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Coup-TFII activates γ-globin
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Figure 2. Coup-TFII overexpression increases γ- (and βh1)-globin expression. (A) Quantitative real-time polymerase reaction (RTqPCR) analysis of globin gene expres- sion using cDNA from erythroblasts derived from E13.5 ex-vivo fetal liver erythroid cultures transduced with either empty vector (EV) or with the Coup-TFII-overex- pressing lentiviral particles (Coup-TFII). Left panel: the relative fold-change on the control is shown (error bars: standard error of mean; *P<0.05, **P<0.01). Expression levels on Gapdh are shown in Online Supplementary Figure S2. Middle panel: the same data are expressed as γ to β ratio (γ+β =1). Right panel: RTqPCR analysis for mouse globin genes. In all experiments >90% cells were transduced as assessed by flow cytometry (FC) analysis for DNGFR expression (Online Supplementary Figure S2). (B) Coup-TFII overexpression in both healthy and thalassemic β039 ex-vivo peripheral blood cultures. The analysis was carried out as in (A). In all experiments, cells were magnetically purified on the basis of DNGFR expression (>85% cells were isolated as assessed by FC analysis on DNGFR expression (Online Supplementary Figure S3). Expression levels on Gapdh are shown in Online Supplementary Figure S3. (C) FC analysis on CD71 and CD235ab (GpA).
This result prompted us to overexpress Coup-TFII in the context of human adult cells, in cultures from periph- eral blood from healthy donors and from thalassemic patients carrying the Sardinian β039 mutation, which gen- erates an in-frame stop codon resulting in β-globin mRNA degradation by nonsense mediated decay.31,32 As shown in Figure 2B, Coup-TFII overexpression in normal cells led to a 7-fold increase in γ-globin RNA expression, with no significant change in β-globin expression. Similarly, a 3- fold activation of γ-globin was also seen in β039 cells fol- lowing COUP-TFII overexpression. Of interest, in β039 thalassemic, but not in wild-type cultures, the increase in γ expression was accompanied by a decrease in the level of the residual β RNA produced from the β039 mutation- carrying alleles, which we tested as a control. Overexpressing Coup-TFII in these cells did not result in statistically significant maturational changes (CD71/GpA profiles in Figure 2C).
Generation of overexpressing and knockout β-K562 cells Together, the above data indicate that Coup-TFII is an activator of γ-globin. To better characterize the role of Coup-TFII at the molecular level, we took advantage of β- K562 cells, a K562 subclone that, to our knowledge, is the only erythroid cell model system that expresses Coup- TFII together with e-, γ- and β-globins18 (Online Supplementary Figure S4). We used these cells to either knockout by CRISPR/Cas9 (KO cells) or overexpress by lentiviral transduction (OE cells) Coup-TFII (Figure 3A). In OE cells, we observed an increase in embryonic HBE and HBG and a decrease in adult HBA and HBB (Figure 3B). By contrast, in KO cells there was a general induction of all globin genes transcription, resulting in increased hemoglo- binization (Figure 3B and Online Supplementary Figure S6A). In this context, β-globin expression increased by an aver- age of 45-fold, whereas the increases in α-, e- and γ-glo- bins was about 10 times lower (Figure 3B). This differen-
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