The GATA and PAX-SIX-EYA-DACH transcriptional networks (PSEDNs) are essential for
proper development across taxa. Here, we demonstrate novel PSEDN roles in vivo in
Drosophila hematopoiesis and in human erythropoiesis in vitro. Using Drosophila genetics,
we show that PSEDN members function with GATA to block lamellocyte differentiation
and maintain the prohemocyte pool. Overexpression of human SIX1 stimulated erythroid
differentiation of human erythroleukemia TF1 cells and primary hematopoietic stem-progenitor
cells. Conversely, SIX1 knockout impaired erythropoiesis in both cell types. SIX1
stimulation of erythropoiesis required GATA1, as SIX1 overexpression failed to drive
erythroid phenotypes and gene expression patterns in GATA1 knockout cells. SIX1 can
associate with GATA1 and stimulate GATA1-mediated gene transcription, suggesting that
SIX1-GATA1 physical interactions contribute to the observed functional interactions.
In addition, both fly and human SIX proteins regulated GATA protein levels. Collectively,
our findings demonstrate that SIX proteins enhance GATA function at multiple levels,
and reveal evolutionarily conserved cooperation between the GATA and PSEDN networks
that may regulate developmental processes beyond hematopoiesis.