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

Supplemental figure S1PMIPS1 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 S3Distribution 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+ GFP+

GFP- GFP- GFP-

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WCL MIP

GFPIP

IgGMIP

GST

Col PMIPS1::MIPS1-GFP ATXR6-GST

Anti-GFP

Anti-GST

80 kD

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Supplemental figure S4

Supplemental figure S4MIPS1-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).

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ATXR6 activity

ATXR6 inhibition assay

ATXR6 / IP_IgGATXR6 / IP_MIPS1

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ATXR6 quantity

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Supplemental figure S5

Supplemental figure S5MIPS1 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).

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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

mips1-1PMIPS1::GUS

mips1-1PMIPS1::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

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ddm1PMIPS1::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.

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H3K27me1

H3K9acCol

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Supplemental figure S9

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Supplemental figure S9A. 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.

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0.8Total H3

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Supplemental figure S10Total 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 name DNA sequence (5' to 3')1-for GGACATAATCAAGCGTACGAGA1-rev CATTTTGTATAAAGGGTGATGTTGA2-for CGAGAGCCAAGATCACACGTTT2-rev TCAGATTTGGGTGTGGCATGTT3-for AGGTACCTTGAAATCATCGGCA3-rev TGGGTTTGACACGTGAATTACCTT4-for CCAAACGACCGTTAACACCGAT4-rev TGTCAATTGGTCGCCGTAGGAA5-for AGGTCCCATGTTGACGAAAC5-rev CATTTTTGTGGGGAACGTTAG6-for TCTATCTCCCTTGCTGTCTCGTCT6-rev TCGGAATTTGGATCGGTTTTGGGG7-for GACCGTCAATGGCACTTACC7-rev GACACGAATATCGGTTTTGAAAT8-for GAAGAGATCTATGCTCCTTTCAAGA8-rev AAAAGCTAGCTAATCACCAACCA9-for TCATGGAGTCTGTGGATAGGG9-rev CACAAGCAATCGCATAAAGTG10-for GGGCTATGCTGGAGAATATTATGA10-rev ATTCATGCTTCTTCATGTTCACTTGUS_for TGCTGTCGGCTTTAACCTCTGUS_rev TGAGCGTCGCAGAACATTAC

Supplemental table I : List of primers used in this study.