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Pathogenic gene fusions have been identified in several histologic types of salivary gland neoplasia, but not previously in acinic cell carcinoma (AcCC). To discover novel gene fusions, we performed whole-transcriptome sequencing surveys of three AcCC archival cases. In one specimen we identified a novel HTN3-MSANTD3 gene fusion, and in another a novel PRB3-ZNF217 gene fusion. The structure of both fusions was consistent with the promoter of the 5' partner (HTN3 or PRB3), both highly expressed salivary gland genes, driving overexpression of full-length MSANTD3 or ZNF217. By fluorescence in situ hybridization of an expanded AcCC case series, we observed MSANTD3 rearrangements altogether in 3 of 20 evaluable cases (15%), but found no additional ZNF217 rearrangements. MSANTD3 encodes a previously uncharacterized Myb/SANT domain-containing protein. Immunohistochemical staining demonstrated diffuse nuclear MSANTD3 expression in 8 of 27 AcCC cases (30%), including the three cases with MSANTD3 rearrangement. MSANTD3 displayed heterogeneous expression in normal salivary ductal epithelium, as well as among other histologic types of salivary gland cancer though without evidence of translocation. In a broader survey, MSANTD3 showed variable expression across a wide range of normal and neoplastic human tissue specimens. In preliminary functional studies, engineered MSANTD3 overexpression in rodent salivary gland epithelial cells did not enhance cell proliferation, but led to significant upregulation of gene sets involved in protein synthesis. Our findings newly identify MSANTD3 rearrangement as a recurrent event in salivary gland AcCC, providing new insight into disease pathogenesis, and identifying a putative novel human oncogene.

Replication-independent histone variants can replace the canonical replication-dependent histones. Vertebrates have multiple H2A variant histones, including H2AZ and H2AX that are present in most eukaryotes. H2AZ regulates transcriptional activation as well as maintenance of gene silencing, while H2AX is important in DNA damage repair. The fruit fly Drosophila melanogaster has only one histone H2A variant (H2AV), which is a chimera of H2AZ and H2AX. In this study we found that lack of H2AV led to the formation of black melanotic masses in the third instar larvae of Drosophila. The formation of these masses was found in conjunction with a loss of a majority of the primary lymph gland lobes. Interestingly, the cells of the posterior signaling center were preserved in these mutants. Reduction of H2AV levels by RNAi knockdown caused a milder phenotype that preserved the lymph gland structure, but that included precocious differentiation of the prohemocytes located within the medullary zone and secondary lobes of the lymph gland. Mutant rescue experiments suggest that the H2AZ-like rather than the H2AX-like function of H2AV is primarily required for normal hematopoiesis.

In Drosophila, mutation of the oncogene Myb reduced the expression of mitotic genes, such as polo and ial, and caused multiple mitotic defects, including disrupted chromosome condensation and abnormal spindles. We now show that binucleate cells, the hallmark phenotype of cytokinesis failure, accumulate in Myb-null ovarian follicle cell and wing disc epithelia. Myb functions as an activator in the generally repressive Drosophila RBF, E2F2, and Myb (dREAM)/Myb-MuvB complex. Absence of the dREAM subunit Mip130 or E2F2 suppressed the Myb-null cytokinesis defect. Therefore, we used Myb-null binucleate cells as a quantitative phenotypic readout of transcriptional repression by the dREAM complex. In the absence of Myb, the complex was sensitive to the dose of the subunits E2F2, Mip120, Caf1, and Lin-52 but not Mip130 or Mip40. Surprisingly, reduction of the dose of His2Av/H2A.z also suppressed the Myb-null binucleate cell phenotype, suggesting a novel role for this variant histone in transcriptional repression by the dREAM complex.

The Drosophila melanogaster Myb-MuvB/dREAM complex (MMB/dREAM) participates in both the activation and repression of developmentally regulated genes and origins of DNA replication. Mutants in MMB subunits exhibit diverse phenotypes, including lethality, eye defects, reduced fecundity, and sterility. Here, we used P-element excision to generate mutations in lin-52, which encodes the smallest subunit of the MMB/dREAM complex. lin-52 is required for viability, as null mutants die prior to pupariation. The generation of somatic and germ line mutant clones indicates that lin-52 is required for adult eye development and for early embryogenesis via maternal effects. Interestingly, the maternal-effect embryonic lethality, larval lethality, and adult eye defects could be suppressed by mutations in other subunits of the MMB/dREAM complex. These results suggest that a partial MMB/dREAM complex is responsible for the lethality and eye defects of lin-52 mutants. Furthermore, these findings support a model in which the Lin-52 and Myb proteins counteract the repressive activities of the other members of the MMB/dREAM complex at specific genomic loci in a developmentally controlled manner.

The Drosophila Myb oncoprotein, the E2F2 transcriptional repressor, and the RBF and Mip130/LIN-9 tumor suppressor proteins reside in a conserved Myb–MuvB (MMB)/dREAM complex. We now show that Myb is required in vivo for the expression of Polo kinase and components of the spindle assembly checkpoint (SAC). Surprisingly, the highly conserved DNA-binding domain was not essential for assembly of Myb into MMB/dREAM, for transcriptional regulation in vivo, or for rescue of Myb-null mutants to adult viability. E2F2, RBF, and Mip130/LIN-9 acted in opposition to Myb by repressing the expression of Polo and SAC genes in vivo. Remarkably, the absence of both Myb and Mip130, or of both Myb and E2F2, caused variegated expression in which high or low levels of Polo were stably inherited through successive cell divisions in imaginal wing discs. Restoration of Myb resulted in a uniformly high level of Polo expression similar to that seen in wild-type tissue, whereas restoration of Mip130 or E2F2 extinguished Polo expression. Our results demonstrate epigenetic regulation of gene expression by Myb, Mip130/LIN-9, and E2F2–RBF in vivo, and also provide an explanation for the ability of Mip130-null mutants to rescue the lethality of Myb-null mutants in vivo.

