Gene body methylation can alter gene expression and is a therapeutic target in cancer

Abstract

DNA methylation in promoters is well known to silence genes and is the presumed therapeutic target of methylation inhibitors. Gene body methylation is positively correlated with expression, yet its function is unknown. We show that 5-aza-2'-deoxycytidine treatment not only reactivates genes but decreases the overexpression of genes, many of which are involved in metabolic processes regulated by c-MYC. Downregulation is caused by DNA demethylation of the gene bodies and restoration of high levels of expression requires remethylation by DNMT3B. Gene body methylation may, therefore, be an unexpected therapeutic target for DNA methylation inhibitors, resulting in the normalization of gene overexpression induced during carcinogenesis. Our results provide direct evidence for a causal relationship between gene body methylation and transcription.

Figure 1. Transient 5-Aza-CdR treatment shows prolonged effects on cell growth and DNA methylation

A. Population doubling times (left panel) for HCT116 cells after 5-Aza-CdR treatment (red) and vehicle treatment (black). The y-axis denotes doubling time (hours); x-axis denotes time (days) after 5-Aza-CdR was withdrawn. Colony formation assay (right panel) for HCT116 cells at the indicated time points that the cells were seeded after the withdrawn of 5-Aza-CdR. Colonies were stained 10 days after seeding. B. Smooth Kernel scatter plots showing global DNA methylation patterns after 5-Aza-CdR treatment and at the indicated time points after treatment. The x-axis indicates beta values for untreated control, the y-axis indicates beta values for 5-Aza-CdR treated cells at indicated time points. C. Probes that were heavily methylated (beta value > 0.8) before 5-Aza-CdR treatment were classified, using consensus clustering, into four groups according to their rates of demethylation and remethylation after treatment. The solid line is the median value, while the dotted lines are the lower quartile (lower dotted line) and the upper quartile (upper dotted line). Distribution of probes: Locations of all probes on the 450K platform compared to probes in Group I to IV. See also Figure S1.

Figure 2. DNA methylation and gene expression of fast rebound probes (Group I) in HCT116 cells and uncultured normal/tumor colon samples

(A, D). Kernel density plots showing the correlation between DNA methylation at the promoter region (A) or gene body (D) and gene expression in HCT116 cells. The x-axis indicates the Pearson correlation (r) between DNA methylation and gene expression for a given probe at all the measured time-points. The y-axis indicates the density of probes. The 25 of 105 probes (r<−0.75) at the promoter region showing a significant inverse correlation between DNA methylation and gene expression are highlight in pink (A). Most of the probes in Group I (2467) are located in gene bodies (D). Among them, the 498 probes at gene bodies exhibit a positive correlation between DNA methylation and gene expression are highlighted in green (D). The small windows in A and D represent the average dynamic changes between DNA methylation (red, Met.) and expression (green, Exp.) from the shaded highlighted regions. The left y-axis represents percentage of DNA methylation and the right y-axis represents relative expression, while the x-axis represents times (from day 0 to day 42). (B , E). Supervised cluster analysis of DNA methylation in TCGA colon samples using the available probes identified in HCT116 (highlighted by shading in A and D). Twenty out of 25 are available for B, while 399 out of 498 are available for E. Green represents normal colon tissues (n=38); brown represents colon tumors (n=258). Blue indicates low beta values; yellow indicates high beta values. (C , F). Oncomine™ expression data analysis of 101 colon cancer samples (Brown) versus 19 normal colon samples (Green). The genes corresponding to the probes identified in B and E, 19 genes correspond to 25 probes in A, and 210 genes correspond to 498 probes in D. The expression data for genes was downloaded from Oncomine™ (TCGA Colon Adenocarcinoma vs. Normal set) and the heatmaps generated based on TCGA expression for the indicated groups. Blue denotes low expression and red denotes high expression. See also Figure S2.

Figure 3. DNA methylation and gene expression of slow rebound probes (Group IV) in HCT116 cells and normal/tumor colon samples

