Figure 3.
EZH2 relevance in PLZF-RARA APL. (A) Violin plot showing Ezh2 expression per cluster (top) and in NT or RA-treated cell conditions per cluster (bottom). The black arrow pointing up indicate significant higher expression in the cluster against all the other clusters (average log2|FC| > .25 and adjusted P < .05). (B) Coverage plot of ATAC signal on Ezh2 gene in the ReP cluster. E2f1, E2f4, and Tfdp1 motifs are detected under the highlighted peak. (C) Experimental scheme to ascertain whether Ezh2 activity is required for PLZF-RARA transformation. Lineage negative (Lin−) cells purified from Cre-ERT;Ezh2fl/fl are purified and transduced with an empty-IRES-GFP (IRES) or a PLZF-RARA-IRES-GFP (P-RARA) retroviral construct. Lin− green fluorescent protein (GFP)–positive cells are purified by FACS and Ezh2 deletion in Lin-GFP positive cells is obtained by adding 150 nM 4-hydroxytamoxifen (4-OHT) in the methylcellulose. (D) (Left) Global levels of PLZF-RARA (black arrow: full length, black star: degraded form), Ezh2 and H3K27me3 detected by western blotting after 4-OHT-induced Ezh2 deletion in the second round of plating. Actin and H3 are used as loading controls. (Right) Bar plots representing the signal intensity of PLZF-RARA (P-RARA), EZH2, and H3K27me3 normalized to the loading control (for P-RARA) and to the IRES condition (for EZH2 and H3K27me3). ns, not significant. ∗P < .05 (2 replicates). (E) Replating efficiency is monitored by counting the total colony-forming units (CFU) of nontransformed (IRES) and PLZF-RARA–transformed (P-RARA) cells in presence (fl/fl) or absence (Δ/Δ) of Ezh2. Results are expressed as a mean standard deviation of 3 experiments (n = 3). ∗P < .05. (F) Cell morphology of P-RARA or IRES-transduced cells in presence (fl/fl) or absence (Δ/Δ) of Ezh2. Representative colonies of indicated conditions after 2 rounds of plating. Cells are cytospun and observed after May-Grünwald Giemsa (MGG) staining. Magnification 64×; bar 10 μm. (G) (Left) Experimental scheme to ascertain whether Ezh2 activity is required for PLZF-RARA leukemia development in vivo. PLZF-RARA TG BM is transduced with an shCtrl-GFP or an shEZH2-GFP retroviral construct. GFP-positive cells are purified by FACS and reinjected into recipient mice. (Right) Survival rate of mice transplanted with shCtrl (gray curve) or shEZH2 cells (green curve). Each cohort contains 5 mice. ∗∗∗P < .001. (H) Nuclear extracts of U937 cells treated or not with ZnSO4 (Zn) immunoprecipitated with anti-immunoglobulin G, anti-EZH2, or anti-SUZ12 antibodies. Immunoprecipitations (IPs) are immunoblotted with anti-PLZF (top) or anti-EZH2 antibodies (bottom). Inputs (in) represent 2% of samples processed in each IP. U937 B412: PLZF-RARA Zn-inducible; U937 MT: parental cells; U937 PR9: PML-RARA Zn-inducible. (I) Relative luciferase intensity monitored in HEK293T transduced or not (NT) with a shEZH2 (left) or transfected with a siE2F1 or siCtrl (NT) (right). Cells were transfected with the RARE-Luc and with a GFP (ᴓ P-RARA) or PLZF-RARA (P-RARA) construct. Cells are treated or not for 48 hours with RA (1 μM). AU, arbitrary units. ∗P < .05, ∗∗P < .01 (n = 6).

EZH2 relevance in PLZF-RARA APL. (A) Violin plot showing Ezh2 expression per cluster (top) and in NT or RA-treated cell conditions per cluster (bottom). The black arrow pointing up indicate significant higher expression in the cluster against all the other clusters (average log2|FC| > .25 and adjusted P < .05). (B) Coverage plot of ATAC signal on Ezh2 gene in the ReP cluster. E2f1, E2f4, and Tfdp1 motifs are detected under the highlighted peak. (C) Experimental scheme to ascertain whether Ezh2 activity is required for PLZF-RARA transformation. Lineage negative (Lin−) cells purified from Cre-ERT;Ezh2fl/fl are purified and transduced with an empty-IRES-GFP (IRES) or a PLZF-RARA-IRES-GFP (P-RARA) retroviral construct. Lin− green fluorescent protein (GFP)–positive cells are purified by FACS and Ezh2 deletion in Lin-GFP positive cells is obtained by adding 150 nM 4-hydroxytamoxifen (4-OHT) in the methylcellulose. (D) (Left) Global levels of PLZF-RARA (black arrow: full length, black star: degraded form), Ezh2 and H3K27me3 detected by western blotting after 4-OHT-induced Ezh2 deletion in the second round of plating. Actin and H3 are used as loading controls. (Right) Bar plots representing the signal intensity of PLZF-RARA (P-RARA), EZH2, and H3K27me3 normalized to the loading control (for P-RARA) and to the IRES condition (for EZH2 and H3K27me3). ns, not significant. ∗P < .05 (2 replicates). (E) Replating efficiency is monitored by counting the total colony-forming units (CFU) of nontransformed (IRES) and PLZF-RARA–transformed (P-RARA) cells in presence (fl/fl) or absence (Δ/Δ) of Ezh2. Results are expressed as a mean standard deviation of 3 experiments (n = 3). ∗P < .05. (F) Cell morphology of P-RARA or IRES-transduced cells in presence (fl/fl) or absence (Δ/Δ) of Ezh2. Representative colonies of indicated conditions after 2 rounds of plating. Cells are cytospun and observed after May-Grünwald Giemsa (MGG) staining. Magnification 64×; bar 10 μm. (G) (Left) Experimental scheme to ascertain whether Ezh2 activity is required for PLZF-RARA leukemia development in vivo. PLZF-RARA TG BM is transduced with an shCtrl-GFP or an shEZH2-GFP retroviral construct. GFP-positive cells are purified by FACS and reinjected into recipient mice. (Right) Survival rate of mice transplanted with shCtrl (gray curve) or shEZH2 cells (green curve). Each cohort contains 5 mice. ∗∗∗P < .001. (H) Nuclear extracts of U937 cells treated or not with ZnSO4 (Zn) immunoprecipitated with anti-immunoglobulin G, anti-EZH2, or anti-SUZ12 antibodies. Immunoprecipitations (IPs) are immunoblotted with anti-PLZF (top) or anti-EZH2 antibodies (bottom). Inputs (in) represent 2% of samples processed in each IP. U937 B412: PLZF-RARA Zn-inducible; U937 MT: parental cells; U937 PR9: PML-RARA Zn-inducible. (I) Relative luciferase intensity monitored in HEK293T transduced or not (NT) with a shEZH2 (left) or transfected with a siE2F1 or siCtrl (NT) (right). Cells were transfected with the RARE-Luc and with a GFP (ᴓ P-RARA) or PLZF-RARA (P-RARA) construct. Cells are treated or not for 48 hours with RA (1 μM). AU, arbitrary units. ∗P < .05, ∗∗P < .01 (n = 6).

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