SUPPLEMENTARY INFORMATION Archetypal

SUPPLEMENTARY INFORMATION Archetypal transcriptional blocks underpin yeast gene regulation in response to changes in growth conditions David Talavera1*, Christopher J Kershaw2, Joseph L Costello2,#a, Lydia M Castelli2,#b, William Rowe3,#c, Paul FG Sims4, Mark P Ashe2, Chris M Grant2, Graham D Pavitt2*, Simon J Hubbard3*. * Corresponding authors [email protected] (DT) [email protected] (GDP) [email protected] (SJH)

1

Supplementary Figures in this file S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21 S22 S23 S24 S25 S26 S27

Decision tree in the assignment of instances to new or existing clusters. Relationship between clusters if changing the correlation threshold. Relationship between clusters if changing the linkage method. Relationship between clusters if changing the linkage method. Relationship between clusters if changing the linkage method. Relationship between clusters if applying normalization techniques to the downloaded data. Relationship between clusters if applying normalization techniques to the downloaded data. Analysis of common transcription regulation within and between BTRs. Characterization of PSRs. Response consistency within PSRs (top/bottom 10% RNAs). Response consistency within PSRs (top/ bottom 5% RNAs). Response consistency within PSRs (top/ bottom 1% RNAs). Biological processes affected by consistent stress response (top/ bottom 10% RNAs). Biological processes affected by consistent stress response (top/ bottom 1% RNAs). Characterization of BTRs. Biological processes overrepresented within BTRs (whole heatmap). Biological processes overrepresented within BTRs (zoomed in). Functional similarity within BTRs. Significance of interactions within BTRs. Regulation of transcription within and between BTRs. BTRs suffering extreme expression changes. Multi-dimensional scaling of transcriptomes from yeast grown under different stress conditions. Similarity of transcriptional response to stress (stress vs. control). Common stress responses. Magnitude of transcriptional stress-response. Biological processes affected by stress response. Use of BTRs for functional annotation.

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Other files containing supporting information S1 File. General description of data and clustering results. S2 File. Matrix containing the log-fold changes for each experiment. S3 File. Matrix containing the Pearson correlations between experiments. S4 File. Matrix containing the Pearson correlations between RNAs. S5 File. Alternative PSR clusters. S6 File. In-detail description of PSRs analysed in the paper. S7 File. Composition of PSR subsets analysed in the paper. S8 File. Biological processes associated with subsets of PSRs. S9 File. Composition of BTRs analysed in the paper. S10 File. Biological processes associated with BTRs. S11 File. edgeR results for NGS-based transcriptomic analyses.

3

Yes

Do nothing

Select two instances

Are both instances in the same cluster?

No

Are the members of each cluster connected to all the members of the other cluster

No Do nothing

Yes

Yes

Are any of the instances already in a cluster?

Yes

No

Are both instances already in a cluster?

Merge both clusters

No

Yes

Do nothing

No

Create a new cluster containing the two instances

Is the nonclustered instance connected to all the members of the cluster

Add instance to the existing cluster

4

S1 Fig. Decision tree in the assignment of instances to new or existing clusters.

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● 80 ● 42 ● 142 ● 65 ● 57 ● 81 ● 143 ● 47 ● 21 ● 76 ● 75 ● 177 ● 74 ● 17 ● 93 ● 18 ● 99 ● 34 ● 197 ● 117 ● 151 ● 73 ● 141 ● 69 ● 109 ● 63 ● 85 ● 22 ● 20 ● 19 ● 8 ● 188 ● 59 ● 40 ● 39 ● 43 ● 7 ● 37 ● 36 ● 161 ● 150 ● 35 ● 217 ● 183 ● 33 ● 30 ● 29 ● 28 ● 26 ● 112 ● 24 ● 13 ●

