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A Critical Evaluation of Transactivation and Target Gene Regulation

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A Critical Evaluation of Transactivation and Target Gene Regulation Oncogene (1999) 18, 2916 ± 2924 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $12.00 http://www.stockton-press.co.uk/onc The Myc oncoprotein: a critical evaluation of transactivation and target gene regulation Michael D Cole*,1 and Steven B McMahon1 1Department of Molecular Biology, Princeton University, Princeton, New Jersey, NJ 08544-1014, USA Mutations which disrupt the regulation or expression Functional domains and Myc transactivation level of the c-myc gene are among the most common found in human and animal cancers (reviewed in ref. Like most transcription factors, the Myc protein has Cole, 1986; Henriksson and Luscher, 1996; Marcu et al., two major domains. The C-terminal 90 amino acids are 1992). Ectopic expression studies de®ne numerous required for dimerization with Max and sequence- biological activities of the c-myc gene, including speci®c DNA binding (Blackwood and Eisenman, transformation, immortalization, blockage of cell dier- 1991; Prendergast et al., 1991). Disruption of this entiation and induction of apoptosis (Askew et al., 1991; domain destroys all biological activity (Stone et al., Cole, 1986; Evan and Littlewood, 1993; Freytag et al., 1987), indicating that DNA binding is essential for 1990; Henriksson and Luscher, 1996; Marcu et al., function. The second domain encompasses the remain- 1992). Furthermore, c-myc is required for ecient ing three-fourths of the Myc protein and can be progression through the cell cycle (Goruppi et al., broadly de®ned as involved in `transactivation' (Kato 1994; Prochownik et al., 1988; Yokoyama and Imamoto, et al., 1990). Transactivation of cellular promoters by 1987), although recent studies indicate that it is not Myc is quite low in transient assays, usually about 3 ± 5 absolutely essential (Mateyak et al., 1997). This fold (see Table 1). Reiteration of Myc/Max consensus fascinating array of biological activities makes the c- binding sites in an arti®cial reporter construct can yield myc gene one of the most intriguing oncogenes and up to eightfold transactivation (Amin et al., 1993; Gu presents the challenging question of how a single gene et al., 1993; Kretzner et al., 1992), whereas fusion of can manifest so many dierent eects. The c-Myc the Myc N-terminus to the GAL4 DNA binding protein exhibits sequence-speci®c DNA binding when domain has been reported to yield 20 ± 200-fold dimerized with its partner Max, and DNA binding is transactivation in various studies (Brough et al., mediated through the basic region, which recognizes the 1995; Kato et al., 1990; Resar et al., 1993). The core sequence CACGTG (Berberich et al., 1992; Black- cellular factors that contribute to this transient well et al., 1993; Blackwood and Eisenman, 1991; transactivation remain unknown, although a direct Prendergast and Zi, 1991; Prendergast et al., 1991), interaction with the TATA binding protein has been but exhibits somewhat higher anity for the more reported (Hateboer et al., 1993; Maheswaran et al., extended sequence ACCACGTGGT (Berberich et al., 1994). The c-Myc N-terminus has short `acidic', 1992; Blackwell et al., 1993; Halazonetis and Kandil, `proline-rich' and `glutamine-rich' clusters similar to 1991). There are three closely related Myc family those associated with some transactivation domains proteins (c-Myc, N-Myc and L-Myc), each with (Kato et al., 1990), but the role of these regions in documented oncogenic potential (Birrer et al., 1988; biological activity has not been thoroughly tested. The Schwab et al., 1985; Yancopoulos et al., 1985) and glutamine-rich region of c-Myc is dispensable for similar DNA binding properties (Mukherjee et al., 1992). oncogenic activity (Stone et al., 1987). In addition, For simplicity, we will use the term Myc to refer to all the Myc N-terminus can `squelch' transactivation by three proteins, but delineate any distinct activities where the herpes virus transactivator VP16 (Resar et al., they apply. The goal of this review is to discuss Myc as a 1993), suggesting that the two proteins compete for transcriptional activator and critically evaluate the common cellular factors in transient assays. GAL4 evidence for the transactivation of speci®c target genes fusion proteins with the L-Myc N-terminus have as direct downstream eectors. Since excellent compre- reduced transactivation compared to fusions with c- hensive reviews on Myc have been published recently Myc, which correlates with the weaker oncogenic (Facchini and Penn, 1998; Henriksson and Luscher, potential of L-Myc in most assays (Barrett et al., 1996), we will focus on the latest observations that oer 1992). Speci®c regions responsible for these dierences mechanistic insight into transactivation and oncogenic have not been mapped, nor has there been a transformation. comparison of cellular promoter transactivation