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. 2017 Jan;27(1):118-132.
doi: 10.1101/gr.207522.116. Epub 2016 Dec 20.

The Epigenetic Landscape of Alu Repeats Delineates the Structural and Functional Genomic Architecture of Colon Cancer Cells

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Free PMC article

The Epigenetic Landscape of Alu Repeats Delineates the Structural and Functional Genomic Architecture of Colon Cancer Cells

Mireia Jordà et al. Genome Res. .
Free PMC article

Abstract

Cancer cells exhibit multiple epigenetic changes with prominent local DNA hypermethylation and widespread hypomethylation affecting large chromosomal domains. Epigenome studies often disregard the study of repeat elements owing to technical complexity and their undefined role in genome regulation. We have developed NSUMA (Next-generation Sequencing of UnMethylated Alu), a cost-effective approach allowing the unambiguous interrogation of DNA methylation in more than 130,000 individual Alu elements, the most abundant retrotransposon in the human genome. DNA methylation profiles of Alu repeats have been analyzed in colon cancers and normal tissues using NSUMA and whole-genome bisulfite sequencing. Normal cells show a low proportion of unmethylated Alu (1%-4%) that may increase up to 10-fold in cancer cells. In normal cells, unmethylated Alu elements tend to locate in the vicinity of functionally rich regions and display epigenetic features consistent with a direct impact on genome regulation. In cancer cells, Alu repeats are more resistant to hypomethylation than other retroelements. Genome segmentation based on high/low rates of Alu hypomethylation allows the identification of genomic compartments with differential genetic, epigenetic, and transcriptomic features. Alu hypomethylated regions show low transcriptional activity, late DNA replication, and its extent is associated with higher chromosomal instability. Our analysis demonstrates that Alu retroelements contribute to define the epigenetic landscape of normal and cancer cells and provides a unique resource on the epigenetic dynamics of a principal, but largely unexplored, component of the primate genome.

