H3K4me3 inversely correlates with DNA methylation at a large class of non-CpG-island-containing start sites

Abstract

Background: In addition to mutations, epigenetic silencing of genes has been recognized as a fundamental mechanism that promotes human carcinogenesis. To date, characterization of epigenetic gene silencing has largely focused on genes in which silencing is mediated by hypermethylation of promoter-associated CpG islands, associated with loss of the H3K4me3 chromatin mark. Far less is known about promoters lacking CpG-islands or genes that are repressed by alternative mechanisms.

Methods: We performed integrative ChIP-chip, DNase-seq, and global gene expression analyses in colon cancer cells and normal colon mucosa to characterize chromatin features of both CpG-rich and CpG-poor promoters of genes that undergo silencing in colon cancer.

Results: Epigenetically repressed genes in colon cancer separate into two classes based on retention or loss of H3K4me3 at transcription start sites. Quantitatively, of transcriptionally repressed genes that lose H3K4me3 in colon cancer (K4-dependent genes), a large fraction actually lacks CpG islands. Nonetheless, similar to CpG-island containing genes, cytosines located near the start sites of K4-dependent genes become DNA hypermethylated, and repressed K4-dependent genes can be reactivated with 5-azacytidine. Moreover, we also show that when the H3K4me3 mark is retained, silencing of CpG island-associated genes can proceed through an alternative mechanism in which repressive chromatin marks are recruited.

Conclusions: H3K4me3 equally protects from DNA methylation at both CpG-island and non-CpG island start sites in colon cancer. Moreover, the results suggest that CpG-rich genes repressed by loss of H3K4me3 and DNA methylation represent special instances of a more general epigenetic mechanism of gene silencing, one in which gene silencing is mediated by loss of H3K4me3 and methylation of non-CpG island promoter-associated cytosines.

Figure 1

ChIP-chip of H3K4me3. (a) Enrichment of H3K4me3 within 2 kb of all human TSSs. (b) Maximum enrichment of H3K4me3 within 1 Kb of the TSS for each human gene. Genes are ordered similarly to (a). (c) Histogram of H3K4me3 signals at all human TSSs. The vertical lines correspond to thresholds used to distinguish genes containing high levels of H3K4me3 (right-side of the distribution) from genes containing low H3K4me3 levels (left side of the distribution). Genes falling between the two vertical lines are indeterminate and not designated as K4-absent or K4-present genes. The bottom panel shows an example of a gene lacking H3K4me3 (CD247), and a gene containing H3K4me3 (UCK2). (d) Maximum H3K4me3 signal at all TSSs in the five colorectal cell lines (SW480, V432, V425, V429, V441) and normal colon mucosa. Columns represent individual samples, and rows represent H3K4me3 signals at TSSs. Dark blue corresponds to high H3K4me3 enrichment; light blue corresponds to little or no enrichment.

Figure 2

Designation of K4-dependent and K4-independent genes in colon cancer. (a) Bar plot showing proportions of K4-dependent and K4-independent genes in each of the five colon cancer cell lines. (b) Heatmap integrating H3K4me3 ChIP-chip signals and corresponding transcript levels. Blue, expressed and H3K4me3 present; red, not expressed and H3K4me3 present; green, not expressed and H3K4me3 absent; white, H3K4me3 status not classifiable.

Figure 3

Loss of H3K4me3 is associated with resistance to DNaseI digestion. (a) Representative DNase-seq signals at a K4-independent (top) and K4-dependent (bottom) gene in cell line SW480. (b) Box plots depicting relative levels of DNase I hypersensitivity among expressed (O), K4-independent (I), and K4-dependent (D) genes.

Figure 4

K4-dependent promoters are DNA hypermethylated. (a-c) Percentage DNA methylation of indicated K4-dependent (K4-D) and K4-independent (K4-I) genes in SW480 (a), V432 (b), and V441 (c). For comparison, methylation levels of genes were also quantified in normal colon crypts. Error bars indicate the standard deviation of pyrosequencing assays performed on three independent preparations of normal colon crypts. All K4-independent genes analyzed contain a CpG island at the TSS. All K4-dependent genes, except PTGDR, lack a CpG island at the TSS. (d) Quantitative RT-PCR analyses of genes from 5-azacytidine (Aza) treated (+) and untreated (-) SW480 cells. Data were normalized to GAPDH. RHBDL2, UBA7, FUT3, and GUCY2C are K4-dependent genes that lack a CpG island. ZFP42 was previously found in this cell line to be DNA hypermethylated and reversible upon treatment with 5-azacytidine, and thus serves as a positive control. OVOL1 is a K4-independent gene with a promoter-associated CpG island and serves as a negative control. Error bars indicate the standard deviation of quantitative RT-PCR reactions performed in triplicate. *P ≤ 0.01.

Figure 5

ChIP-chip analysis of multiple histone marks at the promoters of repressed genes. Plotted in the heatmap are -log10 P-values corresponding to ChIP-chip signal intensities of H3K4me3, H3K27me3, H3K9me2, and H4K20me3 at the promoters of 80 genes as measured in SW480 versus normal colon mucosa. The heatmap reveals two main classes of genes. Most repressed genes designated as K4-independent retain H3K4me3 (blue color) and gain repressive histone marks, including H3K27me3 and H4K20me3, which move from mostly red in normal to blue in the tumor. In contrast, most repressed genes designated as K4-dependent show loss of H3K4me3 (with H3K4me3 going from blue in tumor to red in SW480) and have not acquired any other repressive marks (with the other marks displayed as red in both the normal and tumor). In addition, the majority of genes within K4-dependent lack a CpG island (***no CpG island), while all K4-independent genes have a CpG island.

Figure 6

Model of K4-dependent and K4-independent gene silencing in colon cancer. Expressed genes in normal colon mucosa have high levels of H3K4me3, are DNA hypomethylated at the TSS, and are contained within open chromatin (grey ovals). K4-independent repressed genes retain appreciable levels of H3K4me3, often acquire repressive histone marks such as H3K27me3, and are often not DNA hypermethylated. Also, the chromatin at the TSS of K4-independent repressed genes is more condensed than in normal colon. Virtually all of these genes have a CpG island at the TSS. In comparison, K4-dependent repressed genes lack H3K4me3, are located in highly compacted chromatin, and are DNA hypermethylated, regardless of the presence of a CpG island. K4-dependent genes also do not acquire repressive chromatin marks.