Biomedical subjects

J E Darnell

Publications and source records attributed to J E Darnell.

At least 91 records · Page 5Linked to original sources

PubMed1993

A common nuclear signal transduction pathway activated by growth factor and cytokine receptors.

Growth factors and cytokines act through cell surface receptors with different biochemical properties. Yet each type of receptor can elicit similar as well as distinct biological responses in target cells, suggesting that distinct classes of receptors activate common gene sets. Epidermal growth factor, interferon-gamma, and interleukin-6 all activated, through direct tyrosine phosphorylation, latent cytoplasmic transcription factors that recognized similar DNA elements. However, different ligands activated different patterns of factors with distinct DNA-binding specificities in the same and different cells. Thus, unrelated receptors may activate a common nuclear signal transduction pathway that, through differential use of latent cytoplasmic proteins, permits these receptors to regulate both common and unique sets of genes.

Base Sequence↗

PubMed1993

Induction of the Ly-6A/E gene by interferon alpha/beta and gamma requires a DNA element to which a tyrosine-phosphorylated 91-kDa protein binds.

The murine Ly-6A/E gene is transcriptionally induced in cells exposed to interferon alpha/beta or gamma (IFN-alpha/beta or IFN-gamma). Analysis of the 5' flanking sequence using reporter plasmids that contain upstream elements of the Ly-6E gene has previously identified an approximately 850-base-pair IFN-responsive region that lacked an IFN-alpha-stimulated response element (ISRE), the element present and required for an IFN-alpha response of a number of genes. Analysis by deletion and stable transfection of the IFN-responsive region of the Ly-6E promoter has defined an 80-base-pair region containing an IFN-gamma activation site (GAS) but no ISRE that allows IFN-gamma and IFN-alpha inducibility of the Ly-6E gene. As tested by specific antiserum, a 91-kDa protein known to be activated in IFN-alpha- or IFN-gamma-treated cells binds to the GAS element from the Ly-6E promoter. The 91-kDa protein exists as an inactive cytoplasmic precursor and depends on tyrosine phosphorylation for its activation. Thus the same 91-kDa protein appears to act in the signal transduction pathways of both types of IFN for the Ly-6-A/E gene.

Animals↗

PubMed1993

Interferon gamma-induced transcription of the high-affinity Fc receptor for IgG requires assembly of a complex that includes the 91-kDa subunit of transcription factor ISGF3.

A 39-nt DNA sequence, the interferon gamma (IFN-gamma) response region (GRR), is necessary for the IFN-gamma-induced transcription of the high-affinity Fc receptor for IgG (Fc gamma RI) and sufficient for the IFN-gamma-induced transcription of transfected plasmids. By using extracts from IFN-gamma-treated cells, three protein complexes will assemble in vitro on a 9-nt core region in the 3' domain of the GRR. The sequence of this core resembles the IFN-gamma-activated sequence (GAS) described for the GBP gene. Mutations in this GAS core region prevent complex assembly and result in the loss of IFN-gamma induction of reporter constructs containing the mutation. In addition to the GAS core region, a 5' region of the GRR is necessary for optimal IFN-gamma induction and for formation of one of the DNA-protein complexes. By antibody reactivity, we show that a 91-kDa protein, first identified as a component of ISGF3, the IFN-alpha-induced transcription complex, is present in at least two of the DNA-protein complexes. IFN-alpha can induce the formation of the faster-migrating 91-kDa protein-GAS complex but not the slower-migrating complex. Furthermore, IFN-alpha does not result in appreciable transcriptional activation of Fc gamma RI or constructs containing the GRR. Thus, these data demonstrate that the IFN-gamma-activated 91-kDa protein is required for IFN-gamma induction of Fc gamma RI and suggest that an additional complex may be required for optimal expression and specificity.

Antibody Specificity↗

PubMed1993

The expression pattern of a Drosophila homolog to the mouse transcription factor HNF-4 suggests a determinative role in gut formation.

A Drosophila gene, HNF-4(D), was selected by cross-hybridization with a probe to rat HNF-4 (hepatocyte nuclear factor 4), a steroid hormone receptor super-family member that plays an important role in liver-specific gene expression. The Drosophila gene matched the mouse gene in 60 out of 66 amino acids in the zinc finger DNA binding domain and in 140 out of 206 amino acids in the domain that specifies dimerization and ligand binding. HNF-4(D) is expressed in developing Drosophila embryos in mid-gut, fat bodies and malpighian tubules, a striking similarity to its limited expression in the adult intestine, liver and kidney of the mouse. Furthermore, Drosophila mutant that has a chromosome deletion spanning the HNF-4(D) locus fails to develop tissues where HNF-4(D) is expressed during late embryogenesis. These findings together with the earlier realization that the rat hepatocyte nuclear factor 3 (HNF-3) and forkhead, a Drosophila gene required for anterior and posterior gut formation, had virtually the identical DNA binding domain, lead us to speculate that a group of genes that participate in gut formation of invertebrates has survived in evolution to perform similar functions in mammals.

