Induction of RNA‐directed DNA methylation upon decondensation of constitutive heterochromatin

Abstract

Centromeric constitutive heterochromatin is marked by DNA methylation and dimethylated histone H3 Lys 9 (H3K9me2) in Arabidopsis. RNA-directed DNA methylation (RdDM) is a process that uses 24-nucleotide (nt) small interfering RNAs (siRNAs) to induce de novo methylation to its homologous DNA sequences. Despite the presence of centromeric 24-nt siRNAs, mutations in genes required for RdDM do not appreciably influence the methylation of centromeric repeats. The mechanism by which constitutive heterochromatin is protected from RdDM remains puzzling. Here, we report that the vegetative cell nuclei (VN) of the male gametophyte (pollen) invariably undergo extensive decondensation of centromeric heterochromatin and lose centromere identity. VN show greatly reduced H3K9me2, phenocopying nuclei carrying a mutation in the chromatin remodeller DECREASE IN DNA METHYLATION 1 (DDM1). However, unlike the situation in ddm1 nuclei, the decondensed heterochromatin retains dense CG methylation and transcriptional silencing, and, unexpectedly, is subjected to RdDM-dependent hypermethylation in non-CG contexts. These findings reveal two assembly orders of silent heterochromatin and implicate the condensed form in blocking the RdDM machinery.

The AAA-ATPase molecular chaperone Cdc48/p97 disassembles sumoylated centromeres, decondenses heterochromatin, and activates ribosomal RNA genes

“…Total RNA from 2-wk-old leaves or mature pollen of A. thaliana WT (Col-0) plants was isolated using an RNeasy Plant Mini Kit (Qiagen) as previously described (9). FACS-sorted vegetative cell nuclei (9,28) were directly collected into TRIzol LS Reagent (Life Technologies), and total RNA was isolated according to the manufacturer's protocol. RNA samples were treated with DNase (Thermo Scientific) and precipitated.…”

“…Active rRNA genes transcribed in the nucleolus are demethylated at promoter CG sites in the 5′ external transcribed spacer (ETS) region, whereas silent nucleoplasmic copies are heavily methylated, and loss-of-function met1 DNA methyltransferase mutant alleles disrupt rRNA gene silencing (24). To test whether the expression of full-set rRNA variants in pollen vegetative cells correlates with rDNA demethylation, we analyzed previously published methylome data (27) derived from purified sperm and vegetative cell nuclei by fluorescence-activated cell sorting (FACS) (9,28) (Fig. 5G).…”

“…The primary role of the vegetative cell in plant reproduction is to germinate and form a rapid tip-growing pollen tube, sometimes centimeters in length, which transports two sperm cells to the female gametophyte for double fertilization. We reported previously that the Arabidopsis thaliana pollen vegetative cell invariably undergoes CenH3 removal from centromeres, coincident with loss of the demethylated histone H3 Lys9 (H3K9me2) heterochromatin mark and centromeric heterochromatin decondensation (9), although the mechanism and biological significance of these processes remain unclear.…”

Repeat elements and the Arabidopsis DNA methylation landscape

“…Specifically, in the vegetative nucleus of the pollen grain, where the level of DDM1 protein is particularly low, TEs are reactivated, producing a pattern of siRNA accumulation similar to that seen in ddm1 (Slotkin et al, 2009). These siRNAs are then transported by still unknown mechanisms to the two sperm cells (Slotkin et al, 2009) where they presumably participate in either RdDM or post-transcriptional silencing depending on their size (Schoft et al, 2009;Slotkin et al, 2009). Like other transcripts produced in pollen (Bayer et al, 2009), some or all of these siRNAs could also be carried over to the zygote and the endosperm, where they would exert functions similar to those postulated in sperm cells (Figure 2a).…”

The many faces of plant chromatin: Meeting summary of the 4th European workshop onplantchromatin 2015, Uppsala, Sweden

“…During male gametogenesis, the pollen vegetative cell nucleus undergoes heterochromatin decondenzation coupled with active removal of CenH3. 28 Merai and colleagues showed that the AAA-ATPase chaperone CDC48A is required for the unloading of sumoylated CenH3, chromatin decondenzation and global activation of silent rRNA genes. As the cdc48a allele is rarely paternally transmitted due to defective pollen tube formation, the authors suggested that the bulk rRNA gene activation may be essential to meet the increased metabolic demand during pollen tube growth.…”