Vol. 11, No. 4

MOLECULAR AND CELLULAR BIOLOGY, Apr. 1991, p. 2291-2295 0270-7306/91/042291-05$02.00/0 Copyright C 1991, American Society for Microbiology

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Transcriptional Suppression of Cellular Gene Expression by c-Myc BEOM-SEOK

YANG,"2 TIM J. GEDDES,' ROBERT J. POGULIS,"2 BENOIT DE CROMBRUGGHE,3 AND SVEND 0. FREYTAGl 2*

Molecular Biology Research Program, Henry Ford Hospital, 2799 West Grand Boulevard, Detroit, Michigan 482021; Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 481092; and Department of Genetics, The University of Texas System Cancer Center, M. D. Anderson Hospital and Tumor Institute, Houston, Texas 770303 Received 17 July 1990/Accepted 26 December 1990

High levels of c-Myc in mouse 3T3-L1 cells specifically suppress the expression of three collagen genes. This effect is exerted through collagen promoter sequences and requires the leucine zipper motif of c-Myc. Our data suggest that an important aspect of c-Myc transforming activity is the ability to suppress specific cellular gene transcription. genes that respond to growth factor stimulation (21, 22), mRNA was isolated from quiescent cells that were completely arrested in GO/Gl for 4 days. [32P]cDNA corresponding to both cell types was synthesized and used to screen duplicate copies of a 3T3-L1 cDNA library. Of a total of 7,000 recombinants screened, approximately 2,500 (36%) were detected by hybridization, and 150 gave differential signals in the initial screening. Subsequent screening showed that most of the clones identified in the initial screening were false positives, and only four unique clones were truly positive, as determined by Northern (RNA) blot analysis. All four cDNA clones detected distinct mRNAs that were suppressed 3- to 10-fold in Myc-1 cells (Fig. 1, blots A to D). In contrast, expression of the ,-actin gene (blot E) and the pyruvate carboxylase gene (8) was unaffected in Myc-1 cells. Thus, high expression of c-Myc in 3T3-L1 cells does not produce global changes in gene expression. Although several clones gave increased hybridization signals with the Myc-1 probe during the screening procedure, such clones proved to be false positives after Northern analysis (data not shown). To show that suppression of the mRNAs in Myc-1 cells was not the result of a clonal effect of this cell line, we examined other clonal cell lines that also contained high levels of c-Myc. Cell line pM21 contains a recombinant human c-myc gene (35) driven by Moloney murine leukemia virus (MoMuLV) long terminal repeat (LTR) and expresses high levels of the human c-Myc protein (Fig. 1B, right). Northern blot analysis showed that the mRNA detected by clone 2 (Fig. 1A, blot B) was suppressed approximately 10-fold in pM21 cells (Fig. 1B, left). Similar results were obtained with multiple 3T3-L1 cell lines expressing high levels of a transfected human N-myc gene (data not shown). The identity of three of the cDNAs was determined by DNA sequence analysis followed by comparison with all DNA sequences contained in the GenBank data base. This analysis revealed that clone 1 (Fig. 1A, blot A) was a cDNA for pro-alpha 1(I) collagen, clone 2 (Fig. 1A, blot B) was a cDNA for pro-alpha 2(I) collagen, and clone 3 (Fig. 1A, blot C) was a cDNA for pro-alpha 3(VI) collagen. The identity of the fourth clone (Fig. 1A, blot D) is unknown. If production of the collagen mRNAs is tightly linked to

