A Protein Complex Required for Polymerase V Transcripts and RNA- Directed DNA Methylation in Arabidopsis

Abstract

Summary DNA methylation is an epigenetic modification associated with gene silencing. In Arabidopsis, DNA methylation is established by DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), which is targeted by small interfering RNAs through a pathway termed RNA-directed DNA methylation (RdDM)[1, 2]. Recently, RdDM was shown to require intergenic noncoding (IGN) transcripts that are dependent on the Pol V polymerase. These transcripts are proposed to function as scaffolds for the recruitment of downstream RdDM proteins, including DRM2, to loci that produce both siRNAs and IGN transcripts[3]. However, the mechanism(s) through which Pol V is targeted to specific genomic loci remains largely unknown. Through affinity purification of two known RdDM components, DEFECTIVE IN RNA-DIRECTED DNA METHYLATION 1 (DRD1)[4] and DEFECTIVE IN MERISTEM SILENCING 3 (DMS3)[5, 6], we found that they copurify with each other and with a novel protein, RNA-DIRECTED DNA METHYLATION 1 (RDM1), forming a complex we term DDR. We also found that DRD1 copurified with Pol V subunits and that, RDM1, like DRD1[3] and DMS3[7], is required for the production of Pol V-dependent transcripts. These results suggest that the DDR complex acts in RdDM at a step upstream of the recruitment or activation of Pol V.

DOMAINS REARRANGED METHYLTRANSFERASE3 controls DNA methylation and regulates RNA polymerase V transcript abundance in Arabidopsis

“…This analysis indeed detected DRM3 peptides in addition to all known Pol V subunits (Table S2). Consistent with previous published purifications (19,(34)(35)(36), we also identified other Pol V interactors, including AGO4, DMS3, DRD1, and KTF1/SPT5L. To further validate the mass spectrometry data, we performed co-IP experiments from F1 plants expressing both HA-tagged DRM3 and FLAG-tagged NRPE1.…”

“…The AGO4/siRNA complex is thought to base pair with noncoding RNA transcripts produced by a DNA-DEPENDENT RNA POLYMERASE V (Pol V). Production of Pol V transcripts, as well as genome-wide Pol V chromatin occupancy, requires a complex termed DDR consisting of the putative chromatin-remodeling factor DEFECTIVE IN RNA-DIRECTED DNA METHYLATION 1 (DRD1), structural maintenance of chromosome domain protein DEFECTIVE IN MERISTEM SILENCING 3 (DMS3), and RNA-DIRECTED DNA METHYLATION 1 (17)(18)(19)(20). The SUPPRESSOR OF VARIEGATION 3-9 HOMOLOG 2 (SUVH2) and -9 (SUVH9) proteins are required for genome-wide Pol V chromatin association by binding to DNA methylation (21,22).…”

Transcriptional gene silencing by Arabidopsis microrchidia homologues involves the formation of heteromers

“…However, we did not detect DMS3 or other components of the DDR complex in our IP-MS experiments. Also, previous IP-MS experiments using FLAG-tagged DRD1 and DMS3 proteins as bait did not immunoprecipitate AtMORC6 (37). Nevertheless, we cannot rule out that the interactions between components of the DDR complex and AtMORC6 are weak or ephemeral and could not be detected under our IP conditions.…”

“…DMS3 has also been shown to interact with additional components of the DRD1-DMS3-RDM1 (DDR) complex including Defective in RNA-Directed DNA Methylation 1 (DRD1) or RDM1 as well as with the largest subunit of PolV (37). Furthermore, genome-wide association of PolV to chromatin and thus the production of PolV-dependent transcripts and subsequent DNA methylation are dependent on all members of the DDR complex (37,38). However, we did not detect DMS3 or other components of the DDR complex in our IP-MS experiments.…”

“…Gel filtration experiments were performed according to ref. 37. Briefly, 0.5 g of 2-wk-old seedling tissue of each epitope-tagged line were ground in liquid nitrogen and resuspended in 1 mL of ice-cold IP buffer [50 mM Tris·HCl pH 8.0, 150 mM NaCl, 0.1% Nonidet P-40, 10% (vol/vol) glycerol, 1× Protease Inhibitor Mixture (Roche)] and centrifuged for 10 min at 4°C at 16,000 × g. The supernatants were centrifuged again for 10 min at 4°C at 16,000 × g. The supernatants were then centrifuged through a 0.2 μm filter (Millipore), 500 μL were loaded onto a Superdex 200 10/300GL column (GE Healthcare, 17-5175-01) column, and 250 μL fractions were collected.…”