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Genome-wide DNA hypomethylation and RNA:DNA hybrid accumulation in Aicardi-Goutières syndrome.

Lim YW, Sanz LA, Xu X, Hartono SR, Chédin F - Elife (2015)

Bottom Line: Aicardi-Goutières syndrome (AGS) is a severe childhood inflammatory disorder that shows clinical and genetic overlap with systemic lupus erythematosus (SLE).AGS is thought to arise from the accumulation of incompletely metabolized endogenous nucleic acid species owing to mutations in nucleic acid-degrading enzymes TREX1 (AGS1), RNase H2 (AGS2, 3 and 4), and SAMHD1 (AGS5).However, the identity and source of such immunogenic nucleic acid species remain undefined.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, University of California, Davis, Davis, United States.

ABSTRACT
Aicardi-Goutières syndrome (AGS) is a severe childhood inflammatory disorder that shows clinical and genetic overlap with systemic lupus erythematosus (SLE). AGS is thought to arise from the accumulation of incompletely metabolized endogenous nucleic acid species owing to mutations in nucleic acid-degrading enzymes TREX1 (AGS1), RNase H2 (AGS2, 3 and 4), and SAMHD1 (AGS5). However, the identity and source of such immunogenic nucleic acid species remain undefined. Using genome-wide approaches, we show that fibroblasts from AGS patients with AGS1-5 mutations are burdened by excessive loads of RNA:DNA hybrids. Using MethylC-seq, we show that AGS fibroblasts display pronounced and global loss of DNA methylation and demonstrate that AGS-specific RNA:DNA hybrids often occur within DNA hypomethylated regions. Altogether, our data suggest that RNA:DNA hybrids may represent a common immunogenic form of nucleic acids in AGS and provide the first evidence of epigenetic perturbations in AGS, furthering the links between AGS and SLE.

No MeSH data available.


Related in: MedlinePlus

Methylation profiles of AGS and control cells.(A) Percent methylation of lymphoblastoid cells around TSS, as determined by reduced representation bisulfite sequencing (RRBS). AGS2 LCLs are significantly hypomethylated (p < 2.2 × 10−16). (B) Percent methylation of control and AGS fibroblasts at four CpG sites, as surveyed by LINE-1 pyrosequencing. (C) Gene expression levels (measured by RNA-seq) for a range of genes involved in the control of DNA methylation are shown for control and a variety of AGS fibroblasts.DOI:http://dx.doi.org/10.7554/eLife.08007.009
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fig4s1: Methylation profiles of AGS and control cells.(A) Percent methylation of lymphoblastoid cells around TSS, as determined by reduced representation bisulfite sequencing (RRBS). AGS2 LCLs are significantly hypomethylated (p < 2.2 × 10−16). (B) Percent methylation of control and AGS fibroblasts at four CpG sites, as surveyed by LINE-1 pyrosequencing. (C) Gene expression levels (measured by RNA-seq) for a range of genes involved in the control of DNA methylation are shown for control and a variety of AGS fibroblasts.DOI:http://dx.doi.org/10.7554/eLife.08007.009

Mentions: The observation that RNA:DNA hybrids accumulate over intergenic regions is surprising given that they are normally maintained in a transcriptionally quiescent state owing to the deposition of silencing epigenetic marks such as DNA methylation (Yoder et al., 1997). To determine if DNA methylation patterns were altered in AGS, we performed MethylC-seq (Lister et al., 2009) to profile DNA methylation at low coverage (3.21–7.82 X coverage) across the same set of primary fibroblasts studied above (Supplementary file 1). All AGS cells displayed significant, global, DNA hypomethylation (Figure 4A). AGS2 and 4 cells showed profound DNA hypomethylation, with a ∼20% reduction in genomic methylation levels overall. AGS1 and 5 showed a more moderate, but still highly significant, 5–10% reduction in DNA methylation. This reduction affected TSSs and TTSs (Figure 4B,C; Figure 4—figure supplement 1A) and spread along the lengths of entire chromosomes (Figure 4D), significantly impacting nearly all genomic compartments, including genic, intergenic, and repeat regions (Figure 4E). Analysis of repeat classes further revealed that LINE and LTR elements were significantly hypomethylated in all AGS subtypes (Figure 4F). Pyrosequencing assays targeting human-specific LINE-1 elements also revealed a small but significant decrease in DNA methylation over 4 CpG sites carried at the LINE-1 5′-UTR promoter (Figure 4—figure supplement 1B). SINE elements, satellite repeats, and other repeat types were significantly hypomethylated in AGS2 and 4 but not always in AGS1 and 5 (Figure 4F). This profound reduction in DNA methylation was not caused by any detectable deregulation in the expression of genes encoding for the DNA methylation machinery or of components of a putative DNA de-methylation system (Figure 4—figure supplement 1C).10.7554/eLife.08007.008Figure 4.Genome-wide DNA hypomethylation in AGS fibroblasts.


