Figure 5. Ezh2 mutation produces transcriptional repression and spreading of H3K27me3 surrounding TSS sites.

A. Relative abundance of H3.1K27 by liquid chromatography separation and mass spectrometry of histones from SRBC-immunized mice (n=5). Mean ± SEM; unpaired t test.

B. H3K27me3 bound promoters by ChIP-seq (n=3 mice).

C. H3K27me3 normalized read density heat maps at Ezh2^Y641F-specific H3K27me3 promoters (top), and scaled H3K27me3 mean density plots of region between Ezh2^Y641F-specific H3K27me3 TSS and nearest TSS (bottom).

D. RNA-seq (n=4 mice); transcripts in red, fold-change>1.5, q<0.01.

E. H3K27me3 normalized read density heat maps at promoters of differentially expressed genes in CC (top), and mean H3K27me3 profile across loci interval (bottom).

F. Pathway analysis in CC.

G-H. Fuzzy c-means clustering of RNA-seq data: line plot (G) and heatmap (H) of standardized log2 fold-change relative to normal naive B (NB) cells. Black lines in (G) are cluster centroid; each gene is colored by the degree of cluster membership. Heatmap in (H) are z-scores of log2 fold-change values for each gene relative to NB.

I. RNA-seq and H3K27me3 profiles of Ezh2^Y641F vs. WT CC per module. H3K27me3 enrichment, Wilcoxon test.

J. GSEA of murine CC Ezh2^Y641F gene modules against gene expression of EZH2 mutant FL cases vs. human CC.

K. RT-qPCR in NB, CB and CC (n=4). Each dot represents a mouse, mean fold change mRNA levels normalized to Hprt1 (Abi2), Gapdh (Lgr5) or Rpl13 (Tnfrsf13c,Tnfrsf14 and Ltb) ± SEM; unpaired t test, **p<0.01, ***p<0.001.

L. H3K27me3 ChIP-seq tracks and qChIP validation in CC. Each dot represents a mouse (n=3), mean ± SEM; unpaired t test, **p<0.01.

See also Figure S5.