Proteomic profiling reveals distinct phases to the restoration of chromatin following DNA replication
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion
ORGANISM(S): Homo Sapiens (human)
SUBMITTER: 
Vanesa Alvarez
LAB HEAD: Constance Alabert
PROVIDER: PXD038113 | Pride | 2023-02-02
REPOSITORIES: Pride
Dataset's files
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| NCC_batchB_F1.raw | Raw | |||
| NCC_batchB_F10.raw | Raw | |||
| NCC_batchB_F11.raw | Raw | |||
| NCC_batchB_F12.raw | Raw | |||
| NCC_batchB_F13.raw | Raw |
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Project description:Chromatin organization must be maintained during cell proliferation to preserve cellular identity and genome integrity. However, DNA replication results in transient displacement of DNA bound proteins, and it is unclear how they regain access to newly replicated DNA. Using quantitative MS-based proteomics coupled to Nascent Chromatin Capture, we provide time resolved binding kinetics for thousands of proteins behind replisomes of mid-S replicated regions. This shows that most proteins regain access within the first 15 minutes after the passage of the fork. In contrast, 25% of the identified proteins do not, and this delay cannot be inferred from their known function, physicochemical properties, or nuclear abundance. Here, we provide data where we impaired the H3K27me3 restoration to analyse its impact into chromatin restoration. To this aim, we used the EPZ-6438 inhibitor, which blocks the histone methyltransferase EZH2. Two time-points are analysed by NCC along the cell cycle in late replicated regions: nascent chromatin, just after the passage of the fork, and a sample 16 hours later, in late G1 phase. Our data support that H3K27me3 have an important role in chromatin restoration. Data available in this entry are related with the supplementary table 6 from our manuscript.
2023-02-02 | PXD038376 | Pride
Project description:Chromatin organization must be maintained during cell proliferation to preserve cellular identity and genome integrity. However, DNA replication results in transient displacement of DNA bound proteins, and it is unclear how they regain access to newly replicated DNA. Using quantitative MS-based proteomics coupled to isolation of proteins on nascent DNA (iPOND), we provide time resolved binding kinetics for thousands of proteins behind replisomes in the lung fibroblast cell line TIG-3 (JCRB0506, JCRB Cell Bank). Performing hierarchical clustering, we identified four groups of protein behaviour: Transient enriched on nascent, restored within 11min, peaking in G2/M, and delayed restored. Overall, our data show that most proteins (85%) regain access within the first 2h after the passage of the fork. Only a small percentage of proteins is restored after 2h post-replication, with 5.3 % of factors peaking in G2/M and 9.1% factors not restored before the next G1 phase. We provide evidence that DNA replication not only disrupts but also promotes recruitment of transcription factors and chromatin remodelers, providing a significant advance in understanding how DNA replication could contribute to programmed changes of cell memory.
