Myb oncogene-like
Dictyostelium development is induced by starvation. The adenylyl cyclase gene ACA is one of the first genes expressed upon starvation. ACA produces extracellular cAMP that induces chemotaxis, aggregation, and differentiation in neighboring cells. Using insertional mutagenesis, a mutant has been isolated that does not aggregate upon starvation but is rescued by adding extracellular cAMP. Sequencing of the mutated locus reveals a new gene, DdMYB2, whose product contains three Myb repeats, the DNA-binding motif of Myb-related transcription factors. Ddmyb2-null cells show undetectable levels of ACA transcript and no cAMP production. Ectopic expression of ACA from a constitutive promotor rescues the differentiation and morphogenesis of Ddmyb2-null mutants. The results suggest that development in Dictyostelium starts by starvation-mediated DdMyb2 activation, which induces adenylyl cyclase activity producing the differentiation-inducing signal cAMP (Otsuka, 1998).
The avian retroviral v-myb gene and its cellular homologs throughout the animal and plant kingdoms
contain a conserved DNA binding domain. An isolated Dictyostelium insertional mutant was found to be unable to
switch from slug migration to fruiting body formation i.e. unable to culminate. The disrupted gene,
mybC, codes for a protein with a myb-like domain that is recognized by an antibody against the v-myb
repeat domain. During development of myb+ cells, mybC is expressed only in prestalk cells. When
developed together with wild-type cells, mybC- cells are able to form both spores and stalk cells very
efficiently. Their developmental defect is also bypassed by overexpressing cAMP-dependent protein kinase.
However even when their defect is bypassed, mybC null slugs and culminates produce little if any of the
intercellular signaling peptides SDF-1 and SDF-2 that are believed to be released by prestalk cells at
culmination. It is proposed that the mybC gene product is required for an intercellular signaling process
controlling maturation of stalk cells and spores and that SDF-1 and/or SDF-2 may be implicated in this
process (Guo, 1999).
The CyIIIa actin gene of the sea urchin Strongylocentrotus purpuratus is transcribed exclusively in the embryonic aboral ectoderm, under the control of 2.3 kb cis-regulatory domain that contains a proximal module that controls expression in early embryogenesis, and a middle module that controls expression in later embryogenesis. Previous studies demonstrated that the SpRunt-1 target site within the middle module is required for the sharp increase in CyIIIa transcription which accompanies differentiation of the aboral ectoderm, and that a negative regulatory region near the SpRunt-1 target site is required to prevent ectopic transcription in the oral ectoderm and skeletogenic mesenchyme. This negative regulatory region contains a consensus binding site for the myb family of transcription factors. In vitro DNA-binding experiments reveal that a protein in blastula-stage nuclei interacts specifically with the myb target site. Gene transfer experiments utilizing CyIIIa reporter constructs containing oligonucleotide substitutions indicate that this site is both necessary and sufficient to prevent ectopic expression of CyIIIa. Synthetic oligonucleotides containing the myb target site were used to purify a protein from sea urchin embryo nuclear extracts, employing affinity chromatography. This protein is immunoprecipitated by antibodies specific to the evolutionarily conserved myb domain; amino acid sequences obtained from the purified protein were found to be identical to sequences within the myb domain. Sequence information was used to obtain cDNA clones of SpMyb, the S. purpuratus member of the myb family of transcription factors. Through interactions within the middle module, SpMyb functions to repress activation of CyIIIa in the oral ectoderm and skeletogenic mesenchyme (Coffman, 1997).
The C. elegans synthetic multivulva (synMuv) genes act redundantly to antagonize the specification of vulval cell fates, which are promoted by an RTK/Ras pathway. At least 26 synMuv genes have been genetically identified, several of which encode proteins with homologs that act in chromatin remodeling or transcriptional repression. This study reports the molecular characterization of two synMuv genes, lin-37 and lin-54.lin-37 and lin-54 encode proteins in a complex with at least seven synMuv proteins, including LIN-35, the only C. elegans homolog of the mammalian tumor suppressor Rb. Biochemical analyses of mutants suggest that LIN-9, LIN-53, and LIN-54 are required for the stable formation of this complex. This complex is distinct from a second complex of synMuv proteins with a composition similar to that of the mammalian Nucleosome Remodeling and Deacetylase complex. The class B synMuv complex identified in this study is evolutionarily conserved and likely functions in transcriptional repression and developmental regulation (Harrison, 2006; full text of article).
LIN-37 and LIN-54 form a multisubunit protein complex together with at least five other class B synMuv proteins: LIN-9, LIN-35 Rb, LIN-52, LIN-53 RbAp48, and DPL-1 DP. This DP, Rb, and MuvB (DRM) complex is biochemically and genetically distinct from a NuRD-like complex that includes HDA-1 HDAC1, LET-418 Mi2, and LIN-53 RbAp48. These findings suggest that LIN-35 Rb and DPL-1 DP likely have a function in vulval development distinct from recruitment of the NuRD complex (Harrison, 2006).
The DRM complex is similar to two recently described and highly similar complexes that contain several Drosophila homologs of class B synMuv proteins (Korenjak, 2004; Lewis, 2004). The Myb–MuvB complex was purified by immunoprecipitation of the LIN-54 homolog Mip120 or the LIN-9 homolog Mip130 from Drosophila tissue-culture cells and coimmunoprecipitating proteins were identified by mass spectrometry. The Myb–MuvB complex contains stoichiometric levels of Mip130, RBF, Mip40, Mip120, p55, dDP, dE2F2, and dLin52, which are homologs of LIN-9, LIN-35, LIN-37, LIN-54, LIN-53, DPL-1, EFL-1, and LIN-52, respectively. This complex also contains substoichiometric amounts of Rpd3, the fly homolog of HDA-1, and L(3)MBT, a protein similar to the class B synMuv protein LIN-61. The dREAM complex was identified by biochemical purification of Drosophila Rb-containing complexes from embryo extracts followed by mass spectrometry and Western blot analyses. The dREAM complex contains all of the proteins identified in the Myb–MuvB complex at stoichiometric levels except for dLin52 (Korenjak, 2004). The differences between the dREAM and Myb–MuvB complexes might be a consequence of the methods used for purification or might reflect the existence in different tissues or during different developmental stages of multiple subcomplexes with overlapping components. Both the dREAM and Myb–MuvB complexes can mediate transcriptional repression of many E2F-target genes (Harrison, 2006).
