Myb Target Research Articles

Cloning and characterization of cellular senescence-associated genes in human fibroblasts by suppression subtractive hybridization

Cellular senescence marks the end of the proliferative life span of normal cells in tissue culture and occurs after cells have undergone a certain number of population doublings (PDLs). It is accompanied by alterations in the pattern of gene expression. A specific human embryonic lung diploid fibroblast cell line, 2BS, has been studied as a model of senescence in our laboratory. Here, we report a set of cellular senescence-associated genes identified from suppression subtractive cDNA libraries from senescent and young 2BS cells. They include three novel genes and six previously identified genes of unknown function. The genes whose functions are known belong to various functional pathways that have been reported to change with the onset of senescence. These include three pre-mRNA splicing factors with reduced expression in senescent cells, indicating that the regulation of mRNA splicing is altered during cell senescence. In addition, the expression of the gene TOM1 (target of Myb 1), which has not previously been associated with cellular senescence, is shown to increase in senescent cells, and we demonstrate that the expression of antisense TOM1 gene in 2BS cells can delay the progress of senescence.

Tom1 (target of Myb 1) is a novel negative regulator of interleukin-1- and tumor necrosis factor-induced signaling pathways.

The Tom1 (target of Myb 1) protein has VHS and GAT domains in N-terminal and central parts, respectively. The VHS domain has been found in a number of proteins, some of which have been implicated in intracellular trafficking and sorting. We previously showed that Tom1 forms a complex with Tollip, which has been reported to function in interleukin-1 (IL-1)-dependent signaling. In this study, we carried out a reporter gene assay to investigate the function of Tom1 in inflammatory cytokine-dependent signaling. It was found that overexpression of Tom1 suppresses activation of transcription factors, NF-kappaB and AP-1, induced by either IL-1beta or tumor necrosis factor (TNF)-alpha and that the VHS domain of Tom1 is indispensable for its suppressive activity. Thus, we propose that Tom1 is a common negative regulator of the signaling pathways induced by IL-1beta and TNF-alpha.

The transformation suppressor protein Pdcd4 shuttles between nucleus and cytoplasm and binds RNA.

The Pdcd4 gene has originally been isolated in a search for genes that are activated in cells undergoing apoptosis. Independent of these studies, the Pdcd4 gene has been implicated in the suppression of tumor-promoter-mediated transformation of keratinocytes and as a downstream target of Myb in hematopoietic cells. The Pdcd4 protein has weak homology to the eucaryotic translation initiation factor eIF4G and has been shown to interact with certain translation initiation factors. To explore the molecular function of the Pdcd4 protein, we have studied its subcellular localization. We show that the Pdcd4 protein is a predominantly nuclear protein under normal growth conditions and that it is exported from the nucleus by a leptomycin B-sensitive mechanism upon serum withdrawal. The protein contains two nuclear export signals, one of which is very potent. In addition, we demonstrate that the Pdcd4 protein has RNA-binding activity and that the sequences involved in RNA-binding are located in the amino-terminal part of the protein. Taken together, our data raise the possibility that Pdcd4 is involved in some aspect of nuclear RNA metabolism in addition to its suspected role in protein translation.

Phosphorylation-dependent Down-regulation of c-Myb DNA Binding Is Abrogated by a Point Mutation in the v-mybOncogene

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, none of the point mutations in v-Myb has been directly linked to protein modifications. Here we show that the DNA-binding domain of c-Myb can be phosphorylated on serine 116 by the catalytic subunit of protein kinase A. Phosphorylation of Ser(116) differentially destabilizes a subtype of c-Myb-DNA complexes. The V117D mutation of the AMV v-Myb oncoprotein abolishes phosphorylation of the adjacent Ser(116) residue. Modification of Ser(116) was also detected in live cells in c-Myb, but not in AMV v-Myb. Phosphorylation-mimicking mutants of c-Myb failed to activate the resident mim-1 gene. Our data imply that protein kinase A or a kinase with similar specificity negatively regulates c-Myb function, including collaboration with C/EBP, and that the leukemogenic AMV v-Myb version evades inactivation by a point mutation that abolishes a phosphoacceptor consensus site. This suggests a novel link between Myb, a signal transduction pathway, cooperativity with C/EBP, and a point mutation in the myb oncogene.

