Activation of gene transcription by the amino terminus of the N-Myc protein does not require association with the protein encoded by the retinoblastoma suppressor gene RB1.
N-Myc encodes a nuclear phosphoprotein that contains a basic region (BR), a helix-loop-helix (HLH) and a leucine zipper (Zip). These motifs are hallmarks of certain transcription factors. In pursuit of the question if N-Myc can activate transcription, we have employed an experimental model involving the yeast transcription factor Gal4. We have first generated fusion proteins containing the Gal4 DNA-binding domain joined to portions of N-Myc. Subsequently we have analysed if chimeric proteins can transactivate the transcription of a reporter under the control of Gal4 binding sites. Here we show that the amino terminal portion of N-Myc activates transcription. Activation maps to a domain highly conserved among Myc-proteins and to other non-conserved sequences, suggesting functional redundancy. Previous studies had documented in vitro association of the RB1 protein with N-Myc (Rustig et al., 1991). We here confirm this observation and identify the region encompassing the transactivation domain as responsible for RB1 binding. Analyses of N-Myc transactivation in retinoblastoma cell line WERI lacking a function RB1 protein gave results similar to those with cell lines having an intact RB1 protein, showing that RB1 protein is not required for transactivation by N-Myc. The present findings leave open the question if deregulated expression of N-Myc contributes to tumorigenesis by transcriptional activation of as yet unidentified target genes or by functionally inactivating the protein encoded by the tumor suppressor gene RB1, or by a combination of both.
Department of Classical and Molecular Cytogenetics, German Cancer Research Center, Heidelberg.
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Cziepluch
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Wenzel
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Schürmann
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Schwab
D004268:DNA-Binding Proteins; D005656:Fungal Proteins / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000032:analysis; D016271:Proto-Oncogene Proteins c-myc / Q000737:chemistry / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D016160:Retinoblastoma Protein / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; D016160:Retinoblastoma Protein; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D008156:Luciferases