Regulation of the human topoisomerase IIalpha gene promoter in confluence-arrested cells

Abstract

Expression of DNA topoisomerase IIalpha mRNA and protein reflects the proliferative state of mammalian cell lines and tissues with high levels in actively cycling cells but marked down-regulation during serum deprivation or cell density-induced growth arrest. Using stably integrated gene fusions comprising the human topoisomerase IIalpha promoter with a growth hormone reporter gene, we have localized elements required for the differential activity of the topoisomerase IIalpha promoter in proliferating and confluence-arrested cells. Deletion analysis localized the region of the promoter that responded to changes in the cellular growth state to between -101 and -144 base pairs. Mutation analysis identified an inverted CCAAT box (ICB) located at -108 to -104 as necessary for promoter down-regulation in confluence-arrested cells, while several other potential cis-acting elements, including four additional ICBs, were shown not to be required. The critical ICB was recognized in vitro by the CCAAT box binding factor, NF-Y, with levels of binding activity higher in extracts from proliferating cells than from confluence-arrested cells. We conclude that the differential regulation of topoisomerase IIalpha gene expression in cycling and confluence-arrested cells is mediated, at least in part, through proliferation-specific binding of factors to an ICB element in the gene promoter.