Myb Target Research Articles

MYBsuppresses differentiation and apoptosis of human breast cancer cells

IntroductionMYB is highly expressed in estrogen receptor positive (ER + ve) breast tumours and tumour cell lines. We recently demonstrated that MYB is essential for the proliferation of ER + ve breast cancer cells, and have now investigated its role in mammary epithelial differentiation.MethodsMCF-7 breast cancer cells were treated with sodium butyrate, vitamin E succinate or 12-O-tetradecanoylphorbol-13-acetate to induce differentiation as measured by Nile Red staining of lipid droplets and β-casein expression. The non-tumorigenic murine mammary epithelial cell (MEC) line, HC11, was induced to differentiate with lactogenic hormones. MYB levels were manipulated by inducible lentiviral shRNA-mediated knockdown and retroviral overexpression.ResultsWe found that MYB expression decreases following chemically-induced differentiation of the human breast cancer cell line MCF-7, and hormonally-induced differentiation of a non-tumorigenic murine mammary epithelial cell (MEC) line, HC11. We also found that shRNA-mediated MYB knockdown initiated differentiation of breast cancer cells, and greatly sensitised them to the differentiative and pro-apoptotic effects of differentiation-inducing agents (DIAs). Sensitisation to the pro-apoptotic effects DIAs is mediated by decreased expression of BCL2, which we show here is a direct MYB target in breast cancer cells. Conversely, enforced expression of MYB resulted in the cells remaining in an undifferentiated state, with concomitant suppression of apoptosis, in the presence of DIAs.ConclusionsTaken together, these data imply that MYB function is critical in regulating the balance between proliferation, differentiation, and apoptosis in MECs. Moreover, our findings suggest MYB may be a viable therapeutic target in breast cancer and suggest specific approaches for exploiting this possibility.

MicroRNA Mir-155 and Myb Proto-Oncogene Family Members Cooperate in Pathogenesis of Chronic Lymphocytic Leukemia.

Abstract 58 Introduction:Role of small non-coding microRNAs (miRNA) in hematopoiesis has been recently established by studies demonstrating increased levels of miR-155 in chronic lymphocytic leukemia (CLL) (Fulci 2007, Marton 2008). PU.1 is an ETS family transcription factor controling myelo-lymphoid differentiation and is directly negatively regulated by miR-155 (Vigorito 2007). Our aim of this study is to determine mechanisms of miR-155 upregulation in CLL pathogenesis and the role of PU.1 downregulation in the process. Methods: miRNA and mRNA levels were determined by qPCR and Affymetrix mRNA expression profiling in peripheral blood mononuclear cells (PBMC) and in purified B cells. Control (N=13) and CLL patients (N=66) were studied. All patients were subgrouped according to cytogenetics (FISH) and Rai status. Protein-DNA localization assays were done using chromatin immunoprecipitation. Results: miR-155 is significantly upregulated in both CLL patient PBMCs and a subset of sorted B-cells whereas PU.1 and its target genes are repressed in all CLL subgroups. Indeed, expression profiling analysis of CLL samples identified a broad repression of ∼80 miR-155 targets (among them key transcriptional regulators FOS, SATB1, MEF2A, MYBL1, SIRT1, MECP2 and CEBPB) and ∼380 repressed PU.1 target genes, among them regulators of hematopoietic homeostasis (FOS, CSF1R, CSF2R, IL4R, IL21R) and apoptosis (BID, BIRC3). Next, we have studied the mechanism of miR-155 gene (also known as BIC) upregulation in CLL. Wehave newly identified a regulatory CpG island (BIC-CpG) upstream of miR-155 BIC gene that contains DNA binding motifs for E-box transcription factors and is not mutated in CLL patients. Two E-box-binding proteins, MYB and MYBL2, are significantly upregulated in CLL patient PBMCs as well as in a subset of sorted B-cells in all CLL subgroups. Furthermore in primary CLL cells, MYB protein presence is significantly enriched at BIC-CpG alongside a marked enrichment with transcriptionally active chromatin mark histone H3K9Acetyl. To further study the role of MYB in transactivation of the BIC-CpG we have prepared reporter constructs and found that MYB indeed activates BIC-CpG and downstream transcription. Apart from miR-155/BIC, expression profiling analysis identified additional ∼50 upregulated MYB targets, among them cancer-related genes such as CA1, MCM4, BCL2, PDCD4, and CXCR4. Functional assays using siRNA inhibition of PU.1 in normal PBMCs result in upregulation of miR-155 and MYB, indicating that silencing of PU.1 and upregulation of MYB and its target miR-155 may represent an important mechanism of CLL pathogenesis. Conclusion: Our data propose a mechanistic relationship between PU.1 and its negative regulator miR-155 in CLL. Our data also demonstrate that miR-155 is transcriptionally activated by MYB family of E-box binding proteins. Manipulation of these mechanistic relationships may harbor a potential for molecular therapies against CLL. (Grants NR9021-4, 10310-3, 2B06077) Disclosures:No relevant conflicts of interest to declare.

