Regulation of the Flt3 Gene in Haematopoietic Stem and Early Progenitor Cells.

The MYB transcription factor plays critical roles in normal and malignant haematopoiesis. We previously showed that MYB was a direct activator of FLT3 expression within the context of acute myeloid leukaemia. During normal haematopoiesis, increasing levels of FLT3 expression determine a strict hierarchy within the haematopoietic stem and early progenitor compartment, which associates with lymphoid and myeloid commitment potential. We use the conditional deletion of the Myb gene to investigate the influence of MYB in Flt3 transcriptional regulation within the haematopoietic stem cell (HSC) hierarchy. In accordance with previous report, in vivo deletion of Myb resulted in rapid biased differentiation of HSC with concomitant loss of proliferation capacity. We find that loss of MYB activity also coincided with decreased FLT3 expression. At the chromatin level, the Flt3 promoter is primed in immature HSC, but occupancy of further intronic elements determines expression. Binding to these locations, MYB and C/EBPα need functional cooperation to activate transcription of the locus. This cooperation is cell context dependent and indicates that MYB and C/EBPα activities are inter-dependent in controlling Flt3 expression to influence lineage commitment of multipotential progenitors.

Methylation affects transposition and splicing of a large CACTA transposon from a MYB transcription factor regulating anthocyanin synthase genes in soybean seed coats.

We determined the molecular basis of three soybean lines that vary in seed coat color at the R locus which is thought to encode a MYB transcription factor. RM55-r(m) is homozygous for a mutable allele (r(m)) that specifies black and brown striped seeds; RM30-R* is a stable black revertant isoline derived from the mutable line; and RM38-r has brown seed coats due to a recessive r allele shown to translate a truncated MYB protein. Using long range PCR, 454 sequencing of amplicons, and whole genome re-sequencing, we determined that the variegated RM55-r(m) line had a 13 kb CACTA subfamily transposon insertion (designated TgmR*) at a position 110 bp from the beginning of Intron2 of the R locus, Glyma09g36983. Although the MYB encoded by R was expressed at only very low levels in older seed coats of the black revertant RM30-R* line, it upregulated expression of anthocyanidin synthase genes (ANS2, ANS3) to promote the synthesis of anthocyanins. Surprisingly, the RM30-R* revertant also carried the 13 kb TgmR* insertion in Intron2. Using RNA-Seq, we showed that intron splicing was accurate, albeit at lower levels, despite the presence of the 13 kb TgmR* element. As determined by whole genome methylation sequencing, we demonstrate that the TgmR* sequence was relatively more methylated in RM30-R* than in the mutable RM55-r(m) progenitor line. The stabilized and more methylated RM30-R* revertant line apparently lacks effective binding of a transposae to its subterminal repeats, thus allowing intron splicing to proceed resulting in sufficient MYB protein to stimulate anthocyanin production and thus black seed coats. In this regard, the TgmR* element in soybean resembles McClintock's Spm-suppressible and change-of-state alleles of maize. This comparison explains the opposite effects of the TgmR* element on intron splicing of the MYB gene in which it resides depending on the methylation state of the element.

SpMYB overexpression in tobacco plants leads to altered abiotic and biotic stress responses.

The MYB transcription factors are involved in various plant biochemistry and physiology processes and play a central role in plant defense response. In the present study, a full-length cDNA sequence of a MYB gene, designated as SpMYB, was isolated from tomato. SpMYB encodes the R2R3-type protein consisting of 328 amino acids. The expression level of SpMYB was strongly induced by fungal pathogens. Transgenic tobacco plants overexpressing SpMYB had an enhanced salt and drought stress tolerance compared with wild-type plants, and showed significantly improved resistance to Alternaria alternate. Further analysis revealed that transgenic tobaccos exhibited less accumulation of malondialdehyde (MDA) and more accumulation of superoxide dismutase (SOD), peroxidase (POD) and phenylalanine ammonia-lyase (PAL) after inoculation with A. alternate. Meanwhile, changes in some photosynthetic parameters, such as photosynthetic rate (Pn), transpiration rate (Tr) and intercellular CO2 concentration (Ci) were also found in the transgenic tobaccos. Furthermore, transgenic tobaccos constitutively accumulated higher levels of pathogenesis-related (PR) gene transcripts, such as PR1 and PR2. The results suggested that the tomato SpMYB transcription factor plays an important role in responses to abiotic and biotic stress.

