Down-regulation Of C-myb Expression Research Articles

Functions of IDH1 and its mutation in the regulation of developmental hematopoiesis in zebrafish

Isocitrate dehydrogenase 1 mutation (IDH1R132) was recently identified in AML by next generation sequencing, occurring in 10% of AML with normal cytogenetics. The cancer-associated mutation may result in novel enzymatic activity whereby a-ketoglutarate is converted to 2-hydroxyglutarate (2-HG). The latter may inhibit the function of a-ketoglutarate-dependent dioxygenases, e.g. TET2 and PHDs and result in DNA and histone hypermethylation and hence leukemia initiation. In this study, we examined the role of zebrafish idh1 (zidh1) and the phenotypes of human IDH1R132H expression in embryonic hematopoiesis. The amino acid sequence of zidh1 was remarkably similar to human IDH1. z1dh1 gene was expressed around the AGM region at 36 hpf and in the CHT at 48 hpf. Morpholino knockdown of zidh1 significantly increased gene expression associated with early myeloid progenitor (pu.1) and decreased those of late myelomonocytic differentiation (l-plastin, mpeg1 and mpo) at 18 hpf. Definitive hematopoiesis was also reduced, characterized by down-regulation of c-myb expression along the ventral wall of dorsal aorta at 36 hpf and CHT at 48 and 96 hpf and decreased rag1 expression at 96 hpf. To validate our observations, we constructed a TALEN pair targeting exon 1 of zidh1 and injected the TALEN mRNAs into 1-cell embryos, which showed high mutation frequency (more than 95%) of targeted indels by sequencing. These injected embryos also demonstrated similar hematopoietic phenotypes with that of the zidh1 morphants. Microinjection of cDNA encoding human IDH1R132H and zebrafish idh1R146H but not wild-type IDH1 at 1 cell-stage significantly increased pu.1, l-plastin, mpeg1 and mpo expression and 2-HG level at 24 hpf. The effects could be inhibited by AGI-5198, a specific inhibitor of human IDH1R132H. Overexpression of zebrafish tet2 mRNA could partially rescue the hematopoietic phenotypes of increased myeloid expression by human IDH1R132H. Co-overexpression of human IDH1R132H and FLT3-ITD on zebrafish significantly increased myelopoiesis in the embryos. The observations underscored a hitherto undescribed role of idh1 in zebrafish hematopoiesis and the potential of zebrafish in modeling human AML.

Regulation of c‐myb by NF‐kB family members during murine erythroleukemia differentiation

c‐Myb is a transcription factor and proto‐oncogene that is important for hematopoietic cell differentiation. The regulation of the c‐Myb protein and its down stream targets is well studied while the regulation of expression of the c‐myb gene is not well understood. c‐myb expression is controlled by a conditional block in transcriptional elongation in the first intron of the gene. Previous studies have shown that NF‐kB family members are involved in regulating the activation of c‐myb by binding to Rel‐Related Proteins Binding Elements (RRBE) in the first intron. This leads to the hypothesis that NF‐kB regulates expression of c‐myb during development of hematopoietic cells. Here we show using chromatin immunoprecipitations (ChIPs) and real‐time PCR in murine erythroleukemia cells that the down regulation of c‐myb expression via hexamethylene bisacetamide‐induced differentiation can be over‐come by activation of adenyl cyclase by forskolin. ChIP results demonstrate a correlation between occupancy of the RRBE sites by the p65 subunit of NF‐kB with elevated c‐myb mRNA levels. This study demonstrates for the first time a correlation between NF‐kB occupancy of RRBE sites and c‐myb expression in vivo in hematopoietic cells. Research support provided by a Providence College Committee on Aid to Faculty Research Grant.

Involvement of phosphatidylserine externalization in the down-regulation of c-myb expression in differentiating C2C12 cells

Analysis of c-myb gene down-regulation in differentiating C212 cells revealed that in proliferating cells, c-myb expression is high and ceases as the proliferation rate decreases. However, a low level of c-myb mRNA was detected in confluent non-proliferating differentiating cells for an extended period of time before it declined to an undetectable level. The time course of c-myb gene silencing in differentiating cells correlated with exposition of phosphatidylserine (PS) on the cell surface. Moreover, the interaction of exposed PS with exogenously added annexin V perturbed PS-mediated cell signaling and transiently up-regulated the declining c-myb expression. We, therefore, suggest that cell surface-exposed PS, which plays a role in the process of myotube formation, is also involved in the down-regulation of c-myb expression.

