A

S288C

S288C

HA

P1

HAP1+

Ty1 in hap1

!1278b

B

S288C S288C

HAP1+

1 2 3 4

CYC1

SNR190

S288C

hap

1"

5

!1278b

Figure S1. Repair of the HAP1 allele in the S288C strain background. (A) In the S288C strain, a Ty1 element is inserted at the 3’ end the HAP1open reading frame. The Ty1 was replaced with wild-type sequence from the !1287b strain (O807) as described in the Materials and Methods.

Shown are PCR reactions demonstrating that the repaired strain (S288C HAP1+, strain FY2609), like the !1287b strain (O807), contains wild-

type HAP1. The wild-type (HAP1+) reaction contains primers that anneal within the HAP1 ORF and downstream of the HAP1 ORF; there is no

product in the parent hap1 strain (S288C, strain FY2608) because the size of the Ty1 insertion prevents full extension of the product. The

reaction for the mutant hap1 allele (containing Ty1) contains primers that anneal within Ty1 and downstream of the HAP1 ORF. (B) Northern blotshowing expression of the Hap1 target gene, CYC1, in the indicated strain backgrounds. Lane 1: !1287b strain (O807), lane 2: S288C

(FY2607), lanes 3 and 4: two clones of S288C with repaired HAP1 allele (FY2609 and FY2610), lane 5: S288C hap1" (FY2611).

Figure S2. Hap1 directly regulates aerobic genes through both activation and repression. The genes listed here were identified by our analysis

to be aerobic, Hap1-dependent and bound by Hap1.

gene pathway previous reference

Hap1 aerobically activated genes

ZEO1 cell wall integrity

COQ2 electron transport chain

CYT1 electron transport chain Schneider, 19913,4

RIP1 electron transport chain

CYC7 electron transport chain Prezant, 19873,4; Lowry, 19882; Burke, 19975

ERG13 ergosterol biosynthesis Servouse, 19861

ERG19 ergosterol biosynthesis

FRE4 iron uptake

YGR266W membrane protein

SFK1 phospholipid metabolism

PDR16 sterol metabolism

ACS2 transcription van den Berg, 19965

ROX1 transcription, oxygen regulation Lowry, 19882,3; Deckert, 19954

HYP2 translation Turi, 19922

PET10 unknown function, respiration

Hap1 hypoxically repressed genes

CYB5 ergosterol and fatty acid biosynthesis Ter Linde, 20022

ERG5 ergosterol biosynthesis Hata, 19831

ARE2 sterol esterification Valachovic, 20022,3

IZH1 zinc homeostasis

YHB1 oxidative stress response Crawford, 19952,3,4

NDE1 electron transport chain

Hap1 aerobically activated and hypoxically repressed genes

HMG1 ergosterol biosynthesis Thorsness, 19893,4; Ter Linde, 20022,4

ERG8 ergosterol biosynthesis

OLE1 fatty acid biosynthesis Choi, 19964; Vasconcelles, 20012

CYC1 electron transport chain Guarente, 19832,3; Guarente, 19844

1 lower catalytic activity in hypoxia2 lower mRNA expression in hypoxia3 gene expression induced by heme4 Hap1-dependent expression5 higher mRNA expression in hypoxia

ERG11 Hap1 binding site 1

ERG11 Hap1 binding site 2

ERG2 Hap1 binding site 1

Scer -636 ATCGTACGGCTTAGTCGGGAATAA

Spar -628 ATCGTACGGCTTAGTCGGGAATAA

Skud -627 ATCGTACGGCTTAGTCGGGAATAA

Sbay -634 ATCGTACGGCTTAGTCGGGAATAA

Smik -618 ATCGTACGGCTTAGTCGGGAATAA

Scer -709 AAAATACGAAATTTCCGGCAAAAT

Spar -702 AAAATACGAAATTTCCGGCAGAGT

Skud -700 AAAGAACGAAATTTCCGGCGCGCT

Sbay -717 AAAAAACGAAAATTCCGGCTGCGA

Smik -691 AAAGTACGAAATTTCCGACAGAGT

Scer -166 AACTACCGGTGCTATCGTTCTCGT

Spar -167 AACTACCGGTGCTATCGTTCTCGT

Skud -183 AACTACCGGTGCTATCGTTCTCGT

Sbay -182 AATTACCGGTACTATCGTTCTCGT

Smik -180 ATCTACCGGTGCTATCGTTCTCGT

Figure S3. Putative Hap1 binding sites in the ERG2 and ERG11 promoters. Alignment of Hap1 binding sites of the sequenced sensu stricto

