AIMMS Seminar Series Lecture

Concentration (a.u.)

A = 100 → 10 1.5

A = 100 → 1000 7 6 5 4 3 2 1 0

a

1.2

Clb5,6tot

0.9 0.6

Clb1,2tot

Clb3,4tot

0.3 0 0

10

20

30

40

50

60

b

Figure S1

Clb3,4tot Clb1,2tot

Clb5,6tot

0

10

20

30

40

50

60

Time (minutes)

Time (minutes)

B = 1000 → 100

B = 1000 → 10000

Concentration (a.u.)

1.5

c

1.2 0.9

Clb5,6tot

8

Clb3,4tot

6

d

4

0.6

Clb5,6tot

Clb1,2tot

0.3

2

0

0 0

10

20

30

40

50

60

Clb1,2tot

Clb3,4tot

0

10

Time (minutes)

Concentration (a.u.)

30

40

Clb5,6tot

0.9

f

1.6

Clb3,4tot

Clb5,6tot

Clb1,2tot

1.2

Clb3,4tot

Clb1,2tot

0.6

0.8

0.3

0.4

0

0 10

20

60

C = 100 → 1000

e

0

50

Time (minutes)

C = 100 → 10 1.5 1.2

20

30

40

Time (minutes)

50

60

0

10

20

30

40

Time (minutes)

50

60

t1,2 − t5,6 (min) [left branch]

Figure S1 C = 10 ≤ 100 ≤ 1000, D = 10 ≤ 100 ≤ 1000

20

r = 0.322

g

15 10 5 0 0

2

4

6

8

t1,2 − t5,6 (min) [right branch]

t1,2 − t5,6 (min) [fully connected] A = 10 ≤ 100 ≤ 1000, B = 100 ≤ 1000 ≤ 10000, D = 10 ≤ 100 ≤ 1000

20

r = 0.995

h

15 10 5 0 0

2

4

6

t1,2 − t5,6 (min) [fully connected]

8

Figure S2 a Fold change (mRNA)

1,6

NOC treatment P=0.0513 P=0.0013 P=6.1E-6

1,2

P=0.0022 P=0.0009 P=3.2E-4

P=0.0120 P=0.0003 P=0.0018

P=0.0002 P=0.0003

wild type fkh1∆ fkh2∆

0,8

fkh1∆fkh2∆

0,4 0,0 TSA1

ACT1

CLB1 CLB1

CLB2 CLB2

CLB3 CLB3

CLB4 CLB4

b Fold change (mRNA)

1,6

HU treatment P=0.0513 P=0.0894 P=0.0019

1,2

P=0.6443 P=0.0515 P=1.2E-5

P=0.196 P=0.0959 P=0.0053

P=0.4725 P=0.0104

wild type fkh1∆ fkh2∆

0,8

fkh1∆fkh2∆

0,4 0,0 TSA1

ACT1

CLB1 CLB1

CLB2 CLB2

CLB3 CLB3

CLB4 CLB4

Figure S2

Fold change (protein)

c 7 6 5 4 3 2 1 0

Fkh1 binding

TSA1 CLB1 CLB2 CLB2 CLB3 CLB3 CLB4 CLB4 TSA1 ACT1 ACT1 CLB1

Fold change (RNA pol II occupancy)

d 1,2 wild type

0,9

fkh1∆ fkh2∆

0,6 0,3 0,0 TSA1

ACT1

CLB1

CLB2

CLB3

CLB4

0

80

0

0

1023

30 0

0

1023

wild type

delayed

256

dividing

256 0 0 256 0 256 0

0

1023

0

1023

1023

0 256

0

0

1023

0

1023

0

1023

0

1023

0

1023

0

1023

0

1023

0

1023

0 256

130

0 256

120 110 1023

0

0

100 0

1023

0

1023

90

0

80 1023

60

0

0

1023

0

30

60 0

1023

0

1023

0

60

0

0

1023

0

1023

30

0

0

1023

0

0

90

0

0

1023

1023

0

0

0

0

0

1023

1023

140

0

100 0

1023

0256

1023

1023

0

0

110

0

0256

120

0

1023

0 0

1023

fkh1∆

0

1023

160

1023

0 256

0

1023

150 0

130

0

0256

1023

170

0 256

0

1023

0

80

150

0256

0512 0512

1023

0512

0

180

0

140

0

90

256

160

1023

0

100

256

1023

1023

120 110

256

256 256 0

512 0512

130

170

256 0

0

0

140

1023

0 2560

512 0

0

150

180

dividing

160

5120

170

05120

180

256 256

256 256 256 256

512 512

Time (min)

256

256

512 512 512 512

e

Figure S2

fkh2∆

Figure S2

f 3,0

α-factor treatment

Fold change (mRNA)

wild type

2,4

fkh1∆ fkh2∆

1,8

fkh1∆fkh2∆

1,2 0,6 0,0 TSA1

ACT1

CLB5

B = 1000, FL = 1000 6

a

5

Clb3,4tot

4 3 2

Clb5,6tot Clb1,2tot

1 0 0

10

20

30

40

50

60

Concentration (a.u.)

