Nuclear Factor-Y Binding to the Topoisomerase IIα Promoter Is Inhibited by Both the p53 Tumor Suppressor and Anticancer Drugs

Abstract

Expression of the human DNA topoisomerase II␣ (topo II␣) gene is positively regulated by the binding of the nuclear factor Y (NF-Y) transcription factor to four of five inverted CCAAT boxes (ICBs) located in its promoter. We have demonstrated previously that expression of the p53 tumor suppressor inhibits human topo II␣ promoter activity in murine (10)1 cells. In this report, we demonstrate that the inhibition of topo II␣ gene expression by wild-type p53 correlates with the decreased binding of the transcription factor NF-Y to the first four ICBs of the topo II␣ promoter. The expression of mutant p53 does not affect the binding of NF-Y. In NIH3T3 cells, we show that topo II-targeted drugs inhibit the binding of NF-Y to ICB sites in the topo II␣ promoter. This effect is seen not only with drugs that result in DNA strand breaks but also with drugs that inhibit the catalytic activity of topo II, and even with the mitotic spindle inhibitor, vinblastine. Further experiments with p53-null (10)1 cells treated with these same drugs also demonstrate decreased NF-Y binding to the topo II␣ ICBs. The data presented points to the existence of both p53-dependent and -independent mechanisms for regulating NF-Y binding to ICBs in the topo II␣ promoter and thus the modulation of topo II␣ gene expression.The efficient transcription and replication of DNA requires changes in its double-strand topology at specific times during the cell cycle. These topological alterations are carried out by the ubiquitously expressed, homodimeric nuclear protein, topoisomerase II (topo II). Topo II can relieve supercoiling that results during DNA replication and is an essential enzyme for decatenation of sister chromatids at mitosis. The action of topo II involves the cleavage of one DNA double strand, the passage of a transfer double strand through the break, and religation of the cleaved DNA. During the decatenation cycle, topo II is covalently bound to the cleaved DNA strand, forming an intermediate topo II-DNA cleavable complex. The expression of topo II␣ is lowest in the G 1 phase, increases as the cells traverse S phase, and reaches a maximum at the G 2 /M phase interface (Woessner et al., 1991). This observation is consistent with the association of increased topo II␣ with DNA replication, mitosis, and proliferation.Various transcriptional mechanisms regulate the expression of topo II␣. The promoter region of the topo II␣ gene contains five inverted CCAAT boxes (ICBs), two GC boxes, and an ATF site (Hochhauser et al., 1992). Topo II␣ expression is increased in rapidly proliferating cells after exposure to heat shock (Matsuo et al., 1993) and by the ras oncogene (Chen et al., 1999). On the other hand, topo II␣ expression is down-regulated by wild-type p53 (Wang et al., 1997) or confluence-induced growth arrest . Specific ICBs have been implicated in the regulation of topo II␣ expression by confluence arrest heatshock response (Furukawa et al., 1998), p53 (Wang et al., 1997, and cell cycle dependence (Falck et al., 1999).Topo II␣ is ...

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