A idm-3 idm-3 B Physical distance (Mb) 4.6 4.86.6 8.4 C Chr.3 Recom. Rate (%) ATG 3.9.9.9 9.74 Candidate region (.74 Mb) n=4 TAA D idm-3 G3T(E4) G4A(W988) WT idm-3 ATC TCT GGG ACT CAT GAG CAG GAG GCT AGC ATC TCT GGG ACT CAT TAG CAG GAG GCT AGC WT AGT TTA GTG GAA ACT TGG ACT GAA GGT TTT AGT TTA GTG GAA ACT TGA ACT GAA GGT TTT
E X Col- F X idm- F Supplemental Figure. IDM prevents transcriptional gene silencing. (A) LUC luminescence image showing that the IDM mutations cause silencing of the 3S-LUC reporter gene. Seedlings grown in MS plates were imaged after being sprayed with luciferase substrate. (B) Rough mapping of the mutation. Shown are the molecular markers and their positions on chromosome III around the region. About 4 mutant plants from the progenies of a cross between (in Col- ) and Ler were used to define the position of. The recombination rate at these markers is indicated. (C) Schematic diagram showing the positions of the point mutations at the IDM locus. Black rectangles represent exons. (D) DNA sequence alignments between WT and mutated IDM. Point mutations in the mutants are highlighted in red. (E) Allelism analyses of the mutant. Seedlings grown in MS plates were imaged after being sprayed with luciferase substrate.
A AtSN HaeIII Solo-LTR AluI No digest control B drd- nrpe-9 C Physical distance (Mb) Chr. 4..79 7.74 8.4 9.96.96 Recom. Rate (%) 7.4 6.34 6.3.9 6.67 3.8 drd- n=63 Physical distance (Mb) Chr. Candidate region (.9 Mb) nrpe-9 3.93 6.3 7.6 8.4 D Recom. Rate (%) ATG 7.8.4.67 3. Candidate region (.3 Mb) n=96 drd- G33A (R74H) TAA DRD ATG nrpe-9 G779A (W638) TAA NRPE
E drd- drd- + DRD-Flag drd- drd- + DRD-Flag F nrpe-9 nrpe-9 + NRPE-Flag nrpe-9 nrpe-9 + NRPE-Flag suvh- rdr- Supplemental Figure. Identification of RdDM pathway components as IDM suppressors and effects of their mutations on transcriptional gene silencing of the reporter genes. (A) Analysis of DNA methylation levels at the AtSNI and Solo-LTR loci by chop-pcr. Genomic DNA from and the mutants of IDM suppressors was digested by the DNA methylation-sensitive restriction enzyme HaeIII or AluI and then subjected to semi-quantitative PCR. Undigested samples were used as controls. (B) Effects of drd- and nrpe-9 mutations on 3S-LUC expression. (C) Rough mapping of the drd- and nrpe-9 mutations. (D) Schematic diagram showing the positions of the point mutations at the DRD and NRPE loci. Black rectangles represent exons. (E) Complementation assay showing that introduction of the prodrd::drd-flag or pronrpe::nrpe-flag transgene into drd- or nrpe-9 respectively restored 3S-LUC silencing. (F) Effects of rdr- and suvh- mutations on 3S-LUC expression. The 3S-LUC reporter gene was introduced into suvh- and rdr- mutant plants by crossing the suvh- and rdr- mutants with the plants in background.