The c- myb gene is implicated in the differentiation and proliferation of hematopoietic cells. Truncations of the N and/or C terminus of c-Myb, found in v-Myb, can potentiate its transforming ability. Two negative regulatory subregions, located in the C terminus, were mapped previously by using GAL4–c-Myb fusion proteins in transient transfection assays for the transcriptional activation of a GAL4-responsive reporter gene. To dissect the C terminus of c-Myb in terms of its involvement in transcriptional activation and oncogenic transformation, a series of C-terminal deletion mutants of c-Myb were analyzed. In addition, linker insertion mutants within the transactivation domain and/or heptad leucine repeat of c-Myb were examined along with those deletion mutants. In this study, we demonstrated that the removal of both of the two previously mapped negative regulatory subregions from the native form of c-Myb not only supertransactivates a Myb-responsive reporter gene but also potentiates its transforming ability in culture. However, in contrast to previous results, cells transformed by all of the mutants analyzed here except v-Myb itself exhibited the same phenotype as those transformed by c-Myb. The proliferating cells were bipotenial and differentiated into both the granulocytic and monocytic lineages. This result implies that the C terminus of c-Myb alone has no effect on the lineage determination. Finally, the transactivation activities of these mutants correlated with their transforming activities when a mim-1 reporter gene was used but not when a model promoter containing five tandem Myb-binding sites was used. In particular, a very weakly transforming mutant with a linker insertion in the heptad leucine repeat superactivated the model promoter but not the mim-1 reporter gene.

The c-Myb protein binds to DNA, can regulate transcription, and is required for normal hematopoiesis in vertebrates. Either amino- or carboxy-terminal truncation of this protein is required for efficient oncogenic activation. Previous studies have shown that the carboxyl terminus of c-Myb that is deleted in v-Myb contains negative regulatory domains. We now demonstrate that specific mutations within this carboxyl terminus result in greater transcriptional activation than truncation of the entire carboxyl terminus. Furthermore, this increased transcriptional activation depends upon the presence of the highly conserved Myb DNA-binding domain and is also dependent upon the nature of the Myb-binding sites within the target promoter. In a similar fashion, an activating mutation within the heptad leucine repeat region of c-Myb that is also present in v-Myb functions only in conjunction with the Myb DNA-binding domain and with particular Myb-binding sites. These results suggest a model in which multiple domains of the c-Myb protein are highly interdependent for transcriptional regulation. These interactions are promoter-specific and are not well modeled by heterologous fusion proteins.

An alternatively spliced form of c- myb exists that encodes an additional 120 amino acids in chicken and 121 amino acids in human and mouse. These amino acids are encoded by an additional exon, termed exon 9A. This exon is not present in v- myb , and proteins containing these amino acids have never been tested for oncogenic transformation. A series of myb constructs was therefore created in order to compare the functions of Myb proteins on the basis of their inclusion or exclusion of the amino acids encoded by exon 9A (E9A). We found that the presence of E9A resulted in a robust increase in transactivation for full-length c-Myb (CCC), as well as the singly truncated derivatives dCC and CCd, while doubly truncated Myb proteins v-Myb (dVd) and dCd did not exhibit any differences in transactivation. The increase in transactivation requires the Myb DNA-binding domain. When the leukemic transformation by the Myb proteins was tested, it was found that cells transformed by dVd resembled monoblasts, while cells transformed by CCC and its derivatives, dCd, dCC, and CCd, resembled myelomonoblasts. Despite differences in the morphology of the hematopoietic cells, the cell surface phenotypes and cell cycle profiles of transformed cells did not change for each pair of Myb proteins in the presence or absence of E9A. Thus, there was no direct correlation between the level of transcriptional activation and the strength of leukemic transformation.

Schizosaccharomyces pombe cdc5p is a Myb-related protein that is essential for G2/M progression. To explore the structural and functional conservation of Cdc5 throughout evolution, we isolated Cdc5-related genes and cDNAs from Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, and Homo sapiens. Supporting the notion that these Cdc5 gene family members are functionally homologous to S. pombe cdc5(+), human and fly Cdc5 cDNAs are capable of complementing the temperature-sensitive lethality of the S. pombe cdc5-120 mutant. Furthermore, S. cerevisiae CEF1 (S. cerevisiae homolog of cdc5(+)), like S. pombe cdc5(+), is essential during G2/M. The location of the cdc5-120 mutation, as well as mutational analyses of Cef1p, indicate that the Myb repeats of cdc5p and Cef1p are important for their function in vivo. However, we found that unlike in c-Myb, single residue substitutions of glycines for hydrophobic residues within the Myb repeats of Cef1p, which are essential for maintaining structure of the Myb domain, did not impair Cef1p function in vivo. Rather, multiple W-to-G substitutions were required to inactivate Cef1p, and many of the substitution mutants were found to confer temperature sensitivity. Although it is possible that Cef1p acts as a transcriptional activator, we have demonstrated that Cef1p is not involved in transcriptional activation of a class of G2/M-regulated genes typified by SWI5. Collectively, these results suggest that Cdc5 family members participate in a novel pathway to regulate G2/M progression.

