Ectopic expression of a conditional GATA-2/estrogen receptor chimera arrests erythroid differentiation in a hormone-dependent manner.

Abstract

The GATA factors are a family of transcriptional regulatory proteins in eukaryotes that share extensive homology in their DNA-binding domains. One enigmatic aspect of GATA factor expression is that several GATA proteins, which ostensibly share the same DNA-binding site specificity, are coexpressed in erythroid cells. To elucidate the roles of individual GATA factors in erythropoiesis, conditional alleles of GATA-I, GATA-2, and GATA-3 were prepared by fusing each of the factors to the hormone-binding domain of the human estrogen receptor (ER). These GATA/ER chimeric factors were shown to be hormone-inducible trans-activating proteins in transient transfection assays. When stably introduced into primary erythroblasts or conditionally transformed erythroid progenitors cells, exogenous GATA-2/ER promoted proliferation and inhibited terminal differentiation in an estrogen-dependent manner. These phenotypic effects are specifically attributable to the action of ectopically expressed GATA-2/ER because erythroblasts expressing exogenous GATA-2 are constitutively arrested in differentiation and because erythroid progenitors expressing either Gal/ER or GATA-3/ER do not display a hormone-responsive block in differentiation. Thus, the GATA-2 transcription factor appears to play a role in regulating the self-renewal capacity of early erythroid progenitor cells.[Key Words: GATA transcription factors; conditional alleles; erythroid differentiation] Received November 22, 1992; revised version accepted March 19, 1993.The GATA factors are a family of (C4) zinc finger transcriptional regulatory proteins in eukaryotes that bind to the consensus DNA sequence WGATAR (for review, see Orkin 1990). In vertebrate organisms, each member of this family has a distinct tissue distribution pattern. Thus, GATA-1 is expressed in the erythroid, megakaryocytic, mast cell, and germ cell lineages (Tsai et al. 1989; Martin et al. 1990;Romeo et al. 1990;Ito et al. 1993), whereas GATA-2 is expressed more widely in both hematopoietic and nonhematopoietic lineages (Yamamoto et al. 1990;Zon et al. 1991). The expression of GATA-3 is confined principally to definitive erythroid cells, specific neuronal cells, and T lymphocytes (Yamamoto et al. 1990;Ko et al. 1991 Engel, in prep.). In addition, during erythroid cell differentiation the relative abundance of the GATA factors with respect to one another undergoes specific developmentally controlled changes that are highly conserved in several species (M.W. Leonard, K.-C. Lim, and J.D. Engel, in prep.). These data therefore suggested that the regulation of the precise quantitative balance of the GATA factors might play a role in influencing the developmental decisions available to erythroid progenitor cells, for example, to undergo either self-renewal or terminal differentiation. Thus, we hypothesize that the ectopic expression of these regulatory proteins in erythroblasts might describe novel phenotypes that would lend insight into the roles that individual GATA family members might play in erythropoiesis....

Defining the Functional Boundaries of the Gata2 Locus by Rescue with a Linked Bacterial Artificial Chromosome Transgene

“…GATA-2, originally cloned from a chicken cDNA library (1), was shown to be essential for the proliferation and/or differentiation of early hematopoietic progenitors, as Gata2 null mutant mice die around mid-gestation from a block in primitive hematopoiesis (2). Further examination of Gata2 gain-of-function mice and in vitro differentiation of Gata2 null mutant ES cells underscored the fundamental conclusions from the initial loss of function experiments, and showed that GATA-2 plays a pivotal role in the proliferation of very early hematopoietic progenitors (3)(4)(5).…”

The fibroblast growth factor receptor FGFR-4 acts as a ligand dependent modulator of erythroid cell proliferation

“…SCF and TGFa dependent erythroid progenitor cells (referred to as SCF and TGFa progenitors, respectively) were prepared from bone marrow of 3 ± 10-day old SPAFAS chicks essentially as described (Hayman et al, 1993;Briegel et al, 1996;Wessely et al, 1997). Brie¯y, following Ficoll-Hypaque centrifugation (density 1.077 g/cm 3 , Eurobio, Paris, France) bone marrow cells were cultured in modi®ed CFU-E medium (2 ± 4610 6 cells/ml; Radke et al, 1982;Zenke et al, 1988;Beug et al, 1995) containing 100 ng/ ml avian SCF (Hayman et al, 1993;Bartunek et al, 1996).…”

“…After two days cell proliferation was determined by CellTiter 96 TM cell proliferation assay (Promega, Madison, WI, USA). Hemoglobin content of erythroid cells was measured as described (Briegel et al, 1993). All measurements were done in triplicate.…”

“…RNA samples were analysed by gel electrophoresis and blotted to GeneScreen membranes (Dupont) as described (Briegel et al, 1993). Filters were hybridized sequentially with 32 P labeled probes of chicken cDNA encoding the tyrosine kinase domain of FGFR-4 (this work), b-globin and CAII (Disela et al, 1991).…”

“…Avian hematopoietic cells provide useful experimental model systems that are well suited to obtain largely homogeneous populations of erythroid progenitors derived from bone marrow. These progenitors can be ampli®ed in vitro as homogenous cell populations to large cell numbers in the presence of speci®c growth factors, such as stem cell factor (SCF) and transforming growth factor type a (TGFa; Hayman et al, 1993;Briegel et al, 1993Briegel et al, , 1996Schroeder et al, 1993;Beug et al, 1995;Dolznig et al, 1995;Steinlein et al, 1995;Wessely et al, 1997). Additionally, by changing the culture conditions and addition of di erentiation factors such progenitors e ectively undergo terminal di erentiation and thereby faithfully recapitulate erythroid cell di erentiation in vitro.…”