MOLECULAR AND CELLULAR BIOLOGY, Apr. 1994, p. 2755-2766 Vol. 14, No. 4 0270-7306/94/$04.00+0 Copyright ©) 1994, American Society for Microbiology The DNA-Binding Specificity of the Hepatocyte Nuclear Factor 3/forkhead Domain Is Influenced by Amino Acid Residues Adjacent to the Recognition Helix DAVID G. OVERDIER, ANNA PORCELLA, AND ROBERT H. COSTA* Department of Biochemistry, College of Medicine, The University of Illinois at Chicago, Chicago, Illinois 60612-7334 Received 19 November 1993/Returned for modification 4 January 1994/Accepted 19 January 1994 Three distinct hepatocyte nuclear factor 3 (HNF-3) proteins (HNF-3ox, -3Ip, and -3-y) are known to regulate the transcription of liver-specific genes. The HNF-3 proteins bind to DNA as a monomer through a modified helix-turn-helix, known as the winged helix motif, which is also utilized by a number of developmental regulators, including the Drosophila homeotic forkhead (fkh) protein. We have previously described the isolation, from rodent tissue, of an extensive family of tissue-specific HNF-3/fkh homolog (HFH) genes sharing homology in their winged helix motifs. In this report, we have determined the preferred DNA-binding consensus sequence for the HNF-3P protein as well as for two divergent family members, HFH-1 and HFH-2. We show that these HNF-3/fkh proteins bind to distinct DNA sites and that the specificity of protein recognition is dependent on subtle nucleotide alterations in the site. The HNF-3, HFH-1, and HFH-2 consensus binding sequences were also used to search DNA regulatory regions to identify potential target genes. Furthermore, an analysis of the DNA-binding properties of a series of HFH-1/HNF-3p protein chimeras has allowed us to identify a 20-amino-acid region, located adjacent to the DNA recognition helix, which contributes to DNA-binding specificity. These sequences are not involved in base-specific contacts and include residues which diverge within the HNF-3/fkh family. Replacement of this 20-amino-acid region in HNF-3P with corresponding residues from HFH-1 enabled the HNF-3I recognition helix to bind only HFH-1-specific DNA-binding sites. We propose a model in which this 20-amino-acid flanking region influences the DNA-binding properties of the recognition helix. Deciphering mechanisms which lead to transcriptional reg- 23, 41, 48). Each transcription factor draws specificity from its ulation of a distinct array of genes in a particular cell type is DNA-binding domain and thus is able to recognize only critical for understanding cellular commitment during mam- promoters containing the cognate DNA-binding sites. The malian embryogenesis. Differential expression of protein-en- isolation of conserved family members by low-stringency hy- coding genes occurs at the point of transcriptional initiation bridization with DNA-binding-domain probes has facilitated and involves the assembly of several well-characterized basal the isolation of related transcription factors which have dem- factors with TATA-binding protein and RNA polymerase II at onstrated varied developmental stage- and tissue-specific ex- the initiation site of the promoter region (17). Promoter and pression patterns (14, 31, 38, 52). The manner in which enhancer regions are also composed of multiple DNA sites transcription factor families have evolved to share homology that interact with sequence-specific transcription factors which within their DNA-binding domains is clearly advantageous for are believed to enhance the recruitment of basal factors to the organisms. The generation of sequence divergence within initiation complex. Tissue-restricted gene expression thus re- functional DNA-binding structures confers heterogeneity in lies upon the recognition of multiple cis-acting DNA sequences DNA site recognition, which enables different target promoter nuclear some by cell-specific factors that potentiate or, in regulation without the evolution of an unrelated DNA-binding instances, repress transcriptional initiation (23, 28, 32). Be- motif (10, 38, 52). A general grouping of the DNA-binding cause transcription factors play a central role in regulating motifs in known transcription factor families would include cellular differentiation, the analysis of their molecular struc- those with helix-turn-helix (e.g., homeodomain), zinc finger ture and expression patterns has been fruitful in elucidating (e.g., steroid and hormone regulatory pathways involved in thyroid receptor superfamily), establishing tissue-specific b-Zip (e.g., C/EBP, c-Jun, and c-Fos), helix-loop-helix (e.g., gene transcription. MyoD and myogenin), and RelA (e.g., NF-KB) (2, 10, 23, 38, The functional analysis of a number of transcription factors 41, 44, 62). has demonstrated that they are modular in structure, consist- ing of independently functioning protein domains (11, 18-20). The hepatocyte nuclear factor 3 (HNF-3)/forkhead (fkh) The transcription factor domains include those involved in family of tissue-specific and developmental gene regulators specific DNA recognition (DNA binding), in formation of uses a 100-amino-acid winged helix motif, which consists of a homodimeric or heterodimeric proteins (dimerization), and in modified helix-turn-helix, for monomeric recognition of spe- cific DNA target sites The first members stimulation of RNA polymerase II initiation (activation) (11, (5). family included three distinct hepatocyte transcription factors, HNF-3o, -3r, -3-y, two of which (HNF-3ox and -3p) are expressed in the lung * Corresponding author. Mailing address: Department of Biochem- and are essential participants in liver- and lung-specific gene istry (M/C 536), The University of Illinois at Chicago, College of transcription (3, 7, 22, 27, 29, 30, 36, 43, 50, 51, 56). Mouse Medicine, 1819 West Polk St., Chicago, IL 60612-7334. Phone: (312) embryonic expression studies with HNF-3cx and -31 clones 996-0474. Fax: (312) 413-0364. suggest that they also play a role in specifying neural axis 2755 2756 OVERDIER ET AL. MOL. CELL. BIOL. formation during gastrulation as well as facilitating endoderm GGC, which was located 34 amino acids N terminal of the differentiation (55). Following the isolation of the HNF-3 motif, and the antisense oligonucleotide 5'-CTCTCTAGA cDNA, conservation within the DNA-binding and activation GCC AGG CTG TAG GCT CCG, which was situated 22 domains of HNF-3 and the Drosophila homeotic fkh protein amino acids C terminal of the motif. The HNF-31-GST fusion was noted (30, 47, 60, 61). Subsequent to this discovery, several proteins contained amino acid sequence 144 to 279 or 95 to 279 other HNF-3/fkh family members which participate in deter- (used for DNA site selection) and were made by PCR ampli- mination events during embryogenesis have been identified. fications from previously described HNF-3r expression con- These homologs include the Drosophila sloppypaired (Slpl and structs (47). The chimeras HFH-1.WRNS and HNF-3.WQNS Slp2) and fkh domain (FD1 to FD5) genes (15, 16); Xenopus were created by introducing a unique EcoRI site via PCR- laevis fkh domain genes, XFKH1, Pintallavis, and cXFD1 to mediated mutagenesis into the central portion of the HNF-3f cXFD3 (8, 26, 53); zebrafish Axial (58), and Caenorhabditis and HFH-1 DNA-binding-domain coding region. The EcoRI elegans lin-31 (40). A number of mammalian HNF-3/fkh family site maintained the integrity of the amino acid sequence and members have also been isolated from a variety of different cell provided a means by which to fuse the two DNA-binding types and include the brain-specific BF-1 and its avian onco- domains. In the case of HFH-1.WRNS, a BamHI-EcoRI PCR gene homolog, qin (35, 59), those derived from hemopoietic product was made from HFH-1 template with a 5' flanking lineage, ILF, HTLF, H3, H8, and 5-3 (21, 33, 34), those derived primer and antisense mutagenesis primer 5'-ACGGAA1TC from mesoderm/mesenchyme origin, MFH-1 and MF-i to CGC CAG CCC GTG TAG CT, and an EcoRI-XbaI PCR MF-3 (42, 55), and mouse fkh-] to fkh-6 genes (24). product was made from HNF-31 template with the sense In a previous report, we described the isolation of nine mutagenesis primer 5'-GCAGAATTC CAT CCG TCA 1TC additional HNF-3/fkh homologs (HFH) from a diverse panel TCT C and a downstream flanking primer. Equimolar amounts of rat tissue cDNAs, using both low-stringency hybridization of the digested PCR products were ligated together, and the and PCR amplification (6). The HFH genes possess diverse ligation product was redigested with BamHI and XbaI, purified adult expression patterns and exhibited heterogeneity in their again, and then ligated into pGEX2T (54). The chimera winged helix DNA-binding domains (47 to 68% identity). In HNF-3.WQNS was constructed with the BamHI-EcoRI PCR this study, we determine the DNA recognition sequence for product made from the HNF-3p template with 5' flanking three divergent HNF-3/fkh family members by using bacterial primer and antisense mutagenesis primer 5'-GTGGAATTC fusion proteins containing the HFH-1, HFH-2, and HNF-3,B TGC CAG CGC TGC TGG T, and the EcoRI-XbaI PCR DNA-binding domains for site selection. We show that the product was made from the HFH-1 template with sense HNF-3/fkh proteins possess distinct DNA-binding properties mutagenesis primer 5'-GCGGAATTC CGT GCG CCA CAA which are distinguished by subtle nucleotide changes in their CCT C and the downstream flanking primer as for HFH- DNA recognition sites. Although the HNF-3/fkh family mem- 1.WRNS. HFH-1.AEIY and HNF-3.SEIN were constructed by bers described herein bind to different DNA sites, the residues using unique restriction sites in the HNF-3 domain (BglII) and involved in base-specific contacts in DNA recognition helix 3 in the HFH-1 domain (Sau3AI) as fusion points.