MYB AND ETS PROTEINS ARE CANDIDATE REGULATORS OF C-KIT EXPRESSION IN HUMAN HEMATOPOIETIC-CELLS

Kit is a tyrosine kinase receptor that plays an important role in huma n hematopoietic cell growth. The promoter elements that modulate the g ene's expression have not been extensively studied. Because of c-kit's acknowledged importance in hematopoiesis, we sought to address this i ssue in more detail. To perform these studies we analyzed a human c-ki t 5' flanking fragment similar to 1 kilobase in length. Deletion const ructs showed a region similar to 139 nucleotides upstream from the tra nslation initiation site that was critical for promoter activity. A re gion containing a potential silencing element was also identified. Seq uence analysis indicated several potential Myb-and Ets-binding sites. The functional significance of these sites was explored by showing tha t both wild-type Myb and Ets-2 protein, but not a DNA binding-deficien t Myb mutant protein, bound to distinct 5' flanking fragments that inc luded these sites. Furthermore, binding of recombinant Myb and Ets-2 p rotein to these fragments could be competed with an excess of double s tranded oligodeoxynucleotides containing canonical, but not mutated, M yb-or Ets-binding sites. We also showed that the 5' flanking region of c-kit exhibited promoter activity in nonhematopoietic cells only when the cells were transfected with c-myb or ets-2 expression vectors. Mo reover, Myb and Ets-2 coexpression in such cells augmented transactiva tion of c-kit promoter constructs in comparison to that observed in ce lls transfected with either construct alone. Promoter constructs lacki ng various Myb and Ets sites deleted were much less effective in this same system. Finally, Myb and Ets-2 mRNA expression was detected in CD 34(+), Kit(low) as well as CD34(+), Kit(bright) cells. In aggregate, t hese data further define the human c-kit promoter's functional anatomy and suggest that Myb and Ets proteins play an important, perhaps coop erative, role in regulating expression of this critical hematopoietic cell receptor. (C) 1998 by The American Society of Hematology.