Down-regulation of human topoisomerase IIalpha expression correlates with relative amounts of specificity factors Sp1 and Sp3 bound at proximal and distal promoter regions.

ABSTRACT

BACKGROUND: Topoisomerase IIalpha has been shown to be down-regulated in doxorubicin-resistant cell lines. The specificity proteins Sp1 and Sp3 have been implicated in regulation of topoisomerase IIalpha transcription, although the mechanism by which they regulate expression is not fully understood. Sp1 has been shown to bind specifically to both proximal and distal GC elements of the human topoisomerase IIalpha promoter in vitro, while Sp3 binds only to the distal GC element unless additional flanking sequences are included. While Sp1 is thought to be an activator of human topoisomerase IIalpha, the functional significance of Sp3 binding is not known. Therefore, we sought to determine the functional relationship between Sp1 and Sp3 binding to the topoisomerase IIalpha promoter in vivo. We investigated endogenous levels of Sp1, Sp3 and topoisomerase IIalpha as well as binding of both Sp1 and Sp3 to the GC boxes of the topoisomerase IIalpha promoter in breast cancer cell lines in vivo after short term doxorubicin exposure. RESULTS: Functional effects of Sp1 and Sp3 were studied using transient cotransfection assays using a topoisomerase IIalpha promoter reporter construct. The in vivo interactions of Sp1 and Sp3 with the GC elements of the topoisomerase IIalpha promoter were studied in doxorubicin-treated breast cancer cell lines using chromatin immunoprecipitation assays. Relative amounts of endogenous proteins were measured using immunoblotting. In vivo DNA looping mediated by proteins bound at the GC1 and GC2 elements was studied using the chromatin conformation capture assay. Both Sp1 and Sp3 bound to the GC1 and GC2 regions. Sp1 and Sp3 were transcriptional activators and repressors respectively, with Sp3 repression being dominant over Sp1-mediated activation. The GC1 and GC2 elements are linked in vivo to form a loop, thus bringing distal regulatory elements and their cognate transcription factors into close proximity with the transcription start site. CONCLUSION: These observations provide a mechanistic explanation for the modulation of topoisomerase IIalpha and concomitant down-regulation that can be mediated by topoisomerase II poisons. Competition between Sp1 and Sp3 for the same cognate DNA would result in activation or repression depending on absolute amounts of each transcription factor in cells treated with doxorubicin.