In vitro and in vivo methods were combined to determine the function of the three Myb binding sites (NrasI, NrasII and NrasIII) within the promoter region of the mouse N-ras gene. We found that the c-Myb DNA-binding domain can bind with high affinity to NrasI and NrasII, but with a reduced affinity to NrasIII. In contrast, the full length v-Myb protein from BM2 cells only bound to the middle one of the three sites, NrasII. Both c-Myb and v-Myb functioned as repressors and reduce the basal activity of the N-ras promoter by 60%, as determined by transient transfection experiments using different regions of the N-ras promoter. This repression required a functional Myb DNA-binding domain and could not be overcome by fusion to the potent VP16 activation domain. In electrophoretic mobility shift assays, the v-Myb protein is shown to be present in different conformations depending on its binding to the NrasII or the mim-1A site. The v-Myb conformation is thus suggested to play a critical role in the regulation of v-Myb activity.

Retinoic acid (RA) is capable of inducing the differentiation of various myelomonocytic cell lines. During this differentiation process, the levels of c-myb expression decline, suggesting that the RA receptor (RAR) may act in part by down-regulating this proto-oncogene. We have now investigated whether the RAR can also inhibit the function of Myb proteins themselves. We have found that transcriptional activation of a Myb-responsive reporter gene can be inhibited by RA in a human monocytic cell line. This inhibition could not be overcome by the expression of exogenous Myb. The RAR did not interfere with DNA binding by Myb proteins in vitro, suggesting that the functional inhibition occurs at the level of transcriptional activation. To determine the biological relevance of the inhibition of Myb proteins by the RAR, we have used v-myb-transformed monoblasts. These cells differentiate into macrophages in the presence of phorbol ester (tetradecanoyl phorbol acetate [TPA]) but are normally unresponsive to RA treatment. The introduction of an inducible, exogenous RAR alpha into v-myb-transformed monoblasts permitted an RA-dependent differentiation into macrophage-like cells similar to those induced by TPA. These results demonstrate that transformation by v-myb is recessive to RAR alpha and imply that many types of non-RA-responsive leukemia cells may become responsive following the introduction of the RAR.

The v- myb oncogene causes monoblastic leukemia in chickens and transforms avian myelomonocytic cells in vitro . vMyb is a short-lived nuclear protein which binds to DNA in a sequence-specific manner and can activate gene expression in transient DNA transfections. Analysis of a series of v-Myb mutants has shown that the ability to activate transcription appears to be required for leukemic transformation. We have systematically investigated transcriptional activation by vMyb and have made several new observations: (i) v-Myb is a very weak activator when compared to GAL4; (ii) very weak transcriptional activation by v-Myb is sufficient for transformation, whereas very strong transcriptional activation by a vMyb-VP16 fusion protein is not; and (iii) v-Myb can activate transcription by two genetically distinct mechanisms, only one of which requires the presence of Myb-binding sites.

The v-Myb protein encoded by avian myeloblastosis virus causes oncogenic transformation of monoblastic cells committed to the monocyte/macrophage lineage. v-Myb is a doubly truncated form of its normal cellular counterpart, c-Myb. In addition to its N- and C-terminal deletions, v-Myb contains a number of amino acid substitutions relative to c-Myb. We have previously shown that neither overexpression of c-Myb nor introduction of these amino acid substitutions into c-Myb is sufficient for transformation of myelomonocytic cells. However, a doubly truncated form of c-Myb which lacked these substitutions transformed myeloblastic cells that appeared to be committed to the granulocytic pathway. We demonstrate here that mutations in both the DNA-binding and transcriptional activation domains of v-Myb are required for transformation of rapidly growing monoblasts rather than more slowly growing myeloblasts. These rapidly growing monoblasts do not express mim-1, a target gene for the Gag-Myb-Ets protein of E26 leukemia virus, or C/EBP proteins which cooperate with Myb to activate mim-1 expression. Furthermore, v-Myb proteins which contain both sets of these mutations are weaker transcriptional activators relative to proteins which lack these mutations. These results support a model in which amino acid substitutions in v-Myb have been selected for their ability to activate only a subset of those genes which can be activated by a doubly truncated form of c-Myb. In particular, mim-1 appears to represent a class of genes whose expression was selected against during the development of an increasingly virulent strain of avian myeloblastosis virus by passage in animals.

The avian myeloblastosis virus (AMV) induces acute monoblastic leukemia in chickens and transforms only myelomonocytic cells in vitro. The long terminal repeat (LTR) regulatory region of AMV is unique among the known classes of avian retrovirus LTRs. We demonstrate that the substitution of the AMV LTRs by Rous sarcoma virus LTRs did not alter the cell type specificity or the transforming ability of the virus.

We present an expression/selection system designed for the purification of cell lines inducibly expressing genes coding for unselectable proteins by using dicistronic selection for the cell surface marker CD4. This system enabled us to establish and purify c-myb expressing variants of the v-myb transformed chicken monoblast cell line BM2 with high efficiency.

Oncogenic activation of c-Myb in both avian and murine systems often involves N-terminal truncation. In particular, the first of three DNA-binding repeats in c-Myb has been largely deleted during the genesis of the v-myb oncogenes of avian myeloblastosis virus and E26 avian leukemia virus. This finding suggests that the first DNA-binding repeat may have an important role in cell growth control. We demonstrate that truncation of the first DNA-binding repeat of c-Myb is sufficient for myeloid transformation in culture, but deletion of the N-terminal phosphorylation site and adjacent acidic region is not. Truncation of the first repeat decreases the ability of a Myb-VP16 fusion protein to trans activate the promoter of a Myb-inducible gene (mim-1) involved in differentiation. Moreover, truncation of the first repeat decreases the ability of the Myb protein to bind DNA both in vivo and in vitro. These results suggest that N-terminal mutants of c-Myb may transform by regulating only a subset of those genes normally regulated by c-Myb.

