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DNA Binding of USF Is Required for Specific E-Box Dependent Gene Oncogene (1999) 18, 7200 ± 7211 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $15.00 http://www.stockton-press.co.uk/onc DNA binding of USF is required for speci®c E-box dependent gene activation in vivo Astrid Kiermaier1,5, Jonathan M Gawn2,5, Laurie Desbarats1, Rainer Sarich3, Wilhelm Ansorge3, Paul J Farrell2,4, Martin Eilers1 and Graham Packham*,2,4 1Institute for Molecular Biology and Tumour Research, University of Marburg, Emil-Mannkop-Str 2, 35033 Marburg, Germany; 2Ludwig Institute for Cancer Research, Imperial College School of Medicine, St. Mary's Campus, Norfolk Place, London, W2 1PG, UK; 3Biochemical Instrumentation Programme, EMBL, Meyerhofstr 1, 69117 Heidelberg, Germany; 4Virology and Cell Biology, Department of Medical Microbiology, Imperial College School of Medicine, St. Mary's Campus, Norfolk Place, London, W2 1PG, UK Although USF-1 and -2 are the major proteins that bind Repression requires an amino-terminal domain con- to Myc-regulated E-box (CACGTG) elements in many served in Mad-like proteins that recruits histone cells, there is no clear role for USF during Myc- deacetylases via an adapter protein, sin3 (e.g. Alland dependent gene regulation. Using dominant negative et al., 1997; Laherty et al., 1997; Sommer et al., 1997). alleles of USF-1 we now show that DNA binding by Little doubt exists that Myc exerts at least part of its USF at a Myc-regulated E-box limits the ability of biological eects through the transcriptional activation another E-box binding factor, TFE-3, to activate a of speci®c target genes although Myc has other target gene of Myc in vivo and to stimulate S phase functions that may contribute to its biological proper- entry in resting ®broblasts. Similarly, dominant negative ties (Xiao et al., 1998). Indeed, several target genes of alleles of USF-1 relieve the restriction that prevents Myc are known that can account for some of the activation of the IgH enhancer by TFE-3 in non B-cells. biological properties of Myc (for review, see Grandori DNA binding activity of USF complexes is abundant in et al., 1997). primary human B-cells and is signi®cantly downregulated A number of transcription factors closely related to during B-cell immortalization. Re-expression of USF-1 Myc also bind to CAC(A/G)TG sequence elements and in immortalized B-cells retards proliferation. Our data activate transcription; examples include USF-1 and -2 establish an essential role for USF in restricting E-box (e.g. Sirito et al., 1998), TFE-3 (Beckmann et al., 1990) dependent gene activation in vivo and suggest that this and TFEB (Fisher et al., 1991). Fusion of the amino- control is relaxed during cellular immortalization. terminal repression domain of Mad-1 to TFEB generates a protein that is capable of suppressing Keywords: Myc; Max; USF; prothymosin-a; IgH cellular transformation by Myc, demonstrating that enhancer; EBV these proteins have overlapping binding sites on DNA in vivo (Harper et al., 1996). Despite their biochemical similarities, the biological functions of the dierent E- box binding factors dier widely. For example, ectopic Introduction expression of Myc, but not USF-1 or TFE-3, induces proliferation and apoptosis in RAT1 ®broblasts (for The proto-oncogene c-myc encodes a transcription review, see Bouchard et al., 1998) (A Kiermaier, factor of the helix ± loop ± helix/leucine zipper family. unpublished). Conversely, inhibition of Myc function The gene product, Myc, binds to speci®c DNA often causes an inability of cells to proliferate but loss sequences termed E-boxes with a central CAC(A/ of TFE-3 function in B-cells causes a defect in G)TG sequence as part of a heterodimeric complex activation, but not proliferation (Merrell et al., 1997). with a partner protein, Max. The heterodimeric Most likely, therefore, mechanisms exist that discrimi- complex is a potent activator of transcription due to nate between these closely related factors during activation domains located in the amino-terminus of activation of endogenous target genes. the Myc protein. Max also heterodimerizes with a Several genes have been identi®ed that are second group of related proteins termed Mad-1, Mxi-1, speci®cally activated by single members of this family Mad-3, Mad-4 and Mnt/Rox: these complexes bind to of transcription factors. Examples include prothymo- the same sequences on DNA but, in contrast to Myc/ sin-a (Desbarats et al., 1996), ornithine decarboxylase Max complexes, generally repress transcription (for a (ODC) (Tobias et al., 1995) and CAD (Boyd et al., detailed review, see Henriksson and LuÈ scher, 1996). 