Proc. Nati. Acad. Sci. USA Vol. 88, pp. 10227-10231, November 1991 Cell Biology
Factor-binding element in the human c-myc promoter involved in transcriptional regulation by transforming growth factor 131 and by the retinoblastoma gene product (negative growth factor/tumor suppressor gene/protooncogenes)
JENNIFER A. PIETENPOL*, KARL MUNGERt, PETER M. HOWLEYt, ROLAND W. STEIN*t AND HAROLD L. MOSES* Departments of *Cell Biology and of tMolecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232; and tLaboratory of Tumor Virus Biology, National Cancer Institute, Bethesda, MD 20892
Communicated by David M. Prescott, August 5, 1991 (received for review May 8, 1991)
sequence between positions -100 and +71 relative to the P1 transcription start site (5). Skin keratinocytes transformed by simian virus 40 (SV40), human papilloma virus (HPV)-18, or the HPV-16 transforming genes expressed from a heterologous promoter were found to be resistant to the growth inhibitory or c-myc suppressive effects of TGF-,81 (4). Transient expression of the genes encoding HPV-16 E7, adenovirus ElA, and SV40 large tumor antigen (T antigen) blocked the TGF-j31 suppression of transcription from c-myc promoter/chloramphenicol acetyltransferase (CAT) constructs while mutants of ElA and T antigen that were defective in binding the retinoblastoma gene product (pRB) failed to block TGF-31 suppression (4). These studies suggested that the oncoproteins blocked the actions of TGF-,31 by sequestering an essential factor(s) in this growth suppression pathway. However, it had not been determined in this previous study whether it was pRB or another of the cellular proteins that interacts with the conserved domain of the viral oncoproteins (7, 8) that was an essential element in the TGF-/31 suppression of c-myc transcription and cell growth. In the present study, we demonstrate that transient expression of pRB can inhibit c-myc transcription. An analysis of the c-myc promoter revealed that the same element was required for regulation by both TGF-3 and pRB. This indicates that pRB may be involved in mediating the TGF-,Binduced inhibition of c-myc expression. Furthermore, binding studies with this c-myc promoter element implicated cellular factors in addition to pRB in this growth inhibitory pathway.
ABSTRACT Previous studies have shown that transforming growth factor .31 (TGF-j81) inhibition of keratinocyte proliferation involves suppression of c-myc transcription, and indirect evidence has suggested that the retinoblastoma gene product (pRB) may be involved in this process. In this study, transient expression of pRB in skin keratinocytes was shown to repress transcription of the human c-myc promoter as effectively as TGF-131. The same c-myc promoter region was required for regulation by both TGF-fi1 and pRB. These sequences, termed the TGF-,8 control element (TCE), lie between positions -86 and -63 relative to the P1 transcription start site. Oligonucleotides containing the TCE bound to several nuclear factors in mobility-shift assays using extracts from cells with or without normal pRB. Binding of some factors was inhibited by TGF-131 treatment of TGF-fi-sensitive but not TGF-8-insensitive cells. These data indicate that pRB can suppress c-myc transcription and suggest the involvement of cellular factors in addition to pRB in the TGF-,B1 pathway for the inhibition of c-myc transcription and growth inhibition.
Cellular growth control involves a complex interplay between positive- and negative-acting diffusible factors and growth regulatory genes. Of the diffusible growth inhibitory peptides the transforming growth factors type P (TGF-jBs) are the most potent (for review, see ref. 1). TGF-,31 has been shown to be a potent inhibitor for most cell types in culture and growth inhibitory effects of the TGF-pBs have also been demonstrated by in vivo studies (for review, see ref. 2). The mechanism of TGF-,61 inhibition of epithelial cells has been most extensively studied by utilizing skin keratinocyte model systems. Coffey et al. (3) demonstrated that TGF-/31 inhibited either rapidly growing skin keratinocytes or epidermal
MATERIALS AND METHODS Cell Culture. BALB/MK cells, human foreskin keratinocytes (HFK), and SV40-transformed HFK (HFK/SV40) cells were cultured as described (4). DU-145 (obtained from American Type Culture Collection) were cultured in McCoy's 5A medium with 10% fetal calf serum. Y79, WERI, and NGP cells were cultured in RPMI 1640 medium with 20% fetal calf serum. c-myc/CAT Expression Vectors. Deletion mutants pPLFCAT -2290, -100, and +71 were constructed as described (5). Deletion mutants pPLFCAT -86, -63, -44, +12, and +53 were produced by PCR technology. Primers were designed to make successive deletions of the -100 to +71 region of the c-myc promoter. The lower strand primer was the same for all amplifications (5'-TAATATCCAGCTGAACGGTC-3'). The upper strand primers were as follows:
growth factor-stimulated quiescent keratinocytes. TGF-(1 can inhibit growth factor stimulation of DNA synthesis subsequent to the early events induced by growth factors (4). Recent studies indicate that TGF-f31 effects on c-myc (MYC in human gene nomenclature) expression may be mechanistically important in TGF-f31-induced growth inhibition. TGF-f31 can suppress c-myc mRNA abundance in a variety of cell types (5). In the skin keratinocyte, TGF-/31 rapidly reduces c-myc mRNA and protein levels, and this effect can be blocked by cycloheximide (6). Antisense c-myc oligonucleotides that suppress synthesis of c-myc protein inhibit proliferation of skin keratinocytes as effectively as TGF-,1 (5), providing strong evidence that c-myc expression is necessary for keratinocyte proliferation. The TGF-,81 inhibition of c-myc expression occurs at the level of transcription initiation and requires a cis-acting
Abbreviations: TGF, transforming growth factor; RB, retinoblastoma gene; pRB, RB product; CAT, chloramphenicol acetyltransferase; TCE, TGF-(3 control element; TIE, TGF-j3 inhibitory element; SV40, simian virus 40; RSV, Rous sarcoma virus; HFK, human foreskin keratinocyte.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 10227
10228
Proc. Natl. Acad. Sci. USA 88
Cell Biology: Pietenpol et al.
nucleotide (see Fig. 3 for sequence). Each 25-,ul reaction mixture was incubated at 4°C for 40 min. Samples were subjected to electrophoretic separation at 4°C on a 6% nondenaturing polyacrylamide gel at 200 V for 4-5 hr using TBE (0.9 M Tris borate/0.002 M EDTA)/PAGE conditions. After electrophoresis, the gels were dried and labeled DNA was localized by autoradiography.
