The Arabidopsis DEMETER (DME) DNA glycosylase demethylates the maternal genome in the central cell prior to fertilization and is essential for seed viability. DME preferentially targets small transposons that flank coding genes, influencing their expression and initiating plant gene imprinting. DME also targets intergenic and heterochromatic regions, but how it is recruited to these differing chromatin landscapes is unknown. The C-terminal half of DME consists of 3 conserved regions required for catalysis in vitro. We show that this catalytic core guides active demethylation at endogenous targets, rescuing dme developmental and genomic hypermethylation phenotypes. However, without the N terminus, heterochromatin demethylation is significantly impeded, and abundant CG-methylated genic sequences are ectopically demethylated. Comparative analysis revealed that the conserved DME N-terminal domains are present only in flowering plants, whereas the domain architecture of DME-like proteins in nonvascular plants mainly resembles the catalytic core, suggesting that it might represent the ancestral form of the 5mC DNA glycosylase found in plant lineages. We propose a bipartite model for DME protein action and suggest that the DME N terminus was acquired late during land plant evolution to improve specificity and facilitate demethylation at heterochromatin targets.
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Loss of linker histone H1 in the maternal genome influences DEMETER-mediated demethylation and affects the endosperm DNA methylation landscape
The Arabidopsis DEMETER (DME) DNA glycosylase demethylates the central cell genome prior to fertilization. This epigenetic reconfiguration of the female gamete companion cell establishes gene imprinting in the endosperm and is essential for seed viability. DME demethylates small and genic-flanking transposons as well as intergenic and heterochromatin sequences, but how DME is recruited to these loci remains unknown. H1.2 was identified as a DME-interacting protein in a yeast two-hybrid screen, and maternal genome H1 loss affects DNA methylation and expression of selected imprinted genes in the endosperm. Yet, the extent to which H1 influences DME demethylation and gene imprinting in the Arabidopsis endosperm has not been investigated. Here, we showed that without the maternal linker histones, DME-mediated demethylation is facilitated, particularly in the heterochromatin regions, indicating that H1-bound heterochromatins are barriers for DME demethylation. Loss of H1 in the maternal genome has a very limited effect on gene transcription or gene imprinting regulation in the endosperm; however, it variably influences euchromatin TE methylation and causes a slight hypermethylation and a reduced expression in selected imprinted genes. We conclude that loss of maternal H1 indirectly influences DME-mediated demethylation and endosperm DNA methylation landscape but does not appear to affect endosperm gene transcription and overall imprinting regulation.
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- Award ID(s):
- 1715115
- NSF-PAR ID:
- 10389929
- Date Published:
- Journal Name:
- Frontiers in Plant Science
- Volume:
- 13
- ISSN:
- 1664-462X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Background H2A.X is an H2A variant histone in eukaryotes, unique for its ability to respond to DNA damage, initiating the DNA repair pathway. H2A.X replacement within the histone octamer is mediated by the FAcilitates Chromatin Transactions (FACT) complex, a key chromatin remodeler. FACT is required for DEMETER (DME)-mediated DNA demethylation at certain loci in
Arabidopsis thaliana female gametophytes during reproduction. Here, we sought to investigate whether H2A.X is involved in DME- and FACT-mediated DNA demethylation during reproduction.Results H2A.X is encoded by two genes in Arabidopsis genome,
HTA3 andHTA5 . We generatedh2a.x double mutants, which displayed a normal growth profile, whereby flowering time, seed development, and root tip organization, S-phase progression and proliferation were all normal. However,h2a.x mutants were more sensitive to genotoxic stress, consistent with previous reports. H2A.X fused to Green Fluorescent Protein (GFP) under theH2A.X promoter was highly expressed especially in newly developing Arabidopsis tissues, including in male and female gametophytes, where DME is also expressed. We examined DNA methylation inh2a.x developing seeds and seedlings using whole genome bisulfite sequencing, and found that CG DNA methylation is decreased genome-wide inh2a.x mutant endosperm. Hypomethylation was most striking in transposon bodies, and occurred on both parental alleles in the developing endosperm, but not the embryo or seedling.h2a.x -mediated hypomethylated sites overlapped DME targets, but also included other loci, predominately located in heterochromatic transposons and intergenic DNA.Conclusions Our genome-wide methylation analyses suggest that H2A.X could function in preventing access of the DME demethylase to non-canonical sites. Overall, our data suggest that H2A.X is required to maintain DNA methylation homeostasis in the unique chromatin environment of the Arabidopsis endosperm.
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3389/fpls.2022.1070397
