To provide a comprehensive landscape in

To provide a comprehensive landscape in human hematopoiesis, we studied NF-E2 gene expression throughout the hematopoietic system using HemaExplorer (Bagger et al., 2013) and confirmed by RT-PCR that it was also expressed in the stem cell compartment (Figure 1A). Thereafter, we knocked NF-E2 down in hematopoietic stem and progenitor Manumycin A (HSPCs: Lin−/CD34+/CD38−) derived from human umbilical cord blood (hUCB) using two independent NF-E2 knockdown plasmids (KDNF-E2a and KDNF-E2b) and one knockdown control (KDCTRL). We checked NF-E2 expression 4 days after HSPC transduction and obtained a significant decrease at the mRNA level (Figure 1B), which was confirmed at the protein level 6 days after transduction (Figures 1C and S1A). As expected, lack of NF-E2 in HSPCs led to a reduction in megakaryocyte differentiation in vitro, as shown by cell counting (Figure S1B) and fluorescence-activated cell sorting (FACS) analysis (Figure S1C). To test the functionality of the NF-E2-silenced HSPCs, we performed a colony-forming cell (CFC) assay and observed a significant reduction in colony-forming units (CFU; Figure 1D). There was a significant increase in the relative proportion of erythroid colony-forming units (CFU-E), while the percentage of myeloid colony-forming units (granulocytes = CFU-G, macrophages = CFU-M, granulocyte-macrophages = CFU-GM) was unaffected (Figure S1D). This increase appeared paradoxical, as we expected NF-E2 downregulation to reduce erythroid differentiation, yet our data are in line with what has been previously described in an overexpression experiment, where NF-E2 overexpression in human CD34+ cells reduced the number of CFU-E, arguing that it delays erythroid maturation and retains erythroid progenitors in an immature stage with increased proliferation capacity (Mutschler et al., 2009). Next, we examined whether KDNF-E2 HSPCs displayed altered self-renewal capacity by replating colonies (Figure 1E). The reduction of total CFU was even stronger in secondary colonies and was associated with a further decrease in CFU-GM and an increase in CFU-M (Figure S1E). In light of the similar effects generated by the two NF-E2 knockdown plasmids, we continued the study using only KDNF-E2a (which we then simply identified as KDNF-E2). We hypothesized that decreased self-renewal capacity of HSPCs, highlighted by their reduced capacity to generate colonies, could be counterbalanced by an increase in cell proliferation. To investigate cell proliferation dynamics, we checked the cell-cycle status of HSPCs 8 days after KDNF-E2 by Ki67/DAPI staining, and noticed a decrease in G0 and an increase in G2/M/S phase in vitro (Figures 1F and S1F). We also observed a significant increase in cell number 8 days after NF-E2 silencing (Figure S1G) and a concurrent decrease in P21, a negative regulator of the G1/S cell-cycle transition at both the RNA level (Figure S1H) and the protein level (Figure 1I, day 8 and Figure S1A, day 6). It has been reported that NOTCH1 activation favors self-renewal over differentiation in murine HSCs (Stier et al., 2002). We therefore studied whether NF-E2 could interfere with Notch1. Interestingly, we observed a strong reduction of activated NOTCH1 (NOTCH intracellular domain [NICD]) in HSPCs 6 days after NF-E2 silencing (Figures 1H and S1A), and also detected downregulation of its downstream target HES1 (Figure 1I). To further support this, we transduced human T-acute lymphoblastic leukemia (T-ALL) MOLT4 cells with KDNF-E2 and KDCTRL and induced NOTCH1 activation by growing them on the δ1 receptor-expressing MS5 stroma layer (MS5-DL1). We compared the effect of KDNF-E2 with two known γ-secretase inhibitors ((S)-tert-butyl 2-((S)-2-(2-(3,5-difluorophenyl)acetamido)propanamido)-2-phenylacetate [DAPT] and compound XX). We confirmed in MOLT-4 that knockdown of NF-E2 significantly affects P21 and HES1 level (Figure S1I). We also observed a reduction of NICD nuclear localization by ImageStreamX analysis in MOLT4 cells when NF-E2 was silenced (Figures 1J and S1J) comparable with DAPT- and compound XX-treated cells (Figure 1J). We confirmed these results by a comparable reduction in the expression of HES1 between KDNF-E2 and the two γ-secretase inhibitors (Figure 1K).