Amino Acid Sequence↗

PubMed1993

Complementation of a mutant cell line: central role of the 91 kDa polypeptide of ISGF3 in the interferon-alpha and -gamma signal transduction pathways.

Mutants in complementation group U3, completely defective in the response of all genes tested to interferons (IFNs) alpha and gamma, do not express the 91 and 84 kDa polypeptide components of interferon-stimulated gene factor 3 (ISGF3), a transcription factor known to play a primary role in the IFN-alpha response pathway. The 91 and 84 kDa polypeptides are products of a single gene. They result from differential splicing and differ only in a 38 amino acid extension at the C-terminus of the 91 kDa polypeptide. Complementation of U3 mutants with cDNA constructs expressing the 91 kDa product at levels comparable to those observed in induced wild-type cells completely restored the response to both IFN-alpha and -gamma and the ability to form ISGF3. Complementation with the 84 kDa component similarly restored the ability to form ISGF3 and, albeit to a lower level, the IFN-alpha response of all genes tested so far. It failed, however, to restore the IFN-gamma response of any gene analysed. The precise nature of the DNA motifs and combination of factors required for the transcriptional response of all genes inducible by IFN-alpha and -gamma remains to be established. The results presented here, however, emphasize the apparent general requirement of the 91 kDa polypeptide in the primary transcriptional response to both types of IFN.

Base Sequence↗

PubMed1993

Sequential expression of HNF-3 beta and HNF-3 alpha by embryonic organizing centers: the dorsal lip/node, notochord and floor plate.

Axial patterning in the nervous system of vertebrate embryos depends on inductive signals that derive from the organizer region (the dorsal lip in amphibians and the node in birds and mammals) and leter from the notochord and floor plate. Previous studies have shown that Pintallavis, a member of the HNF-3/fork head transcription factor family, is expressed selectively by these cell groups in frog embryos and may be involved in regulating neural development. We report here that in early rat and mouse embryos, the embryonic endoderm, the node, the notochord and the floor plate express two related transcription factors, HNF-3 alpha and HNF-3 beta, which also function in the control of liver cell differentiation. Early embryonic tissues express variant forms of HNF-3 beta which derive from the use of 5' alternative exons. Within the organizer region and notochord, HNF-3 beta and HNF-3 alpha have distinct temporal patterns of expression and appear in partially overlapping domains. The early expression pattern of mammalian HNF-3 beta in the node, notochord and midline neural plate cells is similar to that of Pintallavis in frog embryos. There does not appear to be a Pintallavis homologue in mice. This prompted us to isolate and analyze the expression of the frog HNF-3 beta gene. In frog embryos, HNF-3 beta is expressed in the dorsal lip, pharyngeal endoderm and floor plate. In contrast to mammalian HNF-3 beta, the onset of frog HNF-3 beta expression in neural tissue occurs after neural tube closure. Thus, the combined expression patterns of Pintallavis and HNF-3 beta in frogs is equivalent to that of HNF-3 beta in rats and mice. Within neural tissue, the onset of expression of these regulatory genes define successive stages in the differentiation of floor plate cells. The results reported here show that closely related members of the HNF-3/fork head gene family are expressed by axial midline cell groups involved in neural induction and patterning and suggest the involvement of these genes in the development of the vertebrate neuraxis.

Amino Acid Sequence↗

PubMed1993

The genomic structure of the murine ICSBP gene reveals the presence of the gamma interferon-responsive element, to which an ISGF3 alpha subunit (or similar) molecule binds.

