SUPPLEMENTARY RESEARCH DATA

Transcription

1 SUPPLEMENTARY RESEARCH DATA Supplementary Experimental Procedures Small RNA Analysis RNA was isolated using the mirvana mirna isolation kit (Ambion). Six µg of RNA was resolved by electrophoresis using a 15% (w/v) denaturing polyacryamide gel. Gels were electroblotted on to Nytransupercharge nylon membranes (Schleicher and Schuell Bioscience) using a semidry transfer apparatus. The CEN-DS hybridization probe was synthesized as described above. The CEN-W, CEN-C, mir163 and mir167 riboprobes were generated according to the mirvana probe construction kit (Ambion) and labeled by T7 polymerase transcription in the presence of α- 32 P UTP. DNA oligonucleotides used for construction of riboprobes are presented in Supplementary Table 4. The hybridizations for the CEN probes were performed using PerfectHyb TM plus buffer (Sigma). In the case of mirna probes, the hybridization was done in 50% formamide, 0.25 M Na 2 HPO 4 (ph 7.2), 0.25 M NaCl, 7% SDS at 40 C (14 16 hr). In both cases, the hybridization step was followed by a 5 min wash in 2X SSC/0.1% SDS, a 15 min wash in 1X SSC/0.1% SDS, and a 15 min wash in 0.5X SSC/1% SDS at C. Molecular markers and mapping procedure We mapped the VIM1 locus using segregating F2 populations. Genomic DNA was prepared from 628 F2 plants between Ler and Bor-4 and genotyped with molecular markers. Published PCR-based molecular markers ( were used for the initial mapping. Higher resolution mapping required the development of new markers based on Col genomic sequence, which were then tested empirically for suitable polymorphisms in the parental genotypes. Primer sequences for these markers are listed in Supplementary Table 4.

2 Supplementary Table 1. Interphase organization of HTR12 and 180-bp repeats (CEN) in root meristematic cells of Bor-4, Col, Col ddm1-2 and Col vim1-2 (SALK_050930) Immunolocalization / FISH signal Genotype HTR12 Large Large Small Small Decondensed CEN Condensed Weaker Decondensed Small Decondensed # nuclei Bor-4 9% 0% 70% 16% 5% 151 Col 72% 0% 10% 3% 15% 93 Col ddm1-2 10% 58% 8% 7% 17% 49 Col vim1-2 7% 0% 75% 15% 3% 128 Supplementary Table 2. The VIM1g-FLAG transgene complements the HTR12 interphase organization phenotype of Bor-4 in root tip cells. Genotype Immunolocalization Signal HTR12 Large Small Decondensed # nuclei Bor-4 16% 73% 11% 90 Col 75% 11% 14% 67 Bor-4 (VIM1g-FLAG) 67% 21% 12% 58

3 Supplementary Table 3. VIM1 interphase localization pattern in a Bor-4 (VIM1g-FLAG) transgenic line FLAG Diffuse nuclear staining; excluded from CC Immunolocalization Signal a Colocalized with all CC Colocalized with the majority of CC # nuclei Tissue Roots 24% 23% 53% 173 Leaves 34% 0% 66% 51 a CC, chromocenters; defined by DAPI staining and HTR12 or CEN localization

