DNA hypermethylation of alternatively spliced and repeat sequences in humans

Abstract

DNA methylation is presently accepted as a tentative regulatory parameter in splicing. Recently, we reported significant methylation differences among various exonic splicing-enhancing elements and alternative splicing events, based on CpG methylation data from the Human Epigenome Project for chromosomes 6, 20 and 22. Presently, using a different computational approach and the same database, we report: (a) significant increase of hypermethylation in intronic and exonic sequences close to acceptor sites, relative to overall introns and exons, respectively (1,973 CpGs examined); (b) frequent CpGs, mostly hypomethylated, in donors and infrequent CpGs mostly hypermethylated, in acceptors; and (c) hypermethylation in cassette exons which are occasionally spliced and have weaker average splicing potential, relative to constitutive exons (p < 0.0001). CpGs are hypomethylated in non-coding exons (only 16 % hypermethylation). Single-exon genes, similarly to first exons, frequently contain hypomethylated CpGs, while in internal and last exons CpGs are more frequently hypermethylated. Methylation is also more frequent in strange introns and splice sites processed by the minor spliceosome, e.g., ATAC, (p < 0.0001 in all cases), but not in sites of incomplete processing, e.g., retained introns or bleeding exons, (p = 0.706 and p = 0.313, respectively). Most Alus, which are known to contribute to transcript presentation, are heavily methylated, in contrast with other Alus, e.g., AluJo and mammalian interspersed repetitive elements which have been previously associated with alternative expression. These results elucidate the role of intragenic methylation in association with alternative splicing and facilitate the evaluation of genomic variations/polymorphisms and the development of tools for the prediction of alternative splicing events.

Fig. 1

Methylation frequencies (obtained from data in Table 1) in exons, coding exons, non-coding exons, and introns

Fig. 2

Average methylation per sequence, i.e., the number of identified HmC, ImC and LmC versus the number of analyzed sequences (no. of CpGs/no. of seqs), in total, single, initial, internal and terminal exons and in introns, containing at least one recorded CpG. The number of CpGs per the number of sequences is shown in the embedded table

Fig. 3

a Distribution of methylation at both sides of donors and acceptors in 20-nt sliding windows of 60 nt total length centered at the splice junctions. The exon–intron boundaries are shown in vertical dashed lines. b Hypermethylation frequency corresponding to the same region shown in a. The average HmC frequency in whole exons and introns is shown in horizontal lines (obtained from Fig. 1)

Fig. 4

Hypermethylation frequency in the 130-nt intronic sequences flanking donor (left) and acceptor (right) sites, using 20-nt sliding windows. The average HmC frequency in introns, obtained from Fig. 1, is shown in horizontal lines

Fig. 5

Graphical representation of methylation densities in exons (cassette, constitutive and total), introns and 100 nt 5′, 3′ intronic sites. The relative widths of the different grey tones correspond to methylation frequencies. The shown frequency scale is enlarged 10× for introns

Fig. 6

Distribution of the studied a HmC, b ImC and c LmC with respect to their co-occurrence with alternative splicing events. The frequency of all reported alternatively spliced introns and exons in chromosomes 6, 20 and 22 is shown in d

Fig. 7

Relative frequencies of different types of SINEs in chromosomes 6, 20 and 22. Some Alus (AluSq/x, AluSg1, Alu, FAM, AluYd2, AluYc2, AluJ/FLAM, and AluYd3a1) generally denoted as ‘other’ have frequency greater than 0.3 % (not shown)

Fig. 8

Distribution of methylated CpGs among different types of SINEs. The total number of SINEs in chromosomes 6, 20 and 22 is 167,304