Characterization of the human topoisomerase IIbeta (TOP2B) promoter activity: essential roles of the nuclear factor-Y (NF-Y)- and specificity protein-1 (Sp1)-binding sites

Abstract

Eukaryotic topoisomerase II (topo II) catalyses topological genomic changes essential for chromosome segregation, chromatin reorganization, DNA replication and transcription. Mammalian topo II exists as two isoforms, designated alpha and beta. Human topo IIalpha is an important cancer drug target, and an established determinant of drug sensitivity and resistance. Human topo IIbeta is also the target of anticancer drugs but its role in drug resistance is less clear. The two human topo II proteins are encoded by the TOP2A and TOP2B genes, respectively, which despite their highly conserved structural organization, are subject to distinctly different modes of regulation. In the present study, we have cloned and characterized the human TOP2B promoter containing a 1.3 kb fragment of the 5'-flanking and untranslated region (-1067 to +193). We found that the promoter activity of this TOP2B fragment was constant throughout the cell cycle, in contrast to the activity of the proximal promoter of TOP2A which was low in resting cells and enhanced during proliferation. Analyses of 5'-serially and internally deleted luciferase reporter constructs revealed that 80% of the TOP2B promoter activity could be attributed to the region between -533 and -481. Mutational analyses of putative regulatory elements indicated that two inverted CCAAT boxes (ICBs) within this region were essential for TOP2B promoter activity and gel mobility-shift assays indicated these sites bound the transcription factor nuclear factor-Y (NF-Y). Co-transfection experiments using a dominant-negative form of subunit A of NF-Y suggested that TOP2B promoter activity required direct interaction of NF-Y with the ICBs. In addition, a specificity protein-1 (Sp1)-binding GC box located just upstream of the ICBs was shown to contribute to TOP2B promoter activity in a synergistic manner with the ICBs. Our results suggest that the binding sites for NF-Y and Sp1 are critical for TOP2B transcription.