1998) that are speci®cally activated by Myc, and the immunoglobulin (IgH) enhancer which is transacti- vated by TFE-3 only in B-cells (Carter et al., 1997). Speci®c mutations in the prothymosin-a gene allow activation by TFE-3; similarly, mutations in the IgH *Correspondence: G Packham, Virology and Cell Biology, enhancer allow activation by TFE-3 in non B-cells, Department of Medical Microbiology, Imperial College School of demonstrating that TFE-3 is actively prevented from Medicine, St. Mary's Campus, Norfolk Place, London, W2 1PG, UK 5The ®rst two authors contributed equally to the paper activating these genes in vivo (Desbarats et al., 1996; Received 30 March 1999; revised 12 August 1999; accepted 16 Genetta et al., 1994). We now demonstrate that DNA August 1999 binding by USF is required for inhibiting transactiva- USF restricts E-box dependent gene activation AKiermaieret al 7201 tion by TFE-3 in vivo. Our results de®ne a novel and cells in addition to the known binding sites for Myc/ unexpected role for the transcription factor USF in Max and AP-2 proteins (see Gaubatz et al., 1995 and restricting inappropriate activation of E-box dependent Figure 2a); in particular, the E2 element is protected by gene activation in vivo and suggest that this control is nuclear extracts, but not by USF-1, AP-2 or Max. relaxed during cellular immortalization. One potential mechanism by which factors binding to E2 could restrict activation by TFE-3 is by stabilizing cellular factors that bind to the TFE-3/ Results Myc/USF-binding site. Since USF proteins constitute the most abundant E-box binding activity, we Previous work has shown that ectopic expression of speculated that USF may have a function in TFE-3 failed to activate the IgH or prothymosin-a restricting activation of target genes of Myc. To test enhancers; in both cases, a mutation in a second E-box the hypothesis that USF interacts with a Myc-activated element close to the TFE-3 binding site relieved the E-box in vivo, chromatin immune-precipitation experi- restriction, suggesting that factors binding to these ments were carried out. RAT1-MycER cells were elements actively prevent transcriptional activation by harvested and proteins cross-linked to DNA by TFE-3 (Desbarats et al., 1996; Genetta et al., 1994) exposure to UV-light. Cross-linked DNA was im- (Figure 1a). In order to test whether this restriction mune-precipitated with anti-Myc, Max or USF targets the transcriptional activation domain of TFE-3, antibodies, or as control, protein-A-sepharose alone. we generated a chimeric protein that carries the The immune-precipitates were subjected to a polymer- transactivation domain of the viral Vp16 protein and ase-chain reaction using nested primers that amplify a a nuclear localization signal of SV40 large T antigen fragment surrounding E1 and E2 (Figure 2b). We fused to the open reading frame of TFE-3 (Figure 1b). detected binding of all three proteins, Myc, Max and Plasmids encoding this chimera under the control of USF to the genomic fragment containing the the CMV immediate early promoter were transfected prothymosin-a TFE-3/Myc/USF binding site in vivo. into HeLa cells together with a reporter plasmid that Similar results have recently been obtained for the carries four E-box elements in front of a minimal Myc-regulated E-Box of the CAD gene (Boyd et al., promoter ((E-box)4-tkluc). Vp16-TFE-3 strongly acti- 1998). vated this promoter (Figure 1b); a mutant derivative, To assess the potential function of USF, we in which the E-boxes had been altered to CACTCA, generated dominant negative alleles that interfere with (E-boxmut)4-tkluc, was not activated by Vp16-TFE-3. DNA binding by USF and by Max (Figure 3a). For In contrast, Vp16-TFE-3 was unable to activate a each protein, the amino-terminus up to and including reporter plasmid in which 36 bp surrounding the Myc- the basic region was deleted, generating USF-1Dbr and regulated E-box from the prothymosin-a gene (E1) had MaxDbr. For USF-1 (but not Max), this deletes the been placed in front of a minimal promoter (36E-tkluc; nuclear import signal and therefore a nuclear import Figure 1a). This is surprising, as recombinant TFE-3 signal derived from SV40 was fused to the amino- binds to this site with high anity in vitro (Desbarats terminus of USF-1Dbr for expression in mammalian et al., 1996). Mutation of the second E-box element cells. In a second mutant, amino acids predicted to (E2) in this 36 bp enhancer fragment (in mutE2-tkluc) make critical DNA contacts based on the crystal allowed both TFE-3 (Figure 1a) and Vp16-TFE-3 to structures of USF-1 (Ferre D'Amare et al., 1994) and activate prothymosin-a (Figure 1b). The experiment Max (Ferre D'Amare et al., 1993) were mutated, shows that the failure of TFE-3 to induce expression of generating USF-1mutbr and Maxmutbr (Figure 3a). prothymosin-a is neither due to a particular transacti- To exclude the possibility that deletion or mutation vation domain nor due to inecient nuclear import of of the basic region of USF-1 generates aberrantly TFE-3.
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