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 decreased enhancer activity 30-fold in 32D cl3 myeloid cells cultured in granulocyte 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 required for optimal enhancer activity and synergy with CBF in 32D cells. These sites cooperated in isolation and independent of a precise spacing. 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 synergized with CBF to activate the enhancer. A GA-rich region at -162 contributed to cooperation with CBF when the adjacent c-Myb-binding site was intact. Mutation of both c-Myb-binding sites in the context of the entire enhancer greatly impaired activation by endogenous CBF in 32D cells. Similarly, activation by c-Myb was impaired in constructs lacking the CBF-binding site. CBF and c-Myb were required for induction of MPO proximal enhancer activity when 32D cells differentiated in response to G-CSF. A fusion protein containing the Gal4 DNA-binding domain and the AML-1B activation domain, amino acids 216 to 480, activated transcription alone and cooperatively with c-Myb in nonmyeloid CV-1 cells. Determining how CBF and c-Myb synergize in myeloid cells might contribute to our understanding of leukemogenesis by the AML1-ETO, AML1-MDS1, CBFbeta-SMMHC, and v-Myb oncoproteins.
Britos-Bray
M
M
Johns Hopkins Oncology Center, Division of Pediatric Oncology, Baltimore, Maryland 21287, USA.
Friedman
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AD
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002454:Cell Differentiation; D002478:Cells, Cultured; D050660:Core Binding Factors; D004268:DNA-Binding Proteins / Q000494:pharmacology*; D004742:Enhancer Elements, Genetic; D004789:Enzyme Activation; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins; D009195:Peroxidase / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D020598:Proto-Oncogene Proteins c-myb; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors / Q000494:pharmacology*; D014158:Transcription, Genetic
D050660:Core Binding Factors; D004268:DNA-Binding Proteins; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D020598:Proto-Oncogene Proteins c-myb; D015534:Trans-Activators; D014157:Transcription Factors; D009195:Peroxidase