ABSTRACT
NFIB (nuclear factor I B) is a NFI transcription factor family member, which is essential for the development of a variety of organ systems. Salivary gland development occurs through several stages, including prebud, bud, pseudoglandular, canalicular, and terminal. Although many studies have been done to understand mouse submandibular gland (SMG) branching morphogenesis, little is known about SMG cell differentiation during the terminal stages. The goal of this study was to determine the role of NFIB during SMG development. We analyzed SMGs from wild-type and Nfib-deficient mice (Nfib (-/-)). At embryonic (E) day 18.5, SMGs from wild-type mice showed duct branching morphogenesis and differentiation of tubule ductal cells into tubule secretory cells. In contrast, SMGs from Nfib (-/-) mice at E18.5 failed to differentiate into tubule secretory cells while branching morphogenesis was unaffected. SMGs from wild-type mice at E16.5 displayed well-organized cuboidal inner terminal tubule cells. However, SMGs from Nfib (-/-) at E16.5 displayed disorganized inner terminal tubule cells. SMGs from wild-type mice at E18.5 became fully differentiated, as indicated by a high degree of apicobasal polarization (i.e., presence of apical ZO-1 and basolateral E-cadherin) and columnar shape. Furthermore, SMGs from wild-type mice at E18.5 expressed the protein SMGC, a marker for tubule secretory cells. However, SMGs from Nfib (-/-) mice at E18.5 showed apicobasal polarity, but they were disorganized and lost the ability to secrete SMGC. These findings indicate that the transcription factor NFIB is not required for branching morphogenesis but plays a key role in tubule cell differentiation during mouse SMG development.
Subject(s)
NFI Transcription Factors/physiology , Submandibular Gland/embryology , Animals , Aquaporin 5/analysis , Biomarkers/analysis , Cadherins/analysis , Cell Differentiation/physiology , Cell Polarity/physiology , Cell Shape , Embryonic Development , Immunohistochemistry , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Confocal , Morphogenesis/physiology , Mucins/analysis , NFI Transcription Factors/genetics , Salivary Ducts/cytology , Salivary Ducts/embryology , Submandibular Gland/cytology , Zonula Occludens-1 Protein/analysisABSTRACT
The distal region (-830 to -720 bp) of the rat whey acidic protein (WAP) gene contains a composite response element (CoRE), which has been demonstrated previously to confer mammary gland-specific and hormonally regulated WAP gene expression. Point mutations in the binding sites for specific transcription factors present within this CoRE have demonstrated the importance of both nuclear factor I (NFI) and STAT5 as well as cooperative interactions with the glucocorticoid receptor (GR) in the regulation of WAP gene expression in the mammary gland of transgenic mice. This study reports the characterization of NFI gene expression during mammary gland development and the identification and cloning of specific NFI isoforms (NFI-A4, NFI-B2, and NFI-X1) from the mouse mammary gland during lactation. Some but not all of these NFI isoforms synergistically activate WAP gene transcription in cooperation with GR and STAT5, as determined using transient cotransfection assays in JEG-3 cells. On both the WAP CoRE and the mouse mammary tumor virus long terminal repeat promoter, the NFI-B isoform preferentially activated gene transcription in cooperation with STAT5A and GR. In contrast, the NFI-A isoform suppressed GR and STAT cooperativity at the WAP CoRE. Finally, unlike their interaction with the NFI consensus binding site in the adenovirus promoter, the DNA-binding specificities of the three NFI isoforms to the palindromic NFI site in the WAP CoRE were not identical, which may partially explain the failure of the NFI-A isoform to cooperate with GR and STAT5A.