In spite of the intense interest in c-Myc, the specific biochemical function of this protein is unknown. The structure of c-Myc resembles that of several other nuclear proteins, some of which are known transcription factors (19, 20, 24, 25). Such proteins include c-Jun, c-Fos, E/CBP, MyoD, and others. The structural similarity between c-Myc and other known transcription factors has led to the suggestion that c-Myc might also be a transcription factor that controls the expression of specific genes (5). The fact that c-Myc can recognize and bind to a specific sequence in DNA lends support to this hypothesis (3). High levels of the Myc proteins (either c-Myc or N-Myc) have been correlated with reduced expression of specific cellular genes (2, 4, 17, 27, 28, 36), including the major histocompatibility complex (MHC) class I antigen genes in human melanoma (36) and neuroblastoma (2) cells. In the latter case, this effect of N-Myc is thought to be mediated through a specific transcription factor, H2TF1, which binds directly to the MHC class I antigen enhancer (23). Other studies have shown that c-Myc can activate gene expression (17, 18, 29) and that it binds directly to enhancers and putative origins of replication (1, 15). To investigate whether c-Myc controls the transcription of specific cellular genes in 3T3-L1 cells, we sought to identify genes whose expression was affected by high levels of c-Myc and then determined whether the effect of c-Myc was exerted at the transcriptional level. We used the procedure of differential filter hybridization to identify genes that were differentially expressed between cells having low levels of c-Myc and those having high levels. Two aspects of our strategy are worth noting. First, since we wanted to detect genes that responded primarily to c-Myc, mRNA was prepared from normal cells and cells that highly express a recombinant c-myc gene. The c-Myc-containing cell line used in this study, Myc-1 (8), is derived from 3T3-L1 cells (11, 12) and expresses high levels of a normal mouse c-Myc mRNA (8) and c-Myc protein (unpublished data). Myc-1 cells grow as a monolayer in culture and do not exhibit anchorage-independent growth or form tumors in athymic mice. Second, since we did not want to detect the myriad of *

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FIG. 1. Suppression of specific mRNAs in Myc-1 cells. (A) Top: Northern blots. One microgram of polyadenylated mRNA was applied in each lane. L, 3T3-L1 cell RNA; M, Myc-1 cell RNA. The filters were probed with [32P]cDNAs from the various clones. Blot A, clone 1; blot B, clone 2; blot C, clone 3; blot D, clone 4; blot E, cDNA for human ,-actin cDNA. Bottom: Serial dilutions of a [32P] DNA fragment, which serve as standards for the Northern blots. (B) Quantification of clone 2 mRNA in a cell line expressing human c-Myc. Left: Northern blot. Ten micrograms of total RNA was applied in each lane. The filter was probed with a [32P]cDNA corresponding to clone 2 (blot B in panel 1A). Right: Western immunoblot of human c-Myc. Protein from 5 x 105 cells was applied to each lane. The filter was probed with the 3C7 c-Myc monoclonal antibody (7) and an alkaline phosphatase conjugate. The numbers on the left indicate the positions of protein molecular mass markers (in kilodaltons).

c-Myc expression, collagen mRNA levels should be restored to normal by reducing the concentration of c-Myc protein. We previously showed that expression of c-myc antisense RNA in Myc-1 cells reduces the level of pRSVmyc mRNA in the cytoplasm and alleviates the myc-induced block to differentiation (8). Northern blot analysis showed that expression of the pro-alpha 2(I) collagen mRNA was restored to at least normal levels in cell lines expressing abundant amounts of c-myc antisense RNA (Fig. 2). Cell lines Mas-21 and Mas-25, which express very high levels of c-myc antisense RNA (data not shown), have 2 times as much pro-alpha 2(I) collagen mRNA as normal 3T3-L1 cells and 10 times as much as Myc-1 cells. In vitro transcription assays showed that pro-alpha 2(I) collagen gene transcription was markedly reduced in Myc-1 cells relative to 3T3-L1 cells (data not shown). Thus, we examined whether the effect of c-Myc was exerted through collagen promoter sequences by testing the ability of c-Myc to suppress the expression of a recombinant fusion gene under the transcriptional control of the mouse pro-alpha 2(I) collagen promoter. We focused our attention on pro-alpha

