Two Components of the RNA-Directed DNA Methylation Pathway Associate with MORC6 and Silence Loci Targeted by MORC6 in Arabidopsis

Abstract

The SU(VAR)3-9 homolog SUVH9 and the double-stranded RNA-binding protein IDN2 were thought to be components of an RNA-directed DNA methylation (RdDM) pathway in Arabidopsis. We previously found that SUVH9 interacts with MORC6 but how the interaction contributes to transcriptional silencing remains elusive. Here, our genetic analysis indicates that SUVH2 and SUVH9 can either act in the same pathway as MORC6 or act synergistically with MORC6 to mediate transcriptional silencing. Moreover, we demonstrate that IDN2 interacts with MORC6 and mediates the silencing of a subset of MORC6 target loci. Like SUVH2, SUVH9, and IDN2, other RdDM components including Pol IV, Pol V, RDR2, and DRM2 are also required for transcriptional silencing at a subset of MORC6 target loci. MORC6 was previously shown to mediate transcriptional silencing through heterochromatin condensation. We demonstrate that the SWI/SNF chromatin-remodeling complex components SWI3B, SWI3C, and SWI3D interact with MORC6 as well as with SUVH9 and then mediate transcriptional silencing. These results suggest that the RdDM components are involved not only in DNA methylation but also in MORC6-mediated heterochromatin condensation. This study illustrates how DNA methylation is linked to heterochromatin condensation and thereby enhances transcriptional silencing at methylated genomic regions.

Fig 1. The involvement of MORC6 in DNA methylation at a small subset of RdDM target loci.

(A) Box plots showing DNA methylation levels of suvh2/9 hypo-DMRs in the indicated mutants. (B) Venn diagram of overlap between hypo-DMRs in morc6 and suvh2/9. (C) Heat maps showing DNA methylation changes within suvh2/9 hypo-DMRs in the wild type and the mutants. Different scales were used for CG, CHG, and CHH methylation. In the scales, the DNA methylation levels of the wild type were set as 0 (light yellow lines) and absolute DNA methylation changes were represented by lines whose colors are ranged from light yellow to black. (D) DNA methylation levels of morc6-specific hypo-DMRs, suvh2/9-specific hypo-DMRs, and hypo-DMRs shared by morc6 and suvh2/9 are indicated at three cytosine contexts in the wild type, morc6, and suvh2/9. (E) Validation of bisulfite sequencing results by PCR-based DNA methylation analysis. Genomic DNA was cleaved by McrBC, a restriction enzyme that specifically recognizes methylated DNA. Class I represents hypo-DMRs shared by morc6 and suvh2/9; Class II represents suvh2/9-specific hypo-DMRs. The RdDM pathway contributes to CHH methylation and to a lesser extent to CG and CHG methylation [5], but it is unknown how MORC6 contributes to DNA methylation at the three cytosine contexts. Our analysis indicated that morc6 affects DNA methylation at all the three cytosine contexts (CG, CHG, and CHH) in a small subset of the 6514 suvh2/9 hypo-DMRs (Fig 1C). In the 527 hypo-DMRs shared in morc6 and suvh2/9, DNA methylation is reduced at all three cytosine contexts in both morc6 and suvh2/9 (Fig 1D). In the 218 morc6 specific hypo-DMRs, DNA methylation is reduced at all three cytosine contexts in the morc6 mutant (Fig 1D). Although the 218 morc6 specific hypo-DMRs do not overlap with the suvh2/9 hypo-DMRs, the DNA methylation level of the 218 hypo-DMRs is reduced at CHG and CHH sites in suvh2/9 (Fig 1D). However, CG methylation of the 218 hypo-DMRs is markedly reduced in morc6 but not in suvh2/9 (Fig 1D). Thus, these 218 hypo-DMRs could be either RdDM-independent loci or RdDM-dependent loci that are not dependent on SUVH2/9.

Fig 2. Identification and characterization of target loci shared by SUVH2/9 and MORC6.

(A) Venn diagrams of overlap between up-regulated (2-fold increase; p<0.01 by Cufflinks) TEs and genes in morc6 and suvh2/9. (B) Heat maps of differentially expressed TEs and genes in suvh2/9 and morc6 relative to the wild type. Up- and down-regulated loci are shown by red and blue lines, respectively. Color scales represent log₂ (mutant/WT). (C) The RNA transcript levels of MORC6 target loci were determined by quantitative RT-PCR in the wild type, suvh2/9, morc6, and suvh2/9;morc6. ACT2 was used as an internal control. Error bars are standard deviation of three biological replicates. (D) Effect of the RdDM mutations nrpd1, rdr2, nrpe1, drm1/2, and idn2 on the transcript levels of SUVH2/9 and MORC6 common target TEs as determined by quantitative RT-PCR analysis. Error bars are standard deviation of three biological replicates. (E) The effect of nrpd1 and nrpe1 on the abundance of 24-nt siRNAs at SUVH2/9 and MORC6 target TEs. Based on small RNA deep sequencing data, the 24-nt siRNA abundance in nrpd1 and nrpe1 is compared with that in the wild type and fold changes are indicated by y-axis.

Fig 3. Determination of effect of suvh2/9 and morc6 on DNA methylation of SUVH2/9 and MORC6 target loci.

