Relationship between gene body DNA methylation and intragenic H3K9me3 and H3K36me3 chromatin marks

Abstract

To elucidate the relationship between intragenic DNA methylation and chromatin marks, we performed epigenetic profiling of chromosome 19 in human bronchial epithelial cells (HBEC) and in the colorectal cancer cell line HCT116 as well as its counterpart with double knockout of DNMT1 and DNMT3B (HCT116-DKO). Analysis of H3K36me3 profiles indicated that this intragenic mark of active genes is associated with two categories of genes: (i) genes with low CpG density and H3K9me3 in the gene body or (ii) genes with high CpG density and DNA methylation in the gene body. We observed that a combination of low CpG density in gene bodies together with H3K9me3 and H3K36me3 occupancy is a specific epigenetic feature of zinc finger (ZNF) genes, which comprise 90% of all genes carrying both histone marks on chromosome 19. For genes with high intragenic CpG density, transcription and H3K36me3 occupancy were not changed in conditions of partial or intensive loss of DNA methylation in gene bodies. siRNA knockdown of SETD2, the major histone methyltransferase responsible for production of H3K36me3, did not reduce DNA methylation in gene bodies. Our study suggests that the H3K36me3 and DNA methylation marks in gene bodies are established largely independently of each other and points to similar functional roles of intragenic DNA methylation and intragenic H3K9me3 for CpG-rich and CpG-poor genes, respectively.

Figure 1. Clustering analysis of epigenetic profiles of genes located on chr19.

Individual genes are indicated by single vertical lines depicting the epigenetic status from the 5' end (top) to the 3' end (bottom) of each gene. Each gene body was divided into 20 bins. The 5 kb upstream of the TSS and 5 kb downstream of the 3' gene end were divided into 10 bins. The average signal for each single bin is indicated. Green, red and black colors represent low, high and average occupation by epigenetic marks in comparison to the average signal on the chromosome, respectively. Zinc finger (ZNF) genes represent a unique epigenetic class.

Figure 2. Crosstalk between DNA methylation and chromatin modifications on chr19 in human bronchial epithelial cells.

A. Gene bodies are marked by DNA methylation and H3K36me3 on human chr19 in HBEC. The numbers of genes with gene bodies marked by H3K36me3, DNA methylation or both, with at least 20% of gene body length covered by the respective mark, are indicated (p<0.001; Chi square test). B. Representative epigenetic profile of genes harboring DNA methylation and H3K36me3 in the gene body. Signals plotted as negative log10(p-values) are shown for the chromatin marks H3K9me3, H3K27me3 and H3K36me3. Methylated CpG density mapped by MIRA is shown along with the mapping of unmethylated CpGs by UnmethylCollector (UMC) C. Composite profile of genes with gene bodies marked by H3K36me3 and DNA methylation over at least 20% of gene body length. Each gene body was divided into 20 bins. Sequences up to 5 kb upstream of the TSS and 5 kb downstream of the 3' gene end were divided into 10 bins. Average of occupation for each single bin is indicated. The chromatin marks histone H3 acetylation (K9/K14), H3K9me3, H3K27me3 and H3K36me3 were analyzed along with methylated CpGs (MIRA) and unmethylated CpGs (UMC).

Figure 3. Epigenetic profile of ZNF genes on chr19 in human bronchial epithelial cells.

A. Genes located on chr19 sorted by CpG density in gene bodies and by percentage of gene body length covered by H3K9me3. B. Distribution of ZNF genes in the human genome. The number of ZNF genes on individual chromosomes is shown. C. Representative epigenetic profile of ZNF genes. Gene names, directions of transcription and gene coordinates on the chromosome are indicated. Note the simultaneous occupation of ZNF gene bodies by H3K9me3 and H3K36me3. D. Composite profile of genes with gene bodies marked by H3K36me3 and H3K9me3 over at least 20% of gene body length. Each gene body was divided into 20 and the 5 kb upstream of the TSS and 5 kb downstream of the 3' gene end were divided into 10 bins. The average signal for each single bin is indicated. E. Gene bodies marked by H3K9me3 and H3K36me3 on human chr19 in HBEC. The number of genes with gene bodies marked by H3K36me3 or H3K9me3 or by both marks over at least 20% of gene body length is shown (p = 0.02; Chi square test). Ninety percent of the dual-occupied genes are zinc finger genes. F. Genes marked by H3K36me3 on chr19 in HBEC. The diagram represents the distribution of H3K9me3 and DNA methylation in gene bodies containing H3K36me3. Genes were assumed to carry a specific modification if at least 20% of gene body length was covered by the analyzed mark. Co-occupancy of H3K9me3 and H3K36me3 is a hallmark of ZNF genes.