by c- Myc and L-Myc themselves. Keywords: Myc; oncoprotein; target gene; transactiva- Like the DNA binding domain, early reports tion; chromatin remodelling indicated that the N-terminal transactivation domain is also required for Myc's biological activities. Myc proteins containing deletions within the N-terminus have reduced abilities to cooperate with the ras oncogene, to induce foci in a rat ®broblast line, and to block the dierentiation of adipocytes (Stone et al., 1987; Sarid et al., 1987; Freytag et al., 1990). However, these deletions with arbitrary boundaries appear to *Correspondence: MD Cole contrast with a naturally occurring variant of c-Myc Myc oncoprotein transactivation MD Cole and SB McMahon 2917 Table 1 Summary of experimental evidence implicating Myc in the regulation of potential target genes cad ODC MrDb LDH-A prothy cdc25a ISGF3g RCC1 p53 elF-4E ECA39 E2F2 Regulation in log phase myc null + 7 7 7 7 7 7 7 nd 7 7 nd cellsa Fold regulation in serum stimulated 8 1.5 2.2 nd 7 1.8 nd nd +/7 7 nd nd myc null cells; G0?S onlya In vivo footprinting +b nd ?c nd nd nd nd nd nd nd nd nd Fold activation by MycER nd 1.7 ± 3.6d 1.8e 2.0f 4.2g 4h +i 2.2g 1.2f nd nd nd Misregulation from stable ectopic expression of Myc nd +f nd +f +g nd nd +j +k nd nd nd Transient transfections; promoter fusions vs isolated binding sites +l +m +e +n +o +h +i +j +p 19 +r +s All genes contained in the table meet the criteria set forth in the text, i.e. reported activation by Myc (rather than repression) and the presence of a Myc binding site with regulatory region. aBush et al., 1998; bBoyd and Farnham, 1997; Boyd et al., 1998; cAs noted in the text, Myc,Max dimers have been reported to bind an MrDb pseudogene in vivo Grandori et al., 1996; dTavtigian et al., 1994; Tsuneoka et al., 1997; Wagner et al., 1993; eGrandori et al., 1996; fTavtigian et al., 1994; Shim et al., 1997; gEilers et al., 1991; hGalaktionov et al., 1996; iWeihua et al., 1997; jTsuneoka et al., 1997; kReisman et al., 1993; Tavtigian et al., 1994; lMiltenberger et al., 1995; mBello-Fernandez et al., 1993; nShim et al., 1997; oGaubatz et al., 1994; pReisman et al., 1993; qJones et al., 1996; rBenvenisty et al., 1992; sSears et al., 1997. nd=not determined called MycS. The MycS protein originates from al., 1994; MacGregor et al., 1996). Interestingly, translation initiation at either of two methionines at MbII mutants have unaltered or even enhanced amino acids 100 ± 110 in relation to the M1 position of transactivation in most reported promoter fusion the most abundant Myc2 protein, which means that constructs with one exception (Bello-Fernandez et MycS retains approximately 260 amino acids N- al., 1993; Brough et al., 1995; Desbarats et al., terminal to the DNA binding domain (Spotts et al., 1995). The MbII domain has recently been shown to 1997). Even though MycS is devoid of all transactiva- facilitate Myc binding to a novel large nuclear tion activity assayed with reporter constructs, ectopic cofactor called TRRAP (TRansactivation/tRansfor- expression of MycS can induce anchorage-independent mation-domain Associated Protein) (McMahon et growth and apoptosis as well as rescue the cell cycle al., 1998). TRRAP is a 3830 amino acid protein delay of Myc-de®cient ®broblasts (Xiao et al., 1998). with limited homology to the PI3 kinase/ATM Conversely, replacing the c-Myc N-terminus with the family, although TRRAP lacks the kinase catalytic potent transactivation domain from the herpesvirus residues present in other members of the family. VP16 protein fails to reconstitute an oncogenic TRRAP binding to the N-terminus is directly function (Brough et al., 1995). Thus, transactivation correlated with Myc oncogenic activity, since by the Myc N-terminus (as de®ned by transient deletions or mutations in Myc that disrupt TRRAP reporter assays) is neither necessary nor sucient in binding are transformation-defective and the weakly many biological assays. On the other hand, it remains transforming L-Myc protein exhibits poor TRRAP entirely possible that the transactivation of speci®c binding. Furthermore, the disruption of endogenous cellular promoters is required for Myc function and TRRAP pools using antisense and dominant that the apparent dichotomy is only a consequence of inhibitory constructs severely impairs Myc-mediated the inability of transient reporter assays to recapitulate oncogenic transformation. These data imply that the the regulation of chromosomal targets. Alternately, the recruitment of TRRAP to cellular promoters is biological functions of Myc may be linked to gene essential for Myc-mediated oncogenic transformation. repression rather than activation, although the The identi®cation of TRRAP as an essential mechanism of repression and its direct association cofactor provided an important mechanistic insight with Myc remain unclear (Claassen and Hann, 1999). into the function of the Myc N-terminal domain when TRRAP was found to be part of the SAGA complex (Saleh et al., 1998).
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