Similar Datasets
Project description:The versatility of Ascorbate Peroxidase-aided mapping uncovers insights of the nuclear lamina interactions and function
| PRJNA669042 | ENA
Project description:The nucleus is highly compartmentalized through the formation of distinct classes of membraneless domains, yet the composition and function of many of these structures is not well understood. Using APEX2-mediated proximity labelling and RNA sequencing, we surveyed transcripts associated with nuclear speckles, several additional domains, and the lamina. Remarkably, speckles and lamina are associated with distinct classes of retained introns enriched in genes that function in RNA processing, translation, and the cell cycle. In contrast to the lamina-proximal introns, retained introns associated with speckles are relatively short, GC-rich, and enriched for functional sites of RNA binding proteins that are concentrated in these domains. They are also highly differentially regulated across diverse cellular contexts, including the cell cycle. Our study thus provides a resource of nuclear domain-associated transcripts and further reveals speckles and lamina as hubs of distinct populations of retained introns linked to gene regulation and cell cycle progression.
2022-02-18 | GSE176439 | GEO
Project description:Proper genome functionality is underpinned by the non-random, spatial or ganisation of chromatin. At the periphery of the nucleus, the association of chr omatin with the nuclear lamina is thought to facilitate both structural organisa tion and regulation of gene expression. Except for a small number of individual loci, the regions of the human genome that locate at the nuclear lamina have not been identified. Here we used DamID to map the interaction sites of lamin B1 at high resolution in human fibroblasts. We show that almost half of the human gen ome preferentially associates with the nuclear lamina. Chromatin-lamina interact ions occur via sharply demarcated domains of up to several megabases in size, wh ich are bordered by similar sized domains that do not associate with the lamina. Lamina associated domains (LADs) are typified by low gene density and low gene expression levels. This is reflected by low levels of PolII binding and histone modification H3K4me2, in combination with enrichment of H3K27me3 (and H3K9?). A considerable subset of LADs is flanked by active promoters pointing outwards, or by the insulator protein CTCF, suggesting possible mechanisms of LAD confinemen t. Finally, we found that chromosome re-arrangements occur more frequently betwe en loci located either within or outside LADs. The identification of enormous, s harply defined stretches of the human genome that associate with the nuclear lam ina provides unprecedented insights into the 3D organisation of chromatin within the nucleus. Our results reveal the division of the human genome into periphera l and internal domains, which is echoed in genome functionality. Keywords: DamID
2008-05-07 | GSE8854 | GEO
Project description:The nuclear lamina is a proteinaceous network of filaments that provide both structural and gene regulatory functions by tethering proteins and large domains of DNA, so-called lamin associated domains (LADs), to the periphery of the nucleus. LADs are a large fraction of the mammalian genome that are repressed, in part, by their association to the nuclear periphery. The genesis and maintenance of LADs is poorly understood as are the proteins that participate in these functions. In an effort to identify proteins that reside at the nuclear periphery and potentially interact with LADs, we have taken a two-pronged approach. First, we have undertaken an interactome analysis of the inner nuclear membrane bound LAP2β to further characterize the nuclear lamina proteome. To accomplish this, we have leveraged the BioID system, which previously has been successfully used to characterize the nuclear lamina proteome. Second, we have established a system to identify proteins that bind to LADs by developing a chromatin directed BioID system. We combined the BioID system with the m6A-tracer system which binds to LADs in live cells to identify LAD proximal and nuclear lamina proteins. In combining these datasets, we have further characterized the protein network at the nuclear lamina as well as identified putative LAD proximal proteins. Our analysis identifies many heterochromatin related proteins related to H3K9 methylation processes as well as many proteins related to cell cycle regulation identifying important proteins essential for LAD function.
2021-03-09 | PXD012943 | Pride
Project description:Characterization of the proteome of organelles and subcellular domains is essential for understanding cellular organization and identifying protein complexes as well as networks of protein interactions. We established a proteomic mapping platform in live Drosophila tissues using an engineered ascorbate peroxidase (APEX). Upon activation, the APEX enzyme catalyzes the biotinylation of neighboring endogenous proteins that can then be isolated and identified by mass spectrometry. We demonstrate that APEX labeling functions effectively in multiple fly tissues for different subcellular compartments and maps the mitochondrial matrix proteome of Drosophila muscle to demonstrate the power of APEX for characterizing subcellular proteomes in live cells. Further, we generate "MitoMax," a database that provides an inventory of Drosophila mitochondrial proteins with subcompartmental annotation. Altogether, APEX labeling in live Drosophila tissues provides an opportunity to characterize the organelle proteome of specific cell types in different physiological conditions.
| S-EPMC4593093 | biostudies-literature
Project description:Regulation of gene expression is highly conserved between vertebrates, yet the genomic binding patterns of transcription factors are poorly conserved, suggesting that other mechanisms may contribute. The spatial organization of chromosomes in the nucleus is known to affect gene activity, but it is unclear to what extent this organization is conserved in evolution. Genome-wide maps of nuclear lamina (NL) interactions show that human and mouse chromosomes have highly similar folding patterns inside the nucleus. Breaks in synteny are often located at transition points between NL interacting and intra-nuclear regions. Data were compared against data from Peric-Hupkes, Meuleman et al. (Molecular Cell, 2010). LaminB1-chromatin interactions were assayed in human ESCs and human HT1080 cells. LaminA-chromatin interactions were assayed in human HT1080 cells. For the all samples there were 2 biological replicates, that were hybridized in a dye-swap design.