Myb transcription factors and light regulate sporulation in the oomycete Phytophthora infestans.

Life cycle progression in eukaryotic microbes is often influenced by environment. In the oomycete Phytophthora infestans, which causes late blight on potato and tomato, sporangia have been reported to form mostly at night. By growing P. infestans under different light regimes at constant temperature and humidity, we show that light contributes to the natural pattern of sporulation by delaying sporulation until the following dark period. However, illumination does not permanently block sporulation or strongly affect the total number of sporangia that ultimately form. Based on measurements of sporulation-induced genes such as those encoding protein kinase Pks1 and Myb transcription factors Myb2R1 and Myb2R3, it appears that most spore-associated transcripts start to rise four to eight hours before sporangia appear. Their mRNA levels oscillate with the light/dark cycle and increase with the amount of sporangia. An exception to this pattern of expression is Myb2R4, which is induced several hours before the other genes and declines after cultures start to sporulate. Transformants over-expressing Myb2R4 produce twice the number of sporangia and ten-fold higher levels of Myb2R1 mRNA than wild-type, and chromatin immunoprecipitation showed that Myb2R4 binds the Myb2R1 promoter in vivo. Myb2R4 thus appears to be an early regulator of sporulation. We attempted to silence eight Myb genes by DNA-directed RNAi, but succeeded only with Myb2R3, which resulted in suppressed sporulation. Ectopic expression studies of seven Myb genes revealed that over-expression frequently impaired vegetative growth, and in the case of Myb3R6 interfered with sporangia dormancy. We observed that the degree of silencing induced by a hairpin construct was correlated with its copy number, and ectopic expression was often unstable due to epigenetic silencing and transgene excision.

The oncoprotein v-Myb activates transcription of Gremlin 2 during in vitro differentiation of the chicken neural crest to melanoblasts.

The neural crest (NC) is a transient dynamic structure of ectodermal origin, found in early vertebrate embryos. The multipotential NC cells migrate along well defined routes, differentiate to various cell types including melanocytes and participate in the formation of various permanent tissues. As there is only limited information about the molecular mechanisms controlling early events in melanocyte specification and development, we exploited the AMV v-Myb transcriptional regulator, which directs differentiation of in vitro chicken NC cells to the melanocyte lineage. This activity is strictly dependent on v-Myb specifically binding to the Myb recognition DNA element (MRE). The two tamoxifen-inducible v-Myb alleles were constructed one which recognizes the MRE and one which does not. These were activated in ex ovo NC cells, and the expression profiles of resulting cells were analyzed using Affymetrix microarrays and RT-PCR. These approaches revealed up-regulation of the BMP antagonist Gremlin 2 mRNA, and down-regulation of mRNAs encoding several epithelial genes including KRT19 as very early events following the activation of melanocyte differentiation by v-Myb. The enforced v-Myb expression in neural tubes of chicken embryos resulted in detectable presence of Gremlin 2 mRNA. However, expression of Gremlin 2 in NC cells did not promote formation of melanocytes suggesting that Gremlin 2 is not the master regulator of melanocytic differentiation.

MicroRNA-195 inhibits non-small cell lung cancer cell proliferation, migration and invasion by targeting MYB.

