Abstract
RNA-directed DNA methylation (RdDM) is required for transcriptional silencing of transposons and other DNA repeats in Arabidopsis thaliana. Although previous research has demonstrated that the SET domain-containing SU(VAR)3–9 homologs SUVH2 and SUVH9 are involved in the RdDM pathway, the underlying mechanism remains unknown. Our results indicated that SUVH2 and/or SUVH9 not only interact with the chromatin-remodeling complex termed DDR (DMS3, DRD1, and RDM1) but also with the newly characterized complex composed of two conserved Microrchidia (MORC) family proteins, MORC1 and MORC6. The effect of suvh2suvh9 on Pol IV-dependent siRNA accumulation and DNA methylation is comparable to that of the Pol V mutant nrpe1 and the DDR complex mutant dms3, suggesting that SUVH2 and SUVH9 are functionally associated with RdDM. Our CHIP assay demonstrated that SUVH2 and SUVH9 are required for the occupancy of Pol V at RdDM loci and facilitate the production of Pol V-dependent noncoding RNAs. Moreover, SUVH2 and SUVH9 are also involved in the occupancy of DMS3 at RdDM loci. The putative catalytic active site in the SET domain of SUVH2 is dispensable for the function of SUVH2 in RdDM and H3K9 dimethylation. We propose that SUVH2 and SUVH9 bind to methylated DNA and facilitate the recruitment of Pol V to RdDM loci by associating with the DDR complex and the MORC complex.
Highlights
DNA methylation is an important epigenetic modification that contributes to transposable element silencing, genome stability, and gene regulation in plants and animals [1,2,3]
Two RNA-directed DNA methylation (RdDM) components DMS3 and DRD1 were identified in affinity purification of SUVH2-3xMyc, whereas MORC6/DEFECTIVE IN MERISTEM SILENCING 11 (DMS11) and DMS3 were identified in affinity purification of SUVH9-3xFlag (Table 1)
The presence of RdDM components DMS3 and DRD1 in affinity purification of SUVH2-3xMyc and SUVH9-3xFlag suggests that SUVH2 and SUVH9 are involved in RdDM by physically associating with RdDM components
Summary
DNA methylation is an important epigenetic modification that contributes to transposable element silencing, genome stability, and gene regulation in plants and animals [1,2,3]. The RdDM pathway is involved in the silencing of transposable elements and DNA repeats [1,2], the stability of the genome [4,5], the development of gametophytes and embryos [6,7,8], and the inheritance of stress-induced transcriptional silencing [9]. In the RdDM pathway, the multi-subunit DNA-dependent RNA polymerase IV (Pol IV) collaborates with RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) to produce doublestranded RNAs, which are subsequently cleaved into 24-nt siRNAs (small interfering RNAs) by DICER-LIKE 3 (DCL3) [10,11,12,13].
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