We have described a genetic polymorphism of NKX3.1, a homeobox gene with organ-specific expression confined to the prostate in the adult. The polymorphism changes codon 154 by substituting a T for a C, thereby resulting in a missense codon 52 that replaces arginine with cysteine (R52C). In a survey of DNA samples we found that NKX3.1 R52C was present in 4 percent of normal donors, but in 12 percent of prostate cancer patients, suggesting that NKX3.1 R52C may confer 3-fold risk for prostate cancer. Moreover, the amino acid alteration coded by the polymorphism increases the avidity of DNA binding by the NKX3.1 protein. Therefore, this polymorphism represents a gain-of-function mutation that appears to affect prostate cancer risk. The proposal focuses on the DNA binding and transcriptional activation by NKX3.1 wild type and polymorphic proteins. We will identify the true DNA binding site for NKX3.1. We will construct reporter plasmids with the NKX3.1 binding site in order to study differences between mutant and polymorphic proteins and to perform deletion analysis of NKX3.1. We will construct chimaeric NKX3.1/GAL4 fusion proteins to study the effects of NKX3.1 sequences flanking the homeodomain on the activity of DNA binding and activation of a known transcription factor. To enhance the in vitro phenotypic impact of the amino acid 52 mutation, we generate other amino acid substitutions and characterize them in reporter gene assays. Lastly, through differential display technology, we will identify genes that are regulated by NKX3.1 to begin to understand the signaling pathways that are activated by NKX3.1 and to study NKX3.1-responsive promoters that are in vivo targets for the protein.
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