Figure 7: Bcl6 expression in HSPCs induced broad epigenetic changes.
(a) Unsupervised hierarchical clustering of methylation ratio data from 11,258 promoter regions revealed that HSPCs and mature B-cells (Mat. B) from Sca1-Bcl6Δ mice are epigenetically more similar to each other than to their respective counterparts from WT mice, again suggesting that Bcl6 acts via an epigenetic mechanism that persists from HSPCs to mature B-cells. Each condition represents a pool of biological replicates from 6 to 9 mice. (b) HSPCs and mature B-cells from Sca1-Bcl6Δ mice show significant hypermethylation of a large number of genes compared with the same subsets from WT mice (Supplementary Data 3). These included a significant overlap of 470 genes, suggesting that Bcl6 creates an epigenetic signature in the HSPCs that is maintained through differentiation and can be found in the mature B-cell compartment. (c) Kernel density plot of the number of BCL6 DNA-binding sequence motifs identified in genomic regions with significant hypermehtyaltion (yellow) or hypomethylation (purple) in both HSPCs and mature B-cells from Sca1-Bcl6Δ compared with WT mice (P value<0.05) and in regions with no change in methylation (black) between Sca1-Bcl6Δ and WT mice (P>0.95). Hypermethylated regions showed a significant (P value <0.001) increase in the abundance of BCL6-binding motifs compared with regions with no change in methylation. Hypomethlated regions also showed a significant, but less dramatic, increase in abundance of BCL6-binding motifs (P value <0.001). P values calculated by Mann–Whitney U-test. (d) Hypergeometric GSEA of the list of genes that were hypermethylated in both HSPCs and mature B-cells of Sca1-Bcl6Δ mice showed significant enrichment (hypergeometric enrichment FDR <0.25) of multiple gene sets, including those associated with murine and human stem cells, and with poor outcome in DLBCL. The number of overlapping genes between those with conserved hypermethylation and those present in listed genes sets are shown by grey bars (bottom x axis), with the corresponding FDR for the hypergeometric enrichment shown by the red line (top x axis).
