HEMATOPOIESIS AND STEM CELLS Graded repression of PU.1/Sfpi1 gene transcription by GATA factors regulates

etic transcription factors that control erythromegakaryocytic and myelolym-phoid differentiation, respectively. These proteins antagonize each other through direct physical interaction to repress alter-nate lineage programs. We used immortal-ized Gata1 erythromegakaryocytic pro-genitor cells to study how PU.1/Sfpi1 expression is regulated by GATA-1 and GATA-2, a related factor that is normally expressed at earlier stages of hematopoi-esis. Both GATA factors bind the PU.1/ Sfpi1 gene at 2 highly conserved regions. In the absence of GATA-1, GATA-2 bind-ing is associated with an undifferentiated state, intermediate level PU.1/Sfpi1 ex-pression, and low-level expression of its downstream myeloid target genes. Resto-ration of GATA-1 function induces erythro-megakaryocytic differentiation. Concomi-tantly, GATA-1 replaces GATA-2 at the PU.1/Sfpi1 locus and PU.1/Sfpi1 expres-sion is extinguished. In contrast, when GATA-1 is not present, shRNA knock-down of GATA-2 increases PU.1/Sfpi1 ex-pression by 3-fold and reprograms the cells to become macrophages. Our find-ings indicate that GATA factors act se-quentially to regulate lineage determina-tion during hematopoiesis, in part by exerting variable repressive effects at the PU.1/Sfpi1 locus. (Blood. 2009;114: 983-994