NCU01005 1 k b NCU07896 NCU07897 1 k 8:A6 Y63...

34
D D D S S S WT Δdim-2 Δdmm-1 D D D S S S WT Δdim-2 Δdmm-1 D D D S S S WT Δdim-2 Δdmm-1 D D D S S S WT Δdim-2 Δdmm-1 D D D S S S WT Δdim-2 Δdmm-1 D D D S S S WT Δdim-2 Δdmm-1 D D D S S S WT Δdim-2 Δdmm-1 D D D S S S WT Δdim-2 Δdmm-1 8:F10 NCU00344 8:A6 NCU07897 NCU07896 Ψ63 NCU01005 1 kb 1 kb 1 kb 1 2 1.6 3 5 0.5 10 1 2 1.6 3 5 0.5 10 1 2 1.6 3 5 0.5 10 Honda_Supplementary Fig. 1

Transcript of NCU01005 1 k b NCU07896 NCU07897 1 k 8:A6 Y63...

Page 1: NCU01005 1 k b NCU07896 NCU07897 1 k 8:A6 Y63 8:F10genesdev.cshlp.org/content/suppl/2010/01/29/gad.1893210... · 2010. 1. 29. · DSDSDS S D m - 2 D m - 1 D D m 2 T WT Ddmm-1 Ddmm-2

D D DS S SW

T∆dim

-2

∆dmm-1

D D DS S SW

T∆dim

-2

∆dmm-1

D D DS S SW

T∆dim

-2

∆dmm-1

D D DS S SW

T∆dim

-2

∆dmm-1

D D DS S SW

T∆dim

-2

∆dmm-1

D D DS S SW

T∆dim

-2

∆dmm-1

D D DS S SW

T∆dim

-2

∆dmm-1

D D DS S SW

T∆dim

-2

∆dmm-1

8:F10NCU00344

8:A6NCU07897NCU07896

Ψ63NCU01005 1 kb

1 kb

1 kb

1

21.6

35

0.5

10

1

21.6

35

0.5

10

1

21.6

35

0.5

10

Honda_Supplementary Fig. 1

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8G3

NCU03486NCU03488NCU03489 NCU03487 NCU03485366 aa486 aa43 aa397 aa1016 aa

1 kb

1

21.6

3

105

0.5

D D DS S SW

T∆dim

-2

∆dmm-1

D D DS S SW

T ∆dim-2

∆dmm-1

D D DS S SW

T∆dim

-2

∆dmm-1

D D DS S SW

T∆dim

-2

∆dmm-1

D D DS S SW

T∆dim

-2

∆dmm-1

D D DS S SW

T∆dim

-2

∆dmm-1

D D DS S SW

T∆dim

-2

∆dmm-1

D D DS S SW

T∆dim

-2

∆dmm-1

D D DS S SW

T∆dim

-2

∆dmm-1

D D DS S SW

T∆dim

-2

∆dmm-1

D D DS S SW

T∆dim

-2

∆dmm-1

D D DS S SW

T∆dim

-2

∆dmm-1

D D DS S SW

T∆dim

-2

∆dmm-1

D D DS S SW

T∆dim

-2

∆dmm-1

D D DS S SW

T∆dim

-2

∆dmm-1

kb

A G A C A T A G A A A A C G G G G A T A T A C T G T A T T G T A C A C A T T G C T

WT

∆dm

m-1

G C C T T G C G A T G C G A T G C A T T A C C T T G G C C G C A A T G C A C G

A

B

Honda_Supplementary Fig. 2

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Page 4: NCU01005 1 k b NCU07896 NCU07897 1 k 8:A6 Y63 8:F10genesdev.cshlp.org/content/suppl/2010/01/29/gad.1893210... · 2010. 1. 29. · DSDSDS S D m - 2 D m - 1 D D m 2 T WT Ddmm-1 Ddmm-2

Aspergillus clavatus

Aspergillus fumigatus

Aspergillus nidulans

Aspergillus niger

Aspergillus terreus

Chaetomium globosum

Gibberella zeae

Magnaporthe grisea

Neosartorya fischeri

Phaeosphaeria nodorum

Neurospora crassa JmjC c c 14581

JmjC c c 7631

757JmjC c c1

JmjC c c 7201

JmjC c c 7551

JmjC c c 7531

JmjC c c 13511

JmjC c c12691

JmjC c c7551

JmjC c c13451

JmjC c c13651

JmjC c c1 653Aspergillus oryzae

JmjC c c1 1073Botryotinia fuckeliana

JmjC JmjC domain c Cysteine-rich region AT-hook motif

A

BDMM-1(CysR2) KG-DVYRCHVCQHKDYTYKLAFCSNPGCSEAYCYGVLYRAFDL-MPQEVLQNERWECPRCLKDNMT3L(CysR) DGYQSY-CSICCSGET---LLICGNPDCTRCYCFECVDSLVGPGTSGKVHAMSNWVCYLCLP .* : * * :* : * :*.**.*:..**: : .. . :* ..* * **

Honda_Supplementary Fig. 4

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R1060A R1150A

∆3x AT-hook

DMM-1 JmjC c c 14581

JmjC JmjC domain c Cys-rich region AT-hook motif

R1090A

WT

∆dmm-1

+R10

60A

+R10

90A

+R11

50A

+R10

60A &

R10

90A

+R10

60A &

R11

50A

+R10

90A &

R11

50A

+3x m

utatio

n

+∆3x AT-h

ook

digestion: Hpa II, probe: 8:G3

∆dim-2

A

B

+WT

∆dmm-1

1

2

1.6

3

5

0.5

kb

10

Honda_Supplementary Fig. 5

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8:G3probe:

