Distinct and diverse chromatin proteomes of ageing mouse organs reveal protein signatures that correlate with physiological functions
- Department of Biochemistry & Molecular Biology and VILLUM Center for Bioanalytical Sciences. University of Southern Denmark, Denmark
Figures
Figure 1 with 2 supplements
Strategy for the enrichment of chromatin-associated proteins in mouse embryonic stem cells.
(A) Western blot analysis of mouse embryonic cell fractionation. The cytoplasmic, nuclei, chromatin, and histone compartments are probed (respectively) by GAPDH, Laminin A/C, SUZ12, and H3 …
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Figure 1—source data 1
Western blot data.
- https://cdn.elifesciences.org/articles/73524/elife-73524-fig1-data1-v2.pdf
Figure 1—figure supplement 1
Comparison of annotated chromatin‐associated proteins from recently deposited proteomics data libraries of chromatin studies.
UpSet plot measurement of the number of chromatin-associated proteins detected in different studies (Medvedeva et al., 2015; van Mierlo et al., 2019; Christoforou et al., 2016; Xu et al., 2016). …
Figure 1—figure supplement 2
Evaluation of the enrichment of chromatin-associated proteins in mouse organs.
Brain organ fractionation was assayed by Western blot demonstrating the separation between nuclear and chromatin fraction in mouse brain. The heterochromatin marker HP1BP3 and histone H2A variant …
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Figure 1—figure supplement 2—source data 1
Western blot results (original scans).
- https://cdn.elifesciences.org/articles/73524/elife-73524-fig1-figsupp2-data1-v2.pdf
Figure 2
Comparative and quantitative proteomics of chromatin proteins in mouse organs.
(A) High-resolution LC-MS/MS brain proteomics during mouse lifespan. Dynamic range plot of the brain tissue fractionation obtained from a mouse. Chromatin lysate and whole total lysate proteomes are …
Figure 3 with 1 supplement
Chromatin proteomics by high mass resolution mass spectrometry demonstrated distinct organ ageing profiles.
(A) Principal component analysis (PCA) of the proteomics datasets. Each data point represents a single replicate (n = 3). Colour subgroups represent each mouse lifespan point, being 3, 5, 10, and 15 …
Figure 3—figure supplement 1
Evaluation of robustness and reproducibility of quantitative proteomics LC-MS/MS strategy.
The biological reproducibility of each organ proteome over time is displayed as a scatter plot and measured by the Pearson correlation coefficient. Red dots indicate high reproducibility, and blue …
Figure 4
Functional proteome analysis of common and distinct organ ageing profiles during the mouse lifespan.
(A) Overlap of proteome data sets across six mouse organs using an UpSet plot. The number of common (orange) and unique (red) organ-specific proteins detected is shown, while various inter-organ …
Figure 5 with 2 supplements
Identification of unique protein abundance changes in organ proteome profiles during ageing.
(A) UpSet plot measurement of the amount of nuclear and relative chromatin-associated proteins differentially expressed during the mouse lifespan across all organs. Unique chromatin-associated …
Figure 5—figure supplement 1
Evaluation of protein expression changes in mouse organs during ageing.
(A) Workflow of the proteome data analysis. (B) Box plots of protein expression profile before and after normalisation across six organs (brain, heart, kidney, lung, liver, and spleen) over time. …
Figure 5—figure supplement 2
Distinct organ ageing profiles are defined by unique differential protein expression.
(A) Volcano plots of differentially regulated proteins across six organs (brain, heart, kidney, lung, liver, and spleen) between the early (3 months) and the late (15 months) adult mouse lifespan. …
Figure 6
Identification of differentially regulated organ-specific profiles that map to ageing, epigenetic processes, and other biochemical processes.
Dot plot of functional annotation Gene Ontology (GO) analysis of the biological processes (BP) showing the enrichment pathway terms among ageing. GO categories are sorted by four group labels: gene …
Figure 7
Distinct organ ageing profiles are defined by unique chromatin-associated proteins.
