Figure 4.
AEL-associated transcription factors transform erythroid progenitors and impair GATA1 activity. (A) Experimental design: mouse erythroid progenitors (CD71+Ter119+KIT+/low) were sorted from lineage marker–depleted BM, transduced with retrovirus encoding SKI, ERG, ETO2, GATA1s, EPO, SPI1, or B4GALNT3 combined with IRES-GFP (GFP expression is a surrogate marker for transgene expression), or an empty vector (Ctrl), and maintained in StemSpan SFEM with cytokines (mSCF, mIL3, mIL6, hEPO, cholesterol, and dexamethasone). (B) A total of 5 × 104 transduced mouse erythroid progenitors were cultured for 15 days and viable cells were enumerated by trypan-blue exclusion. Mean plus or minus SD number of cells is represented. SKI (n = 5), ERG (n = 4), ETO2 (n = 3), GATA1s (n = 3), EPO (n = 3), SPI1 (n = 6), B4GALNT3 (n = 3), or empty vector (Ctrl) (n = 5). (C) Experimental design: erythroid progenitors (CD71+ Ter119+ KIT+/low) from WT, Tet2−/−, Gata1s (G1s), or Tet2−/−+ Gata1s (Tet2−/−+G1s) mice were sorted from lineage marker–depleted BM and maintained for 15 days in StemSpan SFEM with cytokines (mSCF, mIL3, mIL6, hEPO, cholesterol, and dexamethasone). (D) A total of 3.5 × 104 sorted erythroblasts from WT, Tet2−/−, Gata1s (G1s), or Tet2−/−+ Gata1s (Tet2−/−+G1s) mice were grown in liquid cultures over 15 days and viable cells were counted by trypan-blue exclusion. Mean plus or minus SD (n = 3) is shown. (E) Dot-plot showing the log(fold changes) of the percentage of sequence for a given motif found under ATAC-seq peaks, between normal and transformed erythroblasts (expressing ERG, ETO2, SKI, and SPI1). (F) Histogram representation of the percentage of sequence with GATA1 (top) or ERG (bottom) motif found under ATAC-seq peaks of normal (Ctrl) and transformed erythroblasts. Statistical differences were calculated using the χ2 test. For GATA1 motif, Ctrl vs ERG: P = .0013; Ctrl vs ETO2: P = .0001; Ctrl vs SKI: P < .00001; Ctrl vs SPI1: P = .0032. For ERG motif, Ctrl vs ERG: P = .00001; Ctrl vs ETO2: P < .00001; Ctrl vs SKI: P = .0498; Ctrl vs SPI1: P = .0415. (G) Heatmap representing the hierarchical clustering of ATAC-seq signals, performed using normal (Ctrl) and transformed erythroblast by either ERG, ETO2, SKI, or SPI1, focused on GATA1-binding sites in normal Ter119+ erythroblast (ENCODE). Heatmaps were focused on peak centers with ±5 kb. (H) Profile plot representing ATAC-seq signals performed using normal (Ctrl) and transformed erythroblast expressing either ERG, ETO2, SKI, or SPI1 on ATAC-seq specific peaks previously identified in mouse MEP, CFU-E, or proerythroblasts (proE).35 Profile-plot were focused on peaks centers with ±5 kb. (I) Visualization of GATA1 ChIP-seq peaks performed in normal erythroblast (ENCODE, first lane, gray) and ATAC-seq peaks of normal MEP, proerythroblast, orthochromatic erythroblast, and transformed erythroblasts expressing ERG, ETO2, SKI, or SPI1, focused on Nfe2, Hba-a1, and Hbq-a1 genes, using IGV software (v 2.3.88). (J) Quantitative Nfe2 mRNA expression measured by RT-qPCR in WT erythroblast (Ctrl) or erythroblast transformed with either SKI, ERG, ETO2, or SPI1 overexpression. Expression levels were also compared with the erythroleukemia MEL or the Ba/F3 myelolymphoid cell line. (K) Experimental design: G1E cell lines were cotransduced with a GATA1 doxycycline-inducible vector and with MSCV-vector expressing either ERG, ETO2, SKI, SPI1, or empty. (L) Flow cytometry histogram analysis of Ter119 expression in G1E cells expressing ERG (green), ETO2 (orange), SKI (blue), SPI1 (red), or empty control (gray), without (left) or with (right) induction of GATA1 expression. (M) Histogram representation of Ter119 expression detected by flow cytometry analysis of G1E cells expressing ERG, ETO2, SKI, SPI1, or empty control, without (gray) or with (red) induction of GATA1 expression. Statistical significance (in panels B, D, J, and M) is indicated as P values (Student t test except when otherwise specified). *P < .05; **P < .01; ***P < .001.

