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. 2018 Nov 5;14(11):e1007469.
doi: 10.1371/journal.pgen.1007469. eCollection 2018 Nov.

A variably imprinted epiallele impacts seed development

Daniela Pignatta  1 , Katherine Novitzky  1 , P R V Satyaki  1 , Mary Gehring  1   2
Affiliations

A variably imprinted epiallele impacts seed development

Daniela Pignatta et al. PLoS Genet. .

Abstract

The contribution of epigenetic variation to phenotypic variation is unclear. Imprinted genes, because of their strong association with epigenetic modifications, represent an opportunity for the discovery of such phenomena. In mammals and flowering plants, a subset of genes are expressed from only one parental allele in a process called gene imprinting. Imprinting is associated with differential DNA methylation and chromatin modifications between parental alleles. In flowering plants imprinting occurs in a seed tissue - endosperm. Proper endosperm development is essential for the production of viable seeds. We previously showed that in Arabidopsis thaliana intraspecific imprinting variation is correlated with naturally occurring DNA methylation polymorphisms. Here, we investigated the mechanisms and function of allele-specific imprinting of the class IV homeodomain leucine zipper (HD-ZIP) transcription factor HDG3. In imprinted strains, HDG3 is expressed primarily from the methylated paternally inherited allele. We manipulated the methylation state of endogenous HDG3 in a non-imprinted strain and demonstrated that methylation of a proximal transposable element is sufficient to promote HDG3 expression and imprinting. Gain of HDG3 imprinting was associated with earlier endosperm cellularization and changes in seed weight. These results indicate that epigenetic variation alone is sufficient to explain imprinting variation and demonstrate that epialleles can underlie variation in seed development phenotypes.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Natural variation in imprinting is associated with differences in HDG3 expression levels.
(A) HDG3 expression is decreased in Col x Cvi endosperm compared to Col, as determined by mRNA-seq. (B) Schematic of the HDG3 locus. DMR, differentially methylated region. (C) In situ hybridization of HDG3 (purple) in F1 seeds from the indicated crosses. Female parent is written first. In crosses where Col is the male parent, HDG3 is detected in the micropylar (MCE), peripheral (PEN), and chalazal (CZE) endosperm. Arrowheads indicate nuclear-cytoplasmic domains in the PEN. Number of seeds with shown pattern out of total seeds assayed is in corner of each image. Scale bars, 50 μm. (D) RT-qPCR analysis of relative HDG3 transcript abundance in F1 endosperm. Values are the average of 3 biological replicates, bars represent upper and lower range. (E) Schematic representation of relationship between HDG3 methylation, expression, and imprinting in endosperm. Thickness of arrows denotes relative expression level. Lollipops represent methylated (filled) and unmethylated (open) cytosines.
Fig 2
Fig 2. Phenotypic effects of mutation of HDG3 in Col.
(A) In situ hybridization of HDG3 (purple) in seeds from the indicated crosses. Female parent is written first. Scale bars, 50 μm. (B) Endosperm cellularization is slightly delayed in hdg3 compared to wild-type at 5 DAP. For each seed the embryo stage was determined and then the stage of endosperm cellularization was defined as normal, early, or delayed given that embryo stage. (C) Seed weight of wild-type and hdg3 seeds. Individual data points and mean +/- SD shown. P-value from unpaired two-tailed t-test. (D) Seed area is significantly reduced in hdg3 seed (n = 275) compared to wild-type siblings (n = 376) (p = 8.51e-11 by Welch’s two tailed t-test). Seeds were quantified with ImageJ.
Fig 3
Fig 3. Transcriptional effects associated with low HDG3 expression.
(left panel) Genes downregulated in hdg3 mutant endosperm also have reduced expression in Col x Cvi endosperm compared to Col. The plot shows the expression profile of genes with significantly altered expression in hdg3-1 endosperm (p<0.05). Genes were hierarchically clustered by Euclidean distance and complete linkage using Gene-E. (right panel) A subset of putative developmental regulators with reduced expression in hdg3 and Col x Cvi endosperm.
Fig 4
Fig 4. Gain of Cvi HDG3 paternal allele methylation in endosperm.
Total methylcytosine 5’ of HDG3 in F1 endosperm, determined by bisulfite-PCR. Maternally inherited Col allele in orange, paternally inherited Cvi allele in blue. Scale to 100%, tick marks below line indicate unmethylated cytosines. Col x Cvi data published in [20].
Fig 5
Fig 5. HDG3 is imprinted in Cvi HDG3 IR lines.
(A) HDG3 in situ hybridization for indicated genotypes. Arrowheads indicate regions of in situ signal. Right panels shows magnification of chalazal region. Scale bars, 50 μm. (B) RT-qPCR of relative HDG3 transcript abundance in F1 endosperm at 6–7 DAP. Dashed line separates experiments done at different times. Left, avg of 3 technical replicates. Right, avg of biological duplicates. Bars show upper and lower range. (C) % of HDG3 from Cvi allele in endosperm by TaqMan RT-qPCR assay.
Fig 6
Fig 6. Effects of HDG3 imprinting on Cvi seed development.
(A) Aniline blue and safranin O staining of seed sections at 5 DAP from the indicated F1 seeds. Scale bars, 50 μm. (B) Phenotypic characterization of sectioned seeds, assaying degree of endosperm cellularization relative to embryo stage. (C) Seed weight in selfed Cvi and Cvi HDG3 IR plants. Individual data points and mean +/- SD shown. P-value from unpaired two tailed t-test. (D) Seed area for self-fertilized Cvi (n = 287 seeds), Cvi HDG3 IR 2–5 (n = 496) and Cvi HDG3 IR 3–4 (n = 386). Differences between IR seeds and Cvi are significant at p < 2.2e-16 as determined by Welch’s two-tailed t-test.
Fig 7
Fig 7. Schematic summary of relative seed development at 5 DAP.
Shapes represent phenotypic space occupied by the indicated genotypes.
See this image and copyright information in PMC

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References

    1. Gehring M, Huh JH, Hsieh T-F, Penterman J, Choi Y, Harada JJ, et al. DEMETER DNA glycosylase establishes MEDEA polycomb gene self-imprinting by allele-specific demethylation. Cell. 2006; 124(3):495–506. 10.1016/j.cell.2005.12.034 - DOI - PMC - PubMed
    1. Gehring M, Bubb KL, Henikoff S. Extensive demethylation of repetitive elements during seed development underlies gene imprinting. Science. 2009; 324(5933):1447–51. 10.1126/science.1171609 - DOI - PMC - PubMed
    1. Hsieh T-F, Ibarra CA, Silva P, Zemach A, Eshed-Williams L, Fischer RL, et al. Genome-wide demethylation of Arabidopsis endosperm. Science. 2009; 324(5933):1451–4. 10.1126/science.1172417 - DOI - PMC - PubMed
    1. Satyaki PRV, Gehring M. DNA methylation and imprinting in plants: machinery and mechanisms. Crit Rev Biochem Mol Biol. 2017; 52(2):163–75. 10.1080/10409238.2017.1279119 - DOI - PubMed
    1. Haig D. The kinship theory of genomic imprinting. Annu Rev Ecol Syst. 2000; 31: 9–32.

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Grants and funding

This work was funded by the National Science Foundation, Division of Molecular and Cellular Biosciences (https://www.nsf.gov/div/index.jsp?div=MCB) CAREER grant 1453459 to MG. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.