NCU01005 1 k b NCU07896 NCU07897 1 k 8:A6 Y63...
Transcript of NCU01005 1 k b NCU07896 NCU07897 1 k 8:A6 Y63...
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
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
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
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
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
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
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
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
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
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
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
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
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
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
WT
hpo
dim-5
∆dim-2
WB: αH3K4me2
WB: αH3K9me3
∆dmm-1
∆dmm-2
∆dmm-1,
∆dim-2
Honda_Supplementary Fig. 16
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
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
+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
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
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
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
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
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
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.
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.
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
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
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)
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)
Honda et al.
#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’
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’
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’
Honda et al.
dmm-1CysR2
#2389 5’-AGAAGGGCGATGTTTACCGTCTCAAGATCTGCAACTGCGG-3’
#2390 5’-CCGCAGTTGCAGATCTTGAGACGGTAAACATCGCCCTTCT-3’
dmm-1C484A
#2415 5’-CATTACCTGCTCGTACGCCAGAGCCAACATCTTC-3’
#2416 5’-GAAGATGTTGGCTCTGGCGTACGAGCAGGTAATG-3’