Alteration of C-MYB DNA binding to cognate responsive elements in HL-60 variant cells

Aims: To establish whether the MYB protein expressed in HL-60 variant cells, which are cells resistant to 12-O-tetradecanoylphorbol-13-acetate (TPA) induced differentiation, is able to bind MYB recognition elements (MREs) involved...

Identification and characterization of the Myb-inducible promoter of the chicken adenosine receptor 2B gene.

Numerous studies have shown that the retroviral oncogene v-myb encodes a transcription factor (v-Myb) which interferes with the differentiation program of myelomonocytic cells. It is generally thought that v-Myb deregulates the expression of specific target genes and thereby causes transformation of these cells. By using an estrogen-inducible version of v-Myb we have previously identified the gene for the chicken A2B adenosine receptor (A2B-AR), a member of the seven-pass transmembrane receptor superfamily, as a bona fide target gene for v-Myb. The chicken A2B-AR gene is expressed in v-myb transformed myeloblasts as well as in c-myb expressing erythroblasts, offering the opportunity to study how Myb transcription factors activate a target gene in two different hematopoietic lineages. Here, we report the characterization of the promoter of the A2B-AR gene. We show that the A2B-AR promoter region contains an exceptionally large number of Myb binding sites, many of which contribute to the Myb-inducibility of the promoter. The same sites were required for promoter activity in myelomonocytic and erythroid cells. In contrast to the promoters of other Myb target genes the A2B-AR promoter was not activated synergistically by Myb and other lineage-specific transcription factors that have been identified as Myb cooperation partners before. Taken together, our data suggest that the activation of the A2B-AR promoter by Myb depends on the simultaneous binding of a large number of Myb molecules.

Targeted disruption of c-myb in the chicken pre B-cell line DT40.

The c-myb proto-oncogene is highly expressed in a wide variety of immature hematopoietic cells and plays a key role in the development of the hematopoietic system. c-myb and its retroviral counterpart v-myb encode transcription factors which have been implicated in the regulation of certain target genes. Targeting of c-myb in mouse embryonic stem cells by homologous recombination has provided clear evidence that c-myb is necessary for the proper development of most myeloid lineages of the hematopoietic system as well as of T-lymphocytes. Here we have explored the function of c-myb in the B-lymphoid lineage. We have used the chicken DT40 cells, a pre B-cell line which shows extremely high efficiencies of homologous recombination, as a model system to disrupt c-myb. DT40 cells lacking a functional c-myb gene are viable and show only minor perturbations of their growth parameters, indicating that c-myb is not an essential gene in these cells. We have used the c-myb null DT40 cells to analyse the expression of genes which have been previously been identified as myb target genes. Neither c-myc nor bcl-2, two putative myb targets, showed altered expression in the cells lacking c-myb. However, expression of the Pdcd4 gene, a myb target gene originally identified in a myelomonocytic cell line expressing a conditional form of v-myb, was diminished in the absence of c-myb. The c-myb knock-out cells described here should provide a useful model system for the identification and characterization of c-myb target genes in B-lymphoid cells.

Identification of the myb-inducible promoter of the chicken Pdcd4 gene

The retroviral oncogene v- myb encodes a transcription factor (v-Myb) which disrupts the myelomonocytic differentiation program and 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. c- myb, the cellular progenitor of v- myb, is expressed in all immature hematopoietic cells and is presumed to regulate the expression of genes that are essential for the development of the hematopoietic system. Recently, we have identified the chicken Pdcd4 gene as a novel v- myb target gene. Pdcd4 has originally been identified in a screen for genes upregulated in apoptotic cells and, more recently, has been implicated in tumor progression. As a myb-regulated gene Pdcd4 is of interest because unlike most other myb target genes it is expressed in a broad spectrum of hematopoietic cells. As a first step to study the regulation of Pdcd4 expression in more detail, we here report the identification and preliminary characterization of the myb-inducible promoter of the Pdcd4 gene.

