Mitosis-specific phosphorylation of the nuclear oncoproteins Myc and Myb.

The c-myc and c-myb proto-oncogenes encode phosphorylated nuclear DNA binding proteins that are likely to be involved in transcriptional regulation. Here we demonstrate that both Myc and Myb proteins are hyperphosphorylated during mitosis. In the case of Myb, hyperphosphorylation is accompanied by the appearance of three M phase-specific tryptic phosphopeptides. At least one of these phosphopeptides corresponds to a phosphopeptide generated after phosphorylation of Myb in vitro by p34cdc2 kinase. By contrast, the mitotic hyperphosphorylation of Myc does not correlate with the appearance of unique phosphopeptides, suggesting that M phase and interphase sites may be clustered within the same peptides. In addition Myc does not appear to be a target for p34cdc2 phosphorylation. The hyperphosphorylated forms of Myc and Myb from mitotic cells are functionally distinct from the corresponding interphase proteins in that the former have reduced ability to bind nonspecificially to double-stranded DNA cellulose. Furthermore, mitotic Myb binds poorly to oligodeoxynucleotides containing an Myb response element. We surmise that the decreased DNA binding capacity of hyperphosphorylated Myb and Myc during M phase may function to release these proteins from chromatin during chromosome condensation.

Max: a helix-loop-helix zipper protein that forms a sequence-specific DNA-binding complex with Myc.

The myc protooncogene family has been implicated in cell proliferation, differentiation, and neoplasia, but its mechanism of function at the molecular level is unknown. The carboxyl terminus of Myc family proteins contains a basic region helix-loop-helix leucine zipper motif (bHLH-Zip), which has DNA-binding activity and has been predicted to mediate protein-protein interactions. The bHLH-Zip region of c-Myc was used to screen a complementary DNA (cDNA) expression library, and a bHLH-Zip protein, termed Max, was identified. Max specifically associated with c-Myc, N-Myc, and L-Myc proteins, but not with a number of other bHLH, bZip, or bHLH-Zip proteins. The interaction between Max and c-Myc was dependent on the integrity of the c-Myc HLH-Zip domain, but not on the basic region or other sequences outside the domain. Furthermore, the Myc-Max complex bound to DNA in a sequence-specific manner under conditions where neither Max nor Myc exhibited appreciable binding. The DNA-binding activity of the complex was dependent on both the dimerization domain and the basic region of c-Myc. These results suggest that Myc family proteins undergo a restricted set of interactions in the cell and may belong to the more general class of eukaryotic DNA-binding transcription factors.

New findings on the congenital transmission of avian leukosis viruses.

RAV-0, an endogenous avian leukosis virus, does not undergo congenital transmission in infected K28 chickens. In contrast, avian leukosis viruses of exogenous origin undergo highly efficient congenital transmission. The relative abilities of endogenous and exogenous viruses to undergo congenital transmission appear to be determined by the p27 capsid proteins of these viruses.

Sequence-specific DNA binding by the c-Myc protein.

While it has been known for some time that the c-Myc protein binds to random DNA sequences, no sequence-specific binding activity has been detected. At its carboxyl terminus, c-Myc contains a basic--helix-loop-helix (bHLH) motif, which is important for dimerization and specific DNA binding, as demonstrated for other bHLH protein family members. Of those studied, most bHLH proteins bind to sites that contain a CA- -TG consensus. In this study, the technique of selected and amplified binding-sequence (SAAB) imprinting was used to identify a DNA sequence that was recognized by c-Myc. A purified carboxyl-terminal fragment of human c-Myc that contained the bHLH domain bound in vitro in a sequence-specific manner to the sequence, CACGTG. These results suggest that some of the biological functions of Myc family proteins are accomplished by sequence-specific DNA binding that is mediated by the carboxyl-terminal region of the protein.

Myc and Max homologs in Drosophila.

