An Analysis of the Transcriptional Control Domains of the Human c-myc Proto-Oncogene

Transfection of mammalian cells with recombinant plasmid DNAs containing the bacterial chloramphenicol acetyl transferase (CAT) gene as a reporter has been used to analyse genomic sequences regulating the transcription of the human c-myc proto-oncogene. Several regulatory domains 5' to the c-myc coding region have been identified, and their locations defined by deletion analysis. Each of these sites encompass previously identified DNase I in vivo hypersensitive sites. Published data suggested that the c-myc gene may be regulated in vivo by a repressor. The mapping of translocation breakpoints for Burkitt's lymphoma and murine plasmacytoma which involve the c-myc gene, suggests that the cis-acting recognition sequence for this putative repressor is located within the 5' flanking region. I have identified a negative regulatory element (NRE-2) in the 5' flanking region of the gene and localised it to a region between -1052 and -607bp 5' to the PI start site of the c-myc mRNA, by deletion analysis. Subsequent competition experiments showed a 270bp sub-fragment to contain an essential component of the negative regulatory element. This element can function in an orientation independent manner, and has the ability to repress heterologous promoters (both viral and eukaryotic), but to a lesser degree than when acting in cis upon its homologous promoter. My data from both DNA titration and competition transfection analysis indicates that this repression is mediated by at least one trans-acting factor. Since the repression was observed in every cell line used as the transfection recipients, a certain promiscuity in the tissue- and species- specificity of the trans-acting repressor(s) is implied. In vivo footprint analysis tentatively identified two sequence-specific DNA-binding proteins which interact with this domain. Both the CCAAT-binding Transcription Factor (CTF) and Spl bind within the NRE-2 domain. This is the first time either of these DNA-binding factors have been implicated in the transcriptional repression of a gene. In addition, deletion analyses identified an Upstream Promoter Element (UPE), located between the NRE-2 and the c-myc mRNA major cap sites, which is responsible for activation of the high levels of CAT expression observed in cells transfected with the recombinant plasmids. This UPE appears to be a highly complex domain which was shown, by DNase I in vivo footprint analysis, to bind several Spl-like factors. In addition, the UPE is somehow involved in the control of the repression function, although it is not required for the repression of heterologous promoters by the NRE-2. Other data (assayed preliminary) suggested also that two other distal regulatory domains are involved in the control of c-myc expression. The more distal element (PRE) has an activating activity, and was localised to a region which showed sequence homology to enhancer elements. A second element (NRE-1) was tentatively identified which had a negative effect on CAT activity. I conclude that the removal, and/or the rearrangement, of these transcription regulatory domains may play a crucial role in the deregulation of the expression of c-myc that is observed in some neoplastic cells