An Amino-Terminal Domain of Mxil Mediates Anti-Myc Oncogenic Activity and Interacts with a Homolog of the Yeast Transcriptional Repressor SIN3
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CORE Metadata, citation and similar papers at core.ac.uk Provided by Elsevier - Publisher Connector Cell, Vol, 80, 777-786, March 10, 1995, Copyright © 1995 by Cell Press An Amino-Terminal Domain of Mxil Mediates Anti-Myc Oncogenic Activity and Interacts with a Homolog of the Yeast Transcriptional Repressor SIN3 Nicole Schreiber-Agus,*t Lynda Chin,*tt Ken Chen,t et al., 1990), and a carboxy-terminal a-helical domain re- Richard Torres, t Govinda Rao,§ Peter Guida,t quired for dimerization with another basic region-helix- Arthur h Skoultchi,§ and Ronald A. DePinhot Ioop-helix-leucine zipper (bHLH-LZ) protein, Max (Black- "rDepartments of Microbiology and Immunology wood and Eisenman, 1991; Prendergast et al., 1991). and of Medicine Many of the biochemical and biological activities of Myc §Department of Cell Biology appear to be highly dependent upon its association with ~Division of Dermatology Max (Blackwood and Eisenman, 1991 ; Prendergast et al., Albert Einstein College of Medicine 1991; Kretzner et al., 1992; Amati et al., 1993a, 1993b). Bronx, New York 10461 In addition to its key role as an obligate partner in transacti- vation-competent Myc-Max complexes, Max may also re- press Myc-responsive genes through the formation of Summary transactivation-inert complexes that are capable of bind- ing the Myc-Max recognition sequence (Blackwood et al., Documented interactions among members of the Myc 1992; Kato et al., 1992; Kretzner et al., 1992; Makela et superfamily support a yin-yang model for the regula- al., 1992; Mukherjee et al., 1992; Prendergast et al., 1992; tion of Myc-responsive genes in which t ransactivation- Ayer et al., 1993; Zervos et al., 1993). These complexes competent Myc-Max heterodimers are opposed by re- include Max-Max homodimers, whose DNA-binding activ- pressive Mxil-Max or Mad-Max complexes. Analysis ity is subject to regulation by casein kinase II phosphoryla- of mouse taxi1 has led to the identification of two mxil tion (Berberich and Cole, 1992), and the recently de- transcript forms possessing open reading frames that scribed heterodimers Mad-Max (Ayer et al., 1993) and differ in their capacity to encode a short amino- Mxil-Max (Zervos et al., 1993). Together, these function- terminal e-helical domain. The presence of this seg- ally interactive and structurally related bHLH-LZ proteins ment dramatically augments the suppressive potential comprise an expanding Myc superfamily. of Mxil and allows for association with a mammalian The biochemical properties of the highly homologous protein that is structurally homologous to the yeast MAD and MXll have led to a model for their regulation of transcriptional repressor SIN3. These findings provide Myc activity in which these two proteins compete with Myc a mechanistic basis for the antagonistic actions of for binding both to Max and to common target sequences Mxil on Myc activity that appears to be mediated in part (Ayer et al., 1993; Zervos et al., 1993). As such, the relative through the recruitment of a putative transcriptional intracellular levels of Mad and Mxil in comparison to those repressor. of Myc influence the transcriptional activation of Myc- responsive genes through a dynamic interchange be- Introduction tween the formation of transactivation-inert (Mad-Max or Mxil-Max) and transactivation-competent (Myc-Max) Members of the myc family of nuclear proto-oncogenes (c-, complexes (Ayer et al., 1993; Zervos et al., 1993). The N-, and L-myc) play central roles in the control of normal opposing relationship between Myc and Mad or Mxil de- growth and development and in genetic pathways linked rives further support from biological studies demonstrating to cellular transformation and apoptotic cell death (Evan that, during the approach of many cell lineages to the ter- and Littlewood, 1993; Morgenbesser and DePinho, 1994). minally differentiated state, MXl and MAD mRNA and pro- Accumulating structural, biochemical, and genetic evi- tein levels increase, while those of c-MYC decrease (Ayer dence affords the view that the function of Myc familyonco- et al., 1993; Ayer and Eisenman, 1993; Zervos et al., 1993; proteins in these diverse processes relates in part to their Larsson et al., 1994; Schreiber-Agus et al., 1994), and that roles as sequence-specific transcription factors (for re- overexpression of MXll and MAD can antagonize myc views see Kato and Dang, 1992; Torres et al., 1992). Myc activity in cellular transformation assays (Lahoz et al., family oncoproteins appear to influence the expression of 1994). The anti-oncogenic activity of Mad and Mxil ac- growth-promoting genes, such as those involved in DNA quires particular significance with the localization of MAD synthesis (Bello-Fernandez et al., 1993) and cell cycle reg- and MXI1 genes to chromosomal loci implicated in the ulation (Jansen-Durr et al., 1993), in a positive manner. genesis