Myeloblastin is an Myb target gene: mechanisms of regulation in myeloid leukemia cells growth-arrested by retinoic acid

AbstractA pivotal role has been assigned to Myb in the control of myeloid cell growth. Although Myb is a target of retinoic acid, little is known about the mechanisms by which it may contribute to induced growth arrest in leukemia cells. Indeed, few Myb target genes are known to be linked to proliferation. Myeloblastin is involved in the control of proliferation in myeloid leukemia cells. It is expressed early during hematopoiesis and is a granulocyte colony-stimulating factor–responsive gene. Myeloblastin can confer factor-independent growth to hematopoietic cells, an early step in leukemia transformation. The myeloblastin promoter contains PU.1, C/EBP, and Myb binding sites, each of which are critical for constitutive expression in myeloid cells. Inhibition of myeloblastin expression in leukemia cells growth-arrested by retinoic acid is demonstrated to depend on Myb down-regulation. Myb is shown to induce myeloblastin expression and abolish its down-regulation by retinoic acid. Altogether, the data offer a clue as to how a myeloid-specific transcriptional machinery can be accessible to regulation by retinoic acid and point to myeloblastin as a novel target of Myb. This link between Myb and myeloblastin suggests a previously nonidentified Myb pathway through which growth arrest is induced by retinoic acid in myeloid leukemia cells.

Identification and Validation of Candidate Myb Target Genes

ABSTRACTWhile a considerable number of candidate Myb target genes have been reported to date, most of these are likely to play little or no role in transformation by myb oncogenes. Here we have used a conditionally myb-transformed myeloid cell line (ERMYB) to further examine Myb regulation of one candidate target gene—c-myc—that has the potential to affect cell proliferation. It was found that the major influence on c-myc expression was the presence of cytokine (GM-CSF) rather than Myb activity. We also describe the application of PCR-based subtractive hybridization and low-density cDNA array screening, in conjunction with the ERMYB line, to the identification of additional Myb target genes. Preliminary identification of a number of candidates is reported; these include myeloperoxidase, which is known to have essential Myb-binding sites in its regulatory region.

A Novel System to Identify Myb Target Promoters in Friend Murine Erythroleukemia Cells

ABSTRACTFriend murine erythroleukemia (MEL) cells provide an early erythroid precursor model that can be induced to terminally differentiate in cell culture and has been used to study erythroid differentiation as well as multistage tumorigenesis. During the chemically induced differentiation of MEL cells, expression of the c-myb protooncogene is downregulated in a biphasic fashion and forced expression of c-myb is able to block the differentiation process, suggesting that c-myb activity may be limiting for differentiation in MEL cells. We have recently produced stable transfectants in the C19 MEL cell line that carry a dominant interfering myb allele (MEnT) under the control of an inducible mouse metallothionein I (MTH) promoter. Upon inducing expression of MEnT, transfected cells enter a differentiation program and begin to produce α-globin mRNA, assemble hemoglobin, and stop proliferating. Differential display was used to compare mRNA expression between parental C19 MEL cells induced to differentiate with hexamethylene bisacetamide (HMBA) and stable transfectants induced to differentiate via expression of MEnT to identify potential Myb target promoters. We identified six candidate cDNAs in this fashion and present evidence that two of these represent genes that are dependent on c-Myb activity for maximal expression in MEL cells.

