Myelo-Erythroid Lineage Segregation Is Regulated By the GATA-2 Interactome

Hematopoiesis arises from hematopoietic stem cell differentiating into downstream progenitors through a multistep process of sequential lineage potential restriction leading to the final production of >10 hematopoietic cell type.The regulation of hematopoietic lineage development involves synergistic and antagonistic interactions between transcription factors (TF) with lineage-restricted expression patterns, both through protein-protein interaction and direct transcriptional activation.Eosinophils, basophils and mast cells are key myeloid cell types involved in allergic inflammation and have been proposed to arise from a granulocyte-macrophage progenitor (GMP), that also generates neutrophils and macrophages, with GATA-2 up-regulation as a critical determinant. However, recent work identified a distinct basophil/eosinophil/mast cells progenitor (BEMP) that arises from multi-potent erythroid-primed multipotent progenitors (EMPP) where GATA-2 is already expressed (Drissen et al., 2016). Here, we show that the GATA-2 interactome, rather than GATA-2 expression, controls myeloid lineage choice in EMPPs.

To investigate the molecular mechanism instructing lineage decision in EMPP, we performed gene-expression profiling (RNA-seq) comparing its downstream progeny, early restricted erythroid/megakaryocyte progenitor (preMegE) and BEMP. This analysis identified differential expressed genes, including antagonist gene-expression pattern between the LIM-only protein 2 (Lmo2) and protein 4 (Lmo4), both previously shown as GATA-2 interactor. While Lmo2 function in hematopoiesis have been exhaustively studied, the role of Lmo4 remains unknown.

In colony forming unit (CFU)-assay, overexpression of Lmo4 in EMPP mediated by lentiviral delivery, resulted in an increased number of mast-colonies associated with a decreased of erythroid/megakaryocyte colonies, while the total colony number was unchanged. In addition, transplantation of hematopoietic lineage-negative cells transduced with Lmo4/empty(Ctrl) lentiviral particles, showed an increased frequency of basophils and eosinophils associated with a decrease of erythroid cells in Lmo4 overexpressing cells compared to Ctrl, in both bone marrow and spleen of recipient mice, 8 weeks after transplantation. Analysis of transduced Lineage-negative Kit⁺ (LK) compartment of recipient mice, also revealed an increased frequency of BEMP associated with a decreased frequency of preMegE in Lmo4overexpressing cells compared to Ctrl. Taken together, these data suggested that Lmo4 overexpression instruct lineage decision of EMPP toward basophil/eosinophil/mast lineages.Over-expression followed by co-immunoprecipitation in HEK-293T cell line revealed that LMO4 compete with LMO2 binding to GATA-2, suggesting a mutually exclusive transcriptional complex. More importantly, lack of interaction between LMO4 and GATA-2 mediated by a point mutation in the zinc-finger 1 domain (ZnF1) of GATA-2 (GATA2-G320D), frequently identified in AML/AEL, resulted in an absence of mast lineage potential of EMPP compared to GATA2^+/+. Moreover, over-expression of Lmo4 in EMPP from GATA2^G320D/G320D mice compared to GATA2^+/+, failed to instruct myeloid fate in vitro.

Overall, our data identified a specific molecular mechanism of hematopoietic cell fate decision driven by protein-protein interaction with GATA-2. Perturbation of GATA-2 interactome through over-expression of the cofactor Lmo4 or specific point mutation in GATA2 ZnF1 domain results in lineage fate switch of EMPP toward the erythroid/megakaryocyte or basophil/eosinophil/mast lineages.