Evaluation of a potential regulatory role for inverted CCAAT boxes in the human topoisomerase IIα promoter

Several chemotherapeutic agents act via inhibition of topoisomerase (topo) II activity. Topo II levels appear to correlate with drug sensitivity in vivo. The DNA immediately 5' to the topo IIα coding region contains five potentially regulatory inverted CCAAT boxes (ICB). Electrophoretic mobility shift assays (EMSA) using oligomers containing the wild type forms of these ICBs show specific DNA-protein binding. Mutations in these ICBs result in loss of protein binding. EMSA competition studies indicate that the four most 3' ICBs (1-4) bind to the same protein(s), while the most 5' ICE (5) binds to a different protein(s). EMSA supershift assays with antibodies to two known CCAAT binding proteins, CBF and CEB/P, indicate that ICBs 1-4 are binding to CBF, but ICE 5 is not bound by either of these proteins.