Identification of a Myb-responsive enhancer of the chicken C/EBPbeta gene.

The retroviral oncogene v-myb encodes a transcription factor (v-Myb) that disrupts myelomonocytic differentiation and transforms myelomonocytic cells. It is thought that the biological effects of v-Myb are caused by deregulation of specific target genes. The CCAAT box/enhancer binding protein beta (C/EBPbeta), a member of the basic region-leucine zipper (bzip) class of transcription factors, which itself plays an important role during myelomonocytic differentiation, has previously been shown to be regulated by Myb. Here we have addressed the mechanism by which v-Myb affects C/EBPbeta expression. We have employed the mapping of DNase I hypersensitive sites (DHSs) in chromatin as a tool to detect in vivo target sites of v-Myb. Our data identify a DHS downstream of the C/EBPbeta gene that appears to be specific for v-myb-transformed myeloblasts. We have confirmed by chromatin immunoprecipitation that v-Myb is bound to this region in vivo. Furthermore, we have found that ectopic expression of v-Myb in a myelomonocytic cell line is able to induce a DHS downstream of the C/EBPbeta gene, showing for the first time that v-Myb can affect chromatin structure. Reporter gene experiments demonstrate that the downstream DHS acts as a Myb-dependent enhancing element in transiently as well as in stably transfected myelomonocytic cells. Previous work has shown that v-Myb acts on the C/EBPbeta promoter; it now appears that Myb stimulates C/EBPbeta expression by acting on the promoter as well as on an enhancer of the C/EBPbeta gene. Interestingly, the mechanisms by which Myb acts on both elements differ; while Myb activation of the promoter requires the cooperation with C/EBPbeta, activation of the enhancer by Myb is independent of C/EBPbeta. Apart from the identification of a novel Myb-dependent enhancer, our work demonstrates the potential of chromatin structure analysis for the identification of Myb target sites.