1,127 results on '"NUCLEAR matrix"'
Search Results
2. Quantification of Epigenetic DNA Modifications in the Subchromatin Structure Matrix Attachment Regions by Stable Isotope Dilution UHPLC-MS/MS Analysis
- Author
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Hailin Wang, Rui Zhang, and Weiyi Lai
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DNA ,Matrix Attachment Regions ,Nuclear matrix ,Epigenesis, Genetic ,Analytical Chemistry ,Chromatin ,chemistry.chemical_compound ,genomic DNA ,DNA demethylation ,Isotopes ,Biochemistry ,chemistry ,Tandem Mass Spectrometry ,A-DNA ,Epigenetics ,Scaffold/matrix attachment region ,Chromatography, High Pressure Liquid - Abstract
To date, subchromatin structure-based quantification of epigenetic DNA modifications is limited. Matrix attachment regions (MARs), an important subchromatin structure, contain DNA elements that specifically bind chromatin to the nuclear matrix in eukaryotes and are involved in a number of diseases. Here, we exploited a high-salt extraction-based subchromatin fractionