Supplemental Material

Legends for Supplemental Figures

Figure S1. Characterization of Wapl in Xenopus egg extracts. (A) Cytostatic factor

(CSF)-arrested extract of Xenopus eggs was prepared as described in Murray (1991). In this particular experiment, interphase extracts were prepared by addition of 0.4 mM

CaCl2 and 100 µg/ml cyclohexamide to CSF-arrested extracts followed by incubation at

22ºC for 30 min. Immunoprecipitation was performed from CSF-arrested (M) and interphase (I) extracts by using a set of antibodies raised against three different antigens for human Wapl (Gandhi et al. 2006). Aliquots of the extracts (lanes 1-4) and the precipitates (lanes 5-12) were analyzed by immunoblotting. Notably, all of three antibodies precipitated a 160-kDa band (indicated by an arrow) from egg extracts that was recognized with an antibody against a recombinant C-terminal fragment of hWapl

(hereafter referred to as anti-Wapl). This antibody also cross-reacted a single band with the same mobility in extracts (lanes 1-4), suggesting strongly that this 160-kDa band is the Xenopus ortholog of Wapl. Based on the observations described above, we have used anti-Wapl throughout the current study. (B) An aliquot of interphase egg extracts was overlaid onto a 5%-20% sucrose density gradient and spun at 36,000 rpm for 15 hr in an SW55Ti rotor (Beckman Coulter). Fractions were subjected to SDS-PAGE and analyzed by immunoblotting with the antibodies indicated. The positions of standards

1

(BSA [4.5S], aldolase [7.3S] and catalase [11.3S]) are shown. The majority of Wapl was fractionated in a single peak with a sedimentation coefficient of ~7S, which was distinct from the peaks of Pds5 (~9S) or the holocomplex of cohesin (~14S). (C)

Immunoprecipitation was performed from an interphase egg extract with control rabbit

IgG (lane 3), anti-Smc3 (lane 4) and anti-Wapl (lane 5). 1% and 5% of the input extract

(lanes 1 and 2) and the precipitates (lanes 3-5) were subjected to SDS-PAGE and analyzed by immunoblotting with the antibodies indicated. It should be noted that anti-Wapl precipitated not only Wapl itself but also small amounts of cohesin subunits and two isoforms of Pds5 (lane 5). Conversely, anti-Smc3 precipitated cohesin subunits along with small amounts of Wapl and Pds5 (lane 4) (D) Sperm chromatin was mixed with an extract that had been released into interphase to allow nuclear assembly and

DNA replication, and sea urchin cyclin B was added at 100 min to drive the cell cycle into mitosis. Aliquots were taken at the indicated time points, and chromatin fraction was isolated and analyzed by immunoblotting with the antibodies indicated (lanes 4-13).

Aliquots of the extract at the indicated time points (lanes 1 and 2), and a sample from a mock-assembly reaction without sperm chromatin (lane 3) was included. Accumulation of Cdc45 and the XCAP-H subunit of condensin I on chromatin indicates the timing of

DNA replication and chromosome condensation, respectively. The lower part of the gel was stained with Coomassie brilliant blue to determine the level of core histones as a control for chromatin recovery (bottom row)

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Figure S2. Immunodepletion of cohesin and its regulators from Xenopus egg extracts.

(A) An interphase egg extract was immunodepleted with control IgG (Δmock; lanes 1-4), a mixture of antibodies raised against recombinant fragments of Pds5A and Pds5B

(ΔPds5 (rec); lane 5), or a mixture of antibodies raised against synthetic peptides corresponding to the C-terminal sequences of Pds5A and Pds5B (ΔPds5 (pep); lane 6).

To estimate the efficiency of depletion, aliquots of each extract, along with decreasing amounts of the Δmock extract, were analyzed by immunoblotting with the antibodies indicated. (B) Sperm chromatin was incubated with interphase egg extracts that had been depleted of Pds5 as described in (A). 120-min after sperm addition, cyclin B was added to trigger entry into mitosis and the reaction was incubated for another 100 min.

