The SET domain proteins SUVH2 and SUVH9 are required for Pol V occupancy at RNA-directed DNA methylation loci

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.

Figure 1. Detection of the protein interaction by co-IP and gel filtration analyses.

(A) Detection of the interaction between SUVH2-3xMyc and DMS3 by co-IP. The protein extract of SUVH2-3xMyc transgenic plants was immunoprecipitated by anti-Myc antibody. The precipitate was subjected to Western blotting with the antibodies against the Myc epitope and endogenous DMS3. (B) The interaction between SUVH9-3xFlag and MORC6-3xMyc as determined by co-IP. The F1 offspring generated from the cross between SUVH9-3xFlag and MORC6-3xMyc transgenic plants were used for co-IP. (C) The elution profile of MORC6-3xFlag, SUVH2-3xMyc, SUVH9-3xFlag, and DMS3 as determined by gel filtration. Antibodies specific for the Flag epitope and endogenous DMS3 were used for Western blotting. The arrow indicates the fraction of a 440-KDa standard protein. (D) The protein extracts from the wild type and MORC6-3xFlag transgenic plants were affinity purified by anti-Flag antibody. The purified proteins were run on an SDS-PAGE gel and subjected to silver staining. The three separated bands at ∼70–100 KDa were cut out for mass spectrometric analysis. (E) The interaction between MORC1 and MORC6 as determined by co-IP.

Figure 2. Detection of the interaction of SUVH2 and SUVH9 with RdDM components by yeast two-hybrid assay.

The yeast strains harboring GAL4-AD and GAL4-BD fusion constructs were streaked on plates with indicated media. SD-TLH, the synthetic dropout medium without Trp, Leu, and His. SD-TL, the synthetic dropout medium without Trp and Leu. “Vec” represents the empty pGADT7 or pGBKT7 vector. (A) The interaction of SUVH2 with SUVH9, AGO4, IDN2, and RDM1. (B) The interaction of SUVH9 with SUVH2, DMS3, AGO4, IDN2, and RDM1. (C) The interaction of SUVH2 with the MORC family proteins MORC1, MORC2, and MORC6. (D) The interaction of SUVH9 with the MORC family proteins.

Figure 3. Small RNA analyses by small RNA Northern blotting and small RNA deep sequencing.

(A) Diagrams show the distribution of Pol IV-dependent 24-nt siRNAs across all five Arabidopsis chromosomes in the wild type, suvh2, suvh9, suvh2suvh9, nrpe1, and dms3. Normalized 24-nt siRNA reads per ten million in consecutive 500-Kb genomic regions of each chromosome are indicated by the Y coordinate, whereas all five Arabidopsis chromosomes are shown along the X coordinate. (B–D) Venn diagrams indicate the numbers and overlaps of siRNA regions that show decreased siRNAs in dms3 (B), suvh2 (C), suvh9 (D), and suvh2suvh9. (E) compared to nrpd1 and nrpe1. The results are from the small RNA deep sequencing data for the wild type, nrpd1, nrpe1, dms3, suvh2, suvh9, and suvh2suvh9.

Figure 4. RNA transcript analysis by semi-quantitative RT-PCR and RNA deep sequencing.

(A) The effect of suvh2, suvh9, and svuh2suvh9 on transcriptional silencing as determined by quantitative RT-PCR. The transcript levels of RdDM loci solo LTR, AtGP1, SDC, AtSN1 was tested. The actin gene was amplified as an internal control. (B) Pol V-dependent noncoding RNAs from IGN5B, IGN23, and IGN25 loci were measured by semiquantitative RT-PCR. No RT indicates the amplification of the actin gene using total RNAs as template without reverse transcription. (C) The genome-wide effect of suvh2suvh9, nrpe1, and dms3 on gene expression as determined by RNA deep sequencing. Heat map of log₂ (Mutant/WT) was shown for each mutant. (D) The numbers and overlaps of the upregulated genes in suvh2suvh9, nrpe1, and dms3 relative to the wild type are shown in Venn diagram.

