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.

supplementary information table s1. cell lines used in...

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