ATBF1 is a 306-kDa protein containing four homeodomains, 17 zinc finger motifs, and several segments potentially involved in transcriptional regulation (T. Morinaga, H. Yasuda, T. Hashimoto, K. Higashio, and T. Tamaoki, Mol. Cell. Biol. 11:6041-6049, 1991). At least one of the homeodomains of ATBF1 binds to an AT-rich element in the human a-fetoprotein (AFP) enhancer (enhancer AT motif). In the present work, we analyzed the transcriptional regulatory activity of ATBF1 with respect to the enhancer AT motif and similar AT-rich elements in the human AFP promoter and the human albumin promoter and enhancer. Gel retardation assays showed that ATBF1 binds to the AFP enhancer AT motif efficiently; however, it binds weakly or not at all to other AT-rich elements in the AFP and albumin regulatory regions studied. Alterations of the enhancer AT motif by site-specific mutagenesis resulted in the loss of binding of ATBF1. Cotransfection experiments with an ATBF1 expression plasmid and the chloramphenicol acetyltransferase (CAT) gene fused to AFP promoter or enhancer fragments showed that ATBF1 suppressed the activity of AFP enhancer and promoter regions containing AT-rich elements. This suppression was reduced when the mutated AT motifs with low affinity to ATBF1 were linked to the CAT gene. The ATBF1 suppression of AFP promoter and enhancer activities appeared to be due, at least in part, to competition between ATBF1 and HNF1 for the same binding site. In contrast to the AFP promoter and enhancer, the albumin promoter and enhancer were not affected by ATBF1, although they contain homologous AT-rich elements. These results show that ATBF1 is able to distinguish AFP and albumin AT-rich elements, leading to selective suppression of the AFP promoter and enhancer activities.The regulatory regions of all genes so far studied contain multiple cis-acting elements which interact with trans-acting factors (22,32). Liver-specific gene expression is also achieved through a combinatorial action of regulatory DNA elements and interacting transcription factors (20,21,30). One characteristic feature of a number of liver-specific promoters is the presence of DNA sequences having a well-organized array of 10 to 11 adenine (A) and thymine (T) residues sandwiched by a guanine (G) and a cytosine (C) (AT-rich element) (4-7, 11, 15, 23, 26, 40). These elements have been shown to be the binding sites of a liver-enriched transcription factor, HNF1 (also called LF-B1 and APF) (3-6, 12, 31). Mutations of the AT-rich element cause drastic reduction of liver-specific transcriptional activity both in vitro and in vivo (21, 28, 33, 42), indicating that the AT-rich element and HNF1 play a crucial role in liver-specific promoter activation.In the promoters of a-fetoprotein (AFP) genes of mice, rats, and humans, two HNF1-binding sites are conserved. In the enhancers, on the other hand, the AT-rich element is present in the human AFP gene but not in the mouse and rat genes (13). The human AFP enhancer AT motif is also unique in that HNF1 binds to it 10-fold...