DNA-BINDING THROUGH DISTINCT DOMAINS OF ZINC-FINGER-HOMEODOMAIN PROTEIN AREB6 HAS DIFFERENT EFFECTS ON GENE-TRANSCRIPTION

Transcription factor AREB6 has a unique structure composed of two zinc -finger cluster sin N- and C-terminal regions, and one homeodomain in the middle. AREB6 has been known to regulate the expression of the Na, K-ATPase alpha 1 subunit, interleukin 2 and delta-crystallin genes. W e determined the optimal binding sites for the N-terminal zinc-finger cluster as GTCACCTGT or TGCACCTGT and for the C-terminal zinc-finger c luster as C/TACCTG/TT by the CASTing method (cyclic amplification and selection of targets). The additional consensus sequence GTTTC/G, in c onjunction with the CACCTGT sequence, was selected by the second CASTi ng for the entire coding region. The N-terminal zinc-finger cluster bi nds to DNA strongly when the DNA has GTTTC/G in conjunction with the C ACCTGT sequence. The homeodomain had no specific DNA binding activity but was found to interact with the N-terminal zinc-finger cluster. Ana lyses of zinc-finger mutation proteins revealed that the contribution to DNA binding of each N-terminal zinc-finger motif is altered dependi ng on the presence of the additional consensus. Transient transfection assays showed that AREB6 repressed the human 70-kDa heat-shock gene p romoter harboring the CACCTGT sequence together with the additional co nsensus, and the AREB6 activated the promoter harboring the CACCTGT se quence without the additional consensus. These results suggest that AR EB6 has multiple conformational states, leading to positive and negati ve regulations of gene transcription.