Proc. Natl. Acad. Sci. USA Vol. 93, pp. 1308-1313, February 1996 Genetics

Antiproliferative properties of the USF family of helix-loop-helix transcription factors Xu LuO AND MICHiELE SAWADOGO* Department of Molecular Genetics, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030 Communicated by Phillip A. Sharp, Massachusetts Institute of Technology, Cambridge, MA, November 7, 1995 (received for review March 1, 1995)

ABSTRACT USF is a family of transcription factors USF may play an important role in the regulation of gene characterized by a highly conserved basic-helix-loop-helix- expression. However, other groups of bHLH-zip transcription leucine zipper (bHLH-zip) DNA-binding domain. Two differ- factors display DNA-binding specificities similar to those of ent USF genes, termed USF1 and USF2, are ubiquitously USF. These include Myc (11) and its DNA-binding partners expressed in both humans and mice. The USF1 and USF2 Max/Myn (12, 13) and Mad/Mxi (14, 15), as well as the TFE3 proteins contain highly divergent transcriptional activation family (16). It is therefore difficult to assess the direct involve- domains but share extensive homologies in the bHLH-zip ment of USF in the regulation of a particular gene, although region and recognize the same CACGTG DNA motifs. Al- the recent development of dominant-negative mutants of USF though the DNA-binding and transcriptional activities of may provide a means (17, 18). these proteins have been characterized, the biological function Although the DNA-binding and transcriptional activities of of USF is not well understood. Here, focus- and colony- USF have been characterized, its biological function remains formation assays were used to investigate the potential in- unclear. In contrast, substantial evidence, including the im- volvement of USF in the regulation of cellular transformation portance of the myc genes in cancer progression, points to a and proliferation. Both USF1 and USF2 inhibited the trans- role of Myc in the processes of cellular transformation, pro- formation of rat embryo fibroblasts mediated by Ras and liferation, and apoptosis (19). Interaction of c-Myc with Max, c-Myc, a bHLH-zip transcription factor that also binds which is also a bHLH-zip protein, results in the formation of CACGTG motifs. DNA binding was required but not fully heterodimers that bind DNA at CACGTG motifs and activate sufficient for inhibition of Myc-dependent transformation by transcription (12, 20, 21). This heterodimer formation is also USF, since deletion mutants containing only the DNA-binding required for the function of Myc in both cellular transforma- domains of USF1 or USF2 produced partial inhibition. While tion (21, 22) and apoptosis (23), strengthening the idea that the the effect of USF1 was selective for Myc-dependent transfor- biological activity of Myc results at least in part from its role mation, wHd-type USF2 exerted in addition a strong inhibition as a transcription factor. of ElA-mediated transformation and a strong suppression of In light of the structural similarities between USF and Myc, HeLa cell colony formation. These results suggest that mem- as well as their shared DNA-binding specificities, it is conceiv- bers of the USF family may serve as negative regulators of able that USF plays a role in the regulation of some of the same cellular proliferation in two ways, one by antagonizing the cellular processes in which Myc is involved. To gain insights transforming function of Myc, the other through a more into the biological function of USF, we carried out focus- and general growth-inhibitory effect. colony-formation assays to investigate the potential roles of USF in the regulation of cellular transformation and prolif- USF was originally identified as a cellular transcription factor eration. important for expression of the adenovirus major late pro- moter (1-3). Purification of human USF from HeLa cells METHODS revealed two different polypeptides with apparent molecular MATERIALS AND masses of43 and 44 kDa (4). These two forms of USF displayed Plasmid Constructs. Construction of the expression plas- identical DNA-binding specificities and transcriptional activ- mids pSV-USF1 and pSV-USF2, which contain, respectively, ities (5). Isolation of USF cDNA clones from both humans and the human USF1 or murine USF2 cDNA under the control of mice revealed that the 43- and 44-kDa USF polypeptides were the simian virus 40 early promoter, has been previously encoded by two different genes, now termed USFI and USF2, described (17). USFlAB was constructed by subcloning a PCR respectively (6-8). The USF1 and USF2 polypeptide are highly fragment spanning the region between residues 212 and the divergent in their N-terminal sequences but share a highly Sac I site at residue 293 of USFi into pSV-USF1 cut with Esp conserved C-terminal basic-helix-loop-helix-leucine zipper I (residue 203) and Sac I. USFlAN was generated by inserting (bHLH-zip) dimerization and DNA-binding domain (7). USF a PCR fragment spanning the region between residue 193 of binds DNA as a dimer at specific sites that are characterized USF1 and the Nsi I site of pSV-USF1 into the Nco I- and Nsi by a central CACGTG motif (1, 6). Homodimers of USF1 and I-cut pSV-USF2 plasmid. The resulting construct therefore USF2, as well as USF1-USF2 heterodimers, are present in contains at its N terminus the first 6 amino acid residues of various ratios in many different cell types (8). The transcrip- USF2 adjacent to the normal USF1 sequences between resi- tional activation domains identified in both USF1 and USF2 dues 193 and 310. Derivation of the USF2AB construct, which are located in the divergent N-terminal regions, suggesting that contains an internal deletion of amino acids 228-247 in the the cellular functions of the different USF family members basic region of USF2, has been previously reported (17). may not be completely redundant (9, 10, 47). Plasmid USF2AN, which expresses a USF2 protein lacking USF-binding sites have been identified in a variety of amino acids 7-231, was constructed by digestion of pSV-USF2 cellular and viral genes, including several tissue-specific genes with Sma I and Xho I, followed by Klenow end-filling and (see ref. 10 and references cited therein). This suggests that religation.

