Current Biology, Volume 22

Supplemental Information

DNA Methylation Dynamics

during Sexual Reproduction

in Arabidopsis thaliana

Pauline E. Jullien, Daichi Susaki, Ramesh Yelagandula, Tetsuya Higashiyama,

and Frédéric Berger

Supplemental Inventory

Supplemental Figures and Tables Figure S1, related to Figure 1 Figure S2, related to Figure 2 Figure S3, related to Figure 3 Table S1, related to Figure 1 Table S2, related to Figures 1–4

Supplemental Experimental Procedures

Supplemental References

Figure S1.

Figure S1. Characterization of Expression of DNA Methyltransferase Expression during

Female Gametophyte Development

(A) pDRM2-DRM2:GFP and pMET1-MET1:RFP expression during female gametophyte development (Stages are indicated according to [1]). The name of the construct is indicated in each picture. oi (ovule integuments), an (antipodal cells), cc (central cell), ec (egg cell). Scale bars = 20 μm.

(B) Complementation of the met1-3 mutant by the pMET1-MET1:RFP construct. Bisulfite sequencing of FWA loci in six independent plants homozygote for both the mutation and the transgene (DNA was extracted from leaves). It is important to note that the pMET1-MET1:RFP construct is prone to silencing, leading to loss of complementation over successive generations.

(C) Complementation of the drm2-2 mutant by the pDRM2-DRM2:GFP construct. RT-PCR of AtSN1 on eight independent lines. AtSN1 is ectopically expressed in leaves of drm2-2 mutant but not in the drm2-2 mutant carrying the transgene.

(D) Expression of non canonical DNA methyltransferases in mature ovules. Confocal sections Scale bars = 20 μm.

(E) Complementation of the cmt3-11 mutant by the pCMT3-CMT3:CFP construct. Bisulfite sequencing of AtSN1 loci in three independent lines homozygote for the mutation and the transgene (DNA was extracted from leaves). Recovery of wild-type CHG methylation can be seen for line#39 but not for #28 and #46. At least 14 clones were sequenced. Line #39 was used for imaging.

(F) DRM1 expression in seeds and roots. Confocal sections showing lack of pDRM1-DRM1:GFP in both seed and root. Roots are labeled with PI to outline the cell wall. Scale bars = 20 μm.

(G) Quantification of pMET1-MET1:RFP fluorescence. Fluorescence intensity was measured using ImageJ. LIG1GFP was used to mark all nuclei. EC-Egg Cell nucleus, BkEC/Bk2-Background around EC, CC-Central Cell nucleus, BkCC/Bk1-Background around CC, T1-Integument nucleus.

(H) Quantification of pCMT3-CMT3:CFP fluorescence. Fluorescence intensity was measured using ImageJ. HISTONE2A.10:RFP (HTA10RFP) was used to mark all nuclei. EC-Egg Cell nucleus, BkEC-Background around EC, CC-Central Cell nucleus, BkCC-Background around CC, T2-Integument nucleus.

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Figure S2. Characterization of DNA methyltransferase Expression in Sporophytic Tissues

(A) Expression of DNA methyltransferases in developing embryos. Confocal sections of globular, heart and early torpedo stage embryos expressing the reporters for DNA methyltransferases. The embryo stage is indicated above the figure and the name of the different fluorescent reporter constructs on the side of the figure. Scale bars = 10 μm.

(B) Expression of DNA methyltransferases in roots. Confocal sections of root tips expressing reporter for each DNA methyltransferase. Each square is further magnified below each corresponding section to show nuclear localization. Roots were co-labeled with propidium iodide (PI) to outline the cells for GFP and CFP fusions but not for RFP fusion. Scale bars = 25 μm.

(C) LCM microarray data extracted from http://seedgenenetwork.net/ showing expression levels of MET1, DRM2, DRM1 and CMT3 in the pre-globular embryo in comparison to peripheral endosperm. The y axis represents the Average Signal Intensity.

(D) RNA Deep sequencing expression data of dissected embryo from (25).

Figure S3. Raw Data of Bisulfite Sequencing during Embryo Development Shown

in Figure 3

(A) Raw data from the bisulfite analysis presented in Figures 3A - 3C including the number of CG, CHG and CHH analyzed and the number of clones sequenced.

(B) Details of bisulfite analysis for two data points: AtSN1 in globular Columbia embryo and in Columbia rosette leaves. Filled circles indicate methylated cytosine and empty circles unmethylated cytosine. CG site are represented in red, CHG in blue and CHH in green.

