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.

Figure 1

Cytological characterization of centromeric heterochromatin in sperm and vegetative nuclei of wild-type (Col-0) mature pollen. (A) A DAPI-stained mature pollen grain. (B) FISH image of SN and VN of pollen with a probe for 180CEN repeats. Approximately 1,000 vegetative nuclei were examined and all of them showed extremely dispersed FISH signals. Immunostaining of (C) bulk histone H3; (D) CenH3 (HTR12) and H3K9me2; and (E) H3K9me2 and H3K27me1 in SN and VN. Immunostaining patterns similar to those shown were observed in all approximately 100 nuclei examined. (B–E) Nuclei were counterstained with DAPI (blue). Scale bars, 3 μm. DAPI, 4',6-diamidino-2-phenylindole; FISH, fluorescence in situ hybridization; SN, sperm nuclei; VN, vegetative nuclei.

Figure 2

Bisulphite sequencing analysis of cytosine methylation in wild-type (Col-0), drm2 and cmt3 lines. The graphs show the percentage of methylation (%^mC) at individual cytosines in cloned 180CEN (left) and Athila (right) sequences from (A) sorted wild-type vegetative (top) and sperm (middle) nuclei, and seedings (bottom); (B) sorted drm2; and (C) cmt3 vegetative (top) and sperm (bottom) nuclei. Black lines, CG methylation; blue lines, CHG methylation; and red lines, CHH methylation. We define ‘hypermethylated non-CG sites in VN in pollen' as the CHH or CHG sites that are methylated in more cloned DNA sequences from VN than from SN. Note that all CHH sites in both the 180CEN (39 sites) and Athila (30 sites) sequences are hypermethylated in the wild-type VN. Red and blue arrows above the methylation lines indicate the CHH and CHG sites, respectively, at which hypermethylation is lost in mutant VN compared with wild-type VN. Red and blue asterisks mark the CHH and CHG sites, respectively, showing more than a 50% reduction in %^mC in mutant VN compared with wild-type VN. Cytosine positions are indicated by numbers. The results are from 30 and 20 cloned sequences for wild-type pollen nuclei and seedlings, respectively, and 10 cloned sequences for drm2 and cmt3 pollen nuclei. Original data are shown in supplementary Fig S4 online. Cmt3, chromomethylase 3; drm2, domains rearranged methyltransferase 2; SE, seedlings; SN, sperm nuclei; VN, vegetative nuclei.

Figure 3

Southern hybridization analysis of cytosine methylation at bulk 180CEN repeats in sperm and vegetative nuclei of wild-type (Col-0) mature pollen. A subclass of 180CEN repeats contains one MspI/HpaII (CCGG) site and two CviAII/NlaIII (CATG) sites. Genomic DNA (30 ng) extracted from sorted SN or VN was digested using MspI, HpaII, CviAII or NlaIII, separated by gel electrophoresis and probed with 180CEN DNA. MspI and HpaII both cut CCGG but differ in that MspI is inhibited by methylation of outer C, whereas HpaII is inhibited by methylation of inner C. CviAII and NlaIII both cut CATG, but differ in that only NlaIII is inhibited by methylation. SN, sperm nuclei; VN, vegetative nuclei.

Figure 4

Bisulphite sequencing analysis of cytosine methylation in nrpd1 and nrpe1 lines. The graphs show the percentage of methylation (%^mC) at individual cytosines in cloned 180CEN (left) and Athila (right) sequences from sorted vegetative (top) and sperm (bottom) nuclei of (A) nrpd1 and (B) nrpe1 pollen. Black lines, CG methylation; blue lines, CHG methylation; and red lines, CHH methylation. Red and blue arrows above the methylation lines indicate the CHH and CHG sites, respectively, at which hypermethylation is lost in mutant VN compared with wild-type VN. Red and blue asterisks mark the CHH and CHG sites, respectively, showing more than a 50% reduction in %mC in mutant VN compared with wild-type VN. The results are from 10 cloned sequences for both mutants. Original data are shown in supplementary Fig S4 online. nrpd1, nuclear rna polymerase 4 subunit 1; nrpe1, nuclear rna polymerase 5 subunit 1; SN, sperm nuclei; VN, vegetative nuclei.