1

21.6

35

0.5

kb10

D D DS S SDSD D DS S SW

T ∆dim

-2+P

dmm-1

::dmm-1

H216A -H

AT-FLA

G

∆dmm-1

+Pdm

m-1::d

mm-1-HAT-F

LAG

+Pac

tin::d

mm-1-HAT-F

LAG

+Pac

tin::d

mm-1H21

6A -HAT-F

LAG

Honda_Supplementary Fig. 6

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MPAASHPQAK FDPIPPDLDL HALVEKTPNF HWVLRVSAAQ IRNIGPQEFE RLVYLHVIRG GKPLVIEKWN DRLPKTLFSA EWLEGTYNKK QENVRDIVAQTDIPMTMGHY LRSMKQLTNQ WTPSNFRDER RQRLYLKDID CPPEWHERLQ KIIPTNLFYM NDNLDKKVSR RFDDYDYVEE KSCAPAGDLM SSLPEEMRAQNLMCYVGHEG TYTPAHREMC ASLGQNIMVD ASGDENGEKP GSSIWFMTET KDREVVREYF LSILGHDIEI EKHFAQINAW KKANFPVYIV EQKVGDFVLVPPLAPHQVWN RGTRTIKVAW NRTTAETLEM ALHEALPKAR LVCRDEQYKN KAIIYFTLQK YYNQMIEMEQ NADIGLLGFG QDLMKNSFRM TQIAKDFKALFRLFTEILVD EMFATKEKEI EYIEFDSCIT CSYCRANIFN RFLTCKHCVR TLVTGDEDTY DICMECYAMG RSCLCVSNLT WAEQWKWSDL VDRYEEWRALIIKNDGYVDY DTSPLPLELA RRKSGKKSVA QICQEQLRRR PWKDISKLDK PSEPENSEPE VDENGKPKKK PRRKKKKGDV YRCHVCQHKD YTYKLAFCSNPGCSEAYCYG VLYRAFDLMP QEVLQNERWE CPRCLKICNC GACRRLKNGN VPYHPKNTLL GHDTRPIADD RSVETLVDFK VHNLNWLKTS GDDGRNVQSKRLQKLQQQAE AEKAKNADVH IQVPALPSGE QATGAENLDD ALQQSIADGL GEDQERPDVE MTGASADQPN VVQPAGPAEV ADMSVAPEDH DQSAYPDPSMFGQERMLGMG YYQQDDDSPD KILFDPYQRP TAENYVVDEE PELMSEYLKK QLRLAKRRAR HAEDDDPDFA APRSHHRKKP KLDKTSQPPP TRDGQDDALENMDPALFDQT MTDVGGDIRD GSAQAGASQP ANAQSADEAA EESRQARMYS PNVPTLRHAR PKVSYRVDED NEEEFNDILI PRSQRPEYRA AAAPASVVQSIEGPTGPKDP LDLASAAVLA ITGAGGNRSN EASQEPGSAR AAAGSTGPSK LRGPHKKRGR PPGRPRKSEA HHTEDEESHE APEPTRRRGR PPRASLAAKEPADSSHKSGE ENDDEDIDAQ LAQELDGFDA DGEVVERDAG VPQAPKRRGR PPKNGVKKVV VEVTTKAPML SLAERMKLKG KKFKIGERKS TGGTPGKVIAKPVDSVEKSA SPAGSGRVAR DAEKQTSLPV RESPRMTRTR TTSATRHTVQ PAEEESHHSE SEPEAEPEKE MENKKSPEPP ARRSASQASV RISPPRDEASPNKSPERSPS VSASPEPPNR APSTAAAPSA TRISPSPSPS PVRSPSPPPP PPKPAGPTVV RLMDTEDEES DYGGSYSGSR SPSQRSESER SGSVASGTGSEDEPSSDSDS DDEDIPARKE SVRQSFASRG RGGGVGVVSR GRGGIRGRGR GRGRPRGS

DMM-1, 162,990.6 Da (1458 aa)66 unique peptides, 638/1458 amino acids (44% coverage)

MEALLGASNS NPSCGSILEL REQLRSVFST KVTEKRAEHV SVTLELRSTS VFHIPVTEPE NEQLEQAASI DPSLGGAQSS VAPASGEGGA NGNVNGNQPTRQINAIDALI NQPSDDHVLQ KSVAKHIITT LGEIDGSSWA VRQVSRGEQG WTFGYICKDS WQAWSRQNAK NPGRAVIGEW SQKPDSMDAV NLIGQLIELLVPPPAPREPK HALQRREPKK PKEPKPPKPP KPPKQPKPPK DPNAPKPPRK KRPAGEGGAG DGTQPKKRRK KKDAAAVGPD GSILPPEMPG GDTQNGYSTYPEGLVGQAPA AADEGVHVHS ILNIPPAEAA RRRTVAIDLL ESGGIDPKTL SPEQFNIFAN QAPELQQESL DMLKRYGAER LRIVHPDKEQ GDGAQAAPATQAATAPSTAP INPLKKKSKK KKSGVVDSEE GVAASGGKKK KKKNKEKLTR GKCDPCRDRK IPCTKEKPSC SQCTTSGLQC HYALERKVKD ISPESEAEPEAQSEPVVVEA DEEPEGLGSP GFHNEPAHHE PASHEPFNQE QDSHDSIDNN MPHGLPTSGL VSPQPESATT YHQPSDLYQQ FNSSVDASAT PEVAHTGVTPAAMDYMHSSV TDNSLHNFSY TQPQSEPEPA SAPVPEPEPI PEPVPVVEQP PPVQHTSNYA QQSSSTQAGG RTRRSLPSGP SVQSGAMNGH TTHAPTESWTPVPVPTIPAA YQNKPSPRAS RARKPAPAAP QTSMQNMTQH RTQNTVQQPA SQAFDDLRQA SSWSSVPQPT IPAQKTSQQQ ARTSPYQAAA QAVRASSRQGNRSQNHTPVQ QQTSAPGRHS QLQASQSHTD NSNYQSNTGV AASTSVGNYD SYSQYPTSSS RTEATNTRMN YEPYTNPSST TSNSYSSYDT YNTRSSTKTPVASLSAAQPT ASSYNTNTAT ATPSTSQWGS STSVPQTRSS ARSTYDTNPT TSASNQYNMT PSNPPQSSSL QGFNVRPQSM AQTRSSTAAY AQQQQQHHQQQSQAQQQQRQ QVQQQPSYNS YTSQSHTTNT QQQQHQQQQQ TQNWGYGSFG STTNASSTGY GSTGASSSAN NSYSTGTSAS SHAHNAAPAA TNAAAYSHQQPHHRSMNLSS NTYSSHIGNE DQVLYDLLRH PGA