Defective Ribosomal Protein Gene Expression Alters Transcription, Translation, Apoptosis, and Oncogenic Pathways in Diamond‐Blackfan Anemia

Diamond-Blackfan anemia (DBA) is a broad developmental disease characterized by anemia, bone marrow (BM) erythroblastopenia, and an increased incidence of malignancy. Mutations in ribosomal protein gene S19 (RPS19) are found in approximately 25% of DBA patients; however, the role of RPS19 in the pathogenesis of DBA remains unknown. Using global gene expression analysis, we compared highly purified multipotential, erythroid, and myeloid BM progenitors from RPS19 mutated and control individuals. We found several ribosomal protein genes downregulated in all DBA progenitors. Apoptosis genes, such as TNFRSF10B and FAS, transcriptional control genes, including the erythropoietic transcription factor MYB (encoding c-myb), and translational genes were greatly dysregulated, mostly in diseased erythroid cells. Cancer-related genes, including RAS family oncogenes and tumor suppressor genes, were significantly dysregulated in all diseased progenitors. In addition, our results provide evidence that RPS19 mutations lead to codownregulation of multiple ribosomal protein genes, as well as downregulation of genes involved in translation in DBA cells. In conclusion, the altered expression of cancer-related genes suggests a molecular basis for malignancy in DBA. Downregulation of c-myb expression, which causes complete failure of fetal liver erythropoiesis in knockout mice, suggests a link between RPS19 mutations and reduced erythropoiesis in DBA.

Mml1,a New Common Integration Site in Murine Leukemia Virus-Induced Promonocytic Leukemias Maps to Mouse Chromosome 10

MuLV-induced myeloid leukemias (MML) having promonocytic characteristics are produced with high incidence in some strains of adult mice that are undergoing chronic peritoneal inflammation. Previously we showed that many leukemias have rearrangements of the c-myblocus due to insertional mutagenesis, however, we also identified a number of leukemias that had proviral integrations in the absence of c-mybrearrangement. In the present study, a new locus,Mml1,was found to be a target of insertional mutagenesis in 10 of the promonocytic leukemias that lacked c-mybalterations. Chromosomal mapping studies, performed using progeny from interspecies backcross mice generated by mating (BALB/cAn ×M. spretus)F1females to BALB/cAN males, determined thatMml1is located on the proximal end of mouse chromosome 10. Interestingly, there were no recombinants between c-mybandMml1in 101 backcross progeny andMml1was mapped approximately 20–25 kb upsteam ofc-myb.Interestingly, c-mybmRNA and Myb protein are expressed at levels similar to the levels observed in myeloid progenitor cells, but are not overexpressed. It is anticipated that future experiments will determine whetherMml1integration prevents down regulation of c-mybexpression or activates another gene on chromosome 10.

C-myb Proto-Oncogene Is Expressed by Quiescent Scleroderma Fibroblasts and, Unlike B-myb Gene, Does Not Correlate With Proliferation

Systemic sclerosis (scleroderma) is characterized by excessive deposition of extracellular matrix constituents. Although it has been proposed that tissue fibrosis is due to increased fibroblast synthesis of various collagen polypeptides, there is some experimental evidence that patients with systemic sclerosis have a defect in the control of fibroblast growth. The myb family of genes includes, among others, the c-myb proto-oncogene and the structurally related gene, B-myb, which are both implicated in the regulation of differentiation and/or proliferation of hematopoietic and nonhematopoietic cells. To elucidate the molecular basis responsible for scleroderma fibroblast proliferation, we therefore elected to investigate the expression of c-myb and B-myb genes in scleroderma and control cells. Using the reverse transcriptase polymerase chain reaction technique, we detected c-myb transcripts in scleroderma skin fibroblasts rendered quiescent by serum deprivation. Under the same experimental conditions, c-myb message was not found in normal skin fibroblasts, but, after serum stimulation, c-myb RNA was clearly evident from 3 to 72 h in both normal and pathologic cells. Treatment of these cells with c-myb antisense oligonucleotides caused downregulation of c-myb expression, and the inhibition of scleroderma fibroblast proliferation was 42%, whereas in normal fibroblasts the inhibition was weaker (22%). In contrast to c-myb, in normal and scleroderma fibroblasts the level of expression of B-myb correlated with cell proliferation assessed by cell count, and densitometric analysis showed that B-myb message was 1.5-5 times higher in most of pathologic cells studied. The antisense B-myb oligonucleotides had a weaker antiproliferative effect compared with antisense c-myb, inhibiting scleroderma and normal fibroblasts by 23% and 13%, respectively. These data suggest that the B-myb and c-myb genes may play a role in scleroderma fibroblast proliferation and function.

