The DNA-binding specificity of the hepatocyte nuclear factor 3/forkhead domain is influenced by amino-acid residues adjacent to the recognition helix.

Abstract

Three distinct hepatocyte nuclear factor 3 (HNF-3) proteins 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 regulation of a distinct array of genes in a particular cell type is critical for understanding cellular commitment during mammalian embryogenesis. Differential expression of protein-encoding genes occurs at the point of transcriptional initiation and involves the assembly of several well-characterized basal factors with TATA-binding protein and RNA polymerase II at the initiation site of the promoter region (17). Promoter and enhancer regions are also composed of multiple DNA sites that interact with sequence-specific transcription factors which are believed to enhance the recruitment of basal factors to the initiation complex. Tissue-restricted gene expression thus relies upon the recognition of multiple cis-acting DNA sequences by cell-specific nuclear factors that potentiate or, in some instances, repress transcriptional initiation (23,28,32). Because transcription factors play a central role in regulating cellular differentiation, the analysis of their molecular structure and expression patterns has been fruitful in elucidating regulatory pathways involved in establishing tissue-specific gene transcription.The functional analysis of a number of transcription factors has demonstrated that they are modular in structure, consisting of independently functioning protein domains (11,(18)(19)(20).

The nude gene encodes a sequence-specific DNA binding protein with homologs in organisms that lack an anticipatory immune system

“…Furthermore, most other species-specific sequence signatures occur in the two putative wings at the C terminus of the DNA binding domain, whereas the amino acid residues in the N-terminally located presumptive helical and sheet structures are more strongly conserved. We also note that the amino acids TAPDG (just preceding the third helix and including its N terminus), thought to be responsible for sequence-specific DNA binding by forkhead͞winged-helix domains (18,19), are identical in all species (see below; Fig. 2).…”

“…The invariant core sequence in the 11-bp Whn consensus binding sites is rather short and nonpalindromic. The binding sites recognized by different members of the forkhead͞winged-helix family of proteins are quite distinct (18,19), unlike the situation with homeodomains or bHLH domains that all appear to bind to an invariant core sequence (5Ј-TAAT or 5Ј-CANNTG, respectively) despite their very variable amino acid compositions (20,21). The identification of a consensus Whn binding site sequence will not immediately be helpful to pinpoint likely target genes, because the frequency of the invariant tetranucleotide in genomic DNA is rather high.…”

BRCA1 and FOXA1 proteins coregulate the expression of the cell cycle-dependent kinase inhibitor p27Kip1

“…The FOXA1 and FOXA2 proteins share 93% homology in the winged-helix domain, bind similar target sequences and potentially regulate the transcription of numerous genes critical for liver function (Lai et al, 1991;Overdier et al, 1994). X-ray crystallography has determined that the structure of the FOXA FH domain resembles the globular domain of linker histone H1 (Clark et al, 1993).…”

Survey of forkhead domain encoding genes in the Drosophila genome: Classification and embryonic expression patterns

“…Sequences flanking this core motif provide differential DNA-binding specificity, and the amino acid stretch between helix 2 and helix 3 of the forkhead domain appears to be important in dictating site-selective binding (Overdier et al, 1994). In addition, binding to DNA results in DNA bending (Pierrou et al, 1994;Gajiwala and Burley, 2000).…”