Myb oncogene-like

EVOLUTIONARY HOMOLOGS (part 2/3)

Transcriptional targets of Myb

The c-myb proto-oncogene is preferentially expressed in hematopoietic cells and is required for cell cycle progression at the G1/S boundary. Because c-myb encodes a transcriptional activator that functions via DNA binding, it is likely that c-myb exerts its biological activity by regulating the transcription of genes required for DNA synthesis and cell cycle progression. One such gene, cdc2, encodes a 34-kDa serine-threonine kinase that appears to be required for G1/S transition in normal human T-lymphocytes. To determine whether c-myb is a transcriptional regulator of cdc2 expression, a segment of a cdc2 gene containing extensive 5'-flanking sequences and part of the first exon was cloned. Sequence analysis reveals the presence of two closely spaced Myb binding sites that interact with bacterially synthesized Myb protein within a region extending from nucleotides -410 to -392 upstream of the transcription initiation site. A 465-base pair segment of 5'-flanking sequence containing these sites is linked to the CAT reporter gene and was shown to have promoter activity in rodent fibroblasts. Cotransfection of this construct with a full-length human c-myb cDNA driven by the early simian virus 40 promoter results in a 6-8-fold enhancement of CAT activity, which is abrogated by mutations in the Myb binding sites. These data suggest that c-myb participates in the regulation of cell cycle progression by activating the expression of the cdc2 gene (Ku, 1993).

The B-myb gene is expressed in many cell types at the G1/S transition of the cell cycle. Inhibition of B-myb expression in BALB/c 3T3 fibroblasts due to the introduction of a B-myb antisense construct greatly diminishes cell proliferation, whereas constitutive expression of a human B-myb cDNA in these cells reduces their growth factor requirements and induces a transformed phenotype. Constitutive expression of B-myb cDNA is accompanied by activation of cyclin D1 and cdc2 expression but not the activation or expression of of cyclin A and cyclin B. Transfection of BALB/B-myb cells (a cell line expressing high levels of exogenous human B-myb) with a cyclin D1 antisense construct drastically reduces the cloning efficiency of these cells. These results suggest that the B-myb-encoded product regulates fibroblast proliferation by activating cdc2 and cyclin D1 gene expression and that abnormal expression of cyclin D1 might be a step in the process of transformation (Sala, 1992).

The homeobox gene GBX2 has been identified as a target gene of the v-Myb oncoprotein encoded by the avian myeloblastosis virus (AMV). GBX2 activation by c-Myb requires signal transduction emanating from the cell surface, and the leukemogenic AMV v-Myb constitutively induces the GBX2 gene. Mutations in the DNA binding domain of AMV-Myb render it independent of signaling events and concomitantly abrogate the collaboration between Myb and CCAAT Enhancer Binding Proteins (C/EBP), which are involved in granulocyte differentiation. Ectopic expression of GBX2 in growth factor-dependent myeloblasts induces monocytic features and independence from exogenous cytokines, reflecting distinct features of AMV-transformed cells. These results suggest that Myb, or factors with which Myb interacts, contribute to hematopoietic lineage choice and differentiation in a signal transduction-dependent fashion (Kowenz-Leutz, 1997).

ZEB, a vertebrate homolog of Drosophila Zfh-1, binds a subset of E boxes and blocks myogenesis through transcriptional repression of muscle genes. ZEB also has an important role in controlling hematopoietic gene transcription. Two families of transcription factors that are required for normal hematopoiesis are c-Myb and Ets. These factors act synergistically to activate transcription, and this synergy is required for transcription of at least several important hematopoietic genes. ZEB blocks the activity of c-Myb and Ets individually, but together the factors synergize to resist this repression. Such repression imposes a requirement for both c-Myb and Ets for transcriptional activity, providing one explanation for why synergy between these factors is important. The balance between repression by ZEB and transcriptional activation by c-Myb/Ets provides a flexible regulatory mechanism for controlling gene expression in hematopoietic cells. One target of this positive/negative regulation in vivo is the alpha4 integrin, which plays a key role in normal hematopoiesis and the function of mature leukocytes (Postigo, 1997).

The c-Myb transcription factor is required for the production of most hemopoietic lineages, but information is sparse about its mode of action and the particular genes it regulates. An inducible dominant interfering Myb protein was prepared, by creating a chimera composed of the DNA binding domain of c-Myb, the Drosophila Engrailed repressor domain, and a modified estrogen receptor hormone binding domain. When expressed in the murine thymoma cell line EL4, activation of this mutant results in a significant proportion of the cell population undergoing apoptosis, as assessed by nuclear breakdown and DNA fragmentation, but no apparent effect on cell-cycle progression is noted. The apoptotic phenotype is mirrored during thymopoiesis in transgenic mice expressing dominant interfering Myb mutants; their T cells are fragile both in vivo and in vitro. Induction of the Myb dominant interfering mutant in EL4 cells correlates with down-regulation of bcl-2 (Drosophila homolog: death executioner Bcl-2 homologue), but does not affect transcription of other bcl-2 family members; conversely, overexpression of bcl-2 in the transgenic mouse model rescues thymocytes from death. Analysis of the bcl-2 promoter by run-on transcription, bandshifting, and transient expression assays shows that it is a direct target of Myb. These data suggest a new and important role for Myb proteins as regulators of cell survival during hemopoiesis (Taylor, 1996).