The publication costs of this article were defrayed in part by page charge Abbreviations: bHLH-zip, basic-helix-loop-helix-leucine zipper; payment. This article must therefore be hereby marked "advertisement" in REF, rat embryo fibroblast. accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 1308 Downloaded by guest on October 4, 2021 Genetics: Luo and Sawadogo Proc. Natl. Acad. Sci. USA 93 (1996) 1309

Other expression plasmids used in the focus-formation assay Table 1. Effects of USF on cellular transformation by Myc included pT24 (24), pElA (25), and pSV-c-myc-1 (26), which and Ras contain, respectively, the transforming c-Ha-Ras gene, the Number of foci adenovirus type 5 EJA gene, and the murine c-Myc cDNA. Focus-Formation Assay. Second-passage rat embryo fibro- Transfected plasmids Exp. 1 Exp. 2 Exp. 3 blast (REF) cells prepared from 12- to 13-day-old Sprague- Ras 0 ND ND Dawley rat embryos (1.2 x 106 cells per plate) were transfected Ras + USF1 0 ND ND by the calcium phosphate precipitation method. The following Ras + USF2 0 0 ND amounts of DNA were used, when indicated, for the various Ras + Myc 22 23 17 expression plasmids: S ,tg each for pT24 and pElA, and 6 ,tg Ras + Myc + USF1 0 0 4 each for pSV-c-myc-1, pSV-USF1, and pSV-USF2. The total Ras + Myc + USF2 0 2 0 amount of plasmid DNA in each transfection was kept con- Early-passage REF cells were transfected with combinations of stant at 20-25 ,tg by addition of pSG5 vector (Stratagene). The plasmids encoding the various proteins indicated, and the cultures precipitates were washed 12 h after transfection, and 24 h later were monitored for the appearance of foci of morphologically trans- the cells from each plate were expanded onto three plates. Foci formed cells. The numbers shown are the sum of transformed foci were scored 9-12 days after transfection by visual inspection scored for each transfection on all three plates. ND, not determined. and microscopic verification, after which the plates were stained with crystal violet. For establishing stable cell lines, on each plate (15-25 per transfection). In contrast, no foci individual foci were seeded into 24-well plates. Each cell line were observed on either the USF1- or USF2-transfected was subsequently expanded into 5-cm-diameter dishes, and plates. Also, consistent with previous studies (28), Myc was mini nuclear extracts were prepared as described (27). absolutely required for transformation by Ras in this assay, Transient Transfection Assays. REFs (1.2 x 106 cells per since cells transfected with Ras alone did not produce foci. plate) were transfected by the calcium phosphate precipitation Taken together, these initial results indicated that, despite method with a total of 20 ,ug of plasmid DNA including 6 Ag their structural and functional similarities, USF and Myc did of the specified expression plasmid. The precipitates were not play interchangeable roles in the cellular transformation washed with sodium phosphate-buffered saline (pH 7.1) 12 h assay. after transfection and the cells were harvested 24-30 h later. An alternative possibility, also suggested by the similar Whole cell extracts were prepared by lysing the cells in 250 mM DNA-binding specificities displayed by USF and Myc in vitro, Tris HCl buffer (pH 7.9) by four cycles of freezing and thawing. is that USF competes in vivo for the binding of Myc to the DNA Analysis of Ectopic USF Expression. For the electrophoretic and thereby impedes its action. If this were true, one would mobility shift assays, a double-stranded oligonucleotide with predict that USF overexpression would inhibit the transform- the sequence 5'-CTGAATTCCTGGTCACGTGACCG- ing ability of Myc. We therefore carried out additional exper- CAGCTGT-3', which includes the consensus USF-binding iments in which the USF-expressing plasmids were cotrans- site, was labeled with 32P by Klenow end-filling. The DNA- fected with the Myc and Ras plasmids. As shown in Table 1, binding reaction mixtures (15 ,l) contained 10 mM Tris-HCl addition of either USF1 or USF2 almost entirely prevented the (pH 7.8), 50 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 2.5 appearance of transformed foci. This result demonstrated that ,ug of poly(dIldC), 3.3% (vol/vol) glycerol, 0.3 ng of probe, and USF somehow antagonized the