B = 1000, FL = 10 Clb5,6tot

c

Clb3,4tot

0.9

Clb1,2tot

0.6 0.3 0 0

10

20

30

40

50

60

Time (minutes)

B = 1000, FL = 0 Concentration (a.u.)

1.5

Clb5,6tot

d

1.2

Clb3,4tot

0.9

Clb1,2tot

0.6 0.3 0 0

10

20

30

40

Time (minutes)

b

5

Clb3,4tot

4 3 2

Clb1,2tot

1 0

Clb5,6tot

0

10

20

30

40

Time (minutes)

Time (minutes)

1.2

Concentration (a.u.) ₓ 103

Concentration (a.u.) ₓ 1018

6

Figure S3

B = 1000, FL = 100

50

60

50

60

e

4

Clb1,2tot

3 2 Clb3,4 tot

Clb5,6tot

1 0 0

10

20

30

40

50

60

B = 100, FL = 10 Concentration (a.u.)

Clb5,6tot

g

1.2

Clb3,4tot

0.9 0.6 Clb1,2tot

0.3 0 0

10

20

30

40

50

60

Time (minutes)

B = 100, FL = 0 Concentration (a.u.)

1.5

Clb5,6tot

h

1.2

Clb3,4tot

0.9 0.6

Clb1,2tot

0.3 0 0

10

20

30

40

Time (minutes)

2.5

f

2.0

Clb3,4tot

1.5 1.0

Clb1,2tot

0.5

Clb5,6tot

0 0

10

20

30

40

Time (minutes)

Time (minutes)

1.5

Figure S3

B = 100, FL = 100 Concentration (a.u.) ₓ 103

Concentration (a.u.) ₓ 1020

B = 100, FL = 1000

50

60

50

60

B = 10, FL = 1000 Concentration (a.u.) ₓ 1023

i

1.5

Clb1,2tot

1.0 Clb3,4tot

Clb5,6tot

0.5 0 0

10

20

30

40

50

60

B = 10, FL = 10 Concentration (a.u.)

Clb5,6tot

k

1.2

Clb3,4tot

0.9 0.6 Clb1,2tot

0.3 0 0

10

20

30

40

50

60

Time (minutes)

B = 10, FL = 0 1.5

Clb5,6tot

l

1.2

Clb3,4tot

0.9 0.6

Clb1,2tot

0.3 0 0

10

20

30

40

Time (minutes)

j

2.0

Clb3,4tot

1.5 1.0

Clb1,2tot

0.5

Clb5,6tot

0 0

10

20

30

40

Time (minutes)

Time (minutes)

1.5

Concentration (a.u.) ₓ 103

2.5

2.0

Concentration (a.u.)

Figure S3

B = 10, FL = 100

50

60

50

60

Figure S3

B = 0, FL = 100

B = 0, FL = 1000

m

Clb1,2tot

1.5 1.0

Clb3,4tot

Clb5,6tot

0.5 0 0

10

20

30

40

50

60

Concentration (a.u.) ₓ 103

Concentration (a.u.) ₓ 1020

2.5 2.0

n

2.0

Clb3,4tot

1.5 1.0

Clb1,2tot Clb5,6tot

0.5 0 0

10

20

30

40

50

60

Time (minutes)

Time (minutes)

B = 0, FL = 10 Clb5,6tot

p

1.2

Clb3,4tot

0.9 0.6 Clb1,2tot

0.3 0 0

10

20

30

40

50

60

B = 0, FL = 0 Concentration (a.u.)

Clb5,6tot

q

1.2

Clb3,4tot

0.9 0.6 Clb1,2tot

0.3 0 0

10

20

30

40

Time (minutes)

o

1.2

Clb5,6tot

0.9 0.6

Clb1,2tot

0.3 0 0

10

20

30

40

Time (minutes)

Time (minutes)

1.5

Concentration (a.u.)

Concentration (a.u.)

