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RNA-directed modification of histones is essential for maintenance of heterochromatin in higher eukaryotes. In plants, cytosine methylation, especially in non-CG sequence contexts, is tightly related to inactive chromatin, but the mechanisms regulating the coexistence of cytosine methylation and repressive histone modification remain obscure. We recently revealed that MORPHEUS' MOLECULE1 (MOM1) of Arabidopsis thaliana silences endogenous loci related to transposons and homologous to the 24-nt siRNAs accumulated in wild type plants, and suggested that MOM1 transduces RNA-directed DNA methylation (RdDM) signals to repressive histone modification. In this addendum, we focus on the involvement of MOM1 in multiple transcriptional gene silencing (TGS) pathways.
Hallmarks of the silent state of heterochromatin in plants are methylated cytosines and di-methylated histone H3 lysine 9 (H3K9me2). The RNA-directed DNA methylation (RdDM) pathway plays an essential role in transducing signals of regional or repetitive characteristics of heterochromatin to methylation of cytosines through 24-nt siRNAs.1 The process of 24-nt siRNA production includes RNA-DEPENDENT RNA POLYMERASE2 (RDR2) and DICERLIKE 3 (DCL3), and the resulting siRNAs are incorporated into ARGONAUTE4 (AGO4), which directs DNA methylation in regions homologous to the siRNAs by DOMAINS REARRANGED METHYLTRANSFERASE2 (DRM2) at cytosines in all contexts (CG, CHG, CHH; H = A, T or C).1,2 Methylated CG and CHG co-localize with H3K9me2, but it is not clear how signals of cytosine methylation are transduced to methylation of H3K9 in plants. A bridge between cytosine methylation and H3K9me2 includes CHROMOMETHYLASE3 (CMT3) and KRYPTONITE (KYP).3,4 CMT3 binds to LIKE HETEROCHROMATIN PROTEIN 1 (LHP1), an Arabidopsis protein that binds to H3K9me2 methylated by KYP. Methylation of H3K9 and subsequent binding of LHP1 are lost in kyp mutants, resulting in loss of CHG methylation by CMT3.3 KYP carries the SRA domain that binds to methylated cytosines in all three sequence contexts, with a relative preference for CHG and CHH.4 Together, these data raise the possibility that the KYP/CMT3 system works downstream of RdDM through recognition of methylated cytosines, although it is unknown how KYP/CMT3 specifically interacts with the RdDM pathway.
MORPHEUS' MOLECULE1 (MOM1) was identified in a screen for mutants that release TGS of a cluster of transgenes.5 In addition to transgene TGS, mom1 mutants release silencing of remnants of transposons6–8 and silent copies of 5S rRNA genes.9 mom1 mutants have no effect on global cytosine methylation in the genome and in the target genes. Recent studies have shown that loci silenced by MOM1 carry intermediate states of chromatin where no apparent bias to H3K9me2 is observed regardless of the silent states of the loci,7,8 implying that these loci have chromatin properties distinct from those of typical of heterochromatin and euchromatin loci.
In our study,8 we noted that a common character of cytosine methylation in endogenous loci activated in mom1 is a low level of CHH methylation relative to CG and CHG methylation (Fig. 1).8 Similar low levels of CHH methylation were also observed in the cauliflower mosaic virus 35S promoter in a complex transgene locus that is silent in wild type plants but active in mom1.5 AtIS112A and MULE-F19G14, both carrying low levels of CHH methylation, show similar dependency on MOM1 and non-CG methylation for maintenance of their silent state (Fig. 1). They are active in mom1 but only weakly activated in drm2 cmt3—a double mutant deficient in non-CG-methylation. Differential levels of CHG methylation between AtIS112A and MULE-F19G14 do not affect the dependency of their silent states on MOM1 (Fig. 1). In contrast, a transposon-related locus, ROMANIAT5, was recently found as an endogenous locus that is synergistically activated in double mutants deficient in both MOM1 and RdDM.10 Single mutants of MOM1 show only weak activation of ROMANIAT5 (Fig. 1).10 A high level of CHH methylation in ROMANIAT5 coincides with the significant involvement of RdDM in maintenance of the silent state (Fig. 1A), consistent with the idea that a “double lock” of MOM1 and RdDM silences ROMONIAT5.10 That there is no activation of ROMANIAT5 in drm2 cmt3 despite high levels of CHH and CHG methylation is rather surprising (Fig. 1B), and indicates that MOM1 works independently of RdDM and KYP/CMT3 for silencing of ROMANIAT5.
Silencing of another locus carrying low CHH methylation, SUPPRESSOR OF drm1drm2cmt3 (SDC), shows a dependency on non-CG methylation: it is activated strongly in drm2 cmt3 but only weakly in mom1 (Fig. 1).8 CG methylation in SDC has been shown to depend on non-CG methylation, and single mutants in the RdDM or KYP/CMT3 pathway did not release SDC silencing,11 indicating the presence of another “double lock” pathway (RdDM and KYP/CMT3) of non-CG methylation.
In summary, silencing of endogenous loci is maintained by locus-specific combinations of multiple silencing pathways. MOM1 acts on loci carrying non-CG methylation, but the mechanism connecting MOM1 and non-CG methylation remains unknown (Fig. 1). Identification of determining factors for the locus-specific dependency of various silencing pathways is undoubtedly required if we are to understand the complex interplay of plant silencing pathways.
Previously published online: www.landesbioscience.com/journals/psb/article/11663