Figure - available via license: Creative Commons Attribution-NonCommercial 2.0 Generic
Content may be subject to copyright.
Chromatin-immunoprecipitation experiment. (A) Western blot analysis of Myb expression in HD11-E cells grown in the presence or absence of doxycyclin. The black arrow marks the v-MybREV protein. (B) Chromatin immunoprecipitation experiment using HD11-E cells grown in the presence (left part) or absence (right part) of doxycyclin. Immunoprecipitation was carried out using two different Myb-specific antisera (lanes 1 and 2), non-immune serum (lane 3) or no antiserum (lane 4). Lane 5 shows no-template controls and lane 6 shows input controls. PCR primers were specific for the lysozyme gene promoter and the −2.7 kb enhancer. PCR products were analyzed by agarose gel electrophoresis.
Source publication
The oncoprotein v-Myb of avian myeloblastosis virus (AMV) transforms myelomonocytic cells by deregulating specific target
genes. Previous work has shown that the oncogenic potential of v-Myb was activated by truncation of N- and C-terminal sequences
of c-Myb and was further increased by amino acid substitutions in the DNA-binding domain and other p...
Similar publications
The retroviral oncogene v-myb encodes a transcription factor (v-Myb) which is responsible for the transformation of myelomonocytic cells by avian myeloblastosis virus (AMV). v-Myb is thought to exert its biological effects by deregulating the expression of specific target genes. We have recently demonstrated that the chicken Gas41 gene, whose promo...
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...
The viral Myb (v-Myb) oncoprotein of the avian myeloblastosis virus (AMV) is an activated form of the cellular transcription
factor c-Myb causing acute monoblastic leukemia in chicken. Oncogenic v-Myb alterations include N- and C-terminal deletions
as well as point mutations. Whereas truncations in Myb cause loss of various protein modifications, n...
Retroviral transforming genes, v-onc genes, are derived from normal cellular sequences that are called cellular onc (c-onc) genes. DNA from mouse-human somatic cell hybrids that have selectively lost human chromosomes was used in Southern blots to map the chromosomal location of two human onc genes. Cloned human homologues of retroviral onc genes w...
We have identified the product of the human c-myb proto-oncogene as a 80,000-Mr protein, p80c-myb, by using polyclonal and monoclonal antibodies raised against a bacterially synthesized polypeptide from the amino terminus of the viral myb protein. p80c-myb shares at least two distinct antigenic sites with the amino terminal region of the v-myb prot...
Citations
... All reporter studies were performed in at least 3 independent experiments, with replicate transfections in each experiment. Expression vectors for v-Myb [27], chicken C/EBPβ and C/EBPα [28] and mouse and human C/EBPβ [29,30] have been described. A mutant of chicken C/EBPβ carrying replacements of all cysteine residues by alanine has been described [31]. ...
Recent work has shown that deregulation of the transcription factor Myb contributes to the development of leukemia and several other human cancers, making Myb and its cooperation partners attractive targets for drug development. By employing a myeloid Myb-reporter cell line we have identified Withaferin A (WFA), a natural compound that exhibits anti-tumor activities, as an inhibitor of Myb-dependent transcription. Analysis of the inhibitory mechanism of WFA showed that WFA is a significantly more potent inhibitor of C/EBPβ, a transcription factor cooperating with Myb in myeloid cells, than of Myb itself. We show that WFA covalently modifies specific cysteine residues of C/EBPβ, resulting in the disruption of the interaction of C/EBPβ with the co-activator p300. Our work identifies C/EBPβ as a novel direct target of WFA and highlights the role of p300 as a crucial co-activator of C/EBPβ. The finding that WFA is a potent inhibitor of C/EBPβ suggests that inhibition of C/EBPβ might contribute to the biological activities of WFA.