(A) Protein interaction modules (obtained from the STRING database) are shown for Gene Ontology categories found to be significantly up- or downregulated in five organs. Each chord corresponds to a …
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Figure 7—source data 1
Original Western blotting data.
- https://cdn.elifesciences.org/articles/73524/elife-73524-fig7-data1-v2.pdf
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Figure 7—source data 2
Original Western blotting data.
- https://cdn.elifesciences.org/articles/73524/elife-73524-fig7-data2-v2.pdf
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Figure 7—source data 3
Original Western blotting data.
- https://cdn.elifesciences.org/articles/73524/elife-73524-fig7-data3-v2.pdf
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Figure 7—source data 4
Original Western blotting data.
- https://cdn.elifesciences.org/articles/73524/elife-73524-fig7-data4-v2.pdf
Tables
Table 1
Number of chromatin enzymes detected in mouse embryonic stem cell (mESC).
| Chromatin remodelling complex | Whole-cell lysate | Chromatin-enriched lysate |
|---|---|---|
| BHC | 2 | 6 |
| CCR4-NOT | 2 | 6 |
| ING | 1 | 15 |
| INO80 | 2 | 9 |
| MEDIATOR | 8 | 20 |
| MLL | 15 | 56 |
| NUA4HAT | 17 | 8 |
| NURD | 2 | 35 |
| NURF | 10 | 2 |
| ORC | 1 | 30 |
| POLYCOMB | 7 | 30 |
| READER | 8 | 33 |
| SAGA | 8 | 8 |
| SET1 | 3 | 6 |
| SIN3A | 1 | 26 |
| SWI/SNF | 5 | 43 |
| Other chromatin enzymes | 10 | 80 |
Table 2
Number of proteins detected across six mouse organs over four time points and their annotated subcellular location.
Detected number of unique proteins across all organs are shown for each subcellular location.
| Chromatin proteomes during the mouse lifespan | |||||||
|---|---|---|---|---|---|---|---|
| Organ | Brain | Heart | Liver | Kidney | Lung | Spleen | Unique proteins across all organs |
| Chromatin | 936 | 457 | 648 | 714 | 713 | 1113 | 1542 |
| Nuclear | 615 | 323 | 355 | 426 | 442 | 556 | 930 |
| Other | 1559 | 1272 | 1483 | 1465 | 1477 | 1457 | 3456 |
Table 3
‘Core’ proteins shared in all six mouse organs over time sorted to their cell compartments.
| Chromatin proteome during the mouse lifespan | ||||||||
|---|---|---|---|---|---|---|---|---|
| Shared proteins | Unique proteins | |||||||
| Core | Brain | Heart | Liver | Kidney | Lung | Spleen | ||
| Chromatin | 289 | 119 | 18 | 27 | 22 | 17 | 242 | |
| Nuclear | 157 | 69 | 21 | 33 | 16 | 15 | 54 | |
| Other | 417 | 605 | 175 | 329 | 196 | 211 | 286 | |
| Total | 863 | 793 | 214 | 389 | 234 | 243 | 582 | |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | HRP goat anti- mouse(mouse polyclonal) | Merck | Cat# 401253;RRID:AB_437779 | WB (1:10,000) |
| Antibody | HRP goat anti-rabbit(rabbit polyclonal) | Sigma | Cat# A0545;RRID:AB_257896 | WB (1:10,000) |
| Antibody | HRP anti-guinea pig (guinea pig polyclonal) | Gift from Benny Garfinkel | WB (1:10,000) | |
| Antibody | Anti-SIRT3(rabbit monoclonal) | Cell Signaling Technology | Cat# 2627S;RRID:AB_2188622 | WB (1:1000) |
| Antibody | Anti-NEDD4 (rabbit polyclonal) | Cell Signaling Technology | Cat# 2740S;RRID:AB_2149312 | WB (1:500) |
