Genome-wide DNA methylation analysis reveals novel epigenetic changes in chronic lymphocytic leukemia

Abstract

We conducted a genome-wide DNA methylation analysis in CD19 (+) B-cells from chronic lymphocytic leukemia (CLL) patients and normal control samples using reduced representation bisulfite sequencing (RRBS). The methylation status of 1.8-2.3 million CpGs in the CLL genome was determined; about 45% of these CpGs were located in more than 23,000 CpG islands (CGIs). While global CpG methylation was similar between CLL and normal B-cells, 1764 gene promoters were identified as being differentially methylated in at least one CLL sample when compared with normal B-cell samples. Nineteen percent of the differentially methylated genes were involved in transcriptional regulation. Aberrant hypermethylation was found in all HOX gene clusters and a significant number of WNT signaling pathway genes. Hypomethylation occurred more frequently in the gene body including introns, exons, and 3'-UTRs in CLL. The NFATc1 P2 promoter and first intron was found to be hypomethylated and correlated with upregulation of both NFATc1 RNA and protein expression levels in CLL suggesting that an epigenetic mechanism is involved in the constitutive activation of NFAT activity in CLL cells. This comprehensive DNA methylation analysis will further our understanding of the epigenetic contribution to cellular dysfunction in CLL.

Figure 1. DNA methylation changes in CLL. (A) Pair-wise correlation coefficients matrix comparing DNA methylation between normal B-cells, CLL cell lines and primary CLL B-cells. A high similarity was observed among normal CD19⁺ B-cells (CD19), naïve (NBC) and memory B-cells (MBC), while CLL cell lines displayed significant differences when compared with the primary cells. (B) The number of DMRs identified in 3 CLL cell lines and 11 purified CLL B-cell samples when compared with three normal control samples. (C) Genome-wide distribution of 8703 DMRs that were identified in primary CLL samples. (D) Number of DMRs that are common among 11 CLL samples.

Figure 2. DNA hypermethylation of HOX genes. (A and B) UCSC genome browser screenshot illustrating the RRBS results in the HOXA and HOXD gene clusters. The tracks shown from the top to bottom in each figure are: the DNA methylation level at each CpG site derived from the bisulfite sequencing reads, RefSeq genes and annotated CpG islands in the UCSC genome browser. Red and green colors indicate methylated and unmethylated CpG sites, respectively. (C) Methylation profiles of individual HOX genes. In each panel, each row is the result of an individual patient sample. Each box represents a CpG site. Yellow, no methylation; blue, methylation. The proportion of yellow and blue in each box represents the methylation level. Only common CpGs shared by all samples are shown.

Figure 3. Aberrant DNA methylation changes in WNT pathway genes. (A) IPA analysis highlights DNA methylation alteration in WNT pathway genes. Hypermethylated genes are indicated by red and hypomethylated genes are highlighted by green. (B) Single-base CpG methylation patterns in WNT pathway genes in CLL. Each row is the result of an individual patient sample. Each box represents a CpG site. The color indicates the methylation level. Yellow, no methylation; blue, methylation. The proportion of yellow and blue in each box represents the methylation level. Only common CpGs shared by all samples are shown.

Figure 4. Cluster analysis of 160 genes that associated with DMRs in the 5′ end regulatory regions (see text). The average methylation values of each DMR were subjected to hierarchical clustering with Pearson dissimilarity and average linkage method in Partek Genomics Suites. The IGHV mutation status and CD38 expression of each sample are given above the heatmap.

Figure 5. Validation of DNA methylation results. (A) Methylation profiles of two frequently hyper-methylated genes generated by RRBS. Each box represents a CpG site. The color indicates the methylation level. Yellow, no methylation; blue, methylation. The proportion of the yellow and blue color indicates the levels of methylation. (B) Validation of DNA hypermethylation in the promoter and first exon of FOXA2 and SOX11 by bisulfite pyrosequencing, respectively. Each filled circle represents a CLL sample. The methylation values represent the average methylation levels of all CpG sites measured by pyrosequencing. Representative pyrosequencing results can be found in Figures S6 and S7. (C) Expression analysis of four selected genes in 2 lymphoma cell lines: total RNA isolated from treated (A, 5′-Aza; T, TSA; AT, 5′-Aza+TSA;) or untreated (C) cells was used to generate cDNA for RT-PCR analysis. β-actin was amplified for 20 cycles, while the other four genes were amplified for 32 cycles.

Figure 6. Hypomethylation and upregulation of NFATc1 expression in CLL. (A) RRBS results of NFATc1. The tracks show CpG sites that are covered by sequencing reads. The red and green colors indicate methylated and unmethylated CpGs, respectively. (B) Confirmation of DNA methylation in NFATc1 DMR using bisulfite clone sequencing. The methylation status of 36 CpGs from two regions (one in the promoter P2 and one in first intron) was determined from the bisulfite treated DNA of 4 CLL patients, one CLL cell line (Mec-1), and one normal CD19+ B-cell sample. Each row is the result of an individual clone. The same DNA samples used for RRBS sequencing were used in the confirmation study. (C) Quantitative pyrosequencing analysis of the NFATc1 P2 region in normal and CLL B-cells. Six CpGs in the promoter P2 region were analyzed by pyrosequencing. Each row is the result of an individual patient sample. Each box represents a CpG site. Yellow: no methylation; Blue: methylation. The proportion of yellow and blue in each box represents the methylation level. (D) Quantitative RT-PCR analysis of NFATc1 expression in normal and CLL B-cells samples. (E) Western blot analysis of NFATc1 expression in normal and CLL B-cells.