Martin, Laetitia Da Silva, Ivan Montoliu, Sebastiano Collino, Francois-Pierre. Martin, Joanna Ratajczak, Carles Cantó, Martin Kussmann, Felix Naef, and Frédéric.
Cell Reports, Volume 20
Supplemental Information
Circadian and Feeding Rhythms Orchestrate the Diurnal Liver Acetylome Daniel Mauvoisin, Florian Atger, Loïc Dayon, Antonio Núñez Galindo, Jingkui Wang, Eva Martin, Laetitia Da Silva, Ivan Montoliu, Sebastiano Collino, Francois-Pierre Martin, Joanna Ratajczak, Carles Cantó, Martin Kussmann, Felix Naef, and Frédéric Gachon
Supplemental Information Supplemental Figures Figure S1. Experimental design and data quality check, related to Figure 1
A
B 1062
C
236
297
R=0.79
R=0.81
R=0.83
R=0.78
R=0.70
R=0.78
R=0.73
R=0.62
D 0.2
Nucleus Nucleus & Cytoplasm Mitochondria Nucleus & Cytoplasm
TE
NE
Non Nuclear localized
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A. Workflow of the SILAC-based MS analysis of acetylated proteins from total mouse liver extracts (TE) and purified nuclei (NE). The lower graph represents the number of acetylation sites for each time point in TE (black bars, left Y axis) and NE (red bars, right Y axis). B. Number of acetylated sites quantified in TE (red circle, 1298 acetylated sites identified) and NE (green circle, 533 acetylated sites identified). C. Pearson correlation analysis of biological replicates at the eight time points. All values are log2 ratios to the common reference samples. The biological replicates are well correlated (76% average Pearson correlation) and showed similar spread, indicating that the quality of the protein purifications was fairly homogenous. D. Fractions of raw intensity signals quantified with SILAC-MS for nuclear and non-nuclear located proteins in TE (left graph) and NE (right graph).
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Figure S2. Overlap of rhythmic acetylation sites and expression of Acetyl-CoA synthesizing enzymes, related to Figure 2
A
B ZT 0
6
12
18
6
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18
98
n=34
24
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HMGCS2 K83; HMGCS2 K310; STIP1 K325 CPS1 K55; CPS1 K527; CPS1 K915; SOD2 K122; SOD2 K68
5 14
C ZT 0
n=17
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O LK
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Average Absolute MS intensities (relative to ACSS2)
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A. Venn diagram showing the overlap of rhythmic acetylation sites between the TE dataset (106 rhythmic acetylation sites), the NE dataset (27 rhythmic acetylation sites), and the dataset of (Masri et al., 2013) (19 rhythmic acetylation sites). B-C. Heat maps showing 12 hours rhythmic acetylation sites normalized by their corresponding total protein amount in (B) TE (n=34) and (C) NE (n=17) under light-dark and night-restricted feeding conditions. Data were standardized by rows and gray blocks indicate missing protein data. The polar plots on the right of each heatmap display peak phase distribution of the 12 hours rhythmic acetylation sites in each extract. Colors indicate acetylation sites with a corresponding total protein having a defined mitochondrial (n=17 sites; red) or cytoplasmic (n=20 sites; blue) localization in TE, or acetylation sites from NE (n=17; green). D-E. Temporal protein expression of ACLY (black), ACSS2 (blue), and ACSS3 (orange) in TE (D) and NE (E) (ACSS3 not detected in NE). The values represent the mean ± SEM from two independent biological samples. F-G. Raw intensity signals quantified by SILAC-MS for ACLY (black), ACSS2 (blue), and ACSS3 (orange) in TE (F) and NE (G) (ACSS3 not detected in NE). Data are expressed relative to ACSS2 average signal, error bars represent min to max. H. Differential protein expression level of ACLY, ACSS2, and ACSS3 in TE in Cry1/2 DKO mice (red bars) and Bmal1 KO mice (green bars) compared to respective WT animals. Data show average level ± SEM in four samples collected around the clock. Data of panels D, F, H, and E, G are from (Mauvoisin et al., 2014) and (Wang et al., 2017), respectively.
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Figure S3. Temporal nuclear expression of SIRT1 and SIRT7, related to Figure 3
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24 hour-Rhythms 12 hour-Rhythms N=106 N=34
SILAC (L/H) ratio log2
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A. Proportion of SIRT3 targets (orange) versus non-SIRT3 targets (blue) in rhythmic acetylation sites with 24 hourrhythms and 12 hour-rhythms. B. Temporal nuclear expression of SIRT1 (blue) and its coactivator RPS19BP1 (black). C. Temporal nuclear expression of SIRT2. D. Temporal nuclear expression of SIRT7. For B, C, and D, the values represent the mean ± SEM from two independent biological samples. Data are extracted from (Wang et al., 2017).
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Figure
S4.
Additional
metabolic
pathways
affected
by
rhythmic
acetylation,
related to Figure 5
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°* ATP5B °* ATP5C1 * NDUFA2 NDUFA4 NDUFA5
Oxida/ve Phosphoryla/on
ATP5A1
Qu a Ide n3fie n3 d fie d
Rhythmic
Ac-sites
°* NDUFA10 NDUFAB1 ATP5J °* ATP5H °* NDUFB10 NDUFS1 CYB5;CYB5A UQCRC1 ATP5O * ATP5L °* NDUFV1 NDUFV2 UQCRB °* UQCRC2 MT-ATP8 UQCRCQ
ATP5D ATP5F1
COX6B1
ATP5J2
Ac-level different in Cry 1/2 DKO° and Bmal1 KO*mice Lysine AASS
I
V
III
°*
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Succinyl-CoA Synthase Succinate SUCLG2 SUCLA2 SUCLG1 dehydrogenase
°*
α-aminoadipate-6-semialdehyde
°*
SDHA
a-ketoglutarate dehydrogenase DLST DLD Succinyl-CoA
°*
Succinate
AASDH
Lysine degrada/on
COX4I1 COX5A
Fumarate
Fumarate hydratase
°*
α-ketoglutarate
FH
Isocitrate dehydrogenase
α-aminoadipate
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TCA cycle
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Malate dehydrogenase
Malate
D-isocitrate
*
MDH2
Aconitase ACO2
°*
α-ketoadipate BCKDK BCKDHA
Oxaloacetate
Citrate
Pyruvate carboxylase PC
°*
Citrate Synthase Acetyl-CoA CS
°*
Glutaryl-CoA
Pyruvate Pyruvate dehydrogenase complex
GCDH
*
PDHA1 PDHB DLAT
°*
DLD
Crotonyl-CoA ECHS1
β-Hydroxybutyryl-CoA
*
HADH
acetoacetyl-CoA
to ketogenesis
Rhythmic protein acetylation and their regulation by the circadian clock for lysine degradation, oxidative phosphorylation, and TCA cycle pathways. Each dot represents a unique acetylation site within the protein of interest. Grey and black dots represent non-rhythmic identified and quantified acetylation sites, respectively, whereas phase of rhythmic acetylation sites are color-coded. Superscripted red dots and green asterisks indicate protein acetylation level significantly different in Cry1/2 DKO and Bmal1 KO mice, respectively.
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Figure S5. Differential acetylation of the enzymes involved in the different metabolic pathways in Cry1/2 and Bmal1 KO mice, related to Figure 5 and S4
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SDHA
FH
MDH2
PC
Lysine degrada6on § § §
*
*
§
0.0 -0.5 -1.0 -1.5
ACO2
AASS
HDAH
*
ZT 0 6 12 18 0 6 12 18 Cry1/2 DKO Bmal1 KO paired t-test KO-WT § p
