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Natural epigenetic polymorphisms lead to intraspecific variation in Arabidopsis gene imprinting.

Pignatta D, Erdmann RM, Scheer E, Picard CL, Bell GW, Gehring M - Elife (2014)

Bottom Line: Imprinting variability could contribute to observed parent-of-origin effects on seed development.We successfully predicted imprinting in additional strains based on methylation variability.We conclude that there is standing variation in imprinting even in recently diverged genotypes due to intraspecific epiallelic variation.

View Article: PubMed Central - PubMed

Affiliation: Whitehead Institute for Biomedical Research, Cambridge, United States.

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Expression and methylation analysis of other variably imprinted genes.(A) AT3G14205 is a PEG except when Cvi is the paternal parent (mRNA-seq data). Blue bars, % paternal allele expression; red bars, % maternal allele expression from combined mRNA-seq data; vertical line, expected % paternal expression for a non-imprinted gene. (B) Methylation of AT3G14205 5′ flanking region in Col embryo, Ler embryo and endosperm, and Cvi embryo. Red track, CG; green track, CHH. (C) Leaf methylation profile of AT3G14205 in Col, Ler, Kz_9, Cvi and Seattle_0 (http://neomorph.salk.edu/1001_epigenomes.html). (D) AT3G14205 is not imprinted when another hypomethylated strain (Seattle_0) is the pollen parent, but is a PEG in a cross with another methylated strain (Kz_9), as determined by sequencing RT-PCR products that span informative SNPs. The number of RT-PCR clones sequenced is indicated. p value, binomial test of whether the observed maternal:total ratio is less than the expected 2:3 ratio. (E) AT2G34890 is a MEG except when Ler is the paternal parent (mRNA-seq data). Vertical line, expected maternal allele expression for a non-imprinted gene. (F) Methylation of AT2G34890 5′ flanking region in Col and Ler embryo and Cvi embryo and endosperm. (G) Leaf methylation profile of AT2G34890 in Col, Cvi, Ler and Es_0 (http://neomorph.salk.edu/1001_epigenomes.html). (H) AT2G34890 is still imprinted when another hypomethylated strain (Es_0) is the pollen parent. p value: binomial test of whether the observed maternal:total ratio is greater than the expected 2:3 ratio.DOI:http://dx.doi.org/10.7554/eLife.03198.021
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fig6s2: Expression and methylation analysis of other variably imprinted genes.(A) AT3G14205 is a PEG except when Cvi is the paternal parent (mRNA-seq data). Blue bars, % paternal allele expression; red bars, % maternal allele expression from combined mRNA-seq data; vertical line, expected % paternal expression for a non-imprinted gene. (B) Methylation of AT3G14205 5′ flanking region in Col embryo, Ler embryo and endosperm, and Cvi embryo. Red track, CG; green track, CHH. (C) Leaf methylation profile of AT3G14205 in Col, Ler, Kz_9, Cvi and Seattle_0 (http://neomorph.salk.edu/1001_epigenomes.html). (D) AT3G14205 is not imprinted when another hypomethylated strain (Seattle_0) is the pollen parent, but is a PEG in a cross with another methylated strain (Kz_9), as determined by sequencing RT-PCR products that span informative SNPs. The number of RT-PCR clones sequenced is indicated. p value, binomial test of whether the observed maternal:total ratio is less than the expected 2:3 ratio. (E) AT2G34890 is a MEG except when Ler is the paternal parent (mRNA-seq data). Vertical line, expected maternal allele expression for a non-imprinted gene. (F) Methylation of AT2G34890 5′ flanking region in Col and Ler embryo and Cvi embryo and endosperm. (G) Leaf methylation profile of AT2G34890 in Col, Cvi, Ler and Es_0 (http://neomorph.salk.edu/1001_epigenomes.html). (H) AT2G34890 is still imprinted when another hypomethylated strain (Es_0) is the pollen parent. p value: binomial test of whether the observed maternal:total ratio is greater than the expected 2:3 ratio.DOI:http://dx.doi.org/10.7554/eLife.03198.021