pPLFCAT-86, 5'-CGCGAAGCTTAGGGCGTGGGGGAAAAGA-3'; pPLFCAT-63, 5 '-CGCGAAGCTTAAGATCCTCTCTCGCTAA-3'; pPLFCAT-44, 5 '-TCTCTCGCTAAGCTTCGCCCAC-3'; pPLFCAT+12, 5'-GACCCCCAAGCTTTGCTGCT-3'; pPLFCAT+53, 5'CGGCCGTCCCGGGCTCCCCT-3'; pPLFCAT-100.m, 5'-
CGACCCGGGTTCCCAAAGCAGAGGTCTTTTGGGAAAAGAAAA-3'. The amplified products were subcloned into pPLFCAT. Underlined nucleotides represent primer mismatch resulting in restriction enzyme cleavage sites used for cloning the PCR fragments and, in the case of pPLFCAT100.m, for introduction of TGF-,8 control element (TCE) mutations. pRB Expression Vectors. To construct the pCMVRB.WT and pCMVRB.AS expression vectors, the 4.1-kilobase HindIII fragment containing the RB cDNA was isolated from pCMV.RB (9). The HindIII fragment from pCMV.RB was cloned in the sense and antisense orientations in the pCMV.4 cloning vector (10), resulting in pCMVRB.WT and pCMVRB.AS, respectively. DNA Transfection and Analysis of CAT Protein. Transfection of BALB/MK and HFK cells was as described (4). The transfected cells were treated with TGF-,B1 (R & D Systems, Minneapolis) 6-18 hr posttransfection. Cell extracts were prepared 24 hr after TGF-,81 treatment as described (11). The amount of protein extract from the HFK and BALB/MK cells used in the CAT assays was normalized to f-galactosidase activity. Both the traditional CAT assay (11) and a CAT ELISA (5 Prime -* 3 Prime, Inc.) were used to determine levels of CAT activity and protein, respectively. Electrophoretic Mobility-Shift Assays. Cells were treated with lysis buffer (250 mM NaCI/0.1% Nonidet P-40/50 mM Hepes, pH 7.0/25 ,uM sodium fluoride/200 ,uM sodium orthovanadate/0.12 unit of aprotinin) at 4°C for 30 min. The cell suspension was centrifuged at 13,600 x g for 5 min. The protein concentration of the clarified supernatant was typically 1 ,ug/,jl. The binding reaction mixtures contained in a 25-Al vol 10 jig of cell extract in 25 mM Hepes (pH 7.9), 1 mM dithiothreitol, 0.04 mM EDTA, 100 mM NaCl, 40 mM KCl, 0.5 ,ug of poly(dI-dC), 0.5 ,ug of single-stranded DNA, 0.04% (vol/vol) Nonidet P-40, 1.2% (vol/vol) glycerol, and 10 fmol of a 32P-end-labeled double-stranded TCE or TCE.m oligo-
RESULTS RB Suppression of c-myc Transcription. To determine whether expression of pRB might have an effect on c-myc transcription similar to that observed with TGF-/31 treatment, BALB/MK cells were transfected with pPLFCAT-100 either alone or together with an RB plasmid expressing wild-type RB mRNA (pCMVRB.WT), or a control antisense RB mRNA (pCMVRB.AS). As shown in Fig. 1A, transient expression of wild-type RB in the BALB/MK cells alone (no TGF-,Bl treatment) significantly reduced pPLFCAT-100 expression, with the level of repression being the same as that observed with TGF-,B1 treatment. The effect of TGF-f31 treatment and pRB expression together was the same as either alone. Similar results were obtained with HFK cells transfected with pCMVRB.WT under the same conditions (Fig. 1B). The control plasmid (pCMVRB.AS) had no effect on c-myc transcription and did not alter the TGF-,B1 effect on transcription (Fig. 1A). To further address the question of specificity of the pRB effect, CMVRB.WT was cotransfected with a Rous sarcoma virus (RSV) enhancer-driven CAT expression plasmid, RSV/CAT. Production of CAT activity from the RSV promoter was not reduced by TGF-,B1 treatment or by cotransfected CMVRB.WT (Fig. 1C). The results indicate that pRB can inhibit c-myc transcription. Sequences Required for pRB and TGF-l1 Control of c-myc Transcription. Previous studies had indicated that the TGF-,31 response element was within the -100 to +71 region of the c-myc promoter (5). Thus, finer resolution deletion mutants were subcloned into the CAT expression vector (pPLFCAT). The c-myc promoter/CAT chimeras, pPLFCAT -2290, -100, -86, -63, -44, +12, and +71 are designated according to the 5' nucleotide position relative to the P1 transcription start site (+1). All deletion mutants used
B
A
(1991)
( I
mm-lr
I.a
i
X
H ill!!,;[:,
1
-
.
I.m
I
*6a*
*
.i
13
*(R ; 1
0
Q il I',' L: T' ,I0
U
p( C\I RH\I1
00
p( 'I (
Cont.
f
.1
* a
' as
VVI
pCMVRB.WT pCMVRB.AS
FIG. 1. Effect of treatment with TGF-,B1 or cotransfection with RB expression plasmid on myc/CAT transcription in mouse and human keratinocytes. (A) Rapidly growing BALB/MK cells were cotransfected with equimolar amounts of pPLFCAT-100 (a human c-myc promoter-CAT construct containing the sequences -100 to +510) and pBR322 or an RB expression plasmid (pCMVRB.WT) or a control vector (pCMVRB.AS). The CAT protein produced was quantitated by an ELISA without (open bars) or with (hatched bars) treatment with TGF-,81 (10 ng/ml). Each bar represents the mean of duplicate determinations and the results are representative of three separate experiments, each with duplicate determinations. (B) Rapidly growing HFK cells were cotransfected with the same plasmids described above for the BALB/MK cell transfection and control and TGF-/31-treated (10 ng/ml) cultures were assayed. CAT activity was quantitated by determining the percentage conversion of ['4C]chloramphenicol to the acetylated form by liquid scintillation counting. NA, not applicable. (C) Rapidly growing BALB/MK cells were cotransfected with equimolar amounts of RSV promoter-CAT construct and pBR322 or pCMVRB.WT. Control and TGF-B31-treated (10 ng/ml) cultures were assayed. CAT activity was quantitated as described above.
Cell
_
Biology: Pietenpol et al.
Proc. Natl. Acad. Sci. USA 88 (1991) t/ Inhibitiln by p CM V R BAIV . I" ReIaitie PTromo0tCrl- T G F-B I Activitv ii1caiment (Co...t rarlsfc'cii'
tslcs/CATF Constructs i)PLICAT-2-29()
,)pl1FCA IX-
()(
i
1.AX) .,(l -A I .... t.~~~~~~~~~~~~~~~~~~~~~~~~~
pIILFCIA-l-86
i
-axo-
CAT
CAT I
+ 34 1.37±7
482
1_ 1 .C=AT
-ur
p)IIl(FCAT+ l2
In T-, AT -X CiCAT
pIlFC'AIT+7 1
..
(-Yi 6+tl
7 1; 71 + .'2()
.512
pIllFCAT'-63 pPILF-CAT-4-4
10229
98 ±
8 x.
.
.53
8
t
+)A4
f-,
1
I 27 ±+,N'
FIG. 2. Deletion analysis of c-myc promoter to identify TGF-,3 and pRB response element. BALB/MK cells were cotransfected with the indicated pPLFCAT constructs and either pBR322 or the RB expression plasmid pCMVRB.WT and the effect of TGF-f31 treatment (10 ng/ml) or pCMVRB.WT cotransfection on CAT production was determined. CAT protein was quantitated by ELISA. The promoter activity is expressed relative to the mean of the activity obtained with pPLFCAT-2290. The remaining data are expressed as percentage inhibition of CAT protein production from the pPLFCAT constructs by TGF-,B1 treatment or by cotransfection with pCMVRB.WT. Each value represents the mean SEM of four or five determinations with the exception of the data from the pPLFCAT+12 construct, which represents the mean of duplicate determinations.