Subject(s)
CCAAT-Enhancer-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Milk Proteins/genetics , Milk Proteins/metabolism , Receptors, Glucocorticoid/metabolism , Trans-Activators/metabolism , Transcription Factors , Transcription, Genetic , Adenoviridae/genetics , Alternative Splicing , Animals , Binding Sites , Binding, Competitive , Blotting, Western , Breast/metabolism , CCAAT-Enhancer-Binding Proteins/metabolism , Cells, Cultured , Cloning, Molecular , DNA/metabolism , Female , Gene Expression Regulation , Lactation , Luciferases/metabolism , Mice , Models, Genetic , NFI Transcription Factors , Nuclear Proteins , Plasmids/metabolism , Promoter Regions, Genetic , Protein Binding , Protein Isoforms , Protein Structure, Tertiary , RNA/metabolism , Receptors, Glucocorticoid/genetics , Response Elements , Reverse Transcriptase Polymerase Chain Reaction , Ribonucleases/metabolism , STAT5 Transcription Factor , Time Factors , Transfection , Y-Box-Binding Protein 1 , beta-Galactosidase/metabolismABSTRACT
Although the existence of repressor protein(s) involved in the regulation of highly inducible metallothionein-I (MT-I) gene expression has been postulated, none has been identified to date. We considered nuclear factor I (NFL) protein as a potential repressor, as three half-sites for NFI binding are present on MT-I promoter and NFI is known to downregulate several cellular gene promoters. Overexpression of all four isoforms of mouse NFI protein (NFI-A, -B, -C, and -X) suppressed both constitutive and heavy metal-induced activation of the MT-I promoter in HepG2 cells. However, unlike other target genes of NFI, direct interaction of NFI with MT-I promoter is not necessary to mediate its repression. Point mutation of the NFI binding sites within the MT-I promoter that abrogates NFI binding in vitro could not alleviate the repression. Similarly, NFI proteins also repress activity of minimal MT-I promoter deficient in the NFI binding sites. Further, an NFI-C deletion mutant lacking the DNA binding domain continued to repress MT-I promoter. Overexpression of MTF-1, the key trails-acting factor involved in MT-I gene transcription, surmounted NFI-mediated repression of the basal and zinc-induced MT-I promoter activity. These data demonstrate that NFI is a repressor of MT-I expression, where its DNA binding activity is not essential to downregulate the MT-I promoter. Interaction of NFI with another protein(s), probably MTF-I, may be involved in this repression.
Subject(s)
CCAAT-Enhancer-Binding Proteins/metabolism , DNA-Binding Proteins , Down-Regulation , Metallothionein/genetics , Metals, Heavy/pharmacology , Animals , Base Sequence , CCAAT-Enhancer-Binding Proteins/genetics , Cadmium/pharmacology , Cells, Cultured , DNA/genetics , DNA/metabolism , Down-Regulation/drug effects , Electrophoretic Mobility Shift Assay , Luciferases/genetics , Luciferases/metabolism , Mice , Mutation/genetics , NFI Transcription Factors , Nuclear Proteins , Promoter Regions, Genetic/genetics , Rats , Repressor Proteins/metabolism , Response Elements/genetics , Transcription Factors/metabolism , Transfection , Y-Box-Binding Protein 1 , Transcription Factor MTF-1ABSTRACT
T-box genes encode a family of phylogenetically conserved DNA-binding proteins that regulate gene expression during embryogenesis. While the developmental importance of many T-box genes has been well documented, little is known about how family members differ in their DNA binding properties and ability to modulate transcription. Here we show that although TBX1, TBX2 and the Xenopus T protein (Xbra) share only 50-60% identity within their DNA-binding domains they can bind the same DNA sequence in vitro. However, the proteins differ in three important respects. While TBX1 protein binds a palindromic T oligonucleotide as a dimer, as had been previously reported for Xbra, TBX2 appears to bind the same DNA sequence as a monomer. Also, T protein/DNA complexes are stabilized in vitro by the addition of specific antibodies, whereas TBX2/DNA complexes are not stabilized by antibodies. Most importantly, TBX2 represses while Xbra activates transcription of the same chimeric reporter plasmid. TBX1, although capable of binding to the chimeric promoter, has no effect on transcription. Thus, while the DNA binding domains of T-box proteins share substantial homology, these proteins differ in both their DNA binding and transcriptional modulation properties. These results suggest that the various T-box proteins, while highly conserved, likely use different mechanisms to modulate transcription and may have different targets in vivo.
Subject(s)
DNA/metabolism , T-Box Domain Proteins/metabolism , Amino Acid Sequence , Amino Acid Substitution , Animals , Base Sequence , Binding Sites , Cell Line , DNA/genetics , Gene Expression Regulation , Glutathione Transferase/genetics , Glutathione Transferase/metabolism , Green Fluorescent Proteins , Humans , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Models, Molecular , Molecular Sequence Data , Molecular Structure , Mutation , Promoter Regions, Genetic/genetics , Protein Binding , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , T-Box Domain Proteins/genetics , Transcription, Genetic , Transfection , Xenopus , Xenopus ProteinsABSTRACT
The Nuclear Factor I (NFI) family of site-specific DNA-binding proteins (also known as CTF or CAAT box transcription factor) functions both in viral DNA replication and in the regulation of gene expression. The classes of genes whose expression is modulated by NFI include those that are ubiquitously expressed, as well as those that are hormonally, nutritionally, and developmentally regulated. The NFI family is composed of four members in vertebrates (NFI-A, NFI-B, NFI-C and NFI-X), and the four NFI genes are expressed in unique, but overlapping, patterns during mouse embryogenesis and in the adult. Transcripts of each NFI gene are differentially spliced, yielding as many as nine distinct proteins from a single gene. Products of the four NFI genes differ in their abilities to either activate or repress transcription, likely through fundamentally different mechanisms. Here, we will review the properties of the NFI genes and proteins and their known functions in gene expression and development.