A morc6- morc6-6 B Physical distance (Mb).7.99 4..89 6.78 7.6 8.6 Chr. Recom. Rate (%).4 8.8 4.3.6.7. 3.4 n=96 Candidate region (.84 Mb) WT morc6-4 TCC AAT GCT ACC TCA CAT AAA TGG GCT TTT GGA TCC AAT GCT ACC TCA CAT AAA TGA GCT TTT GGA C morc6-3 (GK-99B6) morc6-6 (G37A) ATG () TAG (6) ATG9 morc6-4 (G48A) morc6- (G46A) D morc6-4 morc6- (W8) (W3) 36 morc6-6 (G43E) MORC6 morc6-3
E WT nrpe-9 morc6-4 nrpe-9 + NRPE-Flag morc6-4 + MORC6-Flag morc6-4 morc6-4 + MORC6-Flag Supplemental Figure 3. Map-based cloning of morc6-4. (A) The morc6- and morc6-6 mutations cause partial release of the LUC reporter genes. Seedlings grown in MS plates were imaged after being sprayed with luciferase substrate. (B) Rough mapping of the morc6-4 mutation. Shown are the molecular markers and their positions on chromosome I around the morc6-4 region. Ninety-six mutant plants from the progenies of a cross between morc6-4 (in Col-) and ros4/idm-3 (in C4) were used to define the position of morc6-4. The recombination rate at these markers is indicated. A point mutation (G/A) was identified in MORC6. (C) Schematic diagram showing the positions of the point mutations and T- DNA insertion at the MORC6 locus. Black rectangles represent exons. Gray rectangles represent UTRs. (D) Schematic diagram of MORC6 protein domain structure. MORC6 contains a GHKL domain, a S ATPase domain and a putative Coiled-Coil (CC) domain. The point mutations are in the GHKL domain and S domain. (E) Complementation assay showing that introduction of the promorc6::morc6-flag transgene into morc6-4 restored 3S-LUC silencing.
A Physical distance (Mb) Chr.4 6. 7.97.3 Recom. Rate (%). 8.8 6.6 n=93 B Candidate region (4. Mb) C ATG () TAA (93) AT4G346 suvh9- (salk_4833) suvh9- (C66T) D suvh9- (Q36) SUVH9
E suvh9- suvh9-+suvh9-flag suvh9- suvh9-+suvh9-flag Supplemental Figure 4. Map-based cloning of suvh9-. (A) Rough mapping of the suvh9- mutation. Shown are the molecular markers and their positions on chromosome IV around the suvh9- region. Ninety-three mutant plants from the progenies of a cross between suvh9- (in Col-) and ros4/idm-3 (in C4) were used to define the position of suvh9-. The recombination rate at these markers is indicated. (B) Genome sequencing of suvh9-. A point mutation (G/A) was identified in SUVH9. (C) Schematic diagram showing the positions of the point mutation and T-DNA insertion at the SUVH9 locus. Black rectangles represent exons. (D) Schematic diagram of SUVH9 protein domain structure. SUVH9 contains a SRA domain, a preset domain and a SET domain. The point mutation is in the SRA domain. (E) Complementation assay showing introduction of the prosuvh9::suvh9- Flag transgene into suvh9- restored 3S-LUC silencing.
A 3 7 4 6 8 B. nluc + cluc;. nluc + SUVH9-cLUC; 3. MORC-nLUC + cluc; 4. MORC-nLUC + SUVH9-cLUC;. MORC-nLUC + cluc; 6. MORC-nLUC + SUVH9-cLUC; 7. IDN-nLUC + cluc; 8. IDN-nLUC + SUVH9-cLUC. SD-TL SD-TLH AD/BD SUVH9/IDN IDN/SUVH9 SUVH9/SWI3B SWI3B/SUVH9 C MORC6/SUVH9 3 4 7 4 9 8 6. nluc + cluc. nluc + SUVH9-cLUC 3. SWI3A-nLUC + cluc 4. SWI3A-nLUC + SUVH9-cLUC. SWI3B-nLUC + cluc 6. SWI3B-nLUC + SUVH9-cLUC 7. SWI3C-nLUC + cluc 8. SWI3C-nLUC + SUVH9-cLUC 9. SWI3D-nLUC + cluc. SWI3D-nLUC + SUVH9-cLUC Supplemental Figure. SUVH9 interacts with SWI/SNF chromatin-remodeling proteins. (A) Detection of the interactions of SUVH9 with MORC, MORC and IDN by split-luc assays. (B) Detection of the interactions of SUVH9 with IDN and SWI3B by Y--H assays. (C) Detection of the interactions of SUVH9 with SWI/SNF chromatin-remodeling proteins by split-luc assays. nluc, C- terminal region of firefly luciferase; cluc, N-terminal region of firefly luciferase. Three independent experiments were done with similar results obtained. Red indicates positive protein interaction.