TOM1Genes Map to Human Chromosome 22q13.1 and Mouse Chromosome 8C1 and Encode Proteins Similar to the Endosomal Proteins HGS and STAM

The aviantom1(target of myb 1) gene has been previously characterized from v-myb-transformed cells. We report here cloning of the human and mousetom1orthologs. Both genes are expressed ubiquitously, with the highest levels in skeletal muscle, brain, and intestines, as assessed by Northern blot and mRNAin situhybridization. The N-terminal domain of the TOM1 protein shares similarity with HGS (hepatocyte growth factor-regulated tyrosine kinase substrate) and STAM (signal-transducing adaptor molecule), which are associated with vesicular trafficking at the endosome. A putative coiled-coil domain was also detected in the central part of the TOM1 protein. This domain structure suggests thatTOM1is another member of a family of genes implicated in the trafficking regulation of growth-factor-receptor complexes that are destined for degradation in the lysosome. We also show that a human paralog ofTOM1(TOM1-like gene 1) exists. Furthermore, we provide a transcription map over a 190-kb contig of theTOM1region. This map includes its distal neighborsHMOX1andMCM5and two proximal novel genes, one of which is a HMG-box-containing gene (HMG2L1), and the other of unknown function. Using a genomic PAC clone, we demonstrate that the mouseTom1andHmox1genes are part of an as yet undescribed syntenic group between mouse chromosome 8C1 and human chromosome 22q13.1.

Physical interaction between retinoic acid receptor and the oncoprotein myb inhibits retinoic acid-dependent transactivation.

The c-myb protooncogene is predominantly expressed in hematopoietic cells and plays a vital role in hematopoiesis. Retinoic acid (RA) is able to induce differentiation of several hematopoietic cells. This differentiation is linked to decreased c-myb expression, suggesting that retinoid receptors (RAR/RXR) may down-regulate c-myb gene expression. Furthermore, recent data indicate that RAR inhibits the function of the Myb protein itself. In addition, the Myb-Ets oncogenic fusion protein has been shown to inhibit transcriptional activation by RAR and thyroid hormone receptor. Myb-Ets also antagonizes the biological response of erythrocytic progenitor cells to RA and thyroid hormone. This prompted us to investigate a possible cross talk between RAR and Myb. Here, we demonstrate that RA inhibits the expression of the endogenous Myb target gene tom-1. Conversely, Myb functions as a potent inhibitor of RA-induced biological responses. Functional analysis of Myb mutants in transfection studies revealed that the Myb DNA-binding domain (DBD) is necessary for repression whereas the transactivation domain is dispensable. Furthermore, we show that v-Myb and RAR interact in vitro and in vivo. This interaction requires the DBD of RAR. In contrast, glutathione S-transferase-pulldown assays with v-Myb mutants indicate that the DBD and the C terminus of Myb directly interact with RAR. Our results suggest that the physical interaction between Myb and RAR may play a role in the regulation of hematopoietic gene expression.

Molecular cloning of human and bovine LECT2 having a neutrophil chemotactic activity and its specific expression in the liver

We previously reported the purification and amino acid sequence of a novel neutrophil chemotactic protein termed LECT2 (leukocyte cell-derived chemotaxin 2). In this paper we report molecular cloning of human and bovine LECT2 cDNAs based on the amino acid sequence of the purified protein. The deduced amino acid sequence of human LECT2 (hLECT2) shows an 86% identity to bovine LECT2 (bLECT2). The deduced primary structures of LECT2 were highly homologous to the repeated units of Mim-1 protein (myb induced myeloid protein-1). The mim-1 gene is one of the known myb target genes and is specifically expressed in normal and transformed immature granulocytes in the chicken. Northern blot analysis of normal human tissues demonstrated that the hLECT2 gene is specifically expressed in the adult and fetal livers. In addition, several human hepatoma cell lines also expressed LECT2 mRNA, suggesting that hepatic cells in the liver produce LECT2 protein.

SpMyb functions as an intramodular repressor to regulate spatial expression of CyIIIa in sea urchin embryos.

The CyIIIa actin gene of 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 by affinity chromatography. This protein is immunoprecipitated by antibodies specific to the evolutionarily conserved myb domain, and 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.

Tom-1, a novel v-Myb target gene expressed in AMV- and E26-transformed myelomonocytic cells.