Subject(s)
CCAAT-Enhancer-Binding Proteins , DNA-Binding Proteins/physiology , Gene Expression Regulation, Developmental , Multigene Family , Transcription Factors , Transcription, Genetic/genetics , Animals , DNA-Binding Proteins/genetics , Humans , NFI Transcription Factors , Nuclear Proteins , Y-Box-Binding Protein 1ABSTRACT
Nuclear factor I (NFI) proteins are DNA-binding transcription factors that participate in the tissue specific expression of various genes. They are encoded by four different genes (NFI-A, B, C, and X) each of which generates multiple isoforms by alternative RNA splicing. NFI-like binding sites have been identified in several genes preferentially expressed in olfactory receptor neurons. Our prior demonstration that NFI binds to these elements led to the hypothesis that NFI is involved in the regulation of these genes. To analyse the role of NFI in the regulation of olfactory neuron gene expression we have performed transient transfection experiments in HEK 293 cells using constructs that place luciferase expression under the control of an olfactory marker protein (OMP)-promoter fragment containing the NFI binding site. In vitro mutagenesis of this site revealed a negative modulation of luciferase expression by endogenous NFI proteins in HEK 293 cells. In addition, we have used in situ hybridization to analyse the tissue and cellular distribution of the four NFI gene transcripts during pre- and postnatal mouse development. We have simultaneously characterized the expression of Pax-6, and O/E-1, transcription factors known to regulate the phenotype of olfactory receptor neurons. We demonstrate that all of these transcription factors vary in specific spatio-temporal patterns during the development of the olfactory system. These data on NFI activity, and on transcription factor expression, provide a basis to understand the role of NFI in regulating gene expression in olfactory receptor neurons.
Subject(s)
CCAAT-Enhancer-Binding Proteins , DNA-Binding Proteins/genetics , Homeodomain Proteins , Nerve Tissue Proteins/genetics , Olfactory Mucosa/chemistry , Olfactory Mucosa/embryology , Olfactory Receptor Neurons/chemistry , Transcription Factors , Age Factors , Animals , Cell Line , Eye Proteins , Female , Gene Expression Regulation, Developmental , Genes, Reporter , Hippocampus/chemistry , Hippocampus/embryology , Humans , In Situ Hybridization , Kidney/cytology , Luciferases/genetics , Mice , Molecular Sequence Data , Mutagenesis/physiology , NFI Transcription Factors , Nuclear Proteins , Olfactory Marker Protein , PAX6 Transcription Factor , Paired Box Transcription Factors , Phenotype , Pregnancy , Promoter Regions, Genetic/physiology , RNA, Messenger/analysis , Repressor Proteins , Sequence Homology, Amino Acid , Transfection , Vomeronasal Organ/chemistry , Vomeronasal Organ/embryology , Y-Box-Binding Protein 1ABSTRACT
The phylogenetically conserved nuclear factor I (NFI) family of transcription/replication proteins is essential both for adenoviral DNA replication and for the transcription of many cellular genes. We showed previously that the four murine NFI genes (Nfia, Nfib, Nfic, and Nfix) are expressed in unique but overlapping patterns during mouse development and in adult tissues. Here we show that disruption of the Nfia gene causes perinatal lethality, with >95% of homozygous Nfia(-/-) animals dying within 2 weeks after birth. Newborn Nfia(-/-) animals lack a corpus callosum and show ventricular dilation indicating early hydrocephalus. Rare surviving homozygous Nfia(-/-) mice lack a corpus callosum, show severe communicating hydrocephalus, a full-axial tremor indicative of neurological defects, male-sterility, low female fertility, but near normal life spans. These findings indicate that while the Nfia gene appears nonessential for cell viability and DNA replication in embryonic stem cells and fibroblasts, loss of Nfia function causes severe developmental defects. This finding of an NFI gene required for a developmental process suggests that the four NFI genes may have distinct roles in vertebrate development.
