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Integrative analysis of tissue-specific methylation and alternative splicing identifies conserved transcription factor binding motifs.

Wan J, Oliver VF, Zhu H, Zack DJ, Qian J, Merbs SL - Nucleic Acids Res. (2013)

Bottom Line: More than 14% of alternatively spliced genes were associated with a T-DMR.Novel DNA sequences motifs overrepresented in T-DMRs were identified as being associated with positive and/or negative regulation of alternative splicing in a position-dependent context.Our results suggest that DNA methylation-dependent alternative splicing is widespread and lay the foundation for further mechanistic studies of the role of DNA methylation in tissue-specific splicing regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Wilmer Institute, Johns Hopkins University School of Medicine, 600 North Wolfe Street, 21287 Baltimore, MD, USA, Department of Pharmacology and Molecular Science, Johns Hopkins University School of Medicine, 600 North Wolfe Street, 21287 Baltimore, MD, USA, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, 600 North Wolfe Street, 21287 Baltimore, MD, USA, Department of Neuroscience, Johns Hopkins University School of Medicine, 600 North Wolfe Street, 21287 Baltimore, MD, USA, Institute of Genetic Medicine, Johns Hopkins University School of Medicine, 600 North Wolfe Street, 21287 Baltimore, MD, USA and Institut de la Vision, 17 rue Moreau, 75012 Paris, France.

ABSTRACT
The exact role of intragenic DNA methylation in regulating tissue-specific gene regulation is unclear. Recently, the DNA-binding protein CTCF has been shown to participate in the regulation of alternative splicing in a DNA methylation-dependent manner. To globally evaluate the relationship between DNA methylation and tissue-specific alternative splicing, we performed genome-wide DNA methylation profiling of mouse retina and brain. In protein-coding genes, tissue-specific differentially methylated regions (T-DMRs) were preferentially located in exons and introns. Gene ontology and evolutionary conservation analysis suggest that these T-DMRs are likely to be biologically relevant. More than 14% of alternatively spliced genes were associated with a T-DMR. T-DMR-associated genes were enriched for developmental genes, suggesting that a specific set of alternatively spliced genes may be regulated through DNA methylation. Novel DNA sequences motifs overrepresented in T-DMRs were identified as being associated with positive and/or negative regulation of alternative splicing in a position-dependent context. The majority of these evolutionarily conserved motifs contain a CpG dinucleotide. Some transcription factors, which recognize these motifs, are known to be involved in splicing. Our results suggest that DNA methylation-dependent alternative splicing is widespread and lay the foundation for further mechanistic studies of the role of DNA methylation in tissue-specific splicing regulation.

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Genomic distribution and GO analysis of T-DMRs. (A) Percentage of T-DMR probes (red) and all probes (gray) at each location with respect to protein-coding gene position. Upstream includes up to 4 kb from the TSS. (B) Percentage of T-DMRs and all probes located in upstream, exon or intron regions of non-coding RNA genes. (C) GO analysis of all T-DMRs, the number of genes in each category is noted. Asterisks note significant enrichment (*P < 0.01; ***P < 0.0001).
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gkt652-F1: Genomic distribution and GO analysis of T-DMRs. (A) Percentage of T-DMR probes (red) and all probes (gray) at each location with respect to protein-coding gene position. Upstream includes up to 4 kb from the TSS. (B) Percentage of T-DMRs and all probes located in upstream, exon or intron regions of non-coding RNA genes. (C) GO analysis of all T-DMRs, the number of genes in each category is noted. Asterisks note significant enrichment (*P < 0.01; ***P < 0.0001).

Mentions: DNA methylation profiling of two adult mouse tissues, retina and brain, was conducted using MBD2b/MBD3L1-enrichment and a custom CHARM tiling array (MeKL-chip) and yielded 2498 T-DMRs (17,28). These T-DMRs encompassed 17 965 50-bp probes, just 0.8% of all probes on the CHARM array. To assess the relative location profile of the T-DMRs, they were defined as either intragenic (≤4 kb upstream of the transcription start site (TSS), 5′UTR, coding exon, intron and 3′UTR) or intergenic (all other genomic locations). In total, 22.0% of the 2498 T-DMRs identified between mouse retina and brain were intergenic (i.e. outside of protein-coding genes), demonstrating a significant overrepresentation compared with percentage of all probes within the intergenic region based on the CHARM array design (20.7%; P = 6.4 × 10−6). The majority of T-DMRs were associated with intragenic regions (i.e. protein-coding genes; 78.0%, n = 1596). As might be expected from their proposed regulatory roles, 8.7 and 4.6% of T-DMR probes were in the upstream and 5′UTRs of genes, respectively (Figure 1A). However, this represented fewer probes than expected when compared with the CHARM array probe distributions of upstream and 5′UTR locations (19.6 and 6.4%, respectively). Similarly, T-DMRs were not overrepresented at 3′UTRs. Strikingly, the majority of gene-associated T-DMR probes were located in exons (17.8%) and introns (45.3%), which were significantly overrepresented (12.2 and 39.5%, respectively; P = 0, Figure 1A), suggesting that DNA methylation might play an important role in regulating alternative splicing.Figure 1.