At the indicated time points, chromatin-bound proteins were isolated and analyzed by immunoblotting with the antibodies indicated. It should be noted that, although only a traceable amount of Pds5B was detected in interphase chromatin assembled in the

ΔPds5 (rec) extract (lane 5), a considerably higher amount of Pds5B was detectable in the ΔPds5 (pep) extract (lane 8). For this reason, we have used the protocol for preparing the ΔPds5 (rec) to make “Pds5-depleted” extracts throughout the current study. An example is shown below in (C). A mild phenotype reported in our previous paper was most likely due to incomplete depletion of Pds5 (Losada et al. 2005). (C) An interphase egg extract was immunodepleted with control IgG (Δmock; lanes 1-4), a

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mixture of anti-Smc1 and anti-Smc3 (Δcohesin; lane 5), a mixture of anti-Pds5A and anti-Pds5B (ΔPds5 (rec); lane 6), or anti-Wapl (ΔWapl; lane 7). To estimate the efficiency of depletion, aliquots of each extract were analyzed immunoblotting along with decreasing amounts of the Δmock extract. Since only a small population of Wapl, cohesin and Pds5 is associated with each other (see Fig. 1A and B), immunodepletion of

Wapl did not significantly affect the amount of cohesin and Pds5 present in the extract, and vice versa. (D)-(F) An interphase egg extract was immunodepleted with various antibodies indicated and used for morphological analysis described in Figs. 5 and 6 and

Supplemental Figs S6 and S7. The efficiency of immunodepletion was examined by immunoblotting as above. The asterisk indicates a non-specific band that cross-reacts with the anti-Sgo1 antibody. The signals of Cdc45 (D and F) and XCAP-H (E) serve as loading controls.

Figure S3. Sequence alignment of the N-terminal half of Wapl in vertebrates. Shown here are the sequences of Wapl from three vertebrate species (Homo sapiens [Hs],

Xenopus laevis [Xl] and the zebrafish Danio rerio [Dr]). The Xenopus sequence was assembled from two EST sequences (BC084433 and CA792817). The zebrafish has two isoforms of Wapl encoded by different genes (Dr Wapl1 [XP_688605] and Dr Wapl2

[XP_695007]). Identical and similar residues among the four polypeptides are highlighted in red and blue, respectively. Three FGF-motifs are highlighted in green.

4

Regions displaying high similarities are underscored with the asterisks (shown by the open boxes in Fig. 3A).

Figure S4. The N-terminal half of Wapl is required for its interaction with Pds5 and cohesin. (A) Full-length (F, amino acids 1-1190) or truncated hWapl (N, 1-500; C,

501-1190) with a 3xFLAG-tag at their N-termini was translated in reticulocyte lysates and mixed with interphase egg extracts. After a 30-min incubation at 22ºC, the mixture was subjected to immunoprecipitation with control mouse IgG (ctrl) or the monoclonal antibody anti-FLAG M2 (a-F), and analyzed by immunoblotting. In the bottom panel, the arrowheads and brackets indicate the positions of precipitated hWapl. (B)

Full-length or truncated hWapl was translated individually (lanes 2-4) or simultaneously with hPds5B (lanes 5-10) in reticulocyte lysates, and then recovered on anti-FLAG beads. The beads were subjected to another incubation with a lysate producing hSA1-hRad21 (lanes 1-7) or a control lysate (lanes 8-10), and the beads-bound fractions were analyzed by immunoblotting. (C) Sperm chromatin was incubated with interphase egg extracts that had been supplemented with full-length or truncated hWapl. After 120 min, cyclin B was added to drive the cell cycle into mitosis and the reaction mixtures were incubated for another 100 min. Chromatin was isolated at 120 min and 220 min, and bound fractions were analyzed by immunoblotting (lanes 7-15). To confirm the expression of each fragment, aliquots of the supplemented extracts were examined in

5

parallel (lanes 1-6). (E) Metaphase chromosomes were assembled in a Wapl-depleted extract that had been supplemented with full-length or truncated hWapl. The chromosomes were analyzed as described in Fig. 2.

Figure S5. Sequence alignment of Rad21 and Rec8 in vertebrates. The kleisin α family is composed of two subfamilies, namely, the Rad21 subfamily and the Rec8 subfamily.

