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RNA polymerase II acts to promote the organization of chromatin following DNA replication

ABSTRACT: In mammals, following the passage of the replisome, nucleosomes lose their defined positioning and transcription contributes to their re-organisation. However, whether transcription plays a greater role in the organization of chromatin following DNA replication remains unclear. By performing iPOND-TMT time course experiments in the presents of transcription inhibition, we show that RNAPII promotes the re-association of hundreds of proteins with newly replicated DNA (Data uploaded under PXD046514). To show that this function of RNAPII is specific to newly replicated chromatin, an iPOND-TMT 24hr chase experiment was performed in the presence of transcription inhibition to examine its effect on the protein binding on steady state chromatin.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Susanne Bandau

LAB HEAD: Constance Alabert

PROVIDER: PXD046546 | Pride | 2024-02-14

REPOSITORIES: Pride

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RNA polymerase II promotes the organization of chromatin following DNA replication.

Bandau Susanne S Alvarez Vanesa V Jiang Hao H Graff Sarah S Sundaramoorthy Ramasubramanian R Gierlinski Marek M Toman Matt M Owen-Hughes Tom T Sidoli Simone S Lamond Angus A Alabert Constance C

EMBO reports 20240212 3

Understanding how chromatin organisation is duplicated on the two daughter strands is a central question in epigenetics. In mammals, following the passage of the replisome, nucleosomes lose their defined positioning and transcription contributes to their re-organisation. However, whether transcription plays a greater role in the organization of chromatin following DNA replication remains unclear. Here we analysed protein re-association with newly replicated DNA upon inhibition of transcription u ...[more]

PMID: 38347224

Similar Datasets

RNA polymerase II promotes the organization of chromatin following DNA replication

Project description:Understanding how chromatin organisation is duplicated on the two daughter strands is a central question in epigenetics. In mammals, following the passage of the replisome, nucleosomes lose their defined positioning and transcription contributes to their re-organisation. However, whether transcription plays a greater role in the organization of chromatin following DNA replication remains unclear. Here we have monitored protein re-association to newly replicated DNA upon inhibition of transcription using iPOND coupled to quantitative mass spectrometry. We show that RNAPII acts to promote the re-association of hundreds of proteins with newly replicated chromatin, including ATP-dependent remodellers, transcription factors, DNA repair factors, and histone methyltransferases.

2024-02-14 | PXD046514 | Pride

RNA polymerase II acts to promote the organization of chromatin following DNA replication

Project description:In mammals, following the passage of the replisome, nucleosomes lose their defined positioning and transcription contributes to their re-organisation. However, whether transcription plays a greater role in the organization of chromatin following DNA replication remains unclear. The data uploaded are associated to the iPOND-TMT data uploaded under PXD046514 where protein re-association to newly replicated DNA was monitored upon transcription inhibition. To see whether changes observed in the iPOND-TMT time course experiments (PXD046514) are due to changes in overall protein abundance, whole extract samples were taken for each time point (Nascent, 1h and 2h after EdU labelling) and analysed by TMT-Mass Spec.

2024-02-14 | PXD040888 | 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 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

Dynamics of chromatin maturation after genome replication

Project description:Replication of the eukaryotic genome occurs in the context of chromatin, a nucleoprotein packaging state consisting of repeating nucleosomes. Chromatin is commonly thought to carry epigenetic information from one generation to the next, although it is unclear how such information survives the disruptions of nucleosomal architecture that occur during genomic replication. Here, we sought to directly measure a key aspect of chromatin structure dynamics during replication â how rapidly nucleosome positions are established on the newly-replicated daughter genomes. By isolating newly-synthesized DNA marked with the nucleotide analogue EdU, we characterize nucleosome positions on both daughter genomes of budding yeast during a time course of chromatin maturation. We find that nucleosomes rapidly adopt their mid log positions at highly-transcribed genes, and that this process was impaired upon treatment with the transcription inhibitor thiolutin, consistent with a role for transcription in positioning nucleosomes in vivo. Additionally, experiments in the Hir1Î background reveal a role for HIR in nucleosome spacing. Using strand-specific EdU libraries, we characterize nucleosome positions on the leading and lagging strand daughter genomes, uncovering differences in chromatin maturation dynamics between the two daughter genomes at hundreds of genes. Our data define the maturation dynamics of newly-replicated chromatin, and support a role for transcription in sculpting the chromatin template. We have mapped changes in nucleosome positions on newly replicated DNA in a timecourse after genome replication. We have used Micrococcal Nuclease footprinting of cross linked chromatin to determine nucleosome positions and EdU (ethylene deoxy uridine) to mark nascent DNA strands. EdU incorporated into nascent DNA strands was biotinylated with Click chemistry and nascent DNA strand fragments were subsequently isolated using Streptavidin coated magnetic beads.

2016-08-25 | E-GEOD-74090 | biostudies-arrayexpress

Identification of Elg1 interaction partners and effects on post-replication chromatin re-formation

Project description:Elg1, the major subunit of a Replication Factor C-like complex, is critical to ensure genomic stability during DNA replication, and is implicated in controlling chromatin structure. We investigated the consequences of Elg1 loss for the dynamics of chromatin re-formation following DNA replication. Measurement of Okazaki fragment length and the micrococcal nuclease sensitivity of newly replicated DNA revealed a defect in nucleosome re-assembly in the absence of Elg1. Using a proteomic approach to identify Elg1 binding partners, we discovered that Elg1 interacts with Rtt106, a histone chaperone implicated in replication-coupled nucleosome assembly that also regulates transcription. We find that Rtt106 recruitment to a number of promoters depends on Elg1. A central role for Elg1 is the unloading of PCNA from chromatin following DNA replication, so we examined the relative importance of Rtt106 and PCNA unloading for chromatin reassembly following DNA replication. We find that the major cause of the chromatin assembly defects of an elg1 mutant is PCNA retention on DNA following replication, with Rtt106-Elg1 interaction potentially playing a contributory role.