118 ● 50 ● 68 ● 58 ● 28 52 63 100 ● ● ● ● 48 ● 46 ● 125 51 ● ● 98 139 62 ● ● ● 84 102 ● ● 32 ● 93 56 ● ● 27 50 12 ● ● ● 195 ● 47 25 ● ● 97 92 129 ● ● ● ● 24 46 209 165 174 ● ● ● ● 77 ● 134 ● 94 194 145 ● ● ● 96 45 91 55 96 ● ● ● ● ● 90 ● 209 140 166 ● ● ● 51 86 124 ● ● ● 78 ● 127 119 163 ● ● ● 87 61 138 122 54 ● ● ● ● ● 66 ● 204 115 160 ● ● ● 36 44 106 ● ● ● 77 83 94 175 ● ● ● ● 31 158 ● ● 59 135 ● ● 119 108 ● ● 49 82 157 ● ● ● 162 88 112 155 ● ● ● ● 67 ● 43 48 81 121 ● ● ● ● 149 192 104 ● ● ● 47 80 120 ● ● ● 98 187 145 ● ● ● 32 40 87 ● ● ● 183 105 91 139 180 ● ● ● ● ● 45 ● 131 169 85 ● ● ● 38 22 115 ● ● ● 77 41 168 ● ● ● 52 87 130 ● ● ● 102 ● 46 76 114 ● ● ● 33 34 129 83 167 ● ● ● ● ● 72 ● ● 103 72 133 223 ● ● ● 51 128 ● ● 20 56 ● ● 18 32 43 107 71 ● ● ● ● ● 120 159 75 ● ● ● 40 69 103 ● ● ● 145 ● 89 36 97 52 65 73 117 ● ● ● ● ● ● ● 105 ● 62 ● 29 21 86 63 138 ● ● ● ● ● 53 82 115 ● ● ● 28 15 111 ● ● ● 199 164 ● ● 60 156 ● ● 16 54 89 67 71 114 ● ● ● ● ● ● 4 ● 219 ● 17 26 134 59 131 ● ● ● ● ● 86 74 112 ● ● ● 108 25 130 ● ● ● 226 ● 110 151 153 ● ● ● 224 ● 78 24 30 55 104 66 146 ● ● ● ● ● ● ● 80 ● 118 ● 42 64 101 ● ● ● 50 ● 142 ● 23 14 68 ● ● ● 28 52 65 ● ● ● 58 ● 57 63 100 ● ● ● 13 22 48 ● ● ● 81 125 51 ● ● ● 12 21 46 ● ● ● 84 102 143 ● ● ● 139 20 98 139 62 ● ● ● ● ● 47 ● 18 11 27 50 32 ● ● ● ● ● 21 93 56 ● ● ● 74 12 ● ● 102 47 25 76 ● ● ● ● 16 195 ● ● 24 46 75 ● ● ● 10 154 97 92 129 ● ● ● ● ● 177 ● 77 ● 96 74 45 ● ● ● 94 ● 17 91 55 ● ● ● 90 ● 93 ● ● 51 86 124 ● ● 87 18 61 ● ● ● 127 S2 Fig. Relationship between clusters if changing the correlation threshold. ● 99 138 122 ● ● ● 36 44 66 ● ● ● 34 ● 106 ● 197 77 83 ● ● ● 119 31 ● ● 117 ● 162 108 0.4, in blue; 0.5, in yellow; 0.6 in red. The darkness of the lines represent ● ● 151 49 82 ● ● ● 43 88 ● ● 73 ● 48 81 121 ● ● ● 141 ● 32 40 47 80 120 ● ● ● ● ● 69 ● 87 the similarity of the clusters (Jaccard Index). ● 109 183 105 ● ● ● 38 22 45 ● ● ● 63 ● 115 ● 85 77 41 ● ● ● 52 ● 33 34 22 ● ● ● 46 76 114 ● ● ● 20 ● 72 ● 19 103 72 ● ● ● 18 32 51 ● ● ● 8 ● 43 107 71 ● ● ● 188 ● 89 36 40 69 103 ● ● ● ● ● 59 ● 97 52 65 ● ● ● 29 40 21 ● ● ● 62 ● 39 28 15 86 63 ● ● ● ● ● 53 ● 43 ● 16 54 199 164 ● ● ● ● 7 60 ● ● 89 67 ● ● 37 17 26 ● ● ● 219 ● 108 25 36 134 59 ● ● ● ● ● 86 ● 161 ● 226 ● 78 24 150 ● ● ● 224 ● 35 30 55 ● ● ● 80 ● 217 ● 42 ● 23 183 14 ● ● ● 142 ● 33 ● 28 52 65 ● ● ● 13 30 22 ● ● ● 57 ● 29 12 21 ● ● ● 81 125 51 ● ● ● 139 20 28 ● ● ● 84 102 143 ● ● ● 18 26 11 ● ● ● 47 ● 74 112 27 50 ● ● ● ● 102 21 ● ● 16 24 ● ● ● 47 25 76 ● ● 10 13 154 ● ● ● 24 46 75 ● ● ● 177 ● 96 74 45 ● ● ● 17 ● 93 ● 87 18 61 ● ● ● 99 ● 36 44 ● ● 34 ● 197 ● 119 ● 117 ● 162 ● 151 ● 43 ● 73 ● 141 ● 32 40 ● ● 69 ● 109 ● 38 22 ● ● 63 ● 85 ● 33 34 22 ● ● ● 20 ● 19 ● 18 32 ● ● 8 6 ● 188 ● 89 36 ● ● 59 ● 29 40 21 ● ● ● 39 28 15 ● ● ● 43 ● 64 ●

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● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● S6 Fig. Relationship between clusters if applying normalization techniques ● ● ● ● ● ● ● to the downloaded data. In red, the method described in the main text. In ● ● ● ● ● ● ● blue, clusters generated normalizing data with a Rank-based quantile ● ● ● ● ● ● ● ● normalization approach prior to the correlation calculation. The darkness ● ● ● ● ● ● ● of the lines show how similar are the clusters (Jaccard Index). ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 10 ● ● ● ● ● ● ● ● ● ● ● 209

