Identification of two enhancer elements downstream of the human c-myc gene

Expression of the proto-oncogene c-myc is tightly regulated in vivo. Transcription of c-myc is assumed to be controlled by a number of positive and negative cis-acting control elements located upstream or within exon 1 and intron 1. However, these regulatory elements are not sufficient for c-myc expression after stable transfection or in transgenic mice. Transcription of c-myc in vivo thus requires additional control elements located outside the tested HindIII-EcoRI gene fragment. In order to identify these putative additional control elements, we mapped DNase I hypersensitive sites around the human c-myc gene in nine different tumor cell lines and in primary lymphocytes. Within the coding and 5' region of the gene, an almost identical pattern of DNase I hypersensitive sites was detected in the various cells. In contrast, chromatin analysis of the c-myc 3' region revealed a complex pattern of constitutive and tissue-specific DNase I hypersensitive sites. In enhancer trap experiments we identified two cis-acting control elements, both co-localizing with DNase I hypersensitive sites, that stimulated c-myc transcription after transient transfection in Raji or HeLa cells. Both regulatory elements exerted their enhancer activity in either orientation and regardless of their location within the plasmids. Both elements also conferred activation on a heterologous promoter. The association of these enhancers with DNase I hypersensitive sites, indicating their functional activity in vivo, make them potential candidates for the postulated regulatory control element(s) required for c-myc expression in vivo.