Myb and Ets-2 transactivate c-kitpromoter in a cooperative manner. (A) CAT activity in TK^-ts13 fibroblasts cotransfected with Myb(pCMV-myb), Ets-2 (pCMV-ets-2), orMyb+Ets expression vectors, plus a pCAT B-RsaI/BamHI fragment construct. Transfection of all three vectors into the TK-ts13 cells led to CAT activity ∼threefold greater than that observed with the Myb or Etsexpression constructs alone, and ∼140% that of the pCAT positive control (not shown). (B) Ability of Myb and Ets protein to transactivate wild-type, or mutated forms of theXhoI-BamHI Kit promoter fragment in TK-ts13 cells. In the absence of Myb or Ets, the construct was without CAT activity (lane 1). Cotransfection of Myb orEts-2 expression constructs along with the pCAT BXhoI-BamHI promoter construct significantly increased CAT activity (lanes 2 to 3). Deletion of a single Myb binding site (c-kit-D M1) resulted in loss of Myb's (lane 5), but not Ets's (lane 6) ability to augment CAT activity. Deletion of both Myb sites and two of three Ets sites diminished, but did not entirely abolish the ability of Ets to transactivate the promoter construct (lane 9). Neither c-kit-D M1, nor c-kit-E3 alone elicited CAT activity in the TK-ts13 cells (lanes 4 and 7, respectively). See text for complete details. (Panel C) Schematic representation of Myb andEts-2–binding sites within the XhoI c-kitpromoter region (-481/-24).