The v-myb oncogene and its cellular homolog c-myb encode sequence-specific DNA-binding proteins which regulate transcription from promoters containing Myb-binding sites in animal cells. We have developed a Saccharomyces cerevisiae system to assay transcriptional activation by v-Myb and c-Myb. In yeast strains containing integrated reporter genes, activation was strictly dependent upon both the Myb DNA-binding domain and the Myb recognition element. BAS1, an endogenous Myb-related yeast protein, was not required for transactivation by animal Myb proteins and by itself had no detectable effect on a Myb reporter gene. Deletion analyses demonstrated that a domain of v-Myb C terminal to the previously mapped Myb transcriptional activation domain was required for transactivation in animal cells but not in S. cerevisiae. The same domain is also required for the efficient transformation of myeloid cells by v-Myb. In contrast to results in animal cells, in S. cerevisiae the full-length c-Myb was a much stronger transactivator than a protein bearing the oncogenic N- and C-terminal truncations of v-Myb. These results imply that negative regulation of c-Myb by its own termini requires an additional animal cell protein or small molecule that is not present in S. cerevisiae.

We have analyzed the control of developmental expression of the CD4 gene, which encodes an important recognition molecule and differentiation antigen on T cells. We have determined that the CD4 promoter alone functions at high levels in the CD4+ CD8- mature T cell but not at the early CD4+ CD8+ stage of T-cell development. In addition, the CD4 promoter functions only in T lymphocytes; thus, the stage and tissue specificity of the CD4 gene is mediated in part by its promoter. We have determined that a Myb transcription factor binds to the CD4 promoter and is critical for full promoter function. Thus, Myb plays an important role in the expression of T-cell-specific developmentally regulated genes.

The protein product of the v-myb oncogene of avian myeloblastosis virus, v-Myb, differs from its normal cellular counterpart, c-Myb, by (i) expression under the control of a strong viral long terminal repeat, (ii) truncation of both its amino and carboxyl termini, (iii) replacement of these termini by virally encoded residues, and (iv) substitution of 11 amino acid residues. We had previously shown that neither the virally encoded termini nor the amino acid substitutions are required for transformation by v-Myb. We have now constructed avian retroviruses that express full-length or singly truncated forms of c-Myb and have tested them for the transformation of chicken bone marrow cells. We conclude that truncation of either the amino or carboxyl terminus of c-Myb is sufficient for transformation. In contrast, the overexpression of full-length c-Myb does not result in transformation. We have also shown that the amino acid substitutions of v-Myb by themselves are not sufficient for the activation of c-Myb. Rather, the presence of either the normal amino or carboxyl terminus of c-Myb can suppress transformation when fused to v-Myb. Cells transformed by c-Myb proteins truncated at either their amino or carboxyl terminus appear to be granulated promyelocytes that express the Mim-1 protein. Cells transformed by a doubly truncated c-Myb protein are not granulated but do express the Mim-1 protein, in contrast to monoblasts transformed by v-Myb that neither contain granules nor express Mim-1. These results suggest that various alterations of c-Myb itself may determine the lineage of differentiating hematopoietic cells.

The v-myb oncogene causes acute myelomonocytic leukemia in chickens and transforms avian myeloid cells in vitro. Its product, p48v-myb, is a short-lived nuclear protein which binds DNA. We demonstrate that p48v-myb can function as a trans activator of gene expression in transient DNA transfection assays. trans activation requires the highly conserved amino-terminal DNA-binding domain and the less highly conserved carboxyl-terminal domain of p48v-myb, both of which are required for transformation. Multiple copies of a consensus sequence for DNA binding by p48v-myb inserted upstream of a herpes simplex virus thymidine kinase promoter are strongly stimulatory for transcriptional activation by a v-myb-VP16 fusion protein but not by p48v-myb itself, suggesting that the binding of p48v-myb to DNA may not be sufficient for trans activation.

The p53 protein can control cell cycle progression, programmed cell death, and differentiation of many cell types. Ectopic expression of p53 can resume capability of cell cycle arrest, differentiation, and apoptosis in various leukemic cell lines. In this work, we expressed human p53 protein in v-Myb-transformed chicken monoblasts. We found that even this protein possessing only 53% amino acid homology to its avian counterpart can significantly alter morphology and physiology of these cells causing the G2-phase cell cycle arrest and early monocytic differentiation. Our results document that the species-specific differences of the p53 molecules, promoters/enhancers, and co-factors in avian and human cells do not interfere with differentiation- and cell cycle arrest promoting capabilites of the p53 tumor suppressor even in the presence of functional v-Myb oncoprotein. The p53-induced differentiation and cell cycle arrest of v-Myb-transformed monoblasts are not associated with apoptosis suggesting that the p53-driven pathways controlling apoptosis and differentiation/proliferation are independent.

Completion of chromosome condensation is required before segregation during the mitotic cell cycle to ensure the transmission of genetic material with high fidelity in a timely fashion. In many eukaryotes this condensation is regulated by phosphorylation of histone H3 on Ser 10 (H3S10). This phosphorylation normally begins in the late-replicating pericentric heterochromatin and then spreads to the earlier replicating euchromatin. Here, we show that these phases of condensation are genetically separable in that the absence of Drosophila Myb causes cells to arrest with H3S10 phosphorylation of heterochromatin but not euchromatin. In addition, we used mosaic analysis to demonstrate that although the Myb protein can be removed in a single cell cycle, the failure of chromosome condensation occurs only after many cell divisions in the absence of Myb protein. The Myb protein is normally located in euchromatic but not heterochromatic regions of the nucleus, suggesting that Myb may be essential for a modification of euchromatin that is required for the efficient spread of chromosome condensation.