220 140

194

80

183

196

166

181

163

179

160

106

158

154

155

166

149

148

145

138

139

172

131

123

● ● ● ● ● ● ● ● ● ● ● ●

44

42

90

119

147

74

73

43

92

157

72

87

40

57

110

170

71

78

38

34

104

116

69

72

101

105

117

137

77

84

115

128

76

114

110

112

132

70

98 74

93

102

60

88

92

19

39

32

26

52

29

24

178

28

32

53

26

22

55

25

21

59

50 77

54

176

108

153

24

102

101

27

38

61

134

36

34

41

63

98

87

37

94

65

100

91

182

36

103

200

23

52

49

114

68

109

56

22

47

21

15

86

130

133

129

145

128

112

122

111

120

156

117

137

115

128

114

110

112

157

110

170

104

116

101

105

83

83

51

54

82

91

50

44

81

23

47

58

118

46

33

97

45

35

77

84

44

42

76

90

119

147

18

17

74

73

43

92

16

14

72

87

40

57

10

7

71

78

38

34

69

72

182

36

37

65

70

36

34

41

93

102

19

63

60

39

32

26

52

29

24

178

28

32

53

26

22

55

25

21

88

92

50

91

59

77

54

176

153

87

101

102

108

24

38

27

61

200

114

52

103

23

68

86

49

109

83

83

82

91

18

20

98 74

63

80

100

98

65

55

94

132

174

56

22

47

51

54

21

15

50

44

174

125

● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● S7 Fig. Relationship between clusters if applying normalization techniques ● ● ● ● ● ● ● ● ● ● ● to the downloaded data. In red, the method described in the main text. In ● ● ● ● ● ● ● ● ● ● blue, clusters generated normalizing data with a Spline-based quantile ● ● ● ● ● ● ● ● ● ● ● normalization approach prior to the correlation calculation. The darkness ● ● ● ● ● ● ● ● ● ● of the lines show how similar are the clusters (Jaccard Index). ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 11 ● ● ● ● ● ● ● ● ● ● ● ● ● ●

● ● ● ● ● ● ● ● ● 117

109

47

80

202

209

130 149

194

128

75

183 166

167 155

104 102 101

89

67

53 90

149

172

93

118

145

143

139

181

131

110

79

161

69

117

91

71

102

69

15

65

54

63

52

60

50

59

48

54

44

81

119

86

65

72

28

22

26

16

25

21

177

24

40 37

157

51

50

59

47

64

22

88

46

45

21

18

45

41

204

113

147

87

91

44

57

109

43

23

27

55

80

36

38

61

92 60

80

35

34

104

153

74

77

101

72

141

110

134

71

102

104

97

69

15

102

127

65

54

101

107

63

52

132

60

50

105

59

48

100

54

44

32 29

32

30

36

20

29

72

211

28

22

26

16

25

21

177

24

40 37

126

89

157

52

65

23

27

43

98

90

93

118

51

51

195

50

59

142

47

64

79

46

45

69

45

41

22

88

21

18

204 147

55

114 58

113

71

24

20

165

40

77

24

70

87

91

25

44

57

83

119

20

165

18

114 58

129

76

82

29

51

19

150

83

20

119

95

91

36

83

78

92

30

211

25

33

53

32

70

120

100

32 29

71

83 82

112

141

34

142

92

128

114

72

195

76

115

77

80

43

159

103

74

52

98

47

104

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100

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107

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120

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61

97

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160

38

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130

35

101

132

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36

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150

163

205

60

77

95

112 110

43

103

115 114

92

10

18

10

16

17

10

148

A A

Jaccard Index =

C

BTRi

B B

A∩B A∪B

BTRi

BTRj

A

E

B

F

C

G

D

H

A

B

C

D

Jaccard Indexi = Median value of 6 comparisons

Jaccard Indexi,j = Median value of 16 comparisons

S8 Fig. Analysis of common transcription regulation within and between BTRs. A. Diagram showing the calculation of the Jaccard Index between two sets. B. Analysis of common TFs within a BTR containing 4 RNAs: the black lines represent the pairwise comparisons between the different sets of TFs (one for each RNA). C. Analysis of common TFs between two BTRs: the black lines represent all the pairwise comparisons between sets in different BTRs.

12

50 20 10 5 2

Number of experiments within PSRs

0.5

0.6

0.7

0.8

0.9

1.0

Median correlation within PSRs

S9 Fig. Characterization of PSRs. Scatterplot shows the distribution of PSRs based on their within-group similarity (median of pairwise correlations within a PSR), and number of experiments within. Intensity of grey dots represents the number of overlapping points: the darker, the greater the number. Top and side histograms show the overall distribution of PSRs according to each variable.

13

Top/Bottom 10% RNAs

40

20

300 200

60

100

Number of enriched RNAs

C

0

Number of experiments in PSR

A

20

40

60

80

Number of experiments

D

0

250 200

200 150 0

150

100


300

50

Number of depleted RNAs

250

B 350

100

20

50

40

60


E

200 250 300

150

150

100


100

200

250

50

80

50


0

18 25 51 60 98 110 128 139 155 158 160 209 21 28 46 69 71 76 81 86 117 120 130 131 163 166 183 194 10 16 20 43 72 93 82 101 104 115 129 149 22 29 45 47 74 80 54 77 83 112 119 44 65 145 23 102 38 63 26 36 40 59 24 122 52 114 50 32 91 92 34 87 55 100

0

350

PSRs

0

100

200

300


S10 Fig. Response consistency within PSRs (top/bottom 10% RNAs). A. Number of experiments within each PSR, sorted in increasing order. B. Number of consistently depleted and enriched RNAs within each PSR, ordered as in A. CE. Relationship between number of experiments within PSR, number of enriched transcripts within PSR and number of depleted transcripts within PSR.

