Biomedical subjects
Publications and source records attributed to E B Ziff.
Recent studies centered on the c-Myc basic/helix-loop-helix/leucine zipper (B/HLH/LZ) motifs have led to the identification of a DNA recognition sequence for c-Myc and the isolation of a novel protein that forms a DNA-binding complex with c-Myc in vitro. These advances may make it possible to address directly the long-standing question of c-Myc function in vivo.
In Ras cotransformation assays, Max exhibited a biphasic effect on Myc transformation activity. Cotransfection of low levels of Max expression plasmid stimulated Myc transformation activity, but cotransfection of high levels suppressed it. Mutations in the functionally undefined Max amino- and carboxy-terminal regions outside of the B/HLH/LZ motif partly separated these activities, suggesting various modes of Max regulation. We demonstrate that the Max protein is a nuclear protein in vivo and identify a carboxy-terminal region similar to nuclear localization signals whose integrity is necessary for efficient localization. Two mutants that delete amino- or carboxy-terminal consensus signals for casein kinase II (CKII) exhibited altered gel mobility and DNA-binding potential in vitro and showed modified transforming potential in the Ras cotransformation assay, suggesting that CKII or a CKII-related enzyme may regulate Max function in vivo. Our data suggest that both the ratio of Myc/Max hetero-oligomers to Max homo-oligomers and Max-specific regulation can contribute to determining the biological activity of Myc in vivo.
The peripherin gene, which encodes a neuronal-specific intermediate filament protein, is transcriptionally induced with a late time course when nerve growth factor (NGF) stimulates PC12 cells to differentiate into neurons. We have studied its transcriptional regulation in order to better understand the neuronal-specific end steps of the signal transduction pathway of NGF. By 5' deletion mapping of the peripherin promoter, we have localized two positive regulatory elements necessary for full induction by NGF: a distal positive element and a proximal constitutive element within 111 bp of the transcriptional start site. In addition, there is a negative regulatory element (NRE; -179 to -111), the deletion of which results in elevated basal expression of the gene. Methylation interference footprinting of the NRE defined a unique sequence, GGCAGGGCGCC, as the binding site for proteins present in nuclear extracts from both undifferentiated and differentiated PC12 cells. However, DNA mobility shift assays using an oligonucleotide probe containing the footprinted sequence demonstrate a prominent retarded complex in extracts from undifferentiated PC12 cells which migrates with slower mobility than do the complexes produced by using differentiated PC12 cell extract. Transfection experiments using peripherin-chloramphenicol acetyltransferase constructs in which the footprinted sequence has been mutated confirm that the NRE has a functional, though not exclusive, role in repressing peripherin expression in undifferentiated and nonneuronal cells. We propose a two-step model of activation of peripherin by NGF in which dissociation of a repressor from the protein complex at the NRE, coupled with a positive signal from the distal positive element, results in depression of the gene.
Myn, a novel murine approximately 18 kd basic/helix-loop-helix/"leucine zipper" (B/HLH/LZ) protein, forms a specific DNA-binding complex with the c-Myc oncoprotein through the HLH/LZ motif in both proteins. c-Myc/Myn recognizes a c-Myc-binding site (GACCACGTGGTC) with higher affinity than either protein by itself. CpG methylation of the recognition site greatly inhibits DNA binding, suggesting that DNA methylation may regulate the c-Myc/Myn complex in vivo. In 3T3 fibroblasts, Myn mRNA levels are induced several-fold by serum with delayed early kinetics, suggesting regulation by immediate-early gene products. Coexpression of Myn in a myc/ras rat embryo fibroblast focus formation assay specifically augmented c-myc transforming activity. We suggest that interaction of Myn with c-Myc stabilizes sequence-specific DNA binding in vivo.
