Intricate structure of the interphase chromocenter revealed by proximity based biotinylation
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ABSTRACT:
PROVIDER: PRJNA564796 | ENA |
REPOSITORIES: ENA
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Project description:During interphase centromeres often coalesce into a small number of chromocenters, which can be visualized as distinct, DAPI dense nuclear domains. Intact chromocenters play a major role in maintaining genome stability as they stabilize the transcriptionally silent state of repetitive DNA while ensuring centromere function. Despite its biological importance, relatively little is known about the molecular composition of the chromocenter or the processes that mediate chromocenter formation and maintenance. To shed light on this, we performed confocal and super resolution microscopy and proximity based biotinylation experiments of three proteins associated with the chromocenter in Drosophila: the centromeric H3 variant dCenpA, the heterochromatin protein HP1a and the speciation factor HMR. Our work revealed an intricate internal structure of the chromocenter suggesting a complex multilayered structure of this intranuclear domain.
2020-01-01 | GSE137194 | GEO
Project description: Not available
| S-EPMC7192626 | biostudies-literature
Project description:In higher eukaryotes centromeres often coalesce into a large intranuclear domain called the chromocenter. Chromocenters are important for the organization of pericentric heterochromatin and a disturbance of their formation results in an upregulation of repetitive elements and causes defects in chromosome segregation. Mutations in the gene encoding for the centromere associated Drosophila speciation factor HMR show very similar phenotypes suggesting a role of HMR in chromocenter architecture and function. We performed confocal and super resolution microscopy as well as proximity based biotinylation experiments of HMR, centromeric protein dCenpA and heterochromatic protein HP1a to generate a molecular map of HMR, dCenpA and HP1a bound chromatin. Our work reveals an intricate internal structure of the centromeric chromatin region, which suggests a role of HMR in separating heterochromatin from centromeric chromatin.
2020-03-23 | PXD012551 | Pride
Project description:Proximity biotinylation workflows typically require CRISPR-based genetic manipulation of target cells. To overcome this bottleneck, we fused the TurboID proximity biotinylation enzyme to Protein A. Upon target cell permeabilization, the ProtA-Turbo enzyme can be targeted to proteins of interest using an endogenous antibody against the bait protein. Addition of biotin then triggers bait-proximal protein biotinylation. Biotinylated proteins can subsequently be enriched from crude lysates and identified by mass spectrometry. We demonstrate this workflow by targeting Emerin, the histone modification H3K9me3 and the chromatin remodeler BRG1. Amongst the main findings, our experiments revealed Flywch1 as an essential protein that interacts with H3K9me3-marked centromeric heterochromatin. The ProtA-Turbo enzyme represents an ‘off the shelf’ proximity biotinylation enzyme that facilitates proximity biotinylation experiments in primary cells and can be used to understand how proteins cooperate in vivo and how this contributes to cellular homeostasis and disease.
2021-07-12 | PXD025012 | Pride
Project description:Live-cell mapping of organelle-associated RNAs via proximity biotinylation combined with protein-RNA crosslinking
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Project description:In this study we developed a new assay to perform proximity biotinylation in cells without the requirement for genetic manipulation or transfection to fuse a biotin ligase to a protein of interest. We show the specificity by targeting H3K9me3 and performing ChIP-seq for the biotin modification. Targeting IgG was used as a control. By doing so, we identified flywch1 as a new protein binding to H3K9me3 regions, which we validated using ChIP-seq (IgG ChIP was used as a control)
2021-07-09 | GSE169317 | GEO
Project description:Proximity biotinylation is a commonly used method to identify the in vivo proximal proteome for proteins of interest. This technology typically relies on fusing a bait protein to a biotin ligase using overexpression or CRISPR-based tagging, thus prohibiting such assays in (primary) cell types that are difficult to transfect. We recently developed an ‘off the shelf’ proximity biotinylation method, which makes use of a recombinant enzyme consisting of the biotin ligase TurboID fused to the antibody-recognizing moiety Protein A. In this method, a bait specific antibody and the ProteinA-Turbo enzyme are consecutively added to permeabilized fixed or unfixed cells. Following incubation, during which ProteinA-Turbo-antibody-antigen complexes are formed, unbound molecules are washed away, after which bait-proximal biotinylation is triggered by the addition of exogenous biotin. Finally, biotinylated proteins are enriched from crude lysates using streptavidin beads followed by mass spectrometry-based protein identification. Here, we present a detailed protocol for this method
2022-10-17 | PXD030963 | Pride
Project description: Not available
| S-EPMC7401799 | biostudies-literature
Project description:Proximity biotinylation screens are a widely used strategy for the unbiased identification of interacting or vicinal proteins. The latest generation biotin ligase TurboID has broadened the range of potential applications, as this ligase promotes an intense and faster biotinylation, even in subcellular compartments like the endoplasmic reticulum. On the other hand, the uncontrollable high basal biotinylation rates deny the system´s inducibility and are often associated with cellular toxicity precluding its use in proteomics. We report here an improved method for TurboID-dependent biotinylation reactions based on the tight control of free biotin levels. Blockage of free biotin with a commercial biotin scavenger reversed the high basal biotinylation and toxicity of TurboID, as shown by pulse-chase experiments. Accordingly, the biotin-blockage protocol restored the biological activity of a bait protein fused to TurboID in the endoplasmic reticulum and rendered the biotinylation reaction inducible by exogenous biotin. Importantly, the biotin-blockage protocol was more effective than biotin removal with immobilised avidin and did not affect the cellular viability of human monocytes over several days. The method presented should be useful to researchers interested in exploiting the full potential of biotinylation screens with TurboID and other high-activity ligases for challenging proteomics questions.
2023-03-29 | PXD040302 | Pride
Project description:Both intraflagellar transport (IFT) and lipidated intraflagellar transport (LIFT) pathways are essential for cilia/flagella biogenesis, motility and sensory functions. In the LIFT pathway, lipidated cargoes are transported into the cilia through the coordinated actions of cargo carrier proteins such as Unc119 or PDE6δ, as well as small GTPases Arl13b and Arl3 in the cilium. Our previous studies revealed a single Arl13b ortholog in the evolutionarily divergent Trypanosoma brucei. TbArl13 catalyses two TbArl3 homologs, TbArl3A and TbArl3C, suggesting the presence of a conserved LIFT pathway in these protozoan parasites. Only a single homolog to the cargo carrier protein Unc119 was identified in T. brucei genome, but its function in lipidated protein transport has not been characterized. In this study, we exploited the proximity-based biotinylation approach to identify binding partners of TbUnc119 using an improved version of biotin ligase BioID2. The BioID2 tag was fused to either the N-terminus (3HA-BioID2-TbUnc119) or the C-terminus (TbUnc119-BioID2-HA) of TbUnc119 and expressed using a cumate-inducible expression system in the procyclic cells of T. brucei. We performed LC-MS/MS on the affinity-purified biotinylated samples followed by evaluation of emPAI score.
2020-06-25 | PXD019488 | Pride