The proteins encoded by the myc proto-oncogene family are involved in cell proliferation, apoptosis, differentiation, and neoplasia. Myc acts through dimerization with Max to bind DNA and activate transcription. Homologs of the myc and max genes were cloned from the fruit fly Drosophila melanogaster and their protein products (dMyc and dMax) were shown to heterodimerize, recognize the same DNA sequence as their vertebrate homologs, and activate transcription. The dMyc protein is likely encoded by the Drosophila gene diminutive (dm), a mutation in which results in small body size and female sterility caused by degeneration of the ovaries. These findings indicate a potential role for Myc in germ cell development and set the stage for genetic analysis of Myc and Max.

Myc target genes.

The myc family of proto-oncogenes belongs to the basic helix-loop-helix leucine-zipper (bHLHZ) class of transcription factors. Myc proteins function as transcriptional activators through heterodimerization with Max, but might also act as negative regulators of transcription. Identification of genes directly controlled by Myc-Max has proved difficult, but recent work is producing a growing list of candidates. Results to date suggest that Myc-Max influences cell growth and proliferation through direct activation of genes involved in DNA synthesis, RNA metabolism and cell-cycle progression.

Myc and Max function as a nucleoprotein complex.

The Myc family of oncoproteins are thought to regulate proliferation and differentiation in a wide variety of cell types. Recent studies show that Myc proteins form sequence-specific DNA-binding complexes with Max, a new member of the helix-loop-helix leucine zipper protein class. The properties of the Myc-Max complex suggest a mechanism for Myc's function in both normal and neoplastic cell behavior.

c-erbA encodes multiple proteins in chicken erythroid cells.

To identify and characterize the proteins encoded by the erbA proto-oncogene, we expressed the C-terminal region of v-erbA in a bacterial trpE expression vector system and used the fusion protein to prepare antiserum. The anti-trp-erbA serum recognized the P75gag-erbA protein encoded by avian erythroblastosis virus and specifically precipitated six highly related proteins ranging in size from 27 to 46 kilodaltons from chicken embryonic erythroid cells. In vitro translation of a chicken erbA cDNA produced essentially the same pattern of proteins. Partial proteolytic maps and antigenicity and kinetic analyses of the in vivo and in vitro proteins indicated that they are related and that the multiple bands are likely to arise from internal initiations within c-erbA to generate a nested set of proteins. All of the c-erbA proteins are predominantly associated with chicken erythroblast nuclei. However, Nonidet P-40 treatment resulted in extraction of the three smaller proteins, whereas the larger proteins were retained. During differentiation of erythroid cells in chicken embryos, we found maximal levels of c-erbA protein synthesis at days 7 to 8 of embryogenesis. By contrast, c-erbA mRNA levels remained essentially constant from days 5 to 12. Together, our results indicate that posttranscriptional or translational mechanisms are involved in regulation of c-erbA expression and in the complexity of its protein products.

c-myc and c-myb protein degradation: effect of metabolic inhibitors and heat shock.

The proteins encoded by both viral and cellular forms of the c-myc oncogene have been previously demonstrated to have exceptionally short in vivo half-lives. In this paper we report a comparative study on the parameters affecting turnover of nuclear oncoproteins c-myc, c-myb, and the rapidly metabolized cytoplasmic enzyme ornithine decarboxylase. The degradation of all three proteins required metabolic energy, did not result in production of cleavage intermediates, and did not involve lysosomes or ubiquitin. A five- to eightfold increase in the half-life of c-myc proteins, and a twofold increase in the half-life of c-myb proteins was detected after heat-shock treatment at 46 degrees C. In contrast, heat shock had no effect on the turnover of ornithine decarboxylase. Heat shock also had the effect of increasing the rate of c-myc protein synthesis twofold, whereas c-myb protein synthesis was decreased nearly fourfold. The increased stability and synthesis of c-myc proteins led to an overall increase in the total level of c-myc proteins in response to heat-shock treatment. Furthermore, treatments which reduced c-myc and c-myb protein turnover, such as heat shock and exposure to inhibitors of metabolic energy production, resulted in reduced detergent solubility of both proteins. The recovery from heat shock, as measured by increased turnover and solubility, was energy dependent and considerably more rapid in thermotolerant cells.