Members of the Nuclear Factor κB Family Transactivate the Murine c-myb Gene

Expression of the c-myb proto-oncogene is primarily detected in normal tissue and tumor cell lines of immature hematopoietic origin, and the down-regulation of c-myb expression is associated with hematopoietic maturation. Cell lines that represent mature, differentiated hematopoietic cell types contain 10-100-fold less c-myb mRNA than immature hematopoietic cell types. Differences in steady-state c-myb mRNA levels appear to be primarily maintained by a conditional block to transcription elongation that occurs in the first intron of the gene. The block to transcription elongation has been mapped, using nuclear run-on analysis, to a region of DNA sequence that is highly conserved between mouse and man. Two sets of DNA-protein interactions, flanking the site of the block to transcription elongation, were detected that exhibited DNA-binding activities that strongly correlated with low steady-state c-myb mRNA levels. Several criteria demonstrated that members of the nuclear factor kappa B (NF-kappa B) family of transcription factors were involved in the DNA-protein interactions identified in these two sets. Surprisingly, cotransfection experiments demonstrated that coexpression of members of the NF-kappa B family, specifically p50 with p65 and p65 with c-Rel, transactivated a c-myb/chloramphenicol acetyltransferase reporter construct that contained 5'-flanking sequences, exon I, intron I, and exon II of the c-myb gene. Transactivation by these heterodimer combinations was dependent on regions of the c-myb first intron containing the NF-kappa B-binding sites. These findings suggest that NF-kappa B family members may be involved in either modifying the efficiency of transcription attenuation or acting as an enhancer-like activity to increase transcription initiation. Thus, the regulation of c-myb transcription may be quite complex, and members of the NF-kappa B family likely play an important role in this regulation.

Inhibition of leukaemia cell proliferation by folic acid-polylysine-mediated introduction of c-myb antisense oligodeoxynucleotides into HL-60 cells.

The inhibitory effect of c-myb antisense oligodeoxynucleotides (ODNs) conjugated to folic acid (FA) on HL-60 cell proliferation was examined. Folic acid was covalently linked to a polylysine chain and purified by gel chromatography. Sterile FA-polylysine was complexed with c-myb sense and antisense. Exposure of HL-60 cells to the FA-polylysine-c-myb antisense ODN complex resulted in a down-regulation of c-myb expression and a greater inhibition of proliferation than that obtained using free ODNs. Moreover, FA-polylysine conjugate alone or complexed to c-myb sense ODN was not toxic to cells. The antigenic properties and uptake of the vitamin were not affected by the polylysine chain. These data suggest that this strategy is potentially useful for the selective delivery of anti-oncogene-targeted ODNs into cancer cells.

Regulation of proliferation and cytokine expression of bone marrow fibroblasts: role of c-myb.

The c-myb protooncogene plays a major role in regulating the process of in vitro and in vivo hematopoiesis via its activity as transcriptional regulator in hematopoietic progenitor cells. Since the bone marrow microenvironment appears to regulate in vivo hematopoiesis by maintaining the growth of multipotent progenitors via secretion of specific cytokines, we asked whether c-myb is also required for the proliferation of and/or cytokine production by stromal cells that generate fibroblast-like colonies (fibroblast colony-forming units [CFU-F]). Using the reverse transcriptase polymerase chain reaction technique, we detected low levels of c-myb mRNA transcripts in human normal bone marrow fibroblasts. Treatment of these cells with c-myb antisense oligodeoxynucleotides caused downregulation of c-myb expression, decreased in the number of marrow CFU-F colonies (approximately 54% inhibition) and in the cell number within residual colonies (approximately 80%), and downregulation of granulocyte/macrophage colony-stimulating factor (GM-CSF) and stem cell factor (SCF) mRNA expression. Transfection of T98G glioblastoma cells, in which expression of c-myb, GM-CSF, and SCF mRNAs is undetectable or barely detectable, with a plasmid containing a full-length c-myb cDNA under the control of the SV40 promoter induced the expression of biologically active SCF and GM-CSF in these cells. Regulation of GM-CSF expression by c-myb was due in part to transactivation of the GM-CSF promoter. These results indicate that, in addition to regulating hematopoietic cell proliferation, c-myb is also required for proliferation of and cytokines synthesis by bone marrow fibroblasts.