Mutant cystic fibrosis transmembrane conductance regulator inhibits acidification and apoptosis in C127 cells: possible relevance to cystic fibrosis.

We have shown elsewhere that acidification is an early event in apoptosis, preceding DNA cleavage. Cells expressing the most common mutation (delF508) of the cystic fibrosis transmembrane regulator (CFTR) exhibit a higher resting intracellular pH and are unable to secrete chloride and bicarbonate in response to cAMP. We hypothesized that defective acidification in cells expressing delF508 CFTR would interfere with the acidification that accompanies apoptosis, which in turn, would prevent endonuclease activation and cleavage of DNA. We therefore determined whether the function of the CFTR would affect the process of apoptosis in mouse mammary epithelial C127 cells stably transfected with the wild-type CFTR (C127/wt) or the delF508 mutation of the CFTR (C127/508). C127 cells possessed an acid endonuclease capable of DNA degradation at low pH. Sixteen hours after treatment with cycloheximide, C127/wt cells underwent cytoplasmic acidification. In contrast, C127/508 cells failed to demonstrate acidification. Furthermore, the C127/508 cells did not show nuclear condensation or DNA fragmentation detected by in situ nick-end labeling after treatment with cycloheximide or etoposide, in contrast to the characteristic features of apoptosis demonstrated by the C127/wt cells. Measurement of cell viability indicated a preservation of cell viability in C127/508 cells but not in C127/wt cells. That this resistance to the induction of apoptosis depended upon the loss of CFTR activity is shown by the finding that inhibition of the CFTR with diphenylamine carboxylate in C127/wt cells conferred similar protection. These findings suggest a role for the CFTR in acidification during the initiation of apoptosis in epithelial cells and imply that a failure to undergo programmed cell death could contribute to the pathogenesis of cystic fibrosis.

The virion glycoproteins of Ebola viruses are encoded in two reading frames and are expressed through transcriptional editing.

In late 1994 and early 1995, Ebola (EBO) virus dramatically reemerged in Africa, causing human disease in the Ivory Coast and Zaire. Analysis of the entire glycoprotein genes of these viruses and those of other EBO virus subtypes has shown that the virion glycoprotein (130 kDa) is encoded in two reading frames, which are linked by transcriptional editing. This editing results in the addition of an extra nontemplated adenosine within a run of seven adenosines near the middle of the coding region. The primary gene product is a smaller (50-70 kDa), nonstructural, secreted glycoprotein, which is produced in large amounts and has an unknown function. Phylogenetic analysis indicates that EBO virus subtypes are genetically diverse and that the recent Ivory Coast isolate represents a new (fourth) subtype of EBO virus. In contrast, the EBO virus isolate from the 1995 outbreak in Kikwit, Zaire, is virtually identical to the virus that caused a similar epidemic in Yambuku, Zaire, almost 20 years earlier. This genetic stability may indicate that EBO viruses have coevolved with their natural reservoirs and do not change appreciably in the wild.

The response regulator SprE controls the stability of RpoS.

In Escherichia coli, the sigma factor, RpoS, is a central regulator in stationary-phase cells. We have identified a gene, sprE (stationary-phase regulator), as essential for the negative regulation of rpoS expression. SprE negatively regulates the rpoS gene product at the level of protein stability, perhaps in response to nutrient availability. The ability of SprE to destabilize RpoS is dependent on the ClpX/ClpP protease. Based on homology, SprE is a member of the response regulator family of proteins. SprE is the first response regulator identified that is implicated in the control of protein stability. Moreover, SprE is the first reported protein that appears to regulate rpoS in response to a specific environmental parameter.

A role for the Msx-1 homeodomain in transcriptional regulation: residues in the N-terminal arm mediate TATA binding protein interaction and transcriptional repression.

In a previous study we showed that the murine homeodomain protein Msx-1 is a potent transcriptional repressor and that this activity is independent of its DNA binding function. The implication of these findings is that repression by Msx-1 is mediated through its association with certain protein factors rather than through its interaction with DNA recognition sites, which prompted investigation of the relevant protein factors. Here we show that Msx-1 interacts directly with the TATA binding protein (TBP) but not with several other general transcription factors. This interaction is mediated by the Msx-1 homeodomain, specifically through residues in the N-terminal arm. These same N-terminal arm residues are required for repression by Msx-1, suggesting a functional relationship between TBP association and transcriptional repression. This is further supported by the observation that addition of excess TBP blocks the repressor action of Msx-1 in in vitro transcription assays. Finally, DNA binding activity is separable from both TBP interaction and repression, which further shows that these other activities of the Msx-1 homeodomain are distinct. Therefore, these findings define a role for the Msx-1 homeodomain, particularly the N-terminal arm residues in protein-protein interaction and transcriptional repression, and implicate a more complex role overall for homeodomains in transcriptional regulation.

