Search

We used transgenesis to explore the requirement for downregulation of hepatocyte nuclear factor 6 (HNF6) expression in the assembly, differentiation, and function of pancreatic islets. In vivo, HNF6 expression becomes downregulated in pancreatic endocrine cells at 18. 5 days post coitum (d.p.c.), when definitive islets first begin to organize. We used an islet-specific regulatory element (pdx1(PB)) from pancreatic/duodenal homeobox (pdx1) gene to maintain HNF6 expression in endocrine cells beyond 18.5 d.p.c. Transgenic animals were diabetic. HNF6-overexpressing islets were hyperplastic and remained very close to the pancreatic ducts. Strikingly, alpha, delta, and PP cells were increased in number and abnormally intermingled with islet beta cells. Although several mature beta cell markers were expressed in beta cells of transgenic islets, the glucose transporter GLUT2 was absent or severely reduced. As glucose uptake/metabolism is essential for insulin secretion, decreased GLUT2 may contribute to the etiology of diabetes in pdx1(PB)-HNF6 transgenics. Concordantly, blood insulin was not raised by glucose challenge, suggesting profound beta cell dysfunction. Thus, we have shown that HNF6 downregulation during islet ontogeny is critical to normal pancreas formation and function: continued expression impairs the clustering of endocrine cells and their separation from the ductal epithelium, disrupts the spatial organization of endocrine cell types within the islet, and severely compromises beta cell physiology, leading to overt diabetes.

The reactive oxygen intermediate H2O2 can function as a signaling molecule to activate gene expression. In this study, we demonstrate that oxidant stress induced by tumor necrosis factor alpha (TNFalpha) or H2O2 differentially regulates intercellular adhesion molecule-1 (ICAM-1) and interleukin-8 (IL-8) gene expression in endothelial and epithelial cells. Northern blot analysis revealed that TNFalpha induced both ICAM-1 and IL-8 expression in either the A549 lung epithelial cell line or the human microvessel endothelial cell line (HMEC-1). In contrast, H2O2 selectively induced only ICAM-1 in HMEC-1 and only IL-8 in A549. This cell type-specific pattern of IL-8 expression was also observed in several other endothelial and epithelial cells. TNFalpha induced greater IL-8 gene expression as compared with H2O2, but the kinetics of induction were similar. The induction of epithelial IL-8 message was accompanied by a corresponding increase in functional IL-8 protein secretion as determined by a neutrophil motility assay. The increased neutrophil motility stimulated by conditioned media from H2O2- or TNFalpha-exposed A549 cells was completely inhibited by an anti-IL-8 antibody. TNFalpha and H2O2 also induced a differential pattern of CC chemokine expression in A549. While TNFalpha induced both RANTES and MCP-1, H2O2 induced only MCP-1. These data suggest that epithelial cells under oxidant stress contribute to the inflammatory cytokine network by selective production of IL-8, MCP-1, and RANTES, which may critically influence the site-specific recruitment of leukocyte subsets.

We have previously described the isolation and characterization of genomic clones corresponding to the mouse alpha 1-antitrypsin gene (Krauter et al., DNA 5:29-36, 1986). In this report, we have analyzed the DNA sequences upstream of the RNA start site that direct hepatoma cell-specific expression of this gene when incorporated into recombinant plasmids. The 160 nucleotides 5' to the cap site direct low-level expression in hepatoma cells, and sequences between -520 and -160 bp upstream of the RNA start site functioned as a cell-specific enhancer of expression both with the alpha 1-antitrypsin promoter and when combined with a functional beta-globin promoter. Within the enhancer region, three binding sites for proteins present in hepatoma nuclear extracts were identified. The location of each site was positioned, using both methylation protection and methylation interference experiments. Each protein-binding site correlated with a functionally important region necessary for full enhancer activity. These experiments demonstrated a complex arrangement of regulatory elements comprising the alpha 1-antitrypsin enhancer. Significant qualitative differences exist between the findings presented here and the cis-acting elements operative in regulating expression of the human alpha 1-antitrypsin gene (Ciliberto et al., Cell 41:531-540, 1985; De Simone et al., EMBO J. 6:2759-2766, 1987).

