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Replication forks proteomics reveals ATM-dependent rewiring of broken forks to promote restart

ABSTRACT: The fidelity of DNA replication is governed by a network of DNA repair mechanisms. Defects in replication fork protection can fuel genome instability in cancer, while also creating cancer-specific vulnerabilities. Here, we provide a comprehensive proteomic characterization of the replication fork response to topoisomerase 1 inhibition, a widely used mainstay chemotherapy. We reveal profound changes in the fork proteome and chromatin environment in response to seDSBs and topological stress, and classify fork repair factors according to their specificity for broken and stalled forks. These distinct fork responses also oppositely affect nucleosome occupancy and nuclear membrane interactions. ATM inhibition dramatically rewired the fork breakage response, revealing that ATM promotes HR-dependent fork restart by concomitantly promoting DNA-end resection and suppressing DSB associated protein ubiquitination and NHEJ. Together, this collection of damaged fork proteomes provides an ample resource to understand fork repair mechanisms and identify druggable targets and novel cancer vulnerabilities.

INSTRUMENT(S): Orbitrap Fusion Lumos, Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Diploid Cell

SUBMITTER: Georg Kustatscher

LAB HEAD: Juri Rappsilber

PROVIDER: PXD018092 | Pride | 2021-01-15

REPOSITORIES: Pride

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Analysis of histone PTMs in nascent chromatin in new and old histones upon DNA damage (CAL353, CAL358) using the NCC-SILAC approach

Project description:Dynamic recycling and de novo deposition of histones are fundamental for chromatin restoration. Histone post-translational modifications (PTMs) are assumed to have a causal role in establishing distinct chromatin structures. However, it remains unknown how specific histone PTMs are regulated at broken replication fork. To get better insight into histone PTMs during DNA damage response, we combined NCC (Nascent chromatin capture) with SILAC-MS for identification of histone PTMs at replication forks upon CPT (camptothecin).

2021-01-18 | PXD018220 | Pride

Proteomic profiling reveals distinct phases to the restoration of chromatin following DNA replication

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

Chromatin proteomics experiments for ProteomeHD

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2019-11-11 | PXD008888 | Pride

Proteomic profiling reveals distinct phases to the restoration of chromatin following DNA replication

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2023-02-02 | PXD038113 | Pride

Two distinct modes for propagation of histone PTMs across the cell cycle

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2015-03-30 | PXD001980 | Pride

Unchanged PCNA and DNMT1 dynamics during replication in DNA ligase I-deficient cells but abnormal chromatin levels of non-replicative histone H1

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2023-04-04 | PXD039547 | Pride

Proteomic profiling reveals distinct phases to the restoration of chromatin following DNA replication

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2023-02-02 | PXD035489 | Pride

Avoiding chromosome pathology when replication forks collide

Project description:Chromosome duplication normally initiates via the assembly of replication fork complexes at defined origins. DNA synthesis by any one fork is thought to cease when it meets another travelling in the opposite direction, at which stage the replication machinery may simply dissociate before the nascent strands are finally ligated. But what actually happens is not clear. Here we present evidence consistent with the idea that every fork collision has the potential to trigger re-replication of the already replicated DNA, thus posing a threat to genomic integrity. In Escherichia coli this threat is kept at bay by the RecG DNA translocase. Without RecG, replication initiates where forks meet, establishing new forks with the potential to sustain cell growth and division in the absence of an active origin. The studies reported raise the question of how eukaryotic and archaeal cells are able to exploit multiple origins for the duplication of each chromosome without any apparent ill effect from the consequent multiple fork collisions. Measurement of replication dynamics (marker frequency analysis; MFA) for E. coli strains, including wild-type and various mutants.

2013-07-22 | E-GEOD-41975 | biostudies-arrayexpress

Stably-paused genes revealed through inhibition of transcription initiation by the TFIIH inhibitor Triptolide

Project description:Transcription by RNA Polymerase II (Pol II) in metazoan is regulated in several steps, including preinitiation complex (PIC) formation, initiation, Pol II escape, productive elongation, cotranscriptional RNA-processing and termination. Genome-wide studies have demonstrated that the phenomenon of promoter-bound Pol II pausing is widespread, especially for genes involved in developmental and stimulus-responsive pathways. However, a mechanistic understanding of the paused Pol II states at promoters is limited. For example, at a global level, it’s unclear to what extent the engaged paused Pol II is stably tethered to the promoter or undergoes rapid cycles of initiation and termination. Here we used the small molecule Triptolide (TPL), an XPB/TFIIH inhibitor, to block transcriptional initiation followed by measuring Pol II occupancy by ChIP-seq. This inhibition of initiation enables us to investigate different states of paused Pol II. Specifically, our global analysis reveals that most genes with paused Pol II, as defined by pausing index, show significant clearance of Pol II during the period of TPL treatment. Our study further identifies a group of genes with unexpectedly stably-paused Pol II, with unchanged Pol II occupancy even after one hour of inhibition of initiation. This group of genes constitutes a small portion of all paused genes defined by the conventional criterion of pausing index. These findings could pave the way for evaluating the contribution of different elongation/pausing factors on different states of Pol II pausing in developmental and other stimulus-responsive pathways. ChIP-Seq of total/Ser5P Pol II in HCT116 cells treated with DMSO or TPL in serum starvation/activation or normal conditions. Nascent RNA-seq in HCT116 cells treated with DMSO or TPL in starved condition.

2015-01-05 | E-GEOD-63314 | biostudies-arrayexpress

Allele-specific regulation of gene expression through enhancer function and transcriptional elongation control at imprinted loci

Project description:Genomic imprinting is a critical developmental process characteristic of parent-of-origin- specific gene expression. Here, we have identified the AFF family protein, Aff3, as a factor that functionally interacts with imprinted loci. Indeed, our genome-wide studies demonstrate that Aff3 specifically binds both imprinting control regions (ICRs) and enhancers within imprinted loci in an allele-specific manner. We have identified the molecular regulators involved in the recruitment of Aff3 to ICRs to impede transcription through the ICR, and provide a mechanism requiring Aff3 within the Super Elongation Complex-like 3 (SEC-L3) in the expression of an imprinted polycistronic transcript spanning the Dlk1-Dio3 locus. Our study also shows that DNA methylation at the ICR reinforces silencing of its related enhancers by controlling the binding and activity of Aff3 in an allele-specific manner. This study provides molecular details about the regulation of dosage-critical imprinted gene expression through Aff3âs function in transcriptional elongation control. ChIP-seq of Aff3 in different mES cells. ChIP-seq of Aff3, PolII, H3K9me3 in uniparental MEF cell lines. ChIP-seq of H3K27ac and PolII in mES cells after Aff3 shRNA and non-targeting shRNA. ChIP-seq of H3K27ac in wild type and Zfp57 knockout ES cells. Total RNA-seq and nascent RNA-seq of mES cells after Aff3 shRNA and non-targeting shRNA. Total RNA-seq of uniparental MEF cells after Aff3 shRNA and non-targeting shRNA.

2016-07-03 | E-GEOD-64489 | biostudies-arrayexpress

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