Many oncogenes have been shown to be deregulated transcription factors, yet direct target genes mediating cell transformation remain elusive. A target for v-Myb has been discovered by exploiting a temperature-sensitive mutant of the E26 avian leukemia virus encoding Myb-Ets. Myeloblasts transformed by the mutant differentiate into macrophages or die by apoptosis when shifted to the nonpermissive temperature as a result of inactivation of v-Myb. During this process, mRNA of the antiapoptotic oncoprotein Bcl-2 is down-regulated with kinetics similar to those of Mim-1, a differentiation-related protein whose expression is directly regulated by Myb. Forced expression of bcl-2 rescues the cells from apoptosis, preventing neither their withdrawal from the cell cycle nor their differentiation. v-Myb appears to act directly on the bcl-2 gene, because a bcl-2 promoter-driven reporter is activated by Myb-Ets and v-Myb-VP16 and requires intact Myb binding sites within the promoter. Surprisingly, inactivation of v-Myb in multipotent progenitors transformed by E26 virus does not induce apoptosis, indicating that bcl-2 regulation by the oncoprotein is required for the transformation of some cell types but not others (Frampton, 1996).

The murine neutrophil elastase (NE) gene is expressed specifically in immature myeloid cells. A 91-bp NE promoter region contains three cis elements that are conserved evolutionarily and are essential for activation of the promoter in differentiating 32D cl3 myeloid cells. These elements bind c-Myb (at -49), C/EBPalpha (at -57), and PU.1 (at -82). In NIH 3T3 cells, the NE promoter is activated by c-Myb, C/EBPalpha, and PU.1, via their respective binding sites. Cooperative activation is seen by any combination of c-Myb, C/EBPalpha, and PU.1, including all three together, again via their DNA-binding sites. In CV-1 cells, but not in NIH 3T3 cells, cooperation between Myb and C/EBPalpha depends on the integrity of the PU.1-binding site. In addition to C/EBPalpha, C/EBPdelta strongly activates the NE promoter, alone or with c-Myb, but C/EBPbeta is less active. Either of C/EBPalpha's two transactivation domains cooperatively activate the promoter with c-Myb, in both NIH 3T3 and 32D c13 cells. Synergistic binding to DNA in a gel shift assay between C/EBPalpha, c-Myb, and PU.1 could not be demonstrated. Separation of the C/EBP- and c-Myb-binding sites by 5 or 10 bp did not prevent cooperativity. These results suggest that a coactivator protein mediates cooperative activation of the NE promoter by C/EBP and c-Myb. These factors, together with PU.1, direct restricted expression of the NE promoter to immature myeloid cells (Oelgeschlager, 1996).

Locus control regions (LCRs) are powerful assemblies of cis elements that organize the actions of cell-type-specific trans-acting factors. In the human adenosine deaminase (ADA) gene, a 2.3-kb LCR in the first intron controls expression of ADA in thymocytes This LCR is composed of a 200-bp enhancer domain and extended flanking sequences that facilitate activation from within chromatin. Prior analyses have demonstrated that the enhancer contains a 28-bp core region and local adjacent augmentative cis elements. The core contains a single critical c-Myb binding site. In both transiently cotransfected human cells and stable chromatin-integrated yeast cells, c-Myb strongly transactivates reporter constructs that contain polymerized core sequences. c-Myb protein is strongly evident in T lymphoblasts in which the enhancer is active and is localized within discrete nuclear structures. Fetal murine thymus exhibits a striking concordance of endogenous c-myb expression with that of mouse ADA and human ADA LCR-directed transgene expression. Point mutation of the c-Myb site within the intact 2.3-kb LCR severely attenuates enhancer activity in transfections and LCR activity in transgenic thymocytes. Within the context of a complex enhancer and LCR, c-Myb can act as an organizer of thymocyte-specific gene expression via a single binding site (Ess, 1995).

