Transformation by v-myb correlates with trans-activation of gene expression.

Abstract

The v-myb oncogene of avian myeloblastosis virus causes acute myelomonocytic leukemia in chickens and transforms avian myeloid cells in vitro. Its protein product p48v-myb is a nuclear, sequence-specific, DNA-binding The v-myb oncogene of the avian myeloblastosis virus (AMV) causes acute myelomonocytic leukemia in chickens and transforms avian myeloid cells but not fibroblasts in vitro (for a review, see reference 29). The related E26 avian leukemia virus causes acute erythroid leukemia in chickens and transforms avian erythroid and myeloid cells in vitro (36,43). The c-myb proto-oncogene from which v-myb arose is expressed at high levels predominantly in immature hemopoietic cells including those of the erythroid, lymphoid, and myeloid lineages (8, 16). The expression of c-myb declines as these cells mature (11,48). c-myb itself appears to control this differentiation process because the expression of exogenous c-myb or v-myb can block or reverse the differentiated phenotype of various hemopoietic cells (3,10,12,33,37,47). myb-related genes have also been identified in mammals, Drosophila melanogaster, Zea mays, Saccharomyces cerevisiae, and Dictyostelium discoideum (23,39,41,44,46; R. Firtel, and J. Lipsick, unpublished data).The protein products of v-myb and c-myb are present in the cell nucleus, bind DNA in a sequence-specific fashion, and trans-activate gene expression in transient DNA transfection assays (4-6, 22, 24a, 25, 27, 38, 49). Consistent with these findings, the myb-related BASI gene of S. cerevisiae encodes a genetically defined transcription factor which binds directly to DNA (46). In addition, the myb-related product of the Cl locus ofZ. mays controls several unlinked, developmentally regulated genes involved in the synthesis of anthocyanin pigments (41).A number of oncogenes which encode nuclear proteins (GGGCCC) to the end-filled HindlIlresistant neo fragment of the pUC-NEO plasmid (31). This DNA was digested with SmaI to trim excess linkers, purified by agarose gel electrophoresis, and then cloned into the SmaI site of pUC18 to create the plasmid pANA (Fig. 1). This plasmid was then digested with SmaI to release a 1.4-kilobase neo fragment flanked by single Apal linkers. ApaI was chosen because it does not cut within v-myb; however, the method can easily be adapted for other restriction sites.The plasmid pUC-MYB-dE was used as a target for mutagenesis (21). Plasmid DNA was digested with the desired restriction enzyme, and linear DNA was purified by agarose gel electrophoresis and then ligated to the 1.4-kilobase neo fragment described above. Digestion of the target plasmid with restriction enzymes with multiple recognition sites was limited by titration of ethidium bromide to maximize the yield of linear DNA (40). In rare cases, certain individual sites (e.g., Avall at 960) were never mutagenized by this method, suggesting a strong restriction enzyme site preference during partial digestion in the presence of ethidium bromide. Target DNAs produced by enzymes which generated three-nucleot...

A functional SUMO-interacting motif in the transactivation domain of c-Myb regulates its myeloid transforming ability

“…In studies performed to examine the correlation between transactivation and transformation by v-Myb, Lane et al (1990) in fact generated one insertion mutant interfering with the SIM element as defined in this work (v-Myb 752; I 202 NII-I 202 NGPII). Interestingly, this mutant was able to activate transcription 25-fold more efficiently than AMV v-Myb in QT6 cells (Chen and Lipsick, 1993), suggesting that this might be due to loss of interaction with a unknown cellular inhibitor.…”

“…Thus, deregulation of c-Myb expression is associated with oncogenicity. Moreover, the transcriptional regulatory activity of Myb is crucial for its transforming ability (Gonda et al, 1989;Lane et al, 1990;Hu et al, 1991). Multiple co-factors like p300/CBP, Mi-2a, FLASH and menin/MLL engage in the regulation of the transactivational activity of c-Myb (Dai et al, 1996;Oelgeschlager et al, 1996;Kasper et al, 2002;Saether et al, 2007;Alm-Kristiansen et al, 2008;Jin et al, 2010).…”

Myb binding sites within the N-ras promoter repress transcription

“…When expressing several di erent Myb derivatives (c-Myb, vMyb, a c-Myb/VP16 fusion and a v-Myb/VP16 fusion) a repression of the basal activity by an averge of 60% was observed even for those Myb proteins fused to a strong VP16 activation domain. This observed repression appears to require DNA binding by the di erent Myb proteins which were co-expressed in these transfection experiments, because no repression was observed after coexpression of a v-Myb protein which is de®cient in its DNA binding activity (v-Myb364, contains a linker insertion mutation within the DNA binding domain which abolishes DNA binding) (Lane et al, 1990). Trans-activation studies with the pGLNrasDUM reporter construct further supported that the observed repression with the pGLNras and pGLNrasDU constructs is mostly due to binding of the Myb proteins to the Myb sites, since deletion of all of the three sites resulted in derepression of the N-ras promoter.…”

Inactivation of a c-Myb/estrogen receptor fusion protein in transformed primary cells leads to granulocyte/macrophage differentiation and down regulation of c-kit but not c-myc or cdc2

“…The correlation between transactivation and transformation by Myb (Lane et al, 1990;Kanei-Ishii et al, 1992;Hu et al, 1991) suggests that the ability of Myb to transform hemopoietic cells is dependent on the transactivation of genes critical for this process. We have investigated the regulation of several putative Myb regulated genes in the ERMYB transformed cells and have found that cdc2 and c-myc mRNA levels do not correlate with activation of Myb.…”