The c-Myb oncoprotein

The c-myb gene is the cellular homologue of the v-myb oncogenes carried by the avian leukaemia viruses AMV and E26. It encodes a transcription factor (c-Myb), as does each of the viral oncogenes, which recognises the core DNA sequence C/T-A-A-C-G/T-G via a repeated helix-turn-helix-like motif. c-myb is expressed in immature haemopoietic cells, as well as immature cells of the gastro-intestinal epithelium and is down-regulated with differentiation. Enforced expression of activated or even normal forms of Myb can transform haemopoietic cells, most often of the myeloid lineage, in vitro and in vivo. Although many genes have been identified which are likely to be regulated by c-Myb, the critical target genes involved in Myb's transforming activity are not known. Together with data showing increased c-myb expression in colonic tumours, these observations raise the possibility that c-myb may play a role in human malignant disease. (C) 1998 Elsevier Science Ltd. All rights reserved.

Mutation screening of the c-MYB negative regulatory domain in acute and chronic myeloid leukaemia

Over-expression of the c-myb gene and expression of activated forms of myb are known to transform haemopoietic cells, particularly cells of the myeloid lineage. Truncations or mutations that disrupt the negative regulatory domain (NRD) of the Myb protein confer an increased ability to transform cells. Although it has proved difficult to link mutations in c-MYB to human leukaemia, no studies investigating the presence of mutations within the c-MYB NRD have been reported. Therefore, we have performed mutational analysis of this region, using polymerase chain reaction-single-stranded conformation polymorphism and sequence analysis, in 26 patients with acute or chronic myeloid leukaemia, No mutations were detected, indicating that mutation of this region of the Myb protein is not common in the pathogenesis or progression of these diseases.

Targeting c-Myb expression in human disease

c-Myb is a transcription factor employed in the haematopoietic system and gastrointestinal tract to regulate the exquisite balance between cell division, differentiation and survival. In its absence, these tissues either fail to form, or show aberrant biology. Mice lacking a functional c-myb gene die in utero by day 15 of development. When inappropriately expressed, as is common in leukaemia and epithelial cancers of the breast, colon and gastro-oesophagus, c-Myb appears to activate gene targets of key importance to cancer progression and metastasis. These genes include cyclooxygenase-2 (COX-2), Bcl-2, Bcl-X-L and c-Myc, which influence diverse processes such as angiogenesis, proliferation and apoptosis. The clinical potential for blocking c-Myb expression in malignancies is based upon strong preclinical data and some trial-based evidence. The modest clinical experience to date has been with haematopoietic malignancies, but other disease classes may be amenable to similar interventions. The frontline agents to achieve this are nuclease-resistant oligodeoxynucleotides (ODNs), which are proving to be acceptable therapeutic reagents in terms of tolerable toxicities and delivery. Nevertheless, further effort must be focused on improving their efficacy, eliminating non-specific toxicity and optimising delivery. Optimisation issues aside, it would appear that anti-c-Myb therapies will be used with most success when combined with other agents, some of which will be established cytotoxic and differentiation-inducing drugs. This review will explore the future strategic use of ODNs in vivo, focusing on a wide spectrum of diseases, including several beyond the haematopoietic malignancies, in which c-Myb appears to play a role.

p53 Suppresses c-Myb-induced trans-Activation and Transformation by Recruiting the Corepressor mSin3A

p53 is known to repress transcription of a number of genes, but the mechanism of p53 recruitment to these target genes is unknown. The c-myb proto-oncogene product (c-Myb) positively regulates proliferation of immature hematopoietic cells, whereas p53 blocks cell cycle progression. Here, we demonstrate that p53 inhibits c-Myb-induced transcription and transformation by directly binding to c-Myb. The ability of c-Myb to maintain the undifferentiated state of M1 cells was also suppressed by p53. p53 did not affect the ability of c-Myb to bind to DNA but formed a ternary complex with the corepressor mSin3A and c-Myb. Thus, p53 antagonizes c-Myb by recruiting mSin3A to down-regulate specific Myb target genes.

