A single point mutation in the v-ets oncogene affects both erythroid and myelomonocytic cell differentiation

The v-myb, ets-containing avian leukemia virus E26 is unique in its capacity to transform both erythroblasts and myeloblasts. Previous studies showing that v-myb is sufficient for the transformation of myeloid cells failed to definitively establish the role of the v-ets gene. We have now isolated a mutant of E26, ts1.1, that is temperature-sensitive for erythroid cell transformation and that we found to contain a single mutation in the v-ets gene. Surprisingly, myeloid cells transformed by this mutant showed an altered phenotype relative to wild-type-transformed cells, in that they resemble promyelocytes. In addition, infection of mature macrophages with ts1.1 led to their transformation and conversion into promyelocyte-like cells. We conclude that the v-ets domain of the p135gag-myb-ets protein of E26 has an effect on both erythroid and myeloid cell differentiation, suggesting a possible role for the c-ets/c-myb genes in the commitment of hematopoietic cells towards specific lineages.     

Structural and functional domains of the myb oncogene: requirements for nuclear transport, myeloid transformation, and colony formation.

The v-myb oncogene of avian myeloblastosis virus causes acute myelomonocytic leukemia in vivo and transforms only myeloid cells in vitro. Its product, p48v-myb, is a nuclear protein of unknown function. To determine structure-function relationships for this protein, we constructed a series of deletion mutants of v-myb, expressed them in retroviral vectors, and studied their biochemical and biological properties. We used these mutants to identify two separate domains of p48v-myb which had distinct roles in its accumulation in the cell nucleus. We showed that the viral sequences which normally encode both termini of p48v-myb were dispensible for transformation. In contrast, both copies of the highly conserved v-myb amino-terminal repeat were required for transformation. We also identified a carboxyl-terminal domain of p48v-myb which was required for the growth of v-myb-transformed myeloblasts in soft agar but not for morphological transformation.     

Transformation by v-myb correlates with trans-activation of gene expression.

The v-myb oncogene of avian myeloblastosis virus causes acute myelomonocytic leukemia in chickens and transforms avian myeloid cells in vitro. Its protein product p48v-myb is a nuclear, sequence-specific, DNA-binding protein which activates gene expression in transient DNA transfection studies. To investigate the relationship between transformation and trans-activation by v-myb, we constructed 15 in-frame linker insertion mutants. The 12 mutants which transformed myeloid cells also trans-activated gene expression, whereas the 3 mutants which did not transform also did not trans-activate. This implies that trans-activation is required for transformation by v-myb. One of the transformation-defective mutants localized to the cell nucleus but failed to bind DNA. The other two transformation-defective mutants localized to the cell nucleus and bound DNA but nevertheless failed to trans-activate. These latter mutants define two distinct domains of p48v-myb which control trans-activation by DNA-bound protein, one within the amino-terminal DNA-binding domain itself and one in a carboxyl-terminal domain which is not required for DNA binding.     

Analysis of the v-myb structural components important for transactivation of gene expression.

In order to define the domains of the v-myb protein that are important for transactivation of gene expression, we have studied transactivation by the v-myb gene and a set of v-myb deletion mutants using transient transfection assays in NIH 3T3 cells. Analysis of the set of v-myb deletion products demonstrated that a previously unidentified region in the carboxyl-terminal portion of the protein is required for transactivation. This region lies between amino acids 295-356 with respect to the 5' end of the v-myb gene. Switching the v-myb DNA binding domain with the DNA binding domain of the rat glucocorticoid receptor (rGR) switched the cis-element requirement for v-myb action: only reports containing glucocorticoid response elements were activated by myb-rGR fusion proteins. The carboxyl terminal region essential for transactivation by the intact v-myb gene was also necessary for transactivation by the rGR-fusion gene. Carboxyl-terminal deletion mutations that encompassed the novel transactivation region were able to block wild-type v-myb transactivation when tested in transient co-expression assays. In an unexpected sidelight to our studies, we could demonstrate that the lacZ gene present in the prokaryotic vector sequences contained a DNA element that fortuitously can act as a v-myb-dependent enhancer element, and that v-myb protein can bind to this element in vitro. The lacZ enhancer contains the myb consensus DNA binding site YAAC(G/T)G.     

In vivo cooperation of two nuclear oncogenic proteins, P135gag-myb-ets and p61/63myc, leads to transformation and immortalization of chicken myelomonocytic cells.

To investigate a possible in vivo cooperation between the p61/63myc and P135gag-myb-ets proteins, we used a previously constructed retrovirus, named MHE226, which contains the fused v-myb and v-ets oncogenes of the E26 retrovirus and the v-myc oncogene of MH2. For that purpose, chicken neuroretina cells producing MHE226 and pseudotyped with the Rous associated virus-1 (RAV-1) helper virus were injected in 1-day-old chickens. In control experiments, we also injected chicken neuroretina cells producing E26 (RAV-1), RAV-1 alone, or constructs lacking one of the oncogenes of MHE226. The average life span of MHE226-infected chickens is half that of E26-infected chickens. MHE226-infected chickens harbor tumors scattered in many organs, but compared with E26, MHE226 induced a weak leukemia. Study of integration sites suggests that the majority of the tumors results from clonal or oligoclonal events. Cell cultures were derived from the tumors of MHE226-infected chickens and grown in standard medium without addition of exogenous chicken myelomonocytic growth factor. These cells still divide at high rate after more than 100 passages and can thus be considered immortalized. By using several criteria, these cells were characterized as precursors of the myelomonocytic lineages.     