The function of the c-Myc oncoprotein and its role in cell growth control is unclear. A basic region of c-Myc is structurally related to the basic motifs of helix-loop-helix (HLH) and leucine zipper proteins, which provide sequence-specific DNA binding function. The c-Myc basic region was tested for its ability to bind DNA by attaching it to the HLH dimerization interface of the E12 enhancer binding factor. Dimers of the chimeric protein, termed E6, specifically bound an E box element (GGCCACGTGACC) recognized by other HLH proteins in a manner dependent on the integrity of the c-Myc basic motif. Methylation of the core CpG in the E box recognition site specifically inhibited binding by E6, but not by two other HLH proteins. Expression of E6 (but not an E6 DNA binding mutant) suppressed the ability of c-myc to cooperate with H-ras in a rat embryo fibroblast transformation assay, suggesting that the DNA recognition specificity of E6 is related to that of c-Myc in vivo.
We describe the characterization of a novel cDNA, mbh1 (myc basic motif homolog-1), which was found during a search for candidate factors which might interact with the c-Myc oncoprotein. Embedded within the amino acid sequence encoded by mbh1 is a region distantly related to the basic/helix-loop-helix (B/HLH) DNA-binding motif and a potential nuclear localization signal. Mbh1 encodes a polypeptide structurally similar to the actin-severing proteins gelsolin and severin. Translation of mbh1 RNA in rabbit reticulocyte extracts produces an approximately 45 kd protein capable of binding actin-coupled agarose beads in vitro in a Ca2(+)-dependent manner. Antiserum raised to a trpE/mbh1 bacterial fusion protein recognizes an approximately 45 kb protein in murine 3T3 fibroblasts, suggesting that the cDNA encodes the complete Mbh1 protein. Examination of Mbh1 localization in 3T3 fibroblasts by indirect immunofluorescence reveals a larger cell population showing diffuse staining, and a smaller population exhibiting a distinct nuclear stain. Western analysis corroborates this intracellular localization and indicates that total cellular levels and localization of Mbh1 are not affected by the cell growth state. The data suggest that Mbh1 may play a role in regulating cytoplasmic and/or nuclear architecture through potential interactions with actin.
Olfactory receptor neurons (ORNs) do not express the typical neuronal intermediate filament proteins (IFPs), the neurofilament triplet proteins. Immunocytochemical evidence shows that ORNs coexpress vimentin and peripherin but distribute them differently. Specifically, ORNs contain vimentin in dendrites, cell bodies, and axons, but not in terminals in glomeruli; peripherin is present in axons, but excluded from dendrites, cell bodies, and terminal glomeruli. In adult rats, ORN axon fascicles are variably stained with antisera for peripherin; in juvenile rats, staining of fascicles is uniform. Staining with antibody to vimentin is uniform in both adult and juvenile ORN axon fascicles. The unusual pattern of IFP expression and intracellular sorting may have implications for the unique plastic and regenerative capacities of these neurons.
The expression of the neuronal type III intermediate filament protein peripherin was examined in the rat embryo during and following neuronogenesis in the spinal cord and the peripheral nervous system. In situ hybridization analysis reveals that peripherin mRNA is found in the mid-gestational rat embryo in ventral and lateral motoneurons in the spinal cord, and in neurons of all peripheral ganglia examined, including spinal, sympathetic, and enteric ganglia. Peripherin mRNA is seen only in post-migratory motoneurons or neuronal cells in aggregating ganglia, indicating that precursor cells do not express peripherin. To examine the expression of the protein, an affinity-purified antibody (anti-per) specific for a bacterially produced peripherin fusion protein was generated. Anti-per specifically recognizes a 58 kDa, cytoskeletal-enriched, nerve growth factor (NGF)-inducible protein of the expected tissue distribution. Immunocytodetection with anti-per shows that the initiation of peripherin protein synthesis is coincident with the morphological differentiation of neurons. In development, peripherin is one constituent of a program of gene expression activated at terminal neuronal differentiation.