14

Top/Bottom 5% RNAs

40

20

150 100

60

50


200

C

0


A

20

40

60

80


D

0

140 100 80 60 0

100

40

150

20



B

50 20

40

60

80


E

140 100 80 60

18 25 51 60 98 110 128 139 155 158 160 209 21 28 46 69 71 76 81 86 117 120 130 131 163 166 183 194 10 16 20 43 72 93 82 101 104 115 129 149 22 29 45 47 74 80 54 77 83 112 119 44 65 145 23 102 38 63 26 36 40 59 24 122 52 114 50 32 91 92 34 87 55 100

0

150

40

100

20

50



0

PSRs

0

50

100

150

200


S11 Fig. Response consistency within PSRs (top/ bottom 5% RNAs). A. Number of experiments within each PSR, sorted in increasing order. B. Number of consistently depleted and enriched RNAs within each PSR, ordered as in A. CE. Relationship between number of experiments within PSR, number of enriched transcripts within PSR and number of depleted transcripts within PSR.

15

Top/Bottom 1% RNAs

40

20

30 25 20 15 10

60

5


C

0


A

20

40

60

80


D

0

20 15 10

20 15 10

25

0


30

5


25

B

5

20

40

60

80


E

20 25

15

18 25 51 60 98 110 128 139 155 158 160 209 21 28 46 69 71 76 81 86 117 120 130 131 163 166 183 194 10 16 20 43 72 93 82 101 104 115 129 149 22 29 45 47 74 80 54 77 83 112 119 44 65 145 23 102 38 63 26 36 40 59 24 122 52 114 50 32 91 92 34 87 55 100

0

30

10

15

5

10

20

25

5



0

PSRs

0

5

10

15

20

25

30


S12 Fig. Response consistency within PSRs (top/ bottom 1% RNAs). A. Number of experiments within each PSR, sorted in increasing order. B. Number of consistently depleted and enriched RNAs within each PSR, ordered as in A. CE. Relationship between number of experiments within PSR, number of enriched transcripts within PSR and number of depleted transcripts within PSR.

16

RNA synthesis, modification & transport

Ribosome biogenesis & translation

Environmental stress

transcription from RNA polymerase I promoter RNA modification tRNA processing transcription from RNA polymerase III promoter ribosomal subunit export from nucleus regulation of translation nucleobase−containing compound transport nuclear transport endosomal transport cytokinesis response to starvation membrane invagination vesicle organization DNA−templated transcription, initiation protein glycosylation cellular ion homeostasis snoRNA processing RNA catabolic process vitamin metabolic process response to osmotic stress regulation of DNA metabolic process DNA recombination organelle fission meiotic cell cycle sporulation protein phosphorylation tRNA aminoacylation for protein translation translational initiation chromosome segregation cellular response to DNA damage stimulus mitotic cell cycle telomere organization cytoskeleton organization chromatin organization regulation of organelle organization DNA repair regulation of protein modification process regulation of cell cycle DNA replication protein complex biogenesis amino acid transport organelle fusion nucleus organization signaling cell morphogenesis transposition pseudohyphal growth invasive growth in response to glucose limitation conjugation cell wall organization or biogenesis carbohydrate transport lipid transport lipid metabolic process cellular amino acid metabolic process rRNA processing ribosomal small subunit biogenesis ribosomal large subunit biogenesis ribosome assembly organelle assembly cytoplasmic translation mitochondrion organization mitochondrial translation transmembrane transport ion transport monocarboxylic acid metabolic process generation of precursor metabolites and energy cellular respiration response to oxidative stress response to chemical response to heat oligosaccharide metabolic process proteolysis involved in cellular protein catabolic process protein maturation protein folding nucleobase−containing small molecule metabolic process cofactor metabolic process carbohydrate metabolic process

Yeast modification

Resources limitation

PSRs

PSRs

Enriched

No change

Depleted

17

163 194 34 47 100 32 112 129 149 24 122 50 83 52 45 155 120 92 51 130 10 101 145 166 59 114 76 46 21 20 81 55 60 158 29 36 28 82 119 43 98 104 160 44 87 93 40 22 209 16 74 25 128 38 80 102 23 86 91 65 71 54 63 18 115 69 183 110 77 117 26 131 72 139

163 194 34 47 100 32 112 129 149 24 122 50 83 52 45 155 120 92 51 130 10 101 145 166 59 114 76 46 21 20 81 55 60 158 29 36 28 82 119 43 98 104 160 44 87 93 40 22 209 16 74 25 128 38 80 102 23 86 91 65 71 54 63 18 115 69 183 110 77 117 26 131 72 139

S13 Fig. Biological processes affected by consistent stress response (top/ bottom 10% RNAs). GO Slim terms overrepresented in the set of 10% mostenriched transcripts (red) or the set of 10% most-depleted transcripts (blue). All colouring as in Fig 3. The black squares describe the type of stresses the PSRs suffered. The order of both PSRs and biological processes depends on the hierarchical clustering of Euclidean distances calculated along the columns and rows respectively.

