CORE BINDING-FACTOR CANNOT SYNERGISTICALLY ACTIVATE THE MYELOPEROXIDASE PROXIMAL ENHANCER IN IMMATURE MYELOID CELLS WITHOUT C-MYB

The myeloperoxidase (MPO) gene is transcribed specifically in immature myeloid cells and is regulated in part by a 414-bp proximal enhancer, Mutation of a core binding factor (CBF)-binding site at -288 decrease d enhancer activity 30-fold in 32D cl3 myeloid cells cultured in granu locyte colony-stimulating factor (G-CSF), A novel functional analysis, linking the CBF-binding site to an enhancer deletion series, located at -147 an evolutionarily conserved c-Myb-binding site which was requi red for optimal enhancer activity and synergy with CBF in 32D cells, T hese sites cooperated in isolation and independent of a precise spacin g, Deletional analysis carried out in the absence of the c-Myb-binding site at -147 located at -301 a second c-Myb-binding site which also s ynergized with CBF to activate the enhancer, A GA-rich region at -162 contributed to cooperation with CBF when the adjacent c-Myb-binding si te was intact, Mutation of both c-Myb-binding sites in the context of the entire enhancer greatly impaired activation by endogenous CBF in 3 2D cells, Similarly, activation by c-Myb was impaired in constructs la cking the CBF-binding site, CBF and c-Myb were required for induction of MPO proximal enhancer activity when 32D cells differentiated in res ponse to G-CSF, A fusion protein containing the Gal4 DNA-binding domai n and the AML-IB activation domain, amino acids 216 to 480, activated transcription alone and cooperatively with c-Myb in nonmyeloid CV-1 ce lls, Determining how CBF and c-Myb synergize in myeloid cells might co ntribute to our understanding of leukemogenesis by the AML1-ETO, AML1- MDS1, CBF beta-SMMHC, and v-Myb oncoproteins.