Functional Analysis Of SET Domain Protein SUVH2 And SUVH9 In Transcriptional Gene Silencing

In eukaryotes, RNA-mediated gene silencing plays an important role in regulating gene expression. In Arabidopsis, the silencing of transposable elements and DNA repeats, and the stability of the genome are mainly depended on RNA-directed DNA methylation (RdDM) pathway. Although previous research has demonstrated that the SET domain-containing SU(VAR)3-9 homologs SUVH2 and SUVH9 are required for the RdDM pathway, the underlying mechanism remains unknown.Our results indicated that SUVH2 and SUVH9 (SUVH2/9) interact in vivo with the DDR complex, which is composed of DRD1, DMS3 and RDM1. Yeast two-hybrid assay indicated that the truncated SUVH2 or SUVH9 sequence without its C-terminal SET domain can still interact with DMS3. Besides, the mutation of the conserved SET domain in SUVH2 has no effect on DNA methylation and H3K9me2 at RdDM loci, suggesting that SUVH2 and SUVH9 are not active histone methyltransferases in vivo. Previous research indicated that the DDR complex facilitates global occupancy of RNA polymerase V at RdDM loci. We found that in suvh2suvh9, the occupancy of pol V and the DDR complex at RdDM loci is significantly decreased, indicating that SUVH2 and SUVH9 are involved in RdDM pathway by mediating pol V recruitment at RdDM loci.The MORC family protein MORC1 and MORC6 are involved in transcriptional gene silencing and heterochromatin condensation without altering genome-wide DNA methylation and histone modification patterns. Our result indicated that MORC6 not only interacts with MORC1 and MORC2 to form heteromers, but also associates with SUVH9. By whole-genome DNA methylation analysis, we found that MORC6 contributes to DNA methylation at a small subset of RdDM target loci. By comaring the whole-genome DNA methylation and RNA deep sequencing data, we found that in suvh2suvh9, DNA methylation is notablely reducedin loci whose transcript levels are specifically upregulated in suvh2suvh9 but not in loci whose transcript levels are co-upregulated in suvh2/9 and morc6. Moreover, we demonstrated that MORC6 interact the RdDM component IDN2 and the SWN/SNF chromatin-remodeling complex. These results suggest that SUVH2/9 mediate transcriptional gene silencingnot only throughthe RdDM pathwaybut also thorugh a MORC6-dependent pathway. IDN2 and chromatin-remodeling complex may be required for the function of MORC6 in heterochromatin condensation.Taken together, this study demonstrates that inactive histone methyltransferases SUVH2 and SUVH9 associate with the DDR complex and facilitate the recruitment of pol V to chromatin, thereby acting in the RdDM pathway. Besides, SUVH2 and SUVH9 interact with the MORC family proteins, to mediate transcriptional gene silencing by regulating chromatin superstructure.