have 3' sequences to + 510. The promoter activity for each of the constructs relative to pPLFCAT-2290 is presented in Fig. 2. Although CAT protein levels varied considerably (see standard error) from experiment to experiment, there was not a major loss of promoter activity with sequential deletions of the 5' flanking region. While pPLFCAT-63 had 50% of the activity of pPLFCAT-2290, the -44, +12, and +71 constructs had promoter activity equivalent to pPLFCAT-2290. Treatment with TGF-,S1 inhibited CAT production from 50% to 80% in the -2290, -100, and -86 mutants but did not inhibit transcription from the -63, -44, +12, and +71 constructs (Fig. 2). A very similar pattern of repression was observed when pCMVRB.WT was cotransfected with these c-myc promoter mutants (Fig. 2). These results demonstrated that transcriptional repression by both TGF-,B1 and pRB was mediated through the same region in the human c-myc promoter. This common target sequence, located between bases -86 and -63, has been termed the TCE. The sequence of the TCE in the human c-myc promoter is shown in Fig. 3A in comparison to a previously described element in another gene reported to be repressed by TGF-P1, the TGF-f3 inhibitory element (TIE) in the rat transin/stromelysin promoter (12). A region of similarity between the TCE and TIE is apparent (Fig. 3A). Comparison of the mouse c-myc promoter region, which is also TGF-p responsive, revealed a similar sequence, except for a 34-base-pair (bp) insert between positions -77 and -76 and a 1-bp insertion between positions -68 and -67 in the mouse c-myc promoter relative to the human promoter (13). To determine the necessity of this -86- to -63-bp sequence in the pRB- and the TGF-f31-mediated inhibition of c-myc A
1TI i,] rirnsi/stromelysin
G A
T T C
G1C'G
transcription response in the keratinocytes, four guanines at positions -81, -79, -76, and -77 were substituted by thymines (Fig. 3B). Expression from this mutant construct (pPLFCAT-100.m) was not inhibited by TGF-31 treatment or cotransfection with pCMVRB.WT; rather, the latter gave an unexplained increase in CAT protein levels (Fig. 4). These data corroborate the deletion mutagenesis data and further indicate that the -63 to -86 region (TCE) of the c-myc promoter is necessary for both TGF-P31 and pRB control of myc transcription. TCE Binding to Cellular Proteins. Whole-cell extracts from BALB/MK cells were incubated with an end-labeled oligonucleotide corresponding to the TCE (Fig. 3). Three major retarded species were resolved and are depicted as TCE-I, TCE-II, and TCE-III (Fig. SA). Similar results were obtained with extracts from HFK cells (Fig. SC). Binding of labeled TCE to all three retarded complexes from BALB/MK cells was effectively blocked by competition with unlabeled TCE, but competition with the TCE.m oligonucleotide with 4 base mutations (Fig. 3) had little effect, indicating that TCE binding to all retarded species is specific. Use of labeled TCE.m in the mobility-shift assay gave a pattern of binding completely different from that obtained with wild-type TCE. There was no detectable binding to the retarded species bound by TCE; rather there was binding to a single band migrating between TCE-I and TCE-II (Fig. SA). The effect of TGF-P1 treatment on the capacity of the extracts to bind the TCE was next determined. As shown in Fig. SB, protein-DNA binding corresponding to TCE-II was reduced after TGF-f31 treatment of BALB/MK cells over the time course shown. However, TGF-131 treatment did not FIG. 3. TCE sequence. (A) Sequence of TCE in c-myc is compared to the TIE identified in the rat
G T G A -700
transin/stromelysin promoter (12). Areas of iden-
within shaded boxes. (B) The four mutations -SaGCkGAGGGCsTGGG&GAAAAGAAtity
T'
are
made in the TCE to generate the mutant TCE (TCE.m) used in experiments depicted in Fig. 5 is shown. This mutated promoter region was subB
TCE
-86 G CA GAG G G C G T r G G G G A A A A G A A -63 I
TCE.m
I
I
I
6 G C A G A G G T C T T T T G G G A A A A G AAA63
pPLFCAT-100 vector to give the pPLFCAT-100.m construct used in the expercloned back into the
iment shown in Fig. 4.
10230
Proc. Natl. Acad. Sci. USA 88 (1991)
Cell Biology: Pietenpol et al.
relative to that obtained with HFK extracts. The appearance of binding to HFK extracts normalized for total protein concentration and run simultaneously is shown for comparison. To address the question of potential TCE binding to pRB, assays were carried out with cell extracts isolated from cells with a defective RB gene [WERI and Y79 human retinoblastoma cells (14) and DU-145 human prostate adenocarcinoma cells (15)] in comparison to extracts from cells with an intact RB gene [NGP human retinoblastoma cells (15) and BALB/ MK cells]. By mobility-shift assay, TCE binding to cellular proteins from WERI, Y79, and DU-145 cells was observed, but the binding pattern was different from that obtained with cells containing an intact RB gene (Fig. SD). There was greatly diminished TCE-I binding in the RB-defective cells. However, use of additional protease inhibitors in the extraction buffer resulted in more prominent TCE-I binding in the DU-145 cell extracts (L. Dagnino, H.L.M., and R.W.S., unpublished observations). TCE-II binding in Y79 and WERI cell extracts was equal to or greater than TCE-II binding in extracts from BALB/MK cells. TCE-II binding was greatly enhanced in NGP and DU-145 extracts relative to the BALB/MK extracts and TGF-fll treatment of DU-145 cells had little effect on TCE binding (Fig. 5D).
r. 100
50
0
-100 -100.m pPLFCAT Constucts
FIG. 4. Effect of mutation of TCE on response to TGF-,31 and pRB. Rapidly growing BALB/MK cells were cotransfected with pBR322 or pCMVRB.WT (stippled bars) and the indicated pPLFCAT constructs. Results from untreated plates (open bars) or from plates treated with TGF-f1 (10 ng/ml) after transfection (hatched bars) are shown. Each bar represents the mean of duplicate determinations and the results are representative of three separate experiments.
DISCUSSION Previous studies have implicated a cellular protein(s) interacting with the pRB binding domain of DNA tumor virus oncoproteins in the pathway for TGF-pl-induced suppression of c-myc transcription and cell proliferation in skin keratinocytes (4). Several cellular proteins, in addition to pRB, bind the conserved domain in the viral oncoproteins (7, 8), and any of these proteins could be involved in suppression of c-myc transcription in response to TGF-p81. The RB protein was a good candidate because stable transfection of
appreciably reduce TCE binding to the TCE-I and TCE-III retarded complexes (Fig. SB). Extracts from HFK/SV40 cells untreated and treated with TGF-P1 (10 ng/ml) for 24 hr were bound with labeled TCE. In contrast to the BALB/MK cells, TGF-,B1 treatment had little or no effect on proteinDNA binding in extracts from the HFK/SV40 cells (Fig. SC). This finding correlates with the inability of TGF-P1 to repress c-myc expression and inhibit DNA synthesis in this cell line (4). In addition, binding to the TCE-I and TCE-II retarded complexes was greatly enhanced in the HFK/SV40 extracts B
1)
( t
Fold molar excess unlabeled: °0 TCT 0 25 50100200 0 0 IC E C 0 0 0 25 50 100200* l1('l-' >@.'
()
I l4 I hfrB () 1 24
_
1Ci(l-I---)- *0gg
-
:,e >e: -TCEi-l-- 9,1
_
C r-
7
~L _.
3
^--
TC-F.-.--O 9
T I-CF.-II-1 -El-
rc lI-
II-
4 b
.8
*
T'(T'1'-111-0 4 4 * '1'I{1-LII-_
FIG. 5. Electrophoretic mobility-shift analysis ofTCE binding to cellular proteins. (A) Specificity of TCE binding to BALB/MK cell extracts. Control binding with labeled TCE (left lane) and competition with the indicated -fold molar excess of unlabeled TCE or TCE.m oligonucleotides are shown. The binding of labeled TCE.m to BALB/MK cell extracts is also shown (right lane). (B) TGF-f31 regulation of TCE-protein binding in TGF-,B-sensitive BALB/MK cells. Cellular extracts from rapidly growing BALB/MK cells treated with TGF-,B1 (10 ng/ml) for the indicated times were bound with labeled TCE. (C) Analysis of TCE binding to cellular extracts from TGF-,B-insensitive HFK/SV40 cells without (HFK/SV40) and with (HFK/SV40+T) treatment with TGF-f1 (10 ng/ml) for 24 hr in comparison to control HFK cells. (D) TCE binding to cellular proteins isolated from control BALB/MK and NGP (neuroblastoma) cells with an intact RB gene, retinoblastoma cells with a defective RB gene (Y79 and WERI), and human prostatic adenocarcinoma cells that have an exon 21 deletion in the RB gene without (DU-145) and with (DU-145+T) treatment with TGF-P1 (10 ng/ml) for 24 hr.
