mips1-1/35S::MIPS1- RFP/PMIPS1::MIPS1-GFP mips1-1/PMIPS1::MIPS1- GFP mips1-1/35S::MIPS1- RFP/PMIPS1::MIPS1-GFP mips1-1/PMIPS1::MIPS1- GFP GFPTransmission Supplemental figure S1 PMIPS1 required MIPS1 to be activated. mips1 mutant plantlets were transiently transformed with both PMIPS::MIPS-GFP and 35S::MIPS-RFP or with PMIPS::MIPS-GFP alone. Picture was obtain by using Confocal microscopy.

Supplemental figure S2 Supplemental figure 2: NetNES prediction, which is the output after submission of MIPS1 amino acid sequence. The prediction server calculates the NES score from the HMM and Artificial Neural Network (ANN) scores and all three values are given for each residue.

Supplemental figure S3 Distribution of MIPS1 expression in leaf and root tissues. Plants were transformed with a construct allowing the expression of GFP fused to a nuclear envelope targeting signal downstream of the MIPS1 promoter (MIPS1::NTF-GFP). A: Observation of GFP distribution in roots: GFP fluorescence is observed mainly in the stele and in cortical cells close to the vasculature. B: to quantify the proportion of cells expressing MIPS1 in roots and leaves, we used flow-cytometry. Nuclei were extracted in Galbraith buffer, and analyzed using a MoFlo flow cytometre to monitor Propidium Iodide (PI) fluorescence and GFP fluorescence simultaneously. Whole wild-type plants were used as a control to establish the gate for GFP positive nuclei. GFP fluorescence was observed in 30% of root cells, and 95% of leaf cells. Supplemental figure S3 MIPS1::NTF-GFP GFP transmission MIPS1::NTF-GFP Root MIPS1::NTF-GFP Rosette leaves Col0 GFP+ GFP- A B

WCLM IP GFP IP IgGM IP GST Col PMIPS1::MIPS1-GFPATXR6-GST Anti-GFP Anti-GST 80 kD 50 kD Supplemental figure S4 MIPS1-GFP and GST-ATXR6 interact in vitro. GST-tagged ATXR6 purified from E. coli was mixed with proteins extracted from plants expressing a MIPS1-GFP fusion. Immunoprecipitation was performed using either anti-GFP antibodies or anti-IgG antibodies as negative controls, and immunoprecipitates were analyzed with anti-GFP and anti-GST antibodies. As positive controls, we respectively used WCL (whole cell lysate) of PMIPS1::MIPS1-GFP for MIPS1-GFP and a direct GST immunoprecipitation from the GST-ATXR6 protein production (white arrow indicates MIPS1, black arrow indicates ATXR6).

ATXR6 activity ATXR6 inhibition assay ATXR6 / IP_IgG ATXR6 / IP_MIPS1 ATXR6 activity in arbitrary units ATXR6 quantity ATXR6 activity in arbitrary units Immunoprecipitate quantity A B Supplemental figure S5 MIPS1 could inhibit ATXR6 activity in vitro. ATXR6 was produced in vitro and we measured specifically its HMT activity alone (A) or supplemented with MIPS1-GFP immunoprecipitated from transgenic plant (B).

MIPS1 Colatrx5/6mips1-1 Relative expression Supplemental figure S6 Supplemental figure S6: MIPS1 mRNA accumulation is decreased in atxr5/6 double mutants. Real-time PCR quantification of the MIPS1 mRNA accumulation was performed using Col0 and mips1-1 as controls. Error bars represent sd values from at least three repetitions.

Supplemental figure S7 mips1-1 PMIPS1::GUS mips1-1 PMIPS1::GUS Zebularine -+ Supplemental figure 7: Spatial expression pattern of MIPS1 promoter in mips1- 1 treated or not with zebularine 50µM. Promoter activity was visualized via GUS staining.

Supplemental figure S8 Col PMIPS1::GUS ddm1 PMIPS1::GUS#1 uidA Supplemental figure S8: Real-time PCR quantification of the uidA reporter expression driven by MIPS1 promoter in the wild type and ddm1-2.

H3K27me1 H3K9ac Col Col+flg22 Col Col+flg22 Supplemental figure S9 %of input Supplemental figure S9 A. Acetylation of H3K9 decreased in the MIPS1 promoter after flg22 treatment. H3K9ac state at the whole MIPS1 locus was analyzed by ChIP on chromatin extracted from Col0 treated or not with flg22 using an anti-H3K9ac antibody. t. Error bars represent sd values from at least three repetitions. B. Methylation of H3K27 increases in the MIPS1 promoter after flg22 treatment. H3K27me1 state at the whole MIPS1 locus was analyzed by ChIP on chromatin extracted from Col0 treated or not with flg22 using an anti-H3K27me1 antibody. Error bars represent sd values from at least three repetitions. A B

Total H3 Col mips1-1 Col+flg22 %of input Supplemental figure S10 Total Histone H3 occupancy on MIPS1 locus. The total H3 at the whole MIPS1 locus was analyzed by ChIP on chromatin extracted from mips1-1 and Col0 treated or not with flg22 using an anti-H3 antibody. Error bars represent sd values from at least three repetitions.

Primer nameDNA sequence (5' to 3') 1-forGGACATAATCAAGCGTACGAGA 1-revCATTTTGTATAAAGGGTGATGTTGA 2-forCGAGAGCCAAGATCACACGTTT 2-revTCAGATTTGGGTGTGGCATGTT 3-forAGGTACCTTGAAATCATCGGCA 3-revTGGGTTTGACACGTGAATTACCTT 4-forCCAAACGACCGTTAACACCGAT 4-revTGTCAATTGGTCGCCGTAGGAA 5-forAGGTCCCATGTTGACGAAAC 5-revCATTTTTGTGGGGAACGTTAG 6-forTCTATCTCCCTTGCTGTCTCGTCT 6-revTCGGAATTTGGATCGGTTTTGGGG 7-forGACCGTCAATGGCACTTACC 7-revGACACGAATATCGGTTTTGAAAT 8-forGAAGAGATCTATGCTCCTTTCAAGA 8-revAAAAGCTAGCTAATCACCAACCA 9-forTCATGGAGTCTGTGGATAGGG 9-revCACAAGCAATCGCATAAAGTG 10-forGGGCTATGCTGGAGAATATTATGA 10-revATTCATGCTTCTTCATGTTCACTT GUS_forTGCTGTCGGCTTTAACCTCT GUS_revTGAGCGTCGCAGAACATTAC Supplemental table I : List of primers used in this study.