Mutations in Multiple Domains of c-Myb Disrupt Interaction with CBP/p300 and Abrogate Myeloid Transforming Ability

The c-myb proto-oncogene is a key regulator of hematopoietic cell proliferation and differentiation. MYB mRNA is expressed at high levels in, and is required for the proliferation of, most human myeloid and acute lymphoid leukemias. Recently, chromosomal translocation and genomic duplications of c-MYB have been identified in human T-cell acute leukemia. The present work focuses on the effects of mutations in different domains of the murine c-Myb protein on its transforming ability as defined by suppression of myelomonocytic differentiation and continued proliferation. Using both a novel myeloid cell line-based assay and a primary hematopoietic cell assay, we have shown that mutation of single residues in the transactivation domain important for CBP/p300 binding leads to complete loss of transforming ability. We also simultaneously mutated residues in the DNA-binding domain and the negative regulatory domain of the protein. These double mutants, but not the corresponding single mutants, show a complete loss of transforming activity. Surprisingly, these double mutants show severely impaired transactivation and are also defective for CBP/p300 binding. Our results imply that multiple Myb domains influence its interaction with CBP/p300, highlight the importance of this interaction for myeloid transformation, and suggest an approach for molecular targeting of Myb in leukemia.

Identification of Novel MYB Target Genes

The MYB oncogene encodes a transcription factor, Myb, which is essential for normal haemopoiesis and also for the proliferation of most acute leukaemias (reviewed in ref. 1). While a number of Myb target genes have been reported previously, these do not completely account for key elements of MYB's activity, including its pro-leukaemic and differentiation-suppressing functions. We hypothesised that this reflects the fact that previous screens may have not been sufficiently comprehensive and/or employed the most appropriate cell systems. Thus we have embarked upon a program to identify and validate Myb targets critical for these functions. Here we report results from extensive expression profiling studies using a conditionally myb-transformed myeloid cell line, ERMYB (2). We have used ~44,000-element Illumina Beadchips in conjunction with a kinetic profiling strategy that selects candidates based on rapid, statistically-significant and consistent responses to both activation and inactivation of Myb. This has resulted in the identification of a substantial number of candidate Myb-activated and -repressed genes (381 and 502, respectively). In addition, we have used this cell system to identify candidate Myb-regulated microRNAs.Inspection of the list of candidate Myb-activated genes revealed several previously-described Myb targets including myc, bcl2, gstm1 and mpo, providing additional confidence in our approach. Our focus to date has been on novel candidates that may mediate myb's ability to enhance proliferation, suppress differentiation and possibly suppress apoptosis. Q-RT-PCR was used as an initial validation step for a number of such targets; to date 13/14 genes identified by array screening have been confirmed by this method in ERMYB cells. A second approach to validation is to confirm correlation with Myb over-expression in a second cell system (FDB-1) (3). As in primary cells, enforced Myb expression can suppress differentiation and promote proliferation of these cells in the presence of GM-CSF (4).Amongst the Myb-activated genes are gfi1 and nucleostemin/gnl3, which are involved in stem-cell functions, cellular proliferation and in the case of gfi1, lineage-specific functions. Strikingly, candidate Myb-repressed genes include several important positive regulators of haemopoietic differentiation and/or negative regulators of proliferation, namely gata3, sfpi1/pu.1, cebpb, junb, klf's-3,-6 -13 and btg1. Most of these genes have evolutionarily conserved internal or proximal candidate Myb binding sites. Our progress in validating these by chromatin immunoprecipitation will be presented.Finally, we have identified a number of microRNAs that are potentially regulated by Myb. These include members of the miR-17–92 cluster and mir-146b, which appear to be activated and repressed by Myb, respectively. These have been validated by Q-PCR for both the mature miR and the precursor pri-miR transcript. Interestingly, the mir-17–92 cluster has been strongly implicated in oncogenesis and cell cycle regulation (5), while miR-146a/b may have tumour suppressor activity.