(A, B) Scatter plots showing DNA methylation of transcriptionally up-regulated TEs and genes in suvh2/9 and morc6. Red dots represent TEs and genes that are co-up-regulated in suvh2/9 and morc6. Blue dots represent TEs and genes that are up-regulated in suvh2/9 but not in morc6. Green dots represent TEs and genes that are up-regulated in morc6 but not in suvh2/9. (C) Box plots showing DNA methylation of transcriptionally up-regulated TEs in morc6 and suvh2/9 at total cytosine sites (top panel) and CHH sites (bottom panel). DNA methylation of suvh2/9-specific up-regulated TEs is indicated in the wild type and suvh2/9, and DNA methylation of co-up-regulated TEs in suvh2/9 and morc6 is indicated in the wild type, suvh2/9, and morc6. Asterisks indicate statistical significance (* p<0.05, ** p<0.01; t-test). p values are shown on top of bars. (D) Effect of suvh2/9 and morc6 on DNA methylation of AT1TE45510, AT2TE18240, AT4TE09845, and AT5TE39630 at total cytosine, CG, CHG, and CHH sites. The DNA methylation data were generated from the whole-genome bisulfite sequencing analysis in the wild type, suvh2/9, and morc6.

Fig 4. Determination of effect of RdDM mutations on the transcript and DNA methylation levels of SUVH2/9 and MORC6 target TEs.

(A) Scatter plots showing DNA methylation of SUVH2/9 and MORC6 target TEs in nrpd1, rdr2, nrpe1, drm1/2, and idn2 relative to the wild type. Red dots represent target TEs that are transcriptionally co-up-regulated in suvh2/9 and morc6, whereas blue dots represent TEs that are transcriptionally up-regulated in suvh2/9 but not in morc6. DNA methylation levels at total cytosine and CHH sites are shown in top and bottom panels, respectively. (B) The DNA methylation levels of the SUVH2/9 and MORC6 common target TEs in the wild type, nrpd1, rdr2, nrpe1, drm1/2, suvh2/9, and morc6. The DNA methylation levels of the TEs are separately shown in total cytosine, CG, CHG, and CHH sites.

Fig 5. MORC6 interacts with the coiled-coil domain of IDN2.

(A) The interaction between MORC6 and IDN2 as determined by a yeast two-hybrid assay. IDN2 was fused with GAL4-AD, whereas MORC1, MORC2, and MORC6 were fused with GAL4-BD. “Vec” represents the empty GAL4-AD or GAL4-BD vector. A series of diluted strains were grown on two synthetic dropout media: one lacked Trp and Leu (-Trp-Leu), and the other lacked Trp, Leu, and His (-Trp-Leu-His) and was supplemented with 3-AT. (B) The interaction between MORC6 and IDN2 as determined by a split luciferase complementation assay in tobacco (N. benthamiana). Luciferase activities were determined by luminescence imaging. White circles indicate leaf regions that were infiltrated with the Agrobacterium strains containing the indicated constructs. MORC6 and IDN2 were fused with N-LUC and C-LUC, respectively. The empty N-LUC and C-LUC constructs were transformed as negative controls. (C) The interaction between MORC6 and IDN2 as indicated by co-IP. Transgenic plants harboring MORC6-Flag and IDN2-Myc transgenes were used in the co-IP experiment. (D) Diagrams indicating full-length and truncated forms of IDN2 that were used in yeast two-hybrid assays. (E) Identification of the IDN2 domain required for interaction with MORC6 as indicated by yeast two-hybrid assays. The full-length and truncated forms of IDN2 were fused with GAL4-AD, and the full-length MORC6 was fused with GAL4-BD. “Vec” represents the empty GAL4-BD vector. The growth of four individual colonies is shown for each genotype.

Fig 6. MORC6 and SUVH9 interact with the SWI/SNF chromatin-remodeling complex components SWI3B, SWI3C, and SWI3D.

(A) The interaction of MORC6 and SUVH9 with SWI3B, SWI3C, and SWI3D as indicated by split luciferase complementation assays in tobacco. MORC6 and SUVH9 were fused with N-LUC. SWI3B, SWI3C, and SWI3D were fused with C-LUC. The empty N-LUC and C-LUC constructs were used as negative controls. The indicated constructs were transformed into Agrobacterium and were infiltrated into tobacco leaves. (B) Analysis of the interaction between MORC6 and SWI3D by co-IP. Transgenic plants harboring MORC6-Myc and SWI3D-Flag transgenes were generated and used in the co-IP experiment. (C) The transcript levels of the representative SUVH2/9 and MORC6 common target TEs in the wild type and the swi3d mutant as determined by quantitative RT-PCR. Errors are standard deviation of three biological replicates.

Fig 7. Model for the roles of SUVH2/9 in RNA-directed DNA methylation and chromatin condensation in Arabidopsis.

The methylated-DNA-binding proteins SUVH2/9 interact with the DDR complex to recruit Pol V to chromatin and thereby facilitate a self-reinforcing loop between Pol V transcription and DNA methylation in the RNA-directed DNA methylation pathway. Moreover, SUVH2/9 interact with the MORC1/2-MORC6 complex, which then interacts with IDN2 and the SWI/SNF complex to mediate chromatin condensation. IDN2 is required not only for RNA-directed DNA methylation but also for chromatin condensation. The physical interaction between SUVH2/9, MORCs, IDN2, and the SWI/SNF complex components SWI3/C/D facilitates the interplay between DNA methylation and chromatin condensation, thus enhancing transcriptional silencing at their common target loci.