Faculty Bibliography

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

Human adenovirus 2 grows poorly in monkey cells, partly because of defects in late gene expression. Since deletions in early region 4 (E4) cause similar defects in late gene expression, we examined E4 mRNA expression in abortive infections. Processing of E4 mRNAs was defective during abortive infections, most likely at the level of splicing. At early times in productive infections in HeLa cells, the major E4 species produced is a 2-kb mRNA; at late times, a shift occurs so that smaller spliced E4 mRNAs are also produced. In CV-1 cells, a nonpermissive monkey cell line, this shift did not take place and only the 2-kb species was produced at late times, suggesting a defect in E4 mRNA splicing during abortive infections. The adenovirus DNA-binding protein (DBP) was required for normal processing of E4 mRNAs, since a host range mutant (hr602) containing an altered DBP gene showed a normal late E4 mRNA pattern in CV-1 cells; in addition, DBP was required during infections in HeLa cells for late E4 mRNA expression. DBP was not required for production of the late E4 pattern in transient expression assays in HeLa or 293 cells, suggesting that a second factor in addition to the DBP, present during infection but not transfection, modulates E4 mRNA processing

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

We show that members of two major families of transcription factors, the helix-loop-helix and C/EBP families, interact with the c-fos serum response element (SRE). Two cDNA clones encoding SRE binding factors (clones 9 and 21) were isolated by the direct screening of a PC12 lambda-gt11 cDNA library using SRE oligonucleotide sequences as probes. Clone 9 encodes the rat homolog of the human HLH transcription factor, E12 (called here rE12). Clone 21 encodes a b-zip domain polypeptide that is related to the liver transcription factor C/EBP, and is homologous to the human NFIL-6 transcription factor (called here rNFIL-6). Using in vitro-translated products we show that rNFIL-6 recognizes a 'CCAATT' motif which overlaps the c-fos dyad symmetry element (DSE), the binding site for serum regulatory factor (SRF). Factor rE12 binds to an E-box enhancer sequence, 'CATCTG', immediately adjacent to the rNFIL-6 site, within the SRE. Antibodies specific to rE12 and rNFIL-6 disrupt nucleoprotein complexes with these DNA-binding sites, confirming the interaction of native in vivo factors. We present evidence that rNFIL-6 and SRF binding are mutually exclusive, consistent with the overlap of their binding sites. The demonstration that rE12 and rNFIL-6 bind to the SRE at sites adjacent to the major c-fos regulatory element, the DSE, raises the possibility that helix-loop-helix and C/EBP families regulate the SRE and provide a new basis for the multifunctional properties of the SRE, including possible tissue specificity of expression

The c-fos serum response element (SRE) is a multifunctional regulatory region of the c-fos promoter that responds to a variety of inducers. Recently, we have demonstrated that the SRE binds the C/EBP-related transcription factor rat NFIL-6 (rNFIL-6). In this study we show that rNFIL-6 is regulated by the cAMP second messenger pathway in the rat pheochromocytoma PC12 cell line. Following forskolin treatment, rNFIL-6 binding to the SRE is increased, and the factor becomes phosphorylated and undergoes a trans-location to the nucleus. In transient cotransfection assays, rNFIL-6 is capable of trans-activating the c-fos promoter in a manner dependent on the SRE. These data show that rNFIL-6 undergoes a novel activation in which cAMP-induced nuclear trans-location allows rNFIL-6 to bind to the SRE and contribute to c-fos activation. We propose that rNFIL-6 is an additional regulatory component of the c-fos gene, which provides cAMP responsiveness to the multifunctional SRE

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

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

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

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