ICSBP, a member of the interferon regulatory factor family, is expressed predominantly in lymphoid tissues and is induced by gamma interferon (IFN-gamma). We have studied the genomic organization of the murine ICSBP gene and its 5' upstream region. The murine ICSBP gene (Icsbp) is present as a single copy on chromosome 8 and consists of nine exons. Transcription initiates at two juxtaposed sites downstream from the TATA and CAAT boxes and produces two species of ICSBP mRNA (3.0 and 1.7 kb), presumably by differential usage of poly(A)+ signals. A sequence from -175 to -155 was identified to be an IFN response region that conferred IFN-gamma induction upon a heterologous promoter in lymphoid cell line EL4. This region includes a motif, TTCNNGGAA, designated the palindromic IFN response element (pIRE), to which an IFN-gamma-inducible, cycloheximide-sensitive factor(s) binds. A similar palindromic motif was found in the upstream region of the murine IRF-1 gene, the IFN-gamma activation site of the guanylate-binding protein gene and the IFN-gamma-responsive region of the Fc receptor type I gene, all of which competed with the pIRE for factor binding in gel mobility shift assays. We show that the pIRE binding factor reacts with the antibody against the 91-kDa subunit of ISGF3 alpha recently shown to bind to the IFN-gamma activation site. These results suggest that this factor is related to the IFN-gamma activation factor and contains the 91-kDa subunit of ISGF3 alpha. Taken together, pIRE represents an IRE that is distinct from the classical IFN-stimulated response element and that is capable of conferring IFN-gamma induction through the binding of the 91-kDa ISGF3 alpha subunit (or an antigenically similar molecule).

Animals↗

PubMed1992

Activation of transcription by IFN-gamma: tyrosine phosphorylation of a 91-kD DNA binding protein.

Interferon-gamma (IFN-gamma) induces the transcription of the gene encoding a guanylate binding protein by activating a latent cytoplasmic factor, GAF (gamma-activated factor). GAF is translocated to the nucleus and binds a DNA element, the gamma-activated site. Through cross-linking and the use of specific antibodies GAF was found to be a 91-kilodalton DNA binding protein that was previously identified as one of four proteins in interferon-stimulated gene factor-3 (ISGF-3), a transcription complex activated by IFN-alpha. The IFN-gamma-dependent activation of the 91-kilodalton DNA binding protein required cytoplasmic phosphorylation of the protein on tyrosine. The 113-kilodalton ISGF-3 protein that is phosphorylated in response to IFN-alpha was not phosphorylated nor translocated to the nucleus in response to IFN-gamma. Thus the two different ligands result in tyrosine phosphorylation of different combinations of latent cytoplasmic transcription factors that then act at different DNA binding sites.

Animals↗

PubMed1992

Transiently and stably introduced CCAAT/enhancer-binding-protein genes are constitutively expressed in cultured cells.

CCAAT/enhancer-binding protein (C/EBP) is expressed in certain cell types including hepatocytes and adipocytes. In order to understand the mechanisms that control the expression of the mouse C/EBP gene in the liver as well as in adipocytes, we have studied both the endogenous gene and transfected C/EBP gene constructs. The initiation site of transcription was identified and a strong liver-specific DNase-I hypersensitive site located at -3 kb, which does not appear to contribute functionally to the regulation of the gene in a variety of either transiently or stably transfected cells with constructs which include sequences up to 6-kb upstream of the transcription start. C/EBP gene expression during the transition from preadipocytes to adipocytes was shown to be controlled at the level of transcription. However, adipocytes stably transfected with constructs that include -3.3 kb upstream of the C/EBP gene do not express the reporter genes in a differentiation-specific manner. We detected several DNA-binding proteins that interact with the upstream sites of the C/EBP gene. Those include two labile and two heat-stable site-specific DNA-binding proteins that are present in nuclear extracts from several tissues and cultured cell lines.

Adipose Tissue↗

PubMed1992

Proteins of transcription factor ISGF-3: one gene encodes the 91-and 84-kDa ISGF-3 proteins that are activated by interferon alpha.

ISGF-3 is an interferon-dependent positive-acting transcription factor that is cytoplasmically activated, possibly through direct interaction with the interferon receptor. The factor has been purified, its component proteins have been separated, and its peptide sequences have been obtained. From the sequences, degenerate oligonucleotide probes were constructed to screen for cDNA clones. Sequencing of the selected clones shows that the 91- and 84-kDa components represent two forms of a previously unknown (to our knowledge) protein. Several antibodies raised against these proteins prove that they indeed do encode protein components of ISGF-3. This work provides reagents to explore the modification of this cytoplasmically activated transcription factor.

Amino Acid Sequence↗

PubMed1992

The proteins of ISGF-3, the interferon alpha-induced transcriptional activator, define a gene family involved in signal transduction.