Activation of cMGF expression is a critical step in avian myeloid leukemogenesis.

A non-leukemogenic version of the v-myb oncogene causes in vitro transformation of avian myeloblasts, which are dependent on chicken myelomonocytic growth factor (cMGF). We have shown that this version of v-myb, when combined with the erythroleukemia-inducing v-erbB oncogene, is capable of causing a mixed myeloid and erythroid leukemia. Myeloid leukemic cells transformed by this construct produce cMGF. To test whether autocrine growth stimulation via cMGF is the essential contribution of the tyrosine kinase oncogene v-erbB in avian myeloid leukemogenesis we constructed another retrovirus containing both the non-leukemogenic v-myb and the cMGF cDNA. This virus induced myeloid leukemia at high efficiency. In a third construct we combined v-myb with the human EGF-receptor gene. Myeloid cells transformed by this construct could be stimulated to grow by the addition of cMGF or EGF. Growth stimulation with EGF was blocked by a cMGF antiserum indicating that activation of a normal tyrosine kinase-type receptor induces cMGF expression but does not bypass the cMGF requirement. We conclude that cMGF plays a key role in the growth regulation of normal and transformed avian myeloid cells.     

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.     

The highly conserved amino-terminal region of the protein encoded by the v-myb oncogene functions as a DNA-binding domain.

The retroviral oncogene v-myb encodes a 45,000 Mr nuclear protein (p45v-myb) that is predominantly associated with the chromatin of transformed cells. It has previously been shown that p45v-myb, when released from chromatin by salt-treatment, binds to DNA. To analyse the biochemical properties of p45v-myb in more detail we have expressed the v-myb coding region in Escherichia coli. Our results demonstrate that bacterially expressed myb protein has an intrinsic DNA-binding activity. Using two alternative strategies, (i) inhibition of DNA-binding by monoclonal antibodies and (ii) analysis of DNA-binding activities of partially deleted forms of the bacterial myb protein, we show that the DNA-binding domain is located in the amino-terminal region of the v-myb protein. This region has been highly conserved between myb genes of different species. Our results are therefore consistent with the hypothesis that DNA-binding is an important aspect of myb protein function.     

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.     

Coordinate regulation of myelomonocytic phenotype by v-myb and v-myc.

Both avian myeloblastosis virus (by the action of v-myb) and avian myelocytomatosis virus MC29 (by the action of v-myc) transform cells of the myelomonocytic lineage. Whereas avian myeloblastosis virus elicits a relatively immature phenotype, cells transformed by MC29 resemble mature macrophages. When cells previously transformed by v-myb were superinfected with MC29, their phenotype was rapidly altered to that of a more mature cell. These superinfected cells expressed both v-myb (at a level similar to that found before superinfection) and v-myc. It therefore appears that the expression of v-myc can elicit certain properties of a more differentiated phenotype. In addition, unlike cells transformed by v-myb alone, the cells expressing both v-myb and v-myc could not be induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate to differentiate to fully mature macrophages. Cells with a morphology similar to that of the superinfected cells were elicited by simultaneously infecting yolk sac macrophages with avian myeloblastosis virus and MC29. Such cells expressed both v-myb and v-myc. These results indicate that expression of v-myb and v-myc in infected cells coordinately regulates myelomonocytic phenotype and that the two viral oncogenes vary in their ability to interfere with tumor promoter-induced differentiation. Our findings also sustain previous suggestions that the oncogenes v-myb and v-myc may not transform target cells by simply blocking differentiation.     

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.     

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.     

Multiple domains for the chicken cellular sequences homologous to the v-ets oncogene of the E26 retrovirus.

We have investigated the structure of chicken genomic DNA homologous to v-ets, the second cell-derived oncogene of avian retrovirus E26. We isolated a c-ets locus spanning ca. 30.0 kilobase pairs (kbp) in the chicken genome with homologies to 1,202 nucleotides (nt) of v-ets (total length, 1,508 nt) distributed in six clusters along 18.0 kbp of the cloned DNA. The 5'-distal part of v-ets (224 nt) was homologous to chicken cellular sequences contained upstream within a single 16.0-kbp EcoRI fragment as two typical exons but not found transcribed into the major 7.5-kb c-ets (or 4.0-kb c-myb) RNA species. Between these two v-ets-related cellular sequences we found ca 40.0 kbp of v-ets-unrelated DNA. Finally, the most 3' region of homology to v-ets in the cloned DNA was shown to consist of a truncated exon lacking the nucleotides coding for the 16 carboxy-terminal amino acids of the viral protein but colinear to one of the two human c-ets loci, c-ets-2.     

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.     

v-myb dominance over v-myc in doubly transformed chick myelomonocytic cells

Chick myelomonocytic cells transformed by the v-myc oncogene resemble mature macrophages; those transformed by v-myb or v-myb,ets exhibit an immature phenotype. We have analyzed whether these oncogenes are capable of altering the differentiation phenotype of transformed cells by introducing both v-myc plus either v-myb or v-myb,ets into the same cells. Surprisingly, the doubly transformed cells were found to be essentially indistinguishable from cells transformed by v-myb or v-myb,ets alone even when they expressed a high level of v-myc protein. These results demonstrate that v-myb is dominant over v-myc and that, while v-myc induces cell proliferation without affecting differentiation, v-myb induces in the same target cells both proliferation and a block or reversal of differentiation.