Genome-wide DNA hypomethylation and RNA:DNA hybrid accumulation in Aicardi-Goutières syndrome.

Lim YW, Sanz LA, Xu X, Hartono SR, Chédin F - Elife (2015)

Methylation profiles of AGS and control cells.(A) Percent methylation of lymphoblastoid cells around TSS, as determined by reduced representation bisulfite sequencing (RRBS). AGS2 LCLs are significantly hypomethylated (p < 2.2 × 10−16). (B) Percent methylation of control and AGS fibroblasts at four CpG sites, as surveyed by LINE-1 pyrosequencing. (C) Gene expression levels (measured by RNA-seq) for a range of genes involved in the control of DNA methylation are shown for control and a variety of AGS fibroblasts.DOI:http://dx.doi.org/10.7554/eLife.08007.009
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4528086&req=5

fig4s1: Methylation profiles of AGS and control cells.(A) Percent methylation of lymphoblastoid cells around TSS, as determined by reduced representation bisulfite sequencing (RRBS). AGS2 LCLs are significantly hypomethylated (p < 2.2 × 10−16). (B) Percent methylation of control and AGS fibroblasts at four CpG sites, as surveyed by LINE-1 pyrosequencing. (C) Gene expression levels (measured by RNA-seq) for a range of genes involved in the control of DNA methylation are shown for control and a variety of AGS fibroblasts.DOI:http://dx.doi.org/10.7554/eLife.08007.009
Mentions: The observation that RNA:DNA hybrids accumulate over intergenic regions is surprising given that they are normally maintained in a transcriptionally quiescent state owing to the deposition of silencing epigenetic marks such as DNA methylation (Yoder et al., 1997). To determine if DNA methylation patterns were altered in AGS, we performed MethylC-seq (Lister et al., 2009) to profile DNA methylation at low coverage (3.21–7.82 X coverage) across the same set of primary fibroblasts studied above (Supplementary file 1). All AGS cells displayed significant, global, DNA hypomethylation (Figure 4A). AGS2 and 4 cells showed profound DNA hypomethylation, with a ∼20% reduction in genomic methylation levels overall. AGS1 and 5 showed a more moderate, but still highly significant, 5–10% reduction in DNA methylation. This reduction affected TSSs and TTSs (Figure 4B,C; Figure 4—figure supplement 1A) and spread along the lengths of entire chromosomes (Figure 4D), significantly impacting nearly all genomic compartments, including genic, intergenic, and repeat regions (Figure 4E). Analysis of repeat classes further revealed that LINE and LTR elements were significantly hypomethylated in all AGS subtypes (Figure 4F). Pyrosequencing assays targeting human-specific LINE-1 elements also revealed a small but significant decrease in DNA methylation over 4 CpG sites carried at the LINE-1 5′-UTR promoter (Figure 4—figure supplement 1B). SINE elements, satellite repeats, and other repeat types were significantly hypomethylated in AGS2 and 4 but not always in AGS1 and 5 (Figure 4F). This profound reduction in DNA methylation was not caused by any detectable deregulation in the expression of genes encoding for the DNA methylation machinery or of components of a putative DNA de-methylation system (Figure 4—figure supplement 1C).10.7554/eLife.08007.008Figure 4.Genome-wide DNA hypomethylation in AGS fibroblasts.

Bottom Line: Aicardi-Goutières syndrome (AGS) is a severe childhood inflammatory disorder that shows clinical and genetic overlap with systemic lupus erythematosus (SLE).AGS is thought to arise from the accumulation of incompletely metabolized endogenous nucleic acid species owing to mutations in nucleic acid-degrading enzymes TREX1 (AGS1), RNase H2 (AGS2, 3 and 4), and SAMHD1 (AGS5).However, the identity and source of such immunogenic nucleic acid species remain undefined.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, University of California, Davis, Davis, United States.

ABSTRACT
Aicardi-Goutières syndrome (AGS) is a severe childhood inflammatory disorder that shows clinical and genetic overlap with systemic lupus erythematosus (SLE). AGS is thought to arise from the accumulation of incompletely metabolized endogenous nucleic acid species owing to mutations in nucleic acid-degrading enzymes TREX1 (AGS1), RNase H2 (AGS2, 3 and 4), and SAMHD1 (AGS5). However, the identity and source of such immunogenic nucleic acid species remain undefined. Using genome-wide approaches, we show that fibroblasts from AGS patients with AGS1-5 mutations are burdened by excessive loads of RNA:DNA hybrids. Using MethylC-seq, we show that AGS fibroblasts display pronounced and global loss of DNA methylation and demonstrate that AGS-specific RNA:DNA hybrids often occur within DNA hypomethylated regions. Altogether, our data suggest that RNA:DNA hybrids may represent a common immunogenic form of nucleic acids in AGS and provide the first evidence of epigenetic perturbations in AGS, furthering the links between AGS and SLE.

No MeSH data available.


Related in: MedlinePlus