Proteins encoded by the human c-myc oncogene: differential expression in neoplastic cells.

To examine myc protein products in the wide variety of human tumor cells having alterations of the c-myc locus, we have prepared an antiserum against a synthetic peptide corresponding to the predicted C-terminal sequence of the human c-myc protein. This antiserum (anti-hu-myc 12C) specifically precipitated two proteins of 64 and 67 kilodaltons in quantities ranging from low levels in normal fibroblasts to 10-fold-higher levels in Epstein-Barr virus-immortalized and Burkitt's lymphoma cell lines, to 20- to 60-fold-higher levels in cell lines having amplified c-myc. The p64 and p67 proteins were found to be highly related by partial V8 proteolytic mapping, and both were demonstrated to be encoded by the c-myc oncogene, using hybrid-selected translation of myc-specific RNA. In addition, the p64 protein was specifically precipitated from cells transfected with a translocated c-myc gene. Both p64 and p67 were found to be nuclear phosphoproteins with extremely short half-lives. In tumor cell lines having alterations at the c-myc locus due to amplification or translocation, we observed a significant change in the expression of p64 relative to p67 when compared with normal or Epstein-Bar virus-immortalized cells.

Thyroid hormone receptor can modulate retinoic acid-mediated axis formation in frog embryogenesis.

Thyroid hormone receptor acts as a hormone-dependent transcriptional transactivator and as a transcriptional repressor in the absence of thyroid hormone. Specifically, thyroid hormone receptor can repress retinoic acid-induced gene expression through interactions with retinoic acid receptor. (Retinoic acid is a potent teratogen in the frog Xenopus laevis, acting at early embryonic stages to interfere with the formation of anterior structures. Endogenous retinoic acid is thought to act in normal anterior-posterior axis formation.) We have previously shown that thyroid hormone receptor RNA (alpha isotype) is expressed and polysome-associated during Xenopus embryogenesis preceding thyroid gland maturation and endogenous thyroid hormone production (D. E. Banker, J. Bigler, and R. N. Eisenman, Mol. Cell. Biol. 11:5079-5089, 1991). To determine whether thyroid hormone receptor might influence the effects of retinoic acid in early frog development, we have examined the results of ectopic thyroid hormone receptor expression on retinoic acid teratogenesis. We demonstrate that microinjections of full-length thyroid hormone receptor RNA protect injected embryos from retinoic acid teratogenesis. DNA binding is apparently essential to this protective function, as truncated thyroid hormone receptors, lacking DNA-binding domains but including hormone-binding and dimerization domains, do not protect from retinoic acid. We have shown that microinjections of these dominant-interfering thyroid hormone receptors, as well as anti-thyroid hormone receptor antibodies, increase retinoic acid teratogenesis in injected embryos, presumably by inactivating endogenous thyroid hormone receptor. This finding suggests that endogenous thyroid hormone receptors may act to limit retinoic acid sensitivity. On the other hand, after thyroid hormone treatment, ectopic thyroid hormone receptor mediates teratogenesis that is indistinguishable from the dorsoanterior deficiencies produced in retinoic acid teratogenesis. The previously characterized retinoic acid-responsive gene, Xhox.lab2, can be induced by thyroid hormone in embryos ectopically expressing thyroid hormone receptor and is less responsive to retinoic acid in such embryos. The fact that both thyroid hormone and retinoic acid can affect overlapping gene expression pathways to produce abnormal embryonic axes and can regulate the same early-expressed gene suggests a model in which thyroid hormone receptor blocks retinoic acid receptor-mediated teratogenesis by directly repressing retinoic acid-responsive genes.

Thyroid hormone receptor transcriptional activity is potentially autoregulated by truncated forms of the receptor.

