Faculty Bibliography

The production of the nuclear oncoproteins Fos and Jun is rapidly induced in response to extracellular signals. In the nucleus, the two proteins combine to form a tight complex via leucine zipper domains. The resulting Fos-Jun heterodimer can bind to the TPA-responsive element (TRE) by way of a novel, highly basic motif and can activate the transcription of TPA-responsive genes. The existence of several Fos- and Jun-related proteins with dimerization and DNA binding properties similar to Fos and Jun suggests that these two oncoproteins may be part of a network of related but functionally distinct transcription factors

The products of the fos and jun protooncogenes form a stable heterodimer which binds to the TPA-responsive element (TRE) TGACTCA with high affinity. These two proteins, together with the yeast GCN4 protein, belong to a growing family of transcription factors, including FosB, Fra1, JunB and JunD, whose members share a highly conserved DNA-binding domain. This domain is composed of two structures: a basic motif, which is thought to bind directly to DNA; and a leucine zipper, which provides a dimerization interface. Although this domain is highly conserved in Fos, Jun and GCN4, each of these three proteins has very different relative affinities for the TRE. To understand these differences, we used 'domain-swapping' experiments designed to test the relative contributions of the basic motif and the leucine zipper to TRE-binding affinity. Here we show that fos, jun and GCN4 have different affinities for the TRE due to differences in the hetero- or homo-dimerization capacity of their leucine zipper domains; the basic motifs of these three proteins have comparable DNA binding potential. These results indicate that leucine zippers control the types of protein complexes which can associate with a TRE and regulate gene expression

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

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 investigated the sequences that are necessary and sufficient for the induction of the c-fos gene by serum, TPA or PDGF in different cell types. The dyad symmetry element (DSE) is a regulatory element of the c-fos gene previously shown to be required for induction of c-fos transcription by serum. We show that the DSE is also necessary for the induction of c-fos by either TPA or PDGF in NIH3T3 cells. We also show that in NIH 3T3 cells the DSE is sufficient to confer inducibility on a heterologous promoter, the beta-globin promoter, when serum provides the stimulus. However, it is not sufficient when either TPA or PDGF is the inducer. This suggests a requirement in 3T3 cells for cooperating sequence elements for TPA or PDGF induction but not for serum. Interestingly, the need for cooperating elements for TPA induction is abolished in HeLa cells since the DSE alone is sufficient for TPA inducibility of the beta-globin promoter in these cells. Thus, the highly transformed HeLa cell line displays diminished sequence requirements for TPA induction. We discuss the possibility that mutations which diminish the stringent transcriptional control of protooncogenes such as c-fos may contribute to the transformed state

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

Utilizing F9 embryonal carcinoma cells as a model system for early mammalian development, we have studied the pattern of expression of the endogenous murine homolog of the human K-fgf/hst oncogene, which encodes a new member of the fibroblast growth factors (FGFs) family. The K-fgf mRNA is expressed in undifferentiated F9 cells and its level becomes undetectable upon the induction of differentiation. Furthermore, a growth-promoting activity with properties identical to those of K-FGF is present in the conditioned medium of F9 cells, but absent in that of differentiated cells. Shut-off of K-fgf expression is mediated at the transcriptional level. The acidic FGF gene is also expressed in undifferentiated F9 cells and down modulated once differentiation is induced. In contrast, int-2, another member of the FGF gene family, is transcriptionally induced in differentiated F9 cells. Our data suggest that single members of the FGF gene family may perform distinct functions in vivo, and that the physiological role of K-FGF may be related to early development

Mutagenesis of the fos protein supports the hypothesis that a heptad repeat of leucine residues stabilizes the interaction between the fos and jun proteins. We show that the complex between fos and jun can bind to DNA more tightly than either protein alone and that basic residues adjacent to the leucine repeat of fos contribute to the DNA-binding potential of the complex