Developmental Cell, Volume 32

Supplemental Information

Phosphorylation of Cohesin Rec11/SA3 by Casein

Kinase 1 Promotes Homologous Recombination

by Assembling the Meiotic Chromosome Axis

Takeshi Sakuno and Yoshinori Watanabe

A �

80

60

40

20

0

Patte

rs o

f the

cnt

1-G

FP s

igna

ls (%

)

100

cohered

separated

No exc.

One exc.

cnt1-GFP dh1L-tdTomato

Chromosome 1 5kbp

cnt1-GFP excision

cn t1

RS RS

dh dg dh dg

lacO

cn

(Kinetochore) Peri-

centromere Core centromere

imr1 imr1 lacO tetO

Peri-centromere

equational

reductional

(-) rec8- S412A CK1-so rec11-

5A moa1∆ rec12∆ �He

tero

zygo

us c

en2-

GFP

seg

rega

tion

durin

g m

eios

is I (

%)

0

20

40

60

80

100

D

centromere�

ade6-M26 �

ade6-469 �

Chr.3�plasmid �

B

0

0.2

0.4

0.6

0.8

1

Intra

geni

c re

com

bina

tion

frequ

ency

(%)

1.0

0.8

0.6

0.4

0.2

0

C

0

3

6

9

12

15

18

Reco

mbi

natio

n fre

quen

cy (%

)

0 3 6 9

12 15 18

E

0

2

4

6

8

10

12

14

wt rec8 -S412A

wt

Reco

mbi

natio

n fre

quen

cy (%

)

ura1-lys3 � leu1-mat1 �

0 2

4

6

8

10

12

14

rec8 -S412A

0

0.2

0.4

0.6

0.8

1

wt

Intra

geni

c re

com

bina

tion

frequ

ency

(%)

1.0

0.8

0.6

0.4

0.2

0 rec8 -S412A

Figure S1, related to Figure 1.�(A) (Left) Schematic representation of centromere 1 engineered for excision. (Right) Centromere excision at prophase I was filmed and classified into four categories (‘no exc.’, not excised at all; ‘one exc.’, only one centromere is excised; ‘cohered’, both are excised with cohesion preserved; ‘separated’, both are excised without cohesion (Sakuno et al., 2009)) (n > 90). Only the “separated” and “cohered” populations are presented in Figure 1B. Note that in the CK1-so moa1∆ double mutants cells, the excised cnt1-GFPs were primarily “separated”, similar to the moa1∆ single mutant, suggesting that CK1-so mutation does not inhibit the separation of core centromeric regions if cohesion is not preserved (moa1∆). (B) Intragenic recombination between the genomic ade6-M26 allele and ade6-469 allele provided by a multi-copy plasmid (illustrated at left) was examined in the indicated homozygous zygotes (n > 2,100). The number of Ade+ colonies after spore formation was counted. Error bars show S.D. (n = 3). (C) Intergenic meiotic recombination in the ura1-lys3 (n > 400) intervals was examined in the indicated zygotes. Error bars show S.D. (n = 3). (D) Segregation of heterozygous cen2-GFP during meiosis I was examined in the indicated zygotes with a moa1∆ rec12∆ background (n > 200). Note that due to the Rec8 cleavage defect, 40% of sister chromatid disjunction (equational segregation) in the moa1∆ rec12∆ zygote was inhibited in the rec8-S412A mutant. This phenomenon was also observed to some extent in CK1-so zygote, but not in the rec11-5A zygote, suggesting that Rec8 cleavage occurred normally in the rec11-5A mutant. (E) (Left) Intergenic meiotic recombination in the ura1-lys3 (n > 400) and mat1-leu1 (n > 120) intervals was examined in the rec8+-flag and rec8-S412A-flag zygotes. Error bars show S.D. (n = 3). (Right) Intragenic recombination was examined in the rec8+-flag and rec8-S412A-flag zygotes (n > 3,500). Error bars show S.D. (n = 3). �

wt� rec11-5A� rec11-5D �

DIC

Rec1

1�Re

c11-

GFP�

D �

0 20 40 60 80

100

0 20 40 60 80

100

0 20 40 60 80

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F�

(%)

