Direct DNA crosslinking with CAP-C uncovers transcription-dependent chromatin organization at high resolution
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus Homo sapiens Drosophila melanogaster
PROVIDER: GSE110061 | GEO | 2020/08/22
REPOSITORIES: GEO
Similar Datasets
Project description:Direct DNA crosslinking with CAP-C uncovers transcription-dependent chromatin organization at high resolution
| PRJNA432721 | ENA
Project description: Not available
| S-EPMC8274026 | biostudies-literature
Project description:In this study, we employed the isotopically labeled crosslinkers as the quantitative crosslinking mass spectrometry to investigate the changes in protein-protein interactions during the AD progression.
2024-03-07 | MSV000094259 | MassIVE
Project description:Crosslinking mass spectrometry (XL-MS) is emerging as a method at the crossroads of structural and cellular biology, uniquely capable of identifying protein-protein interactions with residue-level resolution and on the proteome-wide scale. With the development of crosslinkers that can form linkages inside cells and easily cleave during fragmentation on the mass spectrometer (MS-cleavable crosslinks), it has become increasingly facile to identify contacts between any two proteins in complex samples, including in live cells or tissues. Photo-crosslinkers possess the advantages of high temporal resolution and high reactivity, thereby engaging all residue-types (rather than just lysine); nevertheless, photo-crosslinkers have not enjoyed widespread use, and have yet to be employed for proteome-wide studies, because their products are challenging to identify. Here, we demonstrate the synthesis and application of two heterobifunctional photo-crosslinkers that feature diazirines and N-hydroxy-succinimidyl carbamate groups, the latter of which unveil symmetrical MS-cleavable linkages upon acyl transfer to protein targets. Moreover, these crosslinkers demonstrate high water-solubility and cell-permeability. Using these compounds, we demonstrate the feasibility of proteome-wide photo-crosslinking in cellulo. These studies elucidate a small portion of E. coli’s interaction network, albeit with residue-level resolution. With further optimization, these methods will enable the detection of protein quinary interaction networks in their native environment at residue-level resolution, and we expect they will prove useful toward the effort to explore the molecular sociology of the cell.
2023-06-27 | PXD041148 | Pride
Project description:Conformational changes of protein structures depend on their chemical and physical environment. Studying conformational changes depending on environmental parameters is notoriously difficult as many methods of structural biology are affected by parameters like temperature or pH. To make such conformational changes accessible to quantitative crosslinking mass spectrometry (QCLMS) approaches, crosslinking chemistry must be invariant to different conditions. We propose this can be achieved by photo-inducible crosslinkers, which are not influenced by changes in environmental parameters. Here, we introduce a workflow combining photo-crosslinking using 4,4’-azipentanoate (sulfo-SDA) with our recently developed data-independent acquisition (DIA)-QCLMS. In this study, we use this novel photo-DIA-QCLMS approach to quantify pH-dependent conformational changes in human serum albumin and cytochrome C. Both proteins show pH dependent conformational changes resulting in acidic and alkaline transitions. 93% and 95% unique residue pairs (URP) were quantifiable across triplicates for HSA and cytochrome C, respectively. Abundance changes of URPs and hence conformational changes of both proteins, were visualized using hierarchical clustering. For HSA we distinguished the N-F and the N-B form from the native conformation. Additionally, we observed for cytochrome C acidic and basic conformations. In conclusion, photo-DIA-QCLMS distinguished pH-dependent conformers of both proteins.
2019-07-01 | PXD012939 | Pride
Project description:Crosslinking mass spectrometry (XL-MS) is emerging as a unique method at the crossroads of structural and cellular biology, uniquely capable of identifying protein-protein interactions with residue-level resolution and on the proteome-wide scale. With the development of crosslinkers that can form linkages inside cells and easily cleave during fragmentation on the mass spectrometer (MS-cleavable crosslinks), it has become increasingly facile to identify contacts between any two proteins in complex samples, including in live cells or tissues. Photo-crosslinkers possess the advantages of high temporal resolution and high reactivity, thereby engaging all residue-types (rather than just lysine); nevertheless, photo-crosslinkers have not enjoyed widespread use, and have yet to be employed for proteome-wide studies, because their products are challenging to identify, and an MS-cleavable photo-crosslinker has not yet been reported. Here, we demonstrate the synthesis and application of two heterobifunctional photo-crosslinkers that feature diazirines and N-hydroxy-succinimidyl carbamate groups, the latter of which unveil MS-cleavable linkage upon acyl transfer to protein targets. Moreover, these crosslinkers demonstrate high water-solubility and cell-permeability. Using these compounds, we demonstrate the feasibility of proteome-wide photo-crosslinking mass spectrometry (photo-XL-MS), both in extracts and in cellulo. These studies provide a partial interaction map of the E. coli cytosol with residue-level resolution. We find that photo-XL-MS has a propensity to capture protein-protein interactions, particularly involving low-abundance uncharacterized proteins, suggesting it could be a powerful tool to shed light on the “darker” corners of the proteome. Overall, we describe methods that enable the detection of protein quinary interaction networks in their native environment at residue-level resolution proteome-wide, and we expect they will prove useful toward the effort to explore the molecular sociology of the cell.