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.

Recruitment of Tom1L1/Srcasm to Endosomes and the Midbody by Tsg101

Tom1 (target of Myb 1) and its related proteins (Tom1L1/Srcasm and Tom1L2) constitute a protein family, which share an N-terminal VHS (Vps27, Hrs and STAM) domain and a following GAT (GGA and Tom1) domain. Tom1L1 has potential binding sequences for Tsg101, which is one of key regulators of the multivesicular body (MVB) formation. To obtain a clue to the role of Tom1L1 in the MVB formation, we have characterized the Tom1L1-Tsg101 interaction. We have found that not only the PTAP sequence in the GAT domain but also the PSAP sequence in the C-terminal region of Tom1L1 is responsible for its interaction with the UEV domain of Tsg101 and competes with the HIV-1 Gag protein for the Tsg101 interaction. Furthermore, we show that, by means of Tsg101, Tom1L1 associates with the midbody during cytokinesis as well as endosomes. Taken into account the topological equivalency among the events of the MVB formation, viral egress from the cell, and cytokinesis, the data obtained here suggest that Tom1L1 is implicated in these three distinct cellular processes.

Oncogenic point mutations in the Myb DNA-binding domain alter the DNA-binding properties of Myb at a physiological target gene

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 parts of the protein. We have analyzed the activation of the chicken lysozyme gene which is strongly activated by c-Myb but not by its oncogenic counterpart v-Myb. We report that Myb acts on two different cis-regulatory elements, the promoter and an enhancer located upstream of the gene. Interestingly, the activation of the enhancer was abolished by the oncogenic amino acid substitutions. We demonstrated that a single Myb-binding site is responsible for the activation of the lysozyme enhancer by Myb and showed that the v-Myb protein of AMV was unable to bind to this site. Our data demonstrate for the first time that oncogenic activation of Myb alters its DNA-binding specificity at a physiological Myb target gene.

A dual activation mechanism for Myb-responsive genes in myelomonocytic cells

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. Here we have used DNaseI hypersensitive site mapping and reporter gene assays to study the activation of three Myb target genes – mim-1, the lysozyme gene and the C/EBPβ gene – all of which are activated by Myb in myelomonocytic cells but not in other hematopoietic lineages. We have found that these genes are activated by Myb via more than one cis-regulatory region. Our data suggest that all three genes are activated by Myb by dual mechanisms involving the promoters as well as enhancers. Using a cell line that expresses an estrogen-inducible v-Myb/estrogen receptor fusion protein we have also determined the effect of Myb on the expression of the C/EBPα gene. Our results show that C/EBPα expression is down-regulated by v-Myb. Thus, v-Myb affects the expression of two C/EBP family members in opposite directions.

