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X. Yu et al.
sorting (FACS) using TER119 and CD71 expression and the relative ratio normalized versus pLL3.7 control (ctrl), in each population is shown in the Online Supplementary Figure S4B. LDB1 protein decreased by ~60% at D3 (54.3%, for shRNA#1 and 64.3% for shRNA#2 compared to ctrl) which resulted in a ±50% decrease in the P2, P3 and P4 population of cells (see above). In three additional LDB1 KD, a pLL3.7 plasmid containing EGFP was used to determine the RNA levels in GFP+ cells versus the total cells (“All cells”). As expected, the “All cells” result shows a similar ratio in each population as was observed in Figure 3A. The GFP+ cells representing the really infected cells, showed a strong KD effect in P2 to P3 compared to pLL3.7 control, when the level of LDB1 appears highest (Figure 3A). The GATA1 KD showed a decrease of GATA1 pro- tein of ~43.5% at D3 (relative to Scr ctrl) resulting in a ±40% decrease in the P3 population while majority of cells are P1. This result is consistent with the phenotype observed in inducible Gata1 KO mice showing an over- representation of cells in P1 and an under-representation of cells in P3 and P4).28 The difference of the KD effect in LDB1 and GATA1 therefore correlates with their expres- sion in those populations (Figure 3B). It is impossible to conclude whether the decrease in the LDB1 complex has an effect before the proerythroblast stage, because the number of progenitors committed to the erythroid lineage in the P1 is not known, but the 40-50% decrease in P2 to P4 suggests that there is a decrease in the number of proerythroblasts that is propagated to the later cell com- partments. Of note, the cells (P2 to P4) were strongly affected by the KD of LDB1 or GATA1 and were dying.
Discussion
Our study shows a modulation of the levels of the pro- tein complexes from the early stages of erythroid differen- tiation of the systems that we used (erythroid differentia- tion from embryonic stem cells, MEL cells, primary ery- throid cell populations from mouse FL and FL tissue sec- tions). These diverse systems represent different stages and therefore heterogeneous systems of hematopoietic development and differentiation. They show that the same complex is formed at these quite different stages, although these experiments do not directly show that the formation of the complex is essential at all these stages and one system might not directly extrapolate to the other. However previous data using KO or KD experi- ments of individual components of the complex lead to defective hematopoiesis/erythropoiesis.9,10,29 We therefore conclude that GATA1/LDB1 complex formation is essen- tial in these diverse systems and provide novel insight in GATA1 complexes. The result describes the sequential emergence of GATA1/LDB1, GATA1/FOG1 and LDB1/E2A complexes in early stage ES cell differentiation, suggesting dynamic changes of the complexes and their function taking place during the blast colony-forming cell (BL-CFC) stage similar to the hemangioblast stage in vivo. GATA1 as part of the LDB1 complex is absent in undiffer- entiated ES cells and only appears after a few days of dif- ferentiation, while GATA2 is expressed and is part of the LDB1 complex in the very early stages of differentiation where it activates GATA119 in a feed forward type system (Kolovos et al., in revision), which is in agreement with the data on the function of GATA1 and GATA2 in mice or dif- ferentiating ES cells.4,30
GATA2 is present during early erythropoiesis and binds to the Gata1 gene to activate its expression.19 Gata1 expression in turn represses Gata2 expression via the FOG1/MeCP1 complex, while activating its own expres- sion. This "GATA switch" represents a forward drive towards late stage erythroid differentiation through changes in gene expression.19,31 GATA2 regulates impor- tant proliferation genes of stem or progenitor cells where- as GATA1 also regulates the final erythroid fate through the expression of erythroid specific genes. A similar process operates early during embryonic development. GATA2 is present before GATA1 during ES cells differen- tiation to the hematopoietic lineage. The GATA2/LDB1 complex is present in very low concentration binding a small set of hematopoietic specific transcription factors in a co-factor dependent manner (PK, CA-S and FG, manu- script in revision). Unfortunately, the low level of GATA2 and poor quality of its antibodies prevent a PLA signal although the GATA switch must start early as we identi- fied the GATA1/LDB1 and LDB1/E2A complexes already in D4 EB. PLA does not show which cell population con- tains the GATA1/LDB1 and LDB1/E2A complexes in D4 EB, but it is known that LDB1 is expressed in BL-CFC.10 The cells expressing GATA1 and LDB1 in D4 EB are differ- entiating hematopoietic cells. We know from ChIP-seq data that the two factors already form a complex at this stage targeting a core set of genes enriched for categories related to differentiation/quantity of blood/hematopoietic progenitor cells (Kolovos et al., manuscript in preparation). Moreover, we described in Mylona et al. 201310 that blast colony-forming cells deficient for Ldb1 (i.e. Flk1+ cells sort- ed four days after ES cell differentiation) are unable to dif- ferentiate to the hematopoietic lineages with a severe reduction in the number of blast colony forming cells and their failure to give rise to blast colonies.
These cells appear between ES cell differentiation D3.75 to D4.25 expressing vascular endothelial growth factor receptor-2 (VEGFR2, i.e. FLK1). The absence of LDB1 results in less BL-CFC and failure to generate hematopoi- etic and endothelial lineages.10 Of its targets Gata2, Scl/Tal1, Runx1 and Gif1b are down-regulated showing that the complex is essential for activation of early embry- onic hematopoiesis in agreement with our observation here that the GATA1/LDB1 interaction already takes place at this early stage. It has been shown that a KO of Gata1 in ES cells did not affect the formation of clonogenic pro- genitors in chimeric in vitro differentiation, and Gata1 KO colonies contained phenotypically normal macrophages, neutrophils and megakaryocytes,3,4 suggesting that GATA1 is not yet essential at this early stage. Importantly GATA2 is essential for the generation of FLK1+ BL-CFC during in vitro ES cell differentiation32 and Gata2-/- embryos die at E11.5 with severe anemia.30
LDB1 IP experiments in undifferentiated ES cells show that a GATA1/LDB1 complex is absent, but appears at low levels in nuclear extracts (NE) from D4 and D5 ES cell EB (Online Supplementary Figure S1C-D). The expression of FOG1, LDB1 and GATA1 increases from D4 to D5, but is still very low and the binding partner LDB1 could not detected by IP in a GATA1 pulldown (Online Supplementary Figure S1). The E2A protein was detected in the LDB1 IP only at D5, but again the band is very weak. We also applied size-exclusion chromatography to distin- guish the different GATA1 complexes. The expression of LMO2 protein increases at D4 (Online Supplementary Figure
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haematologica | 2020; 105(7)