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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The maize gene maternal derepression of r1 encodes a DNA glycosylase that demethylates DNA and reduces siRNA expression in the endosperm
Demethylation of transposons can activate the expression of nearby genes and cause imprinted gene expression in the endosperm; this demethylation is hypothesized to lead to expression of transposon small interfering RNAs (siRNAs) that reinforce silencing in the next generation through transfer either into egg or embryo. Here we describe maize (Zea mays) maternal derepression of r1 (mdr1), which encodes a DNA glycosylase with homology to Arabidopsis thaliana DEMETER and which is partially responsible for demethylation of thousands of regions in endosperm. Instead of promoting siRNA expression in endosperm, MDR1 activity inhibits it. Methylation of most repetitive DNA elements in endosperm is not significantly affected by MDR1, with an exception of Helitrons. While maternally-expressed imprinted genes preferentially overlap with MDR1 demethylated regions, the majority of genes that overlap demethylated regions are not imprinted. Double mutant megagametophytes lacking both MDR1 and its close homolog DNG102 result in early seed failure, and double mutant microgametophytes fail pre-fertilization. These data establish DNA demethylation by glycosylases as essential in maize endosperm and pollen and suggest that neither transposon repression nor genomic imprinting is its main function in endosperm.
more » « less- Award ID(s):
- 2114797
- NSF-PAR ID:
- 10372269
- Publisher / Repository:
- Oxford University Press
- Date Published:
- Journal Name:
- The Plant Cell
- Volume:
- 34
- Issue:
- 10
- ISSN:
- 1040-4651
- Page Range / eLocation ID:
- p. 3685-3701
- 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
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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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