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 human hematopoietic cell growth. The promoter elements that modulate the gene's expression have not been extensively studied. Because of c-kit's acknowledged importance in hematopoiesis, we sought to address this issue in more detail. To perform these studies we analyzed a human c-kit 5' flanking fragment approximately 1 kilobase in length. Deletion constructs showed a region approximately 139 nucleotides upstream from the translation initiation site that was critical for promoter activity. A region containing a potential silencing element was also identified. Sequence analysis indicated several potential Myb- and Ets-binding sites. The functional significance of these sites was explored by showing that both wild-type Myb and Ets-2 protein, but not a DNA binding-deficient Myb mutant protein, bound to distinct 5' flanking fragments that included these sites. Furthermore, binding of recombinant Myb and Ets-2 protein to these fragments could be competed with an excess of double stranded oligodeoxynucleotides containing canonical, but not mutated, Myb- 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. Moreover, Myb and Ets-2 coexpression in such cells augmented transactivation of c-kit promoter constructs in comparison to that observed in cells transfected with either construct alone. Promoter constructs lacking various Myb and Ets sites deleted were much less effective in this same system. Finally, Myb and Ets-2 mRNA expression was detected in CD34+, Kit low as well as CD34+, Kit bright cells. In aggregate, these data further define the human c-kit promoter's functional anatomy and suggest that Myb and Ets proteins play an important, perhaps cooperative, role in regulating expression of this critical hematopoietic cell receptor.
Ratajczak
M Z
MZ
Department of Pathology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
Perrotti
D
D
Melotti
P
P
Powzaniuk
M
M
Calabretta
B
B
Onodera
K
K
Kregenow
D A
DA
Machalinski
B
B
Gewirtz
A M
AM
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D016466:CHO Cells; D019556:COS Cells; D006224:Cricetinae; D003412:Cricetulus; D004268:DNA-Binding Proteins; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute / Q000473:pathology; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D050787:Proto-Oncogene Protein c-ets-2; D011518:Proto-Oncogene Proteins / Q000502:physiology*; D019009:Proto-Oncogene Proteins c-kit / Q000096:biosynthesis* / Q000235:genetics; D020598:Proto-Oncogene Proteins c-myb; D012097:Repressor Proteins; D017384:Sequence Deletion; D015534:Trans-Activators / Q000502:physiology*; D014157:Transcription Factors; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; C107465:ERF protein, human; C494069:ETS2 protein, human; C494070:Ets2 protein, mouse; D050787:Proto-Oncogene Protein c-ets-2; D011518:Proto-Oncogene Proteins; D020598:Proto-Oncogene Proteins c-myb; D012097:Repressor Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D019009:Proto-Oncogene Proteins c-kit