The similarity between the C. elegans DRM complex and the Drosophila dREAM and Myb–MuvB complexes indicates that the DRM complex likely also acts in transcriptional repression. Given the broad expression patterns of the synMuv genes and the multiple phenotypic abnormalities caused by the loss of individual synMuv proteins, it is proposed that, similar to their Drosophila counterparts, the DRM complex proteins are involved in the repression of many targets important for diverse biological functions (Harrison, 2006).
The DRM complex differs slightly from both the dREAM and the Myb–MuvB complexes. Unlike the dREAM complex, the DRM complex contains a LIN-52 dLin52-like protein. Unlike the Myb–MuvB complex, the DRM complex does not contain HDA-1 Rpd3 or LIN-61 L(3)MBT. The similarities of the DRM, dREAM, and Myb–MuvB complexes suggest that there is a core complex consisting of LIN-35 RBF, EFL-1 E2F2, DPL-1 DP, LIN-9 Mip130, LIN-37 Mip40, LIN-52 dLin-52, LIN-53 p55, and LIN-54 Mip120 and that this complex might associate with other proteins during specific stages of development or in certain cell types (Harrison, 2006).
The dREAM and Myb–MuvB complexes both contain the DNA-binding protein Myb. There is no clear Myb homolog in C. elegans. It is possible that the C. elegans DRM complex does not contain a Myb ortholog or that the functional ortholog of the Drosophila Myb protein found in the dREAM and Myb–MuvB complexes might not be readily identifiable by comparisons of primary sequence (Harrison, 2006).
It is proposed that the DRM complex could be recruited to DNA by multiple DNA-binding factors, including LIN-54 and the heterodimeric transcription factor formed by EFL-1 and DPL-1. The DRM complex could then act with the NuRD-like complex to repress transcription. Alternatively, the DRM complex and the NuRD-like complex could act sequentially. The NURD-like complex could deacetylate the N-terminal tails of histones, and the DRM subsequently could bind these unmodified histone tails, preventing their acetylation. The dREAM complex previously has been shown to bind unmodified histone H4 tails, supporting this model. This binding might be mediated by LIN-53, because the mammalian homolog RbAp48 binds histone H4. Deacetylated histones are associated with transcriptionally repressed areas of the genome. Thus, by protecting histone tails from future acetylation, the DRM complex could act to maintain transcriptional repression of nearby genes (Harrison, 2006).
Although neither the DRM nor the dREAM complexes contains known chromatin-modifying or chromatin-remodeling enzymes, these complexes might require the activity of a histone deacetylase to mediate transcriptional repression. Mutations in genes encoding components of either the DRM or the NuRD-like complex require an additional class A or class C synMuv mutation to produce a highly penetrant Muv phenotype. However, mutations affecting two of the NuRD-like complex components, HDA-1 and LET-418, alone can cause low penetrance Muv phenotypes, suggesting that the chromatin-remodeling and chromatin-modifying activities of this complex might be required more broadly for the transcriptional repression of genes necessary for proper vulval development than is the activity of the DRM complex. Perhaps other class B synMuv proteins not associated with the DRM complex, for example, HPL-2, LIN-36, or LIN-61, act with the DRM complex to maintain the repressed state formed by the activity of the NuRD-like complex (Harrison, 2006).
The high degree of conservation shared by the DRM/Myb-MuvB/dREAM complexes in C. elegans and Drosophila and the important roles that the components of DRM complex play in C. elegans development suggest that a similar complex exists in other organisms, including humans. The core components of these complexes have homologs in humans, and the human homolog of LIN-9, hLin-9, can associate with Rb to specifically promote differentiation (but not to inhibit cell-cycle progression). Perhaps Rb or other Rb-family proteins act within the context of a DRM-like complex to control differentiation. Rb could act as a tumor suppressor through such DRM-mediated regulation of differentiation in addition to its role in cell-cycle regulation. Further biochemical and genetic studies of nematodes, insects, and mammals should elucidate the role that this conserved protein complex plays in development and in carcinogenesis (Harrison, 2006).
The myb gene family has three members, c-myb, A-myb and B-myb. The trans-activating capacity of the B-myb gene product (B-Myb) was examined in various types of cells. B-Myb functions as a transcriptional activator in CV-1 and HeLa cells, but not in NIH3T3 cells, indicating that B-Myb is a cell type-specific transcriptional activator. Deletion analyses of B-Myb have demonstrated that the region conserved between three members of the myb gene family (CR for conserved region) is necessary for trans-activation by B-Myb. An in vivo competition assay suggests that regulatory factor(s) that binds to the CR of B-Myb is required for transactivation. Analyses using an affinity resin show that multiple proteins bind to the CR of B-Myb and that the CR-binding proteins in CV-1 and HeLa cells are different from those in NIH3T3 cells. These results suggest that the CR-binding cofactor(s) is critical for the cell type-specific trans-activation by B-Myb (Tashiro, 1995).
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. 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. Three tryptic phosphopeptides derived from in vitro phosphorylated c-Myb co-migrate 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 (Aziz, 1995).