Inactivation of a c-Myb/estrogen receptor fusion protein in transformed primary cells leads to granulocyte/macrophage differentiation and down regulation of c-kit but not c-myc or cdc2

Primary murine fetal hemopoietic cells were transformed with a fusion protein consisting of the ligand-binding domain of the estrogen receptor and a carboxyl-terminally truncated c-Myb protein (ERMYB), The ERMYB-transformed hemopoietic cells exhibit an immature myeloid phenotype when grown in the presence of beta-estradiol. Upon removal of beta-estradiol, the ERMYB cells display increased adherence, decreased clonogenicity and differentiate to cells exhibiting granulocyte or macrophage morphology, The expression of the c-myc, c-kit, cdc2 and bcl-2 genes, which are putatively regulated by Myb, was investigated in ERMYB cells grown in the presence or absence of beta-estradiol. Neither c-myc nor cdc2 expression was down-regulated after removal of beta-estradiol demonstrating that differentiation is not a consequence of decreased transactivation of these genes by ERMYB. While bcl-2 expression was reduced by 50% in ERMYB cells grown in the absence of beta-estradiol, there was no increase in DNA laddering, suggesting that Myb was not protecting ERMYB cells from apoptosis, In contrast, a substantial (200-fold) decrease in c-kit mRNA level was observed following differentiation of ERMYB cells, and c-kit mRNA could be partially re-induced by the re-addition of beta-estradiol. Furthermore, a reporter construct containing the c-kit promoter was activated when cotransfected with a Myb expression vector, providing further evidence of a role for Myb in the regulation of c-kit.

The transcription factors c-myb and GATA-2 act independently in the regulation of normal hematopoiesis.

The transcription factors c-myb and GATA-2 are both required for blood cell development in vivo and in vitro. However, very little is known on their mechanism(s) of action and whether they impact on complementary or overlapping pathways of hematopoietic proliferation and differentiation. We report here that embryonic stem (ES) cells transfected with c-myb or GATA-2 cDNAs, individually or in combination, underwent hematopoietic commitment and differentiation in the absence of added hematopoietic growth factors but that stimulation with c-kit and flt-3 ligands enhanced colony formation only in the c-myb transfectants. This enhancement correlated with c-kit and flt-3 surface receptor up-regulation in c-myb-(but not GATA-2-) transfected ES cells. Transfection of ES cells with either a c-myb or a GATA-2 antisense construct abrogated erythromyeloid colony-forming ability in methyl cellulose; however, introduction of a full-length GATA-2 or c-myb cDNA, respectively, rescued the hematopoiesis-deficient phenotype, although only c-myb-rescued ES cells expressed c-kit and flt-3 surface receptors and formed increased numbers of hematopoietic colonies upon stimulation with the cognate ligands. These results are in agreement with previous studies indicating a fundamental role of c-myb and GATA-2 in hematopoiesis. Of greater importance, our studies suggest that GATA-2 and c-myb exert their roles in hematopoietic gene regulation through distinct mechanisms of action in nonoverlapping pathways.

Modulation of c-Myb-induced transcription activation by a phosphorylation site near the negative regulatory domain.

The c-myb protooncogene encodes a highly conserved transcription factor that functions as both an activator and a repressor of transcription. The v-myb oncogenes of E26 leukemia virus and avian myeloblastosis virus encode proteins that are truncated at both the amino and the carboxyl terminus, deleting portions of the c-Myb DNA-binding and negative regulatory domains. This has led to speculation that the deleted regions contain important regulatory sequences. We previously reported that the 42-kDa mitogen-activated protein kinase (p42mapk) phosphorylates chicken and murine c-Myb at multiple sites in the negative regulatory domain in vitro, suggesting that phosphorylation might provide a mechanism to regulate c-Myb function. We now report that three tryptic phosphopeptides derived from in vitro phosphorylated c-Myb comigrate with three tryptic phosphopeptides derived from metabolically labeled c-Myb immunoprecipitated from murine erythroleukemia cells. At least two of these peptides are phosphorylated on serine-528. Replacement of serine-528 with alanine results in a 2- to 7-fold increase in the ability of c-Myb to transactivate a Myb-responsive promoter/reporter gene construct. These findings suggest that phosphorylation serves to regulate c-Myb activity and that loss of this phosphorylation site from the v-Myb proteins may contribute to their transforming potential.

Pluripotent hematopoietic stem cells contain high levels of mRNA for c-kit, GATA-2, p45 NF-E2, and c-myb and low levels or no mRNA for c-fms and the receptors for granulocyte colony-stimulating factor and interleukins 5 and 7.

