Regulation of RUNX1 Transcriptional Function by GATA-1 Kinase Recruitment: A Novel Pathway for Megakaryocytic Gene Regulation.

RUNX1 and GATA-1 both play essential roles in the transcriptional programming of normal mammalian megakaryocytic development, deficiencies of either factor having similar phenotypic consequences. We have previously characterized physical and functional interactions between these two factors, and others have confirmed analogous cooperations in Drosophila and Danio homologs. We now present data on molecular mechanisms for the cooperation of these two factors in the transcriptional activation of the megakaryocytic aIIb integrin promoter. In these studies, GATA-2 also physically interacted with RUNX1 but failed to cooperate in transcriptional activation. In fact, increasing amounts of GATA-2 repressed the functional interplay between GATA-1 and RUNX1. Through generation of GATA-2/GATA-1 chimeras, we identified a conserved subdomain within the GATA-1 amino terminus that was both necessary and sufficient for transcriptional cooperation with RUNX1. Coexpression of wild type GATA-1 or of cooperating GATA-2/GATA-1 chimeras, but not of GATA-2 or of non-cooperating chimeras, induced a mobility shift in wild type RUNX1. Using immunoprecipitation followed by immunoblot with a panel of phosphospecific antibodies, we found GATA-1 to induce RUNX1 phosphorylation at recognition sites for cyclin-dependent kinases (cdks). Treatment of cells with roscovitine, a specific cdk inhibitor, blocked the transcriptional cooperation of GATA-1 with RUNX1 and eliminated the RUNX1 mobility shift caused by GATA-1 coexpression. Mutagenesis of RUNX1 identified a cluster of serine/threonine-proline (S/TP) sites collectively required for the transcriptional augmentation and mobility shift induced by GATA-1. In addition, intact DNA binding by RUNX1 was required for cooperation with GATA-1. These results provide a new paradigm for cooperation of interacting transcription factors, in which one partner recruits a kinase leading to phosphorylation and activation of the other partner. Furthermore, these results provide a biochemical basis for the previously inexplicable functional differences between GATA-1, which promotes megakaryocytic maturation, and GATA-2 which promotes proliferation without maturation.