2(I) collagen because its promoter is well characterized with respect to the role of specific cis- and trans-acting elements (13, 14, 26, 31-33). pAZ1003 contains the chloramphenicol acetyltransferase (CAT) gene fused to 2 kb of the mouse pro-alpha 2(I) collagen promoter (31-33). 3T3-L1 cell lines stably expressing pAZ1003 were established by cotransfection with the dominant selectable marker pSV2neo, followed by selection in growth medium containing G418 (34). Cloned cell lines were screened for CAT activity (10), and positive lines were subsequently cotransfected with pSV40myc-dhfr (30) and with a plasmid which confers resistance to hygromycin B. Cloned cell lines expressing high levels of pSV40myc RNA were isolated and examined for CAT activity (Fig. 3A, top). In the examples for which results are shown, the activity of the pro-alpha 2(I) collagen promoter was suppressed 10- to 20-fold in cell lines pAZ1003.2.2 and pAZ1003.2.4, both of which contain abundant levels of pSV2myc RNA (Fig. 3A, bottom). Transfection of cells with a recombinant human c-myc gene (pM21 [35]) driven by an MoMuLV LTR produced similar results (data not shown). These results indicate that the effect of c-Myc on pro-alpha 2(I) collagen gene expression involves cis-acting sequences in the collagen promoter. To show that this effect required a functional c-Myc protein, a mutant of human c-Myc was tested for its ability to suppress collagen promoter activity in 3T3-L1 cells. The c-Myc mutant used in this study, D414-433, lacks the leucine zipper domain which is required for c-Myc transformation activity and for the ability of c-Myc to block 3T3-L1 cell differentiation (6, 9, 35). Cell lines expressing pAZ1003 and containing high levels of D414-433 c-myc RNA were established by cotransfection as described above and then examined for CAT activity. The results show that collagen promoter activity was unaffected in cell lines expressing high levels of the mutant c-myc RNA (Fig. 3B, pAZ1003.2.ml and pAZ1003.2.m2). Thus, the biochemical activity of c-Myc is required for suppression of pro-alpha 2(I) collagen transcription in 3T3-L1 cells. To examine whether the ability to suppress collagen transcription was an acute effect of c-Myc, we performed transient-expression assays in NIH 3T3 cells. For these experiments, a derivative of pAZ1003 (pAZ1003E) containing the simian virus 40 (SV40) enhancer was used because this plasmid gave sufficient expression in NIH 3T3 cells to

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FIG. 3. (A) Suppression of collagen promoter activity by c-Myc. Top: CAT activity in the parental cell line (pAZ1003.2) and in two c-Myc-containing cell lines (pAZ1003.2.2 and pAZ1003.2.4). Bottom: Quantification of pSV40myc RNA by S1 nuclease analysis. Lane b, pAZ1003.2.2 cell RNA; lane c, pAZ1003.2.4 cell RNA. Shown below the autoradiogram is a partial restriction map of pSV40myc-dhfr, with the solid box indicating the SV40 early promoter and the direction of transcription. The S1 nuclease probe yields two protected fragments; one corresponds to the RNA initiating from the SV40 early promoter (440 bases), and the other corresponds to the RNA initiating from the P2 promoter of the endogenous mouse c-myc gene (358 bases). H, HindIll; X, XhoI. (B) Suppression of collagen promoter activity requires the leucine zipper domain of c-Myc. Top: CAT activity in the parental cell line (pAZ1003.2) and in two cell lines expressing the mutant human c-myc gene D414-443 (pAZ1003.2.ml and pAZ1003.2.m2). Bottom: Quantification of D414-433 c-myc RNA by S1 nuclease analysis. Lane b, pAZ1003.2.ml cell RNA; lane c, pAZ1003.2.m2 cell RNA. Shown below the autoradiogram is a partial restriction map of the wild-type human c-myc plasmid pM21 (35), with the solid box indicating the MoMuLV LTR and the open box indicating human c-myc exon 1 sequences. Arrows indicate transcription initiation sites. The S1 nuclease probe yields two protected fragments; one corresponds to the RNA initiating from the MoMuLV LTR in pM21 (448 bases), and the other corresponds to the RNA initiating from the human c-myc P2 promoter in pM21 (349 bases). P, PvuII.

allow the c-Myc suppressing effect to be observed. As seen with stable cell lines, the presence of an active human c-Myc protein (pM21) suppressed the expression of pAZ1003E fivefold (Fig. 4A, compare lanes a and c or b and d). Furthermore, c-Myc did not prevent the induction of proalpha 2(I) collagen promoter activity by transforming growth factor-p (31) (compare lanes a and b), suggesting that these