Figure 5. Effect of suvh2 and suvh9 on the occupancy of Pol V and the DDR complex at RdDM loci.

(A) The effect of suvh2suvh9 on Pol V occupancy at RdDM loci was determined by CHIP assay with anti-Flag antibody. The enrichment of the largest subunit of Pol V, NRPE1, was measured at IGN5, solo LTR, IGN22, IDN25, and IGN26. The abundance of NRPE1 on the actin gene was used as an internal negative control. Enrichment of NRPE1-Flag on RdDM loci and the tublin gene was normalized by the abundance of NRPE1-Flag on the actin gene. (B) The effect of suvh2suvh9 on the occupancy of the DDR complex at RdDM loci was determined by CHIP assay with anti-DMS3 antibody. Enrichment of the DDR complex component DMS3 was determined in the wild type and suvh2suvh9. Relative enrichment of DMS3 is shown.

Figure 6. Effect of suvh2 and suvh9 on DNA methylation and histone H3K9me2 at RdDM targets.

(A) The effect of suvh2, suvh9, and suvh2suvh9 on DNA methylation as indicated by chop-PCR. DNA methylation was determined for AtSN1, IGN5, IGN25, and solo LTR. Genomic DNA from the wild type, nrpd1, nrpe1, dms3, suvh2, suvh9, and suvh2suvh9 was cleaved by the DNA methylation-sensitive restriction enzyme HaeIII or AluI and subjected to semiquantitative PCR. The actin gene, which lacks HaeIII recognition site, was amplified as an internal control. The solo LTR locus was amplified using uncut genomic DNA as template for a control. (B) Effect of suvh2suvh9 on histone H3K9me2 was measured by CHIP assay with anti-H3K9me2 antibody. The RdDM mutants nrpe1 and dms3 were included as controls, in which the H3K9me2 levels at indicated RdDM loci were decreased. The actin gene, which lacks detectable H3K9me2, was used as a negative control.

Figure 7. Mutation of the conserved SET domain in SUVH2 has no effect on DNA methylation and H3K9me2 at RdDM loci.

(A) Alignment of the catalytic active site in the Arabidopsis SET domain proteins SUVH2, SUVH9, SUVH4, SUVH5, and SUVH6, and their homologs including G9a and SUV39H1 in human, Clr4 in fission yeast, and Dim-5 in Neurospora. The conserved residues SUVH2-H596 and SUVH2-P600 that were subjected to point mutation are marked with arrows. (B) Site-directed mutagenesis was carried out to introduce the SUVH2-H596K and SUVH2P600A mutations in the SET domain of SUVH2. The construct harboring either wild-type or mutant SUVH2 sequence was transformed into suvh2suvh9 for complementation assay. For DNA methylation assay, genomic DNA was digested by the DNA methylation-sensitive restriction enzyme HaeIII followed by PCR. The DNA methylation levels at RdDM loci IGN5 and IGN23 were determined. (C) H3K9me2 CHIP assay was performed to test whether the mutant SUVH2 sequences complement the H3K9me2 defect caused by suvh2 in suvh2suvh9 at RdDM loci IGN5 and AtSN1. Enrichment of H3K9me2 was normalized by the abundance of H3K9me2 on the actin gene.

Figure 8. Model for the role of SUVH2, SUVH9 in RdDM.

SUVH2 and SUVH9 bind to methylated DNA at RdDM loci and are at least partially required for the occupancy of the DDR complex at the loci. The DDR complex associates with Pol V and facilitates the recruitment of Pol V to RdDM loci. Pol V-produced noncoding RNAs interact with 24nt-siRNAs bound by AGO4, recruiting the de novo DNA methyltransferase DRM2 for DNA methylation. The methylated DNA is further bound by SUVH2 and SUVH9, resulting in a self-reinforcing loop that facilitates maintenance of DNA methylation at RdDM loci.