DMM-2, 121,736.9 Da (1133 aa)18 unique peptides, 250/1133 amino acids (22% coverage)

Honda_Supplementary Fig. 7

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D D DS S SW

T∆dim

-2

∆dmm-1

D S∆dm

m-2

D D DS S SW

T∆dim

-2

∆dmm-1

D S∆dm

m-2

D D DS S SW

T∆dim

-2

∆dmm-1

D S∆dm

m-2

1

21.6

35

0.5

8:A6 Ψ63 8:F10

kb10

probe:

Honda_Supplementary Fig. 8

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8:G3probe:

1

21.6

35

0.5

kb10

D D DS S SW

T∆dim

-2

∆dmm-1

DS+d

mm-1-HAA B

8:G3probe:

1

21.6

35

0.5

kb10

D D DS S SW

T∆dim

-2

∆dmm-2

DS+F

LAG-dm

m-2

Honda_Supplementary Fig. 9

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8:G3probe:

1

2

1.6

35

0.5

kb10

D DS SW

T∆dim

-2

∆dmm-1

D DS S∆dm

m-1 & ∆dim

-2

EtBr

D DS SW

T∆dim

-2

∆dmm-1

D DS S∆dm

m-1 & ∆dim

-2

Honda_Supplementary Fig. 10

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00 20 40

20

10

15

5

Time (h)

Dis

tanc

e (c

m)

WT

∆dmm-1dim-5

∆dmm-1& dim-5

00 20 40

20

10

15

5

Time (h)

Dis

tanc

e (c

m)

WT

∆dmm-1hpo

∆dmm-1& hpo

A B

Honda_Supplementary Fig. 11

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00 20 40

20

10

15

5

Time (h)

Dis

tanc

e (c

m)

00 20 40

20

10

15

5

Time (h)

Dis

tanc

e (c

m)

∆dim-2∆dim-2+5AC

∆dmm-1 & ∆dim-2∆dmm-1 & ∆dim-2+5AC

A B

Honda_Supplementary Fig. 12

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1

21.6

35

0.5

kb10

D DS SW

T∆dim

-2

∆dmm-1

D DS SDS∆dm

m-1 +5

AC

∆dmm-1

-5AC

A+5AC +5AC +5AC

5AC

Slants (3 days) Liquid culture (5 hrs)

Liquid culture (2 days)

+5AC

+5AC +5AC 5AC

B

D DS SW

T∆dim

-2

∆dmm-1

D DS SDS∆dm

m-1 +5

AC

∆dmm-1

-5AC

8:G3probe:EtBr

Honda_Supplementary Fig. 13

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genes

WT∆dmm-1∆dmm-2

genes

1 2 3 4 5 6 7 8 9 10 11

WT

∆dmm-1

∆dmm-2genes

23 24 25 26 27 28 32 33 34 35 36

WT∆dmm-1

∆dmm-2

12 13 14 15 17 18 19 20 21 2216

WT∆dmm-1

∆dmm-2genes

37 38 39 40 41 42 43 44 45 46

WT

∆dmm-1

∆dmm-2

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 - 28 29 30 31

Centromere32 333435 36 37 38 39 40 4142 43 44 45 46

5

-55

-55

-5

5

-55

-55

-5

5

-55

-55

-5

5

-55

-55

-5

5

-55

-55

-5

100 kb

2 kb

A

B

47

47

Honda_Supplementary Fig. 14

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D D DS S SDSD D DS S SDSW

T∆dim

-2

∆dmm-1

WT

∆dim-2

∆dmm-1

∆dmm-2

∆dmm-2

D D DS S SW

T∆dim

-2

∆dmm-1

DS∆dm

m-2

SD D DS S S∆dim

-2

∆dmm-1

D∆dm

m-2

WT

WT

∆dmm-1

∆dmm-2

genes

peak 11 peak 12 peak 21

5-55-55-5

2 kb 2 kb2 kb

1

21.6

3

105

0.5

kb

peak 35

D D DS S SW

T∆dim

-2

∆dmm-1

D D DS S SW

T∆dim

-2

∆dmm-1

DS∆dm

m-2

DS∆dm

m-2

2 kb

Honda_Supplementary Fig. 15

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WT

hpo

dim-5

∆dim-2

WB: αH3K4me2

WB: αH3K9me3

∆dmm-1

∆dmm-2

∆dmm-1,

∆dim-2

Honda_Supplementary Fig. 16

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genes

genes

1 2 3 4 5 6 7 8 9 10 11

genes

23 24 25 26 27 28 32 33 34 35 36

12 13 14 15 17 18 19 20 21 2216

genes

37 38 39 40 41 42 43 44 45 46

5mC in WT

5mC in ∆dmm-1

K9me3 in ∆dmm-1

1 2 3 4 5 6 78 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 - 28 29 30 31

Centromere32 333435 36 37 38 39 40 4142 43 44 45 46

5

-55

-53

-3

5

-55

-53

-3

5

-55

-53

-3

5

-55

-53

-3

5

-55

-53

-3

5mC in WT5mC in ∆dmm-1

K9me3 in ∆dmm-1

5mC in WT5mC in ∆dmm-1

K9me3 in ∆dmm-1

5mC in WT5mC in ∆dmm-1

K9me3 in ∆dmm-1

5mC in WT5mC in ∆dmm-1

K9me3 in ∆dmm-1

100 kb

2 kb

A

B

47

47

Honda_Supplementary Fig. 17

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1 2 3 4 5 6 7 8 9 10 11 12H3K4me2 (WT)

hH4-1

0.960.06

0.050.02

0.150.11

0.110.14

0.140.16

1.1 1.1

H3K4me2 (∆dmm-1)1 2 3 4 5 6 7 8 9 10 11 12

hH4-1

1.1 0.080.04

0.090.07

0.080.14

0.140.12

0.070.67

1.2

8:G3

8:G3

Honda_Supplementary Fig. 18

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+DMM-1-HA (∆dmm-1)

hH4-1

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

+DMM-1H216A-HA (∆dmm-1)

hH4-11 2 3 4 5 6 7 8 9 10 11 12

1.5 1.1 2.6 5.0 9.0 16 8.7 4.4 1.5 1.1 0.4 0.6

1.2 4.7 3.6 5.9 5.0 1.7 3.1 3.1 3.1 4.9 1.2 1.0

Honda_Supplementary Fig. 19

8:G3

8:G3

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genes

1 2 3 4 5 6 7 8 9 10 11

5mC in WT

HP-GFP

DMM-1-HA

1 2 3 4 5 6 78 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 - 28 29 30 31