Shown here are the sequences of Rad21 and Rec8 from two vertebrate species (Homo sapiens [Hs] and Xenopus laevis [Xl]). Identical and similar residues are highlighted in red and blue, respectively. A highly conserved block (amino acids 372-392 in Hs

Rad21) within the central domain is indicated with the asterisks (Rad21-Rec8 homology, shown by the open box in Fig. 4C). The dashed lines indicate the N-terminal and

C-terminal winged-helix domains.

Figure S6. PP2A does not necessarily co-localize with the inter-sister population of

Sgo1. The chromosomes that had been assembled in mock-depleted and Wapl-depleted extracts were double-stained with anti-PP2A-C (the monoclonal antibody 1D6 [Upstate

Biotech.] against the catalytic subunit of PP2A)(grayscale images, a, d; green, b, e; red, c and f) and anti-XCAP-G (green, c and f). Bulk DNA was visualized with DAPI

(magenta, b and e; blue, c and f). Bar, 10 µm. Note that, although the centromeric population of PP2A-C (most likely co-localizing with Sgo1) was readily detectable in

6

both extracts, no signal was found in the inter-sister regions in the Wapl-depleted chromosomes.

Figure S7. Subchromosomal localization of Sgo1 and INCENP on chromosomes assembled in extracts depleted of Plx1, CPC or Sgo1. Metaphase chromosomes were assembled in the extracts that had been depleted with control IgG (Δmock; a-c), or anti-Plx1 (ΔPlx1; d-f), a mixture of anti-INCENP and anti-aurora B (ΔCPC; g-i), or anti-Sgo1 (ΔSgo1; j-l). The chromosomes were fixed and double-stained with anti-Sgo1

(grayscale images, left; magenta, middle; red, right) and anti-INCENP (green, middle and right). Bulk DNA was visualized with DAPI (blue, right). Bar, 10 µm. The efficiency of depletion in each extract is shown in Supplemental Figs. S2D and S2E.

It should be noted that in an extract depleted of Plx1 alone, a milder set of phenotypes was observed compared to an extract depleted of Plx1 and CPC (Fig. 5A, panels m-p).

On the other hand, subchromosomal localization of Sgo1 and the CPC was dependent on each other, a result consistent with previous reports (Boyarchuk et al. 2007; Rivera and Losada 2009). These results substantiate the notion that the functional contributions of Plx1 and the CPC to the resolution process are additive yet distinct (Losada et al.

2002).

7

Supplemental Materials and Methods

Construction of 3xFLAG tagged vectors

A pair of sense and anti-sense oligonucleotides that encodes the 3xFLAG sequence

(DYKDHDGDYKDHDIDYKDDDK) was annealed, and inserted into the pTnT vector

(Promega). The oligonucleotides used for construction were as follows (the underlined sequences made cohesive ends of the annealed products): pKS103, to insert the 3xFLAG sequence initiated with methionine into the N-ter minus of proteins: KS41, 5’-TCGAGATGGACTACAAAGACCATGACGGTGATT

ATAAAGATCATGACATCGACTACAAGGATGACGATGACAAGG-3’ and KS42,

5’-AATTCCTTGTCATCGTCATCCTTGTAGTCGATGTCATGATCTTTATAATCA

CCGTCATGGTCTTTGTAGTCCATC-3. pKS104, to insert the 3xFLAG sequence terminated with a stop codon into the

C-terminus of proteins: KS44, 5’-TCGACCGACTACAAAGACCATGACGGTGAT

TATAAAGATCATGACATCGACTACAAGGATGACGATGACAAGTAGGC-3’ and KS45, 5’-GGCCGCCTACTTGTCATCGTCATCCTTGTAGTCGATGTCATGA

TCTTTATAATCACCGTCATGGTCTTTGTAGTCGG-3’.

