Supplementary information Table S1. Cell lines used in...
Transcript of Supplementary information Table S1. Cell lines used in...
Supplemental_Data_Kawasumi
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Supplementary information
Table S1. Cell lines used in this study.
Genotype Selective marker Reference
DT40 WT CL18 (Buerstedde and Takeda 1991)
DDX11-/- DDX11/DDX11::KO-Puro/KO-Bsr (Abe et al. 2016)
DDX11-/-
+ESCO1FLAG DDX11/DDX11::KO-Puro/KO-Bsr, +cESCO1-FLAG::His This study
ESCO1-/-/- ESCO1/ ESCO1/ESCO1::KO-Puro/KO-Bsr/KO-His This study
ESCO1-/-/- DDX11-/- +Cre::Neo, ESCO1/ ESCO1/ESCO1::KO-Puro/KO-Bsr/KO-His (all removed), DDX11/DDX11::KO-Puro/KO-Bsr
This study
ESCO1-/-/- ESCO2-
/3xmAID-6xFLAG + Cre1 + TIR19xmyc
+Cre::Neo, ESCO1/ ESCO1/ESCO1::KO-Puro/KO-Bsr/KO-His (all removed), ESCO2/ ESCO2::KO-Puro/3xmAID-6xFLAG FLP-In His, + TIR1-9xmyc::Bsr
This study
ESCO1-/-/- ESCO2-
/3xmAID-6xFLAG WAPL-
/3xmAID-6xFLAG + Cre1 + TIR19xmyc
+Cre::Neo, ESCO1/ ESCO1/ESCO1::KO-Puro/KO-Bsr/KO-His (all removed), ESCO2/ ESCO2::KO-Puro/3xmAID-6xFLAG FLP-In His, + TIR1-9xmyc::Bsr, WAPL/WAPL::KO-Eco/3xmAID-6xFLAG FLP-In Bleo
This study
ESCO1-/-/-ESCO2-
/3xmAID-6xFLAG SMC3-
/K105 106Q + Cre1 + TIR19xmyc
+Cre::Neo, ESCO1/ ESCO1/ESCO1::KO-Puro/KO-Bsr/KO-His (all removed), ESCO2/ ESCO2::KO-Puro/3xmAID-6xFLAG FLP-In His, + TIR1-9xmyc::Bsr, SMC3/SMC3::KO-Hyg/KI-Bleo
This study
ESCO2-/- + Cre1 +Cre::Neo, ESCO2/ESCO2::KO-Puro /cKO-loxP-Bsr-LoxP (removed) This study
ESCO2-/- + ESCO1FLAG
+Cre::Neo, ESCO2/ESCO2::KO-Puro /cKO-loxP-Bsr-LoxP (removed), +cESCO1-FLAG::His This study
ESCO2-/W615G
DDX11-/- + tetoff-DDX11HA
ESCO2/ESCO2::KO-Bsr/KI-Puro, DDX11/DDX11::KO-Bleo/KO-Hyg, +tet off::Neo, +cDDX11-HA::Eco
(Abe et al. 2016)
ESCO2-/W615G
DDX11-/- + tetoff-DDX11HA + ESCO1FLAG
ESCO2/ESCO2::KO-Bsr/KI-Puro, DDX11/DDX11::KO-Bleo/KO-Hyg, +tet off::Neo, +cDDX11-HA::Eco, +cESCO1-FLAG::His
This study
SMC3-/3xmAID-6xFLAG
+TIR1 9xmyc SMC3/SMC3::KO-His/3xmAID-6xFLAG TIR1-9xMyc FLP-In Bsr This study
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SMC3-/3xmAID-6xFLAG
+TIR1 9xmyc
+SMC3HA
SMC3/SMC3::KO-His/3xmAID-6xFLAG TIR1-9xMyc FLP-In Bsr, +SMC3-HA::Neo This study
SMC3-/3xmAID-6xFLAG +TIR1 9xmyc +SMC3HA K105 106Q
SMC3/SMC3::KO-His/3xmAID-6xFLAG TIR1-9xMyc FLP-In Bsr, +SMC3-HA K105 106Q::Neo This study
SMC3-/3xmAID-6xFLAG
+TIR1 9xmyc
+SMC3HA K38I
SMC3/SMC3::KO-His/3xmAID-6xFLAG TIR1-9xMyc FLP-In Bsr, +SMC3-HA K38I::Neo This study
SMC3-/3xmAID-6xFLAG
WAPL-/3xmAID-6xFLAG