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We have studied nerve growth factor (NGF) regulation of the expression of the tyrosine hydroxylase (TH) gene in PC12 cells. The TH gene encodes the initial and rate-limiting enzyme of the catecholamine biosynthetic pathway. We show that the TH gene is transiently transcriptionally induced by a mechanism reliant on new protein synthesis during 1-2 hr of NGF stimulation, a time following the induction of the c-fos gene at 15 min post-NGF treatment. A potential regulatory sequence located within the TH gene promoter, the TH-FSE, shares homology to a known regulatory element, the fat-specific element (FSE), which is found upstream from genes activated during adipocyte differentiation and binds the Fos-Jun transcription factor complex. We show that the TH-FSE DNA sequence elevates the basal level of transcription from the rat TH promoter and is required for NGF inducibility. This DNA element binds authentic Fos-Jun products produced abundance during NGF stimulation and by in vitro translation. We demonstrate further that the TH-FSE can bind proteins present in PC12 nuclear extracts in a sequence-specific manner. The DNA/nucleoprotein complex that forms increases in abundance during NGF stimulation and reaches a maximum level at 4 hr of treatment. Antibody inhibition studies utilizing an anti-Fos antibody indicate that Fos and/or Fos-related antigen(s) associate with the TH-FSE and suggest that the Fos protein family contributes to the regulation of TH in vivo. These results support a model in which NGF-induced immediate early genes, including c-Fos, contribute to the regulation of delayed early genes such as TH and thereby control neuronal differentiation.
In cells transformed by v-raf, an oncogenic counterpart of the serine/threonine kinase Raf-1, regulatory elements of the c-fos promoter were active under conditions of cell growth or stimulation for which they were inactive in untransformed control cells. This suggests that v-raf transforms by deregulating transcription of early response genes.
A DNA element located at positions -295 to -289 of the c-fos promoter (FAP site) is highly homologous to a consensus 12-O-tetradecanoyl phorbol-13-acetate-responsive element (TRE) and to a cyclic AMP (cAMP)-responsive element (CRE). We found that an oligonucleotide containing the FAP element was a transcription regulator which was distinct from both the TRE and CRE. When cloned in multiple copies in front of a reporter gene in HeLa cells, the FAP oligonucleotide was a powerful constitutive activator sequence. Conversely, in the same cells, reporter plasmids containing multiple copies of the TRE of the human metallothionein gene required phorbol esters for their induction. In PC12 cells, the FAP oligonucleotide was cAMP responsive. Its activity was mediated through a cAMP-dependent protein kinase II and did not rely on ongoing protein synthesis for activation. Adenovirus E1a proteins activated viral promoters through ATF (activation transcription factor) consensus binding sequences identical to the CRE. However, E1a repressed the FAP oligonucleotide-associated transcriptional activity in HeLa cells. In PC12 cells, E1a neither transactivated nor transrepressed the basal and cAMP-stimulated FAP activity. In contrast, the CRE of the human c-fos promoter located at -60 was weakly induced by cAMP and E1a in both HeLa and PC12 cells. We suggest that the FAP oligonucleotide acts through a factor(s) distinct from those employed by the TRE and CRE and that the FAP-associated protein factor(s) may differ in HeLa and PC12 cells in expression or posttranslational regulation.
A lambda cDNA library was prepared from polyadenylated RNA isolated from quiescent human diploid FS-4 fibroblasts stimulated with tumor necrosis factor for 3 h. Differential screening was used to isolate cDNA sequences that are stimulated by tumor necrosis factor. Eight distinct tumor necrosis factor-stimulated gene sequences (designated TSG-1, -6, -8, -12, -14, -21, -27, and -37) were partially sequenced and compared with known sequences from GenBank. TSG-1 was identical to the gene for interleukin-8. TSG-8 corresponded to the gene for monocyte chemotactic and activating factor. TSG-21 and -27 were identical to the genes for collagenase and stromelysin, respectively. The other four sequences showed no homologies with known genes. Patterns of induction of mRNAs corresponding to the eight cloned cDNAs by various cytokines, growth factors, and activators of second messenger pathways were analyzed in FS-4 cells.