The A-Myb transcription factor belongs to the Myb family of oncoproteins and is likely to be involved in the regulation of proliferation and/or differentiation of normal B cells and Burkitt's lymphoma cells. To characterize in detail the domains of A-Myb that regulate its function, a series of deletion mutants has been generated and their trans-activation potential investigated as well as their DNA-binding activity. These results have allowed the delineation of the trans-activation domain as well as two separate regulatory regions. The boundaries of the trans-activation domain (amino acid residues 218-319) are centered on a sequence rich in charged amino acids (residues 259-281). A region (residues 320-482) localized immediately downstream of the trans-activation domain and containing a newly identified conserved stretch of 48 residues, markedly inhibits specific DNA binding. Finally, the last 110 residues of A-Myb (residues 643-752), which include a sequence conserved in all mammalian myb genes (region III), negatively regulate the maximal trans-activation potential of A-Myb. The functional interaction between A-Myb and the nuclear adaptor molecule CBP (CREB [cAMP response element-binding protein]-binding protein) has been investigated. CBP synergizes with A-Myb in a dose-dependent fashion; this co-operative effect can be inhibited by E1A and can also be observed with the CBP homolog p300. This functional synergism requires the presence of the A-Myb charged sequence and this involves physical interaction between A-Myb and the CREB-binding domain of CBP (Facchinetti, 1997).
Expression studies as well as the use of transgenic animals have demonstrated that the A-MYB transcription factor plays a central and specific role in the regulation of mature B cell proliferation and/or differentiation. A-MYB is highly expressed in Burkitt's lymphoma cells and may participate in the pathogenesis of this disease. The transcriptional activity of A-MYB and its regulation in several human lymphoid cell lines have been investigated. A-MYB is transcriptionally active in all the B cell lines studied, but not in T cells. In particular the best responder cell line is the Burkitt's cell line Namalwa. The activity of A-MYB in B and not T cells is observed when either an artificial construct or the c-MYC promoter is used as a reporter. The functional domains responsible for DNA binding, transactivation, and negative regulation, previously characterized in a fibroblast context, are found to have similar activity in B cells. The region of A-MYB responsible for the B cell specific activity is the N-terminal 218 amino acids containing the DNA binding domain. A 110-kDa protein has been identified in the nuclei of all the B cell (but not T cell) lines that specifically bind to this A-MYB N-terminal domain. It is hypothesized that this 110-kDa protein may be a functionally important B cell-specific co-activator of A-MYB (Ying, 1997).
Using the yeast two-hybrid system, the transcription factor ATBF1 was identified as v-Myb- and c-Myb-binding protein. Deletion mutagenesis revealed amino acids 2484-2520 in human ATBF1 and 279-300 in v-Myb as regions required for in vitro binding of both proteins. Further experiments identified leucines Leu325 and Leu332 of the Myb leucine zipper motif as additional amino acid residues important for efficient ATBF1-Myb interaction in vitro. In co-transfection experiments, the full-length ATBF1 was found to form in vivo complexes with v-Myb and inhibit v-Myb transcriptional activity. Both ATBF1 2484-2520 and Myb 279-300 regions are required for the inhibitory effect. Finally, the chicken ATBF1 was identified, showing a high degree of amino acid sequence homology with human and murine proteins. These data reveal Myb proteins as the first ATBF1 partners detected so far and identify amino acids 279-300 in v-Myb as a novel protein-protein interaction interface through which Myb transcriptional activity can be regulated (Kaspar, 1999).
c-Myb plays important roles in cell survival and differentiation in immature hematopoietic cells. c-Myb is acetylated at the carboxyl-terminal conserved domain by histone acetyltransferase p300 both in vitro and in vivo. The acetylation sites in vivo have been located at the lysine residues of the conserved domain (K471, K480, K485) by the use of the mutant Myb (Myb-KAmut), in which all three lysine residues are substituted into alanine. Electrophoretic mobility shift assay reveals that Myb-KAmut shows higher DNA binding activity than wild type c-Myb and that acetylation of c-Myb in vitro by p300 causes dramatic increase in DNA binding activity. Accordingly, transactivation activity of both mim-1 and CD34 promoters by Myb-KAmut is higher than that driven by wild type c-Myb. Furthermore, the bromodomain of p300, in addition to the histone acetyltransferase (HAT) domain, is required for effective acetylation of c-Myb; hGCN5 is revealed to be an acetyl-transferase for c-Myb in vitro (Tomita, 2000).
B-MYB is implicated in cell growth control, differentiation, and cancer and belongs to the MYB family of nuclear transcription factors. Evidence exists that cellular proteins bind directly to B-MYB, and it has been hypothesized that B-MYB transcriptional activity may be modulated by specific cofactors. In an attempt to isolate proteins that interact with the B-MYB DNA-binding domain, a modular domain that has the potential to mediate protein-protein interaction, pull-down experiments were performed with a glutathione S-transferase-B-MYB protein and mammalian protein extracts. A 110-kDa protein was isolated associated endogenously with B-MYB in the nuclei of HL60 cells. Microsequence analysis and immunoprecipitation experiments determined that the bound protein was poly(ADP-ribose) polymerase (PARP). Transient transfection assays showed that PARP enhances B-MYB transactivation and that PARP enzymatic activity is not required for B-MYB-dependent transactivation. These results suggest that PARP, as a transcriptional cofactor of a potentially oncogenic protein, may play a role in growth control and cancer (Cervellera. 2000).
c-Myb, but not avian myeloblastosis virus (AMV) v-Myb, cooperates with C/EBPß to regulate transcription of myeloid-specific genes. To assess the structural basis for that difference, the crystal structures were determined for complexes comprised of the c-Myb or AMV v-Myb DNA-binding domain (DBD), the C/EBPß DBD, and a promoter DNA fragment. Within the c-Myb complex, a DNA-bound C/EBPß interacts with R2 of c-Myb bound to a different DNA fragment; point mutations in v-Myb R2 eliminate such interaction within the v-Myb complex. GST pull-down assays, luciferase trans-activation assays, and atomic force microscopy confirm that the interaction of c-Myb and C/EBPß observed in crystal mimics their long range interaction on the promoter, which is accompanied by intervening DNA looping (Tahirov, 2002).