18

transmembrane transport ion transport organelle fusion nucleus organization cell morphogenesis signaling conjugation response to starvation nucleobase−containing small molecule metabolic process regulation of translation carbohydrate transport regulation of protein modification process DNA repair mitotic cell cycle cellular response to DNA damage stimulus protein phosphorylation meiotic cell cycle regulation of cell cycle chromatin organization lipid metabolic process cofactor metabolic process ribosomal subunit export from nucleus nuclear transport ribosomal large subunit biogenesis rRNA processing ribosomal small subunit biogenesis ribosome assembly organelle assembly cytoplasmic translation lipid transport cellular amino acid metabolic process oligosaccharide metabolic process carbohydrate metabolic process monocarboxylic acid metabolic process response to osmotic stress protein folding response to heat generation of precursor metabolites and energy cellular respiration response to oxidative stress response to chemical

Environmental stress Resources limitation Yeast modification

PSRs PSRs

Enriched

No change

Depleted

19

22 45 34 194 32 82 63 183 47 52 69 16 139 18 128 40 72 115 25 77 46 38 87 92 102 50 86 54 71 20 119 145 81 83 24 129 93 10 60 131 23 209

22 45 34 194 32 82 63 183 47 52 69 16 139 18 128 40 72 115 25 77 46 38 87 92 102 50 86 54 71 20 119 145 81 83 24 129 93 10 60 131 23 209

S14 Fig. Biological processes affected by consistent stress response (top/ bottom 1% RNAs). GO Slim terms overrepresented in the set of 1% mostenriched transcripts (red) or the set of 1% most-depleted transcripts (blue). All colouring as in Fig 3. The black squares describe the type of stresses the PSRs suffered. The order of both PSRs and biological processes depends on the hierarchical clustering of Euclidean distances calculated along the columns and rows respectively.

20

50 20 10 5 2

Number of transcripts within BTRs

100

0.5

0.6

0.7

0.8

0.9

1.0

Median correlation within BTRs

S15 Fig. Characterization of BTRs. Scatterplot shows the distribution of BTRs based on their within-group similarity (median of pairwise correlations within a BTR), and number of transcripts within. Intensity of grey dots represents the number of overlapping points: the darker, the greater the number. Top and side histograms show the overall distribution of BTRs according to each variable.

21

Biological Processes Biological Processes

oligosaccharide metabolic process carbohydrate metabolic process response to oxidative stress response to chemical amino acid transport carbohydrate transport regulation of organelle organization cytoskeleton organization mitotic cell cycle protein dephosphorylation cell budding regulation of cell cycle regulation of translation nucleus organization cell morphogenesis sporulation cell wall organization or biogenesis nucleobase−containing compound transport cytoplasmic translation translational elongation tRNA aminoacylation for protein translation cellular amino acid metabolic process cellular ion homeostasis generation of precursor metabolites and energy cellular respiration DNA repair cellular response to DNA damage stimulus meiotic cell cycle organelle fission chromosome segregation regulation of protein modification process DNA−templated transcription, termination DNA−templated transcription, elongation DNA−templated transcription, initiation chromatin organization vitamin metabolic process cofactor metabolic process response to starvation organelle inheritance signaling conjugation protein phosphorylation cytokinesis protein modification by small protein conjugation or removal DNA replication telomere organization DNA recombination lipid transport endocytosis exocytosis organelle fusion membrane fusion Golgi vesicle transport protein acylation peptidyl−amino acid modification histone modification transposition invasive growth in response to glucose limitation regulation of transport nucleobase−containing small molecule metabolic process ion transport monocarboxylic acid metabolic process lipid metabolic process vacuole organization membrane invagination mitochondrion organization mitochondrial translation RNA splicing mRNA processing ribosome assembly organelle assembly nuclear transport ribosomal large subunit biogenesis rRNA processing ribosomal small subunit biogenesis RNA modification ribosomal subunit export from nucleus tRNA processing snoRNA processing transcription from RNA polymerase III promoter transcription from RNA polymerase I promoter translational initiation proteolysis involved in cellular protein catabolic process protein maturation protein complex biogenesis response to heat protein folding protein lipidation protein glycosylation transmembrane transport protein targeting pseudohyphal growth regulation of DNA metabolic process response to osmotic stress RNA catabolic process transcription from RNA polymerase II promoter vesicle organization

0.00

0.01

amino acid transport and metabolism carbohydrates transport and metabolism cell cycle cell polarity/morphogenesis DNA metabolic process

0.02

BTRs

translaAon transport

0.03

signaling/stress response small molecule transport and metabolism transcripAon

BTRs lipid transport and metabolism nucleobase transport and metabolism organelle organisaAon protein modificaAon RNA processing

0.04

0.05

22

1362 1229 1256 1203 1061 1240 958 1109 922 1369 919 967 1264 903 899 895 894 811 767 704 754 1104 653 876 568 554 760 1260 535 861 490 1318 486 649 1210 447 893 378 588 351 864 328 856 318 381 293 646 284 280 566 608 258 915 986 413 886 945 234 538 802 1001 341 857 1245 229 289 252 1204 222 810 1315 267 212 224 228 533 601 207 286 242 534 451 551 202 696 356 407 1010 200 264 487 536 191 443 188 477 763 380 276 209 183 192 411 1136 179 783 173 235 942 1176 160 206 629 156 734 676 406 402 201 310 150 182 215 230 146 587 282 336 376 410 135 791 110 285 187 102 417 148 271 951 909 585 389 495 565 90 371 479 396 375 352 247 279 111 219 333 470 331 347 180 141 60 214 1198 50 330 939 615 631 43 275 29 468 73 68 65 63 19 44 882 841 3 297

S16 Fig. Biological processes overrepresented within BTRs (whole heatmap). Red intensity represents the FDR value. Terms are grouped in 15 categories, and their name coloured accordingly. Zoomed in version of some areas of the heatmap in S13 Fig.