Cell
Biology: Pietenpol et al.
the RB gene in a variety of cancer cell lines resulted in a suppression of tumorigenicity as well as a diminished growth potential of the cells (15). The demonstration in the present study that transient expression of pRB inhibits myc transcription like TGF-p1 and requires the same promoter element (termed TCE) as TGF-/31 provides additional evidence in favor of pRB being a component in this TGF-f31 pathway targeted by the DNA tumor virus oncoproteins. Both positive and negative regulatory elements have previously been identified in the c-myc promoter (for review, see ref. 16). Lipp et al. (17) provided evidence for a large region of the c-myc promoter being involved in negative regulation of c-myc. This region includes the TCE described in the present study. None of the previously described elements has been shown to be involved in growth factor regulation of c-myc expression. Kerr et al. (12) have presented results indicating that TGF-/31 suppression of transin/stromelysin gene transcription is mediated through binding of a foscontaining protein complex to a promoter sequence, termed TIE. TIE shows a region of sequence similarity to a region of the TCE (see Fig. 3). A second region of sequence similarity to TIE within the human c-myc promoter is outside the 5' region shown to be necessary for TGF-f31 suppression of c-myc transcription (2, 5). Robbins et al. (18) have reported that expression of pRB in mouse fibroblasts suppresses transcription of c-fos and have identified an element, termed the retinoblastoma control element (RCE), in the c-fos promoter necessary for this suppression. More recently, sequences homologous to the RCE have been identified in the TGF-,81, -,32, and -P3 promoters by Kim et al. (19). Cotransfection with an RB expression plasmid was shown to regulate expression from TGF-/B1 promoter/CAT constructs positively or negatively depending on the cell type. Kim et al. (19) also reported that sequences similar to the RCE are present in the human c-myc promoter and that RB overexpression enhances c-myc expression in mink lung (MvlLu) cells, which are very sensitive to the growth inhibitory effect of TGF-,8. This indicates that cell type differences also exist in the factors involved in the response pathway for TGF-,8 suppression of c-myc expression and growth inhibition. Studies by Missero et al. (20) have shown that transfection of keratinocytes with ElA renders the cells resistant to growth inhibition by TGF-131. Mutational analysis indicated that these effects correlated with the ability of ElA proteins to bind pRB as well as other cellular proteins. Thus, it is possible that while pRB may be necessary for TGF-f31 suppression of c-myc transcription as indicated by our studies, inactivation of additional cellular proteins may be necessary to give full resistance to the growth inhibitory effects of TGF-f3. Indeed, cellular proteins other than pRB are likely to be involved in the TGF-P1 suppression of c-myc transcription, as indicated by the binding studies. Evidence for factors binding to the TCE was obtained by using electrophoretic gel mobility-shift assays. TCE formed three retarded complexes, TCE-I, TCE-II, and TCE-III. The TCE-II complex, but not the TCE-I or TCE-III complexes, was inhibited by TGF-,81 treatment of the TGF-f3-sensitive BALB/MK cells, but not the TGF-/3-insensitive HFK/SV40 and DU-145 cells. It is possible that the factors binding to the TCE lack physiological relevance to the transcriptional regulatory phenomena described here. However, the observation that some binding is down regulated by TGF-f31 treatment of TGF-/3-sensitive but not TGF-p-insensitive cells argues in favor of at least some of the observed binding reflecting biologically significant protein-DNA interactions. The data further suggest that the TCE-II binding protein(s) may be a positive transcriptional factor(s) for c-myc, since binding is correlated with transcriptional activity of c-myc.
Proc. Natl. Acad. Sci. USA 88 (1991)
10231
Because pRB is a nuclear phosphoprotein of 105-110 kDa that is capable of binding DNA (21) and because pRB appears to be involved in the suppression of c-myc transcription in response to TGF-f31 treatment, the possibility ofTCE binding directly to pRB was examined. No binding of the TCE oligonucleotide was obtained with cellular proteins coprecipitated with antibodies either to DNA tumor viral oncoproteins or to pRB (data not shown). The presence of the TGF-f31-regulated TCE-II complex in RB-defective cells indicates the involvement of cellular protein(s) in addition to pRB in the regulation of c-myc transcription by TGF-,B1. These observations suggest that TCE-II binding may reflect a positive transactivating factor for c-myc. TGF-f31 treatment of cells could cause a reduction in binding to TCE either through mechanisms involving the direct association of pRB with a positive transcription factor or by modification of the factor such that it can no longer interact with the TCE. Alternative models are certainly possible and the further delineation of the molecular events in this pathway will require identification and mechanistic study of the factors involved. The authors wish to thank Dr. Lina Dagnino for helpful advice and Mary Aakre, Muriel Cunningham, and Agnieszka Gorska for excellent technical assistance. This work was supported by Grants CA42572 (H.L.M.), CA-48799 (H.L.M.), and GM-30257 (R.W.S.) from the U.S. Public Health Service and Grant 47095 from the American Cancer Society (R.W.S.). J.A.P. was supported by National Cancer Institute Training Grant CA-09592 and K.M. was supported by an Advanced Training Grant from the Swiss National Science Foundation. R.W.S. is the recipient of a Career Development Award from the Juvenile Diabetes Foundation. 1. Barnard, J. A., Lyons, R. M. & Moses, H. L. (1990) Biochim. Biophys. Acta 1032, 79-87. 2. Moses, H. L., Yang, E. Y. & Pietenpol, J. A. (1990) Cell 63, 245-247. 3. Coffey, R. J., Jr., Sipes, N. J., Bascom, C. C., Graves-Deal, R., Pennington, C. Y., Weissman, B. E. & Moses, H. L. (1988) Cancer Res. 48, 1596-1602. 4. Pietenpol, J. A., Stein, R. W., Moran, E., Yaciuk, P., Schlegel, R., Lyons, R. M., Pittelkow, M. R., Monger, K., Howley, P. M. & Moses, H. L. (1990) Cell 61, 777-785. 5. Pietenpol, J. A., Holt, J. T., Stein, R. W. & Moses, H. L. (1990) Proc. Natl. Acad. Sci. USA 87, 3758-3762. 6. Coffey, R. J., Jr., Bascom, C. C., Sipes, N. J., Graves-Deal, R., Weissman, B. E. & Moses, H. L. (1988) Mol. Cell. Biol. 8, 3088-3093. 7. DeCaprio, J. A., Ludlow, J. W., Figge, J., Shew, J.-Y., Huang, C.-M., Lee, W.-H., Marsilio, E., Paucha, E. & Livingston, D. M. (1988) Cell 54, 275-283. 8. Whyte, P., Williamson, N. M. & Harlow, E. (1989) Cell 56, 67-75. 9. Kaye, F. J., Kratzke, R. A., Gerster, J. L. & Horowitz, J. M. (1990) Proc. Natl. Acad. Sci. USA 87, 6922-6926. 10. Andersson, A., Davis, D. L., Dahlback, H., Jornvall, H. & Russell, D. W. (1989) J. Biol. Chem. 264, 8222-8229. 11. Gorman, C. M., Moffat, L. F. & Howard, B. H. (1982) Mol. Cell. Biol. 2, 1044-1051. 12. Kerr, L. D., Miller, D. B. & Matrisian, L. M. (1990) Cell 61, 267-278. 13. Bernard, O., Cory, S., Gerondakis, S., Webb, E. & Adams, J. M. (1983) EMBO J. 2, 2375-2383. 14. Lee, E. Y.-H. P., Bookstein, R., Young, L.-J., Lin, C.-J., Rosenfeld, M. G. & Lee, W.-H. (1988) Proc. Natl. Acad. Sci. USA 85, 6017-6021. 15. Bookstein, R., Shew, J.-Y., Chen, P.-L., Scully, P. & Lee, W.-H. (1990) Science 247, 712-715. 16. Holt, J. T., Morton, C. C., Nienhuis, A. W. & Leder, P. (1987) in The Molecular Basis of Blood Diseases, eds. Stamatoyannopoulos, G., Nienhuis, A. W., Leder, P. & Majerus, P. (Saunders, Philadelphia), pp. 347-376. 17. Lipp, M., Schilling, R., Wiest, S., Laux, G. & Bornkamm, G. W. (1987) Mol. Cell. Biol. 7, 1393-1400. 18. Robbins, P. D., Horowitz, J. M. & Mulligan, R. C. (1990) Nature (London) 346, 668-671. 19. Kim, S. J., Lee, H. D., Robbins, P. D., Busam, K., Sporn, M. B. & Roberts, A. B. (1991) Proc. Natl. Acad. Sci. USA 88, 3052-3056. 20. Missero, C., Filvaroff, E. & Dotto, G. P. (1991) Proc. Natl. Acad. Sci. USA 88, 3489-3493. 21. Lee, W.-H., Shew, J. Y., Hong, F. D., Sery, T. W., Donosco, L. A., Young, L.-J., Bookstein, R. & Lee, E. Y.-H. P. (1987) Nature (London) 329, 642-644.