2023-02-02 | PXD036236 | Pride
Project description:Chromatin organization must be maintained during cell proliferation to preserve cellular identity and genome integrity. However, DNA replication results in transient displacement of DNA bound proteins, and it is unclear how they regain access to newly replicated DNA. Using quantitative MS-based proteomics coupled to Nascent Chromatin Capture, we provide time resolved binding kinetics for thousands of proteins behind replisomes within euchromatin and heterochromatin in human cells. This shows that most proteins regain access within the first 15 minutes after the passage of the fork. In contrast, 30% of the identified proteins do not, and this delay cannot be inferred from their known function, physicochemical properties, or nuclear abundance. Instead, differential chromatin organization affect their reassociation. We provide evidence that DNA replication not only disrupts but also promotes recruitment of transcription factors and chromatin remodellers, providing a significant advance in understanding how DNA replication could contribute to programmed changes of cell memory.We include here a reference to link the data with our manuscript:Tables SUPP1 and SUPP2:NCC data: PT7988 (Bio-rep1), PT8242 (Bio-rep2), PT8243 (Bio-rep3). 1-24 (24 high-pH RPLC fractions from TMT labeled peptides)Nuclear fraction data: PT7988 (Bio-rep1), PT8242 (Bio-rep2), PT8243 (Bio-rep3). 25-48 (24 high-pH RPLC fractions from TMT labeled peptides)Tables SUPP3-5:NCC data: PT9825 (Bio-rep1), PT9825 (Bio-rep2), PT9825 (Bio-rep3). (11-34, 35-58, 59-82) (24 high-pH RPLC fractions from TMT labeled peptides)Nuclear fraction data: PT9825 (Bio-rep1), PT9825 (Bio-rep2), PT9825 (Bio-rep3). (83-106, 107-130, 131-154). (24 high-pH RPLC fractions from TMT labeled peptides)
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Project description:Long-term perturbation of de novo chromatin assembly during DNA replication has profound effects on epigenome maintenance and cell fate. The early mechanistic origin of these functional defects is unknown. Here, we combine acute degradation of Chromatin Assembly Factor 1 (CAF-1), a key player in de novo chromatin assembly during DNA replication and analyse effects n histone modifications by mass spectrometry. Immediately upon CAF-1 depletion from human TERT-RPE-1 cells, we observe an acute cellular response affecting histone repertoire, global chromatin composition and transcriptional fidelity resulting in a p53-dependent cell cycle arrest. Our work reveals the immediate local and global consequences of defective de novo chromatin assembly during DNA replication, explaining how at later times the epigenome and cell fate can be altered.
2024-11-14 | PXD050618 | Pride
Project description:The ring-shaped cohesin complex links sister chromatids until their timely segregation during mitosis. Cohesin is enriched at centromeres, where it provides the cohesive counter-force to bi-polar tension produced by the mitotic spindle. As a consequence of spindle tension, centromeric sequences transiently split in pre-anaphase cells, in some organisms up to several micrometeres. This M-bM-^@M-^Xcentromere breathingM-bM-^@M-^Y presents a paradox, how sister sequences separate where cohesin is most enriched. We now show that in the budding yeast S. cerevisiae, cohesin binding diminishes over centromeric sequences that split during breathing. We see no evidence for cohesin translocation to surrounding sequences, suggesting that cohesin is removed from centromeres during breathing. Two pools of cohesin can be distinguished. Cohesin loaded before DNA replication, that has established sister chromatid cohesion, disappears during breathing. In contrast, cohesin loaded after DNA replication is partly retained. As sister centromeres re-associate after transient separation, cohesin is re-loaded in a manner independent of the canonical cohesin loader Scc2/Scc4. Efficient centromere re-association requires the cohesion establishment factor Eco1, suggesting that re-establishment of sister chromatid cohesion contributes to the dynamic behaviour of centromeres in mitosis. These findings provide new insights into cohesin behaviour at centromeres. Keywords: ChIP-chip SAMPLES Origin of each biological sample: Saccharomyces cerevisiae (W303background) Growth conditions: Cells containing the MET3-CDC20 allele were grown at 25M-BM-0C in SC medium lacking methionine with 2% raffinose or 2% glucose as the carbon source (Uhlmann et al. 2000). Cultures were synchronized in G1 with M-NM-1-factor (5 M-NM-<g/ml) and