Figure 4.
PLZF-RARA-induced H3K27me3 level at specific enhancers genes that marks RA relapse-initiating cells. (A) Enhancer distribution in PLZF-RARA promyelocytes and normal GMPs. Overlap of Active (left) and Poised enhancers (right) in GMP and PLZF-RARA (P-RARA) conditions. ∗∗∗P < .001. The Poised enhancer dynamics upon PLZF-RARA expression is schematized below. Triangles represent enhancers; colors indicate their activity (Active: blue, Poised: red). Empty triangles: no change in enhancer activity between the 2 conditions. (B) Violin plots showing “switched” and “de novo” Poised signature scores per cluster. Signature score represents the global expression of annotated genes for the signature identified by scRNA-seq. Gray arrow pointing down: significantly lower expression in the cluster against all the others (average score difference > 0.005 and adjusted P < .05). (C) GO analysis of enhancer nearby genes. Gene %, number of genes observed/total number of genes within each GO term, BP, biological processes. (D) Experimental scheme to assess chromatin events associated with RA resistance. PLZF-RARA TG BM is transplanted into recipient mice. Ten days after, mice are injected with corn oil (NT) or with RA for 3 or 7 days (d3 and d7) and sacrificed. Treated BMs are immunophenotyped and reinjected into new recipient mice (tNT, td3, td7). Secondary transplanted mice are not treated (Ø) and sacrificed at day 17 posttransplantation. (E) Leukemia evolution analyzed by MGG staining (magnification 64×), spleen size (bar in centimeter), and FACS (Cd45.2, Cd11b, and Gr1 are monitored). (Top) Impact of RA on PLZF-RARA leukemia (NT, d3, d7). (Bottom) Leukemia relapse evaluation of transplanted untreated (tNT) and RA-treated BM (td3, td7). (F) (Left) Global levels of PLZF-RARA (black arrow: full length, black star: degraded form), EZH2, and H3K27me3. Actin and H3 are used as loading controls. Signal intensity is normalized according to the loading control and to the NT. (Right) Bar plots representing the signal intensity of PLZF-RARA (P-RARA), EZH2, and H3K27me3 normalized to the loading control and to the NT condition. ∗P < .05, ∗∗P < .01 (2 biological replicates). (G) Representative integrative genomics viewer (IGV) tracks of H3K27me3 in NT, d7, and td7 conditions. The gray box underlines enhancer coordinates. (H) Total number of poised enhancers in each condition. (I) Plot heat map of H3K27ac (blue) and H3K27me3 (red) signals in GMP, NT, d7, and td7 conditions at switched enhancers. Signals are plotted 10 Kb (for H3K27ac) and 0.1 Mb (for H3K27me3) upstream and downstream the enhancer center.

PLZF-RARA-induced H3K27me3 level at specific enhancers genes that marks RA relapse-initiating cells. (A) Enhancer distribution in PLZF-RARA promyelocytes and normal GMPs. Overlap of Active (left) and Poised enhancers (right) in GMP and PLZF-RARA (P-RARA) conditions. ∗∗∗P < .001. The Poised enhancer dynamics upon PLZF-RARA expression is schematized below. Triangles represent enhancers; colors indicate their activity (Active: blue, Poised: red). Empty triangles: no change in enhancer activity between the 2 conditions. (B) Violin plots showing “switched” and “de novo” Poised signature scores per cluster. Signature score represents the global expression of annotated genes for the signature identified by scRNA-seq. Gray arrow pointing down: significantly lower expression in the cluster against all the others (average score difference > 0.005 and adjusted P < .05). (C) GO analysis of enhancer nearby genes. Gene %, number of genes observed/total number of genes within each GO term, BP, biological processes. (D) Experimental scheme to assess chromatin events associated with RA resistance. PLZF-RARA TG BM is transplanted into recipient mice. Ten days after, mice are injected with corn oil (NT) or with RA for 3 or 7 days (d3 and d7) and sacrificed. Treated BMs are immunophenotyped and reinjected into new recipient mice (tNT, td3, td7). Secondary transplanted mice are not treated (Ø) and sacrificed at day 17 posttransplantation. (E) Leukemia evolution analyzed by MGG staining (magnification 64×), spleen size (bar in centimeter), and FACS (Cd45.2, Cd11b, and Gr1 are monitored). (Top) Impact of RA on PLZF-RARA leukemia (NT, d3, d7). (Bottom) Leukemia relapse evaluation of transplanted untreated (tNT) and RA-treated BM (td3, td7). (F) (Left) Global levels of PLZF-RARA (black arrow: full length, black star: degraded form), EZH2, and H3K27me3. Actin and H3 are used as loading controls. Signal intensity is normalized according to the loading control and to the NT. (Right) Bar plots representing the signal intensity of PLZF-RARA (P-RARA), EZH2, and H3K27me3 normalized to the loading control and to the NT condition. ∗P < .05, ∗∗P < .01 (2 biological replicates). (G) Representative integrative genomics viewer (IGV) tracks of H3K27me3 in NT, d7, and td7 conditions. The gray box underlines enhancer coordinates. (H) Total number of poised enhancers in each condition. (I) Plot heat map of H3K27ac (blue) and H3K27me3 (red) signals in GMP, NT, d7, and td7 conditions at switched enhancers. Signals are plotted 10 Kb (for H3K27ac) and 0.1 Mb (for H3K27me3) upstream and downstream the enhancer center.

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