Factor-binding element in the human c-myc promoter involved in transcriptional regulation by transforming growth factor 131 and by the retinoblastoma gene product (negative growth factor/tumor suppressor gene/protooncogenes)
JENNIFER A. PIETENPOL*, KARL MUNGERt, PETER M. HOWLEYt, ROLAND W. STEIN*t AND HAROLD L. MOSES* Departments of *Cell Biology and of tMolecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232; and tLaboratory of Tumor Virus Biology, National Cancer Institute, Bethesda, MD 20892
Communicated by David M. Prescott, August 5, 1991 (received for review May 8, 1991)
sequence between positions -100 and +71 relative to the P1 transcription start site (5). Skin keratinocytes transformed by simian virus 40 (SV40), human papilloma virus (HPV)-18, or the HPV-16 transforming genes expressed from a heterologous promoter were found to be resistant to the growth inhibitory or c-myc suppressive effects of TGF-,81 (4). Transient expression of the genes encoding HPV-16 E7, adenovirus ElA, and SV40 large tumor antigen (T antigen) blocked the TGF-j31 suppression of transcription from c-myc promoter/chloramphenicol acetyltransferase (CAT) constructs while mutants of ElA and T antigen that were defective in binding the retinoblastoma gene product (pRB) failed to block TGF-31 suppression (4). These studies suggested that the oncoproteins blocked the actions of TGF-,31 by sequestering an essential factor(s) in this growth suppression pathway. However, it had not been determined in this previous study whether it was pRB or another of the cellular proteins that interacts with the conserved domain of the viral oncoproteins (7, 8) that was an essential element in the TGF-/31 suppression of c-myc transcription and cell growth. In the present study, we demonstrate that transient expression of pRB can inhibit c-myc transcription. An analysis of the c-myc promoter revealed that the same element was required for regulation by both TGF-3 and pRB. This indicates that pRB may be involved in mediating the TGF-,Binduced inhibition of c-myc expression. Furthermore, binding studies with this c-myc promoter element implicated cellular factors in addition to pRB in this growth inhibitory pathway.
ABSTRACT Previous studies have shown that transforming growth factor .31 (TGF-j81) inhibition of keratinocyte proliferation involves suppression of c-myc transcription, and indirect evidence has suggested that the retinoblastoma gene product (pRB) may be involved in this process. In this study, transient expression of pRB in skin keratinocytes was shown to repress transcription of the human c-myc promoter as effectively as TGF-131. The same c-myc promoter region was required for regulation by both TGF-fi1 and pRB. These sequences, termed the TGF-,8 control element (TCE), lie between positions -86 and -63 relative to the P1 transcription start site. Oligonucleotides containing the TCE bound to several nuclear factors in mobility-shift assays using extracts from cells with or without normal pRB. Binding of some factors was inhibited by TGF-131 treatment of TGF-fi-sensitive but not TGF-8-insensitive cells. These data indicate that pRB can suppress c-myc transcription and suggest the involvement of cellular factors in addition to pRB in the TGF-,B1 pathway for the inhibition of c-myc transcription and growth inhibition.
Cellular growth control involves a complex interplay between positive- and negative-acting diffusible factors and growth regulatory genes. Of the diffusible growth inhibitory peptides the transforming growth factors type P (TGF-jBs) are the most potent (for review, see ref. 1). TGF-,31 has been shown to be a potent inhibitor for most cell types in culture and growth inhibitory effects of the TGF-pBs have also been demonstrated by in vivo studies (for review, see ref. 2). The mechanism of TGF-,61 inhibition of epithelial cells has been most extensively studied by utilizing skin keratinocyte model systems. Coffey et al. (3) demonstrated that TGF-/31 inhibited either rapidly growing skin keratinocytes or epidermal
MATERIALS AND METHODS Cell Culture. BALB/MK cells, human foreskin keratinocytes (HFK), and SV40-transformed HFK (HFK/SV40) cells were cultured as described (4). DU-145 (obtained from American Type Culture Collection) were cultured in McCoy's 5A medium with 10% fetal calf serum. Y79, WERI, and NGP cells were cultured in RPMI 1640 medium with 20% fetal calf serum. c-myc/CAT Expression Vectors. Deletion mutants pPLFCAT -2290, -100, and +71 were constructed as described (5). Deletion mutants pPLFCAT -86, -63, -44, +12, and +53 were produced by PCR technology. Primers were designed to make successive deletions of the -100 to +71 region of the c-myc promoter. The lower strand primer was the same for all amplifications (5'-TAATATCCAGCTGAACGGTC-3'). The upper strand primers were as follows:
growth factor-stimulated quiescent keratinocytes. TGF-(1 can inhibit growth factor stimulation of DNA synthesis subsequent to the early events induced by growth factors (4). Recent studies indicate that TGF-f31 effects on c-myc (MYC in human gene nomenclature) expression may be mechanistically important in TGF-f31-induced growth inhibition. TGF-f31 can suppress c-myc mRNA abundance in a variety of cell types (5). In the skin keratinocyte, TGF-/31 rapidly reduces c-myc mRNA and protein levels, and this effect can be blocked by cycloheximide (6). Antisense c-myc oligonucleotides that suppress synthesis of c-myc protein inhibit proliferation of skin keratinocytes as effectively as TGF-,1 (5), providing strong evidence that c-myc expression is necessary for keratinocyte proliferation. The TGF-,81 inhibition of c-myc expression occurs at the level of transcription initiation and requires a cis-acting
Abbreviations: TGF, transforming growth factor; RB, retinoblastoma gene; pRB, RB product; CAT, chloramphenicol acetyltransferase; TCE, TGF-(3 control element; TIE, TGF-j3 inhibitory element; SV40, simian virus 40; RSV, Rous sarcoma virus; HFK, human foreskin keratinocyte.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 10227
10228
Proc. Natl. Acad. Sci. USA 88
Cell Biology: Pietenpol et al.
nucleotide (see Fig. 3 for sequence). Each 25-,ul reaction mixture was incubated at 4°C for 40 min. Samples were subjected to electrophoretic separation at 4°C on a 6% nondenaturing polyacrylamide gel at 200 V for 4-5 hr using TBE (0.9 M Tris borate/0.002 M EDTA)/PAGE conditions. After electrophoresis, the gels were dried and labeled DNA was localized by autoradiography.
