Figure 7.
Figure 7. Alterations in H3K27me3 upon the loss of Ezh2 results in the activation of specific oncogenes. (a) Fold enrichment (ChIP/input) values of H3K27me3 (TSS ± 2.0 kb of RefSeq genes) in WT and JAK2V617F/Ezh2Δ/Δ LSKs 4 wk after the deletion of Ezh2. (b) A scatter plot showing the relationship of fold enrichment (ChIP/input) values of H3K27me3 (TSS ± 2.0 kb of RefSeq genes) between WT and JAK2V617F/Ezh2Δ/Δ LSKs 4 wk after the deletion of Ezh2. The light diagonal line represents the borderlines for twofold changes in H3K27me3 levels. (c) Fold enrichment (ChIP/input) values of H3K27me3 at TSS ± 2.0 kb of MkP signature genes in WT and JAK2V617F/Ezh2Δ/Δ LSKs. (d) A scatter plot showing the relationship of expression of RefSeq genes in JAK2V617F/Ezh2Δ/Δ LSKs relative to WT LSKs and H3K27me3 levels of RefSeq genes in JAK2V617F/Ezh2Δ/Δ LSKs relative to WT LSKs and potential oncogenes activated in JAK2V617F/Ezh2Δ/Δ LSKs (see also Table S2). (e) ChIP-seq view of H3K27me3 levels at the promoter region of Hmga2 in WT and JAK2V617F/Ezh2Δ/Δ LSKs 4 wk after the deletion of Ezh2. (f) qRT-PCR analysis of Hmga2 in LSKs and MEPs from WT, Ezh2Δ/Δ, JAK2V617F, and JAK2V617F/Ezh2Δ/Δ (n = 3–4) mice 1–2 mo after the deletion of Ezh2. (g) The total cell counts of WT (black lines) and JAK2V617F (red lines) HSCs transduced with either a control (straight lines) or an Hmga2 (broken lines) retrovirus were monitored for 8 d. (h) Representative pictures of Hmga2-transduced or control vector–transduced WT and JAK2V617F HSCs on day 8 of the culture observed by May-Grünewald-Giemsa staining. Bars: 10 µm (high magnitude); 100 µm (low magnitude). (i) Proportions of megakaryocytes in Hmga2-transduced or control vector–transduced WT and JAK2V617F HSCs on day 8 of the culture. (a and c) Boxes and whiskers show the mean and minimum to maximum, and asterisks show ***, P < 0.001 by the Student’s t test; experiments used cells from two to four individual mice per genotype. (f, g, and i) Bars and asterisks show the mean ± SEM and *, P < 0.05; **, P < 0.01; and ***, P < 0.001 by the Student’s t test; two independent experiments.

Alterations in H3K27me3 upon the loss of Ezh2 results in the activation of specific oncogenes. (a) Fold enrichment (ChIP/input) values of H3K27me3 (TSS ± 2.0 kb of RefSeq genes) in WT and JAK2V617F/Ezh2Δ/Δ LSKs 4 wk after the deletion of Ezh2. (b) A scatter plot showing the relationship of fold enrichment (ChIP/input) values of H3K27me3 (TSS ± 2.0 kb of RefSeq genes) between WT and JAK2V617F/Ezh2Δ/Δ LSKs 4 wk after the deletion of Ezh2. The light diagonal line represents the borderlines for twofold changes in H3K27me3 levels. (c) Fold enrichment (ChIP/input) values of H3K27me3 at TSS ± 2.0 kb of MkP signature genes in WT and JAK2V617F/Ezh2Δ/Δ LSKs. (d) A scatter plot showing the relationship of expression of RefSeq genes in JAK2V617F/Ezh2Δ/Δ LSKs relative to WT LSKs and H3K27me3 levels of RefSeq genes in JAK2V617F/Ezh2Δ/Δ LSKs relative to WT LSKs and potential oncogenes activated in JAK2V617F/Ezh2Δ/Δ LSKs (see also Table S2). (e) ChIP-seq view of H3K27me3 levels at the promoter region of Hmga2 in WT and JAK2V617F/Ezh2Δ/Δ LSKs 4 wk after the deletion of Ezh2. (f) qRT-PCR analysis of Hmga2 in LSKs and MEPs from WT, Ezh2Δ/Δ, JAK2V617F, and JAK2V617F/Ezh2Δ/Δ (n = 3–4) mice 1–2 mo after the deletion of Ezh2. (g) The total cell counts of WT (black lines) and JAK2V617F (red lines) HSCs transduced with either a control (straight lines) or an Hmga2 (broken lines) retrovirus were monitored for 8 d. (h) Representative pictures of Hmga2-transduced or control vector–transduced WT and JAK2V617F HSCs on day 8 of the culture observed by May-Grünewald-Giemsa staining. Bars: 10 µm (high magnitude); 100 µm (low magnitude). (i) Proportions of megakaryocytes in Hmga2-transduced or control vector–transduced WT and JAK2V617F HSCs on day 8 of the culture. (a and c) Boxes and whiskers show the mean and minimum to maximum, and asterisks show ***, P < 0.001 by the Student’s t test; experiments used cells from two to four individual mice per genotype. (f, g, and i) Bars and asterisks show the mean ± SEM and *, P < 0.05; **, P < 0.01; and ***, P < 0.001 by the Student’s t test; two independent experiments.

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