23

regulation of organelle organization

cellular respiration

generation of precursor metabolites and energy

0.05

0.04

BTRs

0.04

0.05 regulation of organelle organization

0.02

mitotic cell cycle

0.03 cytoskeleton organization 0.01 0.00

protein dephosphorylation cell budding DNA repair

regulation of cell cycle

cellular response to DNA damage stimulus meiotic cell cycle

1001 0.01

0.02

0.03

0.04

0.05

615 0.00

631

341

transport

translaAon

0.05

0.04

0.03

transcripAon

0.05

0.04

0.03

0.02

0.01

0.00

small molecule transport and metabolism

signaling/stress response

RNA processing

protein modificaAon

organelle organisaAon

nucleobase transport and metabolism

lipid transport and metabolism

DNA metabolic process

cell polarity/morphogenesis

cell cycle

carbohydrates transport and metabolism

amino acid transport and metabolism

0.00

0.01

0.02

857

regulation of translation

BTRs

802 939

organelle fission chromosome segregation

538

BTRs

330 60

234

cytoskeleton organization

0.05

141

50 180

0.03

0.04

BTRs

331

mitotic cell cycle

0.03

regulation of protein modification process

470

0.02

0.02

BTRs

ribosome assembly

nuclear transport

organelle assembly

rRNA processing ribosomal small subunit biogenesis RNA modification ribosomal subunit export from nucleus tRNA processing snoRNA processing

333

183 ribosomal large subunit biogenesis

Biological Processes

transcription from RNA polymerase I promoter

219

209 transcription from RNA polymerase III promoter

translational initiation

111

protein dephosphorylation

0.01

375

945 1198 347

0.01

396

cell budding

0.00

479

192


276

Biological Processes 0.05 0.04 0.03 0.02 0.01 0.00

214

0.00

90

regulation of cell cycle

BTRs

oligosaccharide metabolic process carbohydrate metabolic process response to oxidative stress response to chemical amino acid transport carbohydrate transport regulation of organelle organization cytoskeleton organization mitotic cell cycle protein dephosphorylation cell budding regulation of cell cycle regulation of translation nucleus organization cell morphogenesis sporulation cell wall organization or biogenesis nucleobase−containing compound transport cytoplasmic translation translational elongation tRNA aminoacylation for protein translation cellular amino acid metabolic process cellular ion homeostasis generation of precursor metabolites and energy cellular respiration DNA repair cellular response to DNA damage stimulus meiotic cell cycle organelle fission chromosome segregation regulation of protein modification process DNA−templated transcription, termination DNA−templated transcription, elongation DNA−templated transcription, initiation chromatin organization vitamin metabolic process cofactor metabolic process response to starvation organelle inheritance signaling conjugation protein phosphorylation cytokinesis protein modification by small protein conjugation or removal DNA replication telomere organization DNA recombination lipid transport endocytosis exocytosis organelle fusion membrane fusion Golgi vesicle transport protein acylation peptidyl−amino acid modification histone modification transposition invasive growth in response to glucose limitation regulation of transport nucleobase−containing small molecule metabolic process ion transport monocarboxylic acid metabolic process lipid metabolic process vacuole organization membrane invagination mitochondrion organization mitochondrial translation RNA splicing mRNA processing ribosome assembly organelle assembly nuclear transport ribosomal large subunit biogenesis rRNA processing ribosomal small subunit biogenesis RNA modification ribosomal subunit export from nucleus tRNA processing snoRNA processing transcription from RNA polymerase III promoter transcription from RNA polymerase I promoter translational initiation proteolysis involved in cellular protein catabolic process protein maturation protein complex biogenesis response to heat protein folding protein lipidation protein glycosylation transmembrane transport protein targeting pseudohyphal growth regulation of DNA metabolic process response to osmotic stress RNA catabolic process transcription from RNA polymerase II promoter vesicle organization

585

763 279

0.05 0.04 0.03

247

regulation of translation

proteolysis involved in cellular protein catabolic process

protein maturation 0.02 0.01 0.00

371

BTRs

352

945 protein complex biogenesis

857 381

24

1001

341

BTRs

565

318

mitochondrion organization

389

380

1362 1229 1256 1203 1061 1240 958 1109 922 1369 919 967 1264 903 899 895 894 811 767 704 754 1104 653 876 568 554 760 1260 535 861 490 1318 486 649 1210 447 893 378 588 351 864 328 856 318 381 293 646 284 280 566 608 258 915 986 413 886 945 234 538 802 1001 341 857 1245 229 289 252 1204 222 810 1315 267 212 224 228 533 601 207 286 242 534 451 551 202 696 356 407 1010 200 264 487 536 191 443 188 477 763 380 276 209 183 192 411 1136 179 783 173 235 942 1176 160 206 629 156 734 676 406 402 201 310 150 182 215 230 146 587 282 336 376 410 135 791 110 285 187 102 417 148 271 951 909 585 389 495 565 90 371 479 396 375 352 247 279 111 219 333 470 331 347 180 141 60 214 1198 50 330 939 615 631 43 275 29 468 73 68 65 63 19 44 882 841 3 297

495

538 mitochondrial translation

909

802 856


951

234



S17 Fig. Biological processes overrepresented within BTRs (zoomed in). Details of BTRs participating in similar biological processes. Red intensity represents the FDR value. Terms are grouped in 15 categories, and their name coloured accordingly.