ErbA/thyroid hormone receptor is a nuclear receptor that can affect transcription from promoters containing a thyroid hormone response element (TRE) in a thyroid hormone (T3)-dependent manner. We reported earlier that the thyroid hormone receptor is expressed in embryonic avian erythroid cells as a nested set of four proteins with a common C terminus. The full-length receptor is capable of both high-affinity binding to thyroid hormone and specific binding to DNA. We now report that the two smallest ErbA forms, which contain the hormone-binding domain but lack the N-terminal DNA-binding domain, have the same affinity for T3 as does full-length ErbA but are incapable of specific DNA binding. In transactivation assays, these N-terminally truncated proteins are able to specifically suppress both transcriptional repression and hormone-dependent transcriptional activation by the full-length ErbA. We also find that retinoic acid-dependent transactivation by retinoic acid receptors is inhibited by the truncated ErbA proteins. Furthermore, the smaller ErbA forms inhibit binding to TREs by full-length ErbA in vitro. Results from experiments involving site-specific mutagenesis of a conserved region within the hormone-binding domain of the smaller ErbA proteins indicate that the suppressive effect of the smaller receptor forms is independent of hormone binding and that this region is important in mediating protein-hormone as well as protein-protein interactions. We have also found that full-length ErbA homodimers can be detected only in the presence of a specific DNA-binding site. However, no association between full-length and the N-terminally truncated non-DNA-binding ErbA proteins could be detected, indicating that the complex either is unstable or does not form. Our results suggest that inhibition of receptor function occurs through transient formation of heterodimers which lack DNA-binding activity or by competition for factors which positively affect DNA binding by the full-length protein. This finding raises the possibility that thyroid hormone receptor transcriptional activity is autoregulated by means of alternative receptor translation products acting in a dominant negative manner.

Detection of a Myc-associated protein by chemical cross-linking.

A single nuclear protein (Myc-associated protein) can be specifically cross-linked to avian Myc proteins by treatment of nuclei or cells with the reversible cross-linker dimethyl 3,3'-dithiobis-propionimidate. Myc-associated protein has a molecular weight of approximately 500,000, is not detectably phosphorylated and, in contrast to Myc, has a long apparent half-life of greater than 3 h.

Isolation of a thyroid hormone-responsive gene by immunoprecipitation of thyroid hormone receptor-DNA complexes.

Thyroid hormone (T3) receptor (TR) is a ligand-dependent transcription factor that acts through specific binding sites in the promoter region of target genes. In order to identify new genes that are regulated by T3, we used anti-TR antiserum to immunoprecipitate TR-DNA complexes from GH4 cell nuclei that had previously been treated with a restriction enzyme. Screening of the immunopurified, cloned DNA for TR binding sites by electrophoretic mobility shift assay yielded 53 positive clones. A subset of these clones was specifically immunoprecipitated with anti-TR antiserum and may therefore represent biologically significant binding sites. One of these clones, clone 122, was characterized in detail. It includes sequences highly related to the NICER long terminal repeat-like element and contains three TR binding sites as determined by DNase I footprinting. Two of the clone 122 TR binding sites are located upstream of the TATA box, and one is located downstream. The TR binding site downstream from the promoter was necessary and sufficient to confer T3-dependent regulation in transient transfection experiments. Expression of a reporter construct under the control of the clone 122 promoter region was activated by TR in the absence of ligand and returned to basal levels after T3 addition. Clone 122 sequences hybridize to at least two different mRNAs of approximately 6 and 10 kb from GH4 cells. The levels of both of these mRNAs increased upon removal of T3. Our studies suggest that specific immunoprecipitation of chromatin allows identification of binding sites and target genes for transcription factors.

Expression of the c-myc proto-oncogene during development of Xenopus laevis.