Subject(s)
CCAAT-Enhancer-Binding Proteins , Corpus Callosum/physiology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/physiology , Hydrocephalus/genetics , Transcription Factors , Animals , Brain/metabolism , Female , Fertility/genetics , Fibroblasts/metabolism , Glial Fibrillary Acidic Protein/metabolism , Male , Mice , Mice, Inbred C57BL , Models, Genetic , Mutagenesis , NFI Transcription Factors , Nuclear Proteins , Phenotype , Recombination, Genetic , Stem Cells/metabolism , Y-Box-Binding Protein 1ABSTRACT
Olfactory receptor neurons are responsible for the detection and signal transduction of odor ligands. Several genes associated with this activity are preferentially or exclusively expressed in these neurons. Among these genes are those coding for olfactory receptors, adenylyl cyclase type III, the cyclic nucleotide gated olfactory channel 1 (OcNC-1), Galpha(olf) and the olfactory marker protein (OMP). Promoter analyses of these genes identified a binding site for the new transcription factor family O/E whose initial member, Olf-1, is abundantly expressed in olfactory neurons. We report here that the proximal promoters of three of these genes, that are selectively expressed in olfactory neurons, each contains a functional NFI binding site and that the sites have different affinities for NFI proteins indicating a regulatory role for NFI proteins in olfactory gene expression. We further demonstrate, by cloning, that all four NFI genes are expressed in the olfactory nasal mucosa. Analysis by in situ hybridization illustrates that at least three of these gene products are expressed in the neuroepithelium in which the olfactory neurons reside. NFI proteins are capable of functioning as positive or negative regulators of transcription depending on the tissue, cell-type, age, and gene in question. These multivalent functions of NFI could be achieved by temporally and spatially regulated expression of distinct subsets of NFI isoforms. It now remains to characterize the tissue and cell specific patterns of expression of distinct NFI transcription factors during ontogeny and their roles in regulating gene expression.
Subject(s)
DNA-Binding Proteins/physiology , Gene Expression Regulation , Genes , Nerve Tissue Proteins/physiology , Olfactory Receptor Neurons/metabolism , Protein Isoforms/physiology , Repressor Proteins/physiology , Trans-Activators/physiology , Transcription Factors/chemistry , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Cloning, Molecular , DNA, Complementary/genetics , Epithelial Cells/metabolism , In Situ Hybridization , Mice , Molecular Sequence Data , Multigene Family , NFI Transcription Factors , Nerve Tissue Proteins/genetics , Olfactory Mucosa/metabolism , Promoter Regions, Genetic , Protein Isoforms/genetics , Rats , Repressor Proteins/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment , Sequence Homology, Nucleic Acid , Species Specificity , Transcription Factors/geneticsABSTRACT
The Nuclear Factor I (NFI) family of DNA-binding proteins is essential for adenovirus DNA replication and the transcription of many cellular genes. Mammals have four genes encoding NFI proteins, C. elegans has only a single NFI gene, and prokaryotes have none. To assess the relationship between members of this unusually small family of transcription/replication factors, we mapped the chromosomal locations of the four murine NFI genes and analyzed the exons encoding the DNA-binding domains of the mouse, Amphioxus, and C. elegans NFI genes. The four murine NFI genes are on Chrs 4 (Nfia and Nfib), 8 (Nfix), and 10 (Nfic), suggesting early duplication of the genes and dispersal throughout the genome. The DNA-binding domains of all four NFI genes are encoded by large (532 bp) exons with identical splice acceptor and donor sites in each. In contrast, the C. elegans nfi-1 gene has four phased introns interrupting this DNA-binding, domain-encoding exon, and the last exon extends 213 bp past the splice site used in all four murine genes. In addition, the introns present in C. elegans nfi-1 are missing from the NFI genes of Amphioxus and all mammalian genomes examined. This analysis of the exon structure of the C. elegans and murine NFI genes indicates that the murine genes were probably generated by duplication of a C. elegans-like ancestral gene, but that significant changes have occurred in the genomic organization of either the C. elegans or murine NFI genes during evolution.
Subject(s)
CCAAT-Enhancer-Binding Proteins , DNA-Binding Proteins/genetics , Exons/genetics , Animals , Base Sequence , Binding Sites/genetics , Caenorhabditis elegans/genetics , DNA/genetics , Evolution, Molecular , Humans , Mice , Molecular Sequence Data , NFI Transcription Factors , Nuclear Proteins , Transcription Factors/genetics , Y-Box-Binding Protein 1ABSTRACT
To better understand the function of nuclear factor I (NFI) proteins in transcription, we have used transient transfection assays to assess transcriptional modulation by NFI proteins on the NFI-dependent mouse mammary tumor virus (MMTV) promoter. Expression of NFI-C or NFI-X, but not NFI-A or NFI-B proteins, represses glucocorticoid induction of the MMTV promoter in HeLa cells. Repression is DNA binding-independent as a deletion construct expressing the NH2-terminal 160 residues of NFI-C represses but does not bind DNA. Repression by NFI-C is cell type-dependent and occurs in HeLa and COS-1 cells but not 293 or JEG-3 cells. NFI-C does not repress progesterone induction of the MMTV promoter in HeLa cells, suggesting that progesterone induction of the promoter differs mechanistically from glucocorticoid induction. NFI-C-mediated repression is alleviated by overexpression of glucocorticoid receptor (GR), suggesting that NFI-C represses the MMTV promoter by preventing GR function. However, repression by NFI-C occurs with only a subset of glucocorticoid-responsive promoters, as the chimeric NFIGREbeta-gal promoter that is activated by GR is not repressed by NFI-C. Since the coactivator proteins p300/CBP, SRC-1A, and RAC3 had previously been shown to function at steroid hormone-responsive promoters, we asked whether they could influence NFI-C-mediated repression of MMTV expression. Expression of p300/CBP or SRC-1A alleviates repression by NFI-C, whereas RAC3 has no effect. This abrogation of NFI-C-mediated repression by p300/CBP and SRC-1A suggests that repression by NFI-C may occur by interference with coactivator function at the MMTV promoter.