pPLFCAT-86, 5'-CGCGAAGCTTAGGGCGTGGGGGAAAAGA-3'; pPLFCAT-63, 5 '-CGCGAAGCTTAAGATCCTCTCTCGCTAA-3'; pPLFCAT-44, 5 '-TCTCTCGCTAAGCTTCGCCCAC-3'; pPLFCAT+12, 5'-GACCCCCAAGCTTTGCTGCT-3'; pPLFCAT+53, 5'CGGCCGTCCCGGGCTCCCCT-3'; pPLFCAT-100.m, 5'-
CGACCCGGGTTCCCAAAGCAGAGGTCTTTTGGGAAAAGAAAA-3'. The amplified products were subcloned into pPLFCAT. Underlined nucleotides represent primer mismatch resulting in restriction enzyme cleavage sites used for cloning the PCR fragments and, in the case of pPLFCAT100.m, for introduction of TGF-,8 control element (TCE) mutations. pRB Expression Vectors. To construct the pCMVRB.WT and pCMVRB.AS expression vectors, the 4.1-kilobase HindIII fragment containing the RB cDNA was isolated from pCMV.RB (9). The HindIII fragment from pCMV.RB was cloned in the sense and antisense orientations in the pCMV.4 cloning vector (10), resulting in pCMVRB.WT and pCMVRB.AS, respectively. DNA Transfection and Analysis of CAT Protein. Transfection of BALB/MK and HFK cells was as described (4). The transfected cells were treated with TGF-,B1 (R & D Systems, Minneapolis) 6-18 hr posttransfection. Cell extracts were prepared 24 hr after TGF-,81 treatment as described (11). The amount of protein extract from the HFK and BALB/MK cells used in the CAT assays was normalized to f-galactosidase activity. Both the traditional CAT assay (11) and a CAT ELISA (5 Prime -* 3 Prime, Inc.) were used to determine levels of CAT activity and protein, respectively. Electrophoretic Mobility-Shift Assays. Cells were treated with lysis buffer (250 mM NaCI/0.1% Nonidet P-40/50 mM Hepes, pH 7.0/25 ,uM sodium fluoride/200 ,uM sodium orthovanadate/0.12 unit of aprotinin) at 4°C for 30 min. The cell suspension was centrifuged at 13,600 x g for 5 min. The protein concentration of the clarified supernatant was typically 1 ,ug/,jl. The binding reaction mixtures contained in a 25-Al vol 10 jig of cell extract in 25 mM Hepes (pH 7.9), 1 mM dithiothreitol, 0.04 mM EDTA, 100 mM NaCl, 40 mM KCl, 0.5 ,ug of poly(dI-dC), 0.5 ,ug of single-stranded DNA, 0.04% (vol/vol) Nonidet P-40, 1.2% (vol/vol) glycerol, and 10 fmol of a 32P-end-labeled double-stranded TCE or TCE.m oligo-
RESULTS RB Suppression of c-myc Transcription. To determine whether expression of pRB might have an effect on c-myc transcription similar to that observed with TGF-/31 treatment, BALB/MK cells were transfected with pPLFCAT-100 either alone or together with an RB plasmid expressing wild-type RB mRNA (pCMVRB.WT), or a control antisense RB mRNA (pCMVRB.AS). As shown in Fig. 1A, transient expression of wild-type RB in the BALB/MK cells alone (no TGF-,Bl treatment) significantly reduced pPLFCAT-100 expression, with the level of repression being the same as that observed with TGF-,B1 treatment. The effect of TGF-f31 treatment and pRB expression together was the same as either alone. Similar results were obtained with HFK cells transfected with pCMVRB.WT under the same conditions (Fig. 1B). The control plasmid (pCMVRB.AS) had no effect on c-myc transcription and did not alter the TGF-,B1 effect on transcription (Fig. 1A). To further address the question of specificity of the pRB effect, CMVRB.WT was cotransfected with a Rous sarcoma virus (RSV) enhancer-driven CAT expression plasmid, RSV/CAT. Production of CAT activity from the RSV promoter was not reduced by TGF-,B1 treatment or by cotransfected CMVRB.WT (Fig. 1C). The results indicate that pRB can inhibit c-myc transcription. Sequences Required for pRB and TGF-l1 Control of c-myc Transcription. Previous studies had indicated that the TGF-,31 response element was within the -100 to +71 region of the c-myc promoter (5). Thus, finer resolution deletion mutants were subcloned into the CAT expression vector (pPLFCAT). The c-myc promoter/CAT chimeras, pPLFCAT -2290, -100, -86, -63, -44, +12, and +71 are designated according to the 5' nucleotide position relative to the P1 transcription start site (+1). All deletion mutants used
B
A
(1991)
( I
mm-lr
I.a
i
X
H ill!!,;[:,
1
-
.
I.m
I
*6a*
*
.i
13
*(R ; 1
0
Q il I',' L: T' ,I0
U
p( C\I RH\I1
00
p( 'I (
Cont.
f
.1
* a
' as
VVI
pCMVRB.WT pCMVRB.AS
FIG. 1. Effect of treatment with TGF-,B1 or cotransfection with RB expression plasmid on myc/CAT transcription in mouse and human keratinocytes. (A) Rapidly growing BALB/MK cells were cotransfected with equimolar amounts of pPLFCAT-100 (a human c-myc promoter-CAT construct containing the sequences -100 to +510) and pBR322 or an RB expression plasmid (pCMVRB.WT) or a control vector (pCMVRB.AS). The CAT protein produced was quantitated by an ELISA without (open bars) or with (hatched bars) treatment with TGF-,81 (10 ng/ml). Each bar represents the mean of duplicate determinations and the results are representative of three separate experiments, each with duplicate determinations. (B) Rapidly growing HFK cells were cotransfected with the same plasmids described above for the BALB/MK cell transfection and control and TGF-/31-treated (10 ng/ml) cultures were assayed. CAT activity was quantitated by determining the percentage conversion of ['4C]chloramphenicol to the acetylated form by liquid scintillation counting. NA, not applicable. (C) Rapidly growing BALB/MK cells were cotransfected with equimolar amounts of RSV promoter-CAT construct and pBR322 or pCMVRB.WT. Control and TGF-B31-treated (10 ng/ml) cultures were assayed. CAT activity was quantitated as described above.
Cell
_
Biology: Pietenpol et al.
Proc. Natl. Acad. Sci. USA 88 (1991) t/ Inhibitiln by p CM V R BAIV . I" ReIaitie PTromo0tCrl- T G F-B I Activitv ii1caiment (Co...t rarlsfc'cii'
tslcs/CATF Constructs i)PLICAT-2-29()
,)pl1FCA IX-
()(
i
1.AX) .,(l -A I .... t.~~~~~~~~~~~~~~~~~~~~~~~~~
pIILFCIA-l-86
i
-axo-
CAT
CAT I
+ 34 1.37±7
482
1_ 1 .C=AT
-ur
p)IIl(FCAT+ l2
In T-, AT -X CiCAT
pIlFC'AIT+7 1
..
(-Yi 6+tl
7 1; 71 + .'2()
.512
pIllFCAT'-63 pPILF-CAT-4-4
10229
98 ±
8 x.
.
.53
8
t
+)A4
f-,
1
I 27 ±+,N'
FIG. 2. Deletion analysis of c-myc promoter to identify TGF-,3 and pRB response element. BALB/MK cells were cotransfected with the indicated pPLFCAT constructs and either pBR322 or the RB expression plasmid pCMVRB.WT and the effect of TGF-f31 treatment (10 ng/ml) or pCMVRB.WT cotransfection on CAT production was determined. CAT protein was quantitated by ELISA. The promoter activity is expressed relative to the mean of the activity obtained with pPLFCAT-2290. The remaining data are expressed as percentage inhibition of CAT protein production from the pPLFCAT constructs by TGF-,B1 treatment or by cotransfection with pCMVRB.WT. Each value represents the mean SEM of four or five determinations with the exception of the data from the pPLFCAT+12 construct, which represents the mean of duplicate determinations.