The mouse genomic clone for the prealbumin (transthyretin) gene was cloned, and its upstream regulatory regions were analyzed. The 200 nucleotides 5' to the cap site when placed within a recombinant plasmid were sufficient to direct transient expression in HepG2 (human hepatoma) cells, but this DNA region did not support expression in HeLa cells. The sequence of the 200-nucleotide region is highly conserved between mouse and human DNA and can be considered a cell-specific promoter. Deletions of this promoter region identified a crucial element for cell-specific expression between 151 and 110 nucleotides 5' to the RNA start site. A region situated at about 1.6 to 2.15 kilobases upstream of the RNA start site was found to stimulate expression 10-fold in HepG2 cells but not in HeLa cells. This far upstream element was invertible and increased expression from the beta-globin promoter in HepG2 cells. Unlike the simian virus 40 enhancer, the prealbumin enhancer would not stimulate beta-globin synthesis in HeLa cells, and even the simian virus 40 enhancer did not stimulate the prealbumin promoter in HeLa cells. Thus, we identified in the prealbumin gene two DNA elements that respond in a cell-specific manner: a proximal promoter including a crucial sequence between -108 and -151 nucleotides and a distant enhancer element located between 1.6 and 2.15 kilobases upstream.

The hepatocyte nuclear factor 3(alpha) (HNF-3(alpha)), -3(beta), and -3(gamma) proteins share homology in the winged-helix/fork head DNA binding domain and mediate hepatocyte-enriched transcription of numerous genes whose expression is necessary for organ function. In this work, we identify a liver-enriched transcription factor, HNF-6, which recognizes the -138 to -126 region of the HNF-3(beta) promoter and binds the original HNF-3 site of the transthyretin promoter (-94 to -106). We show that HNF-6 and HNF-3 possess different DNA binding specificities by competition and methylation interference studies and are immunologically distinct. Site-directed mutagenesis of the HNF-6 sites in the HNF-3(beta) and transthyretin promoters diminishes reporter gene expression, suggesting that HNF-6 activates transcription of these promoters. Using the HNF-6 binding sequence DHWATTGAYTWWD (where W = A or T, Y = T or C, H is not G, and D is not C) determined by sequence comparison and methylation interference, we predicted that HNF-6 will bind to 22 additional hepatocyte-enriched genes. Of these potential target genes, we selected seven of the HNF-6 binding sequences and demonstrated that they bind the HNF-6 protein. These include promoter sequences from alpha-2 urinary globulin, alpha-1 antitrypsin, cytochrome P-450 2C13, L-type 6-phosphofructo-2-kinase, mouse major urinary protein, tryptophan oxygenase, and alpha-fetoprotein genes. HNF-6 binding activity was also found in the intestinal epithelial cell line HT29, and potential HNF-6 binding sites were present in intestinal sucrase isomaltase, cdx-2 homeodomain protein, and intestinal fatty acid binding protein promoter regions. These studies suggest that HNF-6 may regulate hepatocyte-specific genes and may play a role in epithelial cell differentiation of gut endoderm via regulation of HNF-3(beta).

Transthyretin (TTR) and alpha 1-antitrypsin (alpha 1-AT) are expressed at high levels in the liver and also in at least one other cell type. We report here a detailed analysis of the proximal regulatory region of the TTR gene, which has uncovered two new DNA-binding factors that are present mainly (or only) in hepatocytes. One of these new factors, hepatocyte nuclear factor 3 (HNF-3), binds to two sites that are crucial in TTR expression as well as to two additional sites in the alpha 1-AT proximal enhancer region. The second new factor, HNF-4, binds to two sites in TTR that are required for gene activity. We had previously identified binding sites for another hepatocyte-enriched DNA-binding protein (C/EBP or a relative thereof), and additional promoter-proximal sites for that protein in both TTR and alpha 1-AT are also reported here. From these results it seems clear that cell-specific expression is not simply the result of a single cell-specific factor for each gene but the result of a combination of such factors. The variation and distribution of such factors among different cell types could be an important basis for the coordinate expression of the TTR and alpha 1-AT genes in the liver or the discordant transcriptional activation of these genes in a few other cell types. The identification of such cell-enriched factors is a necessary prelude to understanding the basis for cell specificity.