The v-Myb oncogene causes monoblastic leukemia and transforms only myelomonocytic cells in culture. The v-Myb protein is nuclear and binds to specific DNA sequences. To identify genes regulated by v-Myb, we utilized primary cells transformed by a retrovirus encoding a v-Myb-estrogen receptor (ER) fusion protein. The Ets-2 gene was not expressed in v-Myb-ER transformed cells in the presence of estradiol, but was expressed within 4 h after estradiol withdrawal. The expression of Ets-2 also increased dramatically following phorbol ester-induced differentiation of the v-Myb-transformed BM2 cell line. Conversely, CRYP-alpha, encoding a transmembrane tyrosine phosphatase, was expressed in the presence but not the absence of estradiol in v-Myb-ER transformed cells. CRYP-alpha was downregulated during the phorbol ester-induced differentiation of BM2 cells. Although LIM-3 expression was estradiol-inducible in v-Myb-ER transformed monoblasts, LIM-3 was expressed neither in primary yolk sac cells transformed by unfused v-Myb nor in BM2 cells. We conclude that although v-Myb has been intensively studied as a transcriptional activator, v-Myb can repress biologically relevant genes such as Ets-2, which promotes macrophage differentiation. In addition, we have shown that some genes that are regulated by a v-Myb-ER fusion protein may not be relevant to the biological function of the unfused v-Myb protein.

Three Myb-related genes (A-Myb, B-Myb, and c- Myb) have been found in all vertebrates examined thus far including mammals, birds, and amphibians. Two invertebrates, the sea urchin and the fruit fly, have only one Myb-related gene. Our laboratory has used Drosophila as a model system to explore the function of its sole Myb gene. We have also reintroduced the three different vertebrate Myb genes into Drosophila in order to begin to understand how their different functions may have arisen following gene duplication during evolution.

A minimal transcription activation domain of the v-Myb oncoprotein was initially mapped to a central cluster of charged residues using GAL4-Myb fusion proteins . This region has been proposed to interact directly with the CBP co-activator in animal cells. Regions flanking this central domain of v-Myb are required for transcriptional activation by the native, unfused protein in both mammalian cells and in budding yeast. To identify the critical residues for transcriptional activation, we have now subjected the minimal activation domain and flanking regions including the heptad leucine repeat to random PCR-mediated mutagenesis. We found that the entire region examined can endure extensive substitutions without affecting transcriptional activation by v-Myb in budding yeast. The few mutations that did affect transcriptional activation altered acidic residues within the minimal activation domain or the heptad leucine repeat region, rather than leucine residues. Remarkably, there was a strong concordance between transcriptional activation in animal cells and in budding yeast, even though budding yeast have no known homologue of CBP or related co-activators. In contrast, there was not a strong correlation between transcriptional activation and oncogenic transformation.

The carboxyl terminus of c-Myb contains a negative regulatory domain that is absent in the v-Myb oncoprotein, but conserved among all the known Myb proteins of animals. This domain inhibits transcriptional activation by c-Myb in animal cells, but not in budding yeast, suggesting that additional protein(s) present in animal cells but not yeast are required for this negative regulatory function. A yeast two-hybrid screen identified BS69, an adenovirus E1A-associated protein, as interacting with the carboxy-terminal region of c-Myb. BS69 contains regions of similarity to the PHD finger, the bromodomain, and the MYND domain, all of which are found in other proteins present in high molecular weight complexes that regulate transcription and/or modify chromatin structure. Further study showed that BS69 inhibited the transcriptional activity of c-Myb, that this inhibition was specific, that it mapped to the carboxyl termini of the two proteins and that it was dose-dependent. A direct interaction between these two proteins was observed in vitro. Furthermore, the 289R E1A protein could inhibit the BS69-mediated decrease in transcriptional activation by c-Myb. By analogy with the inhibition of the Rb/E2F regulatory axis by E1A, we propose that a BS69/Myb regulatory circuit may also be a target of disruption during oncogenesis. Oncogene (2001) 20, 125 - 132.

The biological effects of the cellular c-Myb and the viral v-Myb proteins are strikingly different. While c-Myb is indispensable for normal hematopoiesis, v-Myb induces acute leukemia. The v-Myb DNA-binding domain (DBD) differs from that of c-Myb mainly by deletion of the first of three repeats which correlates with efficient oncogenic transformation and a decrease in DNA-binding activity. To investigate the difference in DNA-binding and transcriptional activation, oligonucleotide selection and electrophoretic mobility shift assays were employed. The v-Myb DBD (R2R3) shows an intrinsic DNA-binding specificity for an AT-rich downstream extension of the Myb recognition element (MRE) PyAACT/GG for efficient binding to this site, whereas R1 within the c-Myb DBD allows for more flexibility for this downstream extension. Therefore, due to the presence of repeat R1, c-Myb can bind to a greater number of target sites. The intrinsic DNA-binding specificity of R2R3 is further supported with the results from in vivo transcriptional activation experiments which demonstrated that both the v-Myb and c-Myb DBDs require an extension of the MRE (motif #1) by a downstream T-stretch (motif #2) for full activity. Surprisingly, the T-stretch improves binding when present on either strand, but is required on a specific strand for transcriptional activation.