yeast species: S. cerevisiae (Scer), S. paradoxus (Spar), S. kudravzevii, (Skud), S. bayanus (Sbay), and S. mikitae (Smik). The number denotes

the first nucleotide of the sequence relative to the ATG start codon. Bold and underlined nucleotides represent consensus Hap1 positions, while

the shaded nucleotides denote conserved nucleotides. ERG11 binding site 1 is the reverse complement.

ERG2

CYC1

ERG5

ERG11

region 4

Hap1 half site

Hap1 full site

Figure S4. Distribution of conserved Hap1 binding sites in the promoters of CYC1, ERG2, ERG5 and ERG11. Shown are the positions of half

(red box) and full (black diamond) Hap1 sites relative to the start codon (ATG). Only binding sites fully conserved in the following sensu stricto

yeast species were included: S. cerevisiae, S. paradoxus, S. kudravzevii, S. bayanus and S. mikitae. A half site is defined as CGn (where n is

any nucleotide) while a full site is CG(n)7CGn. Region 4 of the ERG5 promoter, described in Figure 6, is denoted by a gray bar.

-600 -500 -400 -300 -200 -100 0

bp (relative to ATG)

CYC1

SSA1

ERG5

SNR190

G D G D G D G D

ssa2" ssa4"

+O2 -O2 +O2 -O2

ssa2" ssa4"

GAL1pr-SSA1

A

1 2 3 4 5 6 7 8

ERG5

CYC1

SNR190

HSC82

G D G D G D G D

hsp82"

+O2 -O2 +O2 -O2

hsp82"

GAL1pr-HSC82

B

1 2 3 4 5 6 7 8

Figure S5. Hsp70 is partially required for ERG5 repression, while Hsp90 is dispensable. (A) Expression of SSA1, CYC1 and ERG5 wasmonitored by Northern blots in ssa2" ssa4" (FY2631) or ssa2" ssa4" GAL1pr-SSA1 (FY2636) cells grown in 2% galactose (G) or 2% dextrose

(D), and under aerobic (+oxygen) or hypoxic (-oxygen) conditions, as described in the Materials and Methods. (B) Expression of HSC82, CYC1and ERG5 was monitored by Northern blots in hsp82" (FY2622) or hsp82" GAL1pr-HSC82 (FY2635) cells grown as described in Panel A. Note

that the carbon source had an effect on ERG5 and CYC1 expression in the control strain (compare lane 1 to 2, and lane 3 to 4).

Figure S6. List of primers used for real-time PCR analysis.

Primer name Location1 Sequence (5’ to 3’)

FO3024 chr IR CTCTGTAAAAACCAAGGATGTGATGAA

FO3025 chr IR TTAGTCTATTTGATGACGGCTACTGAATCTFO3798 ERG5 (-323) TAGCCCAAACCTGCGTCTAT

FO3799 ERG5 (-189) CAGTCCCTTTGCCTGAAGAC

FO3800 ERG11 (-607) ATACGAGCAGCGCACATACAFO3801 ERG11 (-490) TCGTTTCGTTTCGTTTAGGG

FO3802 ERG2 (-491) GCGTTACGTTCGATACCTTCA

FO3803 ERG2 (-371) CAGCATGTTTGACGTTTGCT

FO3845 CYC1 (-397) ATCTAAAATTCCCGGGAGCAFO3846 CYC1 (-268) CTTGATCCACCAACCAACG

FO4371 ERG5 (-482) CCCACACCCCTGGATTTT

FO4372 ERG5 (-367) AAACGAGGGAAACCTTCTCGFO4810 ERG5 (-2174) CCAGCCAAAATGGACCTTTA

FO4811 ERG5 (-2012) ACTCCGTGCCTATTTGCAGT

FO4812 ERG5 (+1272) GGGCGCTATGTTAATTCCAAFO4813 ERG5 (+1420) GTGGACCACAACCAAAAACC1 Location shown in parentheses relative to ATG.

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