Human stanniocalcin: a possible hormonal regulator of mineral metabolism.

We have isolated a human cDNA clone encoding the mammalian homolog of stanniocalcin (STC), a calcium- and phosphate-regulating hormone that was first described in fishes where it functions in preventing hypercalcemia. STC has a unique amino acid sequence and, until now, has remained one of the few polypeptide hormones never described in higher vertebrates. Human STC (hSTC) was found to be 247 amino acids long and to share 73% amino acid sequence similarity with fish STC. Polyclonal antibodies to recombinant hSTC localized to a distinct cell type in the nephron tubule, suggesting kidney as a possible site of synthesis. Recombinant hSTC inhibited the gill transport of calcium when administered to fish and stimulated renal phosphate reabsorption in the rat. The evidence suggests that mammalian STC, like its piscine counterpart, is a regulator of mineral homeostasis.

Azotobacter vinelandii NIFL is a flavoprotein that modulates transcriptional activation of nitrogen-fixation genes via a redox-sensitive switch.

The NIFL regulatory protein controls transcriptional activation of nitrogen fixation (nif) genes in Azotobacter vinelandii by direct interaction with the enhancer binding protein NIFA. Modulation of NIFA activity by NIFL, in vivo occurs in response to external oxygen concentration or the level of fixed nitrogen. Spectral features of purified NIFL and chromatographic analysis indicate that it is a flavoprotein with FAD as the prosthetic group, which undergoes reduction in the presence of sodium dithionite. Under anaerobic conditions, the oxidized form of NIFL inhibits transcriptional activation by NIFA in vitro, and this inhibition is reversed when NIFL is in the reduced form. Hence NIFL is a redox-sensitive regulatory protein and may represent a type of flavoprotein in which electron transfer is not coupled to an obvious catalytic activity. In addition to its ability to act as a redox sensor, the activity of NIFL is also responsive to adenosine nucleotides, particularly ADP. This response overrides the influence of redox status on NIFL and is also observed with refolded NIFL apoprotein, which lacks the flavin moiety. These observations suggest that both energy and redox status are important determinants of nif gene regulation in vivo.

Transcriptional regulation of human inducible nitric oxide synthase (NOS2) gene by cytokines: initial analysis of the human NOS2 promoter.

The expression of inducible nitric oxide synthase (NOS2) is complex and is regulated in part by gene transcription. In this investigation we studied the regulation of NOS2 in a human liver epithelial cell line (AKN-1) which expresses high levels of NOS2 mRNA and protein in response to tumor necrosis factor alpha, interleukin 1 beta, and interferon gamma (cytokine mix, CM). Nuclear run-on analysis revealed that CM transcriptionally activated the human NOS2 gene. To delineate the cytokine-responsive regions of the human NOS2 promoter, we stimulated AKN-1 cells with CM following transfection of NOS2 luciferase constructs. Analysis of the first 3.8 kb upstream of the NOS2 gene demonstrated basal promoter activity but failed to show any cytokine-inducible activity. However, 3- to 5-fold inductions of luciferase activity were seen in constructs extending up to -5.8 and -7.0 kg, and a 10-fold increase was seen upon transfection of a -16 kb construct. Further analysis of various NOS2 luciferase constructs ligated upstream of the thymidine kinase promoter identified three regions containing cytokine-responsive elements in the human NOS2 gene: -3.8 to -5.8, -5.8 to -7.0, and -7.0 to -16 kb. These results are in marked contrast with the murine macrophage NOS2 promoter in which only 1 kb of the proximal 5' flanking region is necessary to confer inducibility to lipopolysaccharide and interferon gamma. These data demonstrate that the human NOS2 gene is transcriptionally regulated by cytokines and identify multiple cytokine-responsive regions in the 5' flanking region of the human NOS2 gene.

Uptake of fluorescent dyes associated with the functional expression of the cystic fibrosis transmembrane conductance regulator in epithelial cells.

