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. 2015 Mar 11;43(5):2590-602.
doi: 10.1093/nar/gkv103. Epub 2015 Feb 17.

Epigenetic Remodeling in B-cell Acute Lymphoblastic Leukemia Occurs in Two Tracks and Employs Embryonic Stem Cell-Like Signatures

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Free PMC article

Epigenetic Remodeling in B-cell Acute Lymphoblastic Leukemia Occurs in Two Tracks and Employs Embryonic Stem Cell-Like Signatures

Seung-Tae Lee et al. Nucleic Acids Res. .
Free PMC article

Abstract

We investigated DNA methylomes of pediatric B-cell acute lymphoblastic leukemias (B-ALLs) using whole-genome bisulfite sequencing and high-definition microarrays, along with RNA expression profiles. Epigenetic alteration of B-ALLs occurred in two tracks: de novo methylation of small functional compartments and demethylation of large inter-compartmental backbones. The deviations were exaggerated in lamina-associated domains, with differences corresponding to methylation clusters and/or cytogenetic groups. Our data also suggested a pivotal role of polycomb and CTBP2 in de novo methylation, which may be traced back to bivalency status of embryonic stem cells. Driven by these potent epigenetic modulations, suppression of polycomb target genes was observed along with disruption of developmental fate and cell cycle and mismatch repair pathways and altered activities of key upstream regulators.

