Medline] [Order article via Infotrieve]
  26. Okamoto, A., Jiang, W., Kim, S.-J., Spillare, E. A., Stoner, G. D., Weinstein, I. B., and Harris, C. C. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 11576-11580 [Abstract/Free Full Text]
  27. Park, K., Kim, S.-J., Bang, Y.-J., Park, J.-G., Kim, N. K., Roberts, A. B., and Sporn, M. B. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8772-8776 [Abstract]
  28. Roberts, A. B., and M. B. Sporn. (1990) Handbook of Experimental Pharmacology: Peptide Growth Factors and Their Receptors (Sporn, M. B., and A. B. Roberts, eds) pp. 419-472, Springer-Verlag New York Inc., New York
  29. Sporn, M. B., and Roberts, A. B. (1985) Nature 313, 747-749
  30. Sun, L., Wu, G., Willson, J. K. V., Zborowska, E., Yang, J., Rajkarunanayake, I., Wang, J., Gentry, L. E., Wang, X.-F., and Brattain, M. G. (1994) J. Biol. Chem. 269, 26449-26455 [Abstract/Free Full Text]
  31. Wakefield, L. M., and Sporn, M. B. (1990) in Tumor Suppressor Genes (Klein, G., ed) pp. 217-243, Marcel Dekker, Inc., New York
  32. Wang, X.-F., Lin, H. Y., Ng-Eaton, E., Downward, J., Lodish, H. F., and Weinberg, R. A. (1991) Cell 67, 797-805 [Medline] [Order article via Infotrieve]
  33. Wrana, J. L., Attisano, L., Cárcamo, J., Zentella, A., Doody, J., Laiho, M., Wang, X.-F., and Massagué, J. (1992) Cell 71, 1003-1014 [Medline] [Order article via Infotrieve]
  34. Wrana, J. L., Attisano, L., Wieser, R., Ventura, F., and Massagué, J. (1994) Nature 370, 341-347 [CrossRef][Medline] [Order article via Infotrieve]

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