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.     

Role of CREB in transcriptional regulation of CCAAT/enhancer-binding protein beta gene during adipogenesis

The proximal promoter of the C/EBPbeta gene possesses dual cis regulatory elements (TGA1 and TGA2), both of which contain core CREB binding sites. Comparison of the activities of C/EBPbeta promoter-reporter genes with 5'-truncations or site-directed mutations in the TGA elements showed that both are required for maximal promoter function. Electrophoretic mobility shift and chromatin immunoprecipitation (ChIP) analyses with antibodies specific to CREB and ATF1 showed that these CREB family members associate with the proximal promoter both in vitro and ex vivo. Immunoblotting and ChIP analysis revealed that other CREB family members, CREM and ATF1, are up-regulated and associate with the proximal C/EBPbeta promoter in mouse embryonic fibroblasts (MEFs) from CREB(-/-) mice. ChIP analysis of wild-type MEFs and 3T3-L1 preadipocytes revealed that interaction of phospho-CREB, the active form of CREB, with the C/EBPbeta gene promoter occurs only after induction of differentiation of 3T3-L1 preadipocytes and MEFs. Consistent with the interaction of CREB and ATF1 at the TGA regulatory elements, expression of constitutively active CREB strongly activated C/EBPbeta promoter-reporter genes, induced expression of endogenous C/EBPbeta, and caused adipogenesis in the absence of the hormonal inducers normally required. Conversely, expression of a dominant-negative CREB blocked promoter-reporter activity, expression of C/EBPbeta, and adipogenesis. When subjected to the standard adipocyte differentiation protocol, wild-type MEFs differentiate into adipocytes at high frequency, whereas CREB(-/-) MEFs exhibit greatly reduced expression of C/EBPbeta and differentiation. The low level of expression of C/EBPbeta and differentiation in CREB(-/-) MEFs appears to be due to up-regulation of other CREB protein family members, i.e. ATF1 and CREM.     

Identification of new sites of binding and activation of the murine alpha1(I) collagen promoter by CCAAT/enhancer binding protein beta

The CCAAT/enhancer binding protein beta (C/EBPbeta) was previously shown to bind to the alpha(1)(I) collagen promoter at -365 to -335 (site 1) and to activate it. Acetaldehyde also activates the promoter, and this effect is mediated by an increase in stellate-cell C/EBPbeta protein and C/EBPbeta binding. The present study identified two additional distal sites (sites 2 and 3) of binding of C/EBPbeta, in the nuclear extracts of stellate cells, at -399 to -370 and -623 to -592 in the alpha(1)(I) collagen promoter. The C/EBPbeta protein activates the promoter at all three sites. Acetaldehyde increases C/EBPbeta binding to all three sites. Activation by acetaldehyde is abrogated in the transfected promoter mutated at either site 1 or site 3 but is not affected by mutation at site 2. Binding of the 20-kDa C/EBPbeta isoform (p20C/EBPbeta), which is eliminated by mutation at the distal site 3 of C/EBP binding, is necessary for the activation by acetaldehyde of the alpha(1)(I) collagen promoter.