Construction of TnT expression plasmids

DNA fragments encoding the corresponding sequences were amplified by PCR by the

Phusion DNA polymerase (New England Biolab) and inserted into the pTnT, pKS103,

8

or pKS104 vector. The sequences were confirmed after all PCR steps. The primers used for subcloning were as follows: hWapl pWP226 (full-length, 1-1190: 3xFLAG-tagged at the N-terminus): WP5, 5’-CCGG

AATTCATGACATCCAGATTTGGG-3’ (EcoRI site underlined) and KT01, 5’-TC

GGTCGACTAGCAATGTTCCAAATATTC-3’ (SalI site underlined). pWP233 (1-500: 3xFLAG-tagged at the N-terminus): WP5 and KT04, 5’-TCGGT

CGACTAGTCCTGGGAATTATCATTGC-3’ (SalI site underlined). pWP241 (501-1190: 3xFLAG-tagged at the N-terminus): WP06, 5’-CCGGAATTC

ATGAGTCAGTCTGGTACTAACAATGCAG-3’ (EcoRI site underlined) and

KT02, 5’-TCGGTCGACTCGCAATGTTCCAAATATTC-3’ (SalI site underlined). hPds5B pPF102 (full-length, 1-1447): KT15, 5’-CCGGAATTCATGGCTCATTCAAAGAC

TAGGAC-3’ (EcoRI site underlined) and KT20, 5’-ACGGTCGACTCATCGCCGT

TCCCTTTTAGCACTTCG-3’ (SalI site underlined). hRad21 pXP141 (full-length, 1-631: 3xFLAG-tagged at the N-terminus): KT39, 5’-GGGG

TACCATGTTCTACGCACATTTTGTTCTC-3’ (KpnI site underlined) and KT40,

5’-CCGCTCGAGTCATATAATATGGAACCTTGG-3’ (XhoI site underlined). pXP142 (full-length, 1-631: 3xFLAG-tagged at the C-terminus): KT39 and KT41,

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5’-CCGCTCGAGGCTATAATATGGAACCTTGG -3’ (XhoI site underlined). pXP146 (172-631: 3xFLAG-tagged at the C-terminus): KT48, 5’-GGGGTACCAT

GAGAGAAGGCAGTGCTTTTGAGG-3’ (KpnI site underlined) and KT41. pXP147 (450-631: 3xFLAG-tagged at the C-terminus): KT49, 5’-GGGGTACCAT

GCGCCTCCAGGAGTCAGTGATG-3’ (KpnI site underlined) and KT41. pXP151 (287-631: 3xFLAG-tagged at the C-terminus): KT50, 5’-GGGGTACCAT

GCCAACCATGACTGATCAAACAAC-3’ (KpnI site underlined) and KT41. pXP155 (362-631: 3xFLAG-tagged at the C-terminus): KT55, 5’-GGGGTACCAT

GTGGAAAGAGACAGGAGGAG-3’ (KpnI site underlined) and KT41. pXP156 (404-631: 3xFLAG-tagged at the C-terminus): KT56, 5’-GGGGTACCAT

GAGGAGGAAAGGAGGAGAGGCAG-3’ (KpnI site underlined) and KT41. hSA1 pXR143 (full-length, 1-1258): KT42, 5’- ACGCGTCGACATGATTACTTCAGAA

TTACC -3’ (SalI site underlined) and KT43, 5’- TCCCCCGGGTCAGAACATAG

GCATTCCAAATCC -3’ (SmaI site underlined).

Site-directed mutagenesis

For each FGF motif of hWapl, one of two phenylalanine residues was first replaced with glutamic acid, and then the other was replaced. The sequences were confirmed after each step. The primers used for mutagenesis were as follows (only the sense

10

sequences are shown; mutation sites are underlined):

F73E, KS96 (first step): 5’-GAAGAAAGTACTGGAGATCCTGAGGGATTTGAT

AGTGATGATGAG-3’; F73E/F75E, KS97 (second step): 5’-GTACTGGAGATCC

TGAGGGAGAGGATAGTGATGATGAGTCTC-3’.

F429E, KT11 (first step): 5’-GGATGTTAAACTTGAATTTGAGGGTTTTGAAGA

TCATGAGAC-3’; F429E/F431E, KT12 (second step): 5’-GTTAAACTTGAATTT

GAGGGTGAGGAAGATCATGAGACAGG-3’.

F453E, KT13 (first step): 5’-CTAATTACAAAATTAAGTATGAGGGCTTTGATG

ATCTCAGTG-3’; F453E/F455E, KT14 (second step): 5’-CAAAATTAAGTATGA

GGGCGAGGATGATCTCAGTGAAAGCG-3’.

References

Boyarchuk Y, Salic A, Dasso M, Arnaoutov A. 2007. Bub1 is essential for assembly of

the functional inner centromere. J Cell Biol 176: 919-928.

