supplementary material - ec.asm.org€¦ · 8/28/2013 · 1" supplementary material 2"...

Supplementary Material 1

2

SUPPLEMENTARY METHODS 3

4

Single cell GFP analysis. The analysis was performed as shown by Lohse and 5

Johnson (1) with modifications. Fresh opaque single colonies with both allele of WOR1 6

tagged with GFP (AZC90, AZC100, AZC101, and AZC97) were growing in SC+Glu 7

media at 25°C for 4 hrs till OD600 around 0.6. Cells were harvested and washed once 8

with PBS before images with DeltaVision Imaging System (Applied Precision) under the 9

GFP channel. The GFP intensity was calculated as the average GFP intensity in cell 10

nuclei analyzed with ImageJ64 (NIH) and MATLAB (MathWorks) (scripts upon request). 11

12

SUPPLEMENTAL FIGURES 13

14

FIG S1 Noise in WOR1 expression in selective mutants are no different from WT. Wor1 15

protein levels in single WOR1-GFP tagged opaque cells were analyzed by GFP signal 16

intensity. F-statistics analysis were performed and showed that there were no difference 17

in the variance of WOR1-GFP signals between med16Δ/Δ, med1Δ/Δ, med20Δ/Δ and 18

WT. More than 200 single cells were analyzed for each strain. Boxes represent 25% 19

percentile and 75% percentile with median. Whiskers represent minimum and maximum. 20

21

FIG S2 Morphology of cells with or without Wor1 overexpression in WT, med3Δ/Δ and 22

med3Δ/Δ complementation strain. Cells transformed with Pmal-WOR1-3HA cassette 23

were growing in either SC+Glu (WOR1 OFF) or SC+Mal (WOR1 ON) overnight at 25°C. 24

The pictures are representatives of cells analyzed in Fig. 5D. 25

SUPPLEMENTAL TABLES 26

27 TABLE S1 Strains used in this study

Name Genotype MTL Source AZC19 (SN87)

leu2 /leu2 his1 /his1 URA3/ura3 ::imm434 IRO1/iro1 ::imm434 α/α 2

AZC133 (RBY1179) arg4::hisG/arg4::hisG his1::hisG/HIS1* a/a 3

AZC22 (SN152)

arg4/arg4 leu2/leu2 his1 /his1 URA3/ura3 ::imm434 IRO1/iro1 ::imm434 a/a 2

AZC32 arg4/arg4 URA3/ura3::imm434

IRO1/iro1::imm434 med5△::HIS1/med5△::LEU2

a/a This study



a/a This study



a/a This study



a/a This study



a/a This study

AZC51 (yLM95)

arg4/arg4 URA3/ura3::imm434 IRO1/iro1::imm434

med3△::HIS1/med3△::LEU2 a/a 4

cTTR01 arg4/arg4 URA3/ura3::imm434


a/a This study



a/a This study

AZC59 leu2/leu2 URA3/ura3::imm434

IRO1/iro1::imm434 med16△::HIS1/med16△::MED3+ARG4

a/a This study



a/a This study



a/a This study



a/a This study



a/a 4

cTTR02 leu2/leu2 URA3/ura3::imm434


a/a This study



a/a This study

AZC102 arg4/arg4 leu2/leu2 his1 /his1

URA3/ura3::imm434 IRO1/iro1::imm434 ADH1/adh1:Pmal2-WOR1-3HA-SAT1

a/a This study

AZC103


med3△::HIS1/med3△::LEU2 ADH1/adh1:Pmal2-WOR1-3HA-SAT1

a/a This study

AZC104

leu2/leu2 URA3/ura3::imm434 IRO1/iro1::imm434

med3△::HIS1/med3△::MED3+ARG4 ADH1/adh1:Pmal2-WOR1-3HA-SAT1

a/a This study

AZC125


med12△::HIS1/med12△::LEU2 ADH1/adh1:Pmal2-WOR1-3HA-SAT1

a/a This study

AZC134

leu2/leu2 URA3/ura3::imm434 IRO1/iro1::imm434

med12△::HIS1/med12△::MED12+ARG4 ADH1/adh1:Pmal2-WOR1-3HA-SAT1

a/a This study

AZC90 arg4/arg4 leu2/leu2 his1 /his1

URA3/ura3 ::imm434 IRO1/iro1 ::imm434 WOR1:GFP/WOR1:GFP

a/a This study

AZC100


med16△::HIS1/med16△::LEU2 WOR1:GFP/WOR1:GFP

a/a This study

AZC101



a/a This study

AZC97



a/a This study

cTTR03 (BWP17

background)