(C and D) Raw data from the bisulfite analysis presented in Figures 3D (C) and 4E (D) including the number of CG, CHG and CHH analyzed and the number of clones sequenced.

Table S1. Profiling of Transcripts of Genes Encoding DNA Methyltransferases in Egg and

Synergid Cells

total

seq reads 14187146 15475888 20136800 25231163

mapped reads 3489742 8274972 1711275 2273916

mapped reads reads per 1million reads

gene id Model_name egg cell synergid

cell

seedling

1

seedling

2

ec

(per

1milli

on

reads)

sy

(per

1milli

on

reads)

seedlin

g1 (per

1millio

n

reads)

seedlin

g2 (per

1millio

n

reads)

MET1 AT5G49160 AT5G49160.1 57 0 83 74 16 0 49 33

MET2a AT4G14140 AT4G14140.1 1 0 6 6 0 0 4 3

AT4G14140.2 1 0 4 5 0 0 2 2

MET2b AT4G08990 AT4G08990.1 6 1 5 7 2 0 3 3

MET3 AT4G13610 AT4G13610.1 0 0 0 0 0 0 0 0

DRM1 AT5G15380 AT5G15380.1 190 3 0 0 54 1 0 0

DRM2 AT5G14620 AT5G14620.1 206 4 18 32 59 1 11 14

DRM3 AT3G17310 AT3G17310.1 31 1 10 16 9 0 6 7

AT3G17310.2 30 0 9 28 9 0 5 12

CMT1 AT1G80740 AT1G80740.1 0 0 0 0 0 0 0 0

CMT2 AT4G19020 AT4G19020.1 16 2 10 11 5 1 6 5

CMT3 AT1G69770 AT1G69770.1 515 1 36 50 148 0 21 22

AT2G05730.1 0 0 0 0 0 0 0 0

AT2G05740.1 0 0 0 0 0 0 0 0

AT2G47210.1 121 41 74 78 35 12 43 34

AT3G22555.1 0 0 0 0 0 0 0 0

AT5G25480.1 13 0 14 19 4 0 8 8

HIS4 AT2G28740 AT2G28740.1 112 208 361 493 32 60 211 217

RPB1 AT4G35800 AT4G35800.1 104 12 77 135 30 3 45 59

RPB5C AT5G57980 AT5G57980.1 14 0 0 1 4 0 0 0

RPB15.9 AT5G09920 AT5G09920.1 112 44 146 214 32 13 85 94

LIG1 AT1G08130 AT1G08130.1 30 10 43 47 9 3 25 21

Table S2. List of Primers Used in This Study

name sequence

pCMT3-AttB1 Cloning GGGGACAAGTTTGTACAAAAAAGCAGGCTTCatggtgcgtggggtacttag

CMT3-AttB2 Cloning GGGGACCACTTTGTACAAGAAAGCTGGGTGTGCAAGCTCGGAAGGAAGAGTTGGC

CMT1-AttB2 Cloning GGGGACCACTTTGTACAAGAAAGCTGGGTGGATTTGATCCTTTGCCTGCATGCAT

pCMT1-AttB1 Cloning GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGGTCGTGGGAAGTGAAGTGT

CMT2-AttB2 Cloning GGGGACCACTTTGTACAAGAAAGCTGGGTGATGAGGAATGGTTTCTTGAAGCTGA

pCMT2-AttB1 Cloning GGGGACAAGTTTGTACAAAAAAGCAGGCTTCAGGGTGCAACCTAGAACACG

pMET1-attB4 Cloning GGGGACAACTTTGTATAGAAAAGTTGtcatggtaaaatgttagttctcgaa

pMET1-attB1r Cloning GGGGACTGCTTTTTTGTACAAACTTGTTTCAAAATCCCTAGTTTCAAAATC

MET1-attB2r Cloning GGGGACAGCTTTCTTGTACAAAGTGGGGatggtggaaaatggggctaaagctg

MET1-attB3 Cloning GGGGACAACTTTGTATAATAAAGTTGCTAGGGTTGGTGTTGAGGAGACTTC

DRM1-AttB1 Cloning GGGGACAAGTTTGTACAAAAAAGCAGGCTGGAGCTAGGATTAGTATCACGTCAA

DRM1-AttB2 Cloning GGGGACCACTTTGTACAAGAAAGCTGGGTGTCTCCTCATATGTCGTGCTTTGCGA

DRM2-AttB1 Cloning GGGGACAAGTTTGTACAAAAAAGCAGGCTTTCTCGTAGGTTAATCCTCATACGC

DRM2-AttB2 Cloning GGGGACCACTTTGTACAAGAAAGCTGGGTGAGATCCTCTCATCCTCGCACGTACC

DRM3-AttB1 Cloning GGGGACAAGTTTGTACAAAAAAGCAGGCTTCAGTTTCTGTGTTTTGTCATGTCGTA

DRM3-AttB2 Cloning GGGGACCACTTTGTACAAGAAAGCTGGGTGCATCATATCTCTGACACGTTTCGTG

MET2a-attB1 Cloning GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGGATCAACTTTATGTTTATGGAGGA