We isolated and characterized Xenopus laevis c-myc cDNAs from an oocyte-specific library. These cDNA clones encompass 2.35 kilobases of the X. laevis c-myc RNA and contain the entire coding domain of 1,257 nucleotides of the 419-amino acid-long X. laevis c-myc protein. The 2.7-kilobase X. laevis c-myc mRNA is expressed in the oocyte, maintained in the egg, and is present throughout the early cleavage stages of embryogenesis. At the time of transcriptional activation in the embryo the c-myc RNA levels show a significant decline and then reaccumulate continuously throughout the remainder of premorphogenic development. At the early neurula stage of embryogenesis the pattern of c-myc RNA expression is elevated in the mesoderm with respect to the endoderm and ectoderm. In the adult X. laevis the c-myc mRNA is expressed in some (e.g., skin, muscle) but not all differentiated tissues. The X. laevis c-myc protein migrates as a doublet of 61,000- and 64,000-dalton species. Both species are phosphorylated in oocytes and somatic cells, exhibit extremely short half-lives of less than 30 min, and are localized to the nuclear fraction of somatic cells. By contrast, the oocyte protein shows both cytoplasmic and germinal vesicle distribution and appears to be stable.

Delineation of transcriptional control signals within the Moloney murine sarcoma virus long terminal repeat.

We identified three distinct elements within the Moloney murine sarcoma virus long terminal repeat that control transcription. The phenotypes of unidirectional deletion mutants of the long terminal repeat were assayed in microinjected frog oocytes and in transfected mouse fibroblasts. Steady-state levels of RNA bearing the same 5' terminus as the authentic Moloney murine sarcoma viral transcripts were measured by primer extension in assays that included a pseudo-wild-type internal reference. Mutant phenotypes define the boundaries of three functional elements. A region between 21 and 31 base pairs upstream from the mRNA cap site contains AT-rich sequences that function to establish the transcription start site. A second control element, termed the distal signal, lies between 31 and 84 base pairs upstream of the mRNA cap site. A CAT box consensus sequence is located at the 5' boundary of the distal signal. Additional components of the distal signal include a hexanucleotide sequence that is repeated four times. The distal signal augments transcription efficiency in oocytes but contributes only weakly to long terminal repeat-mediated expression in mouse fibroblasts. A third transcriptional control element lies between 156 and 364 base pairs upstream of the mRNA cap site. This element includes the 75-base-pair repeats previously identified as the Moloney murine sarcoma virus enhancer. In contrast to the distal signal, the Moloney murine sarcoma virus enhancer is crucial for significant expression in mouse fibroblasts but does not contribute to transcriptional expression in frog oocytes.

The thyroid hormone receptor gene (c-erbA alpha) is expressed in advance of thyroid gland maturation during the early embryonic development of Xenopus laevis.

The c-erbA proto-oncogene encodes the thyroid hormone receptor, a ligand-dependent transcription factor which plays an important role in vertebrate growth and development. To define the role of the thyroid hormone receptor in developmental processes, we have begun studying c-erbA gene expression during the ontogeny of Xenopus laevis, an organism in which thyroid hormone has well-documented effects on morphogenesis. Using polymerase chain reactions (PCR) as a sensitive assay of specific gene expression, we found that polyadenylated erbA alpha RNA is present in Xenopus cells at early developmental stages, including the fertilized egg, blastula, gastrula, and neurula. By performing erbA alpha-specific PCR on reverse-transcribed RNAs from high-density sucrose gradient fractions prepared from early-stage embryos, we have demonstrated that these erbA transcripts are recruited to polysomes. Therefore, erbA is expressed in Xenopus development prior to the appearance of the thyroid gland anlage in tailbud-stage embryos. This implies that erbA alpha/thyroid hormone receptors may play ligand-independent roles during the early development of X. laevis. Quantitative PCR revealed a greater than 25-fold range in the steady-state levels of polyadenylated erbA alpha RNA across early stages of development, as expressed relative to equimolar amounts of total embryonic RNA. Substantial increases in the levels of erbA alpha RNA were noted at stages well after the onset of zygotic transcription at the mid-blastula transition, with accumulation of erbA alpha transcripts reaching a relative maximum in advance of metamorphosis. We also show that erbA alpha RNAs are expressed unequally across Xenopus neural tube embryos. This differential expression continues through later stages of development, including metamorphosis. This finding suggests that erbA alpha/thyroid hormone receptors may play roles in tissue-specific processes across all of Xenopus development.