4 Supplementary Table 4. Oligonucleotide primers used in this study Name Sequence (5 3 ) Description a Reference b CIW1 F25P12-A T8L23-A T8L23-B F12K22-A T15M6-A F19C14-A m305 1: ACATTTTCTCAATCCTTACTC 2: GAGAGCTTCTTTATTTGTGAT 1: ACATACACTTATTTGACTGTCCA 2: CTTTTAGGTATTACACTGGTATT 1: TGAACTGAACCAAATTGCAGAATTG 2: CTAATCCAGATAAAACCACAATGTG 1: AGGTGTAAGAAATAGAGCCATGA 2: TTGATTTTCTTGAGATGATATTGTA 1: AAGTTAGCGTGGTGGTTTTGTTTTT 2: TTGTAAGAAATTCCTTAGAATATCC 1: GAGAGAGATGATTTTAATAGGTAACC 2: CAGACTGGAGGCTATTGTTGAAC 1: AGTTCTTCCATGACGCGGTGGAT 2: TTTTAATTTCCATTACGCTGGTCCT 1: TGAAGCTAATATGCACAGGAG 2: TTCTCCAGACCACATGATTAT SSLP 1 SSLP CAPS, DraI CAPS, MboI CAPS, TaqI SSLP CAPS, MseI This study This study This study This study This study This study CAPS, HaeIII 2 A ATGGCGCGTGACATCCAACTCCCC Genomic PCR This study B GGATACAGGAAACTAACCTGCTG Genomic PCR This study C CGGTCCCCTGTTGCAAGCCTATT Genomic PCR This study F9M8-F CCTTCTACACCTAAAAACATACAAAAACAT Bisulfite sequencing This study F9M8-R TTAAGTGAATAATTTAGGTAAAGTGTTGG Bisulfite sequencing This study CEN-W CEN-C GGAAATACTACTTAGGCTTTTAAGATGCGGTTGCG TTTTAAGTTCTTATACTCAATCATACACCTGTCTC TGTATGATTGAGTATAAGAACTTAAAACGCAACCG CATCTTAAAAGCCTAAGTAGTATTTCCCCTGTCTC Riboprobe synthesis 3 Riboprobe synthesis 3 mir163t7 TTGAAGAGGACTTGGAACTTCGATCCTGTCTC Riboprobe synthesis 3 mir167t7 TGAAGCTGCCAGCATGATCTACCTGTCTC Riboprobe synthesis 3 CEN-F (JP1623) CEN-R (JP1624) ACCATCAAAGCCTTGAGAAGCA ChIP 4 CCGTATGAGTCTTTGTCTTTGTATCTTCT ChIP 4 5S rrna-f GGATGCGATCATACCAGCACT ChIP This study 5S rrna-r GAGGGATGCAACACGAGGACT ChIP This study Athila-F GAATCATCTATGGATGCCGACTAC ChIP 4 Athila-R ATGACTTAGATGATATGCAGACTC ChIP 4 T5L23.29-F CTCGATGTCGTATTCGCTGA ChIP 5 T5L23.29-R GCAACCTATCAACGCTTCGT ChIP 5 At4g04040-F GCCACGAAAACCAAACAGAC ChIP 5 At4g04040-R CCGGAATTTCGATCAATCCT ChIP 5 a SSLP, simple sequence length polymorphism ~ microsatellite marker; CAPS, cleaved amplified polymorphic sequence marker (Konieczny and Ausubel 1993)

5 b References for Supplementary Table 4 1. Gillmor, S Cao, H., & Baumbusch, L P. Costa Nunes & C.S. Pikaard, personal communication 4. Lindroth et al. (2001) Science 292: Gendrel et al. (2002) Science 297: References Alonso, J.M., A.N. Stepanova, T.J. Leisse, C.J. Kim, H. Chen, P. Shinn, D.K. Stevenson, J. Zimmerman, P. Barajas, R. Cheuk, C. Gadrinab, C. Heller, A. Jeske, E. Koesema, C.C. Meyers, H. Parker, L. Prednis, Y. Ansari, N. Choy, H. Deen, M. Geralt, N. Hazari, E. Hom, M. Karnes, C. Mulholland, R. Ndubaku, I. Schmidt, P. Guzman, L. Aguilar-Henonin, M. Schmid, D. Weigel, D.E. Carter, T. Marchand, E. Risseeuw, D. Brogden, A. Zeko, W.L. Crosby, C.C. Berry, and J.R. Ecker Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301: Konieczny, A. and F.M. Ausubel A procedure for mapping Arabidopsis mutations using co-dominant ecotype-specific PCR-based markers. Plant J 4:

6 Supplementary Figure Legends Supplementary Figure 1. Schematic representation of recombination mapping of the VIM1 locus to a ca. 113-kb interval on chromosome 1 of Arabidopsis. The diagram at the top gives an overview of chromosome 1. The approximate position of the centromere is shown by the black oval. The next line down represents an expanded view of the interval between markers CIW1 and m305. Additional markers are shown above this line and the numbers of recombination breakpoints scored between the vim1 mutation and the markers are indicated below the line. The positions of BAC inserts relative to this region are shown below the recombination map. At the bottom of the figure is shown a physical map of the minimal genetic window containing the vim1 mutation; coordinates are shown in kb. Black boxes indicate predicted genes with ESTs and/or MPSS signatures ( and white boxes indicate presumed hypothetical genes or pseudogenes. The black box highlighted with an asterisk is the VIM1 gene. Supplementary Figure 2. The Bor-4 strain contains a 3.2-kb deletion of A 1g t The top line shows a representation of the annotation in the genomic region including At1g57820 (VIM1). The two genes immediately downstream of At1g57820 are a reverse transcriptase pseudogene (A 1g57810) t and a VIM1 paralog (A 1g57800) t Genomic DNA was isolated from Col, Ler, and Bor-4 plants, and two primer sets (A + B and A + C, see Table S2) were used to amplify the genomic region around A 1g t An EtBr-stained image of the size-fractioned PCR genomic amplification products is shown at the bottom of the figure. Genotypes of the genomic templates and the primer combinations used are shown at the top of the gel image. The open box under the annotation represents the deletion of At1g57820 in Bor-4. The deletion endpoints were confirmed by determining the nucleotide sequence of an amplified Bor-4 PCR product spanning the deletion.