Figures

Figure 1.
Figure 1.
(A) Diagram illustrating the principle of Next-generation Sequencing of UnMethylated Alu repeats (NSUMA) technique. Genomic DNA is represented by a blue line, and the Alu repeat is represented by a gray box. DNA is digested with the restriction enzymes SmaI and MseI (impaired and insensitive to DNA methylation, respectively). The digested DNA fragments are ligated to two adapters with ends compatible with each one of the enzyme cuts. Primers complementary to the adapters are used to generate an amplified representation of the fragments. Amplicons generated from DNA fragments containing the SmaI adapter report unmethylated DNA and constitute the NSUMA canonical universe. Amplicons without the SmaI adapter (MseI-MseI) are also sequenced and used for the analysis of copy number variation. (B) Genetic element distribution according to the occupied fraction of the human genome, the content of SmaI sites, and their representation in the canonical NSUMA universe. (C) Relative unmethylation of different genetic elements in each one of the sample types analyzed by NSUMA in regard to the highly hypomethylated cell line DKO. CpG islands show the highest relative representation in all sample types as most of them are unmethylated, whereas Alu repeats show the lowest representation due to their heavy methylation. (D) Number of differentially methylated elements in the comparison between pairs of tissues (adjusted P-value <0.05 and |log2 FC| >1). Full data summary is reported in Supplemental Table S5, and detailed MA plots are shown in Supplemental Figure S13.
Figure 2.
Figure 2.
Features of genomic compartments based on NSUMA coverage and differential methylation (Supplemental Methods; Supplemental Tables S4, S6). (A) Box plot of the number of CpGs within the Alu (blue boxes) and in the 500-bp flanking regions (yellow boxes). Flanking regions have been arranged by CpG content: low to the left and high to the right. Alu repeats unmethylated in all tissues (UNM All) tend to localize in the boundaries of regions with large differences in CpG content (*). Statistical analyses are shown in Supplemental Table S7. (B) Box plot of the genomic distances between Alu repeats classified in different compartments and TSSs, CpG islands, and LINEs. ANOVA P-values for comparisons between Alu repeat subsets are indicated at the bottom of each graph. (C) Transcription factor binding motif enrichment in different subsets of Alu repeats. Full markers correspond to putative binding sites located inside the Alu sequence, and empty markers indicate motifs located within flanking sequences (500 bp).
Figure 3.
Figure 3.
(A) Visualization of Chromosome 7 NSUMA Alu DMR for HCT116 and three colon cancer tumors (369T, 544T, and 557T) compared with their corresponding normal tissues (HCT116 was compared against the three normal samples). Values below 0 indicate hypomethylation in the tumor against the normal tissue. Additional tracks show the mean differential methylation of three colon tumors analyzed by WGBS (Hansen et al. 2011) and the density of Alu elements, genes, and CpG islands along the genome. (B) Hypomethylated regions obtained from NSUMA profiles in five colon cancer cell lines, and three primary colon cancers (mTUMOR). Hypomethylated regions obtained from WGBS in three colon cancers considering different repeat types (Alu, LINE, and other repeats). Additional tracks show the hypomethylated block (Colon Cancer HB) (Hansen et al. 2011) and the abundance of genes, CpG islands, and Alu repeats. The inset shows a detailed view of the differential methylation profiles in the region enclosed by a red dotted line. (C) Distribution of hypomethylated regions according to the mean differential methylation of Alu repeats determined by NSUMA and WGBS and LINEs determined by WGBS in colorectal cancers and HCT116 cell line. (D) Distribution of differential methylation in Alu (upper panel) and LINE (lower panel) repeat elements determined by WGBS in regard to the mean differential methylation of the enclosing region: HMAR, no HMAR, and SMAR.
Figure 4.
Figure 4.
(A) Density of genomic elements (genes, Alu repeats, CpG islands, and small nuclear RNAs) in genomic segments according to the Alu hypomethylation profiles determined by NSUMA. Overlapping HMAR regions (red line) result from the shared HMARs of three colon tumors and the HCT116 cell line (Supplemental Table S8). Nonoverlapping HMARs (gray line) correspond to hypomethylated regions in tumors or HCT116 but not in both. The rest of the genome (outside HMARs, green line) is neither hypomethylated in tumors nor HCT116 cells. The distribution of other genomic elements is shown in Supplemental Figure S22. (B) Distribution of the Alu differential methylation ratio of HCT116 in regard to Alu and LINE density (elements/100 kb). (C) Distribution of genomic segments according to the Alu mean DMR and the copy number variation in HCT116 and the tumor sample 544T. (D) The upper panel shows the relationship between the extent of the hypomethylated compartment (as percentage of the genome) and the number of regions with chromosomal alterations as detected by array CGH. The lower panel shows the number of chromosome segments with copy number alterations in regard to their location inside or outside HMAR. Tumor samples with a larger hypomethylated compartment show a higher number of alterations in both HMARs and outside HMARs.
Figure 5.
Figure 5.
(A) Relationships in the genomic distribution of colon cancer DNA hypomethylation and histone modification profiles of normal human colonic mucosa. Data have been displayed using the UCSC Genome Browser and represent (from top to bottom): the hypomethylated regions for LINEs analyzed by WGBS in three colorectal carcinomas, for Alu repeats analyzed by NSUMA in three colorectal carcinomas, for Alu repeats analyzed by NSUMA in HCT116 cell line, for Alu repeats analyzed by WGBS in three colorectal carcinomas, and the histone modification profiles for human colonic mucosa produced by the Roadmap Epigenomics Consortium. (B) Distribution of colon cancer hypomethylated regions concurs with nuclear lamina associated domains and late DNA replication. The inset shows a detailed view of genomic elements in the region enclosed by a red dotted line. (C) Alu and LINE differential methylation in colorectal cancer in relation to replication timing in IMR90 cells. Each dot represents a region of 100 kb.
Figure 6.
Figure 6.
Transcriptomic and epigenetic features of Alu elements according to their expression levels in the HCT116 cell line. (A) Distribution of Alu repeats according to the expression levels in regard to DNA methylation, expression of the associated gene, and gene-related location. (B) Enrichment distribution of chromatin functional states in Alu elements according to their expression levels (top) and that of associated gene (middle) and the DNA methylation state. (C) Distribution of Alu repeats according to the expression levels, expression of the associated gene, and DNA methylation in regard to HMAR location. (D) Plotting of 9339 Alu repeats sorted according to their expression (purple dots) and nearest gene expression (black dots). Low DNA methylation state (green bars at the top) and HMAR location (red bars at the bottom) are indicated for each Alu.
Figure 7.
Figure 7.
Diagram illustrating the nuclear epigenetic landscape of Alu repeats in normal and cancer cells. Alu-rich regions (dots) display high transcriptional activity and colocalize with active chromatin functional states (central part of the nucleus). In normal cells, most Alu elements remain methylated (black dots), but a few are unmethylated (gray and light gray dots) and show specific structural and functional features. Global DNA hypomethylation in cancer cells affects Alu repeats localized in late replicating lamin-associated domains, but is largely excluded from Alu-rich domains.

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