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TET2 deficiency reprograms the germinal center B cell epigenome and silences genes linked to lymphomagenesis

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Science Advances  17 Jun 2020:
Vol. 6, no. 25, eaay5872
DOI: 10.1126/sciadv.aay5872
  • Fig. 1 Tet2 deficiency leads to hypermethylation in GC B cells and loss of hypomethylation during the NB-to-GCB transition.

    (A) Principal components analysis based on ERRBS data from three Tet2−/− and three Tet2+/+ GC B cell (GCBs) samples. (B) Hierarchal clustering based on ERRBS data from three Tet2−/− and three Tet2+/+ GCB samples. (C) Comparison of differential methylation events in NB cells and GCBs. (D) Differential methylation events per chromosome. (E) Row-normalized heatmap of the methylation levels of differential methylation events in GCBs (three Tet2−/− replicates and three Tet2+/+ replicates). (F) Differential methylation during transition of NB cells to GCBs in Tet2+/+ mice. Each dot on the scatter plot represents an individual CpG called as differentially methylated. Blue dots represent hypomethylation, and yellow represent hypermethylation. (G) Differential methylation during transition of NB cells to GCBs in Tet2−/− mice. Each dot on the scatter plot represents an individual CpG called as differentially methylated. Blue dots represent hypomethylation, and yellow represent hypermethylation. (H) Venn diagram of the overlap between hypomethylated DMCs in Tet2−/− and in Tet2+/+ mouse models.

  • Fig. 2 Cooperation of 5hmC loss in enhancers and 5mC gain in promoters enhances gene silencing.

    (A) Genomic features within which hyper-DMC distribution was studied. They include promoters, exons, introns, enhancers (defined as H3K27ac peaks from WT GC B cells, excluding promoters), DHMRs losing 5hmC signal, and intergenic regions. (B) This panel depicts an UpSet intersection diagram, showing the total set size and overlaps between the 9043 hyper-DMCs and genomic elements including DHMRs, enhancers (defined as H3K27ac peaks, excluding promoters), exons, promoters, and intergenic and intronic regions. The shaded circles connected by solid black lines in the bottom show the intersecting combinations of genomic elements. This panel illustrates all feature combinations with at least one hyper-DMC. The number of hyper-DMCs overlapping in each region (or regions intersection, e.g., intergenic enhancer) was normalized, by the total number of CpGs covered by ERRBS reads in this region (or intersection of regions), and multiplied the value by 100. (C) Heatmap of the expression levels (row normalized) of 15 down-regulated genes with 5hmC loss in enhancers and 5mC gain in promoters. (D) Heatmap and signal distribution of the 5hmC enrichment at 24 DHMR regions overlapping with enhancer regions of the 15 genes for which expression was visualized in (C). (E) Heatmap of 5mC enrichment at 25 hyper-DMCs overlapping with the promoter regions of 15 genes for which expression (row normalized) was visualized in (C). (F) Genome browser view of the example Jarid2 gene locus, visualizing 5hmC loss in two of its enhancers and 5mC gain in its promoter.

  • Fig. 3 Hypermethylation affects key pathways in B cell biology.

    (A) Result of pathway enrichment analysis of the 930 genes with hyper-DMCs in their promoter regions. Plot shows minus log10 of the FDR scores for the enrichment of genes in seven functional categories important in GC or DLBCL biology. FDR scores were computed using hypergeometric test for these 930 genes. (B) GSEA enrichment plots in Vav-Cre/Tet2−/− versus Vav-Cre/Tet2+/+ GC B cells, against 163 genes with hyper-DMCs in their promoter regions, which were enriched for pathways listed in (A). (C) Row-normalized gene expression of 34 genes, which were identified as “leading-edge genes” in the GSEA analysis shown in (B). Yellow represents high expression, and green represents low expression. NES, normalized enrichment score; Down-DEGs, down-regulated genes; FL, follicular lymphoma. Star (*) indicates the value of FDR below 0.05.

  • Fig. 4 Hypermethylated regions are enriched for binding motives of key TFs in B cell biology.

