The role of NF-Y in the transcriptional regulation of human topoisomerase II¯ : a thesis presented to Massey University in partial fulfilment of the requirement for the degree of Master of Science in Biochemistry

Abstract

DNA topoisomerases are ubiquitous enzymes that catalyse reactions that alter the topological state of DNA during the various processes of DNA metabolism including transcription, recombination, replication and chromosome segregation. Human cells exhibit a Type II enzyme termed DNA topoisomerase IIα. This enzyme is expressed at higher levels in proliferating cells due to an increased demand for chromosome separation. This is advantageous with respect to some of the drugs used in chemotherapy. These drugs can specifically target cancer cells by only being effective at high levels of topoisomerase IIα gene expression. However, the use of such drugs has been limited by both toxicity and the development of resistance. This resistance has been associated with a decrease in topoisomerase IIα at both protein and mRNA levels. The topoisomerase Ila minimal promoter is 650 base pairs in length and includes promoter elements such as inverted CCAAT boxes (ICBs) and GC rich regions. It has been determined that the ICB elements are of the most interest in terms of regulation of the topoisomerase IIα gene expression. Several studies have shown that the transcription factor NF-Y binds to ICB1-4 of the topoisomerase IIα promoter and regulates transcription through these elements. This study aimed to determine the importance of NF-Y in the transcriptional regulation of topoisomerase IIα and to investigate the molecular mechanisms by which NF-Y associates with the topoisomerase IIα promoter with a particular focus on the inverted CCAAT box elements. The binding of NF-Y to oligonucleotides containing selected consensus elements of the topoisomerase IIα promoter was analysed (in vitro) by electrophoretic mobility shift binding assays. The importance of NF-Y in the regulation of topoisomerase IIα expression was analysed by functional assays, using reporter gene constructs in transiently transfected HeLa cells. The binding studies indicated that the flanking sequences affect the affinity of the transcription factor NF-Y for ICB1 and ICB2 and that a regulatory element flanking ICB2 may aid in NF-Y binding to that element. Functional assays showed that NF-Y appears to act a negative regulator of topoisomerase IIα with its effect being entirely -due to interaction with ICB2.