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

Abstract

NKX3.1 is a member of the NK class of homeodomain proteins and is most closely related to Drosophila NK-3. NKX3.1 has predominantly prostate-specific expression in the adult human. Previous studies suggested that NKX3.1 exerts a growth-suppressive effect on prostatic epithelial cells and controls differentiated glandular functions. Using a binding site selection assay with recombinant NKX3.1 protein we identified a TAAGTA consensus binding sequence that has not been reported for any other NK class homeoprotein. By electromobility 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 analyzed the nucleotides in the TAAGTA consensus sequence that are important for NKX3.1 binding. The consensus binding site of a naturally occurring polymorphic NKX3.1 protein with arginine replaced by cysteine at position 52 was identical to the wild-type binding sequence. The binding affinities of wild-type and polymorphic NKX3.1 for the TAAGTA consensus site were very similar, with values of 20 and 22 nM, respectively. Wild-type and polymorphic NKX3.1 specifically repressed transcription of luciferase from a reporter vector with three copies of the NKX3.1-binding site upstream from a thymidine 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 repressor.

Figure 1

Determination of the NKX3.1 consensus DNA binding sequence. Sequences selected by (A) recombinant wild-type or (B) R52C NKX3.1 following five rounds of SAAB are listed. Underlined nucleotides represent non-random sequence. Nucleotides shown in bold represent those used in determination of the consensus binding sequence. The overall consensus binding sequence for each protein is listed below the aligned sequences. The percentage frequencies of each nucleotide for sequences selected by (C) wild-type NKX3.1 and (D) R52C NKX3.1 are shown.

Figure 2

NKX3.1 specifically binds to the TAAGTA consensus binding site. Binding of maltose-binding protein, wild-type NKX3.1 and R52C NKX3.1 to a radiolabeled probe containing either the NKX3.1 consensus sequence (TAAGTA) or an Oct-1 binding site (CTAAAC) was analyzed by gel mobility shift. Free probe and protein-bound complex are indicated with arrows. Where indicated, a 5-fold molar excess of unlabeled TAAGTA sequence was included as competitor. Nucleotide sequences of the probes and competitor are listed at the bottom of the figure.

Figure 3

Wild-type NKX3.1 preferentially binds the NKX3.1 consensus sequence rather than Nkx2.1 and Msx1 binding sites. (A) Binding affinity of NKX3.1 to TAAGTA was compared with NKX3.1 binding to the Nkx2.1 and Msx1 binding sites by a competitive gel shift assay. Wild-type NKX3.1 was incubated with a probe containing the NKX3.1 consensus sequence in the absence or presence of unlabeled competitor DNA. Competitors were included at concentrations of 50, 250 and 500 nM. Free probe and protein-bound complex are indicated with arrows. (B) The data were quantitated and normalized to wild-type NKX3.1 binding to probe with no competitor. Nucleotide sequences of the competitors are listed at the bottom of the graph. closed circles, TAAGTA; closed squares, CAAGTG; closed triangles, TAATTG.

Figure 4

Effect of mutations in the TAAGTA consensus site on NKX3.1 binding. The effects of mutations in the NKX3.1 consensus site were analyzed by competitive gel shift assay. Wild-type NKX3.1 was incubated with a probe containing the NKX3.1 consensus sequence in the absence or presence of unlabeled competitor DNA. Competitors were included at concentrations of 50, 250 and 500 nM. Free probe and protein-bound complex are indicated with arrows. Nucleotide sequences of the competitors are listed at the bottom of the figure.

Figure 5

Wild-type and R52C NKX3.1 exhibit similar DNA binding affinities. The equilibrium dissociation constants for (A) wild-type and (B) R52C NKX3.1 binding to an NKX3.1 consensus site were determined by gel mobility shift using a constant amount of radiolabeled probe (1 × 10^–10 M) with various protein concentrations (0.5–200 × 10^–9 M). A plot of the quantitated data is shown as bound protein–DNA complex concentration as a function of free protein concentration. The data were analyzed by non-linear least squares as described in Materials and Methods.

Figure 6

NKX3.1 represses reporter gene transcription. (A) Diagram representing reporter plasmids used to analyze the effect of NKX3.1 on reporter gene transcription. (B) Reporter plasmids were used to transiently transfect TSU-Pr1 cells co-transfected with wild-type or R52C NKX3.1 expression plasmids or empty expression plasmid (pcDNA3) as described in Materials and Methods. CMV-Renilla plasmid was included as a control for transfection efficiency. (C) TSU-Pr1 cells were co-transfected with expression plasmid and empty reporter vector or reporter vector containing NKX3.1 binding sites in the antisense direction. CMV-Renilla plasmid was included as a control for transfection efficiency.