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.