Impact of Mutant Haemopoietic Transcription Factor GATA1s on Haemopoiesis

Neonates and children with Down Syndrome (DS), with constitutional Trisomy 21 (T21), have a 150-fold increased risk of developing Myeloid Leukaemia (ML-DS), with differentiation arrest of immature megakaryocyte-erythroid cells. Virtually all ML-DS patients have in utero-acquired somatic mutations in the gene encoding the megakaryocyte-erythroid transcription factor GATA1 leading to the production of an 84 amino acid N-terminal truncated form of the GATA1 protein (GATA1s). We, and others, have previously shown that this N-terminal domain is necessary to prevent excessive megakaryocytic proliferation. However, the mechanisms by which GATA1, but not GATA1s, restrains megakaryocyte proliferation are unclear.

We generated knock-in murine ES cell models expressing biotinylated forms of either full length GATA1 or GATA1s protein. We established a large scale in vitro differentiation assay to study embryonic-fetal megakaryocyte differentiation to define the normal megakaryocytic differentiation pathway in GATA1-expressing cells. ES cells were differentiated into embryoid bodies (EBs), which were disaggregated after 6 days and CD41+c-kit+ cells were cultured on OP9 feeder layers with TPO, IL6 and IL11. Detailed examination of the differentiation kinetics including FACS-sorting of specific populations followed by re-culture or Biomark qRT-PCR analysis, showed complex differentiation pathways as wild type cells differentiated into both megakaryocyte and non-megakaryocyte (both erythroid and myeloid) fates. By contrast, GATA1s-expressing cells almost exclusively differentiated into megakaryocyte lineage. Furthermore, as immature CD41+ haemopoietic cells differentiate into the megakaryocyte lineage, they lose c-kit expression and CD41 expression increases. In the GATA1s-expressing cells compared to GATA1-expressing cells, there is a significant accumulation (5 to 10-fold) of a specific immature megakaryocyte CD41++c-kit+ population, partially blocked in differentiation. Cell cycle analysis showed an increase in cells in S-phase specifically in this population in GATA1s-expressing cells (44% compared to 27% in normal cells) together with a decrease in apoptosis (5% compared to 11% in normal cells). To determine the relevance of these findings in vivo , corresponding mouse models were generated and experiments on yolk sacs isolated at different stages of development are ongoing.

Taken together, these data suggest that our in vitro murine ES cell differentiation model is a relevant tool to dissect the molecular mechanisms underlying GATA1s function in abnormal megakaryocytic differentiation and they are likely to provide insight into general mechanisms of how altered function of a critical lineage-affiliated transcription factor can lead to multiple changes in progenitor cell cycle and differentiation.