The C-terminal domain of the β cell homeodomain factor Nkx6.1 enhances sequence-selective DNA binding at the insulin promoter

The Hox-like factor Nkx6.1 is required for the formation and secretory function of insulin-producing beta cells, and has the capacity to activate or repress the transcription of relevant target genes in a DNA-context dependent manner. A key determinant of transcriptional activity by Nkx6.1 may be its C terminus, which has been suggested to interfere with DNA binding. To determine how the C terminus modulates homeodomain binding, we assessed the nature of Nkx6.1-DNA interactions at the insulin promoter. By quantitative gel shift analysis, we demonstrate that although the C terminus of Nkx6.1 mitigates the affinity of the homeodomain for DNA slightly (about 2-fold), it enhances the selectivity of the homeodomain for TAAT DNA sequences nearly 10-fold. By reporter gene analysis, this selectivity is also functionally preserved in mammalian cells in vivo. Based on deletional and mutational studies, the sequence selectivity imparted by the C terminus appears to be mediated by a stretch of highly conserved residues between amino acids 318 and 338. Strikingly, these residues impart minimal changes to the secondary structure of the unbound protein as assessed by circular dichroism spectroscopy, suggesting that conformational adjustments of the homeodomain that occur upon binding to DNA may play a more important role in sequence selectivity. The C terminus of Nkx6.1 also functions in a modular fashion, as it can confer similar DNA binding properties when fused to the heterologous homeodomain of Pdx-1. Taken together, our data suggest a model whereby the Nkx6.1 C terminus may function in a regulatory manner by imposing specific functional constraints upon the protein. These constraints may serve to modulate the potential of Nkx6.1 to both recognize target genes and regulate their transcription.