Figures

Figure 1.
Figure 1.
Overview and statistics of whole-genome bisulfite sequencing data of B-ALLs and normal pre-B cells. (A) Circular barplots of genome-wide average methylation levels in pre-B cells, ETV6-ALL and HD-ALL (from inside to outside, bin size = 5 Mb) showing slight demethylation of HD-ALL throughout genomic location. (B and C) Density plots of DNA methylation levels of all autosomal CpGs between B-ALL and pre-B cells. Individual CpG site methylation of pre-B cell and B-ALL are expressed on the X- and Y-axes. Hotter colors indicate higher density of data. (D) Counts of differentially methylated regions (DMRs; CpGs with methylation difference >20%) within genes or promoters. Most genes have more than one DMR inside the genic region or promoters, with some genes having numerous DMRs. HD-ALL had more de-DMRs than ETV6-ALL. (E) Relative distribution of DMRs according to specified regions. In both tumors, CpGs in promotes, 5′-body (0–0.1 in fractional region of gene body) and CpG island (CGI) are more enriched for de novo DMRs while CGI shelf and repeat regions were so for de-DMRs. Transcription factor (TF) binding sites and DNase hypersensitive sites (HS) are more enriched for de novo methylation in ETV6-ALL and for demethylation in HD-ALL.
Figure 2.
Figure 2.
DNA methylation changes of B-ALLs compared to pre-B cell according to specified regions. (A) Local regression showing methylation difference in DNase HS, stratified by genic location. DNase HS in promoter and 5′-body are more likely to be methylated, while the trend was slight or negligible in main body and downstream regions. (B) Methylation difference in exons and introns in gene main bodies. Exons are less altered while introns, especially the deep portion of long introns, are profoundly demethylated in HD-ALL but not in ETV6-ALL. (C) Genome-wide overview of methylation changes in backbones defined by regions devoid of promoters, 5′-bodies, exons, CGIs, CGI shores, DNase HS, TF-binding sites and enhancer sites. ETV6-ALL demonstrates minimal change when compared with the pre-B cell control. (D) Genome-wide view of methylation changes in backbones of HD-ALL show profound demethylation across the whole genome. Red bars indicate chromosomes with extra copies. (E) Methylation changes of backbones according to location to lamina-associated domain (LAD) show more demethylation of regions inside LADs in HD-ALL. (F) Methylation differences in CGI and DNase HS, stratified by location to LAD show profound de novo methylation of CGI and DNase HS inside LAD.
Figure 3.
Figure 3.
Association between DNA methylation and gene expression. (A) Local regression showing methylation levels of HD-ALL according to genic locations, stratified by expression quintile. Genes in the highest expression quintile tend to have their promoter unmethylated and gene body mostly methylated. (B) Methylation levels of HD-ALL in CGI and DNase HS, stratified by genic location and expression quintile. Methylation in CGI and DNase HS located around promoter and 5′-body apparently correlated with expression while not in the remaining regions.
Figure 4.
Figure 4.
Enrichment of WGBS DMRs according to specific TFs, histone marks or motifs. (A and B) Positional enrichment of de novo DMRs and de-DMRs of HD-ALL against 148 ENCODE TF-binding sites. Two transcription factors, SUZ12 and CTBP2, are highly enriched around de novo DMRs while no specific TFs are enriched around de-DMRs. (C) Enrichment of DMRs of HD-ALL according to H3K4me3 (active) and H3K27me3 (repressive) histone marks of H1 embryonic stem cell (ESC) shows remarkable enrichment of de novo DMRs in bivalent domain characterized by co-occupancy of both histone marks (parallel analysis for ETV6-ALL is presented in Supplementary Figure S5A–C). (D) Positional analysis for histone and TF sites in ESC shows enrichment of H3K4me3 at the center of CTBP2-binding site and dual peak of H3K27me3 around the ±1 kb region from the center, suggesting CTBP2 constitute H3K4me3 part of the bivalent domain. (E) Methylation changes in an exemplary gene, ALX4. In human ESC (H1-hESC), the promoter is poised bivalently with H3K4me3 (active) and H3K27me3 (repressive) histones marks. At differentiation, the gene is activated by H3K4me3 in normal skeletal muscle (HSMM) and suppressed by H3K27me3 in normal lung fibroblasts (NHLF). In ALLs, promoters are mostly methylated (red bars) while gene body is demethylated (blue bars). De novo DMRs frequently coincide with the bivalent regions, CGIs and SUZ12- and CTBP2-binding sites. Note that H3K4me3 marks mostly peak around the CTBP2 sites. (F) Novel and known motifs significantly enriched around DMRs. Regions around de novo DMRs are enriched for REST/NRSF, PU.1 and GATA motifs and others, while those around de-DMRs are so for proto-oncogenes, ERG and MYC and a developmental gene, EBF.
Figure 5.
Figure 5.
Methylation profiles of 227 B-ALL cases analyzed by Illumina 450k array. (A) A hierarchical clustering analysis using 500 most variable CpGs classifies the tumors into four distinct clusters. Some clusters are dominated by specific cytogenetic groups; cluster I by hyperdiploid, cluster II by others, cluster III by ETV6/RUNX1, and cluster IV by hyperdiploid/others. (B) Local regression of the methylation differences illustrates that each methylation cluster has different degrees of CGI methylation in each cytogenetics group. (C) Among the four methylation clusters, many DMRs are shared while some DMRs were cluster-specific. (D) Mean methylation at SUZ12-binding sites significantly correlates with mean expression of polycomb target genes (9) (r = −0.276; P = 0.020), suggesting a role of de novo methylation at polycomb sites for repressing their target genes.
Figure 6.
Figure 6.
Pathway and upstream regulators dysregulated in different clusters. (A) Pathway enrichment analysis for differentially expressed genes (DEGs) compared to pre-B-I and pre-B-II cells were done using the Ingenuity database. Values in boxes indicate -log(FDR-corrected P). B-cell development pathway is more enriched for methylation cluster III, cell cycle and mismatch repair for clusters II and IV, and glucocorticoid receptor signaling for clusters I and II. (B) Upstream regulators significantly dysregulated. Values in boxes display activation z scores calculated from the expression patterns of their downstream target genes. Chromatin modifiers including NUPR1, SMARCB1 and EP400 and cell cycle controllers including CCND1 and TBX2 are significantly dysregulated as well as MYC, RB1, CDKN2A and TP53. Among chemicals, doxorubicin and other investigative drugs for B-ALLs ranked at the top, with different degrees according to methylation clusters.

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