3 4 7 9 8 6. nluc + cluc. nluc + SUVH-cLUC 3. IDN-nLUC + cluc 4. IDN-nLUC+ SUVH-cLUC. MORC-nLUC + cluc 6. MORC-nLUC + SUVH-cLUC 7. MORC-nLUC + cluc 8. MORC-nLUC + SUVH-cLUC 9. MORC6-nLUC + cluc. MORC6-nLUC + SUVH-cLUC. SWI3A-nLUC + cluc. SWI3A-nLUC + SUVH-cLUC 3. SWI3B-nLUC + cluc 4. SWI3B-nLUC + SUVH-cLUC. SWI3C-nLUC + cluc 6. SWI3C-nLUC + SUVH-cLUC 7. SWI3D-nLUC + cluc 8. SWI3D-nLUC + SUVH-cLUC 3 4 7 6 8 Supplemental Figure 6. Detection of the interactions between SUVH and other related proteins by split-luc assays. Interactions of SUVH with IDN, MORC family proteins and SWI/SNF chromatin-remodeling proteins as demonstrated 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. Red indicates positive protein interaction.
A Relative transcript level TE84 TE46 TE487 8 6 4 TE497 8 Solo-LTR COPIA 8 6 4 B Relative transcript level 8 6 4 SDC At3g769 At4g969 3 8 6 4 At4g394 C Relative transcript level At3g769 At4g469 At4g969 At4g394 4 3 3 4 3
D 4 3 morc6-3 Genes DNA methylation level log (morc6/col-) - - -3-4 4 6 8 4 Log (fold change of normalized reads) E DNA methylation level log (suvhsuvh9/col-) 4 3 - - -3-4 - suvhsuvh9 Genes 4 6 8 4 Log (fold change of normalized reads) Supplemental Figure 7. Effects of morc6-4 on RNA transcript level and effects of suvhsuvh9 and morc6-3 on RNA transcript level as determined by RNA-seq. (A) Up-regulated TEs in morc6-3 and morc6-4. (B) Up-regulated genes in morc6-4. (C) Real-time PCR was performed to confirm the RNA-seq results for genes. ACTIN 7 was used as internal control. Standard errors were calculated from three biological replicates, P <.. (D) DNA methylation levels for up-regulated genes in morc6-3. (E) DNA methylation levels for up-regulated genes in suvhsuvh9. Differentially expressed genes were identified when log (fold changes of normalized reads) or - and P value<..
Supplemental Table. Mass-spectrometric analysis of SUVH9 co-purifying proteins. Supplemental Table. Differentially expressed genes in suvhsuvh9 and morc6-3. Supplemental Table 3. Differentially expressed TEs in suvhsuvh9 and morc6-3. Supplemental Table 4. Primers used in this study. Supplemental Table. Mass-spectrometric analysis of SUVH9 co-purifying proteins. Protein Accession Gene Accession Protein Mass Spectra Unique Peptides IPI977 AT4G346 SUVH9 797 7 IPI94 ATG9 MORC6 749 IPI4938 AT3G4867 IDN 777 IPI763 AT4G3443 SWI3B 873 Supplemental Table 4. Primers used in this study. Primer Name Sequence ('-3') Purpose 3S-BS-F