Figure 4. Epigenetic changes in HCT116-DKO cells.

A. Clustering analysis of epigenetic changes on chr19 in HCT116-DKO cells in comparison to HCT116-WT cells. Each vertical line represents one gene. Green, red and black colors represent low, high and average occupation by epigenetic marks, respectively, in comparison to the average signal along the chromosome. Similarly, gene expression is indicated by green, red and black color showing low, high and average expression level, respectively, in comparison to average transcription levels on chr19. For expression changes, red, green and black colors represent activation, repression and no change of transcription activity, respectively, in HCT116-DKO cells in comparison to HCT116-WT cells. Abbreviations WT and DKO are for HCT116-WT and HCT116-DKO cells, respectively. B. Interrelation between genome-wide expression changes in HCT116-DKO cells in comparison to HCT116-WT cells and gene body CpG density (R = -0.02, p  =  0.03). Red color indicates ZNF genes. C. Representative epigenetic profiles of activated ZNF genes in HCT116-DKO and silent state of these genes in HCT116-WT cells. Direction of transcription and gene coordinates on chromosome 19 are indicated. The boxes span promoters of genes that become demethylated and reactivated in DKO cells as indicated by appearance of the H3K36me3 mark in their gene bodies. D. Composite profile of H3K36me3-associated genes in HCT116-DKO and in HCT116-WT cells. The profile was created for genes with gene bodies covered by H3K36me3 and DNA methylation with at least 20% of gene body length covered in HCT116-WT cells. Each gene body was divided into 20 bins and the 5 kb upstream of the TSS and 5 kb downstream of the 3' gene end were divided into 10 bins. The average signal for each single bin is indicated. E. H3K36me3 is not lost in regions with extensive loss of DNA methylation in gene bodies. Despite of loss of DNA methylation (blue tracks) in gene bodies, the H3K36 profile (black tracks) is almost unaltered.

Figure 5. Epigenetic changes after SETD2 knockdown.

A. Western blot for HBEC cells after non-targeting siRNA and SETD2 siRNA transfection using anti-H3K36me3 antibodies and unmodified histone H3 antibodies. B. Composite profile of epigenetic changes after SETD2 siRNA knockdown in HBEC. The profile was created for gene bodies marked by H3K36me3 and DNA methylation over at least 20% of gene body length. Each gene body was divided into 20 bins and the 5 kb upstream of the TSS and 5 kb downstream of the 3' gene end were divided into 10 bins. The average signal for each single bin is indicated. C. Analysis of DNA methylation in the gene body of the NOTCH3 gene in conditions of H3K36me3 deficiency. The H3K36me3 profile of NOTCH3 after non-targeting siRNA and SETD2 siRNA transfections in HBEC cells is shown. The region analyzed by COBRA methylation assays is indicated by a box. Using gene-specific primers, bisulfite-converted DNA was amplified. After cutting with HpyCH4IV recognizing CpG dinucleotides, mock (-) and enzyme-digested (+) PCR products were fractionated on a 2% agarose gel. In vitro CpG-methylated human DNA (M) served as a positive control. Cleavage indicates DNA methylation. D. Analysis of H3K36me3 and DNA methylation in the gene body of RFX2 in conditions of H3K36me3 deficiency. The region analyzed by bisulfite sequencing is marked by a box. Using gene-specific primers, bisulfite-converted DNA was amplified, cloned and 20 individual clones were sequenced. White circles, unmethylated CpG sequences; black circles, methylated CpG sequences. E. Western blot for HCT116-DKO cells after non-targeting siRNA and SETD2 siRNA transfection using anti-H3K36me3 antibodies and unmodified histone H3 antibodies. F. DNA methylation analysis of the gene body of NOTCH3 after non-targeting siRNA and SETD2 siRNA transfection of HCT116-DKO cells. Using gene-specific primers, bisulfite-converted DNA was amplified, cloned and 13 individual clones were sequenced. White circles, unmethylated CpG sequences; black circles, methylated CpG sequences.