Analysis of DNA binding specificity in homeodomains (1-4) of ATBF1

DNA/RNA-dependent ATPase activity is associated with ATBF1, a multiple homeodomain-zinc finger protein (Biochimica et Biophysica Acta). We examined whether ATPase activity is associated with the ATBF1 molecule. A 263-amino acid segment of the ATBF1 molecule, termed AHZ, which contains the ATPase A-motif, homeodomain IV and zinc finger 21, was expressed in E.coli as the form of GST fusion protein and analyzed for ATPase activity. We found that AHZ was able to hydorolyze ATP and had ability to bind both DNA and RNA. The ATPase activity associated with ATBF1 is DNA/RNA-dependent and unique in that it requires both homeodomain and zinc finger motifs.The mouse ZFH-4 protein contains four homeodomains and twenty-two zinc fingers (Biochem. Biophys. Res. Commun). We isolated the mouse zfhi-4 cDNA which is 12 kb long and capable of encoding a 3,550-amino acid protein containing four homeodomains and 22 zinc fingers including two pseudo zinc finger motifs. The mouse ZFH-4 is 51% homologous to the mouse ATBF 1. RT-PCR analysis of zfh-4 transcripts in adult mouse tissues show that zfh-4 expression was detectable in brain, heart, lung and muscle. In these mouse tissues, ATBF1 transcripts were poorly amplified by PCR under the condtions where zfh-4 transcripts were amplified, suggesting that the expression of zfh-4 mRNA is higher than that ofATBF1 mRNA.Alpha-fetoprotein (AFP) producing gastric cancer lacks transcription factor ATBF 1 (Oncogene). We investigated AFP gene regulation in AFP-producing gastric cancer by an active transcription factor, hepatocyte nuclear factor 1 (HNF1) and a repressive transcription factor, ATBF1. Rnase protection assays revealed that the production of AFP in gastric cancer cells did not directly associate with HNF1 expression. An inverse relation between the expressions of ATBF1 and AFP was clearly observed in gastric cancer cells. CAT assays showed the direct inhibition of AFP gene expression by ATBF1. Immunohistochemistry of clinical samples revealed that AFP-producing cells lacked ATBF1 immunoreactivity. Our data suggests that the absence of ATBF1 is responsible for AFP gene expression in gastric cancer, and the absence of ATBF1 is a distinct characteristic of AFP-producing gastric cancer and might be important for its highly malignant nature.