Pluripotent hematopoietic stem cells (PHSCs) were highly enriched from mouse bone marrow by counterflow centrifugal elutriation, lineage subtraction, and fluorescence-activated cell sorting based on high c-kit receptor expression (c-kitBR). We used reverse transcriptase polymerase chain reaction to assay the c-kitBR subset and the subsets expressing low (c-kitDULL) and no (c-kitNEG) c-kit receptor for expression of mRNA encoding hematopoietic growth factor receptors and transcription factors. The c-kitBR cells had approximately 3.5-fold more c-kit mRNA than unfractionated bone marrow cells. The c-kitDULL cells had 47-58% of the c-kit mRNA found in c-kitBR cells and the c-kitNEG cells had 4-9% of the c-kit mRNA present in c-kitBR cells. By comparing mRNA levels in c-kitBR cells (enriched for PHSCs) with those of unfractionated bone marrow, we demonstrated that c-kitBR cells contained low or undetectable levels of mRNA for c-fms, granulocyte colony-stimulating factor receptor, interleukin 5 receptor (IL-5R), and IL-7R. These same cells had moderate levels of mRNA for erythropoietin receptor, IL-3R subunits IL-3R alpha (SUT-1), AIC-2A, and AIC-2B, IL-6R and its partner gp-130, and the transcription factor GATA-1 and high levels of mRNA for transcription factors GATA-2, p45 NF-E2, and c-myb. We conclude from these findings that PHSCs are programmed to interact with stem cell factor, IL-3, and IL-6 but not with granulocyte or macrophage colony-stimulating factor. These findings also indicate that GATA-2, p45 NF-E2, and c-myb activities may be involved in PHSC maintenance or proliferation.

The antiproliferative activity of c-myb and c-myc antisense oligonucleotides in smooth muscle cells is caused by a nonantisense mechanism.

Smooth muscle cell (SMC) proliferation is thought to play a major role in vascular restenosis after angioplasty and is a serious complication of the procedure. Developing antisense (AS) oligonucleotides as therapeutics is attractive because of the potentially high specificity of binding to their targets, and several investigators have reported inhibition of SMC proliferation in vitro and in vivo by using AS strategies. We report here the results of our experiments on vascular SMCs using AS oligonucleotides directed toward c-myb and c-myc. We found that significant inhibition of SMC proliferation occurred with these specific AS sequences but that this inhibition was clearly not via a hybridization-dependent AS mechanism. Rather, inhibition was due to the presence of four contiguous guanosine residues in the oligonucleotide sequence. This was demonstrated in vitro in primary cultures of SMCs and in arteries ex vivo. The ex vivo model developed here provides a rapid and effective system in which to screen potential oligonucleotide drugs for restenosis. We have further explored the sequence requirements of this non-AS effect and determined that phosphorothioate oligonucleotides containing at least two sets of three or four consecutive guanosine residues inhibit SMC proliferation in vitro and ex vivo. These results suggest that previous AS data obtained using these and similar, contiguous guanosine-containing AS sequences be reevaluated and that there may be an additional class of nucleic acid compounds that have potential as antirestenosis therapeutics.

Molecular cloning and chromosomal mapping of the human homologue of MYB binding protein (P160) 1A (MYBBP1A) to 17p13.3

We have previously isolated and characterized murine MYB binding protein (p160) 1a, a protein that specifically interacts with the leucine zipper motif within the negative regulatory domain of the c-Myb proto-oncoprotein, We now describe the molecular cloning of the human MYBBP1A cDNA and chromosomal localization to 17p13.3 by fluorescence in situ hybridization analysis, Given the likely presence of a tumor suppressor gene (or genes) within this region of chromosome 17, the position of MYBBP1A was further mapped by radiation hybrid analysis and was found to lie between markers D17S1828 and D17S938. A P1 artificial chromosome clone containing the 5' region of MYBBP1A was isolated and indicates a physical linkage between MYBBP1A and the 15-lipoxygenase gene (ALOX15), A novel, polymorphic (CA)(25) dinucleotide repeat was also isolated from this PAC and may serve as a useful marker for MYBBP1A and this region of chromosome 17. (C) 1999 Academic Press.

Identification and validation of candidate Myb target genes

While a considerable number of candidate Myb target genes have been reported to date, most of these are likely to play little or no role in transformation by myb oncogenes. Here we have used a conditionally myb-transformed myeloid cell line (ERMYB) to further examine Myb regulation of one candidate target gene-c-myc-that has the potential to affect cell proliferation. It was found that the major influence on c-myc expression was the presence of cytokine (GM-CSF) rather than Myb activity. We also describe the application of PCR-based subtractive hybridization and low-density cDNA array screening, in conjunction with the ERMYB line, to the identification of additional Myb target genes. Preliminary identification of a number of candidates is reported; these include myeloperoxidase, which is known to have essential Myb-binding sites in its regulatory region. (C) 2001 Academic Press.

