2000 4000 6000 8000 10000 12000 14000 16000 18000m/z-21

100

%

-3

100

%

-5

100

%

TOF LD+ 267

7661

511133111514

20552922 3841 4683

565370006199

15307

1129210256 1397513625

TOF LD+ 514x62299

2257

1613

7659

653533112326

2511

2638

5114

435338594417

5655

5699 6668

1532313057

112967882100119033 12768

1314114215

15481

15696 17518 17918

TOF LD+ 58

3310

20132055 1649941623351 1295412108639756524952 6600 7984 1000292788181 10260

1342914820 16231 17709

1+

2+

3+

3+

2+

1+1+

2+

3+

1+

1+

2+

2+

+ =13057+2257 = 1531413057+2299 = 15356

calf H3

Extract on beads + calf H3

Extract on beadsPeptides examined by reflectron

S.1. Endopeptidase activity against calf histone H3.Empty sepharose beads (upper panel) and extract from stationary phase (G0) on sepharose beads (middle panel) were assayed on thymus calf H3 (dark blue circle). Extract from stationary phase (G0) on sepharose beads was assyedin the absence of substrate as control (lowest panel). All reactions were analyzed by MALDI. The two peptides peaksproduct of the reaction are highlighted (red and light blue circle). The light blue peak was analyzed by reflectron (see S2).

Nature Structural & Molecular Biology: doi:10.1038/nsmb.1534

S. 2. Analysis by reflectron of the N-terminal product of calf H3 proteolysis.aa2-aa22 = 2254.322. The idividual peaks correspond to combinations of methylation states on diferent residuesof calf H3. Esi ms/ms was used to confirm the aminoacid sequence of the most prominet peaks marked with X (see S.4) .

Nature Structural & Molecular Biology: doi:10.1038/nsmb.1534

Helena 221-31 peptide + YASL 4-JUL-2008PNAC Facility

1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600m/z0

100

%

0

100

%

helena_221-31yasl 9 (0.373) Cn (Cen,1, 50.00, Ht); Sb (2,40.00 ); Cm (1:39) TOF LD+ 1.75e4x4 1676.86

1676.44

1598.85

1570.871116.60

1128.07 1556.881146.78

1677.39 3353.02

1677.84

2255.382254.37

1678.40

1678.82

1679.38

1696.452099.29

1913.13

3351.94

2256.39

3196.81

3195.932257.54

3167.882468.44 2555.58

3355.02

3355.89

3356.98

3357.86

3358.863427.95

helena_221-31 4 (0.184) Cn (Cen,1, 50.00, Ht); Sb (2,40.00 ); Cm (1:15) TOF LD+ 7.65e3x4 3352.96

1677.41

1676.35

1598.87

1118.37 1568.811326.83

3351.921677.88

1678.30

3197.83

3195.871696.482347.45

1697.34 2278.561843.30

2467.42 3167.813154.852740.56

3353.96

3354.94

3355.89

3356.91

3357.84

3391.893425.93

[M+H]+

∆Arg

∆Arg

-116.02 = loss of thioester group (116.03)

22-31teTKAARKSAPA-SCH2CH2COOCH2CH3

1116.63m/z

1-21ARTKQTARKSTGGKAPRKQLA

2254.32m/z

1-21 [M+2H]2+

[M+H]+

1-31te

1-31teARTKQTARKSTGGKAPRKQLATKAARKSAPA -SCH2CH2COOCH2CH3

3351.92m/z

1-31te [M+2H]2+

1-31te [M+2H]2+

1515

S.3. Endopeptidase activity against a synthetic H3 peptide.Sepharose beads bound extract from yeast grown to stationary phase (G0) was assayed on peptide consistingof aa1-aa30 human H3 (upper panel). Empty sepharose beads were assayed on the same peptide as control(lower panel). Both reactions were analyzed by MALDI. The substrate and the two peptides product are highlighted (red). 1-31te = 1-31thioester