Specific mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), the most common autosomal recessive fatal genetic disease of Caucasians, result in the loss of epithelial cell adenosine 3',5'-cyclic-monophosphate (cAMP)-stimulated Cl- conductance. We show that the influx of a fluorescent dye, dihydrorhodamine 6G (dR6G), is increased in cells expressing human CFTR after retrovirus- and adenovirus-mediated gene transfer. dR6G influx is stimulated by cAMP and is inhibited by antagonists of cAMP action. Dye uptake is ATP-dependent and inhibited by Cl- removal or the addition of 10 mM SCN-. Increased staining is associated with functional activation of CFTR Cl- permeability. dR6G staining enables both the fluorescent assessment of CFTR function and the identification of successfully corrected cells after gene therapy.

Transcriptional activation by protein-induced DNA bending: evidence for a DNA structural transmission model.

Integration host factor (IHF) is a DNA-bending protein that binds to an upstream activating sequence (UAS1) and, on a negatively supercoiled DNA template, activates transcription from the ilvPG promoter of the ilvG-MEDA operon of Escherichia coli. The transcriptional initiation site of the ilvGMEDA operon is located 92 bp downstream of UAS1. Activation is still observed when the orientation of the upstream IHF binding site is reversed. This manipulation places the IHF binding site on the opposite face of the DNA helix, directs the IHF-induced DNA bend in the opposite direction, and presents the opposite face of the nonsymmetrical, heterodimeric, IHF molecule to the downstream RNA polymerase. Lymphoid enhancer-binding factor, LEF-1, is a DNA-bending, lymphoid-specific, mammalian transcription factor that shares no amino acid sequence similarity with IHF. When the IHF site in UAS1 is replaced with a LEF-1 site, LEF-1 activates transcription from the downstream ilvPG promoter in E. coli as well as it is activated by its natural activator, IHF. These results suggest that specific interactions between IHF and RNA polymerase are not required for activation. The results of DNA structural studies show that IHF forms a protein-DNA complex in the UAS1 region that, in the absence of RNA polymerase, alters the structure of the DNA helix in the -10 hexanucleotide region of the downstream ilvPG promoter. The results of in vitro abortive transcription assays show that IIIF also increases the apparent rate of RNA polymerase isomerization from a closed to an open complex. We suggest, therefore, that IHF activates transcription by forming a higher-order protein-DNA complex in the UAS1 region that structurally alters the DNA helix in a way that facilitates open complex formation at the downstream ilvPG promoter site.

Transcriptional activation of the mouse obese (ob) gene by CCAAT/enhancer binding protein alpha.

Like other adipocyte genes that are transcriptionally activated by CCAAT/enhancer binding protein alpha (C/EBP alpha) during preadipocyte differentiation, expression of the mouse obese (ob) gene is immediately preceded by the expression of C/EBP alpha. While the 5' flanking region of the mouse ob gene contains several consensus C/EBP binding sites, only one of these sites appears to be functional. DNase I cleavage inhibition patterns (footprinting) of the ob gene promoter revealed that recombinant C/EBP alpha, as well as a nuclear factor present in fully differentiated 3T3-L1 adipocytes, but present at a much lower level in preadipocytes, protects the same region between nucleotides -58 and -42 relative to the transcriptional start site. Electrophoretic mobility-shift analysis using nuclear extracts from adipose tissue or 3T3-L1 adipocytes and an oligonucleotide probe corresponding to a consensus C/EBP binding site at nucleotides -55 to -47 generated a specific protein-oligonucleotide complex that was supershifted by antibody against C/EBP alpha. Probes corresponding to two upstream consensus C/EBP binding sites failed to generate protein-oligonucleotide complexes. Cotransfection of a C/EBP alpha expression vector into 3T3-L1 cells with a series of 5' truncated ob gene promoter constructs activated reporter gene expression with all constructs containing the proximal C/EBP binding site (nucleotides -55 to -47). Mutation of this site blocked transactivation by C/EBP alpha. Taken together, these findings implicate C/EBP alpha as a transcriptional activator of the ob gene promoter and identify the functional C/EBP binding site in the promoter.

Transcriptional activation of the human proliferating-cell nuclear antigen promoter by p53.

Proliferating-cell nuclear antigen (PCNA) is a DNA damage-inducible protein that performs an essential function in DNA replication and repair as an auxiliary factor for DNA polymerases delta and epsilon. Examination of the human PCNA promoter DNA sequence revealed a site with homology to the consensus DNA sequence bound by p53. PCNA promoter fragments with this site intact bound p53 in vitro and were transcriptionally activated by wild-type p53 in transient expression assays in SAOS-2 cells. The resident p53-binding site could be functionally substituted by a previously described p53-binding site from the ribosomal gene cluster. A plasmid expressing a mutated version of p53 derived from a patient with Li-Fraumeni syndrome failed to activate the PCNA promoter in the cotransfection assay. In different cell types, activation of the PCNA promoter by the p53-binding sequence correlated with the status of p53. Activation of the PCNA promoter by wild-type p53 depends upon the level of p53 expression. This concentration dependence and cell type specificity reconciles the observations presented here with prior results indicating that wild-type p53 represses the PCNA promoter. These findings provide a mechanism whereby p53 modulates activation of PCNA expression as a cellular response to DNA damage.