FIG. 4. (A) Suppression of pAZ1003E expression in NIH 3T3 cells is an acute effect of c-Myc. Ten micrograms of pAZ1003E plasmid DNA was cotransfected with 20 ±Lg of either pM21 (a and b) or D414-433 (c and d) plasmid DNA into NIH 3T3 cells by the CaPO4-DNA precipitation method. Five hours after addition of the CaPO4-DNA precipitate, the medium was changed to Dulbecco's modified Eagle's medium containing 0.5% calf serum to ensure that endogenous c-Myc levels were kept low. Four hours later, half the dishes were either treated (+) or not treated (-) with 3.5 ng of transforming growth factor-p (31) per ml. Cells were harvested 38 h after addition of transforming growth factor-, and assayed for CAT activity. (B) Suppression of pAZ1003E expression in NIH 3T3 cells is dependent upon the concentration of c-Myc. Ten micrograms of each cat plasmid was cotransfected with increasing amounts of c-Myc plasmid (pM21) DNA as indicated. The total amount of plasmid DNA in each transfection was kept constant with pBR322 DNA. Following transfection, cells were maintained in 0.2% calf serum. Cells were harvested 48 h later and assayed for CAT activity. Symbols: 0, pAZ1003E; 0, pSV2cat; A, pLK454. The dashed line (*) represents the fold suppression of pAZ1003E divided by that of pSV2cat with nonsaturating amounts of pM21. This corrects for any effect of c-Myc on the SV40 enhancer contained in pAZ1003E and represents the net effect of c-Myc on collagen promoter activity. CAT activities were quantified by using the Betagen detector and are expressed relative to that in the transfections performed without pM21 DNA. The data were reproduced in three independent experiments.

two regulators of collagen transcription may act through

independent pathways. Figure 4B shows that suppression of pAZ1003E expression in NIH 3T3 cells was a function of the concentration of c-Myc. The sensitivity of the collagen promoter relative to that of other cellular and viral promoters to c-Myc levels was also examined in these experiments. At all nonsaturating concentrations of input c-Myc plasmid (pM21) DNA, pAZ1003E expression was two to five times more sensitive to the concentration of c-Myc than pSV2cat, which contains the SV40 early promoter and enhancer, and four to five times more sensitive than pLK454, which contains 3 kb of the human P-actin promoter. The greater sensitivity of pAZ1003E

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than of pSV2cat to c-Myc strongly argues that most of the effect of c-Myc on pAZ1003E expression was exerted through collagen promoter sequences and not through the SV40 enhancer. Cotransfection experiments with the mutant of c-Myc lacking the leucine zipper motif gave no suppression over all input DNA concentrations (data not shown). The results of this study clearly show that the pro-alpha 2(I) collagen promoter is a target of c-Myc action in 3T3-L1 cells. Several observations support this conclusion. (i) Transcription of the endogenous pro-alpha 2(I) collagen gene is suppressed by c-Myc, and this effect is alleviated by c-myc antisense RNA. (ii) Expression of a recombinant fusion gene under the transcriptional control of the pro-alpha 2(I) collagen promoter is suppressed by c-Myc in both stable and transient assays. (iii) Suppression of the fusion gene (this study) and the endogenous pro-alpha 2(I) collagen gene (unpublished data) requires the biochemical activity of c-Myc. There are many possible mechanisms by which c-Myc could suppress collagen gene transcription. This effect of c-Myc could be direct, either via binding to specific control elements in DNA or by physically interacting with nucleoprotein complexes that bind to DNA, or indirect, by causing a change in the biochemical activity of one or more transcription factors. In vitro DNA-binding assays have revealed that purified c-Myc binds to pro-alpha 2(I) collagen promoter sequences, but no specific binding was detected (unpublished data). Thus, we do not believe that c-Myc affects collagen transcription directly by making specific contacts with collagen transcriptional control sequences. However, it is noteworthy that two other collagen genes in addition to that for pro-alpha 2(I) collagen were also suppressed by high levels of c-Myc. The collagen genes have common transcriptional control elements, and it is possible that the effect of c-Myc is exerted through one of these elements. c-Myc also suppresses its own transcription (27, 28) and that of the fibronectin gene (unpublished data) and inhibits the induction of the mouse mammary tumor virus promoter by glucocorticoids in 3T3-L1 cells (unpublished data). All of the promoters for these genes contain a binding site for the cellular transcription factor CTF/NF-1 (16), and we have found that the DNA-binding activity of CTF/NF-1 is dramatically altered in 3T3-L1 cells expressing high levels of either c-Myc or N-Myc, whereas the DNA-binding activity of a CAAT box-binding protein (13, 14, 26), which also binds to the collagen promoter, is unaltered (unpublished data). Thus, it is possible that c-Myc affects gene transcription indirectly by causing changes in the activities of specific cellular transcription factors, such as CTF/NF-1. Confirmation of this intriguing possibility in 3T3-L1 cells awaits the completion of genetic experiments, which are in progress. We thank William Lee for helpful comments and critical readings of the manuscript. This work was supported by an NIH grant (CA51748) awarded to S.O.F. REFERENCES 1. Ariga, H., Y. Imamura, and S. Iguchi-Ariga. 1989. DNA replication origin and transcriptional enhancer in c-myc gene share the c-myc protein binding sequences. EMBO J. 8:4273-4279. 2. Bernards, R., S. Dessain, and R. Weinberg. 1986. N-myc amplification causes down-modulation of MHC class I antigen expression in neuroblastoma. Cell 47:667-674. 3. Blackwell, K., L. Kretzner, E. Blackwood, R. Eisenman, and H. Weintraub. 1990. Sequence-specific DNA binding by the c-Myc protein. Science 250:1149-1151.

