Scott A. Ness

The v-myb oncogene product binds to and activates the promyelocyte-specific mim-1 gene

The v-myb oncogene induces myeloid leukemias in chickens, transforms myeloid cells in vitro, and encodes a sequence-specific DNA binding protein. We used differential hybridization to screen for v-myb-regulated genes in cells transformed by a temperature-sensitive mutant of the oncogene and identified a new gene, mim-1, which encodes a specifically expressed, secretable protein contained in the granules of both normal and v-myb-transformed promyelocytes. The promoter of the mim-1 gene contains three closely spaced binding sites for v-myb protein and is strongly activated by v-myb in a cotransfection assay. Synthetic copies of the binding sites are both necessary and sufficient to confer v-myb protein-dependent activation to a heterologous promoter. We conclude that mim-1 is a cellular gene that is directly regulated by the product of the v-myb oncogene.

Pim-1 Kinase and p100 Cooperate to Enhance c-Myb Activity

The pim-1 oncogene is regulated by hematopoietic cytokine receptors, encodes a serine/threonine protein kinase, and cooperates with c-myc in lymphoid cell transformation. Using a yeast two-hybrid screen, we found that Pim-1 protein binds to p100, a transcriptional coactivator that interacts with the c-Myb transcription factor. Pim-1 phosphorylated p100 in vitro, formed a stable complex with p100 in animal cells, and functioned downstream of Ras to stimulate c-Myb transcriptional activity in a p100-dependent manner. Thus, Pim-1 and p100 appear to be components of a novel signal transduction pathway affecting c-Myb activity, linking all three to the cytokine-regulated control of hematopoietic cell growth, differentiation, and apoptosis.

C/EBP beta regulation of the tumor necrosis factor alpha gene.

Activated macrophages contribute to chronic inflammation by the secretion of cytokines and proteinases. Tumor necrosis factor alpha (TNF alpha) is particularly important in this process because of its ability to regulate other inflammatory mediators in an autocrine and paracrine fashion. The mechanism(s) responsible for the cell type-specific regulation of TNF alpha is not known. We present data to show that the expression of TNF alpha is regulated by the transcription factor C/EBP beta (NF-IL6). C/EBP beta activated the TNF alpha gene promoter in cotransfection assays and bound to it at a site which failed to bind the closely related protein C/EBP alpha. Finally, a dominant-negative version of C/EBP beta blocked TNF alpha promoter activation in myeloid cells. Our results implicate C/EBP beta as an important regulator of TNF alpha by myelomonocytic cells.

Point Mutations in v-Myb Disrupt a Cyclophilin-Catalyzed Negative Regulatory Mechanism

The c-Myb protein is controlled by intramolecular interactions, and point mutations can enhance its oncogenic activity. We tested whether conformational changes regulate c-Myb and found that Cyp-40, a widely distributed cyclophilin and peptidyl-prolyl isomerase, could inhibit c-Myb DNA binding activity. Inhibition by Cyp-40 required both its C-terminal protein-interaction domain, which bound specifically to c-Myb, and its N-terminal catalytic domain and was blocked by the competitive inhibitor cyclosporin A. Cyp-40 failed to bind or inhibit the oncogenic derivative v-Myb, which has a mutated Cyp-40 binding site. These results suggest that mutations in v-Myb allow it to evade a negative regulatory mechanism mediated by enzymes such as Cyp-40, and implicate peptidyl-prolyl isomerases in the regulation of transcription, transformation, and differentiation.

Tumor Necrosis Factor Alpha Gene Regulation: Enhancement of C/EBPβ-Induced Activation by c-Jun

ABSTRACT Tumor necrosis factor alpha (TNFα) is a key regulatory cytokine whose expression is controlled by a complex set of stimuli in a variety of cell types. Previously, we found that the monocyte/macrophage-enriched nuclear transcription factor C/EBPβ played an important role in the regulation of the TNFα gene in myelomonocytic cells. Abundant evidence suggests that other transcription factors participate as well. Here we have analyzed interactions between C/EBPβ and c-Jun, a component of the ubiquitously expressed AP-1 complex. In phorbol myristate acetate (PMA)-treated Jurkat T cells, which did not possess endogenous C/EBPβ, expression of c-Jun by itself had relatively little effect on TNFα promoter activity. However, the combination of C/EBPβ and c-Jun was synergistic, resulting in greater than 130-fold activation. This effect required both the leucine zipper and DNA binding domains, but not the transactivation domain, of c-Jun, plus the AP-1 binding site centered 102/103 bp upstream of the transcription start site in the TNFα promoter. To determine if C/EBPβ and c-Jun might cooperate to regulate the cellular TNFα gene in myelomonocytic cells, U937 cells that possess endogenous C/EBPβ and were stably transfected with either wild-type c-Jun or the transactivation domain deletion mutant (TAM-67) were examined. U937 cells expressing ectopic wild-type c-Jun or TAM-67 secreted over threefold more TNFα than the control line in response to PMA plus lipopolysaccharide. Transient transfection of the U937 cells expressing TAM-67 suggested that TAM-67 binding to the −106/−99-bp AP-1 binding site cooperated with endogenous C/EBPβ in the activation of the −120 TNFα promoter-reporter. DNA binding assays using oligonucleotides derived from the TNFα promoter suggested that C/EBPβ and c-Jun interact in vitro and that the interaction may be DNA dependent. Our data demonstrate that the TNFα gene is regulated by the interaction of the ubiquitous AP-1 complex protein c-Jun and the monocyte/macrophage-enriched transcription factor C/EBPβ and that this interaction contributes to the expression of the cellular TNFα gene in myelomonocytic cells. This interaction was unique in that it did not require the c-Jun transactivation domain, providing new insight into the cell-type-specific regulation of the TNFα gene.