The retroviral oncogene v-myb is a mutated and truncated version of the c-myb proto-oncogene and encodes a transcription factor (v-Myb) that specifically transforms myelomonocytic cells. Two different variants of v-myb, transduced independently by the oncogenic chicken retroviruses AMV and E26, have been characterized. It is believed that both variants of v-Myb transform myelomonocytic cells by affecting the expression of specific genes; however, no target genes common to both oncogenic viruses have been identified. Here, we describe the identification of a novel v-Myb target gene, designated as tom-1 (target of myb 1). The tom-1 gene has two promoters, one of which is Myb-inducible. tom-1 is expressed at elevated levels in AMV-transformed as well as in E26-transformed myeloid cells. We show that tom-1 activation by v-Myb does not require de novo protein synthesis and that the Myb-inducible tom-1 promoter contains a functional Myb binding site. Thus, tom-1 is the first example of a direct target gene for both oncogenic forms of the v-myb gene. Further analysis of the Myb-inducible tom-1 promoter shows that a C/EBP binding site is juxtaposed to the Myb binding site and that C/EBP is required for the Myb-dependent activation of the promoter. Together with previous work our results suggest that C/EBP may be a general cooperation partner for v-Myb in myelomonocytic cells.

A dominant interfering Myb mutant causes apoptosis in T cells.

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

Interaction of C/EBPβ and v-Myb Is Required for Synergistic Activation of the mim-1 Gene

The retroviral oncogene v-myb encodes a transcription factor (v-Myb) which activates the myelomonocyte-specific mim-1 gene, a natural myb target gene, by cooperating with members of the C/EBP transcription factor family. The finding that v-Myb, together with C/EBP, is sufficient to activate the mim-1 gene in heterologous cell types has implicated Myb and C/EBP as a bipartite molecular switch, which regulates the expression of myelomonocyte-specific genes. To understand the relationship between v-Myb and C/EBP in more detail, we have examined the molecular basis of the activation of the mim-1 promoter by v-Myb and C/EBPbeta, a member of the C/EBP transcription factor family highly expressed in myelomonocytic cells. We have identified a composite Myb and C/EBP response element which mediates synergistic activation of the mim-1 promoter by both factors and consists of closely spaced Myb- and C/EBP-binding sites. In vitro and in vivo protein-binding studies indicate that v-Myb and C/EBPbeta interact with each other via their DNA-binding domains. We show that this interaction is essential for the synergistic activation of the mim-1 promoter by v-Myb and C/EBPbeta. Our work therefore identifies C/EBPbeta as an interaction partner of v-Myb involved in myelomonocyte gene expression.

Bacterial expression, characterization and DNA binding studies on Drosophila melanogaster c-Myb DNA-binding protein.

The Drosophila Myb homologue retains an evolutionarily conserved typical sequence of three imperfect tandem tryptophan repeat units (R1-R2-R3) of 51-53 amino acids towards its N-terminus as its presumptive DNA binding domain. Using PCR amplification and the T7 expression vector pET 11d, we have overproduced this tryptophan repeat domain of Drosophila Myb in Escherichia coli and the protein has been purified. Circular dichroic measurements indicate that the protein has a high helical component (58.6%) in its overall structure. The protein is found to recognize the same cognate target sequence TAACGG, as recognized by the vertebrate proteins. The DNA binding properties of the protein have been investigated in detail by fluorescence spectroscopy taking advantage of the large number of tryptophan residues present in the protein. The fluorescence of the native Drosophila R123 was quenched when synthetic duplex DNA oligomers were added to the protein. The oligomers containing specific Myb target sites quenched the protein fluorescence to a greater extent than the non-specific DNA. Binding constants of the protein to the targets were also length dependent for smaller oligomers. Experiments with the collisional quencher acrylamide and cysteine modification reagent indicated that the specific and non-specific target sequences interact with the protein differently. In the former case both the buried and the exposed tryptophan residues were affected by DNA binding whereas in the latter only the solvent-exposed residues were involved.