ISGF-3 is a multiprotein transcription factor that is very quickly activated in the cell cytoplasm only after attachment of interferon alpha to the cell surface. To understand the specific cytoplasmic activation of proteins that move to the nucleus and direct increased transcription of specific genes, we have purified and now report completion of the cloning of cDNA encoding the four proteins of ISGF-3. With all of the sequences available, it is clear that three of these proteins are encoded by members of a previously unrecognized gene family. We suggest that proteins encoded by this gene family serve the function of interpreting the fact that a cell surface receptor has bound its ligand so that specific signal transduction to the nucleus can occur.

Amino Acid Sequence↗

PubMed1992

Interferon-dependent tyrosine phosphorylation of a latent cytoplasmic transcription factor.

The interferon-alpha (IFN-alpha)-stimulated gene factor 3 (ISGF3), a transcriptional activator, contains three proteins, termed ISGF3 alpha proteins, that reside in the cell cytoplasm until they are activated in response to IFN-alpha. Treatment of cells with IFN-alpha caused these three proteins to be phosphorylated on tyrosine and to translocate to the cell nucleus where they stimulate transcription through binding to IFN-alpha-stimulated response elements in DNA. IFN-gamma, which activates transcription through a different receptor and different DNA binding sites, also caused tyrosine phosphorylation of one of these proteins. The ISGF3 alpha proteins may be substrates for one or more kinases activated by ligand binding to the cell surface and may link occupation of a specific polypeptide receptor with activation of transcription of a set of specific genes.

Cell Membrane↗

PubMed1992

A transcriptional hierarchy involved in mammalian cell-type specification.

Although transcriptional hierarchies have been extensively studied in invertebrates, their involvement in mammalian cell-type specification is poorly understood. Here we report a hepatocyte transcriptional cascade suggested by the expression patterns of hepatic transcription factors in dedifferentiated hepatomas and hepatocyte: fibroblast hybrids in which the liver phenotype was extinguished. These results indicated that the homeoprotein hepatocyte nuclear factor-1 alpha (HNF-1 alpha), and HNF-4, a member of the steroid hormone receptor superfamily, were regulated coordinately or in a hierarchy by a higher-order locus, independently of other hepatic transactivators. HNF-4 was implicated as an essential positive regulator of HNF-1 alpha, as deletion of an HNF-4 binding site in the HNF-1 alpha promoter abolished promoter activity, and HNF-4 potently transactivated the HNF-1 alpha promoter in cotransfection assays. Moreover, genetic complementation of dedifferentiated hepatomas with HNF-4 complementary DNA rescued expression of endogenous HNF-1 alpha messenger RNA and DNA-binding activity. Our studies therefore define an HNF-4----HNF-1 alpha (4----1 alpha) transcriptional hierarchy operative in differentiated hepatocytes but selectively inhibited by an extinguishing locus and somatic mutations which antagonize the liver phenotype.

Animals↗

PubMed1992

Transcriptional control in hepatocytes of normal and c14CoS albino deletion mice.

The transcription rates of the albumin and alpha-fetoprotein (alpha FP) genes were reduced to marginally detectable levels in livers of newborn or fetal c14CoS albino deletion mutant mice, which lack the hepatocyte specific developmental regulation (hsdr-1) locus on chromosome 7 and die shortly after birth. However, steady-state levels of these two mRNAs in livers of mutant mice were similar to those in normal mice, where these genes are actively transcribed. In c14CoS mice, transcription rates of transcription factor genes HNF-1, C/EBP and HNF-4 were reduced, albeit to different extents. These effects are specific because transcription of the HNF-3, DBP, LAP and Jun-B genes remained normal in mutant mice. Steady-state levels of all of these mRNAs reflected the transcriptional activities. Levels of HNF-1 and HNF-4 mRNAs showed much greater depression than that of C/EBP in mutant liver. The availability of this group of transcription factors may be reduced in c14CoS hepatocytes and therefore caused depressed transcription rates of their target genes such as those encoding albumin and alpha FP. However, the normal steady-state levels of albumin and alpha FP mRNAs in mutant mice remains unexplained. Fetal c14CoS hepatocytes in primary culture did acquire competence for glucocorticoid inducible transcription of the albumin, alpha FP, HNF-4 and metallothionein genes but not of the tyrosine aminotransferase (TAT) gene. These results indicate that the hsdr-1 locus is dispensable for the glucocorticoid induced transcription of these genes but not of TAT. The effects caused by the c14CoS deletion are pleiotropic in controlling the expression of numerous genes at distinct levels in the liver.

Albinism↗

PubMed1992

Murine chromosomal location of four hepatocyte-enriched transcription factors: HNF-3 alpha, HNF-3 beta, HNF-3 gamma, and HNF-4.

