Regulatory studies of the homeobox gene NKX3.1 in prostate cancer cells

[Truncated abstract] KX3.1 is a homeobox gene expressed at all stages of development and maturation of the prostate gland and required for the maintenance of differentiation of the adult prostatic epithelium. Expression of NKX3.1 is reduced or undetectable in a high frequency of prostate tumours in association with the deletion of one NKX3.1 allele (loss of heterozygosity), while mechanisms leading to the partial or complete loss of expression from the second allele have not been determined. With the promoter and enhancer regions of the NKX3.1 gene incompletely investigated, this thesis has characterised a ~2kb region of the NKX3.1 5’ proximal promoter, identifying ETS1 regulation of the promoter and of NKX3.1 protein levels in prostate cancer cells that is mediated by an ETS1 binding site (EBS) located within this sequence. In this study, reporter deletion and bioinformatics analyses were performed on the NKX3.1 promoter (-2062 to +6 relative to the transcription start site) to identify regions of transcriptional regulatory function and candidate transcription factors contributing to this activity. The initial 2062, 1421, 993, 487 and 200bp of the promoter were ligated into a luciferase reporter vector and induced luciferase activity assayed following transient transfection of the promoter reporter plasmids into the prostate cancer cell lines LNCaP and DU145 and the monkey kidney cell line Cos7. This approach led to the detection of regions of positive (-2062 to -993; -200 to +6) and negative (-993 to -200) transcriptional activity in these cell lines which may be attributed to the activity of SP1, AP-1, CEBPα, MAX, OCT-1 or CREB candidate binding sites which were located within these promoter regions. Interestingly, reporter deletion assays identified strong positive transcriptional activity between -1421 and -993 in LNCaP and Cos7 cells, but not in the DU145 cell line which does not express NKX3.1. Additional reporter deletion studies of the -1421 to -993 region further mapped this positive activity to between -1069 and -993 in LNCaP and Cos7 cells. Electromobility shift assay (EMSA) analyses of this sequence using a series of 5 overlapping oligonucleotides identified binding between -1057 to -1035 and -1016 to -993 of nuclear proteins from LNCaP, Cos7 and DU145 cells, whilst binding between -1042 to -1020 and -1027 to -1005 was only detected in the presence of DU145 nuclear extracts. Further bioinformatics analyses of the sequences where nuclear factor binding occurred identified putative binding sites for NF-1 and HNF-4 (-1057 to -1035), USF1 (-1042 to -1020 and -1027 to -1005) and STATx and ETS1 (-1016 to -993), however interactions between NF-1, HNF-4, USF1 and STATx and these sequences was not able to be detected. In contrast, competition EMSA studies indicated binding of ETS1 between -1016 and -993 which was verified in subsequent EMSA and supershift assays incorporating a GST-ETS1 protein and an α-GST antibody. Although the bioinformatics analyses had identified 2 ETS1 binding sites (EBS) between -1016 and -993, EBS1 and EBS2, mutagenesis EMSA analyses indicated that ETS1 interacted solely with EBS1 located between -1008 and -999.