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VOL. 11, 1991 26. Oikarinen, J., A. Hatamochi, and B. de Crombrugghe. 1987. Separate binding sites for nuclear factor 1 and a CCAAT DNA binding factor in the mouse pro alpha (I) collagen promoter. J. Biol. Chem. 262:11064-11070. 27. Penn, L., M. Brooks, E. Laufer, and H. Land. 1990. Negative autoregulation of c-myc transcription. EMBO J. 9:1113-1121. 28. Penn, L., M. Brooks, E. Laufer, T. Littlewood, J. Morgenstern, G. Evan, W. Lee, and H. Land. 1990. Domains of human c-Myc protein required for autosuppression and cooperation with ras oncogenes are overlapping. Mol. Cell. Biol. 10:4961-4966. 29. Prendergast, G., and M. Cole. 1989. Posttranscriptional regulation of cellular gene expression by the c-myc oncogene. Mol. Cell. Biol. 9:124-134. 30. Prochownik, E., and J. Kukowska. 1986. Deregulated expression of c-myc by murine erythroleukaemia cells prevents differentiation. Nature (London) 322:848-850. 31. Rossi, P., G. Karsenty, A. Roberts, N. Roche, M. Sporn, and B. de Crombrugghe. 1988. A nuclear factor 1 binding site mediates the transcriptional activation of a type I collagen promoter by

transforming growth

factor-P. Cell 52:405-414.

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32. Schimdt, A., P. Rossi, and B. de Crombrugghe. 1986. Transcriptional control of the mouse pro-alpha 2(I) collagen gene: functional deletion analysis of the promoter and evidence for cellspecific expression. Mol. Cell. Biol. 6:347-354. 33. Schmidt, A., C. Setoyama, and B. de Crombrugghe. 1985. Regulation of a collagen gene promoter by the product of viral mos oncogene. Nature (London) 314:286-289. 34. Southern, P., and P. Berg. 1982. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J. Mol. Appl. Genet. 1:327-341. 35. Stone, J., T. deLange, G. Ramsay, E. Jakobovits, J. M. Bishop, H. Varmus, and W. Lee. 1987. Definition of regions in human c-mvc that are involved in transformation and nuclear localization. Mol. Cell. Biol. 7:1697-1709. 36. Versteeg, R., I. Noordermeer, M. Kruse-Wolters, D. Ruiter, and P. Schrier. 1988. c-myc down-regulates class I HLA expression in human melanomas. EMBO J. 7:1023-1029.