Description of Supplementary Files File Name: Supplementary Information Description: Supplementary Figures, Supplementary Tables and Supplementary References File Name: Supplementary Data 1 Description: RNAseq data related to Figure 1 File Name: Supplementary Data 2 Description: GO term enrichment data related to Figure 1 File Name: Supplementary Data 3 Description: HU sensitivity suppressor screen data related to Figure 2 File Name: Supplementary Data 4 Description: RNAseq data related to Figure 6 File Name: Supplementary Data 5 Description: Fitness data related to Figure 6. File Name: Supplementary Software 1 Description: R scripts and Cellprofiler pipelines used for the analysis of the data generated in this manuscript File Name: Peer Review File
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Description of Supplementary Files
File Name: Supplementary Information Description: Supplementary Figures, Supplementary Tables and Supplementary References
File Name: Supplementary Data 1 Description: RNAseq data related to Figure 1
File Name: Supplementary Data 2 Description: GO term enrichment data related to Figure 1
File Name: Supplementary Data 3 Description: HU sensitivity suppressor screen data related to Figure 2
File Name: Supplementary Data 4 Description: RNAseq data related to Figure 6
File Name: Supplementary Data 5 Description: Fitness data related to Figure 6.
File Name: Supplementary Software 1 Description: R scripts and Cellprofiler pipelines used for the analysis of the data generated in this manuscript
File Name: Peer Review File
1h 2h 4h
Num
ber
of g
enes
050
010
0015
0020
0025
00
553880
1178493
830
1134
increased abundancedecreased abundance
Supplementary Fig. 1. The number of genes induced or repressed upon HU exposure in WT cells. The number of differentially expressed genes was determined using the Tuxedo protocol.
a
-4 -2 0 2 4 6Shalem, MMS 1h
Thi
s st
udy,
HU
1h
Shalem, MMS 2.3h
Thi
s st
udy,
HU
2h
-4 -2 0 2 4Jaehnig, MMS 1h
This
stu
dy, H
U 1
h
-4 -2 0 2 4
-4-2
02
46
Dubacq, 1h HU
Thi
s st
udy,
HU
1h
-6 -4 -2 0 2 4 6Dubacq, 2h HU
Thi
s st
udy,
HU
2h
-4 -2 0 2 4Dubacq, 4h HU
Thi
s st
udy,
HU
4h
-4-2
02
46
-4-2
02
4
-4-2
02
46
-6 -4 -2 0 2 4 6 8
-4-2
02
46
-4-2
02
46
b
R = 0.720 R = 0.610 R = 0.685
R = 0.752R = 0.670R = 0.585
204 67 2011660 290 255178 93 175
Shalem, MMS 1hlog2 ratio < -1 | log2 ratio > +1
DE genes in lsm1∆ and HU 1h
Shalem, MMS 2.3hlog2 ratio < -1 | log2 ratio > +1
Jaehnig, MMS 1hlog2 ratio < -1 | log2 ratio > +1
DE genes in lsm1∆ and HU 2h
DE genes in lsm1∆ and HU 1h
c
Supplementary Fig. 2. Lsm1 regulates RNA abudance of HU-responsive genes as well as more general DNA replication stress genes. (a) Correlation between the expression ratios for the differentially expressed genes identified in WT cells in this study upon HU exposure and the study of Dubacq et al. (2006) at 1, 2 and 4 hours in HU. (b) Correlation between the expression ratios for the differentially expressed genes identified in WT cells in this study upon HU exposure and WT cells in MMS in the study of Shalem et al. (2008) or Jaehnig et al. (2013). (c) Venn Diagram representing the overlap between the genes showing log2 expression ratios inferior to -1 or superior to +1 in the study of Shalem et al. (2008) or Jaehnig et al. (2013) and the genes showing differential expression upon HU exposure and in lsm1∆ cells in this study. DE: Differentially Expressed
1
1C 2C 1C 2C 1C 2C 1C 2C
0h
1h
2h
3h
4h
0h
1h
2h
3h
4h
WT lsm1∆
No Drug HU 200 mM No Drug HU 200 mM
Supplementary Fig. 3. Cell-cycle progression of the WT and lsm1∆ strain under normal growth or in presence of HU. Flow cytometry histograms o NA contents in wild type and lsm1∆ cells. Cells were arrested in G1 and then released into S phase in the absence or presence o HU. The positions o cells with 1C and 2C NA contents are indicated.