The v-myb oncogene of the avian myeloblastosis virus (AMV) is unique among known oncogenes in that it causes only acute leukemia in animals and transforms only hematopoietic cells in culture. AMV was discovered in the 1930s as a virus that caused a disease in chickens that is similar to acute myelogenous leukemia in humans (Hall et al., 1941). This avian retrovirus played an important role in the history of cancer research for two reasons. First, AMV was used to demonstrate that all oncogenic viruses did not contain a single cancer-causing principle. In particular, although both Rous sarcoma virus (RSV) and AMV could replicate in cultures of either embryonic fibroblasts or hematopoietic cells, RSV could transform only fibroblasts whereas AMV could transform only hematopoietic cells (Baluda, 1963; Durban and Boettiger, 1981a). Second, chickens infected with AMV develop remarkably high white counts and therefore their peripheral blood contains remarkably large quantities of viral particles (Beard, 1963). For this reason AMV was often used as a prototypic retrovirus in order to study viral assembly and later to produce large amounts of reverse transcriptase for both research and commercial purposes. Following the discovery of the v-src oncogene of RSV and the demonstration that it arose from the normal c-src proto-oncogene, a number of acute leukemia viruses were analysed by similar techniques and found to also contain viral oncogenes of cellular origin (Roussel et al., 1979). In the case of AMV, it was shown that almost the entire retroviral env gene had been replaced by a sequence of cellular origin (initially called mab or amv, but later renamed v-myb) (Duesberg et al., 1980; Souza et al., 1980). Remarkably, sequences contained in this myb oncogene were shared between AMV and the avian E26 leukemia virus, but were not contained in any other acutely transforming retroviruses. In addition, the E26 virus contained a second sequence of cellular origin (ets) that was unique. The E26 leukemia virus was first described in the 1960s and causes an acute erythroblastosis in chickens, more reminiscent of the disease caused by avian erythroblastosis virus (AEV) than by AMV (Ivanov et al., 1962).

The v-Myb DNA-binding domain differs from that of c-Myb mainly by deletion of the first of three repeats. This truncation correlates with efficient oncogenic transformation and a decrease in DNA-binding activity. Here we demonstrate that the D-type cyclins, cyclin D1 and D2 in particular, specifically inhibit transcription when activated through the v-Myb DNA-binding domain, but not the c-Myb DNA-binding domain. Analysis of a cyclin D1 mutant and a dominant-negative CDK4 mutant implied that this repression is independent of complex formation with a CDK partner. Association of cyclin D1 and D2 with the Myb DNA-binding domain could be demonstrated. Increased levels of cyclin D1 and D2 resulted in a stabilization of the Myb proteins, but not in an alteration in binding of the Myb proteins to DNA. These results highlight an unexpected role for cyclin D as a CDK-independent repressor of transcriptional activation by v-Myb but not c-Myb. This differential effect of D-type cyclins on v-Myb and c-Myb might help to explain the mechanism underlying the oncogenic activity of v-Myb, which appears to be a stronger transcriptional activator following the TPA-induced differentiation of transformed monoblasts when cyclin D1 and D2 are down-regulated.

The nuclear protein v-Myb, encoded by the avian myeloblastosis virus (AMV), can induce acute monoblastic leukemia in vivo and transform chicken myelomonocytic cells in culture. The N terminus of v-Myb functions as the DNA-binding domain, and multiple central and C-terminal regions of this protein have been reported to function in transcriptional activation of model reporter genes. We showed previously that a C-terminal domain (amino acids 296 to 371) is required for transcriptional activation and transformation of primary chicken myelomonocytic cells. In this study, we have now analyzed a series of C-terminal mutants of v-Myb to further investigate this domain. A strong correlation was observed between transcriptional activation and leukemic transformation by this series of mutants. Furthermore, deletion analyses demonstrate that the C-terminal 41 amino acids of v=MybAMV (amino acids 331 to 371 of the Myb portion) are nonessential whereas further deletion of amino acids 321 to 330 (EFAETLQLID) results in a nonfunctional protein. Hence, we defined a 10-amino-acid subregion (the "FAETL" motif) required for transcriptional activation and oncogenic transformation by v-Myb Amv. The FAETL region is part of a putative leucine zipper structure and lies near a cluster of phosphorylation sites. Our analysis of mutants with substitutions of the zipper leucines or multiple adjacent phosphorylation sites demonstrates that the function of the FAETL motif is not dependent on an intact leucine zipper structure or adjacent phosphorylation sites. The study of GAL4-Myb fusions suggests that this region is important in maintaining a fully functional conformation of v-Myb. The putative leucine zipper structure has previously been proposed to exert inhibitory effects on c-Myb because its mutation caused increased transcriptional transactivation and transformation. Interestingly, our results show that this region is essential for the functions of v-Myb without requiring a heptad leucine repeat.

Oncogenic activation of c-Myb in both avian and murine systems often involves N-terminal truncation. In particular, the first of three DNA-binding repeats in c-Myb has been largely deleted during the genesis of the v-myb oncogenes of avian myeloblastosis virus and E26 avian leukemia virus. This finding suggests that the first DNA-binding repeat may have an important role in cell growth control. We demonstrate that truncation of the first DNA-binding repeat of c-Myb is sufficient for myeloid transformation in culture, but deletion of the N-terminal phosphorylation site and adjacent acidic region is not. Truncation of the first repeat decreases the ability of a Myb-VP16 fusion protein to trans activate the promoter of a Myb-inducible gene (mim-1) involved in differentiation. Moreover, truncation of the first repeat decreases the ability of the Myb protein to bind DNA both in vivo and in vitro. These results suggest that N-terminal mutants of c-Myb may transform by regulating only a subset of those genes normally regulated by c-Myb.

The v-myb oncogene and its cellular homolog c-myb encode sequence-specific DNA-binding proteins which regulate transcription from promoters containing Myb-binding sites in animal cells. We have developed a Saccharomyces cerevisiae system to assay transcriptional activation by v-Myb and c-Myb. In yeast strains containing integrated reporter genes, activation was strictly dependent upon both the Myb DNA-binding domain and the Myb recognition element. BAS1, an endogenous Myb-related yeast protein, was not required for transactivation by animal Myb proteins and by itself had no detectable effect on a Myb reporter gene. Deletion analyses demonstrated that a domain of v-Myb C terminal to the previously mapped Myb transcriptional activation domain was required for transactivation in animal cells but not in S. cerevisiae. The same domain is also required for the efficient transformation of myeloid cells by v-Myb. In contrast to results in animal cells, in S. cerevisiae the full-length c-Myb was a much stronger transactivator than a protein bearing the oncogenic N- and C-terminal truncations of v-Myb. These results imply that negative regulation of c-Myb by its own termini requires an additional animal cell protein or small molecule that is not present in S. cerevisiae.