... Reporter studies were performed in at least three independent experiments, with replicate transfections in each experiment. Expression vectors for v-Myb (pCDE26v-Myb) and c-Myb (pCDNA3-chc-Myb) have been described previously (29). A mutant lacking all cysteine residues (pCDE26v-Myb/CallA) was generated by oligonucleotide-directed mutagenesis, converting all cysteine codons to alanine codons. ...
The transcription factor c-Myb is essential for the proliferation of hematopoietic cells and has been implicated in the development of leukemia and other human cancers. Pharmacological inhibition of Myb is therefore emerging as a potential therapeutic strategy for these diseases. By using a Myb reporter cell line we have identified plumbagin and several naphthoquinones as potent low-molecular weight Myb inhibitors. We demonstrate that these compounds inhibit c-Myb by binding to the c-Myb transactivation domain and disrupting the cooperation of c-Myb with the co-activator p300, a major driver of Myb activity. Naphthoquinone-induced inhibition of c-Myb suppresses Myb target gene expression and induces the differentiation of the myeloid leukemia cell line HL60. We demonstrate that murine and human primary acute myeloid leukemia cells are more sensitive to naphtoquinone-induced inhibition of clonogenic proliferation than normal hematopoietic progenitor cells. Overall, our work demonstrates for the first time the potential of naphthoquinones as small-molecule Myb inhibitors that may have therapeutic potential for the treatment of leukemia and other tumors driven by deregulated Myb.
... Transcriptional activation of target genes by Myb has been studied in detail [8][9][10][11][12][13][14][15][16]. Myb activity is highly dependent on the interaction with the coactivator p300. ...
... However, it should be kept in mind that v-Myb in addition to the changes in the hydrophobic region has also suffered amino acid replacements in the DNA-binding domain which contribute to its oncogenicity [1,3,12]. These changes are known to alter the DNAbinding properties of the protein [13] and will affect the spectrum of target genes regulated by Myb. Thus, overall amino acids replacements in v-Myb most likely affect cellular gene expression in a complex manner, both regarding to the spectrum of target genes and the quantitative changes in their expression. ...
... Using TFSEARCH (http://www.cbrc.jp/research/db/TFSEARCH.html), we found that the hypomethylated site at -148 in the Bdnf IV promoter was a potential binding site for c-MYB, a nuclear DNA-binding protein that recognizes a consensus sequence motif and regulates the activity of promoters that contain this motif 18 . A chromatin immunoprecipitation (ChIP) assay using an anti-MYB antibody revealed that the binding of c-MYB was significantly increased in cocaine conditioned group compared to saline conditioned group (t = 3.11, p < 0.05) (Fig. 2C). Figure 2D showed that transcriptional activity of the unmethylated Bdnf IV promoter as measured in a transient transfection luciferase reporter assay was increased by 3-fold in the presence of ectopically expressed c-Myb expression vector, whereas this did not occur with the methylated vector (p < 0.001). ...
... Our study identified a new transcription activator for Bdnf: c-MYB. Previous studies have shown that c-MYB mostly operates as a transcriptional activator by binding to the sequence t/cAACt/gG, which is known as the MYB binding site 18 . It also cooperates with members of the CCAAT-box enhancer-binding protein family to active mim-1 gene 25 . ...
Abnormal BDNF signaling contributes to the structural and behavioral plasticity induced by drugs of abuse. However, the mechanisms regulating expression of Bdnf in drug addiction remain elusive. In the present study, using the conditioned place preference (CPP) model, we showed that expression of Bdnf IV is upregulated in the nucleus accumbens (NAc) of conditioned animals while Bdnf I is upregulated in cocaine-treated mice irrespective of conditioning. The methylation level of a putative c-MYB binding site in the promoter region of Bdnf IV was significantly decreased in the NAc under cocaine CPP conditioning but remained unchanged without conditioning, concurrently with increased binding of c-MYB to this site. Exon IV promoter/luciferase reporter assays revealed that transactivation of Bdnf by c-MYB was blocked by methylation of this c-MYB binding site. Administration of methionine, a precursor of SAM, inhibited cocaine CPP, reversed demethylation of c-MYB binding site and induction of Bdnf IV expression by cocaine CPP. Our results imply that Bdnf IV demethylation at c-MYB binding site is involved in cocaine-triggered seeking behavior, whereas Bdnf I responds to the immediate pharmacological effects of cocaine.