Subject(s)
CCAAT-Enhancer-Binding Proteins , DNA-Binding Proteins/metabolism , Mammary Tumor Virus, Mouse/genetics , Nuclear Proteins/metabolism , Promoter Regions, Genetic , Repressor Proteins/metabolism , Trans-Activators/metabolism , Transcription Factors/metabolism , Animals , Cell Line , Glucocorticoids/pharmacology , Histone Acetyltransferases , Humans , NFI Transcription Factors , Nuclear Receptor Coactivator 1 , Progesterone/pharmacology , Receptors, Glucocorticoid/metabolism , Repetitive Sequences, Nucleic Acid , Y-Box-Binding Protein 1ABSTRACT
Nuclear factor I (NFI) binds to a region of the phosphoenolpyruvate carboxykinase (GTP) (PEPCK) gene promoter adjacent to the cAMP regulatory element (CRE) and inhibits the induction of transcription from the gene promoter caused by the catalytic subunit of protein kinase A. In vivo footprinting studies demonstrated that both the CRE and the NFI-binding site are occupied by transcription factors, regardless of the presence of factors that stimulate (dibutyryl cAMP or dexamethasone) or inhibit (insulin) transcription from the PEPCK gene promoter. The NFI effects on transcription from the PEPCK gene promoter were observed even in the absence of the NFI binding site, suggesting the possibility of other weaker binding sites on the promoter or an interaction of NFI with a transcriptional co-activator. A mammalian two-hybrid system was used to demonstrate direct interaction between the transactivation domain of NFI-C and the CREB binding domain of the CREB-binding protein (CBP). Overexpression of a gene fragment encoding the CREB binding domain of CBP stimulates transcription from the PEPCK gene promoter. The inhibitory effect of NFI on transcription of the PEPCK gene induced by the catalytic subunit of protein kinase A appears to be the result of an interaction between NFI and the CREB-binding protein in which NFI competes with CREB for binding to the CREB-binding site on CBP. In contrast, glucocorticoids and thyroid hormone use the steroid hormone receptor binding domain of CBP to stimulate transcription from the PEPCK gene promoter. NFI-A combines with dexamethasone or thyroid hormone in an additive manner to stimulate PEPCK gene transcription. We conclude that CBP coordinates the action of the multiple factors known to control transcription of the PEPCK gene.
Subject(s)
CCAAT-Enhancer-Binding Proteins , DNA-Binding Proteins/genetics , Nuclear Proteins/metabolism , Phosphoenolpyruvate Carboxykinase (GTP)/genetics , Trans-Activators/metabolism , Transcription Factors/genetics , Transcription, Genetic/genetics , Binding Sites/genetics , CREB-Binding Protein , Cyclic AMP-Dependent Protein Kinases/genetics , DNA Footprinting , Dexamethasone/pharmacology , Humans , NFI Transcription Factors , Promoter Regions, Genetic/genetics , Protein Binding/genetics , Tumor Cells, Cultured , Y-Box-Binding Protein 1ABSTRACT
Promoter-specific differences in the function of transcription factors play a central role in the regulation of gene expression. We have measured the maximal transcriptional activation potentials of nuclear factor I (NFI) proteins encoded by each of the four identified NFI genes (NFI-A, -B, -C, and -X) by transient transfection in JEG-3 cells using two model NFI-dependent promoters: 1) a simple chimeric promoter containing a single NFI-binding site upstream of the adenovirus major late promoter (NFI-Ad), and 2) the more complex mouse mammary tumor virus long terminal repeat promoter. The relative activation potentials for the NFI isoforms differed between the two promoters, with NFI-X being the strongest activator of NFI-Ad and NFI-B being the strongest activator of the MMTV promoter. To determine if these promoter-specific differences in activation potential were due to the presence of glucocorticoid response elements (GREs), we added GREs upstream of the NFI-binding site in NFI-Ad. NFI-X remains the strongest activator of the GRE containing simple promoter, indicating that differences in relative activation potential are not due solely to the presence of GREs. Since NFI proteins bind to DNA as dimers, we assessed the activation potentials of NFI heterodimers. Here, we show that NFI heterodimers have intermediate activation potentials compared with homodimers, demonstrating one potential mechanism by which different NFI proteins can regulate gene expression.