0 � 2 � 4 � 6 � 8 � 10 � (h) �

0 � 2 � 4 � 6 � 8 � 10 � (h) �

0 � 2 � 4 � 6 � 8 � 10 � (h) �

(%)

(%)

wt�

rec11-5A�

CK1-so �

1 nuc.�2 nuc.�

3, 4 nuc.�

Rec11-GFP positive �

1 nuc.�2 nuc.�

3, 4 nuc.�

Rec11-GFP positive �

1 nuc.�2 nuc.�

3, 4 nuc.�

Rec11-GFP positive �

DIC DAPI Rec11�

Rec11- GFP�

0

3

6

9

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wt rec11-5A

leu1-mat1 �Re

com

bina

tion

frequ

ency

(%

)

E�

0

3

6

9

12

100 �80 �

40 �60 �

20 �0 �

100 �80 �

40 �60 �

20 �0 �

100 �80 �

40 �60 �

20 �0 �

untag � wt� 5A � 5D � wt�

wt� CK1-so �

150 �

150 �

150 �anti-GFP

anti-Rec11 pS7

anti-Rec11 pT18pS22

Rec11-GFP:� (KDa) �B �

CBB �

A �

Hhp1 �

Hhp1 �

32P�

meiotic cohesin �

DSB complex � SFT complex� LinE factors�

Rec8

F�

N � C � Rec6

F�

Rec1

2 F�

Rec1

4 F�

Rec1

5 F�

Mde

2 F�

N � M � C � Rec2

5 F�

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Rec7 �

N � C �

Rec24 �

Mug

20 F�

150 �

100 �

75 �

37 �

50 �

(KDa) �

Figure S2, related to Figure 2.�(A) The indicated GST-tagged proteins were incubated with GST-Hhp1 and analyzed for phosphate incorporation (32P) and the protein level (CBB). The red circles indicate the full-length recombinant proteins. The black arrowheads indicate the GST-Hhp1 protein. (B) Whole-cell extracts from cells obtained 4 h after meiosis induction in the indicated cells were prepared for Western blotting with anti-Rec11-pS7 or pT18pS22, phospho-specific antibodies. (C) Whole-cell extracts from cells obtained 3 or 4 h after meiosis induction in the indicated cells were prepared for Western blotting with anti-GFP antibodies (left). Rec11-GFP protein amounts were quantified from three independent experiments (right). Error bars show S.D. (n = 3).�p; unpaired two-tailed t test. (D) Representative images of the Rec11-GFP, Rec11-5A-GFP and Rec11-5D-GFP signals at prophase I in the indicated zygotes. (E) Intergenic meiotic recombination in the ura1-lys3 intervals was examined in the indicated zygotes (n > 400). Error bars show S.D. (n = 3). (F) The progression of meiosis in the cells subjected to PFGE (in Fig. 2F) was monitored by determining the nuclear number and frequency of Rec11-GFP-positive cells (n > 150). A representative picture of the CK1-so mutant at each sampling time point is shown at the bottom. DAPI: 4',6-diamidino-2-phenylindole.�

0 0.2 0.4 0.6 0.8

1 1.2 1.4

rela

tive

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nt o

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GFP�

wt 5A 5D 3 h �

wt 5A 5D

After meiosis induction �4 h �

p=0.463 p=0.108 �

p=0.100 �p=0.687

Rec11 -GFP: �

0.0 �0.2 �0.4 �0.6 �0.8 �1.0 �1.2 �1.4 �

anti- GFP�anti- Tubulin �

150 �

50 �

(untag) �wt 5A 5D Rec11- GFP: �

3 h � 4 h �(untag) �wt 5A 5D

100 �

After meiosis induction �

(KDa) �

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C � DIC�DAPI Rec11�

Rec11 -GFP�

GFP Positive: �

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GFP�

Rec1

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-GFP�

Rec1

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h 4

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Rec1

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wt (3

h 2

0 m

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~78% �

0% �

~100% �

~100% �

~100% �

A � rec11-GFP� rec11-5A-GFP�Rec11 Rec25 �

Rec25- mCherry �GFP�

Rec11 Rec25 �

Rec25- mCherry �GFP�

karyogamy �

prop

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I�

anaphase I �

met

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se I�

0 �

10 �

20 �

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130 �

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150 �

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170 �

180 �

190 �

200 �

210 �

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(min) �

B �

wt� rec11-5A�

rec11-5D � CK1-so �

rec11∆�

rec8∆ �

CK1-so rec11-5D � rec8∆ rec11-5D � rec8-S412A �

Rec25 DIC �

Figure S3, related to Figure 3.�(A) Time-lapse observation (from karyogamy to anaphase I) of Rec11-GFP and Rec25-mCherry signals in the rec11+ (left) and rec11-5A (right) zygotes. (B) Representative images of the Rec25-mCherry signal at prophase I in the indicated zygotes. (C) Representative picture of the indicated cells at the sampling time point used for ChIP in Figure 3B. The frequency of Rec11-GFP-positive cells was shown at right (n > 150).�

B � Rec11 Rec10 DIC � Rec10-mCherry �

rec8

+�re

c8∆�

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1+-G

FP

Rec11-GFP�

rec1

1-5D

-GFP

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c11+

-GFP

Rec10 N / Hop1 F �

Rec25 F / Rec27 F �

Rec27 F / Rec27 F �

Hop1 F / Rec10 F �

Hop1 N / Rec10 F �

Mug20 F / Mug20 F �

T�

p53 / Hop1 F �

p53 / Rec27 F �

p53 / Rec10 F �

p53 / Mug20 F �

p53/ T�

N/S�-Ade -His �

N/S�-Ade -His �

Bait / Prey�Prey �A �

Bait / Prey�

Figure S4, related to Figure 4.�(A) The bait used for the two-hybrid assays in Figure 4A provides an interaction with the positive control, verifying the significance of the assays. (B) Representative images of Rec11-GFP and Rec11-5D-GFP at prophase I in the rec8+ and rec8∆ zygotes expressing Rec10-mCherry. �

A �

Rec25-mCherry �Rec11-GFP-Rec10 �Rec11-Rec10

Rec25 DIC �

Rec10 Rec25 DIC Rec10-GFP Rec25-mCherry

wt�

rec27∆ �

wt�

rec27∆ �

B �

0

3

6

9

12

15

18

Reco

mbi

natio

n fre

quen

cy (%

)

wt� rec27∆ �

rec27∆ �

rec11 -rec10

rec11 -rec10

0

3

6

9

12

15

18

Figure S5, related to Figure 5.�(A) Representative images of Rec11-GFP and Rec25-mCherry (upper) and Rec11-GFP-Rec10 and Rec25-mCherry (lower) at prophase I in the rec27+ and rec27∆ zygotes. (B) Intergenic meiotic recombination in the ura1-lys3 (n > 400) intervals was examined in the indicated zygotes. Error bars show S.D. (n = 3).�

S. pombe Rec11�human SA3 �mouse SA3 �cattle SA3 �

chicken SA3 �

1 �1 �1 �1 �1 �

50 �50 �50 �50 �50 �

Figure S6, related to Figure 2.�The N terminus (1 to 50 a.a.) in the amino acid sequences of fission yeast Rec11 and SA3 in the indicated organisms was aligned using the Clustal W2 program. Red rectangles; CK1-target sites in Rec11. Green rectangles; serine and threonine residues in SA3. �

Table S1, related to Experimental Procedures. S. pombe strains used in this study are listed. (please see the excel file)

Supplemental Experimental Procedures Schizosaccharomyces pombe strain construction