Revisiting a selection of target genes for the hematopoietic transcription factor c-Myb using chromatin immunoprecipitation and c-Myb knockdown

The transcription factor c-Myb is an important regulator of hematopoiesis required for proper development of most blood cell lineages in vertebrates. An increasing number of target genes for c-Myb are being published, although with little or no overlap between the lists of genes reported. This raises the question of which criteria a bona fide c-Myb-target gene should satisfy. In the present paper, we have analyzed a set of previously reported target genes using chromatin immunoprecipitation (ChIP) and siRNA-mediated knockdown. Among the seven well-studied c-Myb target genes that we analyzed by ChIP, only ADA, c- MYC and MAT2A seemed to be occupied by c-Myb under our experimental settings in the Myb-positive cell lines Jurkat and HL60. After siRNA-mediated knockdown of c-Myb expression, the expression levels of two out of three ChIP positive Myb target genes, ADA and c- MYC, were strongly affected. These results clearly demonstrate the importance of combining different methods for target gene validation and suggest that a combination of ChIP and c-Myb knockdown may represent a powerful approach to identify a core collection of c-Myb target genes.

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

BackgroundThe 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.ResultsWe 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.ConclusionOur 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.

GGA function is required for maturation of neuroendocrine secretory granules

Secretory granule (SG) maturation has been proposed to involve formation of clathrin-coated vesicles (CCVs) from immature SGs (ISGs). We tested the effect of inhibiting CCV budding by using the clathrin adaptor GGA (Golgi-associated, gamma-ear-containing, ADP-ribosylation factor-binding protein) on SG maturation in neuroendocrine cells. Overexpression of a truncated, GFP-tagged GGA, VHS (Vps27, Hrs, Stam)-GAT (GGA and target of myb (TOM))-GFP led to retention of MPR, VAMP4, and syntaxin 6 in mature SGs (MSGs), suggesting that CCV budding from ISGs is inhibited by the SG-localizing VHS-GAT-GFP. Furthermore, VHS-GAT-GFP-overexpression disrupts prohormone convertase 2 (PC2) autocatalytic cleavage, processing of secretogranin II to its product p18, and the correlation between PC2 and p18 levels. All these effects were not observed if full-length GGA1-GFP was overexpressed. Neither GGA1-GFP nor VHS-GAT-GFP perturbed SG protein budding from the TGN, or homotypic fusion of ISGs. Reducing GGA3 levels by using short interfering (si)RNA also led to VAMP4 retention in SGs, and inhibition of PC2 activity. Our results suggest that inhibition of CCV budding from ISGs downregulates the sorting from the ISGs and perturbs the intragranular activity of PC2.

Recruitment of clathrin onto endosomes by the Tom1–Tollip complex

Tom1 (target of Myb 1) and its related proteins (Tom1L1/Srcasm and Tom1L2) constitute a protein family and share an N-terminal VHS (Vps27p/Hrs/Stam) domain and a following GAT (GGA and Tom1) domain, both of which are also conserved in the GGA family proteins. However, the C-terminal half is not significantly conserved between the Tom1 and GGA families or even between Tom1 and Tom1L1. We have previously shown that the GAT domain of Tom1 interacts with Tollip (Toll-interacting protein), which is associated with endosomes, to which it recruits Tom1. We here extend the previous data and show that the GAT domains of Tom1L1 and Tom1L2 also interact with Tollip, and the C-terminal regions of all the Tom1 family proteins interact with clathrin. Furthermore, when coexpressed with Tollip, all the Tom1 family proteins recruite clathrin onto endosomes. These results indicate that, in conjunction with Tollip, Tom1 family proteins play an important role in recruiting clathrin onto endosomes and suggest that they modulate endosomal functions.