MicroRNA-195 (miR-195) has been implicated in several other cancers; however, its role in non-small cell lung cancer (NSCLC) remains unclear. In this study, we demonstrated that miR-195 was significantly down-regulated in NSCLC samples and cell lines compared with corresponding normal counterparts. In vitro and in vivo functional assays demonstrated that modulation of miR-195 expression affected NSCLC cell proliferation, migration and invasion. Using miRNA target prediction algorithms and reporter assays, we demonstrated that miR-195 suppressed the expression of MYB both at the mRNA and protein level, and was directly bound to the 3'untranslated region of MYB mRNA. Overexpression of MYB in NSCLC cells using an ectopic expression vector restored the decreased cell proliferation, migration and invasion effects induced by miR-195. Finally, we observed an inverse correlation between MYB and miR-195 in NSCLC. Taken together, our findings indicated that miR-195 functions as tumour suppressor in NSCLC, and the miR-195/MYB axis might represent a potential therapeutic target for NSCLC intervention.

Nuclear factor of activated T-cells 1 increases sensitivity of v-myb transformed monoblasts to all-trans retinoic acid.

Nuclear factors of activated T-cells (NFATs) are important regulators of the cytokine gene expression in activated T-cells. In the last decade, NFATs have been shown to regulate cell cycle, differentiation and apoptosis in cells of various origins revealing their importance for cell homeostasis. In this study, we investigated the effects of NFAT1 on proliferation and differentiation of v-myb-transformed BM2 monoblasts. In contrast to many other leukemic cell lines, BM2 cells do not respond to retinoic acid. However, once overexpressing NFAT1, they became sensitive to all-trans retinoic acid (ATRA). The ATRA-treated BM2NFAT1 cells differentiated along monocyte/macrophage pathway as evidenced by changes in cell morphology, adherence, phagocytic and non-specific esterase activities, reactive oxygen species production, and vimentin expression. Furthermore, overexpressed NFAT1 either alone or in combination with the ATRA-driven signalling pathway deregulated cyclin A and retinoic acid receptor proteins in BM2 cells. Data presented in this study indicate that the NFAT1 and ATRA signalling pathways synergize in control of proliferation and differentiation of BM2 monoblasts.

MYB is essential for mammary tumorigenesis.

MYB oncogene upregulation is associated with estrogen receptor (ER)-positive breast cancer, but disease requirements for MYB function in vivo have not been explored. In this study, we provide evidence of a critical requirement for MYB functions in models of human and murine breast cancer. In human breast cancer, we found that MYB expression was critical for tumor cell growth both in vitro and in vivo in xenograft settings. In transgenic knockout mice, tissue-specific deletion of the murine MYB gene caused a transient defect in mammary gland development that was reflected in delayed ductal branching and defective apical bud formation. In mouse mammary tumor virus (MMTV)-NEU mice where tumors are initiated by activation of HER2, MYB deletion was sufficient to abolish tumor formation. In the more aggressive MMTV-PyMT model system, MYB deletion delayed tumorigenesis significantly. Together, the findings in these transgenic knockout models implied that MYB was critical during an early window in mammary development when it was essential for tumor initiation, even though MYB loss did not exert a lasting impact upon normal mammary function. Two important MYB-target genes that promote cell survival, BCL2 and GRP78/BIP, were each elevated compared with nontransformed mammary epithelial cells, thereby promoting survival as confirmed in colony formation assays in vitro. Taken together, our findings establish a role for MYB at the hub of ER- and HER2-dependent pathways in mammary carcinogenesis.

MYB transcriptionally regulates the miR-155 host gene in chronic lymphocytic leukemia.

Elevated levels of microRNA miR-155 represent a candidate pathogenic factor in chronic B-lymphocytic leukemia (B-CLL). In this study, we present evidence that MYB (v-myb myeloblastosis viral oncogene homolog) is overexpressed in a subset of B-CLL patients. MYB physically associates with the promoter of miR-155 host gene (MIR155HG, also known as BIC, B-cell integration cluster) and stimulates its transcription. This coincides with the hypermethylated histone H3K4 residue and spread hyperacetylation of H3K9 at MIR155HG promoter. Our data provide evidence of oncogenic activities of MYB in B-CLL that include its stimulatory role in MIR155HG transcription.