The genes for rat hepatocyte nuclear factors 3 and 4 (HNF-3 alpha, HNF-3 beta, HNF-3 gamma, and HNF-4) have been mapped in mouse by analysis of restriction fragment length polymorphisms in interspecific backcross mice. These hepatocyte-enriched transcription factors are positive-acting transcription factors with binding sites in regulatory regions of many genes expressed in hepatocytes. Both HNF-3 alpha, beta, and gamma and HNF-4 are also expressed in intestine. They have recently been implicated as potential participants in endodermal development from early gut cells because of their close homology to Drosophila genes, which themselves are expressed in the developing gut. Despite having similar functional roles and highly conserved DNA binding domains, the three loci from the Hnf-3 family of genes mapped to three different mouse chromosomes, suggesting that the Hnf-3 family has become widely dispersed during evolution and implying the necessity for independent activation of each member of the HNF-3 family.

Animals↗

PubMed1992

Mechanisms of liver-specific gene expression.

Significant advances in the field of hepatocyte-specific gene expression have been made during the past year. Several new transcription factors have been cloned and partially characterized. Analyses of the promoter regions of several factors have also been initiated and Drosophila homologs for two of these factors have been found, opening the way for studies on development.

Animals↗

PubMed1992

Mapping of RNA polymerase on mammalian genes in cells and nuclei.

The assembly of an RNA polymerase II initiation complex at a promoter is associated with the melting of the DNA template to allow the polymerase to read the DNA sequence and synthesize the corresponding RNA. Using the specific single-stranded modifying reagent KMnO4 and a new genomic sequencing technique, we have explored the melted regions of specific genes in genomic DNA of whole cells or of isolated nuclei. We have demonstrated for the first time in vivo the melting in the promoter proximal transcribed region that is associated with the presence of RNA polymerase II complexes. An interferon-inducible gene, ISG-54, exhibited KMnO4 sensitivity over approximately 300 nucleotides downstream of the RNA initiation site in interferon-treated cells when the gene was actively transcribed but not in untreated cells where the gene was not transcribed. The extent of KMnO4 modification was proportional to transcription levels. The KMnO4 sensitivity was retained when nuclei were isolated from induced cells but was lost if the engaged polymerases were further allowed to elongate the nascent RNA chains ("run-on"). The sensitivity to KMnO4 in isolated nuclei was retained if the run-on incubation was performed in the presence of alpha-amanitin, which blocks progress of engaged polymerases. A similar analysis identified an open sequence of only approximately 30 bases just downstream of the start site of the transthyretin (TTR) gene in nuclei isolated from mouse liver, a tissue where TTR is actively transcribed. This abrupt boundary of KMnO4 sensitivity, which was removed completely by allowing engaged polymerases to elongate RNA chains, suggests that most polymerases transcribing this gene paused at about position +20. The possibility of mapping at the nucleotide level the position of actively transcribing RNA polymerases in whole cells or isolated nuclei opens new prospects in the study of transcription initiation and elongation.

Animals↗

PubMed1992

Interferon induction of gene transcription analyzed by in vivo footprinting.

The promoters of two interferon-induced genes (the ISG54 and guanylate-binding protein [GBP] genes) have been analyzed in whole cells and in isolated nuclei by using a new genomic sequencing technique. The ISG54 gene contains an interferon-simulating response element (ISRE), earlier shown to be necessary and sufficient for alpha interferon (IFN-alpha) induction, that appeared complexed with proteins in both transcribing and nontranscribing cells. However, the extent of protection and hypersensitivity to DNase I or dimethyl sulfate within the ISRE region was changed upon transcriptional induction, suggesting the binding of different factors in different transcriptional states. In addition to the ISRE, the GBP gene needs a newly recognized DNA element, called the GAS, that partly overlaps the ISRE for full induction by either IFN-alpha or IFN-gamma. This GAS element was transiently protected against DNase I in the nuclei of interferon-treated cells but was not protected at later times when transcription reached maximal levels. Thus, the GAS-binding activity may be necessary only transiently for the initial assembly of a transcription initiation complex on the GBP promoter. Dimethyl sulfate methylation of genomic DNA performed on intact cells showed a characteristic sensitivity over the GAS that correlated with transcription levels and that persisted longer than did DNase I protection over the GAS. These results demonstrate the involvement of the GAS in IFN-alpha and -gamma induction of GBP and suggest the presence of an altered DNA conformation or a small protein in the major groove of the GAS associated with ongoing GBP transcription.

Base Sequence↗