Myb-binding Protein 1a is a Nucleocytoplasmic Shuttling Protein that Utilizes CRM1-dependent and Independent Nuclear Export Pathways

Myb-binding protein 1a (Mybbp1a) is a novel nuclear protein localized predominantly, but not exclusively, in nucleoli. Although initially isolated as a c-Myb interacting protein, Mybbp1a is expressed ubiquitously, associates with a number of different transcription factors, and may play a role in both RNA polymerase I- and II-mediated transcriptional regulation. However, its precise function remains unclear. In this study we show that Mybbp1a is a nucleocytoplasmic shuttling protein and investigate the mechanisms responsible for both nuclear import and export. The carboxyl terminus of Mybbp1a, which contains seven short basic amino acid repeat sequences, is responsible for both nuclear and nucleolar localization, and this activity can be transferred to a heterologous protein. Deletion mapping demonstrated that these repeat sequences appear to act incrementally, with successive deletions resulting in a corresponding increase in the proportion of protein localized in the cytoplasm. Glutathione S-transferase pulldown experiments showed that the nuclear receptor importin-alpha/beta mediates Mybbp1a nuclear import. Interspecies heterokaryons were used to demonstrate that Mybbp1a was capable of shuttling between the nucleus and the cytoplasm. Deletion analysis and in vivo export studies using a heterologous assay system identified several nuclear export sequences which facilitate Mybbp1a nuclear export of Mybbp1a by CRM1-dependent and -independent pathways. (C) 2003 Elsevier Science (USA). All rights reserved.

DNA binding-independent transcriptional activation of the vascular endothelial growth factor gene (VEGF) by the Myb oncoprotein

Myb is a key transcription factor that can regulate proliferation, differentiation, and apoptosis, predominantly in the haemopoietic system. Abnormal expression of Myb is associated with a number of cancers, both haemopoietic and non-haemopoietic. In order to better understand the role of Myb in normal and tumorigenic processes, we undertook a cDNA array screen to identify genes that are regulated by this factor. In this way, we identified the gene encoding vascular endothelial growth factor (VEGF) as being potentially regulated by the Myb oncoprotein in myeloid cells. To determine whether this was a direct effect on VEGF gene transcription, we examined the activity of the murine VEGF promoter in the presence of either wild-type (WT) or mutant forms of Myb. It was found that WT Myb was able to activate the VEGF promoter and that a minimal promoter region of 120 bp was sufficient to confer Myb responsiveness. Surprisingly, activation of the VEGF promoter was independent of DNA binding by Myb. This was shown by the use of DNA binding-defective Myb mutants and by mutagenesis of a potential Myb-binding site in the minimal promoter. Mutation of Sp1 sites within this region abolished Myb-mediated regulation of a reporter construct, suggesting that Myb DNA binding-independent activation of VEGF expression occurs via these Sp1 binding elements. Regulation of VEGF production by Myb has implications for the potential role of Myb in myeloid leukaemias and in solid tumours where VEGF may be functioning as an autocrine growth factor. (c) 2006 Elsevier Inc. All rights reserved.

Regulation of c-fos expression by RNA polymerase elongation competence.

The molecular mechanisms underlying transcription elongation and their role in gene regulation are poorly characterized in eukaryotes. A number of genes, however, have been proposed to be regulated at the level of transcription elongation, including c-myc, c-fos and c-myb. Here, we analyze the control of transcription elongation at the mouse c-fos gene at the nucleotide level in intact cells. We find that RNA polymerases are engaged in the promoter-proximal part of the gene in the absence of gene activation signals and mRNA synthesis. Importantly, we determine that the engaged RNA polymerases originate from a continuous initiation of transcription which, in the absence of gene activation signals, terminate close to the promoter. We also observe that the c-fos gene presents an active chromatin conformation, with the promoter and upstream regulatory sequences constitutively occupied by proteins, accounting for the continuous initiation of RNA polymerase complexes. We propose that activation of c-fos gene expression results primarily from the assembly of elongation-competent RNA polymerases that can transcribe the complete gene. Our results suggest that the engaged RNA polymerases found downstream of a number of other eukaryotic promoters may be associated with transcription termination of elongation-incompetent polymerases in the absence of activating signals.

Evidence that Myb-related CDC5 proteins are required for pre-mRNA splicing

The conserved CDC5 family of Myb-related proteins performs an essential function in cell cycle control at G2/M. Although c-Myb and many Myb-related proteins act as transcription factors, herein, we implicate CDC5 proteins in pre-mRNA splicing. Mammalian CDC5 colocalizes with pre-mRNA splicing factors in the nuclei of mammalian cells, associates with core components of the splicing machinery in nuclear extracts, and interacts with the spliceosome throughout the splicing reaction in vitro. Furthermore, genetic depletion of the homolog of CDC5 in Saccharomyces cerevisiae, CEF1, blocks the first step of pre-mRNA processing in vivo. These data provide evidence that eukaryotic cells require CDC5 proteins for pre-mRNA splicing.