Subject(s)
CCAAT-Enhancer-Binding Proteins , DNA-Binding Proteins/physiology , Promoter Regions, Genetic , Transcription Factors/physiology , Transcriptional Activation , Animals , Binding Sites , DNA-Binding Proteins/genetics , Dimerization , Herpes Simplex Virus Protein Vmw65/metabolism , Mice , NFI Transcription Factors , Nuclear Proteins , Receptors, Glucocorticoid/metabolism , Y-Box-Binding Protein 1ABSTRACT
The reversible oxidative inactivation of transcription factors has been proposed to be important in cellular responses to oxidant stress and in several signal transduction pathways. The nuclear factor I (NFI) family of transcription factors is sensitive to oxidative inactivation due to the presence of a conserved, oxidation-sensitive cysteine residue within the NFI DNA-binding domain. Here we show that restoration of the DNA-binding activity of oxidized NFI-C can be catalyzed in vitro by the cellular enzyme thioltransferase (glutaredoxin) coupled to GSH and GSSG reductase. To test whether GSH-dependent pathways play a role in the maintenance of NFI activity in vivo, we used buthionine sulfoximine, an agent that inhibits GSH synthesis, and N-acetylcysteine, an agent that can replenish intracellular GSH. Pretreatment of HeLa cells with buthionine sulfoximine greatly potentiated the inactivation of NFI by the oxidizing agent diamide. Inclusion of N-acetylcysteine in the culture medium during the recovery period following diamide treatment increased the extent of restoration of NFI activity. These results suggest that maintenance of the DNA-binding activity of NFI proteins during oxidant stress in vivo requires a GSH-dependent pathway, likely involving thioltransferase-catalyzed reduction of the oxidation-sensitive cysteine residue on NFI.
Subject(s)
CCAAT-Enhancer-Binding Proteins , DNA-Binding Proteins/metabolism , Oxidoreductases , Proteins/metabolism , Transcription Factors/metabolism , DNA/metabolism , DNA-Binding Proteins/antagonists & inhibitors , Glutaredoxins , Glutathione/metabolism , HeLa Cells , Humans , NFI Transcription Factors , Nuclear Proteins , Oxidation-Reduction , Transcription Factors/antagonists & inhibitors , Y-Box-Binding Protein 1ABSTRACT
The nuclear factor I (NFI) family of site-specific DNA-binding proteins is required for both the cell-type specific transcription of many viral and cellular genes and for the replication of adenovirus DNA. Although binding sites for NFI proteins within the promoters of several tissue-specific genes have been shown to be essential for their expression, it is unclear which NFI gene products function in specific tissues during development. We have isolated cDNAs from all four murine NFI genes (gene designations Nfia, Nfib, Nfic, and Nfix), assessed the embryonic and postnatal expression patterns of the NFI genes, and determined the ability of specific NFI proteins to activate transcription from the NFI-dependent mouse mammary tumor virus (MMTV) promoter. In adult mice, all four NFI genes are most highly expressed in lung, liver, heart, and other tissues but only weakly expressed in spleen and testis. The embryonic expression patterns of the NFI genes is complex, with NFI-A transcripts appearing earliest-within 9 days postcoitum in the heart and developing brain. The four genes exhibit unique but overlapping patterns of expression during embryonic development, with high level expression of NFI-A, NFI-B, and NFI-X transcripts in neocortex and extensive expression of the four genes in muscle, connective tissue, liver, and other organ systems. The four NFI gene products studied differ in their ability to activate expression of the NFI-dependent MMTV promoter, with the NFI-B protein being most active and the NFI-A protein being least active. These data are discussed in the context of the developmental expression patterns of known NFI-responsive genes. The differential activation of an NFI-dependent promoter, together with the expression patterns observed for the four genes, indicate that the NFI proteins may play an important role in regulating tissue-specific gene expression during mammalian embryogenesis.