25

B

0

0.6 0.0

0.2

0.4

Proportion

40 20

Frequency

60

0.8

80

A

1

2

3

4

5

6+

0

D

3

4

5

6+

0.4 0.3

Proportion

0.0

0.0

0.1

0.2

0.6 0.4 0.2

Proportion

2

Number of GIs per BTR

0.8

C

1

0.5

Number of significant Biological Processes per BTR

0

1

2

3

4

Number of PPIs per BTR

5

6+

2

3

4

5

6+

Number of transcripts per BTR

S18 Fig. Functional similarity within BTRs. A Number of GO Slim Biological Processes that are significantly enriched within each BTR. B. Number of significant (dark grey) and non- significant (light grey) genetic interactions within BTR. C. Number of significant (dark grey) and non-significant (light grey) protein-protein interactions within BTR. D. Number of transcripts per BTR for BTRs having a significant enrichment in A-C (dark grey) or not having any enrichment in A-C (light grey).

26

100

1000 1 2

100

1

10

100

0.8 0.6

Proportion of possible PPIs/BTR

0.0 2

Number of genes/BTR

100

1.0

1.0 0.8 0.6 0.4

Proportion of possible PPIs/BTR

0.0 2

10

Number of GIs/BTR

0.2

1.0 0.8 0.6 0.4 0.2 0.0

Proportion of possible GIs/BTR

10

Number of genes/BTR

0.4

10

Number of genes/BTR

0.2

2

100

Number of PPIs/BTR

100 10

Number of PPIs/BTR

1000

C

1

10 1

Number of GIs/BTR

100

B

10

A

10

Number of genes/BTR

100

0.0

0.2

0.4

0.6

0.8

1.0

Proportion of possible GIs/BTR

S19 Fig. Significance of interactions within BTRs. Number of (and proportion of all possible) genetic (A) and physical (B) interactions per BTR compared to the size of BTR. Significant cases are shown in red. Random expectation was calculated through a resampling process, and is shown as a grey shade (90% confidence intervals for the expectation). C. Number of (and proportion of all possible) physical and genetic interactions within BTRs. BTRs may have a significant overrepresentation of physical (green), genetic (blue) or both interactions (red). BTRs with no overrepresentation are shown in grey. Significance was established as p-value < 0.05. The intensity of the colour is proportional to the number of cases represented by a single point.

27

500 400 0

0

100

100

200

300

Frequency

300 200

Frequency

600

500

B

400

A

0.0

0.2

0.4

0.6

Jaccard Index Median

0.8

1.0

0.0

0.2

0.4

0.6

0.8

1.0

Jaccard Index Median

S20 Fig. Regulation of transcription within and between BTRs. Histograms show the distribution of overlapping sets of transcription factors involved in regulating the expression of transcripts measured as the median of Jaccard Indices (number of TFs controlling the expression of both genes/number of TFs controlling the expression of either of the genes). The grey shades show the expected tip of the histogram bars calculated using resampling. A. Within BTRs. B. Between BTRs.

28

Top 1%

Top 5%

Top 10%

Neither top nor bottom

Bottom 10%

Bottom 5%

Bottom 1%

20 81 24 83 29 36 55 92 82 59 60 44 87 10 145 43 101 98 21 158 129 28 46 119 45 91 114 122 149 104 160 47 50 76 34 120 166 22 100 86 65 80 163 18 139 16 74 102 194 23 40 93 54 38 72 115 52 26 63 131 117 209 25 71 128 51 32 112 69 77 183 155 110 130

381 318 535 204 215 160 168 206 228 149 113 43 371 396 90 565 99 258 209 183 596 337 268 115 214 141 111 60 341 330 487 384 272 38 166 10 263 157 202 244 242 170 176 338 231 29 327 261 373 323 86 80 49 40 26 13 18 82 73 68 4 23 348 54 187 50 25 9 42 28 11 12 46 6 7 635 143 98 97 78 275 285 180 173 108 92

S21 Fig. BTRs suffering extreme expression changes. Heatmap shows if more than half of the members of a specific BTR are in one of the subsets of great changes of a particular PSR. Red shows membership of the subsets of induced RNAs, whereas blue shows membership of the subsets of repressed RNAs. The brighter the colour, the more extreme the percentile. PSRs and BTRs are ordered as they are in Fig 8: PSRs in columns, BTRs in rows. Grey squares show when there was no data available. Black lines divide the heatmap in the different global responses and response layers shown in Fig 8.

29

MDS for all experiments 0.6

●

●

0.2

oxidative stress

● ●

● ● ●

●

amino acid starvation

●

0.0

BCV distance 2

0.4

●

● ●

−0.2

control

−0.4

glucose starvation

−0.5

0.0

0.5

1.0

BCV distance 1 S22 Fig. Multi-dimensional scaling of transcriptomes from yeast grown

under different stress conditions. Control (red); amino acid starvation (green); glucose starvation (blue); and, oxidative stress (purple). The point shapes represent the tags used in the experiment: eIF4E (squares); eIF4G1 (triangles); and, eIF4G2 (circles). It demonstrates that transcriptomes cluster because of stress instead of tag.