combined transforming activ- 1.4 ,tg of protein from either whole cell extracts or mini nuclear ity of Myc and Ras. As mentioned, this inhibitory effect of USF extracts. After 20-min incubation at room temperature, the could be due to a direct competition for the binding of Myc to reaction mixtures were supplemented with 3 ,tl of Ficoll and the DNA. However, the interference exerted by USF could analyzed by electrophoresis on a 4% acrylamide/0.2% bis- also be indirect, perhaps reflecting a functional antagonism. acrylamide/22 mM Tris borate, pH 8.3/0.5 mM EDTA (0.25 x Functional Domains of USF Required for Inhibition of TBE) gel. For Western blot analysis, 7.2 jig of proteins from Myc-Dependent Transformation. If the inhibitory effect of whole cell extracts was resolved by SDS/PAGE on 12% USF on Myc-dependent transformation were due solely to a polyacrylamide gels, transferred to nitrocellulose filters, and competition between USF and Myc for common DNA-binding probed with USF1- (Santa Cruz Biotechnology) and USF2- sites, for instance sites located in the control region of one or specific antisera (7). several growth-related genes, two predictions can be made. Colony Formation Assay. HeLa cells were plated on 10-cm First, USF mutants defective in DNA binding should not diameter dishes (5 x 105 cells per plate) and transfected the inhibit transformation. Second, all USF mutants that retain following day with 6 j,g of the specified plasmid DNA, 12 ,ug full DNA-binding function should inhibit transformation as of pSG5, and 2 ,tg of pSV2neo. After 3 weeks of selection in efficiently as the wild-type USF proteins. We therefore inves- G418 (400 ,ug/ml), resistant colonies were stained with crystal tigated the effect of different USF mutants on cellular trans- violet and counted. formation by Myc and Ras (Fig. 1). DNA-binding-deficient forms of USF1 and USF2 were constructed by introducing small internal deletions into the basic region, which is essential RESULTS for specific DNA recognition (mutants USFlAB and USF2AB, Both USFI and USF2 Can Inhibit Cellular Transformation Fig. 1A). Two other mutants, termed USFIAN and USF2AN, by Myc. Given the structural resemblance between USF and were engineered to contain all of the amino acid sequences of Myc and their similar DNA-binding specificities, we first either USF1 or USF2 minimally required for efficient dimer- considered the possibility that these two families of proteins ization and DNA binding but none of the N-terminal regions could have overlapping functions in cellular transformation. involved in transcriptional activation (6-10, 47). The expres- Since c-Myc has been shown to cooperate with mutant Ras to sion of these various mutants in REFs was first investigated by transform REF cells (28), we began by investigating the effect Western blot analysis using whole cell extracts prepared from of USF in the same focus-forming assay. Early-passage REFs REF cells transiently transfected with the different expression were transfected with expression vectors for USF1, USF2, or vectors. This analysis revealed that all of the USF1 and USF2 Myc, together with a Ras-expressing plasmid. These cultures mutants were expressed efficiently and at a level similar to that were then monitored for the appearance of foci of morpho- of the wild-type proteins (Fig. 1B). The subcellular localization logically transformed cells (Table 1). As expected, cellular of the mutant proteins was investigated by indirect immuno- transformation took place following cotransfection of the Myc fluorescence staining of the transfected REFs with USF1- or and Ras plasmids, resulting in the appearance of multiple foci USF2-specific antibodies. As expected from the known loca- Downloaded by guest on October 4, 2021 1310 Genetics: Luo and Sawadogo Proc. Natl. Acad. Sci. USA 93 (1996) A B DNA binding L Transcri pti on 0 cn 4-) C\N C\)V) activation Dimerization L.L UL- LL. (L) V/) (C) e > < > =, b HLH LZ -~l*.Il\ _ US F1 ==:k) USFIAB IJ1SFlAN US F?