... Expression vectors for v-Myb (pCDE26v-myb) and human c-Myb (pCIneo-HcM-HA) have been described. 23,26 v-Myb C130A is a v-Myb mutant with a substitution of cysteine 130 by alanine. The expression of endogenous mim-1 mRNA in HD11 cells transiently transfected with wild-type or C130A mutant v-Myb expression vectors was analyzed by northern blotting as described before. ...
... To investigate whether parthenolide affects the activation of Myb target genes by Myb, we used the chicken macrophage cell line HD11-E, which was engineered to express a viral version of the Myb protein (v-Myb) in a doxycyclin-inducible manner. 23 These cells show a very strong increase of the expression of several endogenous Myb target genes when Myb expression is induced by doxycyclin, 23 and therefore are a suitable test system to assess the effect of parthenolide on the activity of Myb in a physiological context. The expression of the mim-1 is an extremely sensitive measure of the Myb activity in these cells, because the gene is completely silent in the absence of Myb and reaches very high expression levels in its presence. ...
... To investigate whether parthenolide affects the activation of Myb target genes by Myb, we used the chicken macrophage cell line HD11-E, which was engineered to express a viral version of the Myb protein (v-Myb) in a doxycyclin-inducible manner. 23 These cells show a very strong increase of the expression of several endogenous Myb target genes when Myb expression is induced by doxycyclin, 23 and therefore are a suitable test system to assess the effect of parthenolide on the activity of Myb in a physiological context. The expression of the mim-1 is an extremely sensitive measure of the Myb activity in these cells, because the gene is completely silent in the absence of Myb and reaches very high expression levels in its presence. ...
The c-myb proto-oncogene encodes a transcription factor that is highly expressed in the progenitor cells of the hematopoietic system, where it regulates the expression of genes important for lineage determination, cell proliferation and differentiation. There is strong evidence that deregulation of c-myb expression is involved in the development of human tumors, particularly of certain types of leukemia, and breast and colon cancer. The c-Myb protein is therefore an interesting therapeutic target. Here, we have investigated the potential of natural sesquiterpene lactones (STLs), a class of compounds that are active constituents of a variety of medicinal plants, to suppress Myb-dependent gene expression. We have developed a test system that allows screening of compounds for their ability to interfere with the activation of Myb target genes. Using this assay system, we have identified the STL mexicanin-I as the first cell-permeable, low-molecular-weight inhibitor of Myb-induced gene expression.
... Different c-Myb-mediated synergy on natural chromatin-embedded promoters. (A) Schematic presentation of the regulatory elements of the mim-1 and lysozyme genes according to (35–37). Both genes contain functional MREs in enhancer elements as well as in promoters. ...
... The mim-1 gene is activated by c-Myb through interaction with two distinct regions, a promoter and an enhancer region, both of which contain several MREs (35,36). The lysozyme gene has a similar organization but with fewer active MREs involved (36,37). Hence, we addressed the physiological relevance of the SUMO-mediated SC of c-Myb by comparing the activation of these two endogenous target genes by wild-type c-Myb and c-Myb 2KR, both expressed to similar levels (Figure 3B). ...