+ Cre1 + TIR19xmyc
SMC3/SMC3::KO-His/3xmAID-6xFLAG TIR1-9xMyc FLP-In Bsr WAPL/WAPL::KO-Eco/3xmAID-6xFLAG FLP-In Bleo
This study
SMC3-/3xmAID-6xFLAG, +TIR1 9xmyc
+SMC3HA K105 106A
SMC3/SMC3::KO-His/3xmAID-6xFLAG TIR1-9xMyc FLP-In Bsr, +SMC3-HA K105 106A::Neo This study
SMC3-/3xmAID-6xFLAG, +TIR1 9xmyc
+SMC3HA K105 106R
SMC3/SMC3::KO-His/3xmAID-6xFLAG TIR1-9xMyc FLP-In Bsr, +SMC3-HA K105 106R::Neo This study
WAPL-/- +tetoff-WAPL
WAPL/WAPL::KO-Bleo/KO-Eco, +tetoff::Neo, +cWAPL::His This study
WAPL-/3xmAID-6xFLAG
+ TIR19xmyc WAPL/WAPL::KO-His/3xmAID-6xFLAG FLP-In His, + TIR1-9xmyc::Bsr This study
Plasmid construction and transfection
ESCO1 KO-Bsr, ESCO1 KO-Puro and ESCO1 KO-His were generated from genomic
PCR products combined with Blasticidin S, Puromycin and Histidinol D selection marker
cassettes. Genomic DNA sequences were amplified using primers 5’-
ctatagggcgaattggagctGCTTTGCGTAAGACTTGTCGATCG-3’ and 5’-
gccgccaccgcggtggagctGTTCTCGAGTAACACATGAGCGGAG-3’ (for the right arm of
the KO construct); and 5’-
ctcgagggggggcccggtacCTCCATCTGTACTGGGAGACTTCTG-3’ and 5’-
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aagggaacaaaagctggtacGTTGAGATCAGCAGCAGAACGTCAG-3’ (for the left arm of
the KO construct). Amplified PCR products were purified by gel extraction and cloned
into pre-cutted pLoxP vectors (SacI and KpnI) by using GeneArt® Seamless PLUS
Cloning and Assembly Kit.
WAPL KO-His and WAPL KO-Eco vectors were generated from genomic PCR products
combined with mycophenolic acid and Histidinol D selection marker cassettes. Left arm
and right arm of WAPL KO vectors were amplified using the primers 5’-
AGTGAGCTCCCAGTGTCCCAGTAAGTAGTCTG-3’ (SacI) and 5’-
AGTGCGGCCGCAGGAGCCTTAAAAACTCCATCATC-3’ (NotI) (for the left arm of
the KO construct); and 5’- AGTATCGATACGTTGTGAGTAATCAGCAGCAG-3’
(ClaI) and 5’- AGTGGTACCGTAGCTGTCCTCTGCAGATCTC-3’ (KpnI) (for the right
arm of the KO construct). Amplified PCR products were cut and cloned into pLoxP
vectors (Arakawa et al. 2001) using attached restriction sites. Left arm and right arm of
SMC3 KO vectors were amplified using the primers 5’-
CCTCTCCTTGCTCTGCCTATAGGACGATGC-3’ and 5’-
GAGGATCCGACCCCCCGACCCTATCAGTAC-3’ (BamHI) (for the left arm of the KO
construct); and 5’- GGATCCTTAACAAGGTATGGTCGTGACTTGAC-3’ (BamHI) and
5’- GGTCAGAAGAACCTTCCAGAGAGGGAATCC-3’ (for the right arm of the KO
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construct), and cloned into vector with a Histidinol D marker (Buerstedde and Takeda
1991).
