The oncogenic transcription factor v-Myb disrupts myelomonocytic differentiation and transforms myelomonocytic cells by deregulating the expression of specific target genes. One of these genes, the chicken mim-1 gene, is activated by Myb exclusively in myelomonocytic cells and, therefore, has been an interesting model system to study how Myb activates a target in a lineage-specific manner. Previous work has suggested that Myb activates mim-1 by cooperating with CCAAT box/enhancer binding protein beta (C/EBP␤) or other C/EBP transcription factors at the mim-1 promoter. We have now identified and characterized a powerful Mybdependent enhancer located 2 kb upstream of the mim-1 promoter. The enhancer is preferentially active in myelomonocytic cells, confers Myb responsiveness onto a heterologous promoter, and dramatically increases Myb responsiveness of the mim-1 promoter. Chromatin immunoprecipitation demonstrates that v-Myb and C/EBP␤ are bound to the enhancer in v-Myb-transformed cells; furthermore, cooperation of the enhancer with the mim-1 promoter is greatly stimulated by C/EBP␤ and p300. Taken together, our results show that the regulation of mim-1 expression by v-Myb is more complex than previously assumed and involves two distinct regions of the mim-1 gene. A major function of v-Myb (in addition to its role at the mim-1 promoter) apparently is to activate the mim-1 enhancer and, together with C/EBP␤ and p300, facilitate its cooperation with the promoter. Interestingly, our work also shows that the v-Myb protein encoded by avian myeloblastosis virus is defective in this function, suggesting an explanation for why primary avian myeloblastosis virus-transformed myeloblasts do not express the mim-1 gene.

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CCAAT box/enhancer binding protein beta (C/EBPbeta), a member of the basic region-leucine zipper (bzip) class of transcription factors, plays important roles during differentiation of certain cell types, such as liver cells, fat cells and myelomonocytic cells. C/EBPbeta is highly expressed in these cell types, and activates specific genes during their differentiation. In the hematopoietic system, C/EBPbeta expression is restricted to the myelomonocytic lineage. To investigate the molecular basis of the cell-type specific expression of the C/EBPbeta gene in hematopoietic cells we have cloned the chicken C/EBPbeta gene and mapped DNase I hypersensitive sites (DHS) in the vicinity of the gene, using myelomonocytic as well as other cell types. We show that there are multiple nuclease-sensitive sites, most of which are cell-type specific, suggesting that they might act as cell-type specific cis-regulatory DNA elements. To study the possible function of these elements we have constructed reporter genes containing these sequences and analyzed their activity in different cell types. Our results show that several of the nuclease-sensitive regions act as cis-acting stimulatory elements in myelomonocytic but not in other cells. Taken together, our data suggest that the expression of the C/EBPbeta gene in myelomonocytic cells is controlled by multiple cell-type specific cis-acting sequences located both upstream and downstream of the gene.