of several human cancers (Edelhoff et al., 1994; Myc may also play a repressive role in the regulation of M. F. Seldin and R. A. D., unpublished data). some genes through interaction with an initiation factor of Progress on the elucidation of the precise molecular the general transcriptional machinery (Roy et al., 1993). actions of Myc at the level of Myc-responsive gene targets Myc family proteins possess a multifunctional amino- and on the relation of such actions to growth and differenti- terminal domain with transactivation potential (Kato et al., ation has been hampered by the modest transactivation 1990), a region rich in basic amino acid residues responsi- activity of Myc in available transcription reporter assays ble for sequence-specific DNA-binding activity (Blackwell and by the limited repertoire of bona fide myc gene targets. As an alternative strategy to understanding the nature of the actions of Myc and of the functional relationships *The first two authors contributed equallyto this work. among members of the Myc superfamily, we and others Cell 778 have used the rat embryo fibroblast (REF) cooperation two mRNAs through alternative RNA processing (N. S.-A., assay (Land et al., 1983). This highly quantitative biologi- H.-W. Lee, and R. A. D., unpublished data). cal assay takes advantage of a long-recognized property Nucleic acid sequence analysis of the tmxi-SR cDNA of Myc, namely, its ability to cooperate with activated H-RAS clone revealed an ATG-initiated open reading frame (ORF) (Val-12) to effect the malignant transformation of early- capable of encoding a protein of 228 amino acids with a passage REFs. The REF cooperation assay has proven predicted molecular size of 25,977 Da (Mxi-SR in Figure effective in the evaluation of candidate modulators of myc 1A). That the predicted Mxi-SR protein indeed represents oncogenic potential, including the Max-associated pro- the mouse homolog of human MXll (Zervos et al., 1993; teins MAD and MXll (Lahoz et al., 1994), dominant nega- the human MXl clone was shown to be equivalent to mouse tive mutants of Myc (Mukherjee et al., 1992; Sawyers et al., mxi-SR and not mouse mxi-WR) is supported by their 1992), retinoblastoma (Rb), and other cell cycle regulators shared amino acid identity of 96% compared with only (Serrano et al., 1995). The functional impact of such modu- 61% when aligned with human MAD (Ayer et al., 1993). lators correlates well with their biochemical profiles and The second mxil cDNA clone, tmxi-WR, is identical to postulated mechanisms of action. For instance, Rb, which tmxi-SR in the nucleic acid sequences encoding residues is thought to interact poorly with Myc in vivo, has a minimal 37-228 of the tmxi-SR ORF and in its 3' untranslated re- suppressive effect on myclRAS-induced foci formation gion (3'UTR). However, the 5'-most sequences of the tmxi- (E. G. Lahoz and R. A. D., unpublished data), while overex- SR ORF are absent from tmxi-WR and are replaced by pression of MAD or MXl leads to a profound reduction different sequences that do not encode an in-frame ORF. in transformation activity in a highly Myc-specific manner As a result, the putative protein encoded by tmxi-WR (Lahoz et al., 1994). would likely initiate translation at an ATG that corresponds In the course of investigating the basis of the anti- to a methionine at position 37 in the Mxi-SR protein (Mxi- oncogenic activity of Mxil, we identified two mxil mRNAs WR in Figure 1A). that arise through alternative RNA processing and that The Mxi.SR Amino.Terminal Extension encode proteins with dramatically different abilities to re- While mxi-SR and mxi-WR ORFs encode identical bHLH- press myc-induced transformation. The capacity for strong LZ and carboxy-terminal regions, alternative utilization of repressive activity correlates with an amino-terminal ex- 5' sequences extends the mxil-SR ORF an additional 36 tension of 36 residues that is present in only one of the amino acids beyond the mxil-WR ORF. This extension is two Mxi protein forms. Significantly, use of the yeast two- highly conserved throughout vertebrate evolution, exhib- hybrid interaction system showed that this highly con- iting 100% similaritywith human MXI 1 (Zervos et al., 1993) served (z-helical "repression" domain of Mxil (and MAD) and 72% similarity with zebra fish Mxil (Schreiber-Agus associates with a murine homolog of the yeast transcrip- et al., 1994) (Figure 1B; zebra fish not shown). Notably, tional repressor SIN3 (Nasmyth et al., 1987; Sternberg et this region is also highly homologous (78% similar) to an al., 1987; Strich et al., 1989; Wang et al., 1990; Vidal et analogously positioned domain in human MAD (Ayer et al., al., 1991; Wang and Stillman, 1993). Through coimmuno- 1993) (Figure 1 B). The secondary structure of this Mxil-SR precipitation studies in mammalian cells, Mxil and mouse amino-terminal extension is predicted to be strongly (z-heli- Sin3 (roSin3) were shown to be part of a ternary complex cal, and the potential for (~ helicity is conserved in the human that also included Max.