2012-10-10 | E-GEOD-22428 | biostudies-arrayexpress
Project description:The three-dimensional organization of chromosomes within the nucleus and its dynamics during differentiation are largely unknown. We present a genome-wide analysis of the interactions between chromatin and the nuclear lamina during differentiation of mouse embryonic stem cells (ESCs) into lineage-committed neural precursor cells (NPCs) and terminally differentiated astrocytes. Chromatin in each of these cell types shows a similar organization into large lamina associated domains (LADs), which represent a transcriptionally repressive environment. During sequential differentiation steps, lamina interactions are progressively modified at hundreds of genomic locations. This remodeling is typically confined to individual transcription units and involves many genes that determine cellular identity. From ESCs to NPCs, the majority of genes that move away from the lamina are concomitantly activated. Strikingly, a significant amount remain inactive yet become primed for activation by further differentiation. These results suggest that lamina-genome interactions are widely involved in the control of gene expression programs during lineage commitment and terminal differentiation. laminB1-chromatin interactions were assayed in 4 different mouse cell-types. For each cell-type there were 2 biological replicates, that were hybridized in a dye-swap design.
2010-05-27 | E-GEOD-17051 | biostudies-arrayexpress
Project description:We generated high-resolution maps of genome-lamina interactions for different stages of the pre-implantation mouse embryo, including oocyte, zygote, 2cell stage and 8cell stage and additionally mouse ES cells. We find that LAD domains are already present at the zygote stage, but not in the oocyte, concluding that genome-nuclear lamina interactions are not inherited from the maternal germline, but instead are established de novo rapidly after fertilisation. By using a hybrid genetic background, we were able to identify parental-specific genome-nuclear lamina interaction, allowing us to show that the parental genomes have different features, which are globally resolved at the 8-cell stage. We blocked replication in zygotes by treatment with aphidicolin and found that LAD establishment was unaffected. Moreover, we ectopically expressed Kdm5b in zygotes and show that paternal LAD formation is impaired, suggesting that this mechanism is dependent upon de novo H3K4 methylation. Altogether, our data suggest a step-wise assembly model whereby early LAD formation precedes consolidation of TADs and reveals non-inheritance of nuclear organisation from the germline.
2019-03-13 | GSE112551 | GEO
Project description:Cytochrome c peroxidase (CCP) and ascorbate peroxidase (APX) have very similar structures, and yet neither CCP nor APX exhibits each other's activities with respect to reducing substrates. APX has a unique substrate binding site near the heme propionates where ascorbate H-bonds with a surface Arg and one heme propionate (Sharp et al. (2003) Nat. Struct. Biol. 10, 303-307). The corresponding region in CCP has a much longer surface loop, and the critical Arg residue that is required for ascorbate binding in APX is Asn in CCP. In order to convert CCP into an APX, the ascorbate-binding loop and critical arginine were engineered into CCP to give the CCP2APX mutant. The mutant crystal structure shows that the engineered site is nearly identical to that found in APX. While wild-type CCP shows no APX activity, CCP2APX catalyzes the peroxidation of ascorbate at a rate of approximately 12 min (-1), indicating that the engineered ascorbate-binding loop can bind ascorbate.
| S-EPMC2770236 | biostudies-literature
Project description:UnlabelledAlthough Mn(2+) is the most abundant substrate of versatile peroxidases (VPs), repression of Pleurotus ostreatus vp1 expression occurred in Mn(2+)-sufficient medium. This seems to be a biological contradiction. The aim of this study was to explore the mechanism of direct oxidation by VP1 under Mn(2+)-deficient conditions, as it was found to be the predominant enzyme during fungal growth in the presence of synthetic and natural substrates. The native VP1 was purified and characterized using three substrates, Mn(2+), Orange II (OII), and Reactive Black 5 (RB5), each oxidized by a different active site in the enzyme. While the pH optimum for Mn(2+) oxidation is 5, the optimum pH for direct oxidation of both dyes was found to be 3. Indeed, effective in vivo decolorization occurred in media without addition of Mn(2+) only under acidic conditions. We have determined that Mn(2+) inhibits in vitro the direct oxidation of both OII and RB5 while RB5 stabilizes both Mn(2+) and OII oxidation. Furthermore, OII was found to inhibit the oxidation of both Mn(2+) and RB5. In addition, we could demonstrate that VP1 can cleave OII in two different modes. Under Mn(2+)-mediated oxidation conditions, VP1 was able to cleave the azo bond only in asymmetric mode, while under the optimum conditions for direct oxidation (absence of Mn(2+) at pH 3) both symmetric and asymmetric cleavages occurred. We concluded that the oxidation mechanism of aromatic compounds by VP1 is controlled by Mn(2+) and pH levels both in the growth medium and in the reaction mixture.ImportanceVP1 is a member of the ligninolytic heme peroxidase gene family of the white rot fungus Pleurotus ostreatus and plays a fundamental role in biodegradation. This enzyme exhibits a versatile nature, as it can oxidize different substrates under altered environmental conditions. VPs are highly interesting enzymes due to the fact that they contain unique active sites that are responsible for direct oxidation of various aromatic compounds, including lignin, in addition to the well-known Mn(2+) binding active site. This study demonstrates the limits of versatility of P. ostreatus VP1, which harbors multiple active sites, exhibiting a broad range of enzymatic activities, but they perform differently under distinct conditions. The versatility of P. ostreatus and its enzymes is an advantageous factor in the fungal ability to adapt to changing environments. This trait expands the possibilities for the potential utilization of P. ostreatus and other white rot fungi.
| S-EPMC4959207 | biostudies-literature