Hepatocyte nuclear factor (HNF)-3α, -3β, and -3γ are liver transcription factors that mediate the coordinate expression of a number of hepatocyte-specific genes. The HNF-3 proteins share ...DNA-binding-domain homology among themselves and with the Drosophila homeotic protein forkhead (fkh). The HNF-3/fkh DNA-binding domain constitutes an uncharacterized protein motif that recognizes its cognate DNA binding site as a monomer. Additional HNF-3/fkh-related proteins are known to be required for determination events during embryogenesis in Drosophila and Xenopus. In this report, we describe the isolation of nine additional HNF-3/fkh homologue (HFH) clones from rodent tissue cDNAs by using both low-stringency hybridization and a polymerase chain reaction protocol. Many of the HFH genes exhibit a tissue-restricted expression pattern and are transcribed in tissues other than liver, including brain, kidney, lung, and intestine. The HNF-3/fkh motif therefore comprises a large gene family of transcription factors that play a role in tissue-specific gene regulation and development.
The hepatocyte nuclear factor-3 (HNF-3)/ fork head homolog (HFH) proteins are an extensive family of transcription factors, which share homology in the winged helix DNA binding
domain. Members of the ...HFH/winged helix family have been implicated in cell fate determination during pattern formation, in
organogenesis, and in cell-type-specific gene expression. In this study we isolated a full-length HFH-3 cDNA clone from a
human kidney library which encoded a 351-amino acid protein containing a centrally located winged helix DNA binding domain.
We demonstrate that HFH-3 is a potent transcriptional activator requiring 138 C-terminal residues for activity. We used in situ hybridization to demonstrate that HFH-3 expression is restricted to the epithelium of the renal distal convoluted tubules.
We determined the HFH-3 DNA binding consensus sequence by in vitro DNA binding site selection using recombinant HFH-3 protein and used this consensus sequence to identify putative HFH-3 target
genes expressed there. These putative HFH-3 target genes include the Na/K-ATPase, Na/H and anion exchangers, E-cadherin, and
mineralocorticoid receptor genes as well as genes for the transcription factors HNF-1, vHNF-1, and HNF-4.
The proU operon of Salmonella typhimurium is induced by conditions of high osmolality. The cis-acting sequences that mediate osmotic control of transcription were characterized by deletion analysis. ...The nucleotide sequence between -60 and +274 (relative to the transcription start point) is sufficient for normal osmotic control. Deletions that removed sequences upstream of position +274 but left the promoter intact resulted in greatly increased expression from the proU promoter in the absence of osmotic stress. Thus, the transcription control region of the proU operon consists of two discrete components: (i) the promoter and (ii) a negatively acting site that overlaps the coding sequence of the first structural gene of the operon, proV. That this negative regulatory element is a transcriptional terminator or mRNA processing site was ruled out. Our results suggest that the negative regulatory element behaves as a transcriptional silencer that inhibits transcription initiation at the proU promoter in medium of low osmolality by some action at a distance. We propose several possible mechanisms for the function of this regulatory site.
The hepatocyte nuclear factor 3 (HNF-3)/fork head (fkh) proteins consist of an extensive family of tissue-specific and developmental gene regulators which share homology within the winged helix DNA ...binding motif. We report on the isolation of a new family member, HNF-3/forkhead homolog 8 (HFH-8), from lung cDNA libraries and the derivation of the complete amino acid sequences for the HFH-8 protein as well as previously identified HFH-1 and HFH-4 proteins. The HFH proteins contain several sequence motifs found in activation domains of other transcription factors and HNF-3/fkh family members. In situ hybridization with the HNF-3, HFH-4, and HFH-8 probes in adult lung demonstrate that the HNF-3/fkh cellular expression patterns are regionally specified. Whereas HNF-3 alpha and HNF-3 beta are normally coexpressed in the hepatocyte, their expression patterns in the lung are different. The HNF-3 beta and HFH-4 genes are coexpressed in the bronchiolar epithelium (clara cells), whereas the HNF-3 beta probe exhibits prominent hybridization with the smooth muscle surrounding arterioles and bronchioles. In contrast, HFH-8 probes labeled the type II pneumocyte cells lining the respiratory surfaces of terminal bronchioles and alveolar sac. We have identified an HNF-3 consensus DNA binding sequence in the proximal surfactant protein B (SPB) promoter region (SPB-f2, -78 to -88). SPB gene transcription is restricted to bronchiolar and alveolar epithelium which colocalizes with the expression pattern of the HNF-3 alpha and HFH-8 genes, respectively. We show that the SPB-f2 sequence is recognized by both HNF-3 alpha and HFH-8 proteins and that these cDNA expression vectors activate the SPB promoter in cotransfection assays through the HNF-3 consensus sequence. Our results suggest that SPB promoter activity is regulated by HNF-3 alpha and HFH-8 proteins in a cell type-specific manner.
