Figure 4
Global changes in H3K27me3 are associated with cell context-specific gene expression changes. (a) Immunoblotting of EZH2 and H3K27me3 from whole-cell lysates following 6 days’ incubation with EPZ005687 across the panel of eight cell lines. Actin and total H3 were used as the loading controls, respectively. (b) Fold change in mRNA levels measured by qRT-PCR in EPZ005687-treated cell lines at 6 days, compared with DMSO control. Graphs show mean and s.e.m. for at least three independent replicate experiments. GAPDH was used as the internal control. CDKN2B in JIM3 cells and IFIT3 in KMS12BM cells were not expressed at levels to allow reliable quantification of any change in the expression. Additional gene expression results are shown in Supplementary Figure S8. (c) Putative mechanism of action of EZH2 in myeloma cell lines. The upper part of the diagram demonstrates the change in chromatin structure in the presence of active vs inhibited EZH2, resulting in a change in the methylation status of H3K27. Where EZH2 is not inhibited, H3K27me3 is high and chromatin structure is closed preventing gene transcription. EZH2 inhibition removes methyl marks, chromatin relaxes and genes affected by the H3K27me3 mark are able to be transcribed. We show that CDKN1A and CDKN2B may be directly under the control of H3K27me3 or their transcription might be altered as a downstream result of the expression of another gene being altered. The lower part of the diagram demonstrates the cyclin D/CDK and cyclin E/CDK complexes driving cell proliferation in myeloma at the G1/S checkpoint. Upregulation of the CDK inhibitors can inhibit these complexes preventing passage of cells from G1 to S phase as shown.
