Many eukaryote promoters, particularly those for so-called housekeeping genes, have multiple GC boxes which are the binding sites of the transcription factor, Spl. It has been proposed that Spl binds to the multiple GC boxes, and then the GC box-bound Spl interact with each other to synergistically stimulate transcription. Here, we describe a Spl-dependent promoter which does not necessarily fit the synergistic activation mechanism. The promoter of the human NADH-cytochrome b5 reductase-encoding gene (CYTB5R) possesses five potential GC box sequences. Deletion and mutagenesis studies coupled with CAT assays revealed that three out of five GC box-like sequences were functionally active and activated transcription additively (rather than synergistically). Our results suggested that Spl-mediated activation of transcription occurs in a promoter context-dependent manner.
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