Ubiquitin-binding proteins: similar, but different

Covalent modification of proteins with ubiquitin is a common regulatory mechanism in eukaryotic cells. Typically, ubiquitinated proteins are targeted for degradation by the 26 S proteasome. However, more recently the ubiquitin signal has also been connected with many other cell processes, including endocytosis, vesicle fusion, DNA repair and transcriptional silencing. Hence ubiquitination may be comparable with phosphorylation in its importance as an intracellular switch, controlling various signal-transduction pathways. Similar to the regulation of the extent of phosphorylation by kinases and phosphatases, specific sets of ubiquitinating/deubiquitinating enzymes control the degree of ubiquitination. A large number of ubiquitin-binding proteins act at different steps in the downstream pathways, followed by the ubiquitinated protein. Different families of ubiquitin-binding proteins have been described. UBA (ubiquitin-associated) domain-containing proteins is the largest family and includes members involved in different cell processes. The smaller groups of UIM (ubiquitin-interacting motif), GAT [GGA (Golgi-associated gamma-adaptin homologous) and Tom1 (target of Myb 1)], CUE (coupling of ubiquitin conjugation to endoplasmic reticulum degradation), UEV [ubiquitin E2 (ubiquitin-conjugating enzyme) variant] and NZF (nuclear protein localization gene 4 zinc finger) domain-containing proteins appear to have more specialized functions. Here we discuss functional and structural properties of ubiquitin-binding proteins.

Ubiquitin-binding proteins: similar, but different

Covalent modification of proteins with ubiquitin is a common regulatory mechanism in eukaryotic cells. Typically, ubiquitinated proteins are targeted for degradation by the 26 S proteasome. However, more recently the ubiquitin signal has also been connected with many other cell processes, including endocytosis, vesicle fusion, DNA repair and transcriptional silencing. Hence ubiquitination may be comparable with phosphorylation in its importance as an intracellular switch, controlling various signal-transduction pathways. Similar to the regulation of the extent of phosphorylation by kinases and phosphatases, specific sets of ubiquitinating/deubiquitinating enzymes control the degree of ubiquitination. A large number of ubiquitin-binding proteins act at different steps in the downstream pathways, followed by the ubiquitinated protein. Different families of ubiquitin-binding proteins have been described. UBA (ubiquitin-associated) domain-containing proteins is the largest family and includes members involved in different cell processes. The smaller groups of UIM (ubiquitin-interacting motif), GAT [GGA (Golgi-associated γ-adaptin homologous) and Tom1 (target of Myb 1)], CUE (coupling of ubiquitin conjugation to endoplasmic reticulum degradation), UEV [ubiquitin E2 (ubiquitin-conjugating enzyme) variant] and NZF (nuclear protein localization gene 4 zinc finger) domain-containing proteins appear to have more specialized functions. Here we discuss functional and structural properties of ubiquitin-binding proteins.

Structural mechanism for ubiquitinated-cargo recognition by the Golgi-localized, γ-ear-containing, ADP-ribosylation-factor-binding proteins

The Golgi-localized, gamma-ear-containing, Arf (ADP-ribosylation factor)-binding (GGA) proteins are clathrin adaptors that mediate the sorting of transmembrane-cargo molecules at the trans-Golgi network and endosomes. Cargo proteins can be directed into the GGA pathway by at least two different types of sorting signals: acidic cluster-dileucine motifs and covalent modification by ubiquitin. The latter modification is recognized by the GGAs through binding to their GAT [GGA and TOM (target of Myb)] domain. Here we report the crystal structure of the GAT domain of human GGA3 in a 1:1 complex with ubiquitin at 2.8-A resolution. Ubiquitin binds to a hydrophobic and acidic patch on helices alpha1 and alpha2 of the GAT three-helix bundle that includes Asn-223, Leu-227, Glu-230, Met-231, Asp-244, Glu-246, Leu-247, Glu-250, and Leu-251. The GAT-binding surface on ubiquitin is a hydrophobic patch centered on Ile-44 that is also responsible for binding most other ubiquitin effectors. The ubiquitin-binding site observed in the crystal is distinct from the Rabaptin-5-binding site on helices alpha2 and alpha3 of the GAT domain. Mutational analysis and modeling of the ubiquitin-Rabaptin-5-GAT ternary complex indicates that ubiquitin and Rabaptin-5 can bind to the GAT domain at two different sites without any steric conflict. This ability highlights the GAT domain as a hub for interactions with multiple partners in trafficking.