^ FIG. 1. Effects of various USF proteins on ~XUSF2 c-Myc-dependent cellular transformation. (A) Schematic representation of the struc- L ==:3* USF2AB tures of USF1, USF2, and their deletion mu- tants with the location of functional domains 0 _- USF2AN indicated at the top (b, basic region; HLH, helix-loop-helix; LZ, leucine zipper). (B) De- tection of ectopic USF expression in REFs by C D Western blot analysis. REFs were transiently Z co transfected with the indicated plasmids and o < < whole 4-) C\N C\) C\) the overexpressed proteins present in ULL) L- U- LL. LL. .L cell extracts were detected with USFI- and a) V) L U) V, U- U-C/) s_> zD CD C/D USF2-specific antisera. (C) Electrophoretic mobility-shift assay for DNA binding by the ectopically expressed USF proteins. The same 0 cellular extracts used in B were analyzed for 4- USF DNA-binding activity, using as the ra- dioactive probe an oligonucleotide containing ~04a) the USF consensus binding site. (D) Summary of the results obtained in the focus-formation -0 assay. Shown are the percentage of REF foci ~0aL) transformed by Myc and Ras in the presence I-, of various USF constructs relative to the number of foci obtained in the absence of USF. For each construct, the averages and standard deviations (error bars) were calcu- Cotransfected plasmid lated from 3-8 independent experiments.

tion of nuclear localization signals in USF2 (47), these exper- Ras-expressing plasmids induced frequent transformation iments revealed identical nuclear localization patterns for all of events, with 40-150 foci observed in each transfection. Inter- the mutant and wild-type proteins (data not shown). Finally, estingly, addition of the same USFI expression vector that the DNA-binding abilities of the different proteins in REFs abolished Myc-dependent transformation affected only were measured by electrophoretic mobility-shift assays. This slightly the appearance of ElA-transformed foci (less than analysis confirmed that the N-terminally truncated mutants 30% inhibition). Cotransfection of the USF1zIB and USF1AN (USFIAN and USF2AN) bound DNA as efficiently as the mutants also had little effect on ElA-dependent transforma- wild-type proteins, while the basic region deletion mutants tion (Fig. 2A). (USFIAvB and USF2AB) were totally inactive in DNA binding To verify that the inability of USFI to inhibit the growth of (Fig. 1C). ElA-transformed foci was not due to a lack of expression of Cotransfection in REFs of either USFIAB or USF2AB with the USFI protein in the ElA/Ras/USFI-transfected cells, we the Myc and Ras expression vectors had basically no effect on expanded several of these particular foci. Overexpression of focus formation (Fig. ID), demonstrating that the DNA- USFI in these various cell lines was evidenced by a significant binding function of USF was essential for the inhibition of increase in the overall USF DNA-binding activity detectable in Myc-dependent transformation. This result also indicated that nuclear extracts (Fig. 2B). As compared with control foci the inhibition was probably not due to a nonspecific effect lacking the USFI expression vector, a 2.5- to 4.2-fold increase caused by the overexpression of USF RNAs or proteins. In in USF activity was observed. This result demonstrated that contrast to the result obtained with the basic region deletions, USF1 could indeed be overexpressed in ElA-transformed cells cotransfection of either USF1AN or USF2AN yielded a sig- without significantly affecting their proliferative properties. It nificant decrease in the number of Myc-transformed foci (Fig. also indicated that the inhibitory effect of USFI on cellular ID). However, this inhibition was clearly not as complete as it transformation was probably not a general phenomenon. was with the wild-type USF proteins, indicating that the Instead, USF1 seemed capable of selectively repressing Myc- domains of USF involved in transcriptional control also played dependent transformation, but not ElA-dependent transfor- an important role. Thus it would seem that, to efficiently mation. inhibit Myc-dependent transformation, USF must function not In contrast to the selectivity displayed by USFI, ectopic only as a DNA-binding protein but also as a transcription expression of wild-type USF2 abolished focus formation in factor. REFs not only in the case of Myc-dependent transformation Effects of Various USF Proteins on ElA-Dependent Trans- but also in the case of the transformation induced by EIA and formation. To assess the specificity of the suppression by USF Ras (Fig. 2A) or mutant p53 and Ras (data not shown). of