Centromere32 333435 36 37 38 39 40 4142 43 44 45 46

5

-53

-34

-4

5

-53

-34

-4

100 kb

2 kb

5mC in WT

HP-GFPDMM-1-HA

23 24 25 26 27 28 32 33 34 35 36

12 13 14 15 17 18 19 20 21 2216

37 38 39 40 41 42 43 44 45 46

5

-53

-34

-4

5

-53

-34

-4

genes

5mC in WT

HP-GFPDMM-1-HA

genes

5mC in WT

HP-GFPDMM-1-HA

genes

5mC in WT

HP-GFPDMM-1-HA

5

-53

-34

-4

A

B

47

47

Honda_Supplementary Fig. 20

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peak 332 kb

5

-53

-34

-4

1 2 3 4

1 2 3

genes

5mC in WT

HP-GFP

DMM-1-HA

5

-53

-34

-45

-5genes

5mC in WT

HP-GFP

DMM-1-HA

1 2 3hH4-1

Total input

2.0

1 2 3DMM-1-HA

3.5 1.1

1 2 3H3K9me3

2.6 6.4 6.1

1 2 3HP1-GFP

1.5 2.1 0.6

5

1 2 3 4hH4-1

Total input5

H3K9me31 2 3 4 5

14 14 5.3 3.3 1.8 2.2 2.8 2.1 1.5 1.1

HP1-GFP1 2 3 4 5

DMM-1-HA1 2 3 4 5

2.3 3.3 2.4 1.5 1.1

FLAG-DMM-21 2 3 4 5

0.8 2.8 1.8 1.2 0.8

2.2

1 2 3FLAG-DMM-2

3.2 1.1

peak 112 kb

Honda_Supplementary Fig. 21

peak 11

peak 33

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ChIP: DMM-1-FLAG

8:G3

hH4-18:A6

untag

WT

dim-5

∆dim-2

5.336.85

1.141.41

0.701.61

8:A68:G3

0.651.42

2.413.70

∆dmm-2

hpo

8:G3

hH4-18:A6

untag

WT

dim-5

∆dim-2

2.242.96

0.510.49

0.952.77

8:A68:G3

1.181.77

4.948.21

∆dmm-2

hpo

8:G3

hH4-18:A6

untag

WT

dim-5

∆dim-2

7.3511.8

1.572.25

0.772.34

8:A68:G3

0.670.81

4.989.09

∆dmm-1

hpo

ChIP: FLAG-DMM-2

8:G3

hH4-18:A6

untag

WT

dim-5

∆dim-2

7.9712.4

2.503.45

1.556.50

8:A68:G3

1.001.27

8.0015.6

∆dmm-1

hpo

untag

WT

dim-5

∆dim-2

∆dmm-2

hpo

DMM-1-FLAG

WB: αFLAG

A

B

untag

WT

dim-5

∆dim-2

∆dmm-1

hpo

FLAG-DMM-2

WB: αFLAG

Honda_Supplementary Fig. 22

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DMM-1-FLAG (WT)

hH4-11 2 3 4 5 6 7 8 9 10 11 12

1.5 1.5 2.8 1.9 14 17 7.5 6.4 2.2 1.5 1.6 1.4

FLAG-DMM-2 (WT)

hH4-11 2 3 4 5 6 7 8 9 10 11 12

0.9 2.9 3.0 3.4 27 29 17 12 4.4 2.2 4.8 1.9

DMM-1-FLAG (∆dmm-2)

hH4-11 2 3 4 5 6 7 8 9 10 11 12

1.1 2.6 2.2 3.0 4.2 2.7 2.9 4.4 4.5 3.3 1.3 2.1

FLAG-DMM-2 (∆dmm-1)

hH4-11 2 3 4 5 6 7 8 9 10 11 12

1.7 1.4 1.5 1.1 2.1 1.7 1.2 1.9 1.2 1.5 1.3 1.1

A B

8:G3 8:G3

8:G3 8:G3

Honda_Supplementary Fig. 23

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DIC

HP

1-G

FP

WT ∆dmm-1 ∆dmm-2

B

A

HP1-GFP (WT)

hH4-1

1 2 3 4 5 6 7 8 9 10 11 128G3

NCU03486NCU03488 NCU03487

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

HP1-GFP (∆dmm-1)1 2 3 4 5 6 7 8 9 10 11 12

hH4-1

HP1-GFP (∆dmm-2)1 2 3 4 5 6 7 8 9 10 11 12

hH4-1

0.5 1.6 1.8 1.9 7.6 6.1 3.7 1.8 1.6 1.2 1.2 1.3

1.0 2.0 2.1 2.5 4.1 2.9 2.8 3.9 3.8 2.1 1.7 1.1 1.3 1.2 1.9 1.4 4.6 2.6 2.6 3.9 3.6 2.7 1.5 1.1

Honda_Supplementary Fig. 24

8:G3

8:G3 8:G3

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Honda et al.

Supplementary Table I. Peptides used for histone demethylase assay

Lysine methylation

Residues\methylation mono di tri

H3K4 - - -

H3K9 - - -

H3K27 - - -

H3K36 - - -

H3K79 - - -

H4K20 (human) - - -

H4K20 (Neurospora) - - -

Arginine methylation

Residues\methylation symmetric assymmetric

H3R2 - -

H3R8 - -

H3R17 - -

H3R26 - -

H4R3 - -

* Peptides are around 22 amino acids long and are C-terminal biotinylated.

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Honda et al.

Supplementary Table II. Peptides used for binding assay

Lysine methylation

Residues\methylation unmodified mono di tri

H3K4 - - - -

H3K9 - - - -

H3K27 - - - -

H3K36 - - - -

H3K79 - - - -

H4K20 (human) - - - -

H4K20 (Neurospora) - - - -

Arginine methylation

Residues\methylation unmodified symmetric assymmetric

H3R2 - - -

H3R8 - - -

H3R17 - - -

H3R26 - - -

H4R3 - - -

* Peptides are around 22 amino acids long and are C-terminal biotinylated.