0
1
2
3
4
untreated 2h HU 4h HU
RNA-Seq, lsm1∆qRT-PCR, lsm1∆qRT-PCR, pat1∆
YO
X1
mR
NA
log2
fold
-cha
nge
(mut
ant:W
T)
Supplementary Fig. 4. YOX1 RNA abudance increases in lsm1∆ and pat1∆ cells. log2 expression ratios comparing YOX1 mRNA levels in WT and lsm1∆ cells obtained by RNA-Seq (grey) or qRT-PCR (red) or log2 expression ratios comparing YOX1 mRNA levels in WT and pat1∆ cells obtained by qRT-PCR (orange). The means and two biological replicates are shown. Each qRT-PCR biological replicate included at least 2 technical replicates.
2
3
untreated 1h HU-2
-1
0
1 RNA-Seq, lsm1∆qRT-PCR, lsm1∆
ALD
6 m
RN
Alo
g2 fo
ld-c
hang
e (ls
m1∆
:WT)
Supplementary Fig. 6. ALD6 mRNA abundance decreases in lsm1∆ cells. log2 expression ratios comparing ALD6 mRNA levels in WT and lsm1∆ cells obtained by RNA-Seq (grey) or qRT-PCR (red). The means and two biological replicates are shown. Each qRT-PCR biological replicate included at least 2 technical replicates.
130 kDa100 kDa
70 kDa55 kDa
35 kDa
25 kDa
anti-FLAG
anti-PGK1
Supplementary Fig. 5. Full immuno-blot image related to Fig. 5b. Yox1-FLAG protein abundance was probed using anti-FLAG antibody. Pgk1 was used as loading control.
Yox1-Flag
Pgk1
lsm1∆WTHU (hours) : 0 2 4 0 2 4
lsm1∆WT0 2 4 0 2 4
unspecific
technical replicate #1
technical replicate #2
Supplementary Table 1. Comparison of RNA-seq results obtained with Cuffdiff, EBSeq or edgeR
FC : Fold-Change; Red shading indicates statistically supported differential expression1 Values superior or equal to 0.95 are considered significant for posterior probability in EBSeq
2h HU
gene
4h HU
cuffdiff
log2 FClog2 FC FDR corrected p-valuep-value
untreated
1h HU
posterior
probability 1
EBSeq
condition
edgeR
log2 FC p-value FDR corrected p-value
Supplementary Table 3. YETFASCO scanning results for ALD6 and ICS2 promoter regions
RLKY110 ald6∆ MATa ald6∆::kanMX leu2∆0 his3∆1 ura3∆0 met15∆0 3
RLKY111 YOX1-FLAG MATa YOX1-FLAG-KanMX leu2∆0 his3∆1 ura3∆0 met15∆0 this study
RLKY112 lsm1∆ YOX1-FLAG
MATa lsm1∆::URA3MX YOX1-FLAG-KanMX leu2∆0 his3∆1 ura3∆0 met15∆0 this study
All strains in Figure 2 have the following genotypes: MATa lsm1∆::natMX xxx∆::kanMX can1∆::STE2pr-his5 lyp1∆ leu2∆0 his3∆1 ura3∆0 lys2∆0�MATa pat1∆::natMX xxx∆::kanMX can1∆::STE2pr-his5 lyp1∆::STE3pr-LEU2 leu2∆0 his3∆1 ura3∆0 lys2∆0
this study
All strains in Figure 6 have the following genotypes: MATa xxx∆::kanMX yyy∆::natMX leu2∆0 his3∆1 ura3∆0 lyp1∆0 can1∆0::STE2pr-SpHIS5
this study
All strains in Figure 7a-b have the following genotypes: MATa lsm1∆::kanMX leu2∆0 his3∆1 ura3∆0 met15∆0 pBY011[GAL1-10pr-] �MATa lsm1∆::kanMX leu2∆0 his3∆1 ura3∆0 met15∆0 pBY011[GAL1-10pr-XXX]
this study
All strains in Figure 7f-g have the following genotypes: MATa xxx∆::NatMX RNR3-GFP-HIS3MX can1pr::RPL39pr-tdTomato-CaURA3 can1∆::STE2pr-LEU2 leu2∆0 his3∆1 ura3∆0 met15∆0 lyp1∆0
this study
References
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S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14, 115–32 (1998).
2. Tkach, J. M. et al. Dissecting DNA damage response pathways by analysing protein localization and abundance changes during DNA replication stress. Nat. Cell Biol. 14, 966–76 (2012).
3. Giaever, G. et al. Functional profiling of the Saccharomyces cerevisiae genome. Nature 418, 387–391 (2002).
4. Hendry, J. A., Tan, G., Ou, J., Boone, C. & Brown, G. W. Leveraging DNA damage response signaling to identify yeast genes controlling genome stability. G3 (Bethesda). 5, 997–1006 (2015).