Page 162 - 2021_02-Haematologica-web

C. Fugazza et al.
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Figure 1. Expression profile of Coup-TFII during the early fetal liver globin switching developmental window. (A) Representative western blot to assay the protein lev- els of Coup-TFII in nuclear extracts from E11.5, E12.5 and E13.5 mouse fetal livers. β-actin was used as a loading control. (B) Quantitative real-time polymerase reac- tion expression analysis relative to glyceraldehyde-3-phosphate dehydrogenase (Gapdh) (n≥4, error bars: standard error of mean; *P<0.05; **P<0.01; ***P<0.001). (C) Flow cytometry (FC) analysis defining Coup-TFII in erythro-myeloid progenitors (CD41+/CD117+/CD16/14+) and “primitive” red blood cells (EryP: ey+γ+) in fetal liver cells from human β-globin locus transgenic mouse embryos at E11.5. (D) FC analysis identifying Coup-TFII in EryP (ey+γ+) in blood at E11.5. FSC: forward scatter; EMP: erythro-myeloid progenitors.
The overall percentage of Coup-TFII+ cells in the E11.5 fetal liver is about 2% (Figure 1C). Only approximately 0.63% of EryP cells (i.e., ey+γ+) cells express Coup-TFII, whereas approximately 12.2% of the rare (0.06%) EMP population, identified as CD41+/CD117+/CD16/32+ cells,7 express it (Figure 1C). In blood, about 97.7% of cells are ey+γ+, but only a small percentage of them (1.79%) also express Coup-TFII (Figure 1D). This suggests asynchrony between Coup-TFII expression and hemoglobin synthesis, Coup-TFII being predominantly expressed at early stages and then declining during maturation and hemoglobiniza- tion. Accordingly, the few ey+γ+ Coup-TFII+ cells found in the fetal liver could possibly represent EryP progenitors undergoing maturation.
Coup-TFII overexpression activates embryonic/fetal glo- bin genes in adult cells
To test whether Coup-TFII activates embryonic/fetal globin genes, we first reintroduced Coup-TFII into fetal liver cells isolated from mouse embryos transgenic for the human β-locus at E13.5, when Coup-TFII expression is already greatly reduced (Figure 1). We transduced the expanded proerythroblasts from E13.5 fetal liver with viral particles carrying either the Coup-TFII-DNGFR overex- pressing vector, or the empty vector EV-DNGFR control (Online Supplementary Figure S1). As shown in Figure 2A, Coup-TFII significantly increased human γ-globin and mouse βH1 expression, without affecting the expression of mouse (Hbb-b1/2) and human (HBB) adult globins. This resulted in a significantly increased γ/(γ+β) globin ratio. No major changes were observed in α-like mouse globin genes (Online Supplementary Figure S2).
Statistical analysis
Data are expressed as the mean ± standard error of mean of ≥3 replicates and were analyzed using a paired two-tailed Student t- test (GraphPad Prism).
Results
Coup-TFII expression declines during the time of hemoglobin switching
To gain insight into the role of Coup-TFII in the process of hemoglobin switching, we first analyzed its expression in mouse fetal liver at E11.5-E13.5, i.e., in the timeframe during which globin switching takes place. Coup-TFII is expressed at E11.5 and both its protein and RNA levels decline sharply (Figure 1A, B) at E12.5-E13.5; its expression profile follows that of the decline of embryonic mouse Hbb-bh1/Hbb-y globins and the parallel accumulation of adult Hbb-b1/2 globins (Figure 1B). This trend is consistent with a potential role of Coup-TFII as an activator of embryonic globins, which is the subject of this work.
In order to investigate the role(s) of Coup-TFII in embry- onic/fetal to adult hemoglobin switching in greater detail, we took advantage of a mouse model that is transgenic for the entire human HBB locus,30 in which the human γ to β switch occurs in parallel to the embryonic/adult switch of the murine genes.
In E11.5 fetal liver, erythropoiesis is sustained by EryP cells derived from the yolk sac which express murine embryonic eY and βH1 globins and human transgenic e- and γ-globins,8 and by EMP that will generate the first definitive red blood cells expressing adult β-globins.7,8
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haematologica | 2021; 106(2)