Subject(s)
CCAAT-Enhancer-Binding Proteins , DNA-Binding Proteins/genetics , Gene Expression Regulation, Developmental , Amino Acid Sequence , Animals , Base Sequence , Blotting, Western , Cloning, Molecular , DNA-Binding Proteins/metabolism , In Situ Hybridization , Mice , Mice, Inbred C57BL , Molecular Sequence Data , NFI Transcription Factors , Nuclear Proteins , Time Factors , Tissue Distribution , Transcription Factors/genetics , Transcription Factors/metabolism , Transcriptional Activation , Tumor Cells, Cultured , Y-Box-Binding Protein 1ABSTRACT
The nuclear factor I (NFI) family of transcription/replication proteins is required for the cell type-specific expression of a number of cellular and viral genes. There are four evolutionarily conserved NFI genes (NFI-A, -B, -C and -X) that encode DNA-binding proteins of similar or identical DNA-binding specificity. To address the role of this family of highly conserved transcription/replication proteins in cell differentiation and development, we have assessed the expression of NFI proteins (by DNA binding) and mRNAs of each of the four NFI genes during the in vitro differentiation of several human leukemic cell lines. These cell lines differ in their pattern of differentiation upon exposure to the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA). In K562 and OCI/AML-3 cells, the levels of the four NFI mRNAs and the mobility of NFI-DNA complexes present in nuclear extracts changed during treatment with TPA. The TPA-induced change in mobility of NFI-DNA complexes in OCI/AML-3 cells was blocked by an inhibitor of protein synthesis, cycloheximide. These changes in NFI protein and mRNA expression appear to be cell type-or lineage-specific since no changes were seen during the TPA-induced differentiation of HL-60 or U937 cells. These studies indicate that although their DNA-binding specificities are similar, the proteins encoded by the four NFI genes may differ in their biological functions and play distinct roles in hematopoietic development.
Subject(s)
CCAAT-Enhancer-Binding Proteins , DNA-Binding Proteins/genetics , Leukemia/genetics , Tetradecanoylphorbol Acetate/pharmacology , Transcription Factors , Base Sequence , Binding Sites , Cell Differentiation/drug effects , Cloning, Molecular , Cycloheximide/pharmacology , DNA Probes/chemical synthesis , DNA-Binding Proteins/metabolism , Humans , Leukemia/pathology , Molecular Sequence Data , NFI Transcription Factors , Nuclear Proteins , Protein Synthesis Inhibitors/pharmacology , RNA, Messenger/biosynthesis , Tumor Cells, Cultured , Y-Box-Binding Protein 1ABSTRACT
The nuclear factor I (NFI) family of site-specific DNA-binding proteins plays a role in both transcription and adenovirus DNA replication. The DNA binding domain of NFI family members contains 4 cysteine residues (Cys-2, Cys-3, Cys-4, and Cys-5) that are conserved in all NFI proteins. Mutation of the Cys-2, Cys-4, and Cys-5 residues in the human NFI-C protein to several other amino acids abolished DNA binding, while 8 of 10 mutations of the Cys-3 residue had little or no effect on binding. Wild-type NFI-C was inactivated by N-ethylmaleimide in vitro, while the active Cys-3 mutant proteins were resistant to N-ethylmaleimide. Treatment of wild-type NFI in vitro with the oxidizing agent diamide also inactivated DNA binding, and subsequent reduction with dithiothreitol restored binding activity. The active Cys-3 mutant NFI proteins were resistant to diamide-inactivation, indicating that the Cys-3 residue is required for modulation of DNA-binding by oxidation state. These studies indicate that oxidative-inactivation can play an important role in the modifying NFI-DNA-protein interactions. The presence of this nonessential Cys-3 residue in all known NFI proteins raises the possibility that it may function in a manner similar to redox-sensitive cysteine residues found in other site-specific DNA-binding proteins.
Subject(s)
CCAAT-Enhancer-Binding Proteins , Conserved Sequence , Cysteine/chemistry , DNA-Binding Proteins/chemistry , Transcription Factors/chemistry , Amino Acid Sequence , Base Sequence , DNA, Complementary , Ethylmaleimide/pharmacology , Humans , Molecular Sequence Data , NFI Transcription Factors , Nuclear Proteins , Oxidation-Reduction , Y-Box-Binding Protein 1ABSTRACT
The role of Cys residues in the site-specific DNA binding activity of the nuclear factor I (NFI) family of proteins was assessed by chemical modification and site-specific mutagenesis. Treatment with the thio-specific reagent N-ethylmaleimide abolished site-specific DNA binding of all forms of NFI present in HeLa nuclear extracts. Preincubation of cell extracts with an oligonucleotide containing an NFI-binding site provided partial protection of NFI from N-ethylmaleimide inactivation. Mutations were made in the cDNA encoding a truncated form of the NFI-C/CAAT box transcription factor-1 protein, converting each of the five Cys residues in the DNA-binding domain of the protein into Ser residues. NFI-C proteins containing mutations in any of four conserved Cys residues, expressed in Escherichia coli or in vitro, did not bind to DNA. NFI-C with a mutation in a nonconserved Cys residue had normal DNA binding activity. Both this active mutant and wild-type NFI-C protein were inactivated by modification of their sulfhydryl residues with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and preincubation with an oligonucleotide containing an NFI-binding site gave partial protection against inactivation. After modification with DTNB, DNA binding activity was partially restored by subsequent incubation with dithiothreitol, indicating that inactivation of NFI by DTNB was reversible. These studies indicate an essential role for free sulfhydryl residues in NFI-DNA binding.