Myb proteins: angels and demons in normal and transformed cells.

A key regulator of proliferation, differentiation and cell fate, the c-Myb transcription factor regulates the expression of hundreds of genes and is in turn regulated by numerous pathways and protein interactions. However, the most unique feature of c-Myb is that it can be converted into an oncogenic transforming protein through a few mutations that completely change its activity and specificity. The c-Myb protein is a myriad of interactions and activities rolled up in a protein that controls proliferation and differentiation in many different cell types. Here we discuss the background and recent progress that have led to a better understanding of this complex protein, and outline the questions that have yet to be answered.

Estrogen and MYB in breast cancer: potential for new therapies.

MYB is highly expressed in almost all estrogen receptor (ER)-positive breast tumours and is a direct target of estrogen/ER signalling. Our recent studies have shown that MYB is also required for the proliferation of ER-positive breast tumour cell lines, and have shed further light on the mechanism of ER regulation of MYB expression. Here we discuss the rationale for therapeutic targeting of MYB in breast cancer and consider a number of approaches to developing an anti-MYB therapeutic.

C/EBPbeta induces chromatin opening at a cell-type-specific enhancer.

We have used the chicken mim-1 gene as a model to study the mechanisms by which transcription factors gain initial access to their target sites in compacted chromatin. The expression of mim-1 is restricted to the myelomonocytic lineage of the hematopoietic system where it is regulated synergistically by the Myb and CCAAT/enhancer binding protein (C/EBP) factors. Myb and C/EBPbeta cooperate at two distinct cis elements of mim-1, the promoter and a cell-type-specific enhancer, both of which are associated with DNase I hypersensitive sites in myelomonocytic cells but not in mim-1-nonexpressing cells. Previous work has shown that ectopic expression of Myb and C/EBPbeta activates the endogenous mim-1 gene in a nonhematopoietic cell type (fibroblasts), where the gene is normally completely silent. Here, we investigated the molecular details of this finding and show that the activation of mim-1 occurs by two independent mechanisms. In the absence of Myb, C/EBPbeta triggers the initial steps of chromatin opening at the mim-1 enhancer without inducing transcription of the gene. mim-1 transcription occurs only in the presence of Myb and is associated with chromatin opening at the promoter. Our work identifies a novel function for C/EBPbeta in the initial steps of a localized chromatin opening at a specific, physiologically relevant target region.

The promoter regions of the Myb-regulated Adora2B and Mcm4 genes co-localize with origins of DNA replication.

BACKGROUND: 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 promoter co-localizes with an origin of DNA replication, is a bona fide Myb target gene. Because of this finding we have asked whether other Myb-regulated genes are also associated with DNA replication origins. RESULTS: We show that the promoter region of the chicken adenosine receptor 2B gene (Adora2B), a known Myb-target gene, acts as a DNA replication origin. Furthermore, we have examined known replication origins for the presence of Myb binding sites. We found that the intergenic region between the genes for the minichromosome maintenance 4 protein (Mcm4) and the catalytic subunit of DNA-dependent protein kinase (Prkdc), whose human counterpart has been identified as a replication origin, contains a number of Myb binding sites. Our data show that this region also acts as an origin of replication in chicken cells. Interestingly, we found that the chicken Mcm4 gene is also Myb-regulated. CONCLUSION: Our work identifies the chicken Mcm4 gene as a novel Myb target gene and presents evidence for the co-localization of two novel origins of DNA replication with Myb-regulated genes. Our work raises the possibility that a fraction of Myb target gene promoters is associated with DNA replication origins.