Integrative analysis of tissue-specific methylation and alternative splicing identifies conserved transcription factor binding motifs.

Wan J, Oliver VF, Zhu H, Zack DJ, Qian J, Merbs SL - Nucleic Acids Res. (2013)

Genomic distribution and GO analysis of T-DMRs. (A) Percentage of T-DMR probes (red) and all probes (gray) at each location with respect to protein-coding gene position. Upstream includes up to 4 kb from the TSS. (B) Percentage of T-DMRs and all probes located in upstream, exon or intron regions of non-coding RNA genes. (C) GO analysis of all T-DMRs, the number of genes in each category is noted. Asterisks note significant enrichment (*P < 0.01; ***P < 0.0001).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3794605&req=5

gkt652-F1: Genomic distribution and GO analysis of T-DMRs. (A) Percentage of T-DMR probes (red) and all probes (gray) at each location with respect to protein-coding gene position. Upstream includes up to 4 kb from the TSS. (B) Percentage of T-DMRs and all probes located in upstream, exon or intron regions of non-coding RNA genes. (C) GO analysis of all T-DMRs, the number of genes in each category is noted. Asterisks note significant enrichment (*P < 0.01; ***P < 0.0001).
Mentions: DNA methylation profiling of two adult mouse tissues, retina and brain, was conducted using MBD2b/MBD3L1-enrichment and a custom CHARM tiling array (MeKL-chip) and yielded 2498 T-DMRs (17,28). These T-DMRs encompassed 17 965 50-bp probes, just 0.8% of all probes on the CHARM array. To assess the relative location profile of the T-DMRs, they were defined as either intragenic (≤4 kb upstream of the transcription start site (TSS), 5′UTR, coding exon, intron and 3′UTR) or intergenic (all other genomic locations). In total, 22.0% of the 2498 T-DMRs identified between mouse retina and brain were intergenic (i.e. outside of protein-coding genes), demonstrating a significant overrepresentation compared with percentage of all probes within the intergenic region based on the CHARM array design (20.7%; P = 6.4 × 10−6). The majority of T-DMRs were associated with intragenic regions (i.e. protein-coding genes; 78.0%, n = 1596). As might be expected from their proposed regulatory roles, 8.7 and 4.6% of T-DMR probes were in the upstream and 5′UTRs of genes, respectively (Figure 1A). However, this represented fewer probes than expected when compared with the CHARM array probe distributions of upstream and 5′UTR locations (19.6 and 6.4%, respectively). Similarly, T-DMRs were not overrepresented at 3′UTRs. Strikingly, the majority of gene-associated T-DMR probes were located in exons (17.8%) and introns (45.3%), which were significantly overrepresented (12.2 and 39.5%, respectively; P = 0, Figure 1A), suggesting that DNA methylation might play an important role in regulating alternative splicing.Figure 1.

Bottom Line: More than 14% of alternatively spliced genes were associated with a T-DMR.Novel DNA sequences motifs overrepresented in T-DMRs were identified as being associated with positive and/or negative regulation of alternative splicing in a position-dependent context.Our results suggest that DNA methylation-dependent alternative splicing is widespread and lay the foundation for further mechanistic studies of the role of DNA methylation in tissue-specific splicing regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Wilmer Institute, Johns Hopkins University School of Medicine, 600 North Wolfe Street, 21287 Baltimore, MD, USA, Department of Pharmacology and Molecular Science, Johns Hopkins University School of Medicine, 600 North Wolfe Street, 21287 Baltimore, MD, USA, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, 600 North Wolfe Street, 21287 Baltimore, MD, USA, Department of Neuroscience, Johns Hopkins University School of Medicine, 600 North Wolfe Street, 21287 Baltimore, MD, USA, Institute of Genetic Medicine, Johns Hopkins University School of Medicine, 600 North Wolfe Street, 21287 Baltimore, MD, USA and Institut de la Vision, 17 rue Moreau, 75012 Paris, France.

ABSTRACT
The exact role of intragenic DNA methylation in regulating tissue-specific gene regulation is unclear. Recently, the DNA-binding protein CTCF has been shown to participate in the regulation of alternative splicing in a DNA methylation-dependent manner. To globally evaluate the relationship between DNA methylation and tissue-specific alternative splicing, we performed genome-wide DNA methylation profiling of mouse retina and brain. In protein-coding genes, tissue-specific differentially methylated regions (T-DMRs) were preferentially located in exons and introns. Gene ontology and evolutionary conservation analysis suggest that these T-DMRs are likely to be biologically relevant. More than 14% of alternatively spliced genes were associated with a T-DMR. T-DMR-associated genes were enriched for developmental genes, suggesting that a specific set of alternatively spliced genes may be regulated through DNA methylation. Novel DNA sequences motifs overrepresented in T-DMRs were identified as being associated with positive and/or negative regulation of alternative splicing in a position-dependent context. The majority of these evolutionarily conserved motifs contain a CpG dinucleotide. Some transcription factors, which recognize these motifs, are known to be involved in splicing. Our results suggest that DNA methylation-dependent alternative splicing is widespread and lay the foundation for further mechanistic studies of the role of DNA methylation in tissue-specific splicing regulation.

Show MeSH