2018-10-31 | E-MTAB-6985 | biostudies-arrayexpress

Genetic and proteomic screens reveal perturbed nucleotide metabolism and DNA-protein crosslink formation as central mediators of decitabine cytotoxicity

Project description:Decitabine (5-aza-dC) is a DNMT1-DPC inducing agent used to treat several hematological cancers. However, response rates vary, relapse is common, and predictive biomarkers are unknown. Here, we develop an adaptation of identification of proteins on nascent DNA (iPOND) by combining EdU with 5-aza-dC incorporation by itself or the presence of either ubiquitin E1 or SUMO E1 inhibitor which we term iPOND-DPC. The purpose of this experiments is to identify proteins that are recruited to DNMT1-DPCs and categorize whether their recruitment is dependent on SUMOylation or ubiquitylation.

2024-04-18 | PXD045071 | Pride

Analysis of RNA polymerase II interacting proteins in wild-type and spt6-1004 yeast cells.

Project description:The histone chaperone Spt6 is involved in promoting elongation of RNA polymerase II (RNAPII), maintaining chromatin structure, regulating co-transcriptional histone modifications, and controlling mRNA processing. These diverse functions of Spt6 are partly mediated through its interactions with RNAPII and other factors in the transcription elongation complex. In this study, we used mass spectrometry to characterize the differences in RNAPII interacting factors between wild-type cells and those depleted for Spt6, leading to the identification of proteins that depend on Spt6 for their interaction with RNAPII. In all, eight samples were processed - four genotypes (1. SPT6, RPB3-untagged; 2. SPT6, RPB3-tagged; 3. spt6-1004, RPB3-untagged; 4. spt6-1004; RPB3-tagged) in biological duplicates.

2021-12-05 | PXD027708 | Pride

Div-seq of axolotl pallium injury time course

Project description:The goal of this experiment is to track cellular regeneration after a dorsal injury to the axolotl pallium. To this end, we employed Div-seq, that is, performed snRNA-seq on cells labelled with EdU, which have thus recently replicated. We performed this in a time course, in order to observed the cell populations that were generated as regeneration progressed.

2022-07-20 | E-MTAB-11638 | biostudies-arrayexpress

Dynamics of de novo heterochromatin assembly and disassembly at replication forks ensures fork stability

Project description:Chromatin is dynamically reorganized when DNA replication forks are challenged. However, the process of epigenetic reorganization and its implication for fork stability is poorly understood. Here, we discover a checkpoint regulated cascade of chromatin signaling that activates 5 the histone methyltransferase EHMT2/G9a to catalyze heterochromatin assembly at stressed replication forks. Using biochemical and single molecule chromatin fiber approaches, we show that G9a together with SUV39h1 induces chromatin compaction by accumulating the repressive modifications, H3K9me1/me2/me3, in the vicinity of stressed replication forks. This closed conformation is also favored by the G9a-dependent exclusion of the H3K9-demethylase 10 JMJD1A/KDM3A, which facilitates heterochromatin disassembly upon fork restart. Untimely heterochromatin disassembly from stressed forks by KDM3A enables PRIMPOL access, triggering ssDNA gap formation and sensitizing cells towards chemotherapeutic drugs. These findings may help explaining chemotherapy resistance and poor prognosis observed in cancer patients displaying elevated level of G9a/H3K9me3.

2023-05-12 | PXD041742 | Pride

FOXO3 regulates gene expression from distal enhancers

Project description:FOXO transcription factors are key players in diverse cellular responses affecting tumorigenesis, stem cell maintenance and lifespan. To gain insight into mechanisms of FOXO regulated gene expression, we studied genome-wide effects of FOXO3 activation. Profiling RNA polymerase II (RNAPII) changes shows FOXO3 regulates gene expression through transcription initiation. Correlative analysis of FOXO3 and RNAPII ChIP-seq profiles demonstrates FOXO3 to act as a transcriptional activator. Furthermore, this analysis reveals a significant part of FOXO3 gene regulation proceeds through enhancer regions. FOXO3 binds to and activates enhancers as shown by the presence of and changes in enhancer-specific histone modifications and RNAPII occupancy. In addition, FOXO3-mediated enhancer regulation correlates with regulation of adjacent genes and existence of chromatin loops between FOXO3 bound enhancers and regulated genes. Combined, our data elucidate how FOXOs regulate gene transcription and provide insight into mechanisms by which FOXOs can induce different gene expression programs depending on chromatin architecture. seven 4C view point were analyzed on DLD1 colon carcinoma cells containing 4OH-Tamoxifen inducible FOXO3A3-ER (DL23 cells, Kops et al., 2002, Mol Cell Biol), to investigate 3D topology around FOXO3 bound regions and FOXO3 regulated genes before and 4 hours after addition of tamoxifen. 4C procedure, as published before (Splinter et al., 2001, Genes Dev). Cells are cross linked using 1% formaldehyde for 10min at room temperature, nuclei are isolated, after which chromatin is digested with DpnII and subsequently ligated under diluted conditions. After reversal of the cross links the DNA is purified and treated with the second restriction enzyme treatment (Csp). After a second re-ligation step the sample is purified and ligated fragments are analyzed by inverse PCR.

2012-12-30 | E-GEOD-36835 | biostudies-arrayexpress

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