Graded Repression of PU.1 Expression by GATA Factors Influence Hematopoietic Progenitor Cell Fate

Hematopoietic cell fate is determined by combinatorial interactions between nuclear proteins that activate and repress transcription. This principle is illustrated by the transcription factors GATA-1 and PU.1, which promote erythro-megakaryocytic and granulocyte-macrophage development respectively. These two proteins interact physically to cross-antagonize each other’s activities, creating regulatory loops for hematopoietic differentiation. Previously, we showed that loss of GATA-1 causes expansion of bipotental megakaryocyte erythroid progenitors (MEPs) from embryonic stem cells or fetal liver derived hematopoietic progenitors. These cells, termed G1ME, for G ATA- 1 -null m egakaryocyte- e rythroid, proliferate continuously in culture and differentiate into committed megakaryocytes and erythroblasts when GATA-1 activity is restored. G1ME cells express GATA-2, a GATA-1-related protein normally found in multipotential hematopoietic progenitors and stem cells. These cells also express moderate levels of PU.1 mRNA, approximately 1/3 of that expressed in the myeloid cell line 416B. Upon retroviral restoration of GATA-1, GATA-2 is downregulated and PU.1 mRNA decreases rapidly. Microarray analysis of GATA-1-rescued G1ME cells revealed repression of PU.1 and many of its downstream target genes, raising the possibility of direct PU.1/Sfpi1 gene repression by GATA-1. Chromatin immunoprecipitation (ChIP) studies identified two GATA factor-binding sites at the PU.1/Sfpi1 locus. In the absence of GATA-1, when the PU.1/Sfpi1 gene is active, these sites are occupied by GATA-2. Retrovirally expressed GATA-1 replaces GATA-2 at these sites, repressing PU.1 transcription during concomitant erythro-megakaryocytic maturation. These findings resemble the “GATA-factor switch” described at other loci such as Gata2 and Kit where GATA-2 and GATA-1 compete for the same cis elements to activate and repress transcription respectively. To test this, we used siRNA to repress GATA-2 expression in G1ME cells by about 60%. Strikingly, this caused PU.1 to be upregulated 4-fold, indicating that GATA-2 also represses PU.1/Sfpi1 , but to a lesser extent than GATA-1. Moreover, G1ME cells expressing GATA-2 siRNA differentiated into macrophages, as evidenced by morphology, expression of numerous cell-type specific markers and massive induction of macrophage specific genes including myeloperoxidase, Mac-1, and C/EBPα. Our findings illustrate two new insights into the transcriptional control of hematopoietic cell differentiation: First, cross-antagonism between GATA-1 and PU.1 not only occurs at the level of protein-protein interaction, but also through direct transcriptional repression. Second, in addition to having opposite effects on transcription of the same target gene as described previously, GATA-2 and GATA-1 can act cooperatively and successively to exert repressive effects of different magnitudes that gradually restrict gene expression during hematopoietic development. In this model, hematopoietic progenitors express GATA-2 and low levels of PU.1 that maintain the multipotential state but are not sufficient for myelopoiesis. Repression of GATA-2 in the absence of GATA-1 raises PU.1 levels to stimulate granulocyte-macrophage development. In contrast, activation of GATA-1 causes PU.1 to be fully repressed, promoting erythrocyte and megakaryocyte differentiation. Our data illustrate how lineage fate and hematopoietic differentiation are influenced by the stoichiometry between GATA-1, GATA-2, and PU.1 in multipotential progenitors.