A second c-myb protein is translated from an alternatively spliced mRNA expressed from normal and 5'-disrupted myb loci.

The major protein encoded by the c-myb oncogene in many species has been identified as an unstable, nuclear DNA-binding protein with an apparent molecular mass of 75 to 80 kilodaltons (p75c-myb). Recently, an alternatively spliced form of c-myb-encoded mRNA has been identified in murine cells containing either normal or rearranged c-myb genes. This mRNA includes a new exon, termed E6A, formed through use of cryptic splice sites located in the large intron between c-myb exons vE6 and vE7. E6A is predicted to contribute an internal 121-residue in-frame insertion into a region C terminal of the DNA-binding domain the c-myb-encoded protein. Here we report the identification of an 85-kilodalton (p85c-myb-E6A) protein as the translation product of the alternatively spliced E6A c-myb mRNA. This protein as well as p75c-myb were precipitated with anti-Myb antibodies raised against the conserved DNA-binding region of c-Myb. Proteolytic mapping studies showed that the two proteins are highly related but not identical. However, only the p85 protein reacted with an antiserum prepared against the E6A region expressed in bacteria, demonstrating that p85 but not p75 contains E6A sequences. In addition, the mobilities of both p85 and p75 were increased in myeloid tumor cell lines containing proviral integrations upstream of the 5' coding exons of v-myb, indicating that both proteins are truncated forms of c-Myb expressed from the same disrupted allele. p75c-myb and p85c-myb-E6A were indistinguishable with respect to nuclear localization and protein half-life. Furthermore, both forms of Myb were synthesized continuously throughout the cell cycle in 70Z ore-B cells. The contribution of the E6A domain to c-myb function remains to be elucidated.

V-myc- and c-myc-encoded proteins are associated with the nuclear matrix.

A series of extraction procedures were applied to avian nuclei which allowed us to define three types of association of v-myc- and c-myc-encoded proteins with nuclei: (i) a major fraction (60 to 90%) which is retained in DNA- and RNA-depleted nuclei after low- and high-salt extraction, (ii) a small fraction (1%) released during nuclease digestion of DNA in intact nuclei in the presence of low-salt buffer, and (iii) a fraction of myc protein (less than 10%) extractable with salt or detergents and found to have affinity for both single- and double-stranded DNA. Immunofluorescence analysis with anti-myc peptide sera on cells extracted sequentially with nucleases and salts confirmed the idea that myc proteins were associated with a complex residual nuclear structure (matrix-lamin fraction) which also contained avian nuclear lamin protein. Dispersal of myc proteins into the cytoplasm was found to occur during mitosis. Both c-myc and v-myc proteins were associated with the matrix-lamin, suggesting that the function of myc may relate to nuclear structural organization.

Targeted deletion of the S-phase-specific Myc antagonist Mad3 sensitizes neuronal and lymphoid cells to radiation-induced apoptosis.

The Mad family comprises four basic-helix-loop-helix/leucine zipper proteins, Mad1, Mxi1, Mad3, and Mad4, which heterodimerize with Max and function as transcriptional repressors. The balance between Myc-Max and Mad-Max complexes has been postulated to influence cell proliferation and differentiation. The expression patterns of Mad family genes are complex, but in general, the induction of most family members is linked to cell cycle exit and differentiation. The expression pattern of mad3 is unusual in that mad3 mRNA and protein were found to be restricted to proliferating cells prior to differentiation. We show here that during murine development mad3 is specifically expressed in the S phase of the cell cycle in neuronal progenitor cells that are committed to differentiation. To investigate mad3 function, we disrupted the mad3 gene by homologous recombination in mice. No defect in cell cycle exit and differentiation could be detected in mad3 homozygous mutant mice. However, upon gamma irradiation, increased cell death of thymocytes and neural progenitor cells was observed, implicating mad3 in the regulation of the cellular response to DNA damage.