The v-myb- and v-ets-containing E26 retrovirus induces the proliferation of chicken neuroretina (CNR) cells in minimal medium. Proliferation of E26 CNR cells is strongly stimulated by basic fibroblast growth factor (bFGF). The v-myb-containing avian myeloblastosis virus also induces the proliferation of infected CNR cells stimulated by bFGF. Both E26 CNR and avian myeloblastosis virus CNR cells are able to form colonies in soft agar in the presence of bFGF. This suggests that the v-myb product, a nuclear sequence-specific DNA-binding protein which activates gene expression in transient transfection assays, plays a role in the proliferative response of the infected CNR cells. To determine the structure-function relationships of P135gag-myb-ets and p48v-myb, we have used deletion mutants expressed in retroviral vectors and have analyzed their effect on CNR cell proliferation as well as their effect on the CNR cell response to bFGF. We show that v-ets is not required for bFGF stimulation but increases the proliferation of CNR cells in minimal medium. In the v-myb mutants, the gag sequences derived from the helper virus increase the potency of the myb gene. The carboxyl-terminal domain required for the growth and transformation of myeloid cells and needed for maximal trans-activation in transient DNA transfection assays in fibroblasts was not required for the growth and bFGF response of CNR cells. In contrast, the domain encompassing amino acids 240 to 301 (containing part of the transcriptional activation domain of v-myb) was absolutely required for the response of CNR cells to bFGF and could be functionally replaced by the carboxyl-terminal transcriptional activation domain of the VP16 protein of herpes simplex virus.

A biologically active myeloblastosis-associated virus (MAV) provirus was cloned from a bacteriophage recombinant library constructed from leukemic chicken myeloblast DNA. The restriction endonuclease map of this clone was consistent with that of a type 1 MAV (MAV-1). Interference assays of virus recovered from cultured chicken embryo fibroblasts after DNA transfection established that the provirus was infectious and confirmed that it belonged to avian retrovirus subgroup A (type 1). Antipeptide antibodies raised against the env-encoded carboxyl terminus of p48myb, the transforming protein of avian myeloblastosis virus, specifically immunoprecipitated the gp37env from quail cells transfected with MAV-1 proviral DNA but not from cells infected with MAV-2. This suggests that MAV-1 rather than MAV-2 is the progenitor helper virus from which avian myeloblastosis virus arose by the transduction of cellular proto-oncogene sequences.

The product of the v-myb oncogene of avian myeloblastosis virus is a nuclear protein with an associated DNA-binding activity. We demonstrated that the highly conserved amino-terminal domain of p48v-myb is required for its associated DNA-binding activity. This activity is not required for the nuclear localization of p48v-myb. Furthermore, the associated DNA-binding activity and nuclear localization of p48v-myb together are not sufficient for transformation.

Interleukin 2 (IL-2), a product of activated T lymphocytes but not of T-cell-deficient spleen cells of congenitally athymic (nu/nu) mice, is shown to be strongly mitogenic for spleen cells from both nu/nu and nu/+ mice in the absence of serum. This response does not depend on the presence of additional mitogen or antigen as has been previously reported and is dose dependent with respect to IL-2. Plots of the logarithm of cell number versus logarithm of response indicate that IL-2-induced mitogenesis of nu/nu spleen cells is a one-cell event (slope = 1.28 +/- 0.21, mean +/- SD), whereas the response of nu/+ spleen cells to concanavalin A is a three-cell event (slope = 3.18 +/- 0.16). The nu/nu spleen cells proliferating in response to IL-2 are at least 80% T lymphocytes by the third day of culture, as demontrated by lysis with monoclonal anti-Thy-1 antibody and complement. These results suggest that one of the major roles of the thymus may be to induce IL-2 production in a subpopulation of lymphocytes that is then persistent.

The defective acute leukemia viruses avian myeloblastosis virus (AMV) and E26 virus each contain an inserted cellular sequence related to the same highly conserved cellular gene, proto-amv. The oncogenes of these two retroviruses differ from this cellular proto-oncogene in gene structure, transcript structure, and gene product. The product of the AMV oncogene (myb) is a 48,000 Mr protein, p48myb, encoded by a transduced segment (amv) of proto-amv flanked by short helper-virus-derived terminal sequences. The E26 virus oncogene product is a 135,000 Mr protein, p135gag-amve-ets, encoded by significant portions of a viral structural gene (gag), sequences related to proto-amv (amve), and additional E26-specific sequences (ets) transduced from cellular proto-ets. Both p48myb and p135gag-amve-ets transforming proteins are located in the nucleus of cells transformed by these viruses. A protein of 110,000 Mr which is specifically immunoprecipitated by antisera to amv peptides and may be the product of the normal cellular gene (proto-amv) has been located in the cytoplasm of cells that express proto-amv mRNA.

A flavokinase preparation from Bacillus subtilis is described which catalyzes the phosphorylation of reduced, but not oxidized, riboflavin. The enzyme is distinguished from other known flavokinases also in having an unusually low Km for the flavin substrate, 50 to 100 nM. ATP is the obligatory phosphate donor; one ATP is utilized for each FMNH2 formed. Mg2+ or Zn2+ is required for the reaction; Co2+ and Mn2+ will substitute, but less effectively. The same enzyme preparation catalyzes the synthesis of FADH2 from FMNH2 and ATP, but not the synthesis of FAD from FMN and ATP. FADH2 is also formed from reduced riboflavin, presumably by sequential flavokinase and FAD synthetase action. Zn2+ cannot replace Mg2+ in FADH2 formation. The reverse reaction, formation of FMN from FAD, occurs only with reduced FAD, giving rise to FMNH2, and is dependent on the presence of inorganic pyrophosphate. The enzyme thus appears to be an FADH2 pyrophosphorylase. The two enzymatic activities, flavokinase and FADH2 pyrophosphorylase, although not separated during the purification procedure, are distinguished by differences in metal ion specificity, in concentration dependence for ATP (apparent Km for ATP = 300 microM for FADH2 synthesis and 6.5 microM for flavokinase), and in the inhibitory effects of riboflavin analogues.

