H3 Lysine 9 Methylation Is Maintained on a Transcribed Inverted Repeat by Combined Action of SUVH6 and SUVH4 Methyltransferases

Abstract

Transcribed inverted repeats are potent triggers for RNA interference and RNA-directed DNA methylation in plants through the production of double-stranded RNA (dsRNA). For example, a transcribed inverted repeat of endogenous genes in Arabidopsis thaliana, PAI1-PAI4, guides methylation of itself as well as two unlinked duplicated PAI genes, PAI2 and PAI3. In previous work, we found that mutations in the SUVH4/KYP histone H3 lysine 9 (H3 K9) methyltransferase cause a loss of DNA methylation on PAI2 and PAI3, but not on the inverted repeat. Here we use chromatin immunoprecipitation analysis to show that the transcribed inverted repeat carries H3 K9 methylation, which is maintained even in an suvh4 mutant. PAI1-PAI4 H3 K9 methylation and DNA methylation are also maintained in an suvh6 mutant, which is defective for a gene closely related to SUVH4. However, both epigenetic modifications are reduced at this locus in an suvh4 suvh6 double mutant. In contrast, SUVH6 does not play a significant role in maintenance of H3 K9 or DNA methylation on PAI2, transposon sequences, or centromere repeat sequences. Thus, SUVH6 is preferentially active at a dsRNA source locus versus targets for RNA-directed chromatin modifications.In eukaryotic genomes, condensed transcriptionally silent heterochromatin domains serve key roles in gene regulation and genome stability. In mammals and plants, heterochromatin is associated with cytosine methylation, as well as with histone tail modifications including H3 methylated at lysine 9 (H3 mK9). In many cases, these heterochromatin-associated modifications are directed to target sequences by a doublestranded RNA (dsRNA)-derived signal (26). For example, in plants, sources of dsRNA that can be converted into small RNAs by dicer RNase action, such as RNA viruses, products of RNA-dependent RNA polymerase synthesis, or transcripts derived from inverted repeat templates, direct DNA methylation and H3 mK9 of identical genomic sequences. However, the factors that connect RNA signals with DNA methylation and H3 mK9 remain to be elucidated.Plant RNA-directed DNA methylation typically affects cytosines in all sequence contexts (26). Genetic studies in Arabidopsis thaliana indicate that methylation in the symmetric context 5Ј CG 3Ј is maintained by the MET1 cytosine methyltransferase (MTase) (16, 37). Maintenance of methylation in non-CG contexts is more complex, with contributions from both the DRM1/DRM2 and CMT3 cytosine MTases. DRM1 and DRM2 control the initiation of new methylation imprints (7) but have different roles in the maintenance of non-CG methylation at different genomic regions (6). For example, although DRM1 and DRM2 are required to maintain non-CG methylation at direct repeat sequences that lie 3Ј to the MEA gene, they do not obviously contribute to maintenance of non-CG methylation at the Ta3 retrotransposon or at centromere-associated repeats. Instead, CMT3 is the major enzyme involved in maintenance of non-CG methylation at these targets (2,6,20), as well as at other transposon sequences ...

Ectopic assembly of heterochromatin in Drosophila melanogaster triggered by transposable elements

“…Their presence overlaps the early stages of heterochromatin formation (cycles [11][12][13][14] and of zygotic transcription (nuclear cycle 14) (25). The latter coincides with the appearance of the histone H3K4 demethylase SU(VAR)3-3, without which HP1a and H3K9me2 levels in the heterochromatin become substantially reduced, whereas levels for the activating H3K4me2 mark become high (immunofluorescent staining of nuclei in cycle 14 embryos), resulting in suppression of PEV.…”

“…Possibilities include the terminally inverted repeats; putative transcription start sites (TSSs) found within the right half of the element; and regions with similarity to piRNA reads. Terminally inverted repeats can contribute secondary structure or serve as binding sites for transposon-derived proteins, both mechanisms used in plants (13,14). Present within the right half of 1360 are three putative TSSs, with similarity to the 1360 transcription initiation sites suggested to produce antisense Suppressor of stellate transcripts in the D. melanogaster male germ line (15).…”

Repeat elements and the Arabidopsis DNA methylation landscape

“…A reinforcing loop between these modifications is suggested by the fact that the chromodomain of CMT3 and the SRA domain of SUVH4 bind H3K9me2 and methylated CHG sites, respectively ( Figure 1b; Lindroth et al, 2004;Johnson et al, 2007). Other histone methyltransferases, including SUVH5 and SUVH6, may be involved in similar reinforcing loops (Ebbs et al, 2005;Ebbs and Bender, 2006). To date it is not known whether the hemi-methylated status of CHG sites after passage of the replication fork serves as an additional cue for maintaining methylation at these sites.…”

Plant SET domain-containing proteins: Structure, function and regulation

“…Naumann et al [91] reported that the N-terminus and YDG domain in SUVH2 are involved in directing DNA methylation and subsequent histone methylation at its target locus so that heterochromatic methylation marks are affected in suvh2 mutant plants. Recently, Ebbs et al [92,93] showed that there is a hierarchical and locusspecific control of H3K9 di-methylation as well as non-CG DNA methylation by SUVH4, SUVH5 and SUVH6 HMT in Arabidopsis. The involvement of this class of proteins in heterochromatin formation was demonstrated for NtSET1 in tobacco [94], and reinforced by the subsequent finding that NtSET1 interacts with LHP1 (Arabidopsis homolog of HP1) [95].…”