Gandhi R, Gillespie PJ, Hirano T. 2006. Human Wapl is a cohesin-binding protein that

promotes sister-chromatid resolution in mitotic prophase. Curr Biol 16:

2406-2417.

Losada A, Hirano M, Hirano T. 2002. Cohesin release is required for sister chromatid

resolution, but not for condensin-mediated compaction, at the onset of mitosis.

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Genes Dev 16: 3004-3016.

Losada A, Yokochi T, Hirano T. 2005. Functional contribution of Pds5 to

cohesin-mediated cohesion in human cells and Xenopus egg extracts. J Cell Sci

118: 2133-2141.

Murray AW. 1991. Cell cycle extracts. Methods Cell Biol 36: 581-605.

Rivera T, Losada A. 2009. Shugoshin regulates cohesion by driving relocalization of

PP2A in Xenopus extracts. Chromosoma 118: 223-233.

12 Shintomi_Fig S1

input precipitates with A 2% 10% ctrl IgG α -C-pep α -N-rec α -C-rec

M I M I M I M I M I M I Mr (kDa) 197

Wapl

117 blot with α-C-rec 89

1 2 3 4 5 6 7 8 9 10 11 12

4.6S 7.3S 11.3S B top bottom Wapl Smc3 Rad21 Pds5B Pds5A 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

precipitates extract chromatin fraction C with D cyclin B time input [min] 200 100 (-)sp 5 15 30 50 70 100 115 130 160 200

1 5% ctrl IgG α - Smc3 α - Wapl Wapl Wapl Smc3 SA1 Smc3 Pds5B Rad21 Pds5A Pds5B Cdc45 Pds5A 1 2 3 4 5 XCAP-H (condensin I) core histones 1 2 3 4 5 6 7 8 9 10 11 12 13 Shintomi_Fig S2

∆Pds5 ∆Pds5 A B ∆mock (rec) (pep) time 120 160 220 120 160 220 (-sp)

120 160 220 [min] ∆mock Pds5B 255 50 100% ∆ Pds5 (rec) ∆ Pds5 (pep) Pds5B SA1 Pds5A Smc3 Rad21 Wapl SA1 XCAP-H Smc3 core Rad21 histones 1 2 3 4 5 6 1 8765432 9 10 CPC

C ∆mock D ∆ ∆mock Plx1 CPC Plx1 255 50 100% ∆ cohesin ∆ Pds5 (rec) ∆ Wapl ∆ ∆ ∆ Wapl 5 20 50 100% SA1 Plx1 Smc3 Cdc45 Rad21 aurora B Pds5B INCENP Pds5A 1 2 3 4 5 6 7 1 2 3 4 5 6 7 Sgo1 Sgo1 ∆ E F ∆ ∆mock ∆mock cohesin Sgo1 Plx1 Wapl Sgo1 Plx1 Wapl ∆ ∆ ∆ ∆ ∆ ∆ ∆ 5 20 50 100% 5 20 50 100% Sgo1 Sgo1 SA1 * Smc3 Plx1 Rad21 Cdc45 XCAP-H Wapl 654321 1 8765432 9 Shintomi_Fig S3

******************************************************************************************** Hs Wapl MTSRFGKTYSRKGGNGSSKFDEVFSNKRTTLSTKWGETTFMAKLGQKRPNFKPDIQEIPKKPKVEE-ESTGDPFGFDSDDESLPVSSKNLAQVKCSSYSESSEAAQLEEVTSVLEANSKI 119 Xl Wapl MTSRFGKTYSRRGGNSNSKFDEVFSNKRTTLSTKWGETTFMAKLGQKRPNHKQDVADTPKKPKVDD-DLAEDPFGFDSDEDSIPVSSKNVPQSKNSVQQPLEHLESADNFTSKIEENNKA 119 Dr Wapl2 MTSRFGKTYSRKGGDGTSKFDEVLSNKRAMLSTKWGETTYKAKVGAKRPGLKTEVTDQTKRSKVSEGDGSEDPFGFDSDDESKPVTSRNIAQSKAKVTSSTGGSQASLTLEPNSRVNQPA 120 Dr Wapl1 MTSRFSKTYTRKGGEASSKFDEVFSNKKATLTTKWGESTYKAHLGIKRPSFKPDVSELTKRPRFDDDDSSEDPFGFDSDDESKTVTSRDTAQS--EMDSDTAGP------KNEQL 107

