Genetic inactivation of the polycomb repressive complex 2 in T cell acute lymphoblastic leukemia

Abstract

T cell acute lymphoblastic leukemia (T-ALL) is an immature hematopoietic malignancy driven mainly by oncogenic activation of NOTCH1 signaling. In this study we report the presence of loss-of-function mutations and deletions of the EZH2 and SUZ12 genes, which encode crucial components of the Polycomb repressive complex 2 (PRC2), in 25% of T-ALLs. To further study the role of PRC2 in T-ALL, we used NOTCH1-dependent mouse models of the disease, as well as human T-ALL samples, and combined locus-specific and global analysis of NOTCH1-driven epigenetic changes. These studies demonstrated that activation of NOTCH1 specifically induces loss of the repressive mark Lys27 trimethylation of histone 3 (H3K27me3) by antagonizing the activity of PRC2. These studies suggest a tumor suppressor role for PRC2 in human leukemia and suggest a hitherto unrecognized dynamic interplay between oncogenic NOTCH1 and PRC2 function for the regulation of gene expression and cell transformation.

Figure 1

The PRC2 complex as a tumor suppressor in T-ALL. (a) Structure of the EZH2 protein including 2 SANT DNA binding domains, the cysteine-rich CXC domain and the catalytic SET domain. Overview of all EZH2 mutations identified in primary T-ALL samples. Filled circles: nonsense and frameshift mutations, open circles: missense mutations. (b) Representative chromatograms of paired diagnosis and remission genomic DNA samples showing somatic mutations in exon 8 and exon 10 of EZH2. (c) Structure of the SUZ12 protein including a zinc finger domain and the VEFS domain. (d) Representative DNA sequencing chromatograms showing a frame-shift mutation in exon 10 of SUZ12 and of paired diagnosis and remission genomic DNA samples showing a somatic point mutation in exon 14 of this gene. (e) Pie-chart summarizing the frequencies of homozygous and heterozygous mutations of EZH2 and SUZ12 in adult TALL patients. (f) EZH2 protein levels in samples from patients (#1 and #4) with mutations and deletions on the EZH2 gene compared to WT controls. (g) Silencing of EZH2 and SUZ12 in the Jurkat human T-ALL line. HES1 and DTX1 mRNA expression levels followed silencing of either SUZ12 or EZH2. Knockdown of the luciferase (LUC) gene was used as a control.

Figure 2

Notch1-induced epigenetic changes in an in-vivo model of T-ALL. (a) Comparison of the size of the thymus in leukemic animals and control littermates. (b) Cell populations used in this study. (c) Western blot showing the expression of N1-IC in DP and T-ALL cells. (d) Hes1 cDNA levels between DP and T-ALL. (e, f) ChIP of Notch1 and Pol II on the Hes1 promoter in the indicated cell populations. (g-j) ChIP of the indicated histone marks and S2-P-Pol II in DP and T-ALL cells.

Figure 3

Characterization of T-ALL epigenetic landscape using ChIP-Seq for H3K9ac, H3K4me3 and H3K27me3. (a) Cluster of the major gene expression changes between T-ALL and DP and the accompanied epigenetic changes. Left part: Expression heatmap representing up (red)- and down (blue)-regulated genes with significant epigenetic changes. Right panel: Heatmap representation of the epigenetic marks in T-ALL and DP in TSSs of selected genes. “+”:gain and “−”:loss in the levels of epigenetic mark in T-ALL versus DP. Loss of H3K27me3 (P=6.91x10⁻²², blue bar) and gain of H3K9ac (P=1.52x10⁻⁴, green bar) are enriched in up-regulated genes, whereas loss of H3K9ac (P=2.79x10⁻¹⁹, red bar) is enriched in down-regulated genes. (b) Bar graphs indicate the percentage of genes characterized by each modification in T-ALL cells. The plus and minus signs are used as above. Pink and blue frames: prevalent epigenetic clusters in down-regulated and up-regulated genes, respectively. (c) Functional annotation of epigenetic changes (T-ALL vs DP) in H3K9ac (yellow bars) and H3K4me3 (gray bars) shows enrichment in specific biological processes. (d) ChIP-Seq results for two well-characterized Notch1 targets, Hes1 and Dtx1 (arrows denote the TSS).

Figure 4

Notch1 binding mediates loss of H3K27me3 and eviction of PRC2 in T-ALL. (a) Enrichment of Notch1 binding sites around TSSs characterized by each indicated histone mark. (b) H3K27me3 average signal profiles around TSS areas (blue line: Notch1-bound genes, gray line: genes not-bound by Notch1). (c) ChIP for H3K27me3 in a T-ALL cell line (CUTLL1) treated with γSI. The Loucy T-ALL line is used as a negative control. (d) HES1 expression in the indicated cell lines and normal human thymocytes and ChIP for H3K27me3 in the indicated cell lines and primary cells. (e) High leukemogenic potential of the human T-ALL samples in xenograft models. Spleen sections of recipient mice stained with an hCD45 antibody or an IgG control. (f) qPCR for HES1 expression and the levels of H3K27me3 in primary human T-ALL samples and human thymocytes (P<0.0001 between M69 and the human thymus). (g) ChIP experiments for Ezh2 on the Hes1 promoter in DP (green) and T-ALL (red). (h) Suz12 binding on the Hes1 promoter. (i)γSI-mediated changes of the N1-IC levels modulate JARID2 recruitment to the HES1 promoter (P=0.059).