The Myc family on oncogenes is involved in the pathogenesis of several human tumors. In vitro Myc onco-proteins can trigger unscheduled DNA synthesis and contribute to the transforming ability of other oncogenes. At the molecular level Myc works by binding to specific DNA sequences and by activating transcription. This proposal aims at identifying and characterizing the genes that are directly altered by Myc. Toward this goal, a library of selected human genomic sequences was obtained by immunoprecipitation of chromatin with specific anti-Myc and its dimerization partner Max antibodies. In vivo Myc-Max binding sites allowed identification of several new Myc-responsible mRNAs; among those, one encoding for an RNA helicase, MrDb.
The first aim will focus to further establish a direct transcriptional response of MrDb to Myc. This will be achieved by characterizing the promoter region of MrDb and analyzing the transcriptional role of the Myc-Max binding site identified by immunoprecipitation in its natural context. The genomic clone will also allow monitoring in vivo binding of Myc-Max to the predicted site and other possible sites by exonuclease protection and in vivo footprinting techniques. In addition, the biological role of MrDb as mediator of Myc function will be studied.
The second aim will focus on the remaining 18 potential in vivo binding sites so far sequenced. Preliminary results indicate that Myc-Max heterodimers bind cooperatively to multiple sites present in some of the genomic clones. Cooperativity provides a potential important mechanism for in vivo selection of binding sites among many potential non-specific binding sites. The requirements for cooperativity at the protein level (i.e. Myc and Max mutant) and DNA level (i.e. binding site mutations) will be defined.
The final aim will be to identify additional Myc-responsive genes. This will be achieved by employing the Myc-Max binding sites so far characterized as probes to screen a human genomic library in an exon- trapping vector. These studies will provide several pieces of the puzzle necessary to understand how Myc functions as an oncogene.
|Hirst, S K; Grandori, C (2000) Differential activity of conditional MYC and its variant MYC-S in human mortal fibroblasts. Oncogene 19:5189-97|
|Grandori, C; Cowley, S M; James, L P et al. (2000) The Myc/Max/Mad network and the transcriptional control of cell behavior. Annu Rev Cell Dev Biol 16:653-99|
|Coller, H A; Grandori, C; Tamayo, P et al. (2000) Expression analysis with oligonucleotide microarrays reveals that MYC regulates genes involved in growth, cell cycle, signaling, and adhesion. Proc Natl Acad Sci U S A 97:3260-5|
|Wu, K J; Grandori, C; Amacker, M et al. (1999) Direct activation of TERT transcription by c-MYC. Nat Genet 21:220-4|