GATGTTTTTGTYGATAGTGGTTTTAAAGATGGAT Bisulfite sequencing for 3S-BS-R CTCTTCATAACCTTATACAATTACTCTCCAA 3S promoter SDC-BS-F GAAAAAGTTGGAATGGGTTTGGAGAGTTTAA Bisulfite sequencing for SDC-BS-R CAACAAACCCTAATATATTTTATATTAAAAC SDC Solo LTR-BS-F GATATAAAGGAATGGTTAGATAATATGYGATT Bisulfite sequencing for Solo LTR-BS-R CRATATAACTCAAAATTTATATTACTCTTAA solo-ltr ATCOPIA8-BS-F TATTTATTTYGTTCATTTGGATTAGTTTT Bisulfite sequencing for ATCOPIA8-BS-R ACRATATCAAAATAATTATCATCATCTTAA COPIA 8 LUC-RT-F CTCCCCTCTCTAAGGAAGTCG Real-time PCR for LUC LUC-RT-R CCAGAATGTAGCCATCCATC NPTII-RT-F AGGTTCCATCTGCCAGGTATCA Real-time PCR for NPT II NPTII-RT-R CCCGGTATCCAGATCCACAA SDC-RT-F AATGTAAGTTGTAAACCATTTGAACGTGACC Real-time PCR for SDC SDC-RT-R CAGGCATCCGTAGAACTCATGAGC Solo LTR-F AACTAACGTCATTACATACACATCTTG Real-time PCR for Solo LTR-R AATTAGGATCTTGTTTGCCAGCTA solo-ltr ATCOPIA8-F AGTCCTTTTGGTTGCTGAACA Real-time PCR for COPIA ATCOPIA8-R CCGGATGTAGCAACATTCACT 8 ACTIN 7-LP TCGTGGTGGTGAGTTTGTTA Real-time PCR, loading ACTIN 7-RP CAGCATCATCACAAGCATCC control SUVH-AD-BamHI-F GCGGGATCCATGAGTACATTGTTACC Amplify SUVH CDS for SUVH-AD-XhoI-R TATCTCGAGGTTGCAGATGGCGAGCT
SUVH9-AD-Nde-F AGATTACGCTCATATGATGGGTTCTTCTCAC Amplify SUVH9 CDS for SUVH9-AD-EcoR-R SUVH9-BD-Nde-F SUVH9-BD-EcoR-R CACCCGGGTGGAATTCTTAATTACAAATGGC GGAGGACCTGCATATGATGGGTTCTTCTCAC GGATCCCCGGGAATTCTTAATTACAAATGGC MORC-AD-NdeI-F AGATTACGCTCATATGATGGCGAAAAATTAC Amplify MORC CDS for MORC-AD-EcoRI-R CACCCGGGTGGAATTCCTAAACTTGTTGCATCTCC MORC- AD-NdeI-F AGATTACGCTCATATGATGCCTCCTATGGCG Amplify MORC CDS for MORC- AD-EcoRI-R CACCCGGGTGGAATTCCTAAGCTTGTTGCATC MORC6-AD-in-NdeI-F AGATTACGCTCATATGATGAGTCACGATAG Amplify MORC6 CDS for MORC6-AD-in-EcoRI-R MORC6-BD-EcoRI-F MORC6-BD-SalI-R ACGAGATCTGGTCGACCTACGTATTTACATTTCTTCTG GGCGAATTCATGAGTCACGATAGAAGTG GCCGTCGACCTACGTATTTACATTTC SWI3B-A/BD-NdeI-F GGACATATGATGGCCATGAAAGCTCCCGAT Amplify SWI3B CDS for SWI3B-A/BD-EcoRI-R GGCGAATTCCTAACACTCTATTCTATC SWI3C-BD-NdeI-F GGAGGACCTGCATATGATGCCAGCTTCTGAAG Amplify SWI3C CDS for SWI3C-BD-EcoRI-R GGATCCCCGGGAATTCTTAGTTTAAGCCTAAGC IDN-A/BD-EcoRI-F CCCGAATTCATGGGAAGCACTGTGAT Amplify IDN CDS for IDN-A/BD-BamHI-R ATAGGATCCAGCCATTCCACGCTTGC SUVH-NLUC-BamHI-F CGGTACCCGGGATCCaATGAGTACATTGTTACC Amplify SUVH cdna SUVH-NLUC-SalI- R SUVH-CLUC-BamHI-F SUVH-CLUC-SalI- R ACGAGATCTGGTCGACGTTGCAGATG GCGAGC CGGTACCCGGGATCCaATGAGTACATTGTTACC AGCTCTGCAGGTCGACCTAGTTGCAG ATGGCG for split-luc SUVH9-NLUC-BamHI-F CGGTACCCGGGATCCaATGGGTTCTTCTCAC Amplify SUVH9 cdna