MED8::MED8-6his3FLAG-SAT1/MED8::MED8-6his3FLAG-HIS3

MED3/MED3::MED3-3HA-ARG4 a/α This study

cTTR04

URA3/ura3::imm434 IRO1/iro1::imm434 med16△::HIS1/med16△::LEU2

MED8/MED8::MED8-6his3FLAG-ARG4 MED3/MED3::MED3-3HA-SAT1

a/a This study

cTTR05

URA3/ura3::imm434 IRO1/iro1::imm434 med16△::HIS1/med16△::LEU2

MED15/MED15::MED15-6his3FLAG-SAT1 MED3/MED3::MED3-3HA-ARG4

a/a This study

28

29

TABLE S2 Primers used in this study Number Sequence (5'---3') Name Reference

AZ061 ATTAGATACGTTGGTGGTTC HIS1 left 2

AZ062 AACACAACTGCACAATCTGG HIS1 right 2

AZ063 AAACTTTGAACCCGGCTGCG LEU2 left 2

AZ064 AGAATTCCCAACTTTGTCTG LEU2 right 2

AZ067 AATCGTCAAAAGTACACGACC ARG4 left This study AZ068 CCTGTATTCCATTGTATCGCC ARG4 right This study

AZ069 gcagggatgcggccgctgacAGCTCGGATCCACTAGTAACG universal P2 2

AZ070 ccgctgctaggcgcgccgtgACCAGTGTGATGGATATCTGC universal P5 2

AZ071 GCTATCTCCTTATCATCCACA MED1 P1 This study

AZ072 cacggcgcgcctagcagcggTGGTTCCTGGCATTTGTATTG MED1 P3 This study

AZ073 gtcagcggccgcatccctgcCTGTATGTTTAGAACCGCATAG MED1 P4 This study

AZ074 TATTCGCAACAAATACACGCC MED1 P6 This study

AZ007 GAGAAggtaccTCATCATTGGGATGTTATCTGTTTG MED1 check 5' This study

AZ010 GAGAAgagctcCTTCCGGACCCAATGGAATATG MED1 check 3' This study

AZ148 GAGAAggtaccGCTATCTCCTTATCATCCACA

MED1 complement 5' F (KpnI) This study

AZ149 GAGAActtaagCTATGCGGTTCTAAACATACAG

MED1 complement 5' R (AflII) This study

AZ151 GAGAAgcggccgcCTGTATGTTTAGAACCGCATAG

MED1 complement 3' F (NotI) This study

AZ152 CACCATCATCATctcgagTATAAAAATAT

MED1 complement 3' R (XhoI) This study

AZ075 GATTCAGTTTGGCAGGGCAGAAA MED5 P1 This study

AZ076 cacggcgcgcctagcagcggGCTTGAAACAGCTTGACCCCAA MED5 P3 This study

AZ077 gtcagcggccgcatccctgcCTTGTCTAGTAGTAGTAGGTTTC MED5 P4 This study

AZ078 GTATCACCGGTGGAGAAATAC MED5 P6 This study

AZ055 GAGAAggtacc ATGACTTTATGTCTTCCTTCATC MED5 check 5' This study

AZ030 GCCgagctcATTGGTATTGAACCAACACAAG MED5 check 3' This study

AZ079 GAAGTTGTCAGTTGGGTCAGTA MED16 P1 This study

AZ080 cacggcgcgcctagcagcggTTTGGTTGACTTGGTGGGTTG MED16 P3 This study

AZ081 gtcagcggccgcatccctgcGTGTGGCAATGCATGGGTAAA MED16 P4 This study

AZ082 AATTCTACAGCTGCAAGTCATGG MED16 P6 This study

AZ033 GCGggtaccCAACAAGAAGTTGTTTCTAATGC MED16 check 5' This study

AZ036 GCCgagctcCTACTTCTTTAAGTACCAGTTCAC MED16 check 3' This study

AZ135 GAGAAggtaccGAAGTTGTCAGTTGGGTCAG

MED16 complement 5' F

(KpnI) This study

AZ136 GAGAAggatccATTTACAAACTATTAATTGA

MED16 complement 5' R

(BamHI) This study

AZ137 GAGAAgcggccgcGTTTAATTCCATGTCAAAAT