30

Similarity of transcriptome response to stress

0

5 10

Count

Key+Histo

0

0.2

0.4

Distance eIF4G2 +H2O2 eIF4G1 +H2O2 eIF4E +H2O2 eIF4E −D eIF4G2 −D eIF4G1 −D eIF4G2 −AA eIF4E −AA

eIF4G2 +H2O2

eIF4G1 +H2O2

eIF4E +H2O2

eIF4E −D

eIF4G2 −D

eIF4G1 −D

eIF4G2 −AA

eIF4E −AA

eIF4G1 −AA

eIF4G1 −AA

S23 Fig. Similarity of transcriptional response to stress (stress vs. control). It demonstrates that stress responses cluster because of stress instead of tag.

31

Intersection of up−regulated genes

−D

−AA

97 1255

80 45

138

40 104

Intersection of down−regulated genes

Intersection of no−change genes

−D

−AA

+H2 O2

−D

−AA

40 1264

128 179

93

58 65

237 2365

106

128

2301

87

239

+H2 O2

+H2 O2

S24 Fig. Common stress responses. Overlapping sets of up-regulated genes (A), down- regulated genes (B), and genes with no statistical change (C) following 3 different stresses: amino acid starvation, glucose starvation, and oxidative stress. Statistical significance set at FDR 1%.

32

1000 1500 2000 2500 0

500

Frequency

−AA

−7

−6

−5

−4

−3

−2

−1

0

1

2

3

4

5

6

7

8

9

2

3

4

5

6

7

8

9

2

3

4

5

6

7

8

9

log2 FC

1000 1500 2000 2500 0

500

Frequency

−D

−7

−6

−5

−4

−3

−2

−1

0

1

log2 FC

1000 1500 2000 2500 0

500

Frequency

+H2 O2

−7

−6

−5

−4

−3

−2

−1

0

1

log2 FC

S25 Fig. Magnitude of transcriptional stress-response. Histograms show the distribution of log2 fold-changes. Significant differentially-expressed transcripts are shown in red, the rest are shown in black.

33

−AA

−D

+H2O2 Enriched

monocarboxylic acid metabolic process carbohydrate metabolic process ion transport cellular amino acid metabolic process amino acid transport endosomal transport cellular respiration response to starvation peroxisome organization protein phosphorylation protein maturation membrane invagination generation of precursor metabolites and energy response to oxidative stress response to chemical nucleobase−containing small molecule metabolic process

amino acid transport and metabolism carbohydrates transport and metabolism cell cycle

lipid metabolic process cellular ion homeostasis


transmembrane transport cofactor metabolic process carbohydrate transport protein lipidation mitochondrion organization invasive growth in response to glucose limitation cell wall organization or biogenesis chromatin organization

No change

nucleus organization Golgi vesicle transport RNA catabolic process protein glycosylation peptidyl−amino acid modification protein targeting DNA−templated transcription, initiation

cell polarity/morphogenesis DNA metabolic process lipid transport and metabolism nucleobase transport and metabolism organelle organisaAon protein modificaAon RNA processing signaling/stress response small molecule transport and metabolism transcripAon translaAon transport

DNA−templated transcription, termination response to heat translational initiation transcription from RNA polymerase III promoter snoRNA processing regulation of translation nucleobase−containing compound transport tRNA aminoacylation for protein translation protein folding ribosomal small subunit biogenesis ribosomal large subunit biogenesis ribosome assembly rRNA processing organelle assembly

Enriched

ribosomal subunit export from nucleus nuclear transport RNA modification

No change

transcription from RNA polymerase I promoter cytoplasmic translation

Depleted Depleted All 2x 4x 8x All 2x 4x 8x All 2x 4x 8x

tRNA processing

Transcript variation

S26 Fig. Biological processes affected by stress response. GO Slim terms are that are overrepresented in the set significantly-enriched transcripts (red), the set of significantly-depleted transcripts (blue), or in both sets (black). Terms are grouped in 15 categories, and their name coloured accordingly.

34

MRD1 BUD27

ENP2 NOP53

RPC82

TSR2 TRZ1 UTP14

DBP7

BMT6 NRP1 CMS1

ATC1 YGR160W

NOP4

SSF2

RRP17

SUA5

BCP1 BAS1 MAK16

NUC1

STB5

SLX9 URK1 EFG1

FAL1

ROK1

YML082W

TRM13 BUD22

YCR016W JJJ1

OGG1 NOP12

BFR2 SPB4

PUF6

LTV1

POL1 YOX1YHP1

POL12 CDC9

SPB1

GRC3

YNL120C YHR218W

UTP30

NOP9

BMT5 YFL065C UTP4

SMC3 IRR1

PDS5 TMA16 ACM1 YEL075C MBP1

IPI3

CDC45

CLN1

ESF2

RAD27

GIN4

FAF1

NOP7

CLB6 RCL1

YPR203W

MCD1 YFL064C YLR463C SPT21 YLR464W YPR202W SYO1 YEL076C YLR462WYHL049CCDC21 TOS4 YER189W YBL111C SWI4

SWI6

YAP5

CTF18 POL30

S27 Fig. Use of BTRs for functional annotation. Network of transcription factors associated with BTRs 60 (pink), 263 (yellow), and 330 (green); transcription factors shown as diamonds.

35