SUVH9-NLUC-SalI- R SUVH9-CLUC-BamHI-F SUVH9-CLUC-SalI- R ACGAGATCTGGTCGACATTACAAATGGC CGGTACCCGGGATCCaATGGGTTCTTCTCAC AGCTCTGCAGGTCGACTTAATTACAAATGGC for split-luc MORC-Nluc-KpnI-F CCGGGTACCATGGCGAAAAATTACACAGTC Amplify MORC cdna MORC-Nluc-SalI-R MORC-Cluc-KpnI-F MORC-Cluc-SalI-R CGGGTCGACAACTTGTTGCATCTCCTTCTT CCGGGTACCATGGCGAAAAATTACACAGTC CGGGTCGACCTAAACTTGTTGCATCTCCTT for split-luc MORC-NLUC-BamHI-F CGGTACCCGGGATCCaATGCCTCCTATGGCGA Amplify MORC cdna MORC-NLUC-SalI-R MORC-CLUC-BamHI-F MORC-CLUC-SalI-R ACGAGATCTGGTCGACAGCTTGTTGCATCTCC CGGTACCCGGGATCCaATGCCTCCTATGGCGA AGCTCTGCAGGTCGACCTAAGCTTGTTGCATC for split-luc MORC6-NLUC-BamHI-F CGGTACCCGGGATCCaATGAGTCACGATAGA Amplify MORC6 cdna MORC6-NLUC-SalI-R ACGAGATCTGGTCGACCGTATTTACATTTCTTCTGTGCT CG for split-luc SWI3A-NLUC-BamHI-F CGGTACCCGGGATCCaATGGAAGCCACTGATC Amplify SWI3A cdna for SWI3A-NLUC-SalI-R SWI3A-CLUC-BamHI-F ACGAGATCTGGTCGACTTTCACGTACGTATGAT CGGTACCCGGGATCCaATGGAAGCCACTGATC split-luc
SWI3A-CLUC-SalI-R AGCTCTGCAGGTCGACTCATTTCACGTACGTAT SWI3B-NLUC-BamHI-F CGGTACCCGGGATCCaATGGCCATGAAAGCTC Amplify SWI3B cdna for SWI3B-NLUC-SalI-R SWI3B-CLUC-BamHI-F SWI3B-CLUC-SalI-R ACGAGATCTGGTCGACACACTCTATTCTATCT CGGTACCCGGGATCCaATGGCCATGAAAGCTC AGCTCTGCAGGTCGACCTAACACTCTATTCT split-luc SWI3C-NLUC-BamHI- F CGGTACCCGGGATCCaATGCCAGCTTCTGAA Amplify SWI3C cdna for SWI3C-NLUC-SalI-R SWI3C-CLUC-BamHI- F SWI3C-CLUC-SalI-R ACGAGATCTGGTCGACGTTTAAGCCTAAGCC CGGTACCCGGGATCCaATGCCAGCTTCTGAA AGCTCTGCAGGTCGACTTAGTTTAAGCCTAAGC split-luc SWI3D-NLUC-BamHI-F CGGTACCCGGGATCCaATGGAGGAAAAACGAC Amplify SWI3D cdna for SWI3D-NLUC-SalI-R SWI3D-CLUC-BamHI-F SWI3D-CLUC-SalI-R ACGAGATCTGGTCGACAACCGAAGAAACATTG CGGTACCCGGGATCCaATGGAGGAAAAACGAC AGCTCTGCAGGTCGACCTAAACCGAAGAAAC split-luc SDC-3C-fix CAATCGGTTTGTTCGGATTAGG 3C experiments SDC-3C-Ⅰ SDC-3C-Ⅱ SDC-3C-Ⅲ SDC-3C-Ⅳ SDC-3C-Ⅴ SDC-3C-Ⅵ SDC-3C-Ⅶ SoloLTR-3C-fix SoloLTR-3C-Ⅰ SoloLTR-3C-Ⅱ CGATGTGAAAGATCCGCTGAC GCCGCATTACATGGCTCCAA GGGTGTGCTCCAAATGATTG TGGTCTAGGCTACTACCTAG TGCCCATAACAATCGGAC GGTCTTCAGTCTTGTGTTAGG CGAGTTGGAGAATGTAGATTCC ATATTCGAGGGTGACAGCGT GAATCAGTTTGGCAATGTCG AGGACCGCTAGAAGATTGAG TE84-F CCAACTCAAGATGACCTCCA Real-time PCR for TE TE84-R TE947-F TE947-R TE8-F TE8-R TE46-F TE46-R TE487-F TE487-R TE497-F TE497-R CAGTGGTGGTTCCCTTATCT GGTAGAGCAACAATGGAGAG CTTCTTCCTCCTCTCACCTA TTGCCCAAGCATCTGTGATC CCGCCTGCATTTCTGACTAC GAGGTTGAGGGAAGTTGGTT GTGCATCTGCTCTGGAGTTT CATCGCCGAAAGTGACTGTT GCATTACGAAGAGCTAGATGAG CGGTGCAATACGTAAACCAC CCACAATTGATGAGCATCTGAG transcripts 3