Medulloblastoma is a rare brain tumor usually occurring in late childhood or early adolescence. Little is known regarding the cell of origin or cellular events leading to its malignant transformation. We have studied the expression of developmentally regulated mRNA in tumor samples by the Northern hybridization assay to determine a relative stage at which a block to further differentiation occurs. In a series of five medulloblastoma tumors analyzed, only one of eight markers for cellular differentiation, the glial fibrillary acidic protein mRNA, was expressed at significant levels. Interestingly, three of six medulloblastoma tumor samples were found to have elevated levels of c-myc mRNA. In one of these tumors, we have found evidence of mutation of the c-myc protooncogene. We discuss possible mechanisms of c-myc activation in medulloblastoma tumors.
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The c-fos gene, the cellular homologue of the transforming gene of the FBJ osteosarcoma virus, v-fos, is strongly induced in quiescent BALB/c 3T3 cells by growth factors and in other cell types by a wide variety of transmembrane signalling agents. c-fos is a member of a family of structurally related proteins which includes the fos-related antigens (fra). We have studied the dynamic state of the c-fos protein with an antibody prepared by immunizing rabbits with a plasmid-encoded fos fusion protein. In serum-stimulated BALB/c 3T3 cells, the antibody recognizes a nuclear antigen which resolves on SDS-PAGE as a 60-68-kD group of bands corresponding to c-fos, a doublet at 44-45-kD corresponding to the noncovalently associated p39 protein, as well as an approximately 50-kD band corresponding to a fra. We show that although c-fos protein synthesis is only transiently induced by serum, the c-fos protein persists within the cell after its synthesis has ceased, and it decays with a half-life of 2 hours. Significantly, newly synthesized p39 continues to appear in the immune-precipitated complex even at times when c-fos is no longer synthesized. These kinetics indicate that even following shutoff of c-fos protein synthesis, p39 is newly synthesized and can complex with c-fos protein or a fos-related antigen. During this time, c-fos also undergoes an extensive posttranslational modification. The modification is partially reversed by phosphatase treatment, which implicates protein phosphorylation. Together these results suggest that both interaction with p39 and phosphorylation may progressively modify the properties of c-fos and/or the fos-related antigens over a period of 4-8 hours following the shutoff of fos synthesis. We discuss the implications of the dynamic state of c-fos and fra protein interactions for the function of these proteins.
We have cloned the rat gene encoding peripherin, a neuronal-specific intermediate filament protein that is NGF-regulated. Determination of the complete sequence, including 821 nucleotides of the 5'-flanking region, allows us to make conclusions about the evolutionary origin of the peripherin gene, its homology with other intermediate filament proteins, and possible mechanisms of regulation of peripherin expression in neurons. The positions of the eight peripherin gene introns correspond to the intron patterns of desmin, vimentin, and GFAP, with one example of intron sliding. Together with protein sequence homologies, this conclusively demonstrates that peripherin is a type III intermediate filament protein. The peripherin promoter contains sequences homologous to regions of other NGF-regulated promoters, which may function in peripherin induction by NGF.
F9 embryonal carcinoma (EC) cells serve as a model system for early mammalian development. We have investigated the effect of the E1a gene products on the F9 cell differentiation program by stably introducing into these cells plasmids which express wild type or mutant forms of E1a. We have found that expression of the 12S E1a mRNA product results in the expression of the endoA gene, a marker specific for the differentiated phenotype of F9 cells, as well as an altered cell morphology associated with the differentiated state. These alterations are not observed in F9 cells which express the 13S E1a product. Other markers specific for the differentiated state are regulated normally in the E1a transformants following exposure to retinoic acid (R.A.) and dibutyryl cyclic AMP (dbcAMP). We discuss these results in the context of the well established transcriptional activities of the E1a proteins.