1.5

50

60

50

60

Figure S4

a

Transmitted light

Venus (Ndd1/Fkh2 complex)

DAPI

b Transmitted light

Venus (Ndd1/Fkh2 complex) 0’

10 ’

20 ’

30 ’

40 ’

50 ’

60 ’

70 ’

80 ’

90 ’

Transmitted light

Venus (Ndd1/Fkh2 complex)

Figure S4

Figure S5 a Fkh1 (1455 bp)

(485 aa) 0

76

142

401

303

FHA domain

485

FKH domain

Fkh1360

(125 aa) 360 401

(375 bp)

485

Fkh2 (2589 bp)

(862 aa) 0

82

152

338

429

862

Fkh2387 (1428 bp)

(476 aa) 387 429

862

Fkh2458 (1215 bp)

(405 aa) 458

862

Figure S5

b ß-Galactosidase pBTM + pACT pBTM-Fkh2 + pACT pBTM-Fkh2 + pACT-Clb1 pBTM + pACT-Clb1 pBTM-Fkh2 + pACT-Clb2 pBTM + pACT-Clb2 pBTM-Fkh2 + pACT-Clb3 pBTM + pACT-Clb3 pBTM-Fkh2 + pACT-Clb4 pBTM + pACT-Clb4 pBTM-Fkh2+ pACT-Clb5 pBTM + pACT-Clb5 pBTM-Fkh2 + pACT-Clb6 pBTM + pACT-Clb6 pBTM-F4 + pACT-PABC1

SDII

SDIV

SDIV + 5 mM 3‘AT

Figure S5

c ß-Galactosidase pBTM + pACT pBTM-Fkh2458 + pACT pBTM-Fkh2458 + pACT-Clb1 pBTM + pACT-Clb1 pBTM-Fkh2458 + pACT-Clb2 pBTM + pACT-Clb2 pBTM-Fkh2458 + pACT-Clb3 pBTM + pACT-Clb3 pBTM-Fkh2458 + pACT- Clb4 pBTM + pACT-Clb4 pBTM-Fkh2458 + pACT- Clb5 pBTM + pACT-Clb5 pBTM-Fkh2458 + pACT-Clb6 pBTM + pACT-Clb6 pBTM-F4 + pACT-PABC1

SDII

SDIV

Figure S5

d ß-Galactosidase pBTM + pACT pBTM + pACT-Fkh2 pBTM-Clb1 + pACT-Fkh2 pBTM-Clb1 + pACT pBTM-Clb2 + pACT-Fkh2 pBTM-Clb2 + pACT pBTM-Clb3 + pACT-Fkh2 pBTM-Clb3 + pACT pBTM-Clb4 + pACT-Fkh2 pBTM-Clb4 + pACT pBTM-Clb5 + pACT-Fkh2 pBTM-Clb5 + pACT pBTM-Clb6 + pACT-Fkh2 pBTM-Clb6 + pACT pBTM-F4 + pACT-PABC1

SDII

SDIV

SDIV + 2,5 mM 3‘AT

Figure S5 e

kDa

+

+ – –

+ – –

+ – –

+ – –

GST-Clb2

–

–

+

–

–

–

GST-Clb3

–

–

–

+

–

–

GST-Clb4

–

–

–

–

– –

GST-Clb5

–

–

–

–

+ –

–

–

– –

GST-Clb6

–

+

–

–

–

–

–

+ –

+ –

+ –

+ +

+ –

–

–

–

– –

–

125

Fkh2-Myc GST-Clb1

GST

Fkh2-Myc

95 1

kDa

2

3

4

5

kDa 95

25 75

6

7

8

9

kDa

kDa

95 75 75

55

Figure S5

f ß-Galactosidase pBTM + pACT pBTM + pACT-Ndd1 pBTM-Clb1 + pACT-Ndd1 pBTM-Clb1 + pACT pBTM-Clb2 + pACT-Ndd1 pBTM-Clb2 + pACT pBTM-Clb3 + pACT-Ndd1 pBTM-Clb3 + pACT pBTM-Clb4 + pACT-Ndd1 pBTM-Clb4 + pACT pBTM-Clb5 + pACT-Ndd1 pBTM-Clb5 + pACT pBTM-Clb6 + pACT-Ndd1 pBTM-Clb6 + pACT pBTM-F4 + pACT-PABC1

SDII

SDIV

SDIV + 2,5 mM 3‘AT

Figure S5

g

kDa 75

+

+ – –

+ – –

+ – –

+ – –

GST-Clb2

–

–

+

–

–

–

GST-Clb3

–

–

–

+

–

–

GST-Clb4

–

–

–

–

– –

GST-Clb5

–

–

–

–

+ –

–

–

– –

GST-Clb6

–

+

–

–

–

–

–

+ –

+ –

+ –

+ +

+ –

–

–

–

– –

–

Ndd1-Myc GST-Clb1

GST Ndd1-Myc

55 1

2

3

4

5

6

7

8

9

Figure S5

h Fold change (mRNA)

