The FACT complex is required for DNA demethylation at heterochromatin during reproduction in Arabidopsis

Abstract

The DEMETER (DME) DNA glycosylase catalyzes genome-wide DNA demethylation and is required for endosperm genomic imprinting and embryo viability. Targets of DMEmediated DNA demethylation reside in small, euchromatic, AT-rich transposons and at the boundaries of large transposons, but how DME interacts with these diverse chromatin states is unknown. The STRUCTURE SPECIFIC RECOGNITION PROTEIN 1 (SSRP1), subunit of the chromatin remodeler FAcilitates Chromatin Transactions (FACT), was previously shown to be involved in the DME-dependent regulation of genomic imprinting in Arabidopsis endosperm. Therefore, to investigate the interaction between DME and chromatin, we focused on the activity of the two FACT subunits, SSRP1 and SUPPRESSOR of TY16 (SPT16), during reproduction in Arabidopsis. We find that FACT co-localizes with nuclear DME in vivo, and that DME has two classes of target sites, the first being euchromatic and accessible to DME, but the second, representing over half of DME targets, requiring the action of FACT for DME-mediated DNA demethylation genomewide. Our results show that the FACT-dependent DME targets are GCrich heterochromatin domains with high nucleosome occupancy enriched with H3K9me2 and H3K27me1. Further, we demonstrate that heterochromatin-associated linker histone H1 specifically mediates the requirement for FACT at a subset of DME-target loci. Overall, our results demonstrate that FACT is required for DME targeting by facilitating its access to heterochromatin.

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

“…The functional or expression differences in sperm or egg between rice and Arabidopsis demethylase genes may be caused by divergent evolution of the plant DNA glycosylase family. That the dng mutations affected methylation and expression patterns at distinct genomic regions in the different reproductive cells implies an essential role of the enzymes before and after fertilization, but also raises the question of how the glycosylases target distinct chromatin regions in the different cells (Frost et al, 2018). One possibility would be that their recruitment is conditioned by distinct overall chromatin structures in gametes and zygotes.…”

DNA demethylation affects imprinted gene expression in maize endosperm

“…The ROS1 gene is mainly expressed in vegetative tissues while the DME gene is mainly expressed in reproductive tissues, especially in the central cell of the female gametophyte and the vegetative cell of the male gametophyte [7][8][9]. In addition to these demethylase, there are many other proteins involved in DNA demethylation, including proteins that guide the demethylase to the target regions [10,11]. Besides Arabidopsis, DNA demethylation has been reported in many other species, including tomato, wheat, rice, Medicago, and strawberries [12][13][14][15][16], but it has not been characterized in maize.…”

The maize gene maternal derepression of r1 (mdr1) encodes a DNA glycosylase that demethylates DNA and reduces siRNA expression in endosperm

“…In most maize tissues at most loci, euchromatin is stably depleted of mCHG across development (Oka et al 2017; Crisp et al 2020; Hufford et al 2021). Consistent with both heterochromatic and euchromatic activity of DEMETER in Arabidopsis endosperm (Frost et al 2018), almost half of the DMRs were greater than 2 Kb from the nearest euchromatin, indicating that MDR1 also effectively demethylates deep in heterochromatin, not just at its edges (Supplemental Figure S5A).…”

“…Unifying features of these regions have not been identified, but they exhibit a preference for short transposons in genic areas and for the ends of long transposons. What guides DEMETER to these regions is unknown, but involves FACT, a versatile ATP-dependent chromatin remodeling complex (Frost et al 2018). DEMETER depends on FACT for activity at about half of its ~10,000 identified target regions, which are distinguished by being more heterochromatic than the other half.…”