(A, D). Kernel density plots show the correlation between DNA methylation (promoter region A, and gene body D) and gene expression in HCT116 cells. The x-axis indicates the Pearson correlation (r) between DNA methylation and gene expression for a given probe at all the measured time-points. The y-axis indicates the density of probes. Four hundred eighty-five of 2870 probes (r<−0.75) show a significant inverse correlation between DNA methylation and gene expression (A). A similar number of probes in Group IV (3370) are located in gene bodies (D). Among them, 447 probes (left highlighted region in D) exhibit a negative correlation between DNA methylation and gene expression, while 494 probes (right highlighted region in D) along with a positive correlation. The small windows in A and D represent the average dynamic changes between DNA methylation (red, Met.) and expression (green, Exp.) from the shaded highlighted regions. The left y-axis represents percentage DNA methylation and the right y-axis represents relative expression, while the x-axis represents times (from day 0 to day 42). (B, E, G). Supervised cluster analysis of DNA methylation in TCGA colon samples using the available probes identified in HCT116 cells (highlighted by shading in A and D). Four hundred twenty-three of 485 probes are available for B, while 383 out of 447 probes (negative correlation, E) and 415 out of 494 (positive correlation, G) are available. Green represents normal colon tissues (n=38); Brown represents colon tumors (n=258). Blue indicates low beta values; yellow indicated high beta values. (C, F, H). Oncomine™ expression data analysis of 101 colon cancer samples (Brown) versus 19 normal colon (Green); The genes corresponding to the probes identified in B, E, and G, 294 genes correspond to 485 probes in A, and 209 genes correspond to 447 probes in D, while 219 genes correspond to 494 probes in D. The expression data for genes was downloaded from Oncomine™ (TCGA Colon Adenocarcinoma vs. Normal set); the heatmaps were generated based on TCGA expression data for the indicated groups. Blue denotes low expression and red denotes high expression. See also Figure S3

Figure 4. Comparison of DNA methylation and gene expression prior to and after 5-Aza-CdR treatment of HCT116 and derivative HCT116 1KO and HCT116 3BKO cells

A. Line graphs show the median DNA methylation changes in HCT116, HCT116 1KO and HCT116 3BKO cells for each group of probes previously identified in Figure 1. B. Heatmaps show the remethylation behaviors of Group I probes that were originally heavily methylated in all 3 cell lines. Blue means unmethylated; yellow means fully methylated. The x-axis denotes the days after 5-Aza-CdR treatment. C. Median gene expression and DNA methylation changes of Group I positively correlated probes and the associated genes in HCT116 and HCT116 3BKO cells. See also Figure S4.

Figure 5. Chromatin architecture is disrupted in gene body regions after 5-Aza-CdR treatment

A. ChIP results for H2A.Z, H3K27m3, and H3K36m3 before and after 5-Aza-CdR treatment at two representative gene body regions from Group I (top panel) and two representative gene body regions from Group IV (bottom panel). Each of the four panels shows histone modifications on the left and DNA methylation changes on the right. Fold enrichment of ChIP data was normalized to input and compared to control. Error bars represent standard deviation of the mean from three independent biological experiments. Regarding DNA methylation changes, before and after 5-Aza-CdR treatment, shown on the right side for each gene: the y-axis represents the beta value of DNA methylation, based on data from the 450K Illumina DNA methylation array. For both data sets: ChIP and DNA methylation, the x-axis represents the days after 5-Aza-CdR treatment as indicated in the legend. B. Column scatter plots indicate the NOMe-seq results for changes in chromatin accessibility separating the data, the parent methylated and daughter unmethylated DNA strands before and after 5-Aza-CdR treatment at the two representative gene body regions. The y-axis represents the average sizes of accessible regions where each dot represents the size of accessible regions in individual DNA strand as detected by bisulfite sequencing. Error bars represent standard deviation of the mean of the accessible regions from the independent sequenced DNA strands. NSD represents no significant difference of chromatin accessibility. See also Figure S5.

Figure 6. Differential chromatin signatures are associated with Group I and IV probes

(A , B). Heatmaps showing the enrichment of H3K27m3, H3K36m3 and H2A.Z for each category of probes (promoter and gene body) in A for Group I and B for Group IV. Wiggler was used to normalize the data into a single value for each genomic position and mean wiggler value was calculated in a 10bp-bin. Z-score was then calculated by transforming the wiggler value in each bin as (Xi-Xmean)/Xstdev where Xi is experimental wiggler value subtracted by the input in the same bin and Xmean and Xstdev is an average and standard deviation of Xi in all of the bins in the whole genome. Each methylation probes was then correlated with a z-score (Bernstein et al., 2012; Xie et al., 2013). C-E. Bar charts showing the distribution of probes across a range of z-scores for H3K27m3 (C), H2A.Z (D), and H3K36m3 (E). The x-axes represent the range of z-scores for each modification and y-axes show the number of probes falling in a particular range of z-scores. Z-score of =< 0 indicates no enrichment. See also Figure S6.

Figure 7. GO analyses of gene body probes which exhibit a positive correlation between DNA methylation and gene expression

A and B. Top ten significantly enriched cellular processes in Groups I and IV probes. The lengths of the orange bars denote significance. C and D. Network diagrams indicate the relationship between c-MYC and genes in Group I (C) and in Group IV (D). Ontology analysis was performed using built-in functions of MetaCore™. The networks are constructed from the basic algorithm "analyze networks (Transcription Factors)" of MetaCore™ (Thomson Reuters Inc.). The genes down-regulated by 5-Aza-CdR treatment are marked by blue solid circle. The other symbols used are as seen on the Metacore™ website. See also Table S1 and S2.