Supplemental Data. Lermontova et al. (2013). Plant Cell /tpc

Transcription

1 Supplemental Figure 1. Multiple alignment of the N-terminal parts of plant KNL2 orthologs. Multiple sequence alignment was performed by the MUSCLE method and visualized by the JalView program. Highly conserved amino acids are highlighted in dark blue and less conserved ones in light blue. 1

2 Supplemental Figure 2. Evolutionary relationships of KNL2 protein of 25 taxa. The evolutionary history was inferred using the Neighbor-Joining method (Saitou and Nei, 1987). The optimal tree with the sum of branch length = is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) is shown next to the branches (Felsenstein, 1985). The evolutionary distances were computed using the Poisson correction method (Zuckerkandl and Pauling, 1965) and are in the units of the number of amino acid substitutions per site. All positions containing gaps and missing data were eliminated from the dataset (Complete deletion option). There were a total of 47 positions in the final dataset. Phylogenetic analyses were conducted in MEGA4 (Tamura et al., 2007). - SANTA domain containing proteins of plants, - of Arabidopsis species; - SANTA+KIP domain containing proteins of plants, - of Arabidopsis species. 2

3 3

4 Supplemental Figure 3. FLIP and FRAP on 10-day-old seedlings expressing EYFP- KNL2-C. Roots of 10-day-old seedlings expressing EYFP-KNL2-C were used for FLIP and FRAP analysis. (A) For measuring FLIP, individual nuclei were scanned three times with a 488 nm laserline (2,5% laserpower, scanspeed 6 without averaging) followed by repeated bleaching of a square region of interest within the nucleus measuring 1,4 µm 2, using 100% laserpower and 4 iterations alternated by single recordings. (B) For measuring FRAP, individual nuclei were scanned three times with a 488 nm laserline (2.5% laserpower, scanspeed 6 without averaging). After this a square region of interest measuring 1.4 µm 2 was bleached using 100% laserpower and 4 iterations followed by 50 continuous recordings. Each experiment was run over the time scale of 40 sec, and 5 10 experiments were averaged to produce each curve. KNL2-C recovers to about 80% fluorescence intensity at chromocenters within 10 sec. For both FLIP and FRAP experiments fluorescence intensity was measured in the regions of bleaching (1), and in regions adjacent to the ROI (2, 3). The x axis represents the time scale of the experiment in second, the y axis corresponds to fluorescence intensity (arbitrary units). 4

5 Supplemental Figure 4. Schematic view of the KNL2 gene with the corresponding T- DNA insertions and analysis of KNL2 transcript in SALK mutant. (A) Positions of T-DNA insertions are shown by vertical arrows. The positions of primers used for the RT-PCR analysis are marked by horizontal arrows. (B) RT-PCR analysis (30 cycles) of KNL2 expression in heterozygous (5/6, 5/7) and homozygous (8/1, 8/2) lines. Amplification of products of elongation factor (EF) mrna was used as loading control. 5

6 Supplemental Figure 5. Gene ontology enrichment analysis on A. thaliana genes selected from regulatory network using AgriGO web application. Hierarchical tree graph of overrepresented Gene Ontology (GO) terms in biological process category generated by SEA (Singular enrichment analysis) for genes selected from the gene regulatory network for cenh3 deposition. All genes are listed in Suppl. Table 3. Boxes in the graph represent GO terms labeled by their GO ID, term definition and statistical information. The significant terms (adjusted P 0.05) are marked with color, while non-significant terms are shown as white boxes. The diagram, the degree of color saturation of a box is positively correlated to the enrichment level of the term. Solid, dashed, and dotted lines represent two, one and zero enriched terms at both ends connected by the line, respectively. The rank direction of the graph is set to from top to bottom. 6

7 Supplemental Figure 6. Protein-Protein Interaction network of selected human proteins in STRING. Colored lines indicate different types of the supporting evidences including direct (physical) and indirect (functional) associations from different sources: conserved co-expression, experimental data and previous knowledge (publications and databases). All underlying evidence can be inspected in dedicated viewers that are accessible from the network. 7

