The Catalytic Core of DEMETER Guides Active DNA Demethylation in Arabidopsis

Abstract

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, and how it is recruited to these differing chromatin landscapes is unknown. The C--terminal DME catalytic core consists of three conserved regions required for catalysis in vitro. We show that the catalytic core of DME guides active demethylation at endogenous targets, rescuing the developmental and genomic hypermethylation phenotypes of DME mutants. However, without the N--terminus, heterochromatin demethylation is significantly impeded, and abundant CG--methylated genic sequences are ectopically demethylated. We used comparative analysis to reveal that the conserved DME N--terminal domains are only present in the flowering plants, whereas the domain architecture of DME--like proteins in non--vascular plants mainly resembles the catalytic core, suggesting that it might represent the ancestral form of the 5mC DNA glycosylase found in all 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.

The DME demethylase regulates sporophyte gene expression, cell proliferation, differentiation, and meristem resurrection

“…Of the remaining five genes, three did not change significantly and two were significantly upregulated in the sporophyte, indicating that the transcriptional regulation of these genes may differ between the central cell and the sporophyte (SI Appendix, Table S2). It is possible that the distinct gene expression patterns in the central cell versus the sporophyte might reflect different cell-specific epigenetic profiles (7,69) or different transcriptional complexes required to express the same DME target genes.…”

DNA demethylases remodel DNA methylation in rice gametes and zygote and are required for reproduction

“…It was shown that Arabidopsis DME can access euchromatin but not heterochromatin without the FACT histone chaperone (Frost et al, 2018). In addition, it was shown that the DME catalytic domain could be recruited to heterochromatins but could not process demethylation effectively (Zhang et al, 2019).…”

Loss of Linker Histone H1 in the Maternal Genome Influences DEMETER-Mediated Demethylation and Affects the Endosperm DNA Methylation Landscape

“…The MEDEA promoter-GFP construct contains a 4.238 bp of MEA promoter (upstream of ATG), the HTB2-GFP (nuclear-localized GFP fused with the Arabidopsis histone H2B HTB2 (AT5G22880) protein), and a 2.1 kb MEDEA 3’- end sequence (downstream of the stop codon) concatenated using the Gibson assembly method. The backbone plasmid is a binary plasmid vector, pFGAMh (a hygromycin resistant version of pFGC5941) described before (Zhang et al, 2019).…”

“…The backbone plasmid is a binary plasmid vector, pFGAMh (a hygromycin resistant version of pFGC5941) described before (Zhang et al, 2019).…”