To add 3xmAID-6xFLAG tag by FLP-In system, we generated p3xmAID-6xFLAG
vectors by combining pHyg–AID*–6FLAG (Morawska and Ulrich 2013), pTRE2-
hygro vector (Clontech) and pLoxP vectors (Arakawa et al. 2001). 2-3 kb upstream
DNA sequences of stop codons of WAPL, ESCO2 and SMC3 were amplified with the
primers 5’-AAGTGTCGACAGCCAAGAGAAGCCTGGGAATTGG-3’ (SalI) and 5’-
GATCACTAGTGCAGTGTTCCAAGTATTCAATCACCCTAG-3’ (NheI) (for WAPL
FLP-In construct); and 5’- CGTAGTCGACGAATGCCTCAGTTCACACAGTCATAC-
3’ (SalI) and 5’- TTTACTAGTGTTGCCATAAACAAAGTTGTAG-3’ (NheI)
(for ESCO2 FLP-In construct); and 5’-
TTTTGTCGACAGGAGGAGCTGGACAGGGGCTACAAG-3’ (SalI) and 5’-
TTTGCTAGCACCATGCGTGGTGTCATCTTCTACAAAG-3’ (NheI) (for SMC3 FLP-
In construct). These amplified DNA fragments were cloned into p3xmAID-6xFLAG
vectors at SalI and NheI restriction enzyme sites. The FLP-In vectors were then linearized
at one restriction enzyme site in the middle of the homology region and transfected to
DT40 cells as previously described (Kobayashi et al. 2015).
For Smc3 and ESCO1 cDNA cloning, we used primers 5’-
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TAGACGCGTATGTACATCAAGCAGGTAATCATTCAGGG-3’ (MluI) and 5’-
TAGGCTAGCTTAAGCGTAGTCTGGGACGTCGTATGGGTAACCATGCGTGGTGT
CATCTTCTAC-3’ (NheI + HA) (for SMC3 cDNA); and 5’-
AGTGCGGCCGCCATGGCAGCTCAGAAAAGGAAGTCTGC-3’ (NotI) and 5’-
AGTGCGGCCGCTTACTTGTCGTCATCGTCTTTGTAGTCCATAGCCTGGTGCTG
CCCGTTGAGGAAG-3’ (NotI + FLAG) (for ESCO1 cDNA). HA and FLAG tags were
added to SMC3 and ESCO1 cDNA by PCR, respectively.
To make SMC3 point mutants (SMC3 K105 106Q, K105 106R and K105 106A), we
used WT SMC3 cDNA as a template and inserted mutations using the primers 5’-
CAGGACCAATATTTCTTAGACAAGAAAATGGTGAC-3’ and 5’-
CTGGGCTCCAATGACTCTGCGAAGTGAGACTTC-3’ (for K105Q, K106Q); and
5’-AGGGACCAATATTTCTTAGACAAGAAAATGGTGAC-3’ and 5’-
CCTGGCTCCAATGACTCTGCGAAGTGAGACTTC-3’ (for K105R, K106R); and 5’-
GCGGCGGACCAATATTTCTTAGACAAGAAAATGGTGACG-3’ and 5’-
GGCTCCAATGACTCTGCGAAGTGAGACTTC-3’
(for K105A, K106A).
To establish the SMC3-K105Q K106Q (QQ) Knock-In construct, 2 kb of SMC3
homology sequence with K105Q 106Q mutation and BamHI site on the center of the
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homology sequence was synthesized, Bleo marker cassette was inserted to the BamHI
site, and the left arm was extended by a DNA fragment amplified using the primers 5’-
AAAGGTACCTGAATACAGTGCAGGTAGAGAATTTC-3’ (KpnI) and 5’-
AAAGGTACCGTCAATATCTTATTTGCTCATTGTGTAAGC-3’ (KpnI). These vectors
were transfected to DT40 cells as previously described (Buerstedde and Takeda 1991).
References
Abe T, Kawasumi R, Arakawa H, Hori T, Shirahige K, Losada A, Fukagawa T, Branzei
D. 2016. Chromatin determinants of the inner-centromere rely on replication factors with
functions that impart cohesion. Oncotarget 7: 67934-67947.