MET2a/2b-attB2 Cloning GGGGACCACTTTGTACAAGAAAGCTGGGTGTGATTGGTGTTGAAGAGAACTCTTG

MET2b-attB1 Cloning GGGGACAAGTTTGTACAAAAAAGCAGGCTTCTATTTTTGCCAGCTCCAAAATGTTA

MET3-AttB1 Cloning GGGGACAAGTTTGTACAAAAAAGCAGGCTTCAGTTTCACATTCAAGAGACTTTGCT

MET3-attB2 Cloning GGGGACCACTTTGTACAAGAAAGCTGGGTATGATGTTGCTCGCTTTGG

FWA-TF Bisulfite AAAGAGTTATGGGYYGAAG

FWA-TR Bisulfite CRRRAACCAAAATCATTCTCTAAACA

FIS2-TF Bisulfite AGGTYYAATYGYATATTTATTTAGGGTTTYGGGT

FIS2-TR Bisulfite TCCTACATTTTAATAAAATATTACTRAATCTAARCA

MEA 5F Bisulfite AAAGTGGTTGTAGTTTATGAAAGGTTTTAT

MEA 3R Bisulfite CTTAAAAAATTTTCAACTCATTTTTTTTAAAAAA

ATSN1 5F Bisulfite GTTGTATAAGTTTAGTTTTAATTTTAYGGATYAGTATTAATTT

ATSN1 3R Bisulfite CAATATACRATCCAAAAAACARTTATTAAAATAATATCTTAA

SDC F1 Bisulfite AATYGAAAAAGTTGGAATGGGYTTGGAGAG

SDC 3R Bisulfite CTCATTCTRCTTTAAACCTCTATACTTATA

AT2G20610 BF Bisulfite GTTGYTGATTATATGAAYYGAGATYTT

AT2G20610 BR Bisulfite TTAATTACAACCATARCCACARTRTTCTC

Supplemental Experimental Procedures

Plant Materials and Growth Conditions The met1–3 line was provided by J. Paszkowski [4]. The pFWA-GFP was kindly provided by T. Kinoshita [32]. The mutant lines drm2-2 (SALK_150863), drm1-2 (SALK_031705), nrpd1b-11 (SALK_029919), nrpd2a-1nrpd2b-1 (SALK_095689, SALK_008535) were provided by the Arabidopsis stock center (www.Arabidopsis.org). Unless otherwise stated all plants were in Columbia (Col-0) wild type background.

After 3 days at 4°C in the dark, seeds were germinated and grown on soil. Plants were maintained in a growth chamber under long days at 20°C (16h light/8h night).

Microscopy FWA-GFP fluorescence was imaged using laser scanning confocal microscopy (Zeiss LSM5 Exciter). The expression pattern of DNA methyltransferases in ovules (Figures 1 and S1) and seeds (Figures 2 and S2A) was observed using a laser scanning confocal microscope Zeiss LSM510. The expression pattern of DNA methyltransferases in roots (Figures S2B) was observed using a laser scanning confocal microscope Leica SP5. Brightness and contrast were adjusted using ImageJ (http://rsbweb.nih.gov/ij/).

Plasmid Construction and Transformation All fragments were amplified by PCR using the KOD-plus- PCR kit (TOYOBO), primer sequences can be found in Table S2 and all plasmids were transformed into wild type Columbia plants. pMET1-MET1:RFP was generated using Multisite Gateway technology (Invitrogen). The MET1 promoter was amplified using pMET1-AttB4 and pMET1-AttB1r primers and then recombined into pDONRP4P1r. MET1 cDNA was amplified using MET1-AttB1 and MET1-AttB2 and recombined into pDONRP1P2. Those two plasmids were then recombined into pAlli-R4R3-Tnos together with a pEN-R2/mRFP/L3. MET2a, MET2b and MET3 were fused to H2B-RFP as previously described for MET1 transcriptional fusion [14]. DRMs full locus reporters were generated by single site Gateway technology (Invitrogen). The full genomic locus was amplify using primers containing attB1 and attB2 adapters and recombine into pDONRP1P2. The LR recombination was done using pMDC107-GW-GFP [2] fusing DRMs loci to mGFP6. CMTs were amplified using primers containing attB1 and attB2 adapters and recombine into pDONRP1P2. The LR recombination was performed using pB7CWG2 ([3], http://gateway.psb.ugent.be/) fusing CMTs locus to eCFP. At least ten transgenic lines were analyzed and showed a consistent pattern of expression of the fluorescent reporter. Three lines were used for further detailed analysis.