Mentions: We identified regions of the genome that are subject to DNA demethylation in endosperm in at least one background but that are variably methylated among strains. Overlap between the strain DMRs and embryo-endosperm DMRs ranged from 12% for TEs to 31% for genes (Figure 4E). This suggests that there are sufficient epigenetic polymorphisms at embryo-endosperm DMRs to facilitate the formation of allele-specific imprinting. Differences in imprinting among alleles tied to differences in DNA methylation could be due to genetic or epigenetic differences (Figure 6—figure supplements 1,2, and 3). Of the 12 allele-specific imprinted genes we identified (Figure 2, Figure 2—source data 1), 10 were associated with coincident CG or CHH embryo-endosperm DMRs and strain DMRs, 6 of which occurred at TEs (Figure 2—source data 1, Figure 6, Figure 6—figure supplement 1, Figure 6—figure supplement 2). For all 6 genes we confirmed by sequencing that the TE annotated in Col was present in the same genomic location in Ler and Cvi with no major sequences changes except for a few SNPs. These 6 genes were also included among imprinting validation assays (Figure 1—source data 5, Figure 2—source data 1). We more closely investigated three allele-specific imprinted genes with strong differences in the ratio of maternal to paternal transcripts in imprinted and non-imprinted crosses: AT2G32370, AT2G34890, and AT3G14205 (Figure 2, Figure 6, Figure 6—figure supplement 2). AT2G32370, HDG3, was originally identified as a PEG because of its association with a Col and Ler embryo-endosperm DMR and because it was expressed specifically in the endosperm (Gehring et al., 2009). Our new data showed that HDG3 is not a PEG when Cvi is the male parent (Figure 2, Figure 6). The embryo-endosperm DMR associated with HDG3 is located in a Helitron fragment (ATREP10D) 5′ of the gene, and the methylated paternal allele is predominantly expressed. This region is not methylated in Cvi (Figure 6B, Figure 6—figure supplement 1). In crosses between Cvi females and Col or Ler males, maternal and paternal alleles are differentially methylated and the gene is imprinted (the naturally hypomethylated Cvi maternal allele has the same methylation profile as an actively demethylated maternal allele). But in reciprocal crosses between Col or Ler females and Cvi males, both maternal and paternal alleles are hypomethylated and the gene is biallelically expressed in the expected 2:1 maternal:paternal ratio (Figure 2, Figure 6). This suggests that differential methylation of maternal and paternal alleles, rather than simply demethylation of the maternal allele, is required for imprinted expression. A similar logic applies to AT3G14205; the Cvi allele is hypomethylated at a 5′ RC/Helitron fragment (ATREP1) compared to Col and Ler and the gene is not a PEG when it is transmitted through the Cvi male (Figure 2, Figure 2—source data 1, Figure 6—figure supplement 2). AT2G34890 was a MEG except when Ler is the male parent. The Ler AT2G34890 allele is hypomethylated at a 5′ MuDR element in comparison to Cvi and Col, suggesting that loss of methylation of the paternal allele could lead to its transcription (Figure 6—figure supplement 2). However, consistent with a more ambiguous relationship between DNA demethylation and MEGs (Figure 5), closer inspection of AT2G34890 shows that at a region 5′ of the TE both the Ler and Cvi alleles are hypomethylated compared to Col, suggesting that a clear methylation distinction between imprinted (Col and Cvi) and non-imprinted (Ler) paternal alleles does not exist (Figure 6—figure supplement 2).10.7554/eLife.03198.019Figure 6.Expression and methylation analysis of HDG3, an allele-specific imprinted gene.


Natural epigenetic polymorphisms lead to intraspecific variation in Arabidopsis gene imprinting.

Pignatta D, Erdmann RM, Scheer E, Picard CL, Bell GW, Gehring M - Elife (2014)