A proteomic analysis of protein variations during differentiation of v-myb-transformed monoblasts.

v-myb oncogene of avian myeloblastosis virus (AMV) transforms myelomonocytic cells in vitro and induces acute monoblastic leukemia in vivo. The transforming effect of the v-myb can be suppressed using phorbol ester (TPA) or histone deacetylase inhibitor trichostatin A (TSA), the inducers of cell differentiation that are in clinical trials. In this study, we used proteomics-based approach to identify proteins with variable expression in differentiated BM2 cells. Proteome variations induced by TPA and TSA were compared to examine the mechanism of differentiation-promoting effects of these drugs. We found that expression of several proteins participating in cell cytoskeleton rearrangement, heat shock response, proteosynthesis and cell signaling was altered in TPA- or TSA-treated cells. We present here the first comparative proteome analysis of v-myb-transformed monoblasts BM2 focused on identification of proteins involved in their terminal differentiation.

CBP sensitizes v-myb-transformed monoblasts to differentiation inducers.

CREB-binding protein (CBP) regulates gene expression by binding to certain components of basal transcription machinery and by histone acetylation. In addition, it integrates various cellular signaling pathways through binding to multiple transcription factors, including the Myb proteins. We report in this study that CBP can partially suppress function of the v-Myb oncoprotein in leukemic cells. Although originally described as an activator of v-Myb function, we show that CBP can also act as a v-Myb suppressor. Ectopic expression of murine CBP in v-Myb-transformed chicken monoblasts reduced transcriptional activation abilities of the v-Myb protein and increased sensitivity to differentiation inducers such as phorbol ester or trichostatin A. In addition, exogenous CBP affected morphology of differentiated cells derived from BM2 monoblasts. These results indicate that cellular context is an important factor determining whether CBP will activate or suppress the protein it targets.

v-Myb mediates cooperation of a cell-specific enhancer with the mim-1 promoter.

The oncogenic transcription factor v-Myb disrupts myelomonocytic differentiation and transforms myelomonocytic cells by deregulating the expression of specific target genes. One of these genes, the chicken mim-1 gene, is activated by Myb exclusively in myelomonocytic cells and, therefore, has been an interesting model system to study how Myb activates a target in a lineage-specific manner. Previous work has suggested that Myb activates mim-1 by cooperating with CCAAT box/enhancer binding protein beta (C/EBPbeta) or other C/EBP transcription factors at the mim-1 promoter. We have now identified and characterized a powerful Myb-dependent enhancer located 2 kb upstream of the mim-1 promoter. The enhancer is preferentially active in myelomonocytic cells, confers Myb responsiveness onto a heterologous promoter, and dramatically increases Myb responsiveness of the mim-1 promoter. Chromatin immunoprecipitation demonstrates that v-Myb and C/EBPbeta are bound to the enhancer in v-Myb-transformed cells; furthermore, cooperation of the enhancer with the mim-1 promoter is greatly stimulated by C/EBPbeta and p300. Taken together, our results show that the regulation of mim-1 expression by v-Myb is more complex than previously assumed and involves two distinct regions of the mim-1 gene. A major function of v-Myb (in addition to its role at the mim-1 promoter) apparently is to activate the mim-1 enhancer and, together with C/EBPbeta and p300, facilitate its cooperation with the promoter. Interestingly, our work also shows that the v-Myb protein encoded by avian myeloblastosis virus is defective in this function, suggesting an explanation for why primary avian myeloblastosis virus-transformed myeloblasts do not express the mim-1 gene.

Silent point mutation in an avian retrovirus RNA processing element promotes c-myb-associated short-latency lymphomas.

