Structural organization and nucleotide sequence of mouse c-myb oncogene: activation in ABPL tumors is due to viral integration in an intron which results in the deletion of the 5'' coding sequences.

Bacteriophage libraries of mouse DNA were screened for sequences homologous to the v-myb oncogene and two overlapping clones containing the v-myb related region were isolated. Restriction enzyme mapping, heteroduplex analysis and nucleotide sequence analysis revealed the presence of nine exons. Six of these exons are homologous to the v-myb region while the other three exons are derived from the 5' region which is deleted in the viral oncogene. The sequences downstream to the sixth v-myb exon are not included in the 17 kbp of DNA sequences analyzed in this study. Comparison of the structure of the normal c-myb clone with its rearranged couterpart present in plasmacytoid lymphosarcomas revealed that the rearrangements occur in this locus as a result of viral integration. Present studies demonstrate that such a viral insertion interrupts the c-myb coding region at a region identical to that observed in the generation of the v-myb gene of avian myeloblastosis virus and results in the synthesis of mRNAs that lack the same 5' coding region.     

Subnuclear localization of proteins encoded by the oncogene v-myb and its cellular homolog c-myb.

The retroviral transforming gene v-myb encodes a 45,000-Mr nuclear transforming protein (p45v-myb). p45v-myb is a truncated and mutated version of a 75,000-Mr protein encoded by the chicken c-myb gene (p75c-myb). Like its viral counterpart, p75c-myb is located in the cell nucleus. As a first step in identifying nuclear targets involved in cellular transformation by v-myb and in c-myb function, we determined the subnuclear locations of p45v-myb and p75c-myb. Approximately 80 to 90% of the total p45v-myb and p75c-myb present in nuclei was released from nuclei at low salt concentrations, exhibited DNA-binding activity, and was attached to nucleoprotein particles when released from the nuclei after digestion with nuclease. A minor portion of approximately 10 to 20% of the total p45v-myb and p75c-myb remained tightly associated with the nuclei even in the presence of 2 M NaCl. These observations suggest that both proteins are associated with two nuclear substructures tentatively identified as the chromatin and the nuclear matrix. The function of myb proteins may therefore depend on interactions with several nuclear targets.     

Generation of altered transcripts by retroviral insertion within the c-myb gene in two murine monocytic leukemias

Two murine monocytic leukemia cell lines, WEHI-265 and WEHI-274, were found to carry a rearranged c-myb gene. The rearrangements are due to insertion of a deleted Moloney murine leukemia virus (Mo-MLV) provirus in the 5' region of the c-myb gene and thus are similar to rearrangements in the ABPL tumors (G. L. C. Shen-Ong, M. Potter, J. F. Mushinski, S. Lavu, and E. P. Reddy, Science 226:1077-1080, 1984). In each cell line, the retroviral insertion has induced high levels of two aberrant RNA species, which, as in the ABPL tumors (G. L. C. Shen-Ong, H. C. Morse, M. Potter, and J. F. Mushinski, Mol. Cell. Biol. 6:380-392, 1986), contain both viral (Mo-MLV) and cellular (myb) sequences. Both species lack the sequences encoding the amino terminus of the c-myb protein and thus could encode a protein which, like the v-myb gene products (and the predicted ABPL myb proteins), is truncated at the amino terminus. We have found that the larger (5.3 kilobase [kb]) and more abundant of the tumor-specific myb RNAs was predominantly nuclear, while the smaller species (3.9 kb) was cytoplasmic. Furthermore, our data imply that the 3.9-kb RNA was derived from the 5.3-kb RNA by an additional splice which utilized a cryptic splice acceptor site within the viral gag sequences. On the basis of subcellular distribution and predicted translational potential, we conclude that the 3.9-kb RNA is probably the mRNA which encodes a truncated myb protein. We also show that, due to different insertion points in W265 and W274, the W274 myb RNAs contained sequences from a c-myb exon upstream of the exons represented in the W265 (and ABPL) RNAs. The significance of our findings with regard to transformation by myb in these tumors is discussed.     

v-myb does not prevent the expression of c-myb in avian erythroblasts.

