Fig 2: Schematic of DRAIR dysregulation in diabetes and epigenetic mechanisms of DRAIR actions.Under normal physiological conditions, lncRNA DRAIR reduces enrichment of repressive histone modifications, such as H3K9me2, at key antiinflammatory genes, by preventing recruitment of histone methyltransferase G9a (EHMT2), which allows the normal expression of these antiinflammatory genes. However, under diabetic conditions, downregulation of DRAIR and upregulation of G9a reverses these events, leading to repression of antiinflammatory genes, activation of monocytes, and chronic inflammation.

Fig 3: Activation of ERV gene transcription by EZH2 and G9a inhibition in MM cells.A qRT-PCR analysis of ERV genes in RPMI-8226 cells treated with the indicated inhibitors (1 μM each, 6 days). Results are normalized to cells treated with DMSO. Shown are means of three technical replications; error bars represent SEMs. B ChIP-seq analysis of ERV genes in RPMI-8226 cells. The numbers on the vertical axis indicate the numbers of sequence reads. Regions analyzed by ChIP-PCR are indicated by red arrows on the top, and the locations of ERV genes are indicated at the bottom. Peak, peaks detected by MACS2.0. C, D ChIP-qPCR analysis showing levels of H3K27me3 (C) and H3K9me2 (D) at ERV genes in RPMI-8226 cells treated with the indicated inhibitors. Results are normalized to respective input DNAs. Shown are means of three technical replications; error bars represent SEMs. E qRT-PCR analysis of interferon genes in RPMI-8226 cells treated with the indicated inhibitors. *P < 0.05, **P < 0.01. F Hypothesized mechanism of the antimyeloma effect of EZH2/G9a inhibition.

Fig 4: EHMT2 is a therapeutic target for CRC.A Kaplan–Meier plot of overall survival in CRC samples derived from the GTEx portal. The survival rate of the low EHMT2 group was significantly increased compared with that of the high EHMT2 group (p = 0.043). B qRT-PCR analysis of TNFAIP1 after BIX01294 treatment. Mean ± SD of three independent experiments. p values were calculated using Student’s t-test (***p < 0.001). C Immunocytochemical analysis of TNFAIP1. HCT116 cells treated with BIX01294 were fixed with 100% methanol and stained with anti-TNFAIP1 (Alexa Fluor 488, green) and DAPI (blue). Scale bar, 50 μm. D Cell growth assay after BIX01294 treatment for 24 h. HCT116 and LS174T cells were fixed in 100% methanol and stained with crystal violet solution. Scale bar, 200 μm. E The ChIP assay was performed using anti-H3K9me2 (left) and anti-H3K9ac (right) antibodies. The results are expressed as a percentage of input chromatin compared with the control in HCT116 cells after BIX01294 treatment. Mean ± SD of three independent experiments. p values were calculated using Student’s t-test (***p < 0.001). F Western blot analysis after BIX01294 treatment using anti-PARP and anti-EHMT2 antibodies. ACTB was used as the internal control in HCT116 and LS174T cells. The signal intensities were quantified using ImageJ software. G FACS analysis using Muse Caspase 3/7 working solution was performed after BIX01294 treatment. The upper right panel indicates the apoptotic and dead cell proportions (left). Quantification of caspase 3/7 activity. Mean ± SD of three independent experiments. p values were calculated using Student’s t-tests (***p < 0.001, **p < 0.01) (right). H FACS analysis of Annexin V staining was performed after BIX01294 treatment. The lower right and upper right quadrants indicate early apoptosis and late apoptosis, respectively. Quantification of apoptosis. Mean ± SD of three independent experiments. p values were calculated using Student’s t-test (***p < 0.001) (right). I–K BIX01294 treatment suppressed xenograft nude mouse tumors. Either control or BIX01294 was intraperitoneally injected three times a week after HCT116 cell implantation. I Macroscopic image of tumors on day 23, J tumor volumes [p values were calculated using two-way ANOVA (***p < 0.001)], and K tumor weight [p values were calculated using Student’s t-test (**p < 0.01)].

Fig 5: High-risk neuroblastomas with high euchromatic histone-lysine methyltransferase (EHMT) and EZH2 activity are characterized by MYCN amplification and a T-cell infiltration-poor tumor microenvironment. (A) Outline of bioinformatic strategy. (B, C, D) Generation of drug response signatures from overlap of differentially expressed genes in SK-N-BE and IMR-32 cells treated with UNC-0638 (B), EPZ011989 (C) or both drugs (D). (E) Exemplary visualization of calculation of EHMT activity scores for high-risk NB samples. (F, G) Exemplary heatmap visualization of expression of UNC-0638 drug response genes in high-risk NB samples ranked by increasing EHMT activity score. (H–J) Heatmaps visualizing immune contexture marker genes (eg, CD8A, CXCL10), EHMT2/1, EZH2/1 and MYCN in high-risk NB samples ranked by increasing activity scores of EHMT (H), EZH2 (I) and EHMT+EZH2 (J). Pearson correlation coefficients are indicated besides the names of the transcripts. Statistics: *p<0.05; **p<0.01; ***p<0.001; two-sided t-test for Pearson product moment correlation coefficient. NB, neuroblastoma.