(NotI) This study

AZ138 GAGAAtctagaGGTATACAAAATACCACCAGTAG


(XbaI) This study

AZ095 CTCTCATCTTGAATTCCATAGG MED9 P1 This study

AZ096 cacggcgcgcctagcagcggCACCTTCAAACAAGCAATTG MED9 P3 This study

AZ097 gtcagcggccgcatccctgcCGAAGAGATATGAAGGGCCG MED9 P4 This study

AZ098 AGCAACAACAAGTGACATGGTA MED9 P6 This study

AZ015 GCCggtaccCTTCAAGTCATGATGAGCAAATG MED9 check 5' This study

AZ016 GCCctcgagCACCTTCAAACAAGCAATTGAC MED9 check 3' This study

AZ153 GAGAAggtaccCTCTCATCTTGAATTCCATAGG

MED9 complement 5' F (KpnI) This study

AZ154 GAGAActtaagATACGGCCCTTCATATCTCTTCG

MED9 complement 5' R (AflII) This study

AZ156 GAGAAgcggccgcCGAAGAGATATGAAGGGCCG

MED9 complement 3' F (NotI) This study

AZ157 GAGAAtctagaAGCAACAACAAGTGACATGGTA

MED9 complement 3' R (XbaI) This study

AZ099 CACTCCATTATGACTTTTGCTC MED20 P1 This study

AZ100 cacggcgcgcctagcagcggTGTTGCGAGGTCTGTTAAGA MED20 P3 This study

AZ101 gtcagcggccgcatccctgcGATTGTAAGTTGACCACATA MED20 P4 This study

AZ102 CACTGAATTGAGATACGGATGC MED20 P6 This study

AZ021 GAGTTGGTACCACCAACAGCAACTAAGTAGTT MED20 check 5' This study

AZ022 GCGctcgagTGTTGCGAGGTCTGTTAAGAAG MED20 check 3' This study

AZ158 GAGAAggtaccCACTCCATTATGACTTTTGCTC


(KpnI) This study

AZ159 GAGAActtaagTATGTGGTCAACTTACAATC


(AflII) This study

AZ161 GAGAAgcggccgcGATTGTAAGTTGACCACATA


(NotI) This study

AZ162 GAGAActcgagCACTGAATTGAGATACGGATGC


(XhoI) This study

TRR01 CCACAATACAACCCTACC MED15 P1 This study

TRR02 cacggcgcgcctagcagcggAATAGGCAATCCGGACAC MED15 P3 This study

TRR03 gtcagcggccgcatccctgcAGCTGACTTTTAGTAATC MED15 P4 This study

TRR04 TAACGTTTGATATGTGCC MED15 P6 This study

TRR05 GACAATCAAAATTTCTCATATCAAC MED15 check 5' This study

TRR06 GGAGGTTATGGTTGTTCC MED15 check 3' This study

TRR07 CTCATAggatccCCACAATACAACCCTACC


(BamHI) This study

TRR08 TCAACcttaagTTACTAAAAGTCAGCTTTATTGTATG


(AflII) This study

TRR09 GTTTCTTCCAgcggccgcAGCTGACTTTTAGTAATC


(NotI) This study

TRR10 GCAAATGctcgagTAACGTTTGATATGTGCC


(XhoI) This study

AZ109 GACTGTAGTATACTCTCCGTTGTTATTG MED12 P1 This study

AZ110 cacggcgcgcctagcagcggTAGACATGATACAAGGAATC MED12 P3 This study

AZ111 gtcagcggccgcatccctgcCCCTCGTTAATAGTATGTAG MED12 P4 This study

AZ112 CTCTCTGGCGGTGTTGGACC MED12 P6 This study AZ113 AGCGGGGTCATTAGCAGATGC MED12 check 5' This study

AZ114 GTATCACTTGTATCTGAAACTGCAC MED12 check 3' This study

AZ163 GAGAAggtaccGACTGTAGTATACTCTCCGTTGTTATTG

MED12 1st half complement 5' F

(KpnI) This study

AZ164 GATCAACagtactACCTGGTTC MED12 1st half

complement 5' R (ScaI)