Subject(s)
CCAAT-Enhancer-Binding Proteins , Cysteine/metabolism , DNA-Binding Proteins/metabolism , DNA/metabolism , Transcription Factors , Amino Acid Sequence , Base Sequence , DNA-Binding Proteins/chemistry , Electrophoresis, Polyacrylamide Gel , HeLa Cells , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , NFI Transcription Factors , Nuclear Proteins , Sequence Alignment , Y-Box-Binding Protein 1ABSTRACT
Nuclear factor I (NFI) is composed of a family of site-specific DNA-binding proteins which recognize a DNA-binding site with the consensus sequence TGGC/A(N)5GCCAA. Binding sites for NFI have previously been shown to stimulate mRNA synthesis in vitro when present upstream of the TATA box of the adenovirus major late promoter (AdMLP). We have examined the effect of NFI-binding sites on transcription in vivo in transiently transfected HeLa and COS cells. An NFI-binding site isolated from the human genome activated expression from the minimal AdMLP in vivo in both the absence and presence of the simian virus 40 enhancer. A point mutation that decreased NFI binding affinity for the site in vitro reduced expression to near the basal level of the AdMLP. Several NFI-binding sites which differed in their spacer and flanking sequences were tested for their ability to activate expression in vivo. The ability of these sites to activate expression correlated with the strength of NFI binding in vitro. An NFI-binding site stimulated expression equally well when placed from 33 to 65 bp upstream of the TATA box. However, expression dropped to basal levels when the site was located from 71 to 77 bp upstream of the TATA box. These studies indicate that an NFI-binding site in this chimeric promoter activates expression in vivo only if located within a critical distance of the TATA box.
Subject(s)
CCAAT-Enhancer-Binding Proteins , DNA-Binding Proteins/metabolism , Promoter Regions, Genetic , Transcription Factors , Animals , Base Sequence , Binding Sites , Cell Line , Chimera , Chloramphenicol O-Acetyltransferase/genetics , Chloramphenicol O-Acetyltransferase/metabolism , Genetic Vectors , HeLa Cells/physiology , Humans , Molecular Sequence Data , NFI Transcription Factors , Nuclear Proteins , Transfection , Y-Box-Binding Protein 1 , beta-Galactosidase/genetics , beta-Galactosidase/metabolismABSTRACT
Nuclear factor I (NFI) is a group of related site-specific DNA-binding proteins that function in adenovirus DNA replication and cellular RNA metabolism. We have measured both the levels and forms of NFI that interact with a well-characterized 26-base-pair NFI-binding site. Five different NFI-DNA complexes were seen in HeLa nuclear extracts by using a gel mobility shift (GMS) assay. In addition, at least six forms of NFI were shown to cross-link directly to DNA by using a UV cross-linking assay. The distinct GMS complexes detected were composed of different subspecies of NFI polypeptides as assayed by UV cross-linking. Different murine cell lines possessed varying levels and forms of NFI binding activity, as judged by nitrocellulose filter binding and GMS assays. The growth state of NIH 3T3 cells affected both the types of NFI-DNA complexes seen in a GMS assay and the forms of the protein detected by UV cross-linking.
Subject(s)
CCAAT-Enhancer-Binding Proteins , DNA-Binding Proteins/analysis , Nuclear Proteins/analysis , Transcription Factors , Animals , Cell Division , Cell Line , Cell Nucleus/analysis , DNA/metabolism , DNA-Binding Proteins/metabolism , Deoxyribonucleoproteins/radiation effects , Humans , Mice , NFI Transcription Factors , Nuclear Proteins/metabolism , Ultraviolet Rays , Y-Box-Binding Protein 1ABSTRACT
Nuclear factor I (NFI) is a site-specific DNA binding protein required for the replication of adenovirus DNA in vitro and in vivo. We have examined the effect of natural and synthetic binding sites for NFI (FIB sites) on RNA synthesis in HeLa whole cell extracts. The natural binding site used is the 26bp FIB-2 site previously isolated from the human genome. When present upstream of the TATA box of the adenovirus major late promoter, the FIB-2 site stimulates RNA synthesis 3 to 5-fold. This stimulation occurs with either orientation of the FIB-2 site. A point mutation in FIB-2 that decreases NFI binding at least 100-fold reduces, but does not completely abolish, the stimulation of transcription. A number of synthetic binding sites for NFI were tested for the ability to increase RNA synthesis. The strongest binding sites stimulated transcription the most, while the weakest sites had the least effect. These studies strongly suggest a role for NFI and cellular FIB sites in the control of RNA synthesis.