Hs Wapl SHVVVEDTVVSDKCFPLEDTLLGKEKSTNRIVEDDASISS--CNKLITSDKVENFHEEHEKNSHHIHKNADDSTKKPNAETTVASEIKETNDTWNSQFGKRPESPSEISPIKGSVRTGLF 237 Xl Wapl EQPITAETTVSCKFISFDALHGLREKNTNKIEDGKRQHSS--PKKKNVTGNVQHFAEDS---SYSFPKRSNNDNKKPVVET------LTDTMWDLGNVSGSNSPPKSEQIKDTSTVVLY 227 Dr Wapl2 SVEAVAPSRASFSLSSERSAHKVPEDPGKYFNTSTTGKTPSWPAESTDRHSYSWYKNPSECDTRPLAQTTTLKSEKTEDKD------SYDAWSAVVGLGARSTSLFTAPKESSSMLWA 232 Dr Wapl1 KTQTEKPSRGTLVTSTSSLGSSTNISRG-AVGVNSPAKTS--PASSYVTGTVGTWSKSNVTVSKQLSQVSSLKTPSRGISN------DWDSFGEDSPPGAG------EGSSTQTSN 208

Hs Wapl EWDN-DFEDIRSEDCILSLDSDPLLEMKDDDFKNRLEN---LNEAIEEDIVQSVLRPTNCRTYCRANKTKSSQGASNFDKLMDGTSQALAKANSESSKDGLNQAKKGGVSCGTSFRGTV 352 Xl Wapl EWEELNQENTDISELEMNFDVNSVSETKDDDSPSNMEKNYNEEEHISEETAQSGLKLSNCRTYYRSNKAKQSQGSSNFDKLFDGPSQPATKSNYASEKERSIPVDK---SFSTGTR-ST 342 Dr Wapl2 SSSYSHSNIQGKSSTETRLDN------FHQGEFNVPVEPSESEDHSQSVLRAPNCRTYCRPNKGKQPGG------ADGSGSASSHISTGVLQTIGTDNASK------TTNR--- 325 Dr Wapl1 SSDELKSAEQCPAEPEPPVEQ------PVDFEQLPLLKPS-----SDRVYRRPRGKETESSAAGDTIP------AVSSVSAPAPVMSSNAGVAGSSGVKP------PAGR--- 295

*********************************************************************************************************************** Hs Wapl GRTRDYTVLHPSCLSVCNVTIQDTMERSMDEFTASTPAD-LGEAGRLRKKADIATSKTTTRFRPSNTKSKKDVKLEFFGFEDHETGGDEGGS---GSSNYKIKYFGFDDLSESEDDEDDDC 469 Xl Wapl GRTRDYTVLHPSCLSVCNVTIQDSMERGVDEFTTTPTTD-LGEAGRLRKKADLATTKATTRFRPGNPKSKKDVKLEFFGFDQDDGGSGEGGSKSLGQSNYRIKYFGFDDLSESEEEDDDGW 462 Dr Wapl2 GRTRDFTVLHPSCVSMFNVTFQDSMDRSVEEYTASTQASGPGDEGRLKKKTETES-KPLSRSRPTQSKMK-SSKLELFGFDDTDAVADD-DDSASGSSSYRIKYFGFDDLSESDSEEEEGS 443 Dr Wapl1 GRVRDYTVLHPSCVSVCNVTIQDSIERSVDDFVMPVQAD-IGEAGTFRRKADTQA-PKPTRFRPTQSKSKKETKLEFFGFEENEAQDADGDATGSGSTSYKIKYFGFDDLSESDSEDDDDS 414