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Honda et al.

Supplementary Table SIII. Neurospora crassa strains used in this study

Strain Genotype Reference

N150 mat A FGSC#2489

N623 mat A his-3 FGSC#6103

N1877 mat a his-3; ∆dim-2::hph+ Kouzminova & Selker

(2001)

N2264 mat a his-3; dim-5 leu-2 pan-1 Tamaru & Selker (2001)

N2556 mat a his-3; hpoRIP2

Freitag et al (2004)

N2930 mat A his-3 ∆mus-52::bar+ Honda & Selker (2008)

N3064 mat A his-3; ∆dmm-1::hph+ this study

N3078 mat A his-3+::Pdmm-1::dmm-1-3xHA; ∆dmm-1::hph

+ this study

N3108 mat a his-3+::Pdmm-1::dmm-1-3xHA; ∆dmm-1::hph

+; ∆dim-2::hph

+ this study

N3109 mat A his-3+::Pdmm-1::dmm-1-3xHA; ∆dmm-1::hph

+; ∆dim-2::hph

+ this study

N3110 mat A his-3; ∆dmm-1::hph+; ∆dim-2::hph

+ this study

N3112 mat A his-3; ∆dmm-1::hph+; dim-5 leu-2 pan-1 this study

N3113 mat A his-3+::Pdmm-1::dmm-1-3xHA; ∆dmm-1::hph

+; dim-5 leu-2 pan-1 this study

N3114 mat a his-3+::Pdmm-1::dmm-1-3xHA; ∆dmm-1::hph

+; hpo

RIP2 this study

N3115 mat A his-3; ∆dmm-1::hph+; hpo

RIP2 this study

N3269 mat A his-3+::Pdmm-1::dmm-1-3xHAT-FLAG; ∆dmm-1::hph

+ this study

N3271 mat A his-3+::Pdmm-1::dmm-1

H216A-3xHAT-FLAG; ∆dmm-1::hph

+ this study

N3273 mat A his-3+::Pactin::dmm-1-3xHAT-FLAG; ∆dmm-1::hph

+ this study

N3275 mat A his-3+::Pactin::dmm-1

H216A-3xHAT-FLAG; ∆dmm-1::hph

+ this study

N3319 mat a his-3; hpo-FLAG::loxP::hph+::loxP Honda & Selker (2008)

N3320 mat A his-3; hpo-FLAG::loxP::hph+::loxP Honda & Selker (2008)

N3321 mat a his-3; hpo-gfp::loxP::hph+::loxP Honda & Selker (2008)

N3322 mat A his-3; hpo-gfp::loxP::hph+::loxP Honda & Selker (2008)

N3383 mat a his-3; dmm-1-3xHA::loxP::hph+::loxP this study

N3384 mat A his-3; dmm-1-3xHA::loxP::hph+::loxP this study

N3385 mat a; ∆dmm-2::hph+ FGSC #11100

N3388 mat A his-3; ∆dmm-2::hph+; inl this study

N3389 mat a his-3; ∆dmm-2::hph+; inl this study

N3390 mat A; ∆dmm-1::hph+; ∆dmm-2::hph

+ this study

N3391 mat A his-3+::Pdmm-1::dmm-1-3xFLAG; ∆dmm-1::hph

+; ∆dmm-2::hph

+ this study

N3437 mat a his-3+::Pdmm-2::3xFLAG-dmm-2; ∆dmm-2::hph

+ this study

N3440 mat a his-3+::Pdmm-1::dmm-1

R1060A-3xFLAG; ∆dmm-1::hph

+ this study

N3441 mat A his-3+::Pdmm-1::dmm-1

R1090A-3xFLAG; ∆dmm-1::hph

+ this study

N3442 mat A his-3+::Pdmm-1::dmm-1

R1150A-3xFLAG; ∆dmm-1::hph

+ this study

N3443 mat A his-3+::Pdmm-1::dmm-1

R1060A& R1090A-3xFLAG; ∆dmm-1::hph

+ this study

N3444 mat A his-3+::Pdmm-1::dmm-1

R1060A& R1150A-3xFLAG; ∆dmm-1::hph

+ this study

N3445 mat a his-3+::Pdmm-1::dmm-1

R1090A& R1150A-3xFLAG; ∆dmm-1::hph

+ this study

N3446 mat a his-3+::Pdmm-1::dmm-1

3xATh-mut-3xFLAG; ∆dmm-1::hph

+ this study

N3447 mat A his-3+::Pdmm-1::dmm-1

ATh-3xFLAG; ∆dmm-1::hph

+ this study

N3453 mat a his-3+::Pdmm-2::3xFLAG-dmm-2; ∆dmm-2::hph

+; dmm-1-

3xHA::loxP::hph+::loxP

this study

N3453 mat a his-3+::Pdmm-2::3xFLAG-dmm-2; ∆dmm-2::hph

+; ∆dmm-1::hph

+ this study

N3458 mat a his-3+::Pdmm-2::3xFLAG-dmm-2; ∆dmm-2::hph

+; ∆dim-2::hph

+ this study

N3486 mat a his-3+::Pdmm-2::3xFLAG-dmm-2; ∆dmm-2::hph

+; dim-5 leu-2 pan-1 this study

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Honda et al.