have 3' sequences to + 510. The promoter activity for each of the constructs relative to pPLFCAT-2290 is presented in Fig. 2. Although CAT protein levels varied considerably (see standard error) from experiment to experiment, there was not a major loss of promoter activity with sequential deletions of the 5' flanking region. While pPLFCAT-63 had 50% of the activity of pPLFCAT-2290, the -44, +12, and +71 constructs had promoter activity equivalent to pPLFCAT-2290. Treatment with TGF-,S1 inhibited CAT production from 50% to 80% in the -2290, -100, and -86 mutants but did not inhibit transcription from the -63, -44, +12, and +71 constructs (Fig. 2). A very similar pattern of repression was observed when pCMVRB.WT was cotransfected with these c-myc promoter mutants (Fig. 2). These results demonstrated that transcriptional repression by both TGF-,B1 and pRB was mediated through the same region in the human c-myc promoter. This common target sequence, located between bases -86 and -63, has been termed the TCE. The sequence of the TCE in the human c-myc promoter is shown in Fig. 3A in comparison to a previously described element in another gene reported to be repressed by TGF-P1, the TGF-f3 inhibitory element (TIE) in the rat transin/stromelysin promoter (12). A region of similarity between the TCE and TIE is apparent (Fig. 3A). Comparison of the mouse c-myc promoter region, which is also TGF-p responsive, revealed a similar sequence, except for a 34-base-pair (bp) insert between positions -77 and -76 and a 1-bp insertion between positions -68 and -67 in the mouse c-myc promoter relative to the human promoter (13). To determine the necessity of this -86- to -63-bp sequence in the pRB- and the TGF-f31-mediated inhibition of c-myc A
1TI i,] rirnsi/stromelysin
G A
T T C
G1C'G
transcription response in the keratinocytes, four guanines at positions -81, -79, -76, and -77 were substituted by thymines (Fig. 3B). Expression from this mutant construct (pPLFCAT-100.m) was not inhibited by TGF-31 treatment or cotransfection with pCMVRB.WT; rather, the latter gave an unexplained increase in CAT protein levels (Fig. 4). These data corroborate the deletion mutagenesis data and further indicate that the -63 to -86 region (TCE) of the c-myc promoter is necessary for both TGF-P31 and pRB control of myc transcription. TCE Binding to Cellular Proteins. Whole-cell extracts from BALB/MK cells were incubated with an end-labeled oligonucleotide corresponding to the TCE (Fig. 3). Three major retarded species were resolved and are depicted as TCE-I, TCE-II, and TCE-III (Fig. SA). Similar results were obtained with extracts from HFK cells (Fig. SC). Binding of labeled TCE to all three retarded complexes from BALB/MK cells was effectively blocked by competition with unlabeled TCE, but competition with the TCE.m oligonucleotide with 4 base mutations (Fig. 3) had little effect, indicating that TCE binding to all retarded species is specific. Use of labeled TCE.m in the mobility-shift assay gave a pattern of binding completely different from that obtained with wild-type TCE. There was no detectable binding to the retarded species bound by TCE; rather there was binding to a single band migrating between TCE-I and TCE-II (Fig. SA). The effect of TGF-P1 treatment on the capacity of the extracts to bind the TCE was next determined. As shown in Fig. SB, protein-DNA binding corresponding to TCE-II was reduced after TGF-f31 treatment of BALB/MK cells over the time course shown. However, TGF-131 treatment did not FIG. 3. TCE sequence. (A) Sequence of TCE in c-myc is compared to the TIE identified in the rat
G T G A -700
transin/stromelysin promoter (12). Areas of iden-
within shaded boxes. (B) The four mutations -SaGCkGAGGGCsTGGG&GAAAAGAAtity
T'
are
made in the TCE to generate the mutant TCE (TCE.m) used in experiments depicted in Fig. 5 is shown. This mutated promoter region was subB
TCE
-86 G CA GAG G G C G T r G G G G A A A A G A A -63 I
TCE.m
I
I
I
6 G C A G A G G T C T T T T G G G A A A A G AAA63
pPLFCAT-100 vector to give the pPLFCAT-100.m construct used in the expercloned back into the
iment shown in Fig. 4.
10230
Proc. Natl. Acad. Sci. USA 88 (1991)
Cell Biology: Pietenpol et al.
relative to that obtained with HFK extracts. The appearance of binding to HFK extracts normalized for total protein concentration and run simultaneously is shown for comparison. To address the question of potential TCE binding to pRB, assays were carried out with cell extracts isolated from cells with a defective RB gene [WERI and Y79 human retinoblastoma cells (14) and DU-145 human prostate adenocarcinoma cells (15)] in comparison to extracts from cells with an intact RB gene [NGP human retinoblastoma cells (15) and BALB/ MK cells]. By mobility-shift assay, TCE binding to cellular proteins from WERI, Y79, and DU-145 cells was observed, but the binding pattern was different from that obtained with cells containing an intact RB gene (Fig. SD). There was greatly diminished TCE-I binding in the RB-defective cells. However, use of additional protease inhibitors in the extraction buffer resulted in more prominent TCE-I binding in the DU-145 cell extracts (L. Dagnino, H.L.M., and R.W.S., unpublished observations). TCE-II binding in Y79 and WERI cell extracts was equal to or greater than TCE-II binding in extracts from BALB/MK cells. TCE-II binding was greatly enhanced in NGP and DU-145 extracts relative to the BALB/MK extracts and TGF-fll treatment of DU-145 cells had little effect on TCE binding (Fig. 5D).
r. 100
50
0
-100 -100.m pPLFCAT Constucts
FIG. 4. Effect of mutation of TCE on response to TGF-,31 and pRB. Rapidly growing BALB/MK cells were cotransfected with pBR322 or pCMVRB.WT (stippled bars) and the indicated pPLFCAT constructs. Results from untreated plates (open bars) or from plates treated with TGF-f1 (10 ng/ml) after transfection (hatched bars) are shown. Each bar represents the mean of duplicate determinations and the results are representative of three separate experiments.
DISCUSSION Previous studies have implicated a cellular protein(s) interacting with the pRB binding domain of DNA tumor virus oncoproteins in the pathway for TGF-pl-induced suppression of c-myc transcription and cell proliferation in skin keratinocytes (4). Several cellular proteins, in addition to pRB, bind the conserved domain in the viral oncoproteins (7, 8), and any of these proteins could be involved in suppression of c-myc transcription in response to TGF-p81. The RB protein was a good candidate because stable transfection of
appreciably reduce TCE binding to the TCE-I and TCE-III retarded complexes (Fig. SB). Extracts from HFK/SV40 cells untreated and treated with TGF-P1 (10 ng/ml) for 24 hr were bound with labeled TCE. In contrast to the BALB/MK cells, TGF-,B1 treatment had little or no effect on proteinDNA binding in extracts from the HFK/SV40 cells (Fig. SC). This finding correlates with the inability of TGF-P1 to repress c-myc expression and inhibit DNA synthesis in this cell line (4). In addition, binding to the TCE-I and TCE-II retarded complexes was greatly enhanced in the HFK/SV40 extracts B
1)
( t
Fold molar excess unlabeled: °0 TCT 0 25 50100200 0 0 IC E C 0 0 0 25 50 100200* l1('l-' >@.'
()
I l4 I hfrB () 1 24
_
1Ci(l-I---)- *0gg
-
:,e >e: -TCEi-l-- 9,1
_
C r-
7
~L _.
3
^--
TC-F.-.--O 9
T I-CF.-II-1 -El-
rc lI-
II-
4 b
.8
*
T'(T'1'-111-0 4 4 * '1'I{1-LII-_
FIG. 5. Electrophoretic mobility-shift analysis ofTCE binding to cellular proteins. (A) Specificity of TCE binding to BALB/MK cell extracts. Control binding with labeled TCE (left lane) and competition with the indicated -fold molar excess of unlabeled TCE or TCE.m oligonucleotides are shown. The binding of labeled TCE.m to BALB/MK cell extracts is also shown (right lane). (B) TGF-f31 regulation of TCE-protein binding in TGF-,B-sensitive BALB/MK cells. Cellular extracts from rapidly growing BALB/MK cells treated with TGF-,B1 (10 ng/ml) for the indicated times were bound with labeled TCE. (C) Analysis of TCE binding to cellular extracts from TGF-,B-insensitive HFK/SV40 cells without (HFK/SV40) and with (HFK/SV40+T) treatment with TGF-f1 (10 ng/ml) for 24 hr in comparison to control HFK cells. (D) TCE binding to cellular proteins isolated from control BALB/MK and NGP (neuroblastoma) cells with an intact RB gene, retinoblastoma cells with a defective RB gene (Y79 and WERI), and human prostatic adenocarcinoma cells that have an exon 21 deletion in the RB gene without (DU-145) and with (DU-145+T) treatment with TGF-P1 (10 ng/ml) for 24 hr.