AEL-associated transcription factors transform erythroid progenitors and impair GATA1 activity. (A) Experimental design: mouse erythroid progenitors (CD71+Ter119+KIT+/low) were sorted from lineage marker–depleted BM, transduced with retrovirus encoding SKI, ERG, ETO2, GATA1s, EPO, SPI1, or B4GALNT3 combined with IRES-GFP (GFP expression is a surrogate marker for transgene expression), or an empty vector (Ctrl), and maintained in StemSpan SFEM with cytokines (mSCF, mIL3, mIL6, hEPO, cholesterol, and dexamethasone). (B) A total of 5 × 104 transduced mouse erythroid progenitors were cultured for 15 days and viable cells were enumerated by trypan-blue exclusion. Mean plus or minus SD number of cells is represented. SKI (n = 5), ERG (n = 4), ETO2 (n = 3), GATA1s (n = 3), EPO (n = 3), SPI1 (n = 6), B4GALNT3 (n = 3), or empty vector (Ctrl) (n = 5). (C) Experimental design: erythroid progenitors (CD71+ Ter119+ KIT+/low) from WT, Tet2−/−, Gata1s (G1s), or Tet2−/−+ Gata1s (Tet2−/−+G1s) mice were sorted from lineage marker–depleted BM and maintained for 15 days in StemSpan SFEM with cytokines (mSCF, mIL3, mIL6, hEPO, cholesterol, and dexamethasone). (D) A total of 3.5 × 104 sorted erythroblasts from WT, Tet2−/−, Gata1s (G1s), or Tet2−/−+ Gata1s (Tet2−/−+G1s) mice were grown in liquid cultures over 15 days and viable cells were counted by trypan-blue exclusion. Mean plus or minus SD (n = 3) is shown. (E) Dot-plot showing the log(fold changes) of the percentage of sequence for a given motif found under ATAC-seq peaks, between normal and transformed erythroblasts (expressing ERG, ETO2, SKI, and SPI1). (F) Histogram representation of the percentage of sequence with GATA1 (top) or ERG (bottom) motif found under ATAC-seq peaks of normal (Ctrl) and transformed erythroblasts. Statistical differences were calculated using the χ2 test. For GATA1 motif, Ctrl vs ERG: P = .0013; Ctrl vs ETO2: P = .0001; Ctrl vs SKI: P < .00001; Ctrl vs SPI1: P = .0032. For ERG motif, Ctrl vs ERG: P = .00001; Ctrl vs ETO2: P < .00001; Ctrl vs SKI: P = .0498; Ctrl vs SPI1: P = .0415. (G) Heatmap representing the hierarchical clustering of ATAC-seq signals, performed using normal (Ctrl) and transformed erythroblast by either ERG, ETO2, SKI, or SPI1, focused on GATA1-binding sites in normal Ter119+ erythroblast (ENCODE). Heatmaps were focused on peak centers with ±5 kb. (H) Profile plot representing ATAC-seq signals performed using normal (Ctrl) and transformed erythroblast expressing either ERG, ETO2, SKI, or SPI1 on ATAC-seq specific peaks previously identified in mouse MEP, CFU-E, or proerythroblasts (proE).35  Profile-plot were focused on peaks centers with ±5 kb. (I) Visualization of GATA1 ChIP-seq peaks performed in normal erythroblast (ENCODE, first lane, gray) and ATAC-seq peaks of normal MEP, proerythroblast, orthochromatic erythroblast, and transformed erythroblasts expressing ERG, ETO2, SKI, or SPI1, focused on Nfe2, Hba-a1, and Hbq-a1 genes, using IGV software (v 2.3.88). (J) Quantitative Nfe2 mRNA expression measured by RT-qPCR in WT erythroblast (Ctrl) or erythroblast transformed with either SKI, ERG, ETO2, or SPI1 overexpression. Expression levels were also compared with the erythroleukemia MEL or the Ba/F3 myelolymphoid cell line. (K) Experimental design: G1E cell lines were cotransduced with a GATA1 doxycycline-inducible vector and with MSCV-vector expressing either ERG, ETO2, SKI, SPI1, or empty. (L) Flow cytometry histogram analysis of Ter119 expression in G1E cells expressing ERG (green), ETO2 (orange), SKI (blue), SPI1 (red), or empty control (gray), without (left) or with (right) induction of GATA1 expression. (M) Histogram representation of Ter119 expression detected by flow cytometry analysis of G1E cells expressing ERG, ETO2, SKI, SPI1, or empty control, without (gray) or with (red) induction of GATA1 expression. Statistical significance (in panels B, D, J, and M) is indicated as P values (Student t test except when otherwise specified). *P < .05; **P < .01; ***P < .001.

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