P53 Suppresses c-Myb-induced trans-Activation and Transformation by Recruiting the Corepressor mSin3A

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

Intra-S-Phase Regulator cdc7 Is a Gene Target of the c-Myb Proto-Oncogene in Human Leukemias.

The c-myb proto-oncogene encodes a transcription factor, Myb, which is essential for normal hematopoiesis. Myb may also play a role in leukemogenesis but possible mechanisms remain ill defined. To gain further insights into this issue, we sought to identify Myb regulated genes in human myeloid leukemic cells utilizing a tamoxifen inducible expression system and a microarray approach. Myb function was conditionally abrogated by tamoxifen following infection of K562 cells with a bicistronic retroviral vector MIGR1 which had c-myb's DNA binding domain (DBD) and the Drosophila engrailed protein transcription repressor domain (MERT) fused to a modified estrogen receptor that binds tamoxifen. MERT was subcloned upstream of an IRES-EGFP cassette in MIGR1 to allow for FACS purification on the basis of GFP expression. To identify Myb regulated genes, purified K562-MERT cells were exposed to 1 μM tamoxifen or ethanol (control) for three days, processed for hybridization to the microarray gene chip and analyzed by software algorithms from Incyte and Arrayex. When endogenous Myb activity was suppressed by MERT, 105 genes out of 10,000 genes on the microarray chip changed > 2-fold in expression. Of these 105 genes, 34 increased their expression >2-fold while 70 decreased their expression >2-fold. Since Myb expression is elevated in leukemic cells, we hypothesized that Myb functions in malignant hematopoietic cells to induce the expression of genes that are essential for their maintenance and survival. Therefore, we focused on those genes that decreased in expression when Myb activity was inhibited by MERT. Among the most repressed was cdc7 (2.8-fold decrease), an intra-S-phase regulator. To verify the microarray data, we utilized real-time PCR to quantitate the expression of cdc7 in our K562-MERT cells. Cdc7 expression decreased 5-fold in tamoxifen treated K562-MERT cells relative to control cells, which is consistent with the microarray data. We then performed chromatin immunoprecipitation (ChIP) experiments to determine whether cdc7 is a direct target of Myb. When the chromatin from untreated K562 and K562-MERT cells was immunoprecipitated with anti-c-Myb, we observed one PCR product using a primer pair that flanked the Myb binding sites in the promoter region of cdc7. This same result was observed in our positive control ChIP experiment in which the chromatin was immunoprecipitated with anti-acetyl histone H4, indicating that the region of the cdc7 promoter containing the Myb binding sites is poised for transcription. When Myb transactivation activity was inhibited by MERT in K562-MERT cells, no PCR product was observed following chromatin immunoprepitation with anti-c-Myb. These results strongly suggest that cdc7 is a direct gene target of c-Myb in malignant hematopoietic cells. Investigation of the transcriptional regulation of cdc7 in hematopoietic cells may yield new clues to Myb's role in leukemogenesis.

The glioma-amplified sequence 41 gene (GAS41) is a direct Myb target gene.

The retroviral oncogene v-myb encodes a transcription factor (v-Myb) which transforms myelomonocytic cells in vivo and in vitro. It is thought that v-Myb exerts its biological effects by deregulating the expression of specific target genes, most of which are still unknown. The chicken glioma-amplified sequence 41 gene (GAS41) is located immediately downstream of the lysozyme gene, a known Myb-regulated gene. The GAS41 promoter colocalizes with a CpG island which also functions as an origin of replication. Since the GAS41 promoter contains several potential Myb-binding sites (MBSs) we have investigated whether GAS41 is a v-Myb target gene. Our results show that the GAS41 gene is directly activated by a v-Myb/estrogen receptor fusion protein. Furthermore, our studies reveal that the GAS41 promoter is stimulated by v-Myb in co-transfection experiments and that the DNA-binding activity of v-Myb is crucial for transactivation of the promoter. Electrophoretic mobility-shift assays (EMSA) indicate that several Myb-binding sites, residing approximately 250 bp upstream of the transcriptional start site, are bound by Myb in vitro. Furthermore, chromatin immunoprecipitation assays demonstrate that v-Myb is bound to the GAS41 promoter in vivo. Taken together these findings identify the GAS41 gene as a novel v-Myb target gene. We have also analysed the GAS41 replication origin in myelomonocytic cells and have failed to observe significant differences in origin activity in cells expressing or not expressing v-Myb.