We previously defined two distinct cell-specific DNA elements controlling the transient expression of the transthyretin gene in Hep G2 (human hepatoma) cells: a proximal promoter region (-202 base pairs [bp] to the cap site), and a far-upstream cell-specific enhancer located between 1.6 and 2.15 kilobases (kb) 5' of the cap site (R. H. Costa, E. Lai, and J. E. Darnell, Jr., Mol. Cell. Biol. 6:4697-4708, 1986). In this report, we located the effective transthyretin enhancer element within a 100-bp region between 1.96 and 1.86 kb 5' to the mRNA cap site. In Hep G2 nuclear extracts, three protein-binding sites within this minimal enhancer element were identified by gel mobility and methylation protection experiments. Each binding site was required for full enhancer activity in Hep G2 transient expression assays. Competition experiments in protein-binding assays suggested that two of the three sites were recognized by a similar factor and that the protein interaction with the third site was different. The nuclear protein(s) which bound to the two homologous sites was found mainly or only in cells of hepatic origin, suggesting an involvement of this region in the cell-specific function of this enhancer. The nuclear protein(s) recognizing the third enhancer region was also found in HeLa and spleen cells.

High-resolution transcription mapping localized a spliced 2.7-kilobase herpes simplex virus type 1 mRNA. The 4-kilobase intron of this transcript encodes a nested set of transcripts on the opposite DNA strand. The nucleotide sequence of the DNA encoding the left-hand and right-hand exons of the spliced transcript was determined, and the salient features are presented here. Of major interest is that both exons contained regions within several hundred bases of the splice donor and acceptor sites which showed homology to two regions of the Epstein-Barr virus genome, which are themselves 3 kilobases apart. The spliced herpes simplex virus transcript encoded a translational reading frame which could encode a protein with an approximate size of 75,000 daltons. This value is in agreement with in vitro translation data. The predicted amino acid sequence of the herpes simplex virus protein had significant homology with putative amino acid sequences encoded by the homologous Epstein-Barr virus DNA sequences.

A major, late 6-kilobase (6-kb) mRNa mapping in the large unique region of herpes simplex virus type 1 (HSV-1) was characterized by using two recombinant DNA clones, one containing EcoRI fragment G (0.190 to 0.30 map units) in lambda. WES.B (L. Enquist, M. Madden, P. Schiop-Stansly, and G. Vandl Woude, Science 203:541-544, 1979) and one containing HindIII fragment J (0.181 to 0.259 map units) in pBR322. This 6-kb mRNA had its 3' end to the left of 0.231 on the prototypical arrangement of the HSV-1 genome and was transcribed from right to left. It was bounded on both sides by regions containing a large number of distinct mRNA species, and its 3' end was partially colinear with a 1.5-kb mRNA which encoded a 35,000-dalton polypeptide. The 6-kb mRNA encoded a 155,000-dalton polypeptide which was shown to be the only one of this size detectable by hybrid-arrested translation encoded by late polyadenylated polyribosomal RNA. The S1 nuclease mapping experiments indicated that there were no introns in the coding sequence for this mRNA and that its 3' end mapped approximately 800 nucleotides to the left of the BglII site at 0.231, whereas its 5' end extended very close to the BamHI site at 0.266.

We have isolated as recombinant DNA clones, in the plasmid pBR322, regions of the herpesvirus type 1 genome spanning the region between 0.53 and 0.6 on the prototypical arrangement. This 11,000-base-pair region corresponds to 10% of the large unique region and encodes five major and several minor mRNA species abundant at different times after infection, which range in length from 7 to 1 kilobase. In this report, we have used RNA transfer blots and S1 nuclease digestion of hybrids between viral DNA and polyribosomal RNA to precisely localize (+/- 0.1 kilobase) these mRNA's. Comparison of neutral and alkaline gels of S1 nuclease-digested hybrids indicates no internal introns in the coding sequences of these mRNA's, although noncontiguous leader sequences near (ca. 0.1 kilobase) the 5' ends of any or all mRNA's could not be excluded. The 5' ends of several late mRNA's that are encoded opposite DNA strands map very close to one another, and the 3' ends of a major late and a major early mRNA, which are partially colinear, terminate in the same region. In vitro translation of the viral mRNA's isolated by hybridization with DNA bound to cellulose and fractionation of mRNA species on denaturing agarose gels allowed us to assign specific polypeptide products to each of the mRNA's characterized. Among other results, it was demonstrated unequivocally that two major late mRNA's, which partially overlap, encode the same polypeptide.