Fig 2: Combination of SHR2554 and HBI8000 exhibited synergistic anti-tumor effect in DLBCL models in vivo. Four DLBCL-derived xenograft models (CDXs: SU-DHL-6 and U2932; PDXs: PDX001-EZH2 WT and PDX002-EZH2 Y641N) were used to assess the anti-tumor activity of drug combination in vivo. (a) Tumor size curves derived from four models. (b) Tumor weight derived from four models. (c) Representative immunohistochemistry staining of Ki67 is shown (Scale bar 200 µM). (d) Tumor tissues from four models were used to evaluate cell cycle, apoptosis and histone modification-related pathway by Western blot. Detailed information about Western Blot can be found at supplementary materials. ** p < 0.01, *** p < 0.001, # p < 0.05, ## p < 0.01, ### p < 0.001, compared with vehicle group.

Fig 3: Weaver syndrome mutants are impaired in their histone methyltransferase activity in vitro. Histone methyltransferase reactions were performed using 2 µg purified core histones and 0.67 µM 3H-S-adenosyl-methionine (3H-SAM). Each reaction was incubated with 250 ng of either wild-type (WT) or a mutant HMTase complex (or no enzyme controls). Histone methyltransferase activity was measured based on the incorporation of 3H-labeled methyl groups, represented in scintillation counts per minute. Counts were normalized by subtracting background counts (i.e., no enzyme) from the total counts. A: Incorporation of tritiated methyl groups from 3H-SAM onto core histones is shown for each complex: EZH2 WT •, p.(Phe672Ile) ×, p.(Pro132Ser) ?, p.(Tyr153del) ?, p.(His694Tyr) ?, p.(Glu745Lys) ?, p.(Ala682Thr) ?, p.(Arg684Cys) ?, p.(Tyr133Cys) ?, and p.(Asp185His) ?. Error bars represent standard deviation (SD) within the groups “EZH2 WT” and “EZH2 mutants.” Unpaired t-test showed statistically significant difference between the two groups (P value < 0.0001). B: Incorporation of tritiated methyl groups from 3H-SAM onto core histones is shown for the positive control EZH2 WT, the negative control EZH2 (p.Phe672Ile), and the mutant complex with activity closest to WT, namely, EZH2 (p.Pro132Ser). Error bars represent SD of four independent replicates for the controls, and three independent replicates for the mutant EZH2 (p.Pro132Ser). One-way ANOVA showed statistically significant difference between all groups (overall P value < 0.0001; P values between WT and p.(Phe672Ile), between p.(Phe672Ile) and p.(Pro132Ser), and between WT and p.(Pro132Ser) were all <0.05).

Fig 4: Model of the effect of HMGN-H1 interplay on Olig1&2 expressions. HMGN and H1 continuously compete for nucleosome binding sites (red and green arrows on the single nucleosome in the center). In WT cells (left) the presence of HMGNs reduced the binding of H1 thereby facilitating Olig1&2 expressions. In DKO cells (right) loss of HMGN enhances the binding of H1 thereby recruiting the PRC2 complex including its EZH2 histone methyltransferase, thus leading to increased levels of H3K27me3 and gene silencing at the Olig1&2 genomic loci. Reduced Olig1&2 expressions lead to impaired oligodendrocyte development, reduced myelination in the spinal cords, and altered mouse neurological behaviors.

Fig 5: HMGNs modulate EZH2 binding and H3K27me3 levels around Olig1&2 genes. (A) Genome browser snapshot visualizes decreased mRNA and increased H3K27me3 and EZH2 levels at genic regions encompassing Olig1&2 in DKO ESCs. Numbers in parenthesis indicate the scales of the y-axis, in RPM. Input lanes show number of reads in the absence of antibody. (B–E) ChIP-qPCR analysis of H3K27me3 and EZH2 at the specific loci indicated by arrows in (A). Legend shows cell type. Combined values are shown in C and E. Data were obtained from two independent ESC clones (biological replicates) and presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. IgG controls shown in supplemental Figure 3B. Primers used are listed in Supplementary Table S1. (F) Westerns showing OLIG1&2 expression in DKO ESCs treated with two concentrations of EZH2 specific inhibitor GSK126. (G–H) Quantifications of panel F, normalized to ß-actin.