Dysregulated expression of basic fibroblast growth factor 2 (FGF-2) mediates autocrine growth of melanoma cells. The presence of a consensus Myb binding site in the human FGF-2 promoter prompted an investigation of whether this transcription factor can regulate FGF-2 expression in melanomas. c-MYB mRNA is overexpressed in melanoma cell lines, as compared to normal melanocytes. Ectopic expression of murine c-Myb in SK-MEL-2 human melanoma cells results in increased expression of FGF-2 mRNA and FGF-2 protein. Murine c-Myb transactivates a reporter plasmid containing the human FGF-2 promoter region in contransfected SK-MEL-2 human melanoma cells. Although a functional DNA-binding domain is required for transactivation, responsiveness to c-Myb is independent of the putative Myb binding site and maps to two regions of the FGF-2 promoter that do not bind c-Myb in vitro. It is suggested that c-MYB contributes to FGF-2-mediated autocrine growth of melanomas by indirectly regulating the FGF-2 promoter (Miglarese, 1997).

Enhancers and promoters within TCR loci functionally collaborate to modify chromatin structure and to confer accessibility to the transcription and V(D)J recombination machineries during T cell development in the thymus. Two enhancers at the TCRalphadelta locus, the TCR alpha enhancer and the TCR delta enhancer (Edelta), are responsible for orchestrating the distinct developmental programs for V(D)J recombination and transcription of the TCR alpha and delta genes, respectively. Edelta function depends critically on transcription factors core binding factor (CBF)/polyoma enhancer-binding protein 2 (PEBP2) and c-Myb as measured by transcriptional activation of transiently transfected substrates in Jurkat cells, and by activation of V(D)J recombination within chromatin-integrated substrates in transgenic mice. To understand the molecular mechanisms for synergy between these transcription factors in the context of chromatin, in vivo footprinting was used to study the requirements for protein binding to Edelta within wild-type and mutant versions of a human TCR delta minilocus in stably transfected Jurkat cells. The data indicate that CBF/PEBP2 plays primarily a structural role since it induces a conformational change in the enhanceosome that is associated with augmented binding of c-Myb. In contrast, c-Myb has no apparent affect on CBF/PEBP2 binding, but is critical for transcriptional activation. Thus, these data reveal distinct functions for c-Myb and CBF/PEBP2 in the assembly and function of an Edelta enhanceosome in the context of chromatin in vivo (Hernandez-Munain, 2002).

Previous work has provided evidence for E2F-dependent transcription control of both G1/S- and G2/M-regulated genes. Analysis of the G2-regulated cdc2 and cyclin B1 genes reveals the presence of both positive- and negative-acting E2F promoter elements. Additional elements provide both positive (CCAAT and Myb) and negative (CHR) control. Chromatin immunoprecipitation assays identify multiple interactions of E2F proteins that include those previously shown to activate and repress transcription. E2F1, E2F2, and E2F3 were found to bind to the positive-acting E2F site in the cdc2 promoter, whereas E2F4 binds to the negative-acting site. Binding of an activator E2F is dependent on an adjacent CCAAT site that is bound by the NF-Y transcription factor and binding of a repressor E2F is dependent on an adjacent CHR element, suggesting a role for cooperative interactions in determining both activation and repression. Finally, the kinetics of B-Myb interaction with the G2-regulated promoters coincides with the activation of the genes, and RNAi-mediated reduction of B-Myb inhibits expression of cyclin B1 and cdc2. The ability of B-Myb to interact with the cdc2 promoter is dependent on an intact E2F binding site. These results thus point to a role for E2Fs, together with B-Myb, which is an E2F-regulated gene expressed at G1/S, in linking the regulation of genes at G1/S and G2/M (Zhu, 2004).

The otic placode, a specialized region of ectoderm, gives rise to components of the inner ear and shares many characteristics with the neural crest, including expression of the key transcription factor Sox10. This study shows that in avian embryos, a highly conserved cranial neural crest enhancer, Sox10E2, also controls the onset of Sox10 expression in the otic placode. Interestingly, this study showed that different combinations of paralogous transcription factors (Sox8, Pea3 and cMyb versus Sox9, Ets1 and cMyb) are required to mediate Sox10E2 activity in the ear and neural crest, respectively. Mutating their binding motifs within Sox10E2 greatly reduces enhancer activity in the ear. Moreover, simultaneous knockdown of Sox8, Pea3 and cMyb eliminates not only the enhancer-driven reporter expression, but also the onset of endogenous Sox10 expression in the ear. Rescue experiments confirm that the specific combination of Myb together with Sox8 and Pea3 is responsible for the onset of Sox10 expression in the otic placode, as opposed to Myb plus Sox9 and Ets1 for neural crest Sox10 expression. Whereas SUMOylation of Sox8 is not required for the initial onset of Sox10 expression, it is necessary for later otic vesicle formation. This new role of Sox8, Pea3 and cMyb in controlling Sox10 expression via a common otic/neural crest enhancer suggests an evolutionarily conserved function for the combination of paralogous transcription factors in these tissues of distinct embryological origin (Betancur, 2011).

Myb expression during embryogenesis

Continued: Evolutionary homologs part 3/3 | back to part 1/3 |

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