The DBD of c-Myb consists of three imperfect tandem repeats of 51 or 52 amino acid residues, referred to as R1, R2, and R3 from the N terminus. Each repeat contains three helices (alpha1, alpha2, and alpha3) with the helix-turn-helix variant motif, and that R2 and R3 are involved in specific DNA recognition, while R1 loosely covers the DNA position next to the R2 binding site. The structure of R2 also contains a partially exposed hydrophobic patch; within the corresponding region of the AMV v-Myb DBD, this hydrophobic patch contains the three point mutations responsible for the failure of v-Myb to activate the mim-1 promoter, suggesting that this hydrophobic patch is important for the interaction of c-Myb and C/EBPß and for their synergistic activation of transcription (Tahirov, 2002 and references therein).
To establish the structural basis for the synergy between c-Myb and C/EBPß and its disruption by the mutations found in the AMV v-Myb DBD, the crystal structures were determined of ternary complexes containing the c-Myb or AMV v-Myb DBD, the C-terminal portion of C/EBPß, including the DBD, and a DNA fragment from the tom-1A promoter: c-Myb38-193-C/EBPß259-336-DNA [c-Myb38-193-C/EBPß273-336-DNA, and AMV v-Myb66-193-C/EBPß259-336-DNA are respectively named c-Myb complex I, c-Myb complex II (or generally c-Myb complex without discrimination of complexes I and II)], and v-Myb complex. It was found that the c-Myb complex shows specific intercomplex binding between c-Myb and C/EBPß: this is not shown by the v-Myb complex. This interaction between c-Myb and C/EBPß led to the speculation that, via DNA looping, c-Myb and C/EBPß are able to interact and cooperate despite the fact that they bind to natural promoters at some distance from one another. Atomic force microscopy (AFM) was used to confirm that c-Myb and C/EBPß could interact through looping of the mim-1 promoter and its functional relevance, in vivo, was shown using a luciferase trans-activation assay (Tahirov, 2002).
The B-Myb transcription factor has been implicated in coordinating the expression of genes involved in cell cycle regulation. Although it is expressed in a ubiquitous manner, its transcriptional activity is repressed until the G(1)-S phase of the cell cycle by an unknown mechanism. In this study biochemical and cell-based assays were used to demonstrate that the nuclear receptor corepressors N-CoR and SMRT interact with B-Myb. The significance of these B-Myb-corepressor interactions was confirmed by the finding that B-Myb mutants that were unable to bind N-CoR exhibit constitutive transcriptional activity. It has been shown previously that phosphorylation of B-Myb by cdk2/cyclin A enhances its transcriptional activity. It has now been determined that phosphorylation by cdk2/cyclin A blocks the interaction between B-Myb and N-CoR and that mutation of the corepressor binding site within B-Myb bypasses the requirement for this phosphorylation event. Cumulatively, these findings suggest that the nuclear corepressors N-CoR and SMRT serve a previously unappreciated role as regulators of B-Myb transcriptional activity (Li, 2002).
Transcriptional initiation of eukaryotic genes depends on the cooperative interaction of various transcription factors. Using the yeast two-hybrid assay, the murine Rcd-1 protein has been identified as a cofactor of the c-myb proto-oncogene product. Rcd-1 is evolutionarily conserved among many species, and moreover the yeast homologue CAF40 is part of the carbon catabolite repressor protein transcriptional mediator thought to be involved in the negative regulation of genes transcribed by RNA polymerase II. Rcd-1 is located mainly in the nucleus, and it interacts with c-Myb both in vitro and in vivo. The activation of the myeloid c-myb-specific mim-1 promoter is repressed by Rcd-1. Interestingly, rcd-1 is an erythropoietin regulated gene, which also represses the action of the AP-1 transcription factor on its target genes (Haas, 2004).
The small nuclear RNA (snRNA) activating protein complex (SNAPc) is essential for transcription of genes that encode the snRNAs. Drosophila melanogaster SNAPc (DmSNAPc) consists of three subunits (DmSNAP190, DmSNAP50 and DmSNAP43) that form a stable complex that recognizes an snRNA gene promoter element called the PSEA. Although all three subunits are required for sequence-specific DNA binding activity, only DmSNAP190 possesses a canonical DNA binding domain consisting of 4.5 tandem Myb repeats homologous to the Myb repeats in the DNA binding domain of the Myb oncoprotein. This study used site-specific protein-DNA photo-cross-linking technology followed by site-specific protein cleavage to map domains of DmSNAP190 that interact with specific phosphate positions in the U6 PSEA. The results indicate that at least two DmSNAP190 Myb repeats contact the DNA in a significantly different manner when DmSNAPc binds to a U6 PSEA versus a U1PSEA, even though the two PSEA sequences differ at only 5 of 21 nucleotide positions. The results are consistent with a model in which the specific DNA sequences of the U1 and U6 PSEAs differentially alter the conformation of DmSNAPc, leading to the subsequent recruitment of different RNA polymerases to the U1 and U6 gene promoters (Kang, 2014: PubMed).
