Genome-wide tracking of unmethylated DNA Alu repeats in normal and cancer cells

Abstract

Methylation of the cytosine is the most frequent epigenetic modification of DNA in mammalian cells. In humans, most of the methylated cytosines are found in CpG-rich sequences within tandem and interspersed repeats that make up to 45% of the human genome, being Alu repeats the most common family. Demethylation of Alu elements occurs in aging and cancer processes and has been associated with gene reactivation and genomic instability. By targeting the unmethylated SmaI site within the Alu sequence as a surrogate marker, we have quantified and identified unmethylated Alu elements on the genomic scale. Normal colon epithelial cells contain in average 25 486 +/- 10 157 unmethylated Alu's per haploid genome, while in tumor cells this figure is 41 995 +/- 17 187 (P = 0.004). There is an inverse relationship in Alu families with respect to their age and methylation status: the youngest elements exhibit the highest prevalence of the SmaI site (AluY: 42%; AluS: 18%, AluJ: 5%) but the lower rates of unmethylation (AluY: 1.65%; AluS: 3.1%, AluJ: 12%). Data are consistent with a stronger silencing pressure on the youngest repetitive elements, which are closer to genes. Further insights into the functional implications of atypical unmethylation states in Alu elements will surely contribute to decipher genomic organization and gene regulation in complex organisms.

Figure 1.

Schematic diagram of the QUMA and AUMA methods. DNA is depicted by a solid line, Alu elements are represented by dashed boxes. The QUMA and AUMA recognition sites (AACCCGGG) are represented by dashed/gray boxes. CpGs at SmaI sites are shown as full circles when methylated and as open circles when unmethylated. The methylation-sensitive restriction endonuclease SmaI can only digest unmethylated targets, leaving blunt ends to which adaptors can be ligated. (A) QUMA is performed by real-time PCR of an inner Alu fragment using a primer complementary to the Alu consensus sequence upstream of the SmaI site and the primer complementary to the adaptor to which two Alu homologous nucleotides (TT) have been added. (B) In AUMA, sequences flanked by two ligated adaptors are amplified by PCR using a single primer, the same adaptor primer plus the TT nucleotides. When only a few nucleotides are added to the primer, i.e. TT, as illustrated here, other non-Alu sequences may be amplified. This allows the amplification of a large number of sequences that typically range from 100 to 2000 bp.

Figure 2.

AUMA of normal (N)–tumor (T) pairs of two different patients performed using primer BAu-TT. A highly reproducible band patterning is observed among the four replicates. Representative bands showing gains (hypomethylations) and losses (hypermethylations) are marked with up and down arrowheads, respectively.

Figure 3.

Relative distribution the Alu elements and sequence targets considered in bioinformatic and experimental QUMA and AUMA. Mb: number of megabases occupied by each type of element; elements: number of elements considered (‘Rest’ has been set arbitrarily to 50%); SmaI site: CCCGGG sequence; vQUMA hits: AACCCGGG (or GGGCCCTT) sites in Alu elements; vAUMA hits: AACCCGGG (or GGGCCCTT) sites; vAUMA ends: vAUMA hits considering only putative AUMA products of <1 kb (see Material and Methods section); AUMA: elements at each one of the two ends of actual AUMA products.

Figure 4.

Quantitation of unmethylated Alu's in 17 paired normal mucosa and colorectal carcinoma by QUMA. The values represent the estimated number of unmethylated Alu's per haploid genome. Most tumors exhibited a higher level of hypomethylation when compared with the respective normal.

Figure 5.

(A) Chromosomal origin of AUMA products. A competitive hybridization of AUMA product obtained from normal tissue DNA (red) and genomic DNA (green) to metaphase chromosomes was performed. AUMA products showed an unequal distribution along chromosomes, displaying highest densities at most telomeric regions and some interstitial bands. Chromosomes 16, 17 and 19 yielded the highest AUMA density. (B) Intensity distribution of AUMA products hybridized to BAC arrays in selected chromosomes. The average intensity (X-axis) of the two normal (blue) and tumor samples analyzed (red) for each BAC is shown. BACs are arranged along the Y-axis according to its position in the chromosome. (C) Differential methylation profiles determined by competitive hybridization of AUMA products from normal and tumor tissue to BAC arrays. Illustrative examples are shown for chromosomes 7 and 8 from the two cases analyzed (81 and 151). X-axis indicates log2 ratio of tumor/normal intensities. Positive values (to the right) indicate hypomethylations, negative values (to the left) indicate hypermethylations. Additional examples are shown in Supplementary Figure 5.

Figure 6.

Distribution of hypermethylation and hypomethylation rates in the 110 AUMA tagged bands. Rates were obtained by comparison of the AUMA fingerprints obtained in 50 colorectal tumors as compared to their respective matched normal tissue.

Figure 7.

(A) Detail of the AUMA fingerprints generated from five normal–tumor sample pairs. The presence of the Aq3 band is indicated by an asterisk under the three Aq3 positive cases. (B) The relative position of the AUMA Aq3 band, MLT1A and Alu Y repetitive elements, as well as MYOM2 ninth exon are shown. Each vertical line in the CpG distribution represents a CpG dinucleotide along the DNA sequence. Two different fragments were amplified for the bisulfite sequencing analysis (gray boxes). Sequence is oriented 5′–3′ in regard to MYOM2 3′ end. (C) Methylation status of the CpG nucleotides in the two fragments amplified were ascertained by direct sequencing of bisulfite-treated DNAs of 5 normal–tumor pairs and 5 colon cancer cell lines. (D) ChIP analysis of the AluY element frequently hypomethylated in cancer revealed loss of trimethylation in histone 3 lysine 9 residue (3mH3K9) in LoVo cells (unmethylated at DNA level) as compared to HCT116 (methylated at DNA level). Treatment of HCT116 cells with 5AzaC and TSA produced a moderate decrease in the levels of trimethylation in H3K9.