Volume 9, Issue 8, Pages 1156-1167 (August 2016) SUVH2 and SUVH9 Couple Two Essential Steps for Transcriptional Gene Silencing in Arabidopsis  Yuqing Jing, Han Sun, Wei Yuan, Yue Wang, Qi Li, Yannan Liu, Yan Li, Weiqiang Qian  Molecular Plant  Volume 9, Issue 8, Pages 1156-1167 (August 2016) DOI: 10.1016/j.molp.2016.05.006 Copyright © 2016 The Author Terms and Conditions

Figure 1 Identification of Suppressors of idm1-14. (A) The morc6-4 and suvh9-2 mutations cause partial release of the LUC and NPTII reporter genes. Seedlings grown in MS plates were imaged after being sprayed with luciferase substrate. For the kanamycin resistance test, the seeds were planted on MS medium supplemented with 50 mg/L kanamycin and incubated for 2 weeks before being photographed. (B) Real-time PCR analysis of the expression levels of the LUC and NPTII reporter genes in the selected genotypes. (C) DNA methylation levels at the 35S promoter region in idm1-14, idm1-14morc6-4, and idm1-14suvh9-2. Molecular Plant 2016 9, 1156-1167DOI: (10.1016/j.molp.2016.05.006) Copyright © 2016 The Author Terms and Conditions

Figure 2 Effects of the suvh9-2 Mutation on Gene Transcription and DNA Methylation. (A–C) The left panel shows the real-time PCR analysis of the transcript levels of SDC (A), Solo-LTR (B), and COPIA 28 (C). The right panel shows the bisulfite sequencing results for the corresponding locus in idm1-14 and idm1-14suvh9-2. ACTIN 7 was used as an internal control. Standard errors were calculated from three biological replicates, *P < 0.01. Molecular Plant 2016 9, 1156-1167DOI: (10.1016/j.molp.2016.05.006) Copyright © 2016 The Author Terms and Conditions

Figure 3 Confirmation of Protein–Protein Interactions by Yeast-Two-Hybrid and Split-LUC Assays. (A) Y-2-H analysis of SUVH9 and MORC6 interaction. Yeast cells harboring different fusion protein combinations (listed on the left) in pGBK-T7 (BD) and pGAD-T7 (AD) vectors were plated on the medium lacking Leu and Trp (SD-TL) or the medium lacking Leu, Trp, and His (SD-TLH) but supplemented with 2 mM 3-AT. A yeast colony formed on an SD-LTH plate indicates a positive protein interaction. (B) Interactions of SUVH9 with MORC6 as determined by firefly luciferase complementation imaging assays in Nicotiana benthamiana leaves. nLUC, C-terminal region of firefly luciferase; cLUC, N-terminal region of firefly luciferase. Three independent experiments were done with similar results obtained. (C) Y-2-H analysis of the interactions between MORC proteins and IDN2, SWI3B, and SWI3C. Yeast colony formed on an SD-LTH plate indicates a positive protein interaction. Molecular Plant 2016 9, 1156-1167DOI: (10.1016/j.molp.2016.05.006) Copyright © 2016 The Author Terms and Conditions

Figure 4 SUVH2 and SUVH9 Are Required for Chromocenter Condensation. (A) Examples of the three different patterns of chromocenters. (B) Percentages of nuclei with decondensed, partially decondensed (intermediate), and WT chromocenters in WT, morc6-3, suvh2-1, suvh9-1, and suvh2 suvh9 were calculated after immunostaining of the nuclei using an antibody against H3K9me1. Molecular Plant 2016 9, 1156-1167DOI: (10.1016/j.molp.2016.05.006) Copyright © 2016 The Author Terms and Conditions

Figure 5 Effects of morc6-3 and suvh2 suvh9 on Chromatin Structures at SDC and Solo-LTR Loci. (A) Chromatin interactions in the SDC promoter region in morc6-3 and suvh2 suvh9. Seven regions upstream of the SDC transcription start site (TSS) were selected. Chromatin interactions between those seven regions and the anchor region were calculated. (B) Chromatin interactions in the Solo-LTR region in morc6-3 and suvh2 suvh9. Two regions upstream of Solo-LTR TSS were selected for the detection of chromatin interactions between the upstream regions and gene body of Solo-LTR. Real-time PCR was performed to quantify the amounts of amplification products between two chromatin fragments, and 3C interaction frequencies were calculated based on the real-time PCR data. HindIII restriction sites are indicated with vertical dotted lines, the analyzed regions are numbered with Roman numerals, and the anchor regions are highlighted in gray color. On each fragment, a primer (arrow) was designed at a distance of about 100–200 bp from the restriction site and all the 3C primers were set in one direction. Standard errors were calculated from three biological replicates, *P < 0.05. Molecular Plant 2016 9, 1156-1167DOI: (10.1016/j.molp.2016.05.006) Copyright © 2016 The Author Terms and Conditions

Figure 6 Effects of suvh2 suvh9 and morc6-3 on RNA Transcript Levels as Determined by RNA-Seq. (A) Differentially expressed genes or TEs in morc6-3 and suvh2 suvh9 were compared. Differentially expressed genes were identified when log2(fold changes of normalized reads) ≥ 2 or ≤ −2 and P value <0.05. Differentially expressed TEs were identified when log2(fold changes of normalized reads) ≥ 2 or ≤ −2 and P value <0.1. (B) Real-time PCR was performed to confirm the RNA-seq results for TEs. ACTIN 7 was used as internal control. Standard errors were calculated from three biological replicates. (C) DNA methylation levels in upregulated TEs. Green spots indicate upregulated TEs in both morc6-3 and suvh2 suvh9. Red spots and yellow spots indicate TEs specifically upregulated in morc6-3 and suvh2 suvh9, respectively. DNA methylation levels were examined using the previously published whole-genome bisulfite sequencing data (Moissiard et al., 2012; Johnson et al., 2014). Molecular Plant 2016 9, 1156-1167DOI: (10.1016/j.molp.2016.05.006) Copyright © 2016 The Author Terms and Conditions

Figure 7 Model for SUVH2/9-Dependent Transcriptional Gene Silencing in Arabidopsis. Step 1: DNA methylation-dependent gene silencing pathway. In the RdDM pathway, SUVH2 or SUVH9 binds to methylated DNA at RdDM loci (e.g., the 35S promoter in this study) and is required for the recruitment of the DDR complex. Step 2: Methylated DNA is further bound by SUVH2 or SUVH9, which recruits the MORC-IDN2-SWI/SNF complex to change the 3D structure of the chromatin and thus completely silence the gene. Molecular Plant 2016 9, 1156-1167DOI: (10.1016/j.molp.2016.05.006) Copyright © 2016 The Author Terms and Conditions