********************************** Hs Wapl QVERKTSKKRTKTAPSPS-----LQPPPES--NDNSQDSQSGTNNAENLDFTEDLPGVPESVKKPINKQGDKSKENTRKIFSGPKRSPTKAVYNARHWNHPDSEE 567 Xl Wapl HQKEKKVKNKTRSAVVPS-----ESIPPSSGIAENLKDNQS----TYNSEFSEDQHAVPEVFKKPASKQADKSKDGARKIFSGT-RSPTKAVYNARHWDQPESEE 557 Dr Wapl2 SHRRRAKRMAAAEAEPLLSIDTNVDSPPPREMQSSHSSYTAGHSLKHDPLEFPDIPFPFETVRKPQVKAQEKAKES-RRIFSGPKKSPTKAVYNARHWNQPEAEE 547 Dr Wapl1 AARERKK-NKKAEAEALS---TGTGSP---QFSDSQESQQSQSSLSTDSQESLDETGATGSQKERHRKQSNKSKEIGRKIFSGPKKSPAKAVYNARHWNQPESEE 512

Shintomi_Fig S4

A 5% input precipitates B precipitates with α-FLAG hSA1-hRad21 + - - F N C - F N C 3xF-hWapl hPds5B - + IP with 3F-hWapl ctrl α -F ctrl α -F ctrl α -F ctrl α -F - F N C F N C F N C Pds5B hPds5B Pds5A hSA1 SA1 hRad21 Smc3 Rad21 F F truncation) ( truncation)

( C 3xFLAG- C 3xFLAG- N N hWapl hWapl 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10

∆Wapl C extracts chromatin D +hWapl (F) 3xF-hWapl F N C F N C time [min] 120 220 120 220 120 220 (-sp) 120 220 (-sp) 120 220 (-sp) 120 220

F

3xFLAG- C N ( trucation) hWapl ∆Wapl ∆Wapl 1 2 3 4 5 6 +hWapl (N) +hWapl (C) SA1 Smc3 Rad21 XCAP-H

core histones

7 8 9 10 11 12 13 14 15 10 µm Shintomi_Fig S5

======Hs Rad21 -MFYAHFVLSKRGPLAKIWLAAHWDKKLTKAHVFECNLESSVESIIS------PKVKMALRTSGHLLLGVVRIYHRKAKYLLADCNEAFIKIKMAFRPGVVDLPEENREAAY- 105 Xl Rad21 -MFYAHFVLSKRGPLAKIWLAAHWDKKLTKAHVFECNLESSVESIIC------PKVKMALRTSGHLLLGVVRIYHRKAKYLLADCNEAFIKIKMAFRPGVVDLPEENREAAY- 105 Hs Rec8 MFYYPNVLQRHTGCFATIWLAATRGSRLVKREYLRVNVVKTCEEILNYVLVRVQPPQPGLPRPRFSLYLSAQLQIGVIRVYSQQCQYLAEDIQHILERLHRAQLQIRIDMETELPSLLLP 120 Xl Rec8 MFYYPNVLQRHTGCFATIWLAATKGTKILKREYLKVNVISTCQQIIEYLLHQIPPPHVGLPVPRFSLYLSAQLSYGVVRVYHRQCDLLIEEMKSTLDRIYKAEKQMRIDLLQPEQQALLP 120

Hs Rad21 NAITLPEEFHDFDQPLPDLDDIDVAQQFSLIQSRVEEITMREEVGNISILQENDFGDFGMDDREIMREGSAFEDDDMLVSTTTSNLLLESEQSTSNLNEKI 206 Xl Rad21 NAITLPEEFHDFDQPLPDLDDIDVAQQFSLNQSRVEEITMREEVSNINILQDNDFGDFGMDDREMMREGSAFEDD--MLTTNASNLKLEPEQSTSQLNEKS 204 Hs Rec8 NHLAMMETLEDAPDPFFGMMSVDPRLPSPFDIPQIRHLLEAAIPERVEEIPP----EVPTEPREPERIPVTVLPPEAITILEAEPIRMLEIEGERELPEVS 217 Xl Rec8 DALSLMEMLEDAPDPFFGMMGVPPQPPDPLFIPQIRQLLEAPSPLRREPVTT----PTRPKKAKKRDEGDHVTSPERITLRDVEPITFPSIELIRDLPEAS 217