The avian leukosis virus DeltaLR-9 causes a high frequency of B-cell lymphomas within weeks after injection into 10-day-old chicken embryos. These lymphomas result from proviral integrations into the oncogene c-myb. In contrast, LR-9, which lacks the 42-nucleotide gag gene deletion of DeltaLR-9, does not cause a high frequency of c-myb-associated short-latency lymphomas. Although viral replication rates and spliced env mRNA levels were found to be similar for both viruses, DeltaLR-9 exhibited an increase in readthrough transcription compared to LR-9. The DeltaLR-9 deletion is located in the region of the gag gene corresponding to the matrix (MA) protein as well as in the negative regulator of splicing (NRS) element. To test whether disruption of the NRS or of the MA protein was responsible for inducing short-latency lymphomas, we generated viruses with NRS point mutations that maintained the wild-type Gag amino acid sequence. One of the mutant viruses induced an even higher incidence than DeltaLR-9 of short-latency lymphomas with viral integrations into c-myb. Thus, we propose that disruption of the NRS sequence promotes readthrough transcription and splicing to the downstream myb gene, causing overexpression of a slightly truncated Myb protein, which induces short-latency tumors.

The leucine zipper region of Myb oncoprotein regulates the commitment of hematopoietic progenitors.

The development of blood cells proceeds from pluripotent stem cells through multipotent progenitors into mature elements belonging to at least 8 different lineages. The lineage choice process during which stem cells and progenitors commit to a particular lineage is regulated by a coordinated action of extracellular signals and transcription factors. Molecular mechanisms controlling commitment are largely unknown. Here, the transcription factor v-Myb and its leucine zipper region (LZR) are identified as regulators of the commitment of a common myeloid progenitor and progenitors restricted to the myeloid lineage. It is demonstrated that wild-type v-Myb with the intact LZR directs development of progenitors into the macrophage lineage. Mutations in this region compromise commitment toward myeloid cells and cause v-Myb to also support the development of erythroid cells, thrombocytes, and granulocytes, similar to the c-Myb protein. In agreement with that, the wild-type v-Myb induces high expression of myeloid factors C/EBP beta, PU.1, and Egr-1 in its target cells, whereas SCL, GATA-1, and c-Myb are more abundant in cells expressing the v-Myb LZR mutant. It is proposed that Myb LZR can function as a molecular switch, affecting expression of lineage-specifying transcription factors and directing the development of hematopoietic progenitors into either myeloid or erythroid lineages.

Cytotoxic effects of colicins E1 and E3 on v-myb-transformed chicken monoblasts.

Colicins show a considerable cytostatic activity, which is much less known and understood than their killing activity targeting bacteria of the Enterobacteriaceae family. In this communication, the cytotoxic effects of colicins E1 and E3 on v-myb-transformed chicken monoblasts BM2 are presented. We detected clear reduction of the viable cell number induced by colicins E1 and E3, occurring without apparent changes in cell cycle profiles. The level of inhibition was proportional to the colicin concentration within the limits of 0.5-1.25 microg/ml. This result documents that colicins produced by Enterobacteriaceae exert their cytotoxic effects on leukemic cells.

MafB is an inducer of monocytic differentiation.

The bZip transcription factor MafB is expressed specifically in the myeloid lineage of the hematopoietic system and is up-regulated successively during myeloid differentiation from multipotent progenitors to macrophages. Here we report that this induction reflects an essential role of MafB in early myeloid and monocytic differentiation. We observed that the expression of MafB in transformed chicken hematopoietic precursors dramatically increases the proportion of myeloid colony formation at the expense of multipotent progenitor-type colonies. In addition, the overexpression of MafB in transformed myeloblasts stimulates the rapid formation of macrophages, as judged by morphology, surface marker expression and functional criteria. MafB-induced macrophages exhibit typical levels of phagocytic activity and nitric oxide release after activation by lipopolysaccharide. By contrast, overexpression of the myeloid transcription factor PU.1 in these cells does not induce macrophage differentiation. Furthermore, a dominant-negative allele of MafB inhibits both myeloid colony formation and the differentiation of myeloblasts into macrophages. Taken together, our results indicate that MafB induction is a specific and essential determinant of the monocytic program in hematopoietic cells.