v-Myb Mediates Cooperation of a Cell-Specific Enhancer with the mim-1 Promoter

Abstract

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.

Myb-induced Chromatin Remodeling at a Dual Enhancer/Promoter Element Involves Non-coding RNA Transcription and Is Disrupted by Oncogenic Mutations of v-myb

“…Reporter Genes, Expression Vectors, and Transfections-Luciferase reporter genes containing the mim-1 promoter (p-240-luc) and the mim-1 enhancer (pGL3-tk81-mimwt) have been described (7,24). tk-mim(181-394)-luc and tk-mim(307-612)-luc contain only the indicated part of the enhancer (numbers refer to Ref.…”

“…Previous work has shown that the mim-1 promoter harbors Myb and C/EBP binding sites and that Myb synergizes with C/EBP family members to activate mim-1 expression (22,23). Additional insight into mim-1 gene activation was provided by the identification of a Myb-responsive enhancer located 2-kb upstream of the mim-1 promoter (24). We have recently shown that the transcription factor C/EBP␤ induces opening of the compact chromatin structure at the enhancer in a step preceding the transcription of the gene (25).…”

Inhibition of Myb-dependent gene expression by the sesquiterpene lactone mexicanin-I

“…It was previously shown that the cooperation of both cis-elements is responsible for the strong activation of the mim-1 gene by Myb. 17,19 We then transfected the mim-1/eGFP reporter into the cell line HD11-C3, followed by selection of stable transfectants. HD11-C3 cells are similar to HD11-E cells except that they express chicken c-Myb in a doxycyclin-inducible manner.…”

“…17 The luciferase reporter gene pGL4-5xMRE(GG)-Myc contains five tandem copies of an Myb-binding site upstream of the human c-myc core promoter. 25 The b-galactosidase reporter gene pCMVb was from Clontech (Heidelberg, Germany).…”

“…[11][12][13] One of the most strongly activated Myb target genes is the chicken myb-inducible myeloid-specific gene-1 (mim-1), originally identified by Ness et al 14 Recent work has shown that mim-1 expression is regulated by Myb in a complex manner, involving several cis-acting DNA-elements, as well as several cooperating proteins. [15][16][17][18][19] Although myb was originally identified as the oncogene of the avian myeloblastosis virus, it was suspected that c-myb is also involved in the development of human cancer. Recent work has indeed provided strong evidence that deregulated c-myb expression has a role in the development of certain leukemias, and of breast and colon tumors.…”