Hs Rad21 NHLEYEDQYKDDNFGEGNDGGILDDKLISNNDGGIFDD------PPALSEAGVMLPEQP---AHDDMDEDDNVSMGGPDSPDSVDPVEP 286 Xl Rad21 NHLEYDDQYKDDNFGEGNEGGILDDKLLSNDAGGIFDD------PPAMPEEGVAMPEQP---VHDDLDDDDNVSMGAPDSPDSVDPVEP 284 Hs Rec8 -RRELDLLIAE----EEEAILLEIPR-LPPPAPAEVEG------IGEALGPEELRLTGWEPGALLMEVTPPEELRLPAPPSPERRPPVPP 295 Xl Rec8 -VHDLEFLMSDLPLLEEEPISEPSKKDTPRKKEAEVDGSKLETLEESVDEARSQEGEIPAAELSPSVGPSLEPIPELDAAITLDEVTGEPVVDFPELTSPERLIPAEV 324

.287 .361 ********************** .403 Hs Rad21 MPTMTDQTTLVPNEEEAFALEPIDITVKETKAKRKRKLIVDSVKELDSKTIRAQLSDYSDIVTTLDLAPPTKKLMMWKETGGVEKLFSLPAQPLWNN-RLLKLFTRCLTPLVPEDLRK 403 Xl Rad21 LPTMTDQTTLVPNEEEAFALEPIDITVKETKAKRKRKLIVDSVKELDSKTIRAQLSDYSDIVTTLDLAPPTKKLMMWKETGGVEKLFSLPAQPLWNT-RLLKLFTRCLTPLVLDDLRK 401 Hs Rec8 PP--RRRR------RRRLLFWDKETQISPEKFQEQLQTRAHCWECPMVQPPERT------IRGPAELFRTPTLSGWLPPELLGLWTHCAQPPPKALRRE 380 Xl Rec8 SP--SERISLPASPQLVLKELPAADRSSKVEAVRRGKLIIDKETKIAAKKIQEQIQ-RPDIYTQAAV-PALLP------PKTPASLFQSPTYQYWMAPELTELWTRCALLQP------426

.449 Hs Rad21 RRKGGEADNLDEFLKEFENPEVPREDQQQQHQQRDVIDEPIIEEPSRLQESVMEASRTNIDESAMPPPPP-QGVKRKAGQIDPEPVMPPQQVE-QMEIPP------VELPPEEPPNICQLIPELELLP 523 Xl Rad21 RRKGGEADNLDEFLKEFENPEVPREELRPQ----DVIDQPILEEASHLQESLMEGSRTHLDDTIMPPPPPKQGVKRDSLQMEPEPMPMMQEAEPQIEMPPPPLPPPLELPPEEPQSISDLIPELNLLP 525 Hs Rec8 LPEEAAAEEERRKIEVPSEIEVPREALEPS------VPLMVS----LEISLEAAEEEKSRISLIPPEERWAWPEVEAPEAPALPVVPELPEVPMEMPL------VLPPELELLS 478 Xl Rec8 ---LQYVEEKEREEETMSEVEALRAIAEPS------GSLIMS----SEISLEVSEEERSHPILFTPEERR---AVSVQEERLLPMVSEMPELMFELP------ETGDFS 513

======Hs Rad21 EKEKEKEKEKEDDEEEEDEDASGGDQDQEERRWNKRTQQMLHGLQRALAKTGAESISLLELCRNTNRKQAAAKFYSFLVLKKQQAIELTQEEPYSDIIATPGPRFHII 631 Xl Rad21 EKEKEKDEE----EEEEEEDTTGTEQDQEERRWNKRTQQMLHGLQRVLAKTGAESISLLDLCRNTNRKQAAAKFYSFLVLKKQQAIELTQREPYSDIVATPGPRFHTV 629 Hs Rec8 ------LEAVHRAVALELQANREPDFSSLVSP------LSPRRMAARVFYLLLVLSAQQILHVKQEKPYGRLLIQPGPRFH-- 547 Xl Rec8 ------LDDAERRLRSQIDYLGQTDFLSHAPH------SISRIAASRLFFSSLVLCTQSIICLEQKQPFKQILITPGPLYSQN 584

Shintomi_Fig S6

PP2A-C / XCAP-G PP2A-C PP2A-C / DAPI DAPI a b c mock ∆

d e f Wapl ∆ Shintomi_Fig S7

Sgo1 Sgo1 / INCENP Sgo1 / INCENP / DAPI a b c mock ∆

d e f Plx1 ∆

g h i CPC ∆

j k l Sgo1 ∆