8 Supplemental Table 1A: List of selected A. thaliana genes involved in regulation of cenh3 assembly and their homologues of H. sapiens. Last column shows sequence similarity of corresponding proteins. Arabidopsis thaliana Homo sapiens Protein sequence similarity (%) Gene name Gene Nr Gene name Accession Nr CenH3 At1G01370 CENPA AAH % KNL2 At5G02520 Mis18BP1 NP_ % E2F1 At5G22220 E2F1 AAH % E2F2 At1G47870 E2F2 AAM % E2F3 At2G36010 E2F3 CAI % RBR At3G12280 retinoblastoma-like 1 AAH % SuvH4 At5G13960 SUV39H2 NP_ % Met1 At5G49160 DNMT1 NP_ % 8

9 Supplemental Table 1B: H. sapiens proteins involved in cenh3 assembly and their interacting partners. Score above 0,400 is defined as medium confidence and above 0,700 - as high. Protein Inter. Partner Exp./Bioch. Data (Score) Source CENPA HJURP 0,798 Dunleavy et al. 2009; Foltz et al RBBP4 0,522 Dunleavy et al HJURP CENPA 0,798 Dunleavy et al. 2009; Foltz et al RBBP4 CENPA 0,522 Dunleavy et al RB1 0,981 Qian and Lee 1995 RBBP4 SUV39H1 0,538 Vaute et al E2F1 0,543 Nicolas et al RB1 E2F1 0,999 Helin et al 1992 E2F2 0,987 Wu et al DNMT1 0,845 Robertson et al SUV39H1 0,845 Nielsen et al. 2001; Vandel et al E2F3 0,998 Lees et al RBBP4 0,981 Qian and Lee 1995 E2F1 RB1 0,999 Helin et al 1992 RBBP4 0,543 Nicolas et al DNMT1 0,620 Robertson et al E2F2 RB1 0,987 Wu et al E2F3 RB1 0,998 Lees et al SUV39H1 RBBP4 0,538 Vaute et al RB1 0,845 Nielsen et al. 2001; Vandel et al DNMT1 0,812 Fuks et al DNMT3B 0,620 Geiman et al DNMT1 RB1 0,845 Robertson et al SUV39H1 0,812 Fuks et al DNMT3B 0,814 Kim et al E2F1 0,620 Robertson et al DNMT3B DNMT1 0,814 Kim et al SUV39H1 0,620 Geiman et al

10 Supplemental Table 2: Primers used in this study. Primer name Primer sequence Reference Gateway cloning KNL2-attB1l KNL2-attB1sh KNL2-attB2 KNL2- attb1gensh KNL2prexin-attB2 GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGACGGAACCAAATCTCGAC GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGAATTACTCTGGGACGAAAG GGGGACCACTTTGTACAAGAAAGCTGGGTCTTTGATTTTCAAGTTTCTTCG GGGGACAAGTTTGTACAAAAAAGCAGGCTTCAACTATATGATTGTTTACTAC GGGGACCACTTTGTACAAGAAAGCTGGGTCCTATTAAGGCAAAATTCGAAG RT-PCR qknl2_2081f qknl2_2297r qknl2_27f qknl2_198r qknl2_607f qknl2_799r actin2-rev qcenh-3 -r actin2-for qcenh3-3 -f ATTGGGACAGAAACGGTCAA TCTGTTCCCATGGTTGGTCT TGGTTCCAAGTCGTCTTTCC TTTCCCTTCGAATTCCTTTG CCTTGTTGGGAACGAGTTTG GGGAGGACAAGCTGCTAAGA CAAGAGGCGGCAGAAGATTAC AACGATTCCTGGACCTGCCTC TCCCTCAGCACATTCCAGCAG GCATCACCAAGAGACAAGGAG Analysis of T-DNA insertion mutants SALK_ LP LBb1.3 SALK_ RP TAATACCACTTCCAACCGCTG ATTTTGCCGATTTCGGAAC TTTGTTCATCTGGGTTTTTCG Bisulfite sequencing MEA-ISR-5F MEA-ISR-3R AtSN1-3R AtSN1-5F AAAGTGGTTGTAGTTTATGAAAGGTTTTAT CTTAAAAAATTTTCAACTCATTTTTTTTAAAAAA CAATATACRATCCAAAAAACARTTATTAAAATAATATCTTAA GTTGTATAAGTTTAGTTTTAATTTTAYGGATYAGTATTAATTT Zheng et al., 2007 Zheng et al., 2007 Zheng et al., 2007 Zheng et al.,

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