Differential regulation of the N-myc proto-oncogene by ROR alpha and RVR, two orphan members of the superfamily of nuclear hormone receptors

ROR alpha 1 and RVR are orphan members of the superfamily of nuclear hormone receptors which constitutively activate and repress, respectively, gene transcription by binding to a common DNA sequence, In an attempt to understand the physiological functions of these two transcription factors, we aimed to identify target genes, We have identified a consensus binding site for ROR alpha 1 and RVR in the first intron of the M-myc gene that we designated N-myc RORE (ROR response element), Unlike most of the intronic sequence, the region encompassing the N-myc RORE is highly conserved between human and mouse, underscoring its importance, Our studies revealed that ROR alpha 1 and RVR specifically bind to the human and mouse N-myc ROREs and transactivate and transrepress, respectively, reporter constructs containing the ROREs, Moreover Northern blot analysis demonstrated a direct modulation of an exogenously introduced N-myc gene by ROR alpha 1 and RVR in COS-1 cells, This effect is mediated through the N-myc RORE, since mutation of this site abolished the regulatory effects of both receptors, While transfection of ROR alpha 1 in P19 embryonic carcinoma cells had no effect on the levels of endogenous N-myc mRNA, RVR down-regulated its expression, The regulatory function of the N-myc RORE was further demonstrated by the rat embryonic fibroblast (REF) transformation assay, Mutation of the RORE increased the oncogenic potential of the N-myc gene in the REF assay, The foci were more numerous and significantly larger with the mutated than with the wild-type N-myc gene, regardless of ROR alpha 1 or RVR expression, Moreover, concomitant expression of ROR alpha 1 and wild-type N-myc resulted in a twofold increase in the number of transformed foci, In contrast, RVR expression resulted in the formation of foci that could be established as permanent clones with a very low frequency compared to foci transformed in its absence, These observations show that ablation of the RORE results in a more oncogenic form of N-myc and suggest that deregulation of the activity of the ROR alpha 1 and RVR could contribute to the initiation and progression of certain neoplasias.