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. 2012 Sep 15:13:480.
doi: 10.1186/1471-2164-13-480.

Genome-wide association between DNA methylation and alternative splicing in an invertebrate

Kevin Flores  1 , Florian Wolschin, Jason J Corneveaux, April N Allen, Matthew J Huentelman, Gro V Amdam
Affiliations

Genome-wide association between DNA methylation and alternative splicing in an invertebrate

Kevin Flores et al. BMC Genomics. .

Abstract

Background: Gene bodies are the most evolutionarily conserved targets of DNA methylation in eukaryotes. However, the regulatory functions of gene body DNA methylation remain largely unknown. DNA methylation in insects appears to be primarily confined to exons. Two recent studies in Apis mellifera (honeybee) and Nasonia vitripennis (jewel wasp) analyzed transcription and DNA methylation data for one gene in each species to demonstrate that exon-specific DNA methylation may be associated with alternative splicing events. In this study we investigated the relationship between DNA methylation, alternative splicing, and cross-species gene conservation on a genome-wide scale using genome-wide transcription and DNA methylation data.

Results: We generated RNA deep sequencing data (RNA-seq) to measure genome-wide mRNA expression at the exon- and gene-level. We produced a de novo transcriptome from this RNA-seq data and computationally predicted splice variants for the honeybee genome. We found that exons that are included in transcription are higher methylated than exons that are skipped during transcription. We detected enrichment for alternative splicing among methylated genes compared to unmethylated genes using fisher's exact test. We performed a statistical analysis to reveal that the presence of DNA methylation or alternative splicing are both factors associated with a longer gene length and a greater number of exons in genes. In concordance with this observation, a conservation analysis using BLAST revealed that each of these factors is also associated with higher cross-species gene conservation.

Conclusions: This study constitutes the first genome-wide analysis exhibiting a positive relationship between exon-level DNA methylation and mRNA expression in the honeybee. Our finding that methylated genes are enriched for alternative splicing suggests that, in invertebrates, exon-level DNA methylation may play a role in the construction of splice variants by positively influencing exon inclusion during transcription. The results from our cross-species homology analysis suggest that DNA methylation and alternative splicing are genetic mechanisms whose utilization could contribute to a longer gene length and a slower rate of gene evolution.

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Figures

Figure 1
Figure 1
The distribution of DNA methylation among included exons vs. skipped exons. The plots in (A) and (B) illustrate that exons that are included in transcription are higher methylated than exons that are skipped during transcription. The absolute methylation (mCG/length) around exons was calculated by dividing the regions +/− 200 bp within the start site (A) or end site (B) of either included exons or skipped exons into 20 equal intervals. Plots show the methylation level within each interval. Included exons have significantly more methylation than skipped exons in regions beginning after the exon start site and just before the exon end site (P < .05, Wilcoxon rank-sum test in each interval). Genome-wide DNA methylation data were obtained from BS-seq [18].
Figure 2
Figure 2
Methylated genes are enriched for alternative splicing. Genome-wide DNA methylation data was overlaid with genes and the occurrence of alternative transcripts predicted from a de novo transcriptome assembly. The proportion of methylated genes that were alternatively spliced (triangles) was significantly greater than for unmethylated genes (circles) for several minimum expression thresholds (Fisher’s exact test, P < 1e-10 for each minimum FPKM); expression is measured as the expected number of fragments per kilobase of transcript per million reads (FPKM). We tested several minimum FPKM values to show that enrichment is robust despite the possibility that transcript abundance estimation may be inaccurate at low expression (FPKM). Raw data were the same as for Figure 1.
Figure 3
Figure 3
Alternative splicing and DNA methylation in genes is associated with a longer gene length. The cumulative distribution function (CDF) for the size of a gene (the size of the gene body, including introns, is the number of base pairs (bp) in the gene annotation) is plotted for several categories of gene methylation and alternative splicing. Top left panel: The CDFs of methylated and unmethylated genes show that methylated genes are longer than unmethylated genes. Top right panel: Alternatively spliced genes are longer than non-alternatively spliced genes. Bottom left panel: Methylation is also associated with longer gene size among non-alternatively spliced genes. Bottom right panel: Methylation is associated with longer gene size among alternatively spliced genes. Splice variants were annotated by assembling a de novo transcriptome from RNA-seq data. The length of a gene is defined to be the maximum length from any splice variant (including introns) of that gene. The significance of shifted distributions was quantified by using the Wilcoxon rank sum test with continuity correction (P < 2.2e-16 for each panel). Genome-wide DNA methylation data were obtained from BS-seq [18].
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