In the absence of growth signals, cells exit the cell cycle and enter into G0 or quiescence. Alternatively, cells enter senescence in response to inappropriate growth signals such as oncogene expression. The molecular mechanisms required for cell cycle exit into quiescence or senescence are poorly understood. The DREAM (DP, RB [retinoblastoma], E2F, and MuvB) complex represses cell cycle-dependent genes during quiescence. DREAM contains p130, E2F4, DP1, and a stable core complex of five MuvB-like proteins: LIN9, LIN37, LIN52, LIN54, and RBBP4. In mammalian cells, the MuvB core dissociates from p130 upon entry into the cell cycle and binds to BMYB during S phase to activate the transcription of genes expressed late in the cell cycle. Mass spectroscopic analysis to identify phosphorylation sites that regulate the switch of the MuvB core from BMYB to DREAM. This study reports that DYRK1A can specifically phosphorylate LIN52 on serine residue 28, and that this phosphorylation is required for DREAM assembly. Inhibiting DYRK1A activity or point mutation of LIN52 disrupts DREAM assembly and reduces the ability of cells to enter quiescence or undergo Ras-induced senescence. These data reveal an important role for DYRK1A in the regulation of DREAM activity and entry into quiescence (Litovchick, 2011).
B-MYB expression is associated with cell proliferation. Recent studies have suggested that it promotes the S phase of mammalian cells. Based on its homology to the transcription factors c-MYB and A-MYB, B-MYB was thought to be involved in transcriptional regulation; however, its activity was not detectable in several cell lines. It was postulated that B-MYB function may depend on the presence of a cofactor, and recent studies suggest that B-MYB is phosphorylated specifically during S phase in murine fibroblasts. Evidence suggests that the product of the human B-myb gene can be activated in vivo by coexpression with cyclin A or cyclin E. Transfection studies show that B-MYB is a weak transcriptional activator in SAOS-2 cells and is unable to promote their proliferation. In contrast, overexpression of both B-MYB and cyclin A or cyclin E causes a drastic increase in the number of SAOS-2 cells in S phase. Overexpression of cyclin A and cyclin E in SAOS-2 cells enhances the ability of B-MYB, but not c-MYB, to transactivate various promoters, including the cdc2 promoter, the HIV-1-LTR, and the simian virus 40 minimal promoter. A direct role for cyclin-dependent activation of B-MYB has been demonstrated using an in vitro transcription assay. These observations suggest that one mechanism by which cyclin A and E may promote the S phase is through modification and activation of B-MYB (Sala, 1997).
A-myb, a conserved member of the Myb proto-oncogene family, encodes a sequence-specific DNA binding protein (A-Myb) that binds to and transactivates promoters containing myb-binding sites. Previous work has suggested that the C-terminus of A-Myb functions as a regulatory domain, however, the physiological signals that control the activity of A-Myb have not yet been identified. The presence of potential phosphorylation sites for cyclin-dependent kinases in the C-terminus of A-Myb has prompted an examination of the possibility that the function of A-Myb is controlled by the cell cycle. The transactivation potential of A-Myb is repressed by the C-terminal domain; phosphorylation of A-Myb, induced by cyclins A and E, relieves this inhibitory effect. This work provides the first evidence that the function of A-Myb is regulated by the cell cycle machinery and that the carboxy-terminal domain of A-Myb acts as a cell cycle sensor. In addition, A-myb mRNA expression is also cell cycle regulated and attains maximal levels during late G1- and early S-phase. Thus, A-Myb appears to be controlled by two different mechanisms resulting in maximal A-Myb activity during the G1/S-transition and the S-phase of the cell cycle (Ziebold, 1997).
Precise control of hematopoietic stem cell (HSC) proliferation and differentiation is needed to maintain a lifetime supply of blood cells. Using genome-wide ENU mutagenesis and phenotypic screening, a mouse line has been identified that harbors a point mutation in the transactivation (TA) domain of the transcription factor c-Myb (M303V) that reduces c-Myb-dependent TA by disrupting its interaction with the transcriptional coactivator p300. The biological consequences of the c-MybM303V/M303V mutation include thrombocytosis, megakaryocytosis, anemia, lymphopenia, and the absence of eosinophils. Detailed analysis of hematopoiesis in c-MybM303V/M303V mice reveals distinct blocks in T cell, B cell, and red blood cell development, as well as a remarkable 10-fold increase in the number of HSCs. Cell cycle analyses show that twice as many HSCs from c-MybM303V/M303V animals are actively cycling. Thus c-Myb, through interaction with p300, controls the proliferation and differentiation of hematopoietic stem and progenitor cells (Sandberg, 2005).
The present study supports a critical role for c-Myb-p300 interaction throughout the hematopoietic hierarchy. Importantly, c-Myb is not required at every step and for every lineage; rather, there are distinct steps and lineages that are critically dependent on c-Myb function. These include negative regulation of HSC proliferation, megakaryocyte numbers, and essential roles in erythropoiesis, eosinophil, T, and B cell development. Although previous studies have provided clues that c-Myb may play a role in some of these processes, the severe phenotype of c-Myb-/- animals has precluded a detailed examination of c-Myb function in adult animals (Sandberg, 2004).
Several lines of evidence support a critical role for c-Myb in directing megakaryocyte and erythrocyte development. (1) c-Myb-/- mice die of anemia in utero at day 15 due to a failure to initiate fetal liver hematopoiesis. megakaryocyte/erythrocyte progenitors (MEPs) are likely formed in c-Myb-/- mice, since megakaryocytes are present in the fetal liver of c-Myb-/- animals. c-Myb-/- animals contain low numbers of definitive stem/progenitor cells that can give rise to early thymic precursors and initiate myeloid cell development. Thus, the failure to initiate fetal erythropoiesis is likely to result from an inability of later progenitor cells to differentiate into RBCs. (2) Neonatal mice containing ~20% the normal amount of c-Myb also have anemia and elevated numbers of megakaryocytes and platelets. (3) The c-MybM303V/M303V mice and other ENU-induced alleles of c-Myb contain elevated numbers of platelets and CD41+ megakaryocytes and reduced numbers of RBCs. Thus, multiple independent alleles of c-Myb give an identical phenotype: reduced numbers of RBCs and elevated numbers of megakaryocytes. Since these two cell types arise from a common precursor, the simplest explanation is that c-Myb is required for the MEP to initiate the RBC program and also participates in turning off the megakaryocyte program. In support of this view, only modest reductions in the number of MEPs are found in c-MybM303V/M303V animals compared to normal littermates; however, these cells are unable to make RBCs following culture. Thus, these bipotential cells are present, but their differentiation potential is limited. Collectively, these data suggest that c-Myb normally limits the ability of the HSC to differentiate toward the megakaryocyte lineage while promoting the RBC program (Sandberg, 2004).