Expression and methylation analysis of other variably imprinted genes.(A) AT3G14205 is a PEG except when Cvi is the paternal parent (mRNA-seq data). Blue bars, % paternal allele expression; red bars, % maternal allele expression from combined mRNA-seq data; vertical line, expected % paternal expression for a non-imprinted gene. (B) Methylation of AT3G14205 5′ flanking region in Col embryo, Ler embryo and endosperm, and Cvi embryo. Red track, CG; green track, CHH. (C) Leaf methylation profile of AT3G14205 in Col, Ler, Kz_9, Cvi and Seattle_0 (http://neomorph.salk.edu/1001_epigenomes.html). (D) AT3G14205 is not imprinted when another hypomethylated strain (Seattle_0) is the pollen parent, but is a PEG in a cross with another methylated strain (Kz_9), as determined by sequencing RT-PCR products that span informative SNPs. The number of RT-PCR clones sequenced is indicated. p value, binomial test of whether the observed maternal:total ratio is less than the expected 2:3 ratio. (E) AT2G34890 is a MEG except when Ler is the paternal parent (mRNA-seq data). Vertical line, expected maternal allele expression for a non-imprinted gene. (F) Methylation of AT2G34890 5′ flanking region in Col and Ler embryo and Cvi embryo and endosperm. (G) Leaf methylation profile of AT2G34890 in Col, Cvi, Ler and Es_0 (http://neomorph.salk.edu/1001_epigenomes.html). (H) AT2G34890 is still imprinted when another hypomethylated strain (Es_0) is the pollen parent. p value: binomial test of whether the observed maternal:total ratio is greater than the expected 2:3 ratio.DOI:http://dx.doi.org/10.7554/eLife.03198.021
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fig6s2: Expression and methylation analysis of other variably imprinted genes.(A) AT3G14205 is a PEG except when Cvi is the paternal parent (mRNA-seq data). Blue bars, % paternal allele expression; red bars, % maternal allele expression from combined mRNA-seq data; vertical line, expected % paternal expression for a non-imprinted gene. (B) Methylation of AT3G14205 5′ flanking region in Col embryo, Ler embryo and endosperm, and Cvi embryo. Red track, CG; green track, CHH. (C) Leaf methylation profile of AT3G14205 in Col, Ler, Kz_9, Cvi and Seattle_0 (http://neomorph.salk.edu/1001_epigenomes.html). (D) AT3G14205 is not imprinted when another hypomethylated strain (Seattle_0) is the pollen parent, but is a PEG in a cross with another methylated strain (Kz_9), as determined by sequencing RT-PCR products that span informative SNPs. The number of RT-PCR clones sequenced is indicated. p value, binomial test of whether the observed maternal:total ratio is less than the expected 2:3 ratio. (E) AT2G34890 is a MEG except when Ler is the paternal parent (mRNA-seq data). Vertical line, expected maternal allele expression for a non-imprinted gene. (F) Methylation of AT2G34890 5′ flanking region in Col and Ler embryo and Cvi embryo and endosperm. (G) Leaf methylation profile of AT2G34890 in Col, Cvi, Ler and Es_0 (http://neomorph.salk.edu/1001_epigenomes.html). (H) AT2G34890 is still imprinted when another hypomethylated strain (Es_0) is the pollen parent. p value: binomial test of whether the observed maternal:total ratio is greater than the expected 2:3 ratio.DOI:http://dx.doi.org/10.7554/eLife.03198.021
Mentions: We identified regions of the genome that are subject to DNA demethylation in endosperm in at least one background but that are variably methylated among strains. Overlap between the strain DMRs and embryo-endosperm DMRs ranged from 12% for TEs to 31% for genes (Figure 4E). This suggests that there are sufficient epigenetic polymorphisms at embryo-endosperm DMRs to facilitate the formation of allele-specific imprinting. Differences in imprinting among alleles tied to differences in DNA methylation could be due to genetic or epigenetic differences (Figure 6—figure supplements 1,2, and 3). Of the 12 allele-specific imprinted genes we identified (Figure 2, Figure 2—source data 1), 10 were associated with coincident CG or CHH embryo-endosperm DMRs and strain DMRs, 6 of which occurred at TEs (Figure 2—source data 1, Figure 6, Figure 6—figure supplement 1, Figure 6—figure supplement 2). For all 6 genes we confirmed by sequencing that the TE annotated in Col was present in the same genomic location in Ler and Cvi with no major sequences changes except for a few SNPs. These 6 genes were also included among imprinting validation assays (Figure 1—source data 5, Figure 2—source data 1). We more closely investigated three allele-specific imprinted genes with strong differences in the ratio of maternal to paternal transcripts in imprinted and non-imprinted crosses: AT2G32370, AT2G34890, and AT3G14205 (Figure 2, Figure 6, Figure 6—figure supplement 2). AT2G32370, HDG3, was originally identified as a PEG because of its association with a Col and Ler embryo-endosperm DMR and because it was expressed specifically in the endosperm (Gehring et al., 2009). Our new data showed that HDG3 is not a PEG when Cvi is the male parent (Figure 2, Figure 6). The embryo-endosperm DMR associated with HDG3 is located in a Helitron fragment (ATREP10D) 5′ of the gene, and the methylated paternal allele is predominantly expressed. This region is not methylated in Cvi (Figure 6B, Figure 6—figure supplement 1). In crosses between Cvi females and Col or Ler males, maternal and paternal alleles are differentially methylated and the gene is imprinted (the naturally hypomethylated Cvi maternal allele has the same methylation profile as an actively demethylated maternal allele). But in reciprocal crosses between Col or Ler females and Cvi males, both maternal and paternal alleles are hypomethylated and the gene is biallelically expressed in the expected 2:1 maternal:paternal ratio (Figure 2, Figure 6). This suggests that differential methylation of maternal and paternal alleles, rather than simply demethylation of the maternal allele, is required for imprinted expression. A similar logic applies to AT3G14205; the Cvi allele is hypomethylated at a 5′ RC/Helitron fragment (ATREP1) compared to Col and Ler and the gene is not a PEG when it is transmitted through the Cvi male (Figure 2, Figure 2—source data 1, Figure 6—figure supplement 2). AT2G34890 was a MEG except when Ler is the male parent. The Ler AT2G34890 allele is hypomethylated at a 5′ MuDR element in comparison to Cvi and Col, suggesting that loss of methylation of the paternal allele could lead to its transcription (Figure 6—figure supplement 2). However, consistent with a more ambiguous relationship between DNA demethylation and MEGs (Figure 5), closer inspection of AT2G34890 shows that at a region 5′ of the TE both the Ler and Cvi alleles are hypomethylated compared to Col, suggesting that a clear methylation distinction between imprinted (Col and Cvi) and non-imprinted (Ler) paternal alleles does not exist (Figure 6—figure supplement 2).10.7554/eLife.03198.019Figure 6.Expression and methylation analysis of HDG3, an allele-specific imprinted gene.

Bottom Line: Imprinting variability could contribute to observed parent-of-origin effects on seed development.We successfully predicted imprinting in additional strains based on methylation variability.We conclude that there is standing variation in imprinting even in recently diverged genotypes due to intraspecific epiallelic variation.

View Article: PubMed Central - PubMed

Affiliation: Whitehead Institute for Biomedical Research, Cambridge, United States.

Show MeSH
Related in: MedlinePlus