supplemental figure 1. multiple alignment of the n ... · 1 supplemental figure 1. multiple...
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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.
Supplemental Data. Lermontova et al. (2013). Plant Cell 10.1105/tpc.113.114736
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Supplemental Data. Lermontova et al. (2013). Plant Cell 10.1105/tpc.113.114736
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 = 8.35759732 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.
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Supplemental Data. Lermontova et al. (2013). Plant Cell 10.1105/tpc.113.114736
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Supplemental Data. Lermontova et al. (2013). Plant Cell 10.1105/tpc.113.114736
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 µm2, 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 µm2 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).
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Supplemental Data. Lermontova et al. (2013). Plant Cell 10.1105/tpc.113.114736
Supplemental Figure 4. Schematic view of the KNL2 gene with the corresponding T-
DNA insertions and analysis of KNL2 transcript in SALK-039432 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.
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Supplemental Data. Lermontova et al. (2013). Plant Cell 10.1105/tpc.113.114736
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.
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Supplemental Data. Lermontova et al. (2013). Plant Cell 10.1105/tpc.113.114736
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.
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Supplemental Data. Lermontova et al. (2013). Plant Cell 10.1105/tpc.113.114736
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 AAH02703 52% KNL2 At5G02520 Mis18BP1 NP_060823 56% E2F1 At5G22220 E2F1 AAH50369 46% E2F2 At1G47870 E2F2 AAM54044 45% E2F3 At2G36010 E2F3 CAI21471 56% RBR At3G12280 retinoblastoma-like 1 AAH32247 33% SuvH4 At5G13960 SUV39H2 NP_001180356 41% Met1 At5G49160 DNMT1 NP_001370 37%
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Supplemental Data. Lermontova et al. (2013). Plant Cell 10.1105/tpc.113.114736
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. 2009
RBBP4 0,522 Dunleavy et al. 2009
HJURP CENPA 0,798 Dunleavy et al. 2009; Foltz et al. 2009
RBBP4 CENPA 0,522 Dunleavy et al. 2009 RB1 0,981 Qian and Lee 1995 RBBP4 SUV39H1 0,538 Vaute et al. 2002 E2F1 0,543 Nicolas et al. 2000 RB1 E2F1 0,999 Helin et al 1992 E2F2 0,987 Wu et al. 1995 DNMT1 0,845 Robertson et al. 2000
SUV39H1 0,845 Nielsen et al. 2001; Vandel et al. 2001
E2F3 0,998 Lees et al. 1993 RBBP4 0,981 Qian and Lee 1995 E2F1 RB1 0,999 Helin et al 1992 RBBP4 0,543 Nicolas et al. 2000 DNMT1 0,620 Robertson et al. 2000 E2F2 RB1 0,987 Wu et al. 1995 E2F3 RB1 0,998 Lees et al. 1993 SUV39H1 RBBP4 0,538 Vaute et al. 2002
RB1 0,845 Nielsen et al. 2001; Vandel et al. 2001
DNMT1 0,812 Fuks et al. 2003 DNMT3B 0,620 Geiman et al. 2004 DNMT1 RB1 0,845 Robertson et al. 2000 SUV39H1 0,812 Fuks et al. 2003 DNMT3B 0,814 Kim et al. 2002 E2F1 0,620 Robertson et al. 2000 DNMT3B DNMT1 0,814 Kim et al. 2002 SUV39H1 0,620 Geiman et al. 2004
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Supplemental Data. Lermontova et al. (2013). Plant Cell 10.1105/tpc.113.114736
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_039432-LP
LBb1.3
SALK_039432-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., 2007
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Supplemental Data. Lermontova et al. (2013). Plant Cell 10.1105/tpc.113.114736
Supplemental References
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Foltz, D.R., Jansen, L.E., Bailey, A.O., Yates, J.R., 3rd, Bassett, E.A., Wood, S., Black, B.E., and Cleveland, D.W. (2009). Centromere-specific assembly of CENP-a nucleosomes is mediated by HJURP. Cell 137, 472-484.
Fuks, F., Hurd, P.J., Deplus, R., and Kouzarides, T. (2003). The DNA methyltransferases associate with HP1 and the SUV39H1 histone methyltransferase. Nucleic Acids Res 31, 2305-2312.
Geiman, T.M., Sankpal, U.T., Robertson, A.K., Zhao, Y., and Robertson, K.D. (2004). DNMT3B interacts with hSNF2H chromatin remodeling enzyme, HDACs 1 and 2, and components of the histone methylation system. Biochem Biophys Res Commun 318, 544-555.
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Nicolas, E., Morales, V., Magnaghi-Jaulin, L., Harel-Bellan, A., Richard-Foy, H., and Trouche, D. (2000). RbAp48 belongs to the histone deacetylase complex that associates with the retinoblastoma protein. J Biol Chem 275, 9797-9804.
Nielsen, S.J., Schneider, R., Bauer, U.M., Bannister, A.J., Morrison, A., O'Carroll, D., Firestein, R., Cleary, M., Jenuwein, T., Herrera, R.E., and Kouzarides, T. (2001). Rb targets histone H3 methylation and HP1 to promoters. Nature 412, 561-565.
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Vandel, L., Nicolas, E., Vaute, O., Ferreira, R., Ait-Si-Ali, S., and Trouche, D. (2001). Transcriptional repression by the retinoblastoma protein through the recruitment of a histone methyltransferase. Mol Cell Biol 21, 6484-6494.
Vaute, O., Nicolas, E., Vandel, L., and Trouche, D. (2002). Functional and physical interaction between the histone methyl transferase Suv39H1 and histone deacetylases. Nucleic Acids Res 30, 475-481.
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Zheng, X., Zhu, J., Kapoor, A., and Zhu, J.K. (2007). Role of Arabidopsis AGO6 in siRNA accumulation, DNA methylation and transcriptional gene silencing. EMBO J 26, 1691-1701.
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