Functional analysis and the molecular dissection of zinc-fingers and homeoboxes 1 (ZHX1)

Zinc-fingers and homeoboxes 1 (ZHX1) is a protein that interacts with the activation domain of the A subunit of nuclear factor-Y. The function of ZHX1, as a transcription factor, was characterized and their domains were mapped. To determine the nuclear localization signal, expression vectors, in which various truncated forms of ZHX1 were fused to the C-terminal of green fluorescence protein (GFP), were transfected into human embryonic kidney (HEK) 293 cells. All GFP-ZHX1 fusion proteins including an arginine-rich region that corresponds to the amino acid sequence between 734 and 768 were localized in the nuclei. A dimerization domain of the ZHX1 was also mapped using protein-protein interaction assays. The homeodomain (HD) 1 consisting of the amino acid sequence between 272 and 432 of ZHX1 was necessary and sufficient for dimerization. Lastly, the transcriptional activity of ZHX1 was examined using a mammalian one-hybrid system. ZHX1, fused to the C-terminal of the GAL4 DNA-binding domain, was co-transfected with luciferase reporter plasmids with or without five copies of the GAL4-binding site into HEK293 cells. The luciferase activity was decreased in both concentration- and GAL4-binding site-dependent manner. The acidic region corresponding to the amino acid sequence between 831 and 873 was a repressor domain and dimerization was prerequisited for full repressor activity.