CCAAT Binding Factor (CBF) Binding Mediates Cell Cycle Activation of Topoisomerase IIα

To understand the role of the CCAAT binding factor (CBF) in transcription during the cell cycle, we studied the mouse topoisomerase IIα (topo IIα) promoter, which is activated during the late S and G2/M phases of the cell cycle and contains multiple CBF binding sites. Mutational analysis of the promoter shows that CBF binding to an inverted orientation of the CCAAT motif in the topo IIα promoter, but not to a direct orientation, is required for transcription activation during the cell cycle. In contrast, analysis of the promoter in an in vitroreconstituted transcription system shows that CBF activates transcription of the topo IIα promoter irrespective of the orientation of the CBF binding sites. This analysis demonstrates that only one of the three transcription start sites of the topo IIα promoter is stimulated by CBF, indicating that transcription activation by CBF is dependent on basal promoter structure. Interestingly, mutations of the start site that abolish CBF-dependent transcription activation in vitro do not inhibit activation of the promoter during the cell cycle. Consistent with this observation, expression of a truncated CBF-B subunit lacking a transcription activation domain, which inhibits activity of a collagen promoter, does not affect activity of the topo IIα promoter in fibroblast cells. In contrast, expression of an allele-specific CBF-B mutant that binds high affinity to a mutant CBF binding site containing a CCAAC motif revives transcription activation of an inactive mutant topo IIα promoter containing CCAAC during the cell cycle. Altogether, this study indicates that CBF binding, but not conventional CBF activation domains, are required for activation of the topo IIα promoter during the cell cycle. Considering these results together with results of another recent study, we hypothesize that binding of CBF that disrupts the nucleosomal structure in the topo IIα promoter is a major function of CBF by which it regulates the cell cycle-dependent transcription of the topo IIα promoter and possibly many other cell cycle-regulated promoters containing CBF binding sites.