Graded Repression of PU.1 Expression by GATA Factors Influence Hematopoietic Progenitor Cell Fate
Authors: Chou Stella T. , Bailey Charles , Yao Yu , Huang Zan , Nichols Kim , Crispino John , Hardison Ross , Blobel Gerd , Vakoc Chris , Weiss Mitchell J. 
Published: April 2020

Abstract
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 GATA-1-null megakaryocyte-erythroid, 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.