1,6

NOC treatment P=0.0362 P=0.0231 P=0.0034

1,2

P=0.044 P=7.4E-4 P=0.0053

wild type fkh2∆ clb3∆clb4∆

0,8

clb5∆clb6∆ 0,4 0,0 TSA1

ACT1

CLB2

CLB3

Figure S6

a ß-Galactosidase pBTM + pACT pBTM-Fkh1 + pACT pBTM-Fkh1 + pACT-Clb1 pBTM + pACT-Clb1 pBTM-Fkh1 + pACT-Clb2 pBTM + pACT-Clb2 pBTM-Fkh1 + pACT-Clb3 pBTM + pACT-Clb3 pBTM-Fkh1 + pACT-Clb4 pBTM + pACT-Clb4 pBTM-Fkh1 + pACT-Clb5 pBTM + pACT-Clb5 pBTM-Fkh1 + pACT-Clb6 pBTM + pACT-Clb6 pBTM-F4 + pACT-PABC1

SDII

SDIV

SDIV + 1,2 mM 3‘AT

Figure S6

b ß-Galactosidase pBTM + pACT pBTM + pACT-Fkh1 pBTM-Clb1 + pACT-Fkh1 pBTM-Clb1 + pACT pBTM-Clb2 + pACT-Fkh1 pBTM-Clb2 + pACT pBTM-Clb3 + pACT-Fkh1 pBTM-Clb3 + pACT pBTM-Clb4 + pACT-Fkh1 pBTM-Clb4 + pACT pBTM-Clb5 + pACT-Fkh1 pBTM-Clb5 + pACT pBTM-Clb6 + pACT-Fkh1 pBTM-Clb6 + pACT pBTM-F4 + pACT-PABC1

SDII

SDIV

Figure S6

c ß-Galactosidase pBTM + pACT pBTM-Fkh1360 + pACT pBTM-Fkh1360 + pACT-Clb1 pBTM + pACT-Clb1 pBTM-Fkh1360 + pACT-Clb2 pBTM + pACT-Clb2 pBTM-Fkh1360 + pACT-Clb3 pBTM + pACT-Clb3 pBTM-Fkh1360 + pACT-Clb4 pBTM + pACT-Clb4 pBTM-Fkh1360 + pACT-Clb5 pBTM + pACT-Clb5 pBTM-Fkh1360 + pACT-Clb6 pBTM + pACT-Clb6 pBTM-F4 + pACT-PABC1

SDII

SDIV

Figure S6

d

kDa

+

+ – –

+ – –

+ – –

+ – –

GST-Clb2

–

–

+

–

–

–

GST-Clb3

–

–

–

+

–

–

GST-Clb4

–

–

–

–

– –

GST-Clb5

–

–

–

–

+ –

–

–

– –

GST-Clb6

–

+

–

–

–

–

–

+ –

+ –

+ –

+ +

+ –

–

–

–

– –

–

75

Fkh1-Myc GST-Clb1

GST

Fkh1-Myc

55 1

2

3

4

5

6

7

8

9

Figure S7

a ß-Galactosidase pBTM + pACT pBTM + pACT-Ndd1 pBTM-Fkh1 + pACT-Ndd1 pBTM-Fkh1 + pACT pBTM-Fkh2 + pACT-Ndd1 pBTM-Fkh2 + pACT pBTM-F4 + pACT-PABC1

SDII

SDIV

SDIV + 10 mM 3‘AT

Figure S7

b

kDa

+

+

+

+

Ndd1-Myc

–

–

+

–

GST-Fkh1

–

–

–

+

GST-Fkh2

–

+

–

–

GST

75

Ndd1-Myc

55 1

2

3

kDa

4

5

kDa 130 95

25

75

c

Ndd1/Fkh1 complex

Ndd1/Fkh2 complex

Control

Figure S7

Venus

DAPI

Merged

Transmitted light + Merged

d Transmitted light + Venus Venus (Ndd1/Fkh1 complex) 0’

10 ’

20 ’

30 ’

40 ’

50 ’

60 ’

70 ’

80 ’

90 ’

Transmitted light + Venus Venus (Ndd1/Fkh1 complex)

Figure S7

a Sic1

Clb5

Clb3

Clb2

Figure S8

1,0 0,8 0,6 0,4 0,2 0,0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 Time steps 1,0 0,8 0,6 0,4 0,2 0,0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