Synergy between transcription factors operating together on complex promoters is a key aspect of gene activation. The ability
of specific factors to synergize is restricted by sumoylation (synergy control, SC). Focusing on the haematopoietic transcription
factor c-Myb, we found evidence for a strong SC linked to SUMO-conjugation in its negative regulatory domain (NRD), while
AMV v-Myb has escaped this control. Mechanistic studies revealed a SUMO-dependent switch in the function of NRD. When NRD
is sumoylated, the activity of c-Myb is reduced. When sumoylation is abolished, NRD switches into being activating, providing
the factor with a second activation function (AF). Thus, c-Myb harbours two AFs, one that is constitutively active and one
in the NRD being SUMO-regulated (SRAF). This double AF augments c-Myb synergy at compound natural promoters. A similar SUMO-dependent
switch was observed in the regulatory domains of Sp3 and p53. We show that the change in synergy behaviour correlates with
a SUMO-dependent differential recruitment of p300 and a corresponding local change in histone H3 and H4 acetylation. We therefore
propose a general model for SUMO-mediated SC, where SUMO controls synergy by determining the number and strength of AFs associated
with a promoter leading to differential chromatin signatures.
MYB is a transcription factor which was identified in birds as a viral oncogene (v-MYB). Its cellular counterpart was subsequently isolated as c-MYB which has three functional domains - DNA binding domain, transactivation domain and negative regulatory domain. c-MYB is essential for survival, and deletion of both alleles of the gene results in embryonic death. It is highly expressed in hematopoietic cells, thymus and neural tissue, and required for T and B lymphocyte development and erythroid maturation. Additionally, aberrant MYB expression has been found in numerous solid cancer cells and human leukemia. Recent studies have also implicated c-MYB in the regulation of expression of fetal hemoglobin which is highly beneficial to the β-hemoglobinopathies (beta thalassemia and sickle cell disease). These findings suggest that MYB could be a potential therapeutic target in leukemia, and possibly also a target for therapeutic increase of fetal hemoglobin in the β-hemoglobinopathies.
Key Points
Inhibition of Myb activity by a small molecule blocks proliferation of AML cells and prolongs survival of mice in an in vivo AML model.
The c-myb protooncogene as well as its transforming derivate, the v-myb oncogene code for transcription factors. They regulate transcription of specific target genes thus controlling proliferation, differentiation and apoptosis of hematopoietic cells. Up-regulation of the c-myb expression or rearrangement/amplification of the myb locus are often involved in leukemogenesis. Enforced myb expression blocks differentiation of various leukemic cell lines. Human promonocytes U937 can be induced to differentiate to monocyte/macrophage-like cells using phorbol esters or to granulocytes using retinoic acid. In order to investigate transforming capability of v-myb, we expressed the v-myb oncogene of avian myeloblastosis virus in U937 cells. We found that v-Myb efficiently suppressed formation of macrophages upon treatment with phorbol ester. Some features of granulocytic differentiation of retinoic acid-treated U937 cells were affected by the v-Myb protein as well. These results document that v-Myb is significantly involved in control of myeloid differentiation.
The oncogene v-myb of avian myeloblastosis virus (AMV) encodes a transcription factor (v-Myb) that transforms myelomonocytic cells by deregulating
the expression of specific target genes. v-myb has acquired its oncogenic potential by truncation as well as by a number of point mutations of its cellular progenitor c-myb. As a result of these changes, the target gene spectrum v-Myb differs from that of c-Myb. We recently showed that the chicken
mim-1 gene, a c-Myb target gene that is not activated by v-Myb harbors a powerful cell type-specific and Myb-inducible enhancer
in addition to a Myb-responsive promoter. We now show that Myb-dependent activation of the mim-1 gene is accompanied by extensive remodeling of the nucleosomal architecture at the enhancer. We found that the mim-1 enhancer region also harbors a promoter whose activity is stimulated by Myb and which directs the transcription of an apparently
non-coding RNA. Furthermore, our data show that the oncogenic mutations of AMV have disrupted the ability of v-Myb to induce
remodeling of chromatin structure at the mim-1 enhancer. Together, our results demonstrate for the first time directly that Myb induces alterations of the nucleosomal organization
at a relevant target site and provide new insight into the functional consequences of the oncogenic amino acid substitutions.