    (A) Heatmap of the FDR scores of the motif enrichment analysis conducted using Homer for hyper-DMCs (±50 bp) located in promoter regions. Each TF name is accompanied by a logo sequence of the binding site. (B) Minus log10 of the FDR scores for enrichment of the target genes of 13 TFs, the binding sites of which were identified as hyper-methylated in Tet2−/− GC B cells. FDR scores were computed using GSEA, as described in Materials and Methods. Direction of the enrichment is biased toward Vav-Cre/Tet2+/+ GC B cells, when compared with Vav-Cre/Tet2−/− GC B cells (i.e., target genes are down-regulated in Tet2−/− GC B cells). (C) Normalized gene expression of the 1274 genes identified as leading-edge genes in at least 1 of 13 significantly enriched gene sets shown in (B). (D) Overlap between the 1274 leading-edge genes and 34 hypermethylated leading-edge genes of seven gene signatures important in GC exit or B cell biology. (E) Minus log10 of the FDR scores for the enrichment of 10 gene sets important in B cell biology. FDR scores were computed using hypergeometric test for leading-edge genes presented in (C) and (D). Star (*) indicates the value of FDR below 0.05.

  • Fig. 5 TET2-deficient GC B cells manifest AID loss-of-function signature.

    (A) Examination of 5mC accumulation during NB to GC B cell differentiation in TET2- and AID-deficient mouse models. CpGs (19,111) that are hypomethylated during normal NB to GC B cell differentiation in a WT mouse model were taken as a reference. (B) Venn diagram showing the overlap between CpGs that accumulate 5mC in Aicda−/− and in Tet2−/− NB to GC B cell differentiation. (C) GSEA enrichment analysis of 1238 genes accumulating 5mC during Aicda−/− NB to GC B cell transition, in Tet2−/− versus Tet2+/+ GC B cells. (D) Examination of gene inactivation during NB to GC B cell differentiation in TET2- and AID-deficient mouse models. Genes (4198) up-regulated during normal NB to GC B cell differentiation in a WT mouse model were taken as a reference. (E) Venn diagram showing the overlap between genes that remain silenced during Aicda−/− and Tet2−/− NB to GC B cell differentiation. (F) Enrichment of the 1500 genes that remain silenced during Aicda−/− NB to GC B cells transition, in Tet2−/− versus Tet2+/+ GC B cells.

  • Fig. 6 TET2- and AID-deficient cells are enriched for the same gene signatures.

    (A) Venn diagram showing the overlap between the integrated methylation- and expression-based aberrant gene signatures in Aicda−/− and Tet2−/− mouse models. (B) Enrichment of the aberrant Aicda−/− gene signature (3111 genes) in Tet2−/− versus Tet2+/+ GC B cells. Enrichment plot was generated using GSEA and shows down-regulation in Tet2−/− GC B cells. (C) Result of pathway enrichment analysis of the 3111 and 1949 genes with aberrant Aicda−/− and Tet2−/− signatures, respectively. FDR scores were computed using hypergeometric test. (D) Heatmap shows the FDR score of eight selected gene signatures from (C). FDR scores were computed using hypergeometric test. (E) Per-clone C-to-T mutation frequency at WRC motif, where W = A or T and R = G or A. Mann-Whitney U test was computed between Tet2−/− and Tet2+/+ conditions, combining mutation frequency values from each replicate accordingly to condition type.

  • Fig. 7 TET2 loss of function in human DLBCL manifest Tet2-deficient GC DMC and gene signature.

    (A) Enrichment of the aberrant human TET2MUT gene signature (241 genes with hyper-DMCs in their promoters) in mouse Tet2−/− versus Tet2+/+ GC B cells. (B) Enrichment of the aberrant mouse Tet2−/− gene signature (614 genes with hyper-DMCs in their promoters) in human TET2MUT versus TET2WT DLBCL. (C) First and third columns of the heatmap represent the results of pathway enrichment analysis of 614 mouse and 241 human genes with hyper-DMCs in their promoters, respectively. FDR scores for these results were computed using hypergeometric test. Second and fourth columns represent the results of pathway enrichment analysis of the same gene sets, using GSEA in mouse Tet2−/− versus Tet2+/+ GC B cells and human TET2MUT versus TET2WT DLBCL, respectively. NES scores of all pathways in mouse and human are biased toward Tet2−/−/TET2WT.

  • Table 1 The number and ratio of down-DEGs and up-DEGs with 5hmC loss in enhancer or 5mC gain in promoter.

    5hmC loss in
    enhancer
    5mC gain
    in
    promoter
    Number ofRatio
    (down to
    up-DEGs)
    Down-
    DEGs
    Up-DEGs
    YesYes14114.0:1
    NoYes2263.7:1
    YesNo116383.0:1
    NoNo2431441.7:1

Supplementary Materials

  • Supplementary Materials

    TET2 deficiency reprograms the germinal center B cell epigenome and silences genes linked to lymphomagenesis

    Wojciech Rosikiewicz, Xiaowen Chen, Pilar M. Dominguez, Hussein Ghamlouch, Said Aoufouchi, Olivier A. Bernard, Ari Melnick, Sheng Li

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