1

0

0

0

1

1

1

1

Sic1 attractor

0

1

1

1

1

0

0

0

Clb5 attractor

0

0

1

1

1

1

0

0

Clb3 attractor

0

0

0

1

1

1

1

0

Clb2 attractor

Figure S8

attractor A b

attractor B

attractor C

Sic1

1

1

0

0

0

1

1

Clb5

0

1

1

1

0

0

0

Clb3

0

0

1

1

1

0

0

Clb2

0

0

0

1

1

1

0

Sic1

1

0

0

0

1

1

1

1

Clb5

0

1

1

1

1

0

0

0

Clb3

0

0

1

1

1

1

0

0

Clb2

0

0

0

1

1

1

1

0

Sic1

1

1

1

0

0

1

1

1

Clb5

0

1

1

1

0

0

0

0

Clb3

0

0

1

1

1

1

0

0

Clb2

0

0

0

1

1

1

1

0

Figure S8

attractor A c

attractor B

attractor C

Sic1

1

1

1

1

1

1

1

Clb5

0

1

1

1

0

0

0

Clb3

0

0

1

1

1

0

0

Clb2

0

0

0

1

1

1

0

Sic1

1

1

1

1

1

1

1

1

Clb5

0

1

1

1

1

0

0

0

Clb3

0

0

1

1

1

1

0

0

Clb2

0

0

0

1

1

1

1

0

Sic1

1

1

1

1

1

1

1

1

Clb5

0

1

1

1

0

0

0

0

Clb3

0

0

1

1

1

1

0

0

Clb2

0

0

0

1

1

1

1

0

Figure S8

d

attractor A

Sic1

0

Clb5

1

Clb3

1

Clb2

1

Figure S9 a 1

4

2

3

5

6

b

Sic1

wild type

Clb5

Clb3

Clb2

Figure S9

1,0 0,8 0,6 0,4 0,2 0,0 0 1 2 3 SIC1 overexpression

4

5

6

7

8

9

10

11

12

13

14

15

16

0

1,0 0,8 0,6 0,4 0,2 0,0

1,0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

sic1Δ

0,8 0,6 0,4 0,2 0,0 0

Time steps

c

Figure S9

d

Sic1

wild type

Clb5

Clb3

Clb2

Figure S9

1,0 0,8 0,6 0,4 0,2 0,0 0 1 2 3 SIC1 overexpression

4

5

6

7

8

9

10

11

12

13

14

15

0

1,0 0,8 0,6 0,4 0,2 0,0

1,0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

sic1Δ

0,8 0,6 0,4 0,2 0,0 0

Time steps

e

Sic1

wild type

Clb5

Clb3

Clb2

Figure S9

1,0 0,8 0,6 0,4 0,2 0,0 0 1 2 3 SIC1 overexpression

4

5

6

7

8

9

10

11

12

13

14

15

0

1,0 0,8 0,6 0,4 0,2 0,0

1,0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

sic1Δ

0,8 0,6 0,4 0,2 0,0 0

Time steps

f

Sic1

Clb2 dosage = 1

Clb5

Clb3

Clb2

Figure S9

1,0 0,8 0,6 0,4 0,2 0,0 0 1 2 Clb2 dosage = 5

3

4

5

6

7

8

9

10

11

12

13

14

15

0

3

4

5

6

7

8

9

10

11

12

13

14

15

5,0 4,0 3,0 2,0 1,0 0,0 1

2

Clb2 dosage = 30 30,0 1,0

20,0

0,0

10,0

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

0,0 0

1

2

3

4

5

6

7

8

Time steps

9

10

11

12

13

14

15

a

Figure S10

1,0 all inactive

0,9

Sic1 active Sic1, Clb2 active

0,8

Clb5 active Sic1, Clb5 active

0,7

Sic1, Clb3, Clb2 active

0,6

Clb5, Clb3 active

0,5

Sic1, Clb5, Clb3 active

Clb5, Clb3, Clb2 active

Sic1, Clb5, Clb3, Clb2 active

0,4 0,3 0,2 0,1 0,0 0

1

2

3

4

5

6

7 Time steps

8

9

10

11

12

13

14

b

Figure S10

4 3

H TH

2 1 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 5 5,5 6 6,5 7 7,5 8 8,5 9 9,5 10 10,5 11 11,5 12 12,5 13 13,5 14 14,5

0

Time steps

c 1 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0

Legend as in panel a

0

1

2

3

4

5

6

7 Time steps

8

9

10

11

12

13

14