The glioma amplified sequence 41 gene (GAS41) is a direct Myb target gene

The retroviral oncogene v-myb encodes a transcription factor (v-Myb) which transforms myelomonocytic cells in vivo and in vitro. It is thought that v-Myb exerts its biological effects by deregulating the expression of specific target genes, most of which are still unknown. The chicken glioma-amplified sequence 41 gene (GAS41) is located immediately downstream of the lysozyme gene, a known Myb-regulated gene. The GAS41 promoter colocalizes with a CpG island which also functions as an origin of replication. Since the GAS41 promoter contains several potential Myb-binding sites (MBSs) we have investigated whether GAS41 is a v-Myb target gene. Our results show that the GAS41 gene is directly activated by a v-Myb/estrogen receptor fusion protein. Furthermore, our studies reveal that the GAS41 promoter is stimulated by v-Myb in co-transfection experiments and that the DNA-binding activity of v-Myb is crucial for transactivation of the promoter. Electrophoretic mobility-shift assays (EMSA) indicate that several Myb-binding sites, residing ∼250 bp upstream of the transcriptional start site, are bound by Myb in vitro. Furthermore, chromatin immunoprecipitation assays demonstrate that v-Myb is bound to the GAS41 promoter in vivo. Taken together these findings identify the GAS41 gene as a novel v-Myb target gene. We have also analysed the GAS41 replication origin in myelomonocytic cells and have failed to observe significant differences in origin activity in cells expressing or not expressing v-Myb.

Identification of a Myb-responsive enhancer of the chicken C/EBPbeta gene.

The retroviral oncogene v-myb encodes a transcription factor (v-Myb) that disrupts myelomonocytic differentiation and transforms myelomonocytic cells. It is thought that the biological effects of v-Myb are caused by deregulation of specific target genes. The CCAAT box/enhancer binding protein beta (C/EBPbeta), a member of the basic region-leucine zipper (bzip) class of transcription factors, which itself plays an important role during myelomonocytic differentiation, has previously been shown to be regulated by Myb. Here we have addressed the mechanism by which v-Myb affects C/EBPbeta expression. We have employed the mapping of DNase I hypersensitive sites (DHSs) in chromatin as a tool to detect in vivo target sites of v-Myb. Our data identify a DHS downstream of the C/EBPbeta gene that appears to be specific for v-myb-transformed myeloblasts. We have confirmed by chromatin immunoprecipitation that v-Myb is bound to this region in vivo. Furthermore, we have found that ectopic expression of v-Myb in a myelomonocytic cell line is able to induce a DHS downstream of the C/EBPbeta gene, showing for the first time that v-Myb can affect chromatin structure. Reporter gene experiments demonstrate that the downstream DHS acts as a Myb-dependent enhancing element in transiently as well as in stably transfected myelomonocytic cells. Previous work has shown that v-Myb acts on the C/EBPbeta promoter; it now appears that Myb stimulates C/EBPbeta expression by acting on the promoter as well as on an enhancer of the C/EBPbeta gene. Interestingly, the mechanisms by which Myb acts on both elements differ; while Myb activation of the promoter requires the cooperation with C/EBPbeta, activation of the enhancer by Myb is independent of C/EBPbeta. Apart from the identification of a novel Myb-dependent enhancer, our work demonstrates the potential of chromatin structure analysis for the identification of Myb target sites.