Epigenomic analysis of Alu repeats in human ependymomas

Abstract

Global loss of DNA methylation has been known for decades as an epigenomic aberration associated with carcinogenesis and cancer progression. Loss of DNA methylation affects predominantly repetitive elements, which encompass >50% of the CpG dinucleotides present in the human genome. Because of the lack of an effective approach, no studies have been conducted to reveal such genome-wide methylation changes at a single-base resolution. To precisely determine the CpG sites with methylation loss during progression of pediatric intracranial ependymomas, we exploited a high-throughput bisulfite sequencing approach that simultaneously generates methylation profiles for thousands of Alu elements and their flanking sequences. Comparison of the methylation profiles of normal and tumor tissues revealed that the methylation status of the majority of CpG sites adjacent to or within Alu repeats remain unaltered, while a small set of CpG sites gain or lose methylation in ependymomas. Compared to the CpG sites with stable methylation level between normal control and ependymomas, the differentially methylated CpG sites are enriched in the sequences with low CpG density in the flanking regions of Alu repeats, rather than within the Alu sequences themselves. In addition, the CpG sites that are hypermethylated in ependymomas are proximal to CpG islands, whereas those that are hypomethylated are overrepresented in intergenic regions. Lastly, aberrant methylation of several genomic loci was confirmed to be associated with the aggressive primary tumors and the relapsed ependymomas.

Fig. 1.

Global Alu methylation profile in ependymomas. Each dot represents the methylation level of an individual sample. The x axis represents the sample groups. The y axis represents the global Alu methylation levels determined by pyrosequencing of intra-Alu PCR products. Group comparisons were conducted for normal controls and tumor tissues with P values determined by t test (two-sample assuming unequal variances).

Fig. 2.

Pyrosequencing validations. (A–E) Methylation profiles of five genomic loci in a panel of ependymoma tissues and normal controls. Each dot represents the methylation level of an individual sample. The x axis represents the sample groups. The y axis represents the average methylation level determined by pyrosequencing. NC, PN, PA, and RL represent normal control (n = 10), primary nonaggressive (n = 12), primary aggressive (n = 15), and relapsed ependymoma tissues (n = 25), respectively. Group comparisons were conducted for normal controls and tumor tissues with P values determined by t test (two-sample assuming unequal variances). *, P < 0.01 in comparison with the normal controls; **, P < 0.001 in comparison with the normal controls. (F) Correlation of methylation levels of genomic locus 1 and 2. Each dot represents the methylation levels of genomic locus 1 and 2 in an individual sample.

Fig. 3.

Methylation status of global Alu and five specific loci and the clinical behavior of ependymomas. (A) Classifier distinguishing the ependymomas from normal. Green squares = methylation level of the sample is within the cut-offs, two standard derivations from the average methylation levels of normal controls; red squares = methylation level of the sample is either below the cut-off for hypomethylated loci or above the cut-off for hypermethylated locus. (B) Classifier distinguishing the primary aggressive ependymomas from primary nonaggressive ones. Green squares = methylation level of the sample is within the cut-offs, two standard derivations from the average methylation levels of primary nonaggressive ependymomas; red squares = methylation level of the sample is either below the cut-off for hypomethylated loci or above the cut-off for hypermethylated locus. Each row represents a tissue sample, and each column represents a specific locus (L1 to L5) or global Alu methylation level (GA) indicated on top. NC, PN, and PA represent normal control, primary nonaggressive, and aggressive ependymoma tissues, respectively.