Regulation of the vav proto-oncogene by LKLF.

The transcriptional induction of the vav proto-oncogene coincides with the first appearance of the definitive hematopoietic stem cell in the aorta-gonad-mesonephros region. Vav promoter activity was dependent on a previously identified 23 bp DNA segment containing PU.1 and Runx1/AML-1 binding sites and on a newly identified, highly conserved, 12 bp DNA segment (Box B). The sequence of Box B was identical in the human, mouse and rat species. Mutation of the CACCC core sequence led to diminished vav promoter activity. A protein complex which bound to Box B was found in hematopoietic cells but not in cells which did not express vav. A double-stranded oligonucleotide containing a mutation of the CACCC core was less effective in electro-mobility shift assay competitions than the wild-type sequence. UV crosslinking studies identified a 37 kDa DNA binding protein which interacted with Box B in U937 cells. Antibody supershift assays identified this protein as lung Krüppel-like factor (LKLF). LKLF, expressed as a glutathione S-transferase fusion protein, was capable of binding to Box B. A dominant-negative LKLF was able to inhibit the expression of enhanced green fluorescent protein by the vav promoter and chromatin immunoprecipitations detected LKLF bound to the vav promoter in U937 but not HeLa cells. These in vitro results suggest future in vivo experiments to examine the role of LKLF, a gene required for vasculogenesis, in the induction of vav during the genesis of the definitive hematopoietic stem cell from the vascular endothelium.