Mutations in v-myb alter the differentiation of myelomonocytic cells transformed by the oncogene

Chick myelomonocytic cells transformed by the v-myb oncogene-containing viruses E26 and AMV differ in that the former resemble myeloblasts and express the v-myb-regulated granulocyte-specific mim-1 gene, while the latter resemble monoblasts and are mim-1 negative. We constructed a series of AMV-E26 chimeras and localized the critical differences between these viruses to three point mutations within the second repeat of the v-myb DNA binding domain. These three positions are altered in the v-myb protein of AMV relative to the proteins encoded by c-myb or E26 v-myb. Back mutating AMV v-myb at any of these three sites restored the oncogenes ability to activate the mim-1 gene. Surprisingly, two of these changes led to the transformation, in vitro and in vivo, of cells having a promyelocyte-like phenotype. These results indicate that different forms of v-myb impose alternate phenotypes of differentiation on transformed myeloid cells, probably by regulating unique sets of differentiation-specific genes.

Myb binding proteins: regulators and cohorts in transformation

The c-Myb and v-Myb proteins are transcription factors that regulate cell proliferation and differentiation. Both Myb proteins have been shown to interact with a number of cellular proteins, some of which are transcription factors that cooperate to activate specific promoters, while others regulate the transcriptional activity of Myb in specific contexts. By comparing and analysing the types of proteins that bind Myb, and the conserved domains of Myb that interact with other proteins, conclusions can be drawn regarding the role of specific partner proteins in the regulation of gene expression, cell proliferation and disease.

Distinct changes in gene expression induced by A-Myb, B-Myb and c-Myb proteins

The c-Myb, A-Myb and B-Myb transcription factors have nearly identical DNA-binding domains, activate the same reporter gene constructs in animal cells, but have different biological roles. The Myb proteins are often coexpressed in the same cells, raising questions about whether they activate similar or distinct gene expression profiles, and whether they cooperate or compete in regulating the same promoters. Here, recombinant adenoviruses were used to express each protein in human mammary cells, and then microarray assays were used to assess global changes in gene expression. Each Myb protein induced a unique and specific set of changes, displaying activities far more complex than revealed by standard reporter gene assays. These results have important implications for the roles of various Myb proteins in normal and transformed human cells, for regulatory pathways that might modify their activities and for the importance of acquired mutations that may qualitatively alter their functions in tumors.

Pim-1 phosphorylates the DNA binding domain of c-Myb.

The c-Myb transcription factor regulates cellular differentiation and proliferation and is regulated by complex mechanisms that control its repressed oncogenic activity. The transcriptional activity of c-Myb is regulated by the serine/threonine protein kinase Pim-1. Here, we show that Pim-1 is able to interact with c-Myb and the closely related transcription factor A-Myb, via direct interactions with the highly conserved Myb DNA binding domain. Pim-1 associated with Myb both in cells and in vitro, and phosphorylated the Myb DNA binding domain, suggesting that it regulates Myb protein activity by direct phosphorylation.

Identification and Regulation of c-Myb Target Genes in MCF-7 Cells

BackgroundThe c-Myb transcription factor regulates differentiation and proliferation in hematopoietic cells, stem cells and epithelial cells. Although oncogenic versions of c-Myb were first associated with leukemias, over expression or rearrangement of the c-myb gene is common in several types of solid tumors, including breast cancers. Expression of the c-myb gene in human breast cancer cells is dependent on estrogen stimulation, but little is known about the activities of the c-Myb protein or what genes it regulates in estrogen-stimulated cells.MethodsWe used chromatin immunoprecipitation coupled with whole genome promoter tiling microarrays to identify endogenous c-Myb target genes in human MCF-7 breast cancer cells and characterized the activity of c-Myb at a panel of target genes during different stages of estrogen deprivation and stimulation.ResultsBy using different antibodies and different growth conditions, the c-Myb protein was found associated with over 10,000 promoters in MCF-7 cells, including many genes that encode cell cycle regulators or transcription factors and more than 60 genes that encode microRNAs. Several previously identified c-Myb target genes were identified, including CCNB1, MYC and CXCR4 and novel targets such as JUN, KLF4, NANOG and SND1. By studying a panel of these targets to validate the results, we found that estradiol stimulation triggered the association of c-Myb with promoters and that association correlated with increased target gene expression. We studied one target gene, CXCR4, in detail, showing that c-Myb associated with the CXCR4 gene promoter and activated a CXCR4 reporter gene in transfection assays.ConclusionsOur results show that c-Myb associates with a surprisingly large number of promoters in human cells. The results also suggest that estradiol stimulation leads to large-scale, genome-wide changes in c-Myb activity and subsequent changes in gene expression in human breast cancer cells.