Nature Structural & Molecular Biology: doi:10.1038/nsmb.1534

helena_calfH3beads_4

T:

250 300 350 400 450 500 550 600 650 700 750 800

m/z

0

20

40

60

80

100737.74673.63

685.28

642.30457.19

636.37521.42 672.79574.98 593.69 712.36 783.43

664.80 686.34607.31512.67459.22 738.40554.44454.92314.19228.22 784.41300.87253.87 538.39 746.01326.09 354.04 430.89365.16 792.56387.35

x3

850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400

m/z

0

20

40

60

80

1001014.43

1213.62 1341.68

877.78 899.62 1214.851126.67 1342.62963.42 1015.301005.40 1108.341035.06868.78 1343.441127.64835.52 1324.741195.50955.07 1089.81 1395.231178.79969.53 1075.56 1291.70900.59 1245.24

1354.67

x3

1450 1500 1550 1600 1650 1700 1750 1800 1850 1900 1950 2000

m/z

0

20

40

60

80

100

1464.12

1411.671631.981476.68 1664.001508.71 1566.311417.32 1528.69 1823.551738.971624.491481.04 1756.731675.281583.59 1724.85

x10

2254.3MH+ ms/ms 3+

y12

y19 2+

y13y11

y9

y7

y18 2+y17 2+

ARTKQTARKSTGGKAPRKQLAPredicted y-ions for this sequence

y9-y13 and y172+-y192+ pattern and peptide mass consistent with sequence

S 4. ES ms/ms analysis of the H3 endopeptidase N-terminal product “X” .

“X” :

Nature Structural & Molecular Biology: doi:10.1038/nsmb.1534

250 300 350 400 450 500 550 600 650 700 750 800

m/z

0

20

40

60

80

100747.17

783.54645.95

651.80544.72 640.08594.73741.54590.86547.05

535.40459.15 612.17484.16723.13527.40 798.96548.03436.01 768.71228.98 485.65 657.05335.24 463.28313.11 414.61 712.29365.16295.20

685.85404.34390.75354.62266.01248.33

x5 x3

850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400

m/z

0

20

40

60

80

100

R

e

l

a

t

i

v

e

A

b

u

n

d

a

n

c

e

913.63 1126.58968.381108.62

904.87840.44 1370.55969.37

891.70 978.44 1127.88850.651213.63

868.52 1214.671178.85914.42 1028.98 1160.59999.40947.37 1311.83

834.46 1073.91 1339.821298.31

942.42 1081.87 1351.83 1371.731041.48 1330.781224.36 1265.43

x5 x5 x10

1450 1500 1550 1600 1650 1700 1750 1800 1850 1900 1950 2000

m/z

0

20

40

60

80

100

1424.841577.881515.57

1650.021479.631457.62 1808.731537.70 1606.90 1727.14 1749.971707.98 1920.99

x10

2282.4MH+ ms/ms 3+ +28shift

y12

y19 2+

y13(Me2)

: ARTKQTARKMe2STGGKAPRKQLA

y11y9

y7

y17 2+

y18 2+

y9-y12 m/z unaltered, y13 shifts by +28amu, indicating modification on K9

”“X“

S 5. ES ms/ms analysis of the H3 endopeptidase N-terminal product “X” . Nature Structural & Molecular Biology: doi:10.1038/nsmb.1534

S 6. ES ms/ms analysis of the H3 endopeptidase N-terminal product “X” .