N3487 mat a his-3+::Pdmm-2::3xFLAG-dmm-2; ∆dmm-2::hph

+; hpo

RIP2 this study

N3490 mat A his-3+::Pdmm-1::dmm-1-3xFLAG; ∆dmm-1::hph

+ this study

N3579 1-7 mat a his-3; ∆dmm-1::hph+; dim-5 leu-2 pan-1 this study

N3580 1-2 mat A his-3+::Pdmm-1::dmm-1-3xFLAG; ∆dmm-1::hph

+; dim-5 leu-2 pan-1 this study

N3581 1-2 mat a his-3; ∆dmm-1::hph+; hpo

RIP2 this study

N3582 1-2 mat A his-3+::Pdmm-1::dmm-1-3xFLAG; ∆dmm-1::hph

+; hpo

RIP2 this study

N3583 1-7 mat a his-3; ∆dmm-1::hph+; ∆dim-2::hph

+ this study

N3584 mat a his-3+::Pdmm-1::dmm-1-3xFLAG; ∆dmm-1::hph

+; ∆dim-2::hph

+ this study

N3585 mat A his-3+::Pdmm-1::dmm-1-3xFLAG; ∆dmm-1::hph

+; ∆dim-2::hph

+ this study

N3586 mat A his-3+::Pdmm-1::dmm-1-3xFLAG; ∆dmm-1::hph

+ this study

N3587 mat a his-3+::Pdmm-1::dmm-1-3xFLAG; ∆dmm-1::hph

+ this study

N3588 1-7 mat a his-3; ∆dmm-1::hph+ this study

N3589 1-3 mat a his-3+::Pdmm-1::dmm-1

H216A-3xFLAG; ∆dmm-1::hph

+ this study

N3590 mat A his-3+::Pdmm-1::dmm-1

CysR1-3xFLAG; ∆dmm-1::hph

+ this study

N3591 mat A his-3+::Pdmm-1::dmm-1

CysR2-3xFLAG; ∆dmm-1::hph

+ this study

N3592 mat A his-3+::Pdmm-1::dmm-1

CysR1&2-3xFLAG; ∆dmm-1::hph

+ this study

N3593 mat A his-3+::Pdmm-1::dmm-1

C484A-3xFLAG; ∆dmm-1::hph

+ this study

N3599 mat A his-3; ∆dmm-1::hph+ this study

N3600 1-4 mat a his-3 this study

N3601 1-4 mat A his-3; ∆dim-2::hph+ this study

N3602 1-4 mat A his-3+::Pdmm-1::dmm-1

H216A-3xFLAG; ∆dmm-1::hph

+ this study

N3604 1-4 mat A his-3; dim-5 leu-2 pan-1 this study

N3605 1-2 mat a his-3; dim-5 leu-2 pan-1 this study

N3606 1-9 mat A his-3; ∆dmm-2::hph+ this study

N3607 1-3 mat A his-3; ∆dmm-2::hph+; ∆dim-2::hph

+ this study

N3608 1-4 mat A his-3; ∆dmm-2::hph+; dim-5 leu-2 pan-1 this study

N3609 1-3 mat A his-3; ∆dmm-2::hph+; hpo

RIP2 this study

N3657 mat a his-3+::Pdmm-2::3xFLAG-dmm-2; ∆dmm-2::hph

+; dmm-1-

3xHA::loxP::hph+::loxP; hpo-gfp::loxP::hph

+::loxP

this study

N3676 mat A his-3; ∆dmm-2::hph+; hpo-gfp::loxP::hph

+::loxP this study

N3677 mat A his-3; ∆dmm-2::hph+; hpo-gfp::loxP::hph

+::loxP; ∆dim-2::hph

+ this study

N3678 mat A his-3; ∆dmm-1::hph+; hpo-gfp::loxP::hph

+::loxP this study

N3679 mat A his-3; ∆dmm-1::hph+; hpo-gfp::loxP::hph

+::loxP; ∆dim-2::hph

+ this study

N3682 mat a his-3; dmm-1-3xHA::loxP::hph+::loxP; hpo-FLAG::loxP::hph

+::loxP this study

N3683 mat A his-3; dmm-1-3xHA::loxP::hph+::loxP this study

N3706 mat a his-3; dmm-1-3xHA::loxP::hph+::loxP; hpo-FLAG::loxP::hph

+::loxP;

∆dim-2::hph+

this study

N3707 mat A his-3; dmm-1-3xHA::loxP::hph+::loxP; hpo-FLAG::loxP::hph

+::loxP;

dim-5 leu-2 pan-1

this study

N3708 mat a his-3; dmm-1-3xHA::loxP::hph+::loxP; hpo-FLAG::loxP::hph

+::loxP;

∆dmm-2::hph+

this study

N3709 mat A his-3; dmm-1-3xHA::loxP::hph+::loxP; hpo-FLAG::loxP::hph

+::loxP;

∆dmm-2::hph+

this study

N3710 mat a his-3; dmm-1-3xHA::loxP::hph+::loxP; hpo-FLAG::loxP::hph

+::loxP;

∆dmm-2::hph+; ∆dim-2::hph

+

this study

N3711 mat A his-3; dmm-1-3xHA::loxP::hph+::loxP; hpo-FLAG::loxP::hph

+::loxP;

∆dmm-2::hph+; ∆dim-2::hph

+

this study

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Honda et al.

Supplementary Table SIV. Primers used in this study

Primer Sequence

Gene disruption of dmm-1

hph gene

#1632 5'-CCGTCGACAGAAGATGATATTGAAGGAGC-3'

#1633 5'-AGCTGACATCGACACCAACG-3'

dmm-1 5’

#1707 5’-GTAACGCCAGGGTTTTCCCAGTCACGACGCGGCTAACATGACGATGATG-3’

#1712 5’-ACCGGGATCCACTTAACGTTACTGAAATCATATTCTCGAAGGGGTTCGG-3’

dmm-1 3’

#1717 5’-GCTCCTTCAATATCATCTTCTGTCGACGGCCCAGTAAGCGTTTGTTGTC-3’

#1722 5’-GCGGATAACAATTTCACACAGGAAACAGCCGAATTATCTGCAGCTTGCC-3’

Knock-in

dmm-1 5’

#2060 5’-GAGGTCGACGGTATCGATAAGCTTGATATGGAACCCGCGGACAGTAGTC-3’

#2061 5’-CCTCCGCCTCCGCCTCCGCCGCCTCCGCCCGAACCCCTAGGCCTACCCC-3’

dmm-1 3’

#2062 5’-TGCTATACGAAGTTATGGATCCGAGCTCGAGCGAACGAGGATGAGATATG-3’

#2063 5’-ACCGCGGTGGCGGCCGCTCTAGAACTAGTTGAGTGAATGTGCGGAGACG-3’

his-3 targetting vectors

dmm-1

#1794 5’-GCTGGAGCTCCACCGCGGTGGCGGCCGCTTCCCGCATTCGCGCTGCTTC-3’