Bisulfite Sequencing Before bisulfite treatment gDNA was extracted by CTAB extraction for leaf sample but no DNA extraction was performed for the dissected embryos. Dissected embryos were washed at least six times in water and were ruptured by thaw (95C) and freeze (liquid nitrogen) cycle prior bisulfite treatment. Bisulfite treatment was performed using the EpiTect Bisulfite Kit (Quiagen) for leaf samples and with the EZ DNA Methylation-Direct™ Kit (Zymo) for dissected embryos. Bisulfite treated DNA was then amplified using the HotStarTaq DNA Polymerase (Quiagen). Efficiency of bisulfite conversion was controlled using the unmethylated control sequence At2g20610. Primers used are listed in Table S2. PCR fragments were purified and cloned into pGEM-t-easy (Promega) and individual colonies were sequenced using SP6 or T7 primers. Single clone sequences were selected using the BISMA web interface (http://biochem.jacobs-university.de/BDPC/BISMA/,[4]). Sequences were then exported and analyzed with Kismeth software (http://katahdin.mssm.edu/kismeth/revpage.pl, [5]). Raw data can be found in Figures

S3. The results shown were obtained from at least two or more independent bisulfite experiments.

RNA Sequencing mRNA were extracted from each isolated egg and synergid cells of 30 as described previously [6]. Amplifications of mRNAs were performed using MessageAm II aRNA Amplification Kit (Ambion) following the 2

nd round protocol. Paired-end libraries were generated with mRNA-Seq

Sample Prep Kit (Illumina) and Multiplexing Sample Preparation Oligonucleotide Kit (Illumina), and selecting an average insert size of ~400 bps following manufacturer’s protocols. Sequencing was carried out 100 bps with a Genome Analyzer IIx using standard reagents. The transcriptome of egg and synergid cells were compared to publicly available RNA-seq transcriptome data from seedlings of Arabidopsis thaliana (SRR346552, SRR346553, http://trace.ddbj.nig.ac.jp/DRASearch/SRR~[7]). 50 bps of all reads were mapped to 250 bps at the 3’end of all annotated transcripts (TAIR10, www.arabidopsis.org).

Supplemental References

1. Christensen, C.A., King, E.J., Jordan, J.R., and Drews, G.N. (1997). Megagametogenesis in Arabidopsis wild type and the Gf mutant. Sex Plant Reprod. 10, 49-64.

2. Curtis, M.D., and Grossniklaus, U. (2003). A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol 133, 462-469.

3. Karimi, M., De Meyer, B., and Hilson, P. (2005). Modular cloning in plant cells. Trends Plant Sci 10, 103-105.

4. Rohde, C., Zhang, Y., Reinhardt, R., and Jeltsch, A. (2010). BISMA--fast and accurate bisulfite sequencing data analysis of individual clones from unique and repetitive sequences. BMC Bioinformatics 11, 230.

5. Gruntman, E., Qi, Y., Slotkin, R.K., Roeder, T., Martienssen, R.A., and Sachidanandam, R. (2008). Kismeth: analyzer of plant methylation states through bisulfite sequencing. BMC Bioinformatics 9, 371.

6. Ikeda, Y., Kinoshita, Y., Susaki, D., Ikeda, Y., Iwano, M., Takayama, S., Higashiyama, T., Kakutani, T., and Kinoshita, T. (2011). HMG domain containing SSRP1 is required for DNA demethylation and genomic imprinting in Arabidopsis. Dev Cell 21, 589-596.

7. Schmid, M.W., Schmidt, A., Klostermeier, U.C., Barann, M., Rosenstiel, P., et al. (2012). A Powerful Method for Transcriptional Profiling of Specific Cell Types in Eukaryotes: Laser-Assisted Microdissection and RNA Sequencing. PLoS ONE 7, e29685. doi:10.1371/journal.pone.0029685.