PU.1 inhibits GATA-1 function and erythroid differentiation by blocking GATA-1 DNA binding

The lineage-specific transcription factors GATA-1 and PU.1 can physically i nteract to inhibit each other's function, but the mechanism of repression o f GATA-1 function by PU,I has not been elucidated. Both the N terminus and the C terminus of PU.1 can physically interact with the C-terminal zinc fin ger of GATA-1. It is demonstrated that the PU.1 N terminus, but not the C t erminus, is required for inhibiting GATA-1 function. Induced overexpression of PU.1 in K562 erythroleukemia cells blocks hemin-induced erythroid diffe rentiation. In this system, PU.1 does not affect the expression of GATA-1 m essenger RNA, protein, or nuclear localization. However, GATA-1 DNA binding decreases dramatically. By means of electrophoretic mobility shift assays with purified proteins, it is demonstrated that the N-terminal 70 amino aci ds of PU.1 can specifically block GATA-1 DNA binding. In addition, PU.1 had a similar effect in the G1ER cell line, in which the GATA-1 null erythroid cell line G1E has been transduced with a GATA-1-estrogen receptor fusion g ene, which is directly dependent on induction of the GATA-1 fusion protein to effect erythroid maturation. Consistent with in vitro binding assays, ov erexpression of PU,I blocked DNA binding of the GATA-1 fusion protein as we ll as GATA-1-mediated erythroid differentiation of these G1ER cells. These results demonstrate a novel mechanism by which function of a lineage-specif ic transcription factor is inhibited by another lineage-restricted factor t hrough direct protein-protein interactions. These findings contribute to un derstanding how protein-protein interactions participate in hematopoietic d ifferentiation and leukemogenesis, (C) 2000 by The American Society of Hema tology.