Aberrant silencing of cancer-related genes by CpG hypermethylation occurs independently of their spatial organization in the nucleus

Abstract

Aberrant promoter DNA-hypermethylation and repressive chromatin constitutes a frequent mechanism of gene inactivation in cancer. There is great interest in dissecting the mechanisms underlying this abnormal silencing. Studies have shown changes in the nuclear organization of chromatin in tumor cells as well as the association of aberrant methylation with long-range silencing of neighboring genes. Furthermore, certain tumors show a high incidence of promoter methylation termed as the CpG island methylator phenotype. Here, we have analyzed the role of nuclear chromatin architecture for genes in hypermethylated inactive versus nonmethylated active states and its relation with long-range silencing and CpG island methylator phenotype. Using combined immunostaining for active/repressive chromatin marks and fluorescence in situ hybridization in colorectal cancer cell lines, we show that aberrant silencing of these genes occurs without requirement for their being positioned at heterochromatic domains. Importantly, hypermethylation, even when associated with long-range epigenetic silencing of neighboring genes, occurs independent of their euchromatic or heterochromatic location. Together, these results indicate that, in cancer, extensive changes around promoter chromatin of individual genes or gene clusters could potentially occur locally without preference for nuclear position and/or causing repositioning. These findings have important implications for understanding relationships between nuclear organization and gene expression patterns in cancer.

Figure 1. Association of MLH1 with the H3K27Me3 domains

RKO (A) and SW480 cells (B) immunostained for H3K4Me2 (top) or H3K27Me3 (bottom) and MLH1 and ACTB (shown in Supplementary Fig. S3 A-B, G-H) by DNA-FISH. Line scan plots the intensities of the modified histone (red), gene signal (green) and DNA (blue) along the line. Inset shows the degree of colocalization of the FISH and modified histone signal as white pixels. Only allele(s) in a single z-slice are shown here (all alleles are shown in Supplementary Fig. S3). (C) Boxplots show colocalization (Manders’ coefficient) between the FISH and modified histone signal from a single experiment (n=25 nuclei). X-axis labels shows the genes analyzed in SW480 (prefix S) and RKO (prefix R). (D) Colocalization values normalized to ACTB. The median Manders’ coefficients of MLH1, SFRP4 and HBB colocalization with the modified histones from three experiments (n=25, 10, 10 nuclei) were normalized to that of ACTB and plotted as relative colocalization (y-axis). Error bars indicate standard deviation.

Figure 2. Position of MLH1, SFRP4, HBB, ACTB relative to the perinuclear and perinucleolar domains

(A) Orthogonal sections passing through the three aneuploid alleles of SFRP4 locus (green) in SW480 cells showing proximity to the perinuclear or perinucleolar regions, which are stained with H3K27Me3 (red). Nucleoli are devoid of DNA staining (blue). Scale-bar is 2 μm. (B) Quantitation of gene position relative to perinuclear (PNu) or perinucleolar (PNo) regions in SW480 and RKO cells. The nearest distance to the perinuclear or perinucleolus for 30–45 alleles are plotted (y-axis).

Figure 3. Association of SFRP5 and ICAM1 with the H3K4Me2 domains

HCT116 (A) and SW480 cells (B) immunostained for H3K4Me2 (top) or H3K27Me3 (bottom) and ICAM1 and ACTB (not shown) loci by DNA-FISH. Figure details same as Fig. 1A-B. All alleles are shown in Supplementary Fig. S6. (C) Quantitation of colocalization from a single experiment (n=20 nuclei) as in Fig. 1C. SW480, RKO and HCT116 are labeled with prefix S, R and H respectively. (D) Colocalization values of SFRP5 and ICAM1 normalized to ACTB as in Fig. 1D (n=20, 10, 10 nuclei) plotted as relative colocalization (y-axis). Asterisk indicates that ICAM1 localization was compared between SW480 and HCT116 cells.

Figure 4. Relationship between nuclear position, LRES and gene density

(A) Expression of genes in a 1Mb domain around the genes studied here in HCT116 (H), DKO (D), SW480 (S), SW480 + 5-aza-CdR (S′), RKO (R) and RKO + 5-aza-CdR (R′). The CR genes are highlighted in grey and ACTB control is highlighted in blue; genes are listed top to bottom in the order of their 5′ to 3′ position in the genome (UCSC). Positive expression (green), no expression (red) and weak expression (yellow) are shown. White box in DKO in the ICAM1 locus indicates the absence of DNMT1 gene due to genetic knockout. MLH1, SFRP4 and SFRP5 loci tend to show variable degree of silencing of adjacent genes in RKO, HCT116 and SW480, in that order. (B) Gene densities (y-axis) in a 2 and 6 Mb window centered on the listed genes plotted as genes/Mb.

Figure 5. Relationship between LRES and CIMP

(A and B) Neighborhood expression score (NES) for every gene was calculated as the median expression values of three upstream and three downstream genes. The NES was used as a measure of LRES. A and B shows distribution of the NES values for genes methylated in both SW480 and RKO (A) and genes methylated only in SW480 or only in RKO (B). S and R in legend denote SW480 and RKO.