Buerstedde JM, Takeda S. 1991. Increased ratio of targeted to random integration after
transfection of chicken B cell lines. Cell 67: 179-188.
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Supplementary figures and figure legends
Figure S1. (A) Growth curves. 1 × 105 cells of the indicated genotypes were inoculated
in 1 ml of medium, counted and passaged every 24 h for each mutant. (B), (C) ESCO1
mRNA levels were measured by quantitative polymerase chain reaction. (D)
Chromosomes from metaphase spreads were analyzed for cohesion defects as outlined in
Fig. 1E. Figure S1A relates to Figure 1, Figures S1B-D relate to Figure 3.
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Figure S2. (A) Schematic representation of the SMC3 gene locus and gene-targeting KO
and Knock-In (KI) constructs. Closed boxes indicate exons, and “Marker” indicates drug
resistance genes. Grey triangles indicate Lox-sequence. (B) Knocked-In mutation was
verified by cDNA sequencing. (C) The mutations at SMC3-K105, K106 were verified by
Western blotting, using specific antibody for SMC3 acetylated at K105 and 106. Auxin
was added 6 hr before the sample collection, when indicated. Figure S2 relates to Figure
4.
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Figure S3. (A) Growth curves. 1 × 105 cells of the indicated genotypes were inoculated
in 1 ml of medium, counted and passaged every 12 h. 500 µM of Auxin was added at time
0, when indicated. (B) Depletion of SMC3-3AID-6FLAG protein and comparable protein
levels of SMC3 variants expressed from randomly integrated constructs were confirmed
by Western blotting for indicated smc3 mutants. Figure S3 relates to Figure 4.
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Figure S4. (A) Representation of the WAPL gene locus and gene-targeting KO construct.
Closed boxes indicate exons, and “Marker” indicates drug resistance genes. (B) Depletion
of SMC3-3AID-6FLAG protein and WAPL-3AID-6FLAG protein was confirmed by
Western blotting. Similar degradation was observed in three independent experiments.
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(C) Chromosomes from metaphase spreads were classified in five groups as indicated,
and more than 100 metaphase cells were analyzed for each genotype. The results of two
independent experiments are plotted. Auxin (final: 500 µM) was added 6 hr before cell
collection, when indicated. Figure S4 relates to Figure 5.
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Figure S5. (A) Scheme of the conditional WAPL mutant establishment by Tetoff-system.
(B) Immunostaining using the anti-Smc3 antibody and DAPI. Samples were prepared by
the cytospin method. Auxin (final: 500 µM) was added 6 hr before sample collection;
Doxycycline (Dox, final: 1 µg/mL) was added 24 hr before collecting samples, as
indicated. Scale bars indicate 10 µm in upper panels and 5 µm in bottom panels. Figure
S5 relates to Figure 6.
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Figure S6. RNA-seq analysis of transcriptome alterations caused by disruption of
ESCO1, ESCO2 and WAPL. (A) Duplicates of each mutant were analyzed by RNA seq,
and gene expression levels are visualized as a heatmap. (B, C) Numbers of differentially
expressed genes between the different mutants are summarized in Venn Diagrams (B,
three-pair comparison; C, four-pair comparison). Figure S6 relates to Figure 6.
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Figure S7. Quantification of chromatin-associated cohesin and acetylated-Smc3
using chromatin fractionation assays. (A, B) Left panel: chromatin binding assay.
Western blotting was performed with fractionated protein samples from each of the
indicated cell lines. (A) SMC3, (B) acSMC3 were detected. Tubulin, Histone H3 were
detected as control. WCE = whole cell extract, NUC = nucleoplasmic fraction, CHR =
chromatin bound fraction. Right panel: quantification of SMC3 (A), (B) acSMC3 in each
fraction. The signal intensity of each fraction was normalized to WT, and plotted. Auxin
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(final: 500 µM) was added 6 hr before sample collection. (C) Comparison of SMC3 and
acSMC3 protein level in each fraction in wapl mutants. The signal intensities were
normalized to WCE, and then plotted. Figure S7 relates to Figure 7.