We demonstrated that molecular clones of the v-myb oncogene of avian myeloblastosis virus (AMV) can direct the synthesis of p48v-myb both in avian and mammalian cells which are not targets for transformation by AMV. To accomplish this, we constructed dominantly selectable avian leukosis virus derivatives which efficiently coexpress the protein products of the Tn5 neo gene and the v-myb oncogene. The use of chemically transformed QT6 quail cells for proviral DNA transfection or retroviral infection, followed by G418 selection, allowed the generation of cell lines which continuously produce both undeleted infectious neo-myb viral stocks and p48v-myb. The presence of a simian virus 40 origin of replication in the proviral plasmids also permitted high-level transient expression of p48v-myb in simian COS cells without intervening cycles of potentially mutagenic retroviral replication. These experiments establish that the previously reported DNA sequence of v-myb does in fact encode p48v-myb, the transforming protein of AMV.

The v-myb oncogene of avian myeloblastosis virus induces acute myeloblastic leukemia in chickens and transforms avian myeloid cells in vitro. The protein product of this oncogene, p48v-myb, is partially encoded by the retroviral gag and env genes. We demonstrated that the env-encoded carboxyl terminus of p48v-myb is not required for transformation. Our results showed, in addition, that a coding region of c-myb which is not essential for transformation was transduced by avian myeloblastosis virus.

The expression of p80c-myb was examined during the activation of resting human T lymphocytes. Before activation, no detectable p80c-myb was present. Synthesis of p80c-myb was observed only after initiation of the S phase of the cell cycle.

The genome of the replication-defective avian myeloblastosis virus (AMV) contains an inserted cellular sequence (amv) that is part of the oncogene responsible for acute myeloblastic leukemia in chickens infected with AMV. Three antisera raised against distinct synthetic peptides predicted from the long open reading frame of amv specifically precipitated the same 48-kilodalton protein (p48amv) from leukemic myeloblasts but not from normal hematopoietic tissue, fibroblasts, or from fibroblasts infected with the AMV helper virus, MAV-1 (myeloblastosis-associated virus type 1). p48amv is not glycosylated or phosphorylated and does not appear to act as a protein kinase in vitro. The same three antisera that recognized p48amv also specifically precipitated a common 110-kilodalton protein from normal uninfected hematopoietic tissue. This normal cellular homologue of the AMV leukemogenic protein, p110proto-amv, was not present in normal fibroblasts, MAV-1 infected fibroblasts, or, interestingly, in some leukemic myeloblasts. We conclude that p48amv is the leukemogenic product of an altered, transduced, partial protooncogene. Short helper-virus sequences provide its carboxyl terminus and also may provide the amino terminus.

Antibodies directed against a bacterial fusion protein that contains the domain encoded by the highly evolutionarily conserved 5' one-third of the v-myb oncogene of avian myeloblastosis virus (AMV) detect the protein products of various members of the myb gene family. Immunoprecipitation or immunoblot analyses with these antibodies yielded the following information. First, the products of the v-myb oncogenes of AMV (p48v-myb) and of E26 virus (p135gag-myb-ets) contain this highly conserved amino acid sequence, as previously hypothesized. Second, p75c-myb, the product of the chicken c-myb protooncogene, also contains this protein domain. Third, these antibodies have identified the products of the human, murine, and Drosophila c-myb genes, which were all found to be nuclear proteins of Mr 75,000-80,000. The human c-myb protein product is present in immature cells of the erythroid, myeloid, and lymphoid lineages.

The expression of p80c-myb was examined during the activation of resting human T lymphocytes. Before activation, no detectable p80c-myb was present. Synthesis of p80c-myb was observed only after initiation of the S phase of the cell cycle.

Summary Two chicken lectins with similar sugar specificities are mitogenic for nonadherent, Thy-1 negative mouse spleen cells in serum-free cultures. This effect appears to be due to the sugar-binding properties of these lectins since asialofetuin and lactose, specific inhibitors of lectin activity, inhibit mitogenesis. Among other lectins with similar sugar specificity, purpurin, the lectin from Dictyostelium purpureum , is also mitogenic for these cells whereas peanut lectin is not. The mitogenic lectins work similarly with spleen cells from athymic (nu/nu) mice and their heterozygote (nu/+) littermates.

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.

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.

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.

The c-Myb protein is a transcriptional regulator initially identified by homology to the v-Myb oncoprotein, and has since been implicated in human cancer. The most highly conserved portion of the c-Myb protein is the DNA-binding domain which consists of three imperfect repeats. Many other proteins contain one or more Myb-related domains, including a number of proteins that do not bind directly to DNA. We performed a phylogenetic analysis of diverse classes of Myb-related domains and discovered a highly conserved patch of acidic residues common to all Myb-related domains. These acidic residues are positioned in the first of three alpha-helices within each of the three repeats that comprise the c-Myb DNA-binding domain. Interestingly, these conserved acidic residues are present on a surface of the protein which is distinct from that which binds to DNA. Alanine mutagenesis revealed that the acidic patch of the third c-Myb repeat is essential for transcriptional activity, but neither for nuclear localization nor DNA-binding. Instead, these acidic residues are required for efficient chromatin binding and interaction with the histone H4 N-terminal tail.