Myc-dependent cellular transformation, we next investi- Inhibition of ElA-dependent transformation seemed to re- gated the effects of various USF proteins on cellular transfor- quire both the DNA-binding and transcriptional activation mation mediated by the adenovirus-5 ElA oncoprotein in functions of USF2, since neither the USF2AB nor the USF2zAN cooperation with Ras. The results of this experiment are shown mutant exhibited a similar suppressive effect (Fig. 2A). These in Fig. 2. As expected, cotransfection in REFs of the EIA- and experiments suggested, therefore, that the wild-type USF2 Downloaded by guest on October 4, 2021 Genetics: Luo and Sawadogo Proc. Natl. Acad. Sci. USA 93 (1996) 1311

FIG. 2. Effects of various USF proteins on A B ElA foci ElA-mediated cellular transformation. (A) Focus- - - + + + + (USF1) formation assay in REFs transfected with the c-Ha-ras and adenovirus-5 EIA genes and the C- indicated USF expression vectors. For each con- struct, the results from 3-5 independent experi- ments were plotted as described for Fig. 1D. In the absence of USF, the total number of foci ranged 0 from 40 to 150. (B) Ectopic expression of USF1 in 4- :gu 4-) ElA-transformed cells. Foci of REF cells trans- a) formed by ElA and Ras in the presence or absence -0 of USF1 were individually picked and expanded. Mini nuclear extracts (28) were prepared from the (D resulting cell lines and analyzed for USF DNA- LU binding activity by electrophoretic mobility-shift assay using in each case 1.4 jig of protein per reaction. Lanes 1 and 2, control clones from EIA/ Ras transfections. Lanes 3-6, clones from ElA/ Cotransfected plasmid Ras/USF1 cotransfections.

protein may affect pathways that are not strongly affected by type USF2 was coexpressed with the USF2zXB and USF2AN the USF1 protein. mutants. Since both of these mutant proteins are fully active Growth Inhibition by USF2 in a Transformed Cell Line. The in dimerization, but otherwise inactive in either DNA-binding strong inhibition by USF2 of both Myc- and ElA-mediated or transcriptional activation functions, they can actually serve transformation raised the possibility that this transcription as dominant-negative mutants of USF (17, 18, 47). If the factor could exert a general repressive effect on cellular growth inhibition exerted by USF2 requires a functional growth. To explore this possibility, we used a colony-formation protein, addition of either one of these dominant-negative assay, which allowed us to investigate the effect of USF2 in an mutants would be expected to alleviate the inhibition. The entirely different cell type. USF expression vectors were results of such an experiment are depicted in Fig. 4, where the transfected in HeLa cells together with a plasmid containing same amount of USF2 expression plasmid used in the previous a neomycin-resistance gene (pSV2neo) as selectable marker, experiments was cotransfected with the pSV2neo plasmid and and the number of G418-resistant colonies that formed in each either USF2AB or USF2/N. In both cases, the growth inhi- case was determined. The result of this experiment is shown in bition observed with USF2 alone was partially relieved. The Fig. 3. While expression of either USF1 or USF2AB caused a plating ability increased from 8% of the control in the case of slight inhibition in this assay, none of the other USF1- or USF2 alone to 30% and 45% following addition of USF2AN USF2-derived mutants had any significant effect on the num- and USF2AB, respectively. These results indicate that a high ber of drug-resistant colonies observed. In contrast, ectopic level of the wild-type USF2 polypeptide is by itself insufficient expression of wild-type USF2 inhibited the colony-forming for growth inhibition. Instead, it would seem that USF2 must more This result be present in an active form, in terms of both its DNA-binding ability of HeLa cells than 10-fold (Fig. 3). and transcriptional activation functions, in order to exert its strengthens the idea that USF2 may indeed inhibit growth in effect on HeLa cells. different types of cells. antiproliferative A concern in these experiments is that the USF expression level in the transfected cells may be considerably higher than DISCUSSION the physiological levels. It was therefore important to inves- The USF