The v-myb oncogene of avian myeloblastosis virus transforms myeloid cells exclusively, both in vivo and in vitro. The c-myb proto-oncogene from which v-myb arose is expressed at relatively high levels in immature hematopoietic cells of the lymphoid, erythroid, and myeloid lineages but not in myeloblasts transformed by v-myb. This finding suggested that the nuclear v-myb gene product p48v-myb might act directly to inhibit the normal expression of the c-myb gene. I have therefore used a selectable avian retroviral vector to express p48v-myb in avian erythroblasts which normally express high levels of the c-myb gene product p75c-myb. The results demonstrate that p48v-myb and p75c-myb can be coexpressed in the nuclei of cloned cells. Therefore, p48v-myb does not invariably prevent the expression of p75c-myb.     

Estrogen-dependent alterations in differentiation state of myeloid cells caused by a v-myb/estrogen receptor fusion protein.

The oncogene v-myb and its cellular progenitor c-myb encode nuclear, DNA binding phosphoproteins that are thought to regulate the expression of myb-responsive genes during myeloid differentiation. To identify such myb-regulated genes, and to explore the mechanisms by which v-myb affects their expression, we have established a conditional expression system for v-myb. We have converted the v-myb protein to an estrogen-inducible transactivator by fusing the protein to the hormone binding domain of the human estrogen receptor. Expression of the chimeric protein in a chicken macrophage cell-line causes estrogen-dependent, reversible changes in the differentiation state as well as alterations in the gene expression program of the cells. We have used this estrogen-dependent v-myb expression system to identify a novel v-myb regulated gene.     

Nucleotide sequence of cDNA clones of the murine myb proto-oncogene.

We have isolated cDNA clones of murine c-myb mRNA which contain approximately 2.8 kb of the 3.9-kb mRNA sequence. Nucleotide sequencing has shown that these clones extend both 5' and 3' to sequences homologous to the v-myb oncogenes of avian myeloblastosis virus and avian leukemia virus E26. The sequence contains an open reading frame of 1944 nucleotides, and could encode a protein which is both highly homologous, and of similar size (71 kd), to the chicken c-myb protein. Examination of the deduced amino acid sequence of the murine c-myb protein revealed the presence of a 3-fold tandem repeat of 52 residues near the N terminus of the protein, and has enabled prediction of some of the likely structural features of the protein. These include a high alpha-helix content, a basic region toward the N terminus of the protein and an overall globular configuration. The arrangement of genomic c-myb sequences, detected using the cDNA clones as probes, was compared with the reported structure of rearranged c-myb in certain tumour cells. This comparison suggested that the rearranged c-myb gene may encode a protein which, like the v-myb protein, lacks the N-terminal region of c-myb.     

Functional antagonism between members of the myb family: B-myb inhibits v-myb-induced gene activation.

The oncogene v-myb and its cellular progenitor c-myb encode nuclear, DNA binding phosphoproteins that control the expression of certain target genes in immature hematopoietic cells. Here, we report the isolation of a myb-related chicken gene, chicken B-myb. We show that expression of B-myb, unlike that of c-myb, is not restricted to hematopoietic cells, suggesting that B-myb functions in a broader spectrum of cell types than c-myb. We have identified the authentic chicken B-myb protein as a nuclear protein of approximately 110 kDa. We show that the B-myb protein specifically recognizes v-myb binding sites in vitro and that binding is mediated by an N-terminally located DNA binding domain. Although B-myb protein recognizes myb binding sites, B-myb fails to transactivate several myb-responsive gene constructs as well as the endogenous myb-responsive gene mim-1. Instead, we find that B-myb represses v-myb- and c-myb-mediated activation of the mim-1 gene, most likely by competing with other myb proteins for binding sites. Our results raise the possibility that B-myb is an inhibitory member of the myb family.     