Deletion of the endogenous rec12+, rec11+, sgo1+, moa1+, hhp1+, hhp2+, rec8+, rec10+ and rec27+ genes and tagging of rec10+, rec25+ and rec11+ by GFP, mCherry or 3×Pk were performed according to the PCR-based gene targeting method for S. pombe using

the kanMX6 (kanR), hphMX6 (hygR), natMX6 (natR) and bsdR genes as selection markers (Bahler et al., 1998; Rabitsch et al., 2001; Sato et al., 2005). To generate the rec11-5A or -5D strain, the endogenous rec11+ locus was cloned with its promoter and

terminator into pUC118. S7, S10, S11, T18 and S22 of rec11+ were subsequently changed to alanines or aspartic acids using the PrimeSTAR Max Mutagenesis Kit (TaKaRa). These rec11 mutant alleles were then amplified via PCR, including the

promoter and terminator, and transformed into the ∆rec11::ura4+ ura4-D18 strain and select 5-FOA (5-fluoroorotic acid, SIGMA) resistant colonies. Collect replacement was confirmed using PCR and direct sequencing. The cnt1-GFP (for excision), cen2-GFP

and CK1-so (Puhp1-3×HA-hhp1+ hhp2∆) strains have been described previously (Ishiguro et al., 2010; Sakuno et al., 2009; Yokobayashi and Watanabe, 2005). To visualize tubulin, the Padh13 (a weak version of the adh1+ promoter)-derived

mCherry-tagged atb2+ gene was integrated at the locus adjacent to the zfs1+ gene on chromosome II (designated as the Z locus). To promote the expression of the Rec11-Rec10 fusion protein, the rec11+-GFP-kanR or rec11-5A-GFP-kanR strain was

transformed with the PCR fragment containing GFP-rec10+-bsdR-rec11-3’UTR, and blasticidin S-resistant (Iwai Chemicals comp., 30 mg/l), but G418-sensitive, colonies were selected. Collect integration was confirmed using PCR and direct sequencing.

Chromatin immunoprecipitation (ChIP) assay The procedures were carried out essentially as described previously (Yokobayashi and

Watanabe, 2005). Anti-GFP polyclonal antibodies (Living Colors Full-length A.v. Polyclonal Antibody, BD), monoclonal anti-Pk (Serotec) and anti-Rec8 (Kitajima et al., 2003a) antibodies were used for immunoprecipitation. DNA prepared from the

whole-cell extracts or immunoprecipitated fractions was analyzed via quantitative PCR (qPCR) with the LightCycler 480 system (Roche) using SYBR Green I Master (Roche). The primers used for PCR have all been described previously (Sakuno et al., 2009;

Miyoshi et al., 2012). Immunoprecipitation with control IgG and anti-H3 polyclonal antibodies (Abcam) was performed in each experiment to account for nonspecific binding in the ChIP fractions and verify the significance of immunoprecipitation. Each

percent IP was quantified from three independent qPCR experiments. Two-hybrid assay The constructs of Rec8-M (134-300 a.a.), Rec10-N (1-551 a.a.), Rec10-C (552-792 a.a.), Rec25, Rec27, Hop1, Hop1-N (1-300 a.a.) and Mug20 were amplified via PCR and then cloned into pGBKT7 vectors and used as bait. To construct the prey version of

Rec11-N1 (1-140 a.a.)-wt, -5A and 5D, these fragments were amplified via PCR and cloned into a pGADT7C vector, in which the GAL4 activation domain (AD) was expressed at the C terminus of the cloned gene. The pGADT7C vector was constructed

as follows: the SV40-NLS (nuclear localizing signal) and GAL4AD coding regions in the original pGADT7 (Clontech) were deleted, and the PCR fragment of the GAL4AD-HA tag-SV40 NLS coding region was cloned into the XhoI site in MCS

(multicloning sites). The plasmids were subsequently transformed into the Saccharomyces cerevisiae AH109 strain. Plates lacking both histidine and adenine were used as selective media.

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cohesion within the centromere. Nature 458, 852-858. Sato, M., Dhut, S., and Toda, T. (2005). New drug-registant cassettes for gene disruption and epitope tagging in Schizosaccharomyces pombe. Yeast 22, 583-591.

Yokobayashi, S., and Watanabe, Y. (2005). The kinetochore protein Moa1 enables cohesion-mediated monopolar attachment at meiosis I. Cell 123, 803-817.