#1808 5’-CCTCCGCCTCCGCCTCCGCCGCCTCCGCCCGAACCCCTAGGCCTACCCC-3’

dmm-1 ORF

#1973 5’-GCTCTAGAATGCCGGCCGCATCCCATCC-3’

#1972 5’-CCTTAATTAACGAACCCCTAGGCCTACCC-3’

actin promoter

#1974 5’-CGCGGCCGCCGGGCACTCACTGTCAGT-3’

#1975 5’-GCTCTAGATAGATTAGGTGGTGACGA-3’

dmm-2 promoter

#2095 5’-GGAATTCCTATCTGCAGGTGATTCC-3’

#2094 5’-CGGGATCCGTGCTTGATTCTCAATCAC-3’

dmm-2 ORF

#2096 5’-CGGCGCGCCGGAGGCGTTGCTAGGCGCTTC-3’

#2097 5’-GCTCTAGATGAGACGAGAACAGACATGC-3’

Probe

8:G3-1 (462 bp)

#1829 5’-ATTTGCGATCGGAACACACC-3’

#1830 5’-GTTGATGGGAGTTGATCAGG-3’

8:G3-2 (3773 bp)

#1831 5’-CATCACCCTGATCAACTCCC-3’

#1832 5’-AGTAGCCAGTTCCCGAAAGC-3’

8:G3-3 (562 bp)

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Honda et al.

#1833 5’-ACATCTGGGCCAAGATCAGC-3’

#1834 5’-CACTACGACCTGATCACAGG-3’

8:G3-4 (497 bp)

#1835 5’-CTTGAGGACGACAATCGAGG-3’

#1836 5’-ACTCCGATCTTGGTCTCTCC-3’

8:G3-5 (579 bp)

#1837 5’-TCCCCACTGGGATCCGGATG-3’

#1838 5’-ACAGCTCATCTCCCGAGATC-3’

8:G3-6 (588 bp)

#1839 5’-TGTCCTAGATCTGAGGATCG-3’

#1840 5’-TCGACGTATTGGATGGATCC-3’

8:G3-7 (633 bp)

#1841 5’-GGGATCCATCCAATACGTCG-3’

#1842 5’-AATGACGGCGACAAGGTTCG-3’

8:G3-8 (486 bp)

#1843 5’-GGGTGCAAAGATCTTCGTCG-3’

#1844 5’-GATCAGTATCGGGATCGTCG-3’

8:G3-9 (765 bp)

#1845 5’-CGATTCCTCGGACCTCGATC-3’

#1846 5’-TCATGCCGCTTCGTGATCTG-3’

8:G3-10 (514 bp)

#1847 5’-GGGACAAACGATCCAATGGC-3’

#1848 5’-GATCTCATCTGTGCGGATGG-3’

8:G3-11 (445 bp)

#1849 5’-CATCCGCACAGATGAGATCC-3’

#1850 5’-AGGTAACTCTGAGCTTCGGG-3’

8:G3-12 (1239 bp)

#1922 5’-TCTTCGTGACCTGTCTGACG-3’

#1923 5’-CTTCTAGAACGAGTGGCATCC-3’

8:G3-13 (811 bp)

#1851 5’-GATCTTGAGGATGCCACTCG-3’

#1852 5’-CCGATCGGAAGATTTCGTCG-3’

8:G3-14 (640 bp)

#1853 5’-TGTGGCTGTTGCCTTTTGCG-3’

#1854 5’-CTTGATCTTTGCTCGGTGGG-3’

8:G3-15 (525 bp)

#1855 5’-ACCGAGCAAAGATCAAGAGC-3’

#1856 5’-TGACGATCTCGGTCGCTAGC-3’

8:A6-1 (606 bp)

#1877 5’-TGGTTGGTCGATTGTGGTGG-3’

#1876 5’-TTTTGAGGATCCGCCATCCG-3’

8:A6-2 (721 bp)

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#1879 5’-TATCCCGGGGAATGACAACG-3’

#1880 5’-ACACAGACGCAAGCACAAGC-3’

8:F10-1 (554 bp)

#1892 5’-TCTTTCCAGAATCATGATCG-3’

#1893 5’-ACGGACATTTGATCTTGTCG-3’

8:F10-2 (808 bp)

#1894 5’-TACCCAGGGCGATCCGCTAG-3’

#1895 5’-TCCCTTGGCTTGTATGATCG-3’

63-1 (483 bp)

#1907 5’-TAAGTTTACCCTCTCCGCAGG-3’

#1908 5’-AAGATCTCTCCCGTCTGAGG-3’

63-2 (615 bp)

#1909 5’-ACAATGCCTTGCTAGCCGAG-3’

#1910 5’-CGCTCTCCCATAGATAAGCC-3’

63-3 (790 bp)

#1911 5’-AGGTAACCGGATCTTGCAGG-3’

#1912 5’-CCTCGGACTTCAGAGGCTTA-3’

Peak11-L (602 bp)

#2358 5’-TAGGAGATGCCGTTCGGTTG-3’

#2359 5’-CGGTTCAGGCTGCTATGTCG-3’

Peak11-R (949 bp)

#2360 5’-TTGAGCGGTACGTTAGATCC-3’

#2361 5’-TCCGGATCCTCTTCCTTAGG-3’

Peak12 (461 bp)

#2364 5’-CGCTTAAGGTAGAGATCTCC-3’

#2365 5’-GGACTATTACCGCCGATTAG-3’

Peak21 (1024 bp)

#2366 5’-CTTGATGGTGTTGCTGCTGC-3’

#2367 5’-GTAGCCACGTTCACATACCC-3’

Peak35-a (733 bp)

#2370 5’-GCCACAATCGATCAGATCGG-3’

#2371 5’-GAGAGAAGATGCTCTCCAGG-3’

Peak35-b (842 bp)

#2368 5’-ACGAAGCTCTCCTTGTCGTC-3’

#2369 5’-GTTAGAATTCGCCCTGGAGC-3’

Bisulfite sequencing

#2026 5’-ACATTCACATCTCAATCTC-3’

#2027 5’-GAAAGAGGGATGGGGAAGAAAATAA-3’

ChIP

hH4-1 (425 bp)

#1924 5’-AACCACCGAAACCGTAGAGGGTAC-3’

#1925 5’-ATCGCCGACACCGTGTGTTGTAAC-3’

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Honda et al.