The hepatocyte nuclear factor 3/
fork head homolog (HFH) proteins are an extensive family of transcription factors which share homology in the winged helix DNA binding domain. Members of the winged ...helix family have been implicated in cell fate determination during pattern formation, in organogenesis and in cell type-specific gene expression. In this study, we used in situ hybridization to identify the cellular expression pattern of the winged helix transcription factor, HFH-8, during mouse embryonic development. We showed that HFH-8 expression initiates during the primitive streak stage of mouse embryogenesis in the extraembryonic mesoderm and in the lateral mesoderm which gives rise to the somatopleuric and splanchnopleuric mesoderm. During organogenesis, HFH-8 expression is found in the splanchnic mesoderm in close apposition of the gut endoderm, suggesting a role in mesenchymal–epithelial induction of lung and gut morphogenesis. HFH-8 expression continues in lateral mesoderm-derived tissue throughout mouse development. HFH-8 expression is observed in the mesenchymal cells of the oral cavity, esophagus, trachea, lung, intestine, dorsal aorta and intersomitic arteries, but not in the vasculature of the head, liver, kidney or heart. Consistent with these embryonic expression studies, adult HFH-8 expression is restricted to the endothelium and connective fibroblasts of the alveolar sac and in the lamina propria and smooth muscle of the intestine. We also show that several adult endothelial cell lines maintain abundant HFH-8 expression. Furthermore, we used our determined HFH-8 consensus sequence to identify putative target genes expressed in pulmonary and intestinal mesenchymal cells. Cotransfection assays with one of these target promoters, P-selectin, demonstrated that HFH-8 expression was required for IL-6 stimulation of P-selectin promoter activity and suggest that HFH-8 is involved in mediating its cell-specific transcriptional activation in response to cytokines.
A 100-amino-acid DNA-binding motif, known as the winged helix, was first identified in the mammalian hepatocyte nuclear factor-3 (HNF-3) and
Drosophila fork head family of transcription factors. ...Subsequently, more than 40 different genes that contain the winged helix motif have been identified. In the studies described here, we have determined the murine chromosomal location of eight members of this gene family, HFH-1, HFH-3, HFH-4, HFH-5, HFH-6, HFH-8, BF-1, and BF-2, by interspecific backcross analysis. These genes, designated HNF-3 fork head homolog 1 (
Hfh1),
Hfh3, Hfh4, Hfh5, Hfh6, Hfh8, Hfh9, and
Hfh10, respectively, mapped to 6 different mouse autosomes and are thus well dispersed throughout the mouse genome. Based on this mapping information, we predict the chromosomal location of these genes in humans and discuss the potential of these genes as candidates for uncloned mouse mutations.
We investigated factors influencing the presence of the thiol glutathione (GSH) in estuarine waters. Our study addressed thiol phase-association, the biological release from algal cultures, and the ...role of copper in both thiol release and preservation. Our measurements in three diverse estuaries in the continental United States (San Diego Bay, Cape Fear Estuary, and Norfolk Estuary) show that dissolved GSH, present at sub-nanomolar levels, is preferentially partitioned into the ultra-filtrate fraction (<1 kDa) in comparison with dissolved organic carbon (DOC). Concentrations of GSH generally increased with increases in total copper (Cu) levels, although large variability was observed among estuaries. In 30-h exposure experiments, release of dissolved GSH from the diatom Thalassiosira weissflogii into organic ligand-free experimental media was a strong function of added Cu concentration. The released GSH increased from about 0.02 to 0.27 fmol/cell as Cu was increased from the background level (0.5 nM) to 310 nM in the modified Aquil media. However, excretion of GSH was lower (up to 0.13 fmol/cell) when cells were grown in surface waters of San Diego Bay, despite much higher total Cu concentrations. Experiments conducted in-situ in San Diego Bay water indicated that high concentrations of added Cu destabilized GSH, while both Mn(II) and natural colloids promoted GSH stability. In contrast, laboratory experiments in synthetic media indicated that moderate levels of added Cu enhanced GSH stability.
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