| Antibody | Anti-PSME2 (rabbit polyclonal) | Cell Signaling Technology | Cat# 2409S;RRID:AB_2171085 | WB (1:500) |
| Antibody | Anti-SIRT5 (rabbit monoclonal) | Cell Signaling Technology | Cat# 8782S;RRID:AB_2716763 | WB (1:1000) |
| Antibody | Anti-CHD2 (rabbit polyclonal) | Cell Signaling Technology | Cat# 4170S;RRID:AB_10621947 | WB (1:500) |
| Antibody | Anti-KAT2A/GCN5 (rabbit monoclonal) | Cell Signaling Technology | Cat# 3305;RRID:AB_2128281 | WB (1:500) |
| Antibody | Anti-PSMD14 (rabbit monoclonal) | Cell Signaling Technology | Cat# 4197S;RRID:AB_11178935 | WB (1:1000) |
| Antibody | Anti-RING1B/RNF2 (rabbit monoclonal) | Cell Signaling Technology | Cat# 5694P;RRID:AB_10705604 | WB (1:500) |
| Antibody | Anti-H3 (rabbit polyclonal) | Abcam | Cat# ab1791;RRID:AB_302613 | WB (1:20,000) |
| Antibody | Anti-macroH2A.1 (rabbit polyclonal) | Abcam | Cat# ab37264;RRID:AB_883064 | WB (1:1000) |
| Antibody | Anti-macroH2A2.2 (rabbit polyclonal) | Abcam | Cat# ab4173;RRID:AB_2115110 | WB (1:1000) |
| Antibody | Anti-RNF20 (rabbit polyclonal) | Cell Signaling Technology | Cat# 9425S;RRID:AB_2797700 | WB (1:1000) |
| Antibody | Anti-H3acetyl (rabbit polyclonal antibody) | Active motif | Cat# 39139;RRID:AB_2687871 | WB (1:500) |
| Antibody | Anti-beta actin (mouse monoclonal) | Sigma | Cat# A5441; RRID:AB_476744 | WB (1:5000) |
| Antibody | Anti-FBX041 (rabbit polyclonal) | Protein Tech | Cat# 24519-1-AP; RRID:AB_2879586 | WB (1:500) |
| Antibody | Anti-HP1BP3(guinea pig polyclonal) | Gift from Benny Garfinkel | WB (1:2000) | |
| Software, algorithm | Xcalibur software | Thermo Scientific | RRID:SCR_014593 | |
| Software, algorithm | Progenesis QI v2.2 | Waters | RRID:SCR_018923 | |
| Software, algorithm | Proteome Discoverer v2.1.0.81 | Thermo Scientific | RRID:SCR_014477 | |
| Software, algorithm | PolySTest | RRID:SCR_021942 | http://computproteomics.bmb.sdu.dk:8192/app_direct/PolySTest/ |
Additional files
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Supplementary file 1
Supplemental Table S1.
- https://cdn.elifesciences.org/articles/73524/elife-73524-supp1-v2.xlsx
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Supplementary file 2
Supplemental Table S2.
- https://cdn.elifesciences.org/articles/73524/elife-73524-supp2-v2.xlsx
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Supplementary file 3
Supplemental Table S3.
- https://cdn.elifesciences.org/articles/73524/elife-73524-supp3-v2.xlsx
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Supplementary file 4
Supplemental Table S4.
- https://cdn.elifesciences.org/articles/73524/elife-73524-supp4-v2.xlsx
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Supplementary file 5
Supplemental Table S5.
- https://cdn.elifesciences.org/articles/73524/elife-73524-supp5-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/73524/elife-73524-transrepform1-v2.pdf
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Source data 1
Raw and annotated Western blot images.
- https://cdn.elifesciences.org/articles/73524/elife-73524-data1-v2.zip