This study

AZ165 GAACCAGGTagtactGTTGATC MED12 2nd half complement 5' F

(ScaI) This study

AZ166 GAGAAggatccCTACATACTATTAACGAGGG

MED12 2nd half complement 5' R

(BamHI) This study

AZ168 GAGAAgcggccgcCCCTCGTTAATAGTATGTAG


(NotI) This study

AZ169 GAGAActcgagCTCTCTGGCGGTGTTGGACC


(XhoI) This study

AZ191 GTCGACATGTCAAAGGATTC adh1:Pmal2-

WOR1-3HA check 5' F

This study

AZ192 CAATGTAGGCTGCTCTACACC adh1:Pmal2-

WOR1-3HA check 5' R

This study

AZ193 GGGAGTGGTAAACTCGACTT adh1:Pmal2-

WOR1-3HA check 3' F

This study

AZ194 GTGTCCAATACGTATCTACTC adh1:Pmal2-

WOR1-3HA check 3' R

This study

AZq001 CATCACAACCAGGTTCTACAACCAAT EFG1 RT-qPCR F 5

AZq003 GCTGtTGGTATGGATATGATGATGC EFG1 RT-qPCR R 5

AZq004 CAGAACAATTCAAGGATAAGGTTACTG WH11 RT-qPCR F 5

AZq005 TTGGAGTCACCAAAAATAGCATCAG WH11 RT-qPCR R 5

AZq006 CCTCAAAAGCTGCTACCTC OP4 RT-qPCR F 5

AZq007 GTATCAACAGTTGGAGTAGAAGTAG OP4 RT-qPCR R 5

AZq008 CAAGCACCAATTCATCTTGCC WOR2 RT-qPCR F 6

AZq009 TCTTTCTGCAAGTAAGACAGCCAG WOR2 RT-qPCR R 6

AZq010 GTATTCTGCTGCTGGTA CZF1 RT-qPCR F This study AZq011 TTGTTGCTTGACTTGTTG CZF1 RT-qPCR R This study

AZcp007 CACGAACAACAGGAGTAGG WOR1 RT-qPCR F 6

AZcp008 GCCATTACCACCACTAAC WOR1 RT-qPCR R 6

AZcp013 AGTTTCTGGAAGTGTATTTGTGTT WOR1 5'UTR RT-qPCR F 6

AZcp014 CGACCTTGGATTCTTGATGG WOR1 5'UTR RT-qPCR R 6

AZq012 GCTGGAACAGATGACGCAG WOR1-3HA RT-qPCR F This study

AZq026 TGGTGATGGTGTTACTCACG ACT1 RT-qPCR F 7

AZq027 GACAATTTCTCTTTCAGCAC ACT1 RT-qPCR R 7

AZ195 TAGAAGTGGCGCGCCTCAGC WOR1-3HA RT-qPCR R This study

AZ186 CAACAACAACAGTAGCTCACGAACAACAGG

WOR1 flank check within the ORF F 1

AZ187 GATATGAGAATTAGGGTTATGGTATGATGATTTTCTGGATTTCCG

WOR1 flank check 3'HR R 1

AZ188 GGTTGGCCATGGAACTGGCA GFP check toward the end of WOR1 1

AZ189 GGTGATGGTCCAGTCTTGTTACCAGAC

GFP check toward SAT1 flipper 1

LM017

CAAAGAAAGCAACCAAGAGGGCCTCGGTGAGCAATAAAGATATGGGAAGTAGTGCCAATCAACCAATTGTCATTGGTCGACGGATCCCCG

MED3 tagging forward 4

LM018

TTGACCAGCAAGCTTTCAAACATCTACAAGTACAGTAGGAACTATGTCCAAGGCTTTATGCTCTTTCTCTTCGATGAATTCGAGCTCGTT

MED3 tagging reverse 4

LM025

TGAAAATAACTGCTGGTGGTCAAAAAAGTTTGCATCCAAATCAAGTATTGAAATTTATGTGTCAAGGACAGATAGGTCGACGGATCCCCC


LM014

AACACACAGTAATATTACATAAACCACCATCTTGTTCTTCTTCTACTTCATCCCCCCCTCTCCTAAAAATTTCGATGAATTCGAGCTCGT


LM024

TAGATTTTGATAGATATCTTTATGAACCTGTATCGTTTGATGAGTGGAAAGATTTTGTTGTTTCTTCCATACAAGGTCGACGGATCCCCC


LM012

ACGAAACGAAATATGTGCGAAAATAATATACACAGGTGTAAAGTTCAACCCTAGATTACTAAAAGTCAGCTTCGATGAATTCGAGCTCGTT


30

31

TABLE S3 Plasmid used in this study Name Parent Plasmid desription Target locus Reference pSN69 _ ARG4 marker _ 2

AZE042 pSN69 5'HR-MED16-ARG4-3'HR med16Δ/Δ This study AZE040 pSN69 5'HR-MED9-ARG4-3'HR med9Δ/Δ This study AZE036 pSN69 5'HR-MED20-ARG4-3'HR med2020Δ/Δ This study AZE039 pSN69 5'HR-MED1-ARG4-3'HR med1Δ/Δ This study

AZE030 pSN69 5'HR-MED3-ARG4-3'HR med3Δ/Δ 4

pTTR01 pSN69 5'HR-MED15-ARG4-3'HR med15Δ/Δ This study AZE043 pSN69 5'HR-MED12-ARG4-3'HR med12Δ/Δ This study Ectopic Wor1 _ adh1: Pmal-WOR1-3HA-