Cell
Biology: Pietenpol et al.
the RB gene in a variety of cancer cell lines resulted in a suppression of tumorigenicity as well as a diminished growth potential of the cells (15). The demonstration in the present study that transient expression of pRB inhibits myc transcription like TGF-p1 and requires the same promoter element (termed TCE) as TGF-/31 provides additional evidence in favor of pRB being a component in this TGF-f31 pathway targeted by the DNA tumor virus oncoproteins. Both positive and negative regulatory elements have previously been identified in the c-myc promoter (for review, see ref. 16). Lipp et al. (17) provided evidence for a large region of the c-myc promoter being involved in negative regulation of c-myc. This region includes the TCE described in the present study. None of the previously described elements has been shown to be involved in growth factor regulation of c-myc expression. Kerr et al. (12) have presented results indicating that TGF-/31 suppression of transin/stromelysin gene transcription is mediated through binding of a foscontaining protein complex to a promoter sequence, termed TIE. TIE shows a region of sequence similarity to a region of the TCE (see Fig. 3). A second region of sequence similarity to TIE within the human c-myc promoter is outside the 5' region shown to be necessary for TGF-f31 suppression of c-myc transcription (2, 5). Robbins et al. (18) have reported that expression of pRB in mouse fibroblasts suppresses transcription of c-fos and have identified an element, termed the retinoblastoma control element (RCE), in the c-fos promoter necessary for this suppression. More recently, sequences homologous to the RCE have been identified in the TGF-,81, -,32, and -P3 promoters by Kim et al. (19). Cotransfection with an RB expression plasmid was shown to regulate expression from TGF-/B1 promoter/CAT constructs positively or negatively depending on the cell type. Kim et al. (19) also reported that sequences similar to the RCE are present in the human c-myc promoter and that RB overexpression enhances c-myc expression in mink lung (MvlLu) cells, which are very sensitive to the growth inhibitory effect of TGF-,8. This indicates that cell type differences also exist in the factors involved in the response pathway for TGF-,8 suppression of c-myc expression and growth inhibition. Studies by Missero et al. (20) have shown that transfection of keratinocytes with ElA renders the cells resistant to growth inhibition by TGF-131. Mutational analysis indicated that these effects correlated with the ability of ElA proteins to bind pRB as well as other cellular proteins. Thus, it is possible that while pRB may be necessary for TGF-f31 suppression of c-myc transcription as indicated by our studies, inactivation of additional cellular proteins may be necessary to give full resistance to the growth inhibitory effects of TGF-f3. Indeed, cellular proteins other than pRB are likely to be involved in the TGF-P1 suppression of c-myc transcription, as indicated by the binding studies. Evidence for factors binding to the TCE was obtained by using electrophoretic gel mobility-shift assays. TCE formed three retarded complexes, TCE-I, TCE-II, and TCE-III. The TCE-II complex, but not the TCE-I or TCE-III complexes, was inhibited by TGF-,81 treatment of the TGF-f3-sensitive BALB/MK cells, but not the TGF-/3-insensitive HFK/SV40 and DU-145 cells. It is possible that the factors binding to the TCE lack physiological relevance to the transcriptional regulatory phenomena described here. However, the observation that some binding is down regulated by TGF-f31 treatment of TGF-/3-sensitive but not TGF-p-insensitive cells argues in favor of at least some of the observed binding reflecting biologically significant protein-DNA interactions. The data further suggest that the TCE-II binding protein(s) may be a positive transcriptional factor(s) for c-myc, since binding is correlated with transcriptional activity of c-myc.
Proc. Natl. Acad. Sci. USA 88 (1991)
10231
Because pRB is a nuclear phosphoprotein of 105-110 kDa that is capable of binding DNA (21) and because pRB appears to be involved in the suppression of c-myc transcription in response to TGF-f31 treatment, the possibility ofTCE binding directly to pRB was examined. No binding of the TCE oligonucleotide was obtained with cellular proteins coprecipitated with antibodies either to DNA tumor viral oncoproteins or to pRB (data not shown). The presence of the TGF-f31-regulated TCE-II complex in RB-defective cells indicates the involvement of cellular protein(s) in addition to pRB in the regulation of c-myc transcription by TGF-,B1. These observations suggest that TCE-II binding may reflect a positive transactivating factor for c-myc. TGF-f31 treatment of cells could cause a reduction in binding to TCE either through mechanisms involving the direct association of pRB with a positive transcription factor or by modification of the factor such that it can no longer interact with the TCE. Alternative models are certainly possible and the further delineation of the molecular events in this pathway will require identification and mechanistic study of the factors involved. The authors wish to thank Dr. Lina Dagnino for helpful advice and Mary Aakre, Muriel Cunningham, and Agnieszka Gorska for excellent technical assistance. This work was supported by Grants CA42572 (H.L.M.), CA-48799 (H.L.M.), and GM-30257 (R.W.S.) from the U.S. Public Health Service and Grant 47095 from the American Cancer Society (R.W.S.). J.A.P. was supported by National Cancer Institute Training Grant CA-09592 and K.M. was supported by an Advanced Training Grant from the Swiss National Science Foundation. R.W.S. is the recipient of a Career Development Award from the Juvenile Diabetes Foundation. 1. Barnard, J. A., Lyons, R. M. & Moses, H. L. (1990) Biochim. Biophys. Acta 1032, 79-87. 2. Moses, H. L., Yang, E. Y. & Pietenpol, J. A. (1990) Cell 63, 245-247. 3. Coffey, R. J., Jr., Sipes, N. J., Bascom, C. C., Graves-Deal, R., Pennington, C. Y., Weissman, B. E. & Moses, H. L. (1988) Cancer Res. 48, 1596-1602. 4. Pietenpol, J. A., Stein, R. W., Moran, E., Yaciuk, P., Schlegel, R., Lyons, R. M., Pittelkow, M. R., Monger, K., Howley, P. M. & Moses, H. L. (1990) Cell 61, 777-785. 5. Pietenpol, J. A., Holt, J. T., Stein, R. W. & Moses, H. L. (1990) Proc. Natl. Acad. Sci. USA 87, 3758-3762. 6. Coffey, R. J., Jr., Bascom, C. C., Sipes, N. J., Graves-Deal, R., Weissman, B. E. & Moses, H. L. (1988) Mol. Cell. Biol. 8, 3088-3093. 7. DeCaprio, J. A., Ludlow, J. W., Figge, J., Shew, J.-Y., Huang, C.-M., Lee, W.-H., Marsilio, E., Paucha, E. & Livingston, D. M. (1988) Cell 54, 275-283. 8. Whyte, P., Williamson, N. M. & Harlow, E. (1989) Cell 56, 67-75. 9. Kaye, F. J., Kratzke, R. A., Gerster, J. L. & Horowitz, J. M. (1990) Proc. Natl. Acad. Sci. USA 87, 6922-6926. 10. Andersson, A., Davis, D. L., Dahlback, H., Jornvall, H. & Russell, D. W. (1989) J. Biol. Chem. 264, 8222-8229. 11. Gorman, C. M., Moffat, L. F. & Howard, B. H. (1982) Mol. Cell. Biol. 2, 1044-1051. 12. Kerr, L. D., Miller, D. B. & Matrisian, L. M. (1990) Cell 61, 267-278. 13. Bernard, O., Cory, S., Gerondakis, S., Webb, E. & Adams, J. M. (1983) EMBO J. 2, 2375-2383. 14. Lee, E. Y.-H. P., Bookstein, R., Young, L.-J., Lin, C.-J., Rosenfeld, M. G. & Lee, W.-H. (1988) Proc. Natl. Acad. Sci. USA 85, 6017-6021. 15. Bookstein, R., Shew, J.-Y., Chen, P.-L., Scully, P. & Lee, W.-H. (1990) Science 247, 712-715. 16. Holt, J. T., Morton, C. C., Nienhuis, A. W. & Leder, P. (1987) in The Molecular Basis of Blood Diseases, eds. Stamatoyannopoulos, G., Nienhuis, A. W., Leder, P. & Majerus, P. (Saunders, Philadelphia), pp. 347-376. 17. Lipp, M., Schilling, R., Wiest, S., Laux, G. & Bornkamm, G. W. (1987) Mol. Cell. Biol. 7, 1393-1400. 18. Robbins, P. D., Horowitz, J. M. & Mulligan, R. C. (1990) Nature (London) 346, 668-671. 19. Kim, S. J., Lee, H. D., Robbins, P. D., Busam, K., Sporn, M. B. & Roberts, A. B. (1991) Proc. Natl. Acad. Sci. USA 88, 3052-3056. 20. Missero, C., Filvaroff, E. & Dotto, G. P. (1991) Proc. Natl. Acad. Sci. USA 88, 3489-3493. 21. Lee, W.-H., Shew, J. Y., Hong, F. D., Sery, T. W., Donosco, L. A., Young, L.-J., Bookstein, R. & Lee, E. Y.-H. P. (1987) Nature (London) 329, 642-644.