8:A6 (303 bp)

#1823 5’-GGATGGCGGATCCTCAAAAATA-3’

#1824 5’-TAACCGCCGCTTTTTAAAATTAGGA-3’

8:G3-1 (290 bp)

#1860 5’-ACCGAAACCGACGTCTTGAC-3’

#1834 5’-CACTACGACCTGATCACAGG-3’

8:G3-2 (245 bp)

#1861 5’-CACCAAGACACTGGCAATCC-3’

#1838 5’-ACAGCTCATCTCCCGAGATC-3’

8:G3-3 (562 bp)

#1862 5’-GTTCAATTCCGCGTTTGAGG-3’

#1840 5’-TCGACGTATTGGATGGATCC-3’

8:G3-4 (273 bp)

#1863 5’-GCTCAAGCTCAAAGCCAACC-3’

#1842 5’-AATGACGGCGACAAGGTTCG-3’

8:G3-5 (264 bp)

#1864 5’-AAACGCGTTACGGCTCTTGC-3’

#1865 5’-CGTGCTTTAGCGTTACTCCC-3’

8:G3-6 (231 bp)

#1866 5’-CGTAGAGAAGGGAAGTAGTAGAAGG-3’

#1867 5’-GCACAATACGAAGTCACTTTTCACC-3’

8:G3-7 (232 bp)

#1868 5’-TGGTCATGGTTGGGGAAAGG-3’

#1869 5’-GTCCGGGTAACTTGATGTGG-3’

8:G3-8 (232 bp)

#1843 5’-GGGTGCAAAGATCTTCGTCG-3’

#1870 5’-TCACATTTAGTCGGCAGAGG-3’

8:G3-9 (258 bp)

#1871 5’-CTGCGTCTAAAAGTGCACCG-3’

#1846 5’-CAGATCACGAAGCGGCATGA-3’

8:G3-10 (269 bp)

#1847 5’-GGGACAAACGATCCAATGGC-3’

#1872 5’-CATCTCTCCATTCAGCCAGG-3’

8:G3-11 (210 bp)

#1873 5’-ATAGAGGAACTCCGGGTACG-3’

#1850 5’-AGGTAACTCTGAGCTTCGGG-3’

8:G3-12 (215 bp)

#1851 5’-GATCTTGAGGATGCCACTCG-3’

#1874 5’- GGGTGGACATACCAAACAGC-3’

Peak11-1 (243 bp)

#2485 5’-AGACAGGACAAAGCGTCAGC-3’

#2486 5’-TCGGGATATCCTCAGCAAGG-3’

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Honda et al.

Peak11-2 (218 bp)

#2358 5’-TAGGAGATGCCGTTCGGTTG-3’

#2423 5’-TGGTTATTCGAGCGCGTTCG-3’

Peak11-3 (237 bp)

#2477 5’-GCAATGTGCATTGTGTGATGTGC-3’

#2478 5’-TGCCCAGATGGAGGATTACC-3’

Peak11-4 (297 bp)

#2479 5’-TTATAAGGCTACTAAGTAAAGTATTCG-3’

#2480 5’-CTAGGATCTAACGTACCGCTC-3’

Peak33-1 ( bp)

# 5’-CGACGGAACAAATTACTACTATACACAAC-3’

# 5’-GGTTTTCGTATAGTAAGTTACCCGCTTC-3’

Peak33-2 ( bp)

# 5’-AGGTTTTTGTTGTGTGCAGATG-3’

# 5’-AGGATGGCTGCAAGGCAGAAC-3’

Peak33-3 ( bp)

#2481 5’-GCCCAATTTGGCAAACCTGG-3’

#2482 5’-GACCTTGCATAGACACGACG-3’

Peak33-4 ( bp)

#2483 5’-TGGTTCCAGCCTCATCATCC-3’

#2484 5’-AGAGTAGGCTCGGAAGTTGG-3’

Peak33-5 ( bp)

#2487 5’-AGTTCATGGCGTGCACAACG-3’

#2488 5’-ATGGGGACACTGCTTGTAGG-3’

Mutagenesis

dmm-1H216A

#2058 5’-CATACACCCCGGCCGCTCGCGAAATGTGCG-3’

#2059 5’-CGCACATTTCGCGAGCGGCCGGGGTGTATG-3’

dmm-1R1060A

#2351 5’-CATAAGAAGCGAGGAGCTCCTCCCGGTCGGCC-3’

#2352 5’-GGCCGACCGGGAGGAGCTCCTCGCTTCTTATG-3’

dmm-1R1090A

#1930 5’-ACTCGGAGACGCGGGGCCCCCCCACGCGCATCA-3’

#1931 5’-TGATGCGCGTGGGGGGGCCCCGCGTCTCCGAGT-3’

dmm-1R1150A

#1932 5’-CCTAAGAGGAGAGGGGCCCCACCCAAGAATGGC-3’

#1933 5’-GCCATTCTTGGGTGGGGCCCCTCTCCTCTTAGG-3’

dmm-1ATh

#1926 5’-CCATCAAAGCTACGTGGGCCGAAGGTCGTGGTTGAGGTCACT-3’

#1927 5’-AGTGACCTCAACCACGACCTTCGGCCCACGTAGCTTTGATGG-3’

dmm-1CysR1

#2387 5’-AGTACATTGAGTTTGACTCCGTTTCCAACCTCACTTGGGC-3’

#2388 5’-GCCCAAGTGAGGTTGGAAACGGAGTCAAACTCAATGTACT-3’

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dmm-1CysR2

#2389 5’-AGAAGGGCGATGTTTACCGTCTCAAGATCTGCAACTGCGG-3’

#2390 5’-CCGCAGTTGCAGATCTTGAGACGGTAAACATCGCCCTTCT-3’

dmm-1C484A

#2415 5’-CATTACCTGCTCGTACGCCAGAGCCAACATCTTC-3’

#2416 5’-GAAGATGTTGGCTCTGGCGTACGAGCAGGTAATG-3’