The low numbers of B and T cells in the blood of c-Myb-/- mice suggest that c-Myb may be regulating both B and T cell development. c-MybM303V/M303V mice contained normal numbers of phenotypic Lin-Kit+Sca1+IL7R+ common lymphoid progenitors (CLPs), suggesting that these early precursor cells are present in normal numbers. Consistent with this observation, normal numbers of the earliest pre-pro B cells were found. However, the numbers of pro-B cells were reduced 3-fold, and the numbers of pre-B cells were down more than 10-fold in c-MybM303V/M303V mice. Importantly, the percentage of B cells at subsequent stages of development was not different, although the absolute numbers were reduced in c-MybM303V/M303V mice, and their functional capacity remains to be tested. Reduced numbers of pro-B and pre-B cells in c-MybM303V/M303V mice suggest that c-Myb may play a role in directing IgH chain rearrangement or in the expansion of those cells that have successfully rearranged IgH (Sandberg, 2004).
The ability of c-Myb to regulate T cell development is well established; however, identification of the precise points that c-Myb regulates has been controversial. Early studies demonstrated a requirement for c-Myb at the pre-DN1 stage of T cell development, while later studies with a dominant-negative form of c-Myb showed a role for c-Myb in the regulation of DN3 and DN4 cell expansion post-TCR β chain selection. A recent study with neonatal mice with partial reductions in c-Myb levels showed that in fetal thymus development c-Myb is required for DN1 to DN2 maturation and the transition from DN2 to DN3, with the former step requiring higher threshold levels of c-Myb. Very recent data generated with conditional null alleles in which Myb is deleted early or late in T cell development in the thymus support a roll for c-Myb in directing the DN3-to-DN4 transition, survival of preselection double-positive cells, and differentiation of CD4 SP cells. In agreement with these data, the c-MybM303V/M303V mice described in this study show 2- to 10-fold reductions in thymocyte number with 3-fold reductions in the number of both DN1 and DN4 cells in mature animals. Interestingly, competitive bone marrow reconstitution experiments show that cells harboring the c-MybM303V/M303V allele contain normal numbers of DN1 cells and more than 4-fold reductions in DN4 cells. Based on the current work and previous studies, it seems that c-Myb can regulate T cell development at many points; however, the mechanistic details remain an area of active research (Sandberg, 2004).
Reconstitution studies with mixed bone marrow chimeras show a 10- to 20-fold increase in the number of functional HSCs in bone marrow from c-MybM303V/M303V mice, and 5-fold elevations in the absolute number of HSCs in primary recipients; thus, these phenotypic HSCs are functional. Secondary and tertiary transplant experiments of these bone marrow chimeras show that these HSCs are capable of self-renewal and long-term multilineage reconstitution, thus confirming that these represent bona fide long term HSCs. These experiments also reveal that the defects in hematopoiesis in c-MybM303V/M303V mice are cell intrinsic; c-MybM303V/M303V bone marrow gives rise to normal numbers of myeloid and granulocytes, while the numbers of T and B cells are decreased. Importantly, WT cells cotransplanted into these same animals differentiate normally, showing that the defects in hematopoiesis result from direct effects of c-MybM303V/M303V on the development of HSC and progenitors and not from secondary effects of the lymphopenia, anemia, or megakaryocytosis. HSCs from c-MybM303V/M303V mice contain an increase in the number of actively cycling cells, providing a mechanistic framework for the elevated numbers of HSCs. Thus, c-Myb-p300 normally acts to repress the proliferation of HSCs or links proliferation with subsequent differentiation (Sandberg, 2004).
Previous data have shown that c-Myb is required to generate definitive HSCs, that c-Myb-/- animals die at E14.5 as a result of a lack of definitive erythropoiesis, and that few HSCs are found in early embryos. In contrast, partial loss of c-Myb results in increased numbers of fetal liver progenitor cells expressing CD34+ or Sca-1+, supporting a negative role for c-Myb in the control of early progenitors. Interestingly, many of the phenotypes described in the c-MybM303V/M303V mice are also found in mice homozygous for a triple point mutation in the KIX domain of the transcriptional coactivator p300 (p300KIX/KIX) that disrupts the association of p300 with c-Myb. It will interesting to determine if the p300KIX/KIX and other c-Myb mutants have similar alterations in the number of HSCs and progenitor cells (Sandberg, 2004).
The current study shows that the c-Myb-p300 interaction controls hematopoiesis at many distinct points, both promoting and repressing proliferation and differentiation and thus highlighting c-Myb as a key regulator of hematopoiesis. Determining how a single transcription factor is able to control the diverse processes of selfrenewal, proliferation, and differentiation at distinct points in hematopoiesis and defining the molecular interactions that control c-Myb activity remain important areas of future research (Sandberg, 2004).
Activity of the human B-myb promoter appears to be regulated during the cell cycle by the E2 transcription factor (E2F). Comparison of the human B-myb promoter sequence with that of its murine counterpart reveals an evolutionally conserved sequence that contains an E2F-binding site. In transiently transfected murine NIH3T3 and human HaCaT cells, luciferase (Luc) reporter activity directed by the human B-myb promoter is found to increase significantly in late G1/S phase of the cell cycle. Mutation of the promoter E2F site results in significantly greater Luc activity in NIH3T3 and HaCaT cells made quiescent by serum deprivation, indicating that E2F represses transcription of this gene during G0. Analysis of E2F DNA-binding activity in G0 HaCaT cells reveals a distinct complex that apparently contains neither the retinoblastoma gene protein (pRb) nor the related p107 protein. De-repression of transcription in S phase is accompanied by the disappearance of this G0 E2F complex and the appearance of a distinct complex containing p107. In addition, complexes containing pRb are detected at both stages of the cell cycle (Lam, 1995).