Molecular mechanisms of dexamethasone inhibition of nitric oxide synthase expression in interleukin 1 beta-stimulated mesangial cells: evidence for the involvement of transcriptional and posttranscriptional regulation.

Inducible nitric oxide synthase (iNOS; EC 1.14.13.39) is expressed in rat glomerular mesangial cells upon exposure to the inflammatory cytokine interleukin 1 beta (IL-1 beta). We have reported that nanomolar concentrations of dexamethasone suppress IL-1 beta-induced iNOS protein expression and production of nitrite, the stable end product of NO formation, without affecting IL-1 beta-triggered increase in iNOS mRNA levels. We now have studied the mechanisms by which dexamethasone suppresses IL-1 beta-stimulated iNOS expression in mesangial cells. Surprisingly, nuclear run-on transcription experiments demonstrate that dexamethasone markedly attenuates IL-1 beta-induced iNOS gene transcription. However, this is counteracted by a prolongation of the half-life of iNOS mRNA from 1 h to 2.5 h by dexamethasone. Moreover, dexamethasone drastically reduces the amount of iNOS protein by reduction of iNOS mRNA translation and increased degradation of iNOS protein. These results indicate that glucocorticoids act at multiple levels to regulate iNOS expression, thus providing important insights into the treatment of inflammatory diseases.

The transcriptional transactivator of simian foamy virus 1 binds to a DNA target element in the viral internal promoter.

The transcriptional transactivator (Tas) of simian foamy virus type 1 strongly augments gene expression directed by both the promoter in the viral long terminal repeat and the newly discovered internal promoter located within the env gene. A region of 121 bp, located immediately 5' to the TATA box in the internal promoter, is required for transactivation by Tas. The present study aimed to identify the precise Tas-responsive target(s) in this region and to determine the role of Tas in transcriptional regulation. By analysis of both clustered-site mutations and hybrid promoters in transient expression assays in murine and simian cells, two separate sequence elements within this 121-bp region were shown to be Tas-dependent transcriptional enhancers. These targets, each < 30 bp in length and displaying no apparent sequence homology one to the other, are designated the promoter-proximal and promoter-distal elements. By means of the gel electrophoresis mobility-shift assays, using purified glutathione S-transferase-Tas fusion protein expressed in Escherichia coli, the target proximal to the TATA box exhibited strong binding to glutathione S-transferase-Tas, whereas the distal element appears not to bind. In addition, footprint analysis revealed that 26 bp in the promoter proximal element was protected by glutathione S-transferase-Tas from DNase I. We propose a model for transactivation of the simian foamy virus type 1 internal promoter in which Tas interacts directly with the proximal target element positioned immediately 5' to the TATA box. In this model, Tas attached to this element is presumed to interact with a component(s) of the cellular RNA polymerase II initiation complex and thereby enhance transcription directed by the viral internal promoter.

Selective repression of transcriptional activators by Pbx1 does not require the homeodomain.

PBX1 is a homeobox-containing gene identified as the chromosome 1 participant of the t(1;19) chromosomal translocation of childhood pre-B-cell acute lymphoblastic leukemia. This translocation produces a fusion gene encoding the chimeric oncoprotein E2A-Pbx1, which can induce both acute myeloid and T-lymphoid leukemia in mice. The binding of Pbx1 to DNA is weak; however, both Pbx1 and E2A-Pbx1 exhibit tight binding to specific DNA motifs in conjunction with certain other homeodomain proteins, and E2A-Pbx1 activates transcription through these motifs, whereas Pbx1 does not. In this report, we investigate potential transcriptional functions of Pbx1, using transient expression assays. While no segments of Pbx1 activated transcription, an internal domain of Pbx1 repressed transcription induced by the activation domain of Sp1, but not by the activation domains of VP16 or p53. This Pbx1 domain, which lies upstream of the homeodomain and is highly conserved among Pbx proteins, is thus predicted to bind a specific transcription factor. Surprisingly, the repression activity of Pbx1 did not require homeodomain-dependent DNA binding. Thus, Pbx1 may be able to alter gene transcription by both DNA-binding-dependent and DNA-binding-independent mechanisms.