The hepatocyte nuclear factor 3␣ (HNF-3␣) and 3␤ proteins have homology in the winged helix/fork head DNA binding domain and regulate cell-specific transcription in hepatocytes and in respiratory and intestinal epithelia. In this study, we describe two novel isoforms of the winged helix transcription factor family, HNF-3/fork head homolog 11A (HFH-11A) and HFH-11B, isolated from the human colon carcinoma HT-29 cell line. We show that these isoforms arise via differential splicing and are expressed in a number of epithelial cell lines derived from tumors (HT-29, Caco-2, HepG2, HeLa, A549, and H441). We demonstrate that differentiation of Caco-2 cells toward the enterocyte lineage results in decreased HFH-11 expression and reciprocal increases in HNF-3␣ and HNF-3␤ mRNA levels. In situ hybridization of 16 day postcoitus mouse embryos demonstrates that HFH-11 expression is found in the mesenchymal and epithelial cells of the liver, lung, intestine, renal cortex, and urinary tract. Although HFH-11 exhibits a wide cellular expression pattern in the embryo, its adult expression pattern is restricted to epithelial cells of Lieberkühn's crypts of the intestine, the spermatocytes and spermatids of the testis, and the thymus and colon. HFH-11 expression is absent in adult hepatocytes, but its expression is reactivated in proliferating hepatocytes at 4, 24, and 48 h after partial hepatectomy. Consistent with these findings, we demonstrate that HFH-11 mRNA levels are stimulated by intratracheal administration of keratinocyte growth factor in adult lung and its expression in an adult endothelial cell line is reactivated in response to oxidative stress. These experiments show that the HFH-11 transcription factor is expressed in embryonic mesenchymal and epithelial cells and its expression is reactivated in these adult cell types by proliferative signals or oxidative stress.Cell-specific transcription relies on the combinatorial recognition of multiple cis-acting elements by families of cell-restricted transcription factors (80). One of these regulatory families is represented by the hepatocyte nuclear factor 3␣ (HNF-3␣), HNF-3␤, and HNF-3␥ proteins (43), which have homology in the winged helix DNA binding domain (12) and function in combination with other liver-enriched transcription factors to mediate hepatocyte-enriched transcription (17). The HNF-3␣ and -3␤ proteins also activate the transcription of genes important for respiratory epithelial cell function (7,14,35,40,60,82). The HNF-3 proteins thus appear to play an important transcriptional regulatory role in epithelial cell typespecific gene expression in adult tissues derived from gut endoderm.In the adult intestine, multipotent proliferative stem cells in Lieberkühn's crypts in the mouse intestine give rise to four terminally differentiated cell types: digestive and absorptive columnar enterocytes (representing the most abundant cell type), mucus-producing goblet cells, enteroendocrine cells, and Paneth cells (53). As the postmitotic enterocytes, goblet cells, and ente...

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 hepatocyte nuclear factor 3 (HNF-3) gene family is composed of three proteins (a4, 1, and 'y) that are transcription factors involved in the coordinate expression of several liver genes. All three proteins share strong homology in their DNA binding domains (region I) and are able to recognize the same DNA sequence. They also possess two similar stretches of amino acids at the carboxyl terminus (regions H and I) and a fourth segment of homology at the amino terminus (region 1V amino-terminal sequences defined by conserved region IV also contributed to transactivation, but region IV activity required the participation of the region II-r domain. Region IV is abundant in serine amino acids and contains two putative casein kinase I phosphorylation sites, a feature similar to protein motifs described for the transcription factors Pit-1/GHF-1 and HNF-1. Cellular differentiation during mammalian development is accompanied by the differential expression of tissue-specific genes (27). The identification of genes responsible for homeotic or developmental mutations in Drosophila melanogaster has shown that temporal cascades of transcription factors are involved in establishment of tissue-specific expression patterns (36,55). The existence of homologies between the homeotic proteins and mammalian transcription factors suggests that similar regulatory cascades are employed during mammalian development (20, 55). Since transcriptional initiation is the main regulatory step in the expression of tissue-specific genes, an understanding of transcriptional control will provide insight into mechanisms of cellular differentiation (27, 45). Tissue-specific gene transcription is maintained through the recognition of promoter sites by transcription factors which display restricted cellular activity (27). The functional characterization of these tissue-specific factors will identify protein regions involved in transcriptional activation and may provide insight into their regulatory mechanisms (3,16,25,35,48,50,57,62). The analysis of promoter regions regulating the expression of tissue-specific transcription factors may identify their position in the hierarchy of the developmental regulatory cascade (2,8,31,41,42,49,58,61,63).The analysis of transcriptional initiation has been the focus of considerable research because it is a major regulatory step in gene expression. The initiation complex is * Corresponding author.composed of an array of basal transcription factors (for example, TFII-A, -B, -D, -E, -F, and -G) that usually assemble at the TATA box sequence with RNA polymerase II (for reviews, see references 27, 45, and 52). In addition to the TATA sequences, promoter regions include multiple DNA binding motifs that bind sequence-specific transcription factors and elevate the rate of transcriptional initiation. The functional analysis of a number of transcription factors has demonstrated that these proteins are modular in structure and consist of domains that can function independently of one another. These analyses have identified r...

Hepatocyte nuclear factor (HNF)-3a, -3p8, and -3y are liver transcription factors that mediate the coor-