250 300 350 400 450 500 550 600 650 700 750 800

m/z

0

20

40

60

80

100752.27

746.70

745.70 784.44

656.46 793.15775.60693.57

601.04 613.81 692.78 731.44587.01 806.30422.97 567.15550.95 690.66641.20512.75

468.09439.02413.48224.12 313.16 338.01246.01 370.24286.92

x3 x5

850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400

m/z

0

20

40

60

80

100

R

e

l

a

t

i

v

e

A

b

u

n

d

a

n

c

e

1035.40

984.69

1026.60848.04 898.68

976.63 1108.521036.13856.51 1168.73911.18

890.19 996.61920.58 1384.521255.611213.551178.52831.03 975.33 1324.351067.50876.53 1200.65934.77 1304.631150.741068.55 1127.521046.14 1370.231256.66 1326.40 1385.751244.59

x5 x10

1450 1500 1550 1600 1650 1700 1750 1800 1850 1900 1950 2000

m/z

0

20

40

60

80

100

1524.75

1426.591476.21 1797.471594.471505.83 1620.89 1858.021463.77 1537.20 1570.84 1678.84 1700.001653.35 1982.761722.93 1754.51

x10

2296.3MH+ ms/ms 3+ +42shift

y12 y13(Me3)

y11

y10

y19 2+y18 2+

y17 2+

:ARTKQTARKMe3STGGKAPRKQLA

y9-y12 m/z unaltered, y13 shifts by +42amu, indicating modification on K9

“X”

Nature Structural & Molecular Biology: doi:10.1038/nsmb.1534

Extract prepared from stationa

r

y phase culture was pulled down on sepharose beads.Panel A: Bound proteins (Sepharose) and proteins eluted with 2M NaCL g (NaCl elution)were assayed for activity against calf histone H3. The reactions were analyzed by westernblot with anti C-terminal H3 antibody. The H3 Clipped product is highlighted. Panel B:Silver staining of the fractions containing the endopeptidase activity. Sepharose: beadsbound proteins. NaCl elution: 2M NaCL eluted proteins.

S7.

MW Sepharose

NaCl elu

tion

50

37

25

20

15

75100150250

Silver Staining

B. A.

Ponceau

α-H3 ab

Extract: - + +

NaCl elu

tion

Sepharose

<- H3<- H3 ∆1-21

Nature Structural & Molecular Biology: doi:10.1038/nsmb.1534

0

0.5

1

1.5

2

2.5

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

OD: 0.6 OD: 5 OD: 7 OD: 11

Relative Fluorescense Units

Chromatin Inmunoprecipitation HSP12 promoter

CN

C

N C

N CN

0

50

100

150

200

250

300

350

400

1 2 3 4

OD: 0.6 OD: 5 OD: 7 OD: 11

HSP12 mRNA level

S8. Chromatin immunoprecipitation experiments were performed in yeast cells cultured in glucose to OD600nm: 0.6 (green bars), OD600nm: 5 (read bars), OD600nm: 7 (yellow bars) and OD600nm: 11 (blue bars) using anti-myc antibody to detect the N-terminus of histone H3 (N) and anti H3 to detect the C-terminus of H3 (upper panel). The diagrams represent relative fluorescent units after normalizing the signal at the HSP12 promoter to an intergenic region on chromosome V. Lower panel: RT-PCR analysis was performed on the same cultures using primers specific to HSP12. The expression level of was normalized to the RNA levels of RTG2.

Nature Structural & Molecular Biology: doi:10.1038/nsmb.1534

Supplementary Table 1

S1 NMA111

S8 PRB1

S8 YSP3

S8 KEX2

S8 YCR54C

S9 DAP2

S9 YNL320W

S10 YBR139W

S10 KEX1

S16 PIM1

S26 IMP1

S26 IMP2

S33 YJU3

S33 MET2

S33 ECM18

S33 ICT1

S54 YGR101W

S54 YOL107W

S52 RBD2

S59 NUP100

S9 STE13

S59 NUP145 (unviable)

S59 NSP116 (unviable)

S26 SEC11 (unviable)

S10 PRC1

S: Serine protease

Table 1. Knocked out strains tested for H3 endopeptidase activity.

All strains were BY4741 background from Open Biosystem. Yeasts were grown to

stationary phase (OD600mn=5-7) and proteins bound to sepharose beads were assayed on

recombinant histone H3. The activity was detected by western blot with anti H3 antibody.

Nature Structural & Molecular Biology: doi:10.1038/nsmb.1534