proteins have been well characterized for their tigate whether the presence of high levels of USF2 in cells was DNA-binding and transcriptional activation properties, but sufficient to cause growth inhibition, or whether a functional their biological functions remain largely unexplored. Here, we USF2 protein was actually required. To address this question, used focus- and colony-formation assays to investigate the transfection experiments were performed in which the wild- L- 0 4.) co 8 ~~~~< C. C\J a,) LL 4-) >) L L.. + 4.)C-) .00) C\Ji Ui- U_. C.. C') 0100 ,~~~~~~~~C' ,1 0 0) __V) a C.) 0 050 0 0 Cotransfected plasmids U- a,~~~~~~V FIG. 4. Dominant-negative mutants of USF can relieve the growth 0 inhibition by USF2. HeLa cells were transfected, as indicated, with 6 Cotransfected plasmiid jig of USF2 expression vector (same amount as in Fig. 3) and 10 jig of the plasmids encoding the dominant-negative mutants USF2AN or FIG. 3. Colony-formation assay in HeLa cells. HeLa cells were USF2AB. The height of each bar represents the number of colonies transfected with 2 jig of pSV2neo, 12 ,rg of pSG5, and 6 jig of the observed in each transfection, expressed as percentages relative to the indicated USF expression plasmids. After 3 weeks of selection in G418, number of colonies obtained with transfection of the parent pSG5 resistant colonies were scored. The results shown are the averages and vector alone. The results were averaged from three separate experi- standard deviations of three or more independent experiments. ments. Downloaded by guest on October 4, 2021 1312 Genetics: Luo and Sawadogo Proc. Natl. Acad. Sci. USA 93 (1996) potential involvement of USF in cellular growth and transfor- type. In this respect, the development of USF-null mice could mation. Taken together, the results of this initial set of prove very useful in determining the biological function of experiments indicate that the USF proteins participate in the USF. control of cellular proliferation in more than one way. First, we Our experiments revealed that, besides its effect on Myc- found that the activity of both USF1 and USF2 can somehow dependent transformation, the USF2 protein could also inhibit counteract the transforming ability of Myc. Given the notice- the proliferation of different types of cells. As mentioned able similarity of the preferred DNA-binding sites of Myc and above, this growth inhibition by USF2 may be due to the USF, this functional competition could be due in part to a activation by this transcription factor of one or several other physical competition between the two transcription factors for genes involved in cell cycle control or suppression of trans- some specific sites on the DNA. On the other hand, we found formation. The observation is not entirely surprising, since evidence that the USF2 protein may also possess a more there is increasing evidence for the ability of many transcrip- general growth-inhibitory activity, which could be observed in tion factors to negatively regulate growth. Both MyoD and different types of cells (e.g., ElA-transformed REFs or HeLa C/EBPa have been shown to inhibit cellular proliferation cells). independently of their differentiating activities (38, 39). JunD Further understanding of the exact mechanisms by which was found to negatively regulate fibroblast growth and inhibit these various effects of USF1 and USF2 take place may require transformation by Ras (40). Several bHLH proteins, including a more complete knowledge of the genes that are the key the myogenic transcription factors and the ubiquitous E2A targets of this family of transcription factors. In this respect, it proteins, have been shown to inhibit growth by affecting the is interesting to note that several of the putative USF target cell cycle progression from G1 to S (41, 42). In the case of genes are themselves involved in the control of cellular pro- MyoD, withdrawal from the cell cycle and arrest in Go was liferation and differentiation. Among these are the p53 tumor correlated with the induction by this transcription factor of the suppressor gene (29), several cyclin-encoding genes (30, 31), cyclin-dependent kinase inhibitor p21 (43). Because our evi- and the differentiation-inducing transcription factor C/EBPa dence for a growth inhibition by USF2 is