7 Supplementary Figure 3. DNA blot hybridization (HpaII digest, CEN probe) analysis demonstrating a lack of complementation between Col T-DNA insertion (SALK_000930) and Bor-4 At1g57820 alleles. The Col SALK_ allele is recessive to the wild-type Col At1g57820 allele (no ladder pattern in +/- control F1 hybrid), as is the Bor-4 vim1 allele (data not shown). Genotype designations: +/+, homozygous wildtype allele; +/-, heterozygous for mutant allele, -/-, homozygous for mutant allele. Supplementary Figure 4. VIM1 structure and related proteins. (A) The VIM1 protein sequence determined from the Col accession ( Residues constituting the PHD domain, the RING domains, and the SRA domain are underlined. Sequences for putative nuclear localization signals are bolded. Asterisks denote highly conserved amino acid sequences present in each motif. (B) An unrooted tree (neighbor joining, distance) showing the relationship between VIM1 and four Arabidopsis VIM1-like proteins. Proteins encoded by expressed genes are shown in bold. Supplementary Figure 5. Centromeric repeat hypomethylation phenotype in different vim single mutants. Genomic DNA samples from the indicated genotypes were digested with HpaII and sizefractionated DNA was transferred to a membrane and hybridized with a 180-bp centromere repeat probe (CEN). The vim2-1 and vim3-1 alleles correspond to the insertions in SALK_ and SALK_088570, respectively (Alonso et al. 2003). Supplementary Figure 6. The abundance of centromeric repeat sirnas is not significantly changed in the Col vim1-2 mutant. A small RNA blot was hybridized with a radiolabeled double-stranded CEN probe (CEN-DS), as well as strand-specific probes (CEN-W and CEN-C). As a loading control, the filter was rehybridized sequentially with probes recognizing mir163 and mir167. An ethidium bromide (EtBr) stained image of the samples is shown in the bottom row.

8

9 At1g57820 (VIM1) At1g57810 At1g57800 A A B C Size marker Col AB Ler Bor-4 Col AC Ler Bor-4 kb

10 C ol (+/+) B or-4 (-/-) C ol B or-4 S ALK _000930(-/-) S ALK _ (-/-) +/- -/- female male F 1

11 A PHD domain 1 MARDIQLPCDGDGVCMRCKSNPPPEESLTCGTCVTPWHVSCLSSPPKTLA * * * * * * 51 STLQWHCPDCSGEIDPLPVSGGATGFESAGSDLVAAIRAIEADESLSTEE * * 101 KAKMRQRLLSGKGVEEDDEEEKRKKKGKGKNPNLDVLSALGDNLMCSFCM * * RING domain 151 QLPERPVTKPCGHNACLKCFEKWMGQGKRTCGKCRSIIPEKMAKNPRINS * * * * * * 201 SLVAAIRLAKVSKSAAATTSKVFHFISNQDRPDKAFTTERAKKTGKANAA B At5g39550 [VIM3] At1g66050 [VIM2] At1g66040 [VIM4] SRA domain 251 SGKIYVTIPPDHFGPIPAENDPVRNQGLLVGESWEDRLECRQWGAHFPHV 301 AGIAGQSTYGAQSVALSGGYKDDEDHGEWFLYTGSGGRDLSGNKRTNKEQ 351 SFDQKFEKSNAALKLSCKLGYPVRVVRSHKEKRSAYAPEEGVRYDGVYRI 401 EKCWRKVGVQGSFKVCRYLFVRCDNEPAPWTSDENGDRPRPIPNIPELNM 451 ATDLFERKETPSWDFDEGEGCWKWMKPPPASKKSVNVLAPEERKNLRKAI At1g57820 [VIM1] RING domain 501 KAAHSNTMRARLLKEFKCQICQQVLTLPVTTPCAHNFCKACLEAKFAGKT * * * * * * At1g57800 [VIM5] 551 LVRERSTGGRTLRSRKNVLNCPCCPTDISDFLQNPQVNREVAEVIEKLKT * * 601 QEEDTAELEDEDEGECSGTTPEEDSEQPKKRIKLDTDATVSATIR

12 Col vim1-2 vim2-1 vim3-1

13 CEN-DS Col Col vim1-2 (nt) CEN-W CEN-C mir mir EtBr