Transcription of B-Myb is cell-cycle regulated by an E2F transcription factor-mediated repression mechanism operating in G0/G1. G0-arrest of serum-deprived mouse fibroblasts is achieved without significant reduction in B-Myb levels; moreover, over-expression of B-Myb in stably transfected cells does not prevent their entry into G0. Following serum-induction of arrested fibroblasts, as cells enter S phase, B-Myb abundance increases to levels significantly greater than those found in cycling cells. This increase is accompanied by the appearance of a novel phosphorylated form of B-Myb (112 kDa), with distinctly lower electrophoretic mobility than B-Myb present in G1 (110 kDa). The 112 kDa species is S phase-specific even in transfected cells overexpressing B-Myb. Consistent with modification in the S phase of the cell cycle, preliminary evidence suggested that a cyclin A/cdk2, but not cyclin E/cdk2 or cyclin D1/cdk4, complex can induce a similar electrophoretic mobility change in baculovirus-specified B-Myb. These findings show that B-Myb expression may be subject to two levels of control during the cell cycle: at transcription and during protein phosphorylation (Robinson, 1996).
The retinoblastoma protein family has been implicated in growth control and modulation of the activity of genes involved in cell proliferation, such as B-myb. Recent evidence indicates that the product of the B-myb gene is necessary for the growth and survival of several human and murine cell lines. Upon overexpression, B-myb induces deregulated cell growth of certain cell lines. B-myb overexpression is able to induce DNA synthesis in p107 growth-arrested human osteosarcoma cells (SAOS2). p107 might exert its growth-suppressive activity by regulating B-myb gene transcription. Indeed, p107 down-modulates B-myb promoter activity and drastically decreases E2F-mediated transactivation. B-myb is able to stimulate DNA synthesis of both stably and transiently transfected human glioblastoma cells (T98G). Together, these data provide definitive evidence that the human B-myb protein is involved in growth control of human cells, and that p107 has a significant role in regulating B-myb gene activity (Sala, 1996).
B-myb belongs to a group of cell cycle genes whose transcription is repressed in G0/early G1 through a binding site for the transcription factor E2F. The B-myb repressor element is specifically recognised by heterodimers consisting of DP-1 with either E2F-1, E2F-3 or E2F-4. Surprisingly, E2F-mediated repression is dependent on a contiguous corepressor element that resembles the CHR (cell cycle genes homology region) previously established as a corepressor of the CDE (cell cycle-dependent element) in cell cycle genes derepressed in S/G2, such as cyclin A, cdc2 and cdc25C. A factor binding to the B-myb CHR (cell cycle genes homology region) was identified in fractionated HeLa nuclear extract and found to interact with the minor groove, as previously shown by in vivo footprinting for the cyclin A CHR. The B-myb and cdc25C CHRs are related with respect to protein binding but functionally, are clearly distinct. These results support a model where both E2F- and CDE-mediated repression, acting at different stages in the cell cycle, are dependent on promoter-specific CHR elements (Liu, 1996).
B-myb and cdc25C exemplify different groups of genes whose transcription is consecutively up-regulated during the cell cycle. Both promoters are controlled by transcriptional repression via modules consisting of an E2F binding site (E2FBS) or the related CDE (cell cycle-dependent element) plus a contiguous CHR (cell cycle genes homology region) co-repressor element. The B-myb repressor module, which is derepressed early (mid G1), is preferentially recognized by E2F-DP complexes; a mutation that selectively abolishes E2F binding, impairs regulation. In contrast, the cdc25C repressor module, which is derepressed late (S/G2), interacts selectively with CDE-CHR binding factor-1 (CDF-1). E2F binding, but not CDF-1 binding, requires specific nucleotides flanking the E2FBS/CDE core, while CDF-1 binding, but not E2F binding, depends on specific nucleotides in the CHR. Swapping these nucleotides between the two promoters profoundly changes protein binding patterns and alters expression kinetics. Predominant CDF-1 binding leads to derepression in late S, predominant E2F binding results in up-regulation in late G1, while promoters binding both E2F and CDF-1 with high efficiency show intermediate kinetics. These results support a model where the differential binding of E2F and CDF-1 repressor complexes contributes to the timing of promoter activity during the cell cycle (Lucibello, 1997).
Neuroblastoma cells can undergo neural differentiation once treated with a variety of chemical inducers and growth factors. During this process, many cell cycle-related genes are downregulated while differentiation-specific genes are triggered. The retinoblastoma family proteins (pRb, p107, and pRb2/p130) are involved in transcriptional repression of proliferation genes, mainly through their interaction with the E2F transcription factors. pRb2/p130 expression levels increase during differentiation of neuroblastoma cell line LAN-5. In contrast, both pRb and p107 decrease and undergo progressive dephosphorylation at late differentiation times. The expression of B-myb and c-myb, two targets of the retinoblastoma family proteins, are downregulated in association with the increase of pRb2/p130, which is detected as the major component of the complex with E2F on the E2F site of the B-myb promoter in differentiated cells. Interestingly, E2F4, a preferential partner of p107 and pRb2/p130, is upregulated and undergoes changes in cellular localization during differentiation. The data suggest a major role for pRb2/p130 in the regulation of B-myb promoter during neural differentiation, despite the importance of cofactors in modulating the function of the retinoblastoma family proteins (Raschella, 1997).