released into medium containing 100 mM hydroxyurea (HU) for arrest in early S-phase. For arrest in metaphase, cells were released from M-NM-1-factor or HU arrest by filtration and re-suspension in YP medium supplemented with 5 mM methionine to deplete Cdc20. To inactivate cohesin loading, cells harbouring the temperature sensitive scc2-4 allele (Ciosk et al. 2000) were shifted to the restrictive temperature of 35M-BM-0C for 1 hour before release. To prevent metaphase spindle assembly, or to disassemble already formed spindles, 7.5 M-NM-<g/ml nocodazole was added to the medium for 1 hour. To wash out nocodazole, cells were filtered again, washed, and re-suspended in fresh medium lacking nocodazole for 1 hour. Expression of Scc1 to induce differentially epitope-tagged cohesin under control of the GAL1 promoter, or of Cdc14, was achieved by addition of 2% galactose for 1 hour to cultures grown in raffinose-containing medium. EXPERIMENTAL DESIGN Experiment type: ChIP-chip = Chromatin immunoprecipitation (ChIP) followed by hybridization to a high-density oligonucleotide microarray (chip). Number of hybridizations performed: 13 ChIP-chip samples; 2 SUPernatant samples Analysis: ChIP samples were normalized against SUPernatant fractions Quality control: Confirmation of results by duplication of experiments, by usage of different epitope tags for the same protein, and by usage of different subunits of the same protein complex. Also, whole cell extract, SUPernatant, and ChIP fractions were checked by Western blotting. Protocols: ChIP-chip and hybridization protocols were performed as previously described (Katou et al. 2003; Lengronne et al. 2004; Lengronne et al. 2006). A summary is provided below: ChIP. Cells were grown under the conditions described above and cross-linked with 1% formaldehyde. Cells were disrupted using a Multi-Beads Shocker, and whole cell extracts were sonicated to obtain 400-600 bp genomic DNA fragments. Chromatin immunoprecipitation (ChIP) was performed with anti-HA antibody or anti-PK monoclonal antibody coupled to protein A Dynabeads. Immunprecipates were eluted and incubated overnight at 65M-BM-:C to reverse the cross-link. The immunoprecipitated genomic DNA was then incubated with proteinase K, extracted twice with phenol/chloroform/isoamylalcohol, precipitated, resuspended in TE and incubated with RNaseA. The DNA was purified, concentrated by ethanol precipitation and amplified by PCR after random priming. 10ug of the amplified DNA was digested with DNase I and end-labelled using TdT Terminal Transferase and Biotin-N6-ddATP. Hybridization and scanning. Samples were hybridized in buffer containing SSPE, TritonX-100, denatured salmon sperm DNA and biotin-labelled control oligonucleotide, oligo B2. Samples were denatured at 95M-BM-:C for 10 minutes before being hybridized to the arrays for 16 hours at 42M-BM-:C in a GeneChip Hybridization Oven 640. The washing and scanning protocol (FlexMidi_euk2v3_450), provided by Affymetrix, was performed automatically on a GeneChip Fluidics Station 450. Arrays were scanned using the GeneChip Scanner 3000 7G, and primary analysis of tiling chip data was performed using the Affymetrix GeneChip Operating Software (GCOS). ARRAY DESIGN General array design: in situ synthesized arrays from Affymetrix Location and ID of each spot on arrays: available upon request from Affymetrix Probe type: oligonucleotide Availability of arrays: commercially available from Affymetrix S. cerevisiae (Chromosome VI) rikDACF, P/N# 510636 S. cerevisiae T iling Array (Forward), P/N# 520286 REFERENCES Ciosk R, Shirayama M, Shevchenko A, Tanaka T, Toth A, Shevchenko A, Nasmyth K (2000) Cohesin's binding to chromosomes depends on a separate complex consisting of Scc2 and Scc4 proteins. Mol Cell 5: 1-20 Katou Y, Kanoh Y, Bandoh M, Noguchi H, Tanaka H, Ashikari T, Sugimoto K, Shirahige K (2003) S-phase checkpoint proteins Tof1 and Mrc1 form a stable replication-pausing complex. Nature 424: 1078-1083 Lengronne A, Katou Y, Mori S, Yokobayashi S, Kelly GP, Itoh T, Watanabe Y, Shirahige K, Uhlmann F (2004) Cohesin relocation from sites of chromosomal loading to places of convergent transcription. Nature 430: 573-578 Lengronne A, McIntyre J, Katou Y, Kanoh Y, Hopfner K-P, Shirahige K, Uhlmann F (2006) Establishment of sister chromatid cohesion at the S. cerevisiae replication fork. Mol Cell 23: 787-799 Uhlmann F, Wernic D, Poupart M-A, Koonin EV, Nasmyth K (2000) Cleavage of cohesin by the CD clan protease separin triggers anaphase in yeast. Cell 103: 375-386
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