SAT1 ADH1 8

pMB126 _ WOR1-GFP-SAT1 tagging cassette WOR1 1

pFA-HA-

SAT1 _ 3XHA-SAT1 tagging

cassette MED3 9

pFA-HA-

ARG4 _ 3XHA-ARG4 tagging

cassette MED3 9

pFA-HF-SAT1 _ 6XHIS-FLAG-SAT1 tagging

cassette MED15 9

pFA-HF-ARG4 _ 6XHIS-FLAG-ARG4 tagging

cassette MED8 9

pFA-HF-HIS3 _ 6XHIS-FLAG-HIS3 tagging

cassette MED8 9

32

33

34

TABLE S4 Doubling time (in minutes) of Mediator mutants

White, SC+Glu,

25°C

Opaque, SC+Glu,

25°C

White, YPD, 30°C

WT 103±5 129±2 90±7 med5Δ/Δ 95±6 143±15 95±13 med16Δ/Δ 101±5 121±10 90±11 med9Δ/Δ 97±6 142±17 82±13 med20Δ/Δ 105±15 133±14 92±16 med1Δ/Δ 86±7 133±8 85±13 med12Δ/Δ 105±15 112±9 86±9 med3Δ/Δ 105±8 _ 87±3

35

The doubling time is represented as mean ± SD of three independent experiments 36

performed on different days. 37

38

Supplementary References 39

40

1. Lohse MB, Johnson AD. 2010. Temporal anatomy of an epigenetic switch in cell 41

programming: the white-opaque transition of C. albicans. Mol. Microbiol. 78:331-43. 42

43

2. Noble SM, Johnson AD. 2005. Strains and strategies for large-scale gene deletion 44

studies of the diploid human fungal pathogen Candida albicans. Eukaryot. Cell 45

4:298-309. 46

47

3. Bennett RJ, Johnson AD. 2006. The role of nutrient regulation and the Gpa2 protein 48

in the mating pheromone response of C. albicans. Mol Microbiol. 62:100-19. 49

50

4. Zhang A, Petrov KO, Hyun ER, Liu Z, Gerber SA, Myers LC. 2012. The Tlo 51

proteins are stoichiometric components of C. albicans Mediator anchored via the 52

Med3 subunit. Eukaryot. Cell 11:874-84. 53

54

5. Zordan RE, Galgoczy DJ, Johnson AD. 2006. Epigenetic properties of white-55

opaque switching in Candida albicans are based on a self-sustaining transcriptional 56

feedback loop. Proc. Natl. Acad. Sci. U. S. A. 103:12807-12. 57

58

6. Hnisz D, Schwarzmüller T, Kuchler K. 2009. Transcriptional loops meet chromatin: 59

a dual-layer network controls white-opaque switching in Candida albicans. Mol. 60

Microbiol. 74:1-15. 61

62

7. Lu Y, Su C, Liu H. 2012. A GATA transcription factor recruits Hda1 in response to 63

reduced Tor1 signaling to establish a hyphal chromatin state in Candida albicans. 64

PLoS Pathog. 8:e1002663. 65

66

8. Stevenson JS, Liu H. 2011. Regulation of white and opaque cell-type formation in 67

Candida albicans by Rtt109 and Hst3. Mol. Microbiol. 81:1078-91. 68

69

9. Lavoie H, Sellam A, Askew C, Nantel A, Whiteway M. 2008. A toolbox for epitope-70

tagging and genome-wide location analysis in Candida albicans. BMC Genomics 71

9:578. 72

FIG S1 Noise in WOR1 expression in selective mutants are no different from WT. Wor1 protein levels in single WOR1-GFP tagged opaque cells were analyzed by GFP signal intensity. F-statistics analysis were performed and showed that there were no difference in the variance of WOR1-GFP signals between med16Δ/Δ, med1Δ/Δ, med20Δ/Δ and WT. More than 200 single cells were analyzed for each strain. Boxes represent 25% percentile and 75% percentile with median. Whiskers represent minimum and maximum.

FIG S2 Morphology of cells with or without Wor1 overexpression in WT, med3Δ/Δ and med3Δ/Δ complementation strain. Cells transformed with Pmal-WOR1-3HA cassette were growing in either SC+Glu (WOR1 OFF) or SC+Mal (WOR1 ON) overnight at 25°C. The pictures are representatives of cells analyzed in Fig. 5D.

supplementary material - ec.asm.org€¦ · 8/28/2013 · 1" supplementary material 2"...

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