based on overex- (32, 33). It is also interesting that USF1 and USF2 displayed pression, the possibility of an antiproliferative role of this different behaviors in some of our assays, which indicates that transcription factor at physiological concentrations remains to the functions of these two proteins may not be interchangeable be further substantiated. However, such a role would be and that they may have different targets. consistent with the observation that USF2 homodimers are The possibility that USF and Myc may compete for some essentially absent from most rapidly proliferating cells, includ- common binding sites in vivo is suggested by several different ing HeLa (8). types of studies. These include not only experiments that One noticeable difference between USF2 and other bHLH demonstrated the similarity of the DNA-binding specificities proteins implicated in growth control is that the activity of of Myc and USF in vitro (34) and the ability of the two these other transcription factors can be antagonized by the transcription factors to transactivate the same reporter genes formation of heterodimers with the DNA-binding-deficient Id in transient transfection assays (32) but also in vivo studies in proteins (44). In contrast, the presence of a leucine zipper in yeast. A null mutant of the yeast centromere-binding protein USF is known to prevent its dimerization with other HLH CBP1, another member of the bHLH family, could be rescued proteins, including Id (45). One would therefore predict that, by CBP1 mutants whose basic domain had been replaced with if the activity of USF had to be down-regulated in actively either the USF1 or the c-Myc basic domain, but not by a growing cells, this regulation would take place by mechanisms mutant containing the PH04 basic domain (35). This result other than heterodimerization with Id-like proteins. Such clearly illustrates the structural and functional similarities mechanisms may include posttranslational modifications, al- between the basic domains of Myc and USF. Other studies ternative splicing (6, 10, 47), or interactions with regulatory have already shown that proteins that can compete for the factors outside of the HLH family (46). binding of Myc to the DNA, including Max, Mad, and Mxil, also inhibit cellular transformation by Myc (22, 36). Similarly, We are grateful to G. Lozano and C. V. Dang for advice on setting a hybrid that contained the basic domain of Myc and the up the focus-formation assay. We also thank M. N. Szentirmay and dimerization domain of E12, which could bind the CACGTG M. W. Van Dyke for many useful discussions and critical reading of the motif in vitro, effectively inhibited Myc-dependent transfor- manuscript. This work was supported by National Institutes of Health mation, presumably by interacting with the Myc-binding sites Grant GM38212. in vivo (37). Competition for common binding sites in vivo is therefore an attractive model to explain the inhibition of 1. Sawadogo, M. & Roeder, R. G. (1985) Cell 43, 165-175. Myc-dependent transformation observed here with USF1 or 2. Carthew, R. W., Chodosh, L. A. & Sharp, P. A. (1985) Cell 43, USF2 and their minimal DNA-binding domains. Yet the 439-448. antagonism between USF and Myc may entail additional 3. Miyamoto, N. G., Moncollin, V., Egly, J. M. & Chambon, P. mechanisms besides a direct DNA-binding competition at (1985) EMBO J. 4, 3563-3570. 4. Sawadogo, M., Van Dyke, M. V., Gregor, P. D. & Roeder, R. G. E-box sequences. For instance, opposite effects of Myc and (1988) J. Biol. Chem. 263, 11985-11993. USF on gene transcription were shown to take place through 5. Sawadogo, M. (1988) J. Biol. Chem. 263, 11994-12001. the core promoter of genes that contain an initiator element 6. Gregor, P. D., Sawadogo, M. & Roeder, R. G. (1990) Genes Dev. (32). 