Isolation of monoclonal antibodies specific for products of avian oncogene myb.

We isolated a series of monoclonal antibodies which were raised against a bacterially expressed protein, bp37v-myb, and coded for by part of the avian v-myb gene. These monoclonal antibodies recognized a range of antigenic specificities on bp37v-myb, and this was reflected in their differing specificities for the gene products of the v-myb, c-myb, and E26 viral oncogenes. One monoclonal antibody recognized, in addition to the v-myb and c-myb gene products, a conserved nuclear protein found in all tested cells. We describe the characterization of these monoclonal antibodies.     

Characterization of the v-myb DNA binding domain.

The transforming protein encoded by the v-myb oncogene is a sequence-specific DNA-binding protein that is thought to be involved in the regulation of gene expression. The N-terminal region of the v-myb protein is composed of two highly conserved tandem repeat sequences of unknown function. It has been speculated that the N-terminal v-myb repeats might be crucial for DNA-binding, since N-terminal deletions destroy the DNA-binding activity of the v-myb protein. Here, we have studied the v-myb DNA-binding domain in more detail. Our results show that the N-terminal region of the v-myb protein is sufficient for specific DNA-binding. Dissection of this region suggests that both repeats are required for DNA-binding, but that both repeats play different roles in v-myb protein DNA interaction. We also show that the myb repeats of a drosophila melanogaster homolog of c-myb function as sequence-specific DNA-binding domain. Our results support the view that specific sequence-recognition, mediated by the conserved myb repeats, is a general feature of myb-related proteins.     

Structure of the protein encoded by the chicken proto-oncogene c-myb.

The retroviral oncogene v-myb arose by transduction of the chicken proto-oncogene c-myb. We isolated and sequenced cDNA that represents the entire coding domain of chicken c-myb. By transcribing the cDNA into mRNA in vitro and then translating the RNA, we were able to document the integrity of the cDNA and to identify the codon responsible for initiation of translation from c-myb. Two different alleles of v-myb are extant, one in the genome of avian myeloblastosis virus (AMV) and the other in the genome of erythroblastosis virus 26 (E26V). The proteins encoded by the AMV and E26V alleles of v-myb differ from the product of c-myb in three ways: at their amino termini, they lack 71 and 80 amino acids respectively; at their carboxy termini, they are deficient in 199 and 278 residues; and 11 substitutions of amino acids are scattered throughout the product of AMV allele, whereas the product of the E26V allele contains only a single substitution. The structural origins of tumorigenicity by v-myb and the biological functions of c-myb remain enigmatic. The findings and molecular clones described here should now permit a systematic exploration of these enigmas.     

Specific amino acid substitutions are not required for transformation by v-myb of avian myeloblastosis virus.

The protein product of the v-myb oncogene of avian myeloblastosis virus, p48v-myb, differs structurally in several ways from its normal cellular homolog, p75c-myb. We demonstrated that the 11 specific amino acid substitutions found in two independent molecular clones of this virus were not required for the transformation of myeloblasts by v-myb.     

Mutations in v-myb alter the differentiation of myelomonocytic cells transformed by the oncogene

Chick myelomonocytic cells transformed by the v-myb oncogene-containing viruses E26 and AMV differ in that the former resemble myeloblasts and express the v-myb-regulated granulocyte-specific mim-1 gene, while the latter resemble monoblasts and are mim-1 negative. We constructed a series of AMV-E26 chimeras and localized the critical differences between these viruses to three point mutations within the second repeat of the v-myb DNA binding domain. These three positions are altered in the v-myb protein of AMV relative to the proteins encoded by c-myb or E26 v-myb. Back mutating AMV v-myb at any of these three sites restored the oncogene's ability to activate the mim-1 gene. Surprisingly, two of these changes led to the transformation, in vitro and in vivo, of cells having a promyelocyte-like phenotype. These results indicate that different forms of v-myb impose alternate phenotypes of differentiation on transformed myeloid cells, probably by regulating unique sets of differentiation-specific genes.     

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.