During hexamethylene bisactamide (HMBA)-induced differentiation of murine erythroleukemia (MEL) cells, erythroid genes are transcriptionally activated while c-myb and several other nuclear proto-oncogenes are down-regulated. Differentiation is inhibited by cAMP analogs and the adenyl cyclase-stimulating agent forskolin. These drugs prevent the late down-regulation of c-myb, which is known to be critical for MEL cell differentiation. Since the increase in c-myb mRNA levels is due to increased mRNA transcription, an examination was made of the transcription factors NF-kappaB and AP-1 which have been implicated in the regulation of c-myb expression. Binding of MEL cell nuclear proteins to a NF-kappaB recognition sequence in c-myb intron I is strongly induced by 8-Br-cAMP or forskolin; induction is delayed and correlates with the up-regulation of c-myb. The cAMP-induced NF-kappaB complex contains p50 and RelB; in co-transfection assays, p50 and RelB transactivate a reporter construct containing the c-myb intronic NF-kappaB site upstream of a minimal promoter. 8-Br-cAMP and forskolin also increase the DNA binding activity of AP-1 complexes containing JunB, JunD and c-Fos in MEL cells, which can cooperate with NF-kappaB. It is concluded that inhibition of MEL cell differentiation by pharmacological doses of cAMP can be explained by the up-regulation of c-myb which is mediated, at least in part, by NF-kappaB p50/RelB heterodimers (Suhasini, 1998).
Down-regulation of c-myb mRNA levels by agents that raise the internal level of Ca2+ ions (A23187, thapsigargin, cyclopiazonic acid) and by erythropoietin was comparatively studied in the erythropoietin-responsive murine erythroleukemia cell line, ELM-I-1. The Ca2+-induced suppression of c-myb mRNA can be inhibited by the calmodulin antagonists trifluoperazine and calmidazolium, as well as by cyclosporin A, an inhibitor of the Ca2+/calmodulin-dependent protein phosphatase 2B (calcineurin). KN-62, an inhibitor of Ca2+/calmodulin-dependent protein kinases, does not antagonize the Ca2+-mediated decrease in c-myb mRNA. In cyclosporin A-treated ELM-I-1 cells, a close correlation can be demonstrated between the antagonization of the Ca2+ effect on c-myb mRNA levels and inhibition of the calcineurin phophatase activity. In contrast, FK506, which does not inhibit calcineurin activity in ELM-I-1 cells, fails to prevent the Ca2+-mediated decrease in c-myb mRNA. The erythropoietin-induced down-regulation of c-myb mRNA levels can be demonstrated also in the presence of EGTA and is resistant to calmodulin antagonists and cyclosporin A. No increase in [Ca2+]i is observed in ELM-I-1 cells in response to erythropoietin. Cyclosporin A inhibits the Ca2+-induced hemoglobin production, while the erythropoietin-mediated increase in hemoglobin synthesis is not affected. The Ca2+-induced decrease in c-myb mRNA and increase in hemoglobin synthesis is mediated by calcineurin, while these effects of erythropoietin occur independently of Ca2+ in ELM-I-1 cells. Calcineurin may be involved in the regulation of c-myb expression in erythroid precursor cells and Ca2+ signals via calcineurin may positively modulate the differentiation inducing action of erythropoietin (Schaefer, 1996).
B-Myb is a highly conserved member of the Myb family of transcription factors, which has been implicated in cell cycle regulation. B-Myb is expressed in most proliferating cells and its activity is highly regulated around the G1/S-phase border of the cell cycle. It is generally assumed that B-Myb regulates the expression of genes that are crucial for cell proliferation; however, the identity of these genes, the molecular mechanisms by which B-Myb stimulates their expression and the involvement of other proteins have not been sufficiently clarified. The hamster cell line ts13 was used as a tool to demonstrate a functional link between B-Myb and the coactivator TAF(II)250, a key component of the transcriptional machinery which itself is essential for cell proliferation. ts13 cells express a point-mutated version of TAF(II)250 whose intrinsic histone acetyl transferase activity is temperature sensitive. Transactivation of Myb-responsive reporter genes by B-Myb is temperature-dependent in ts13 cells but not in ts13 cells, which have been rescued by transfection with an expression vector for wild-type TAF(II)250. Furthermore, B-Myb and TAF(II)250 can be coprecipitated, suggesting that both proteins are present in a complex. The formation of this complex is dependent on the DNA-binding domain of B-Myb and not on its transactivation domain. Taken together, these observations provide the first evidence that the coactivator TAF(II)250 is involved in the activation of Myb responsive promoters by B-Myb. The finding that B-Myb transactivation is dependent on a key coactivator involved in cell cycle control is consistent with and strengthens the idea that B-Myb plays a crucial role as a transcription factor in proliferating cells (Bartusel, 2003).
The c-myb proto-oncogene product (c-Myb) regulates both the proliferation and apoptosis of hematopoietic cells by inducing the transcription of a group of target genes. However, the biologically relevant molecular mechanisms that regulate c-Myb activity remain unclear. This study reports that c-Myb protein is phosphorylated and degraded by Wnt-1 signal via the pathway involving TAK1 (TGF-beta-activated kinase), HIPK2 (homeodomain-interacting protein kinase 2), and NLK (Nemo-like kinase). Wnt-1 signal causes the nuclear entry of TAK1, which then activates HIPK2 and the mitogen-activated protein (MAP) kinase-like kinase NLK. NLK binds directly to c-Myb together with HIPK2, which results in the phosphorylation of c-Myb at multiple sites, followed by its ubiquitination and proteasome-dependent degradation. Furthermore, overexpression of NLK in M1 cells abrogates the ability of c-Myb to maintain the undifferentiated state of these cells. The down-regulation of Myb by Wnt-1 signal may play an important role in a variety of developmental steps (Kanei-Ishii, 2004; full text of article).
Transcriptional targets of Myb
Continued: Evolutionary homologs part 2/3 | part 3/3 |
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