DNA-binding sequence of the human prostate-specific homeodomain protein NKX3.1

NKX3.1 is a member of the NK class of homeodomain proteins and is most clos ely related to Drosophila NK-3. NKX3.1 has predominantly prostate-specific expression in the adult human. Previous studies suggested that NKX3.1 exert s a growth-suppressive effect on prostatic epithelial cells and controls di fferentiated glandular functions. Using a binding site selection assay with recombinant NKX3.1 protein we identified a TAAGTA consensus binding sequen ce that has not been reported for any other NK class homeoprotein. By elect romobility shift assay we demonstrated that NKX3.1 preferentially binds the TAAGTA sequence rather than the binding site for Nkx2.1 (CAAGTG) or Msx1 ( TAATTG). Using mutated binding sites in competitive gel shift assays, we an alyzed the nucleotides in the TAAGTA consensus sequence that are important for NKX3.1 binding. The consensus binding site of a naturally occurring pol ymorphic NKX3.1 protein with arginine replaced by cysteine at position 52 w as identical to the wild-type binding sequence. The binding affinities of w ild-type and polymorphic NKX3.1 for the TAAGTA consensus site were very sim ilar, with values of 20 and 22 nM, respectively. Wild-type and polymorphic NKX3.1 specifically repressed transcription of luciferase from a reporter v ector with three copies of the NKX3.1-binding site upstream from a thymidin e kinase promoter. The data show that among NK family proteins NKX3.1 binds a novel DNA sequence and can behave as an in vitro transcriptional repress or.