4, 1730-1740. A competition model, whether direct or indirect, can ac- 7. Sirito, M., Walker, S., Lin, Q., Kozlowski, M. T., Klein, W. H. & commodate most of our results on the inhibition of Myc- Sawadogo, M. (1992) Gene Exp. 2, 231-240. dependent transformation by USF. However, the system that 8. Sirito, M., Lin, Q., Maity, T. & Sawadogo, M. (1994) Nucleic was used in these studies depends on the overexpression of Acids Res. 22, 427. USF to levels that may be much higher than the physiological 9. Kirschbaum, B. J., Pognonec, P. & Roeder, R. G. (1992) Mol. relevant to the Cell. Biol. 12, 5094-5101. levels. Are our results then physiological 10. Lin, Q., Luo, X. & Sawadogo, M. (1994) J. Biol. Chem. 269, situation? We would suggest that, in normal cells, USF could 23894-23903. inhibit some of the activities of Myc, therefore protecting the 11. Blackwell, R. K., Kretzner, L., Blackwood, E. M., Eisenman, cells against its oncogenic potential. In contrast, when Myc R. N. & Weintraub, H. (1990) Science 250, 1149-1152. expression becomes deregulated, this safeguard mechanism 12. Blackwood, E. & Eisenman, R. N. (1991) Science 251, 1211-1217. may be overcome by the excess of Myc protein, which would 13. Prendergast, G. C., Lawe, D. & Ziff, E. B. (1991) Cell 65, then contribute to the development of the oncogenic pheno- 395-407. Downloaded by guest on October 4, 2021 Genetics: Luo and Sawadogo Proc. Natl. Acad. Sci. USA 93 (1996) 1313 14. Ayer, D. E., Kretzner, L. & Eisenman, R. N. (1993) Cell 72, 32. Li, L. H., Nerlov, C., Prendergast, G., MacGregor, D. & Ziff, 211-222. E. B. (1994) EMBO J. 13, 4070-4079. 15. Zervos, A. S., Gyuris, J. & Brent, R. (1993) Cell 72, 223-232. 33. Timchenko, N., Wilson, D. R., Taylor, L. R., Abdelsayed, S., 16. Beckman, H., Su, L. K. & Kadesh, T. (1990) Genes Dev. 4, Wilde, M., Sawadogo, M. & Darlington, G. J. (1995) Mol. Cell. 167-179. Biol. 15, 1192-1202. 17. Meier, J. L., Luo, X., Sawadogo, M. & Straus, S. E. (1994) Mol. 34. Fisher, D. E., Parent, L. A. & Sharp, P. A. (1993) Cell 72, Cell. Biol. 14, 6896-6906. 467-476. 18. Lefrancois-Martinez, A. M., Martinez, A., Antoine, B., Ray- 35. Dang, C. V., Dolde, C., Gillison, M. L. & Kato, G. J. (1992) Proc. mondjean, M. & Kahn, A. (1995) J. Biol. Chem. 270, 2640-2643. Natl. Acad. Sci. USA 89, 599-602. 19. Marcu, K. B., Bossone, S. A. & Patel, A. J. (1992) Annu. Rev. Biochem. 61, 809-860. 36. Lahoz, E. G., Xu, L., Schreiber-Agus, N. & De Pinho, R. A. 20. Amati, B., Dalton, S., Brooks, M. W., Littlewood, T. D., Evan, (1994) Proc. Natl. Acad. Sci. USA 91, 5503-5507. G. I. & Land, H. (1992) Nature (London) 359, 423-426. 37. Prendergast, G. C. & Ziff, E. B. (1991) Science 251, 186-189. 21. Amati, B., Brooks, M. W., Levy, N., Littlewood, T. D., Evan, G. I. 38. Sorrentino, V., Pepperkok, R., Davis, R. L., Ansorge, W. & & Land, H. (1993) Cell 72, 233-245. Philipson, L. (1990) Nature (London) 345, 813-815. 22. Makela, T. P., Koshinen, P. J., Vastrik, I. & Alitalo, K. (1992) 39. Umek, R. M., Friedman, A. D. & McKnight, S. L. (1991) Science Science 256, 373-377. 251, 288-292. 23. Amati, B., Littlewood, T. D., Evan, G. I. & Land, H. (1993) 40. Pfarr, C. M., Mechta, F., Spyrou, G., Lallemand, D., Carillo, S. EMBO J. 12, 5083-5087. & Yaniv, M. (1994) Cell 76, 747-760. 24. Goldfarb, M., Shimizu, K., Perucho, M. & Wigler, M. (1982) 41. Crescenzi, M., Fleming, T. P., Lassar, A. B., Weintraub, H. & Nature (London) 296, 404-409. Aaronson, S. A. (1990) Proc. Natl. Acad. Sci. USA 87,8442-8446. 25. Hearing, P. & Shenk, T. (1985) Mol. Cell. Biol. 5, 3214-3221. 42. Peverali, J. A., Ramqvist, T., Saffrich, R., Pepperkok, R., Barone, 26. Land, H., Chen, A. C., Morgenstern, J. P., Parada, L. F. & M. V. & Philipson, L. (1994) EMBO J. 13, 4291-4301. Weinberg, R. A. (1986) Mol. Cell. Biol. 6, 1917-1925. 43. Halevy, O., Novitch, B. G., Spicer, D. B., Dkapek, S. X., Rhee, J., 27. Schreiber, E., Matthias, P., Muller, M. M. & Schaffner, W. (1989) D. & A. B. Science 267, Nucleic Acids Res. 17, 6419. Hannon, G. J., Beach, Lassar, (1995) 28. Land, H., Parada, L. F. & Weinberg, R. A. (1983) Nature (Lon- 1018-1021. don) 304, 596-602. 44. Benezra, R., Davis, R. L., Lockshon, D., Turner, D. L. & Wein- 29. Reisman, D. & Rotter, V. (1993) Nucleic Acids Res. 21, 345-350. traub, H. (1990) Cell 61, 49-59. 30. Philip, A., Schneider, A., Vasrik, I., Finke, K., Xiong, Y., Beach, 45. Sun, X. H., Copeland, N. C., Jenkins, N. A. & Baltimore, D. D., Alitalo, K. & Eilers, M. (1994) Mol. Cell. Biol. 14,4032-4043. (1991) Mol. Cell. Biol. 11, 5603-5611. 31. Cogswell, J. P., Godlevski, M. M., Bonham, M., Bisi, J. & Babiss, 46. Blanar, M. A. & Rutter, W. J. (1992) Science 256, 1014-1018. L. (1995) Mol. Cell. Biol. 15, 2782-2790. 47. Luo, X. & Sawadogo, M. (1996) Mol. Cell. Biol., in press. Downloaded by guest on October 4, 2021