2023-12-07 | PXD036884 | Pride
Project description:Paclitaxel is a widely used therapeutic chemical for breast cancer treatment, however, cancer recurrence remains obstacle for improved prognosis of cancer patients. In the current study, cold atmospheric plasma (CAP) was examined for its potential use to overcome the drug resistance, which mainly comprised of reactive oxygen and nitrogen species. After developing a paclitaxel (Tx)-resistant MCF-7 (MCF-7/TxR) breast cancer cell, CAP was applied to the cells and its effect on recovery of drug sensitivity was examined in cellular as well as molecular aspect. The MCF-7/TxR cells restored sensitivity against Tx up to 70% by CAP. Comparison of genome-wide expression profiles between the Tx resistance cell and its CAP-treated cell identified 86 genes that commonly appeared with significant change. Notably, 48 genes including DAGLA and CEACAM1, which are known to contribute to the acquisition of Tx resistance, showed opposite expression in the two cellular status. The protein expression level of selected genes, DAGLA and CEACAM1, was recovered to that of their parental cell by CAP. Furthermore, the dysregulation of DAGLA and CEACAM1 in MCF-7/TxR alleviated the drug sensitivity recovery effect of CAP. Taken together, CAP inhibited the growth of Tx-resistant MCF-7 cancer cells and recovered Tx sensitivity by resetting expression of multiple drug resistance–related genes. These findings may contribute to extending the application of CAP to the treatment of Tx-resistant cancer.
2020-01-24 | GSE131479 | GEO
Project description:Paclitaxel is a widely used therapeutic chemical for breast cancer treatment, however, cancer recurrence remains obstacle for improved prognosis of cancer patients. In the current study, cold atmospheric plasma (CAP) was examined for its potential use to overcome the drug resistance, which mainly comprised of reactive oxygen and nitrogen species. After developing a paclitaxel (Tx)-resistant MCF-7 (MCF-7/TxR) breast cancer cell, CAP was applied to the cells and its effect on recovery of drug sensitivity was examined in cellular as well as molecular aspect. The MCF-7/TxR cells restored sensitivity against Tx up to 70% by CAP. Comparison of genome-wide expression profiles between the Tx resistance cell and its CAP-treated cell identified 86 genes that commonly appeared with significant change. Notably, 48 genes including DAGLA and CEACAM1, which are known to contribute to the acquisition of Tx resistance, showed opposite expression in the two cellular status. The protein expression level of selected genes, DAGLA and CEACAM1, was recovered to that of their parental cell by CAP. Furthermore, the dysregulation of DAGLA and CEACAM1 in MCF-7/TxR alleviated the drug sensitivity recovery effect of CAP. Taken together, CAP inhibited the growth of Tx-resistant MCF-7 cancer cells and recovered Tx sensitivity by resetting expression of multiple drug resistance–related genes. These findings may contribute to extending the application of CAP to the treatment of Tx-resistant cancer.
2020-01-24 | GSE131477 | GEO
Project description:Nuclear processing and quality control of eukaryotic RNA is mediated by the multi-subunit RNA exosome, which utilizes accessory factors to regulate its enzymatic activity. However, the mechanism of exosome recruitment to its ribonucleoprotein (RNP) targets remains poorly understood. Here we disclose a physical link between the human nuclear RNA exosome and the cap-binding complex (CBC). The CBC associates with the ARS2 protein to form CBC-ARS2 (CBCA), and then further connects together with the uncharacterized ZC3H18/NHN1 protein to the nuclear exosome targeting (NEXT) complex, forming CBC-NEXT (CBCN). RNA immunoprecipitation analysis using CBCN factors as baits as well as the combinatorial depletion of CBCN and exosome components underscore the functional relevance of CBC-exosome bridging at the level of target RNA. Specifically, CBCA suppresses read-through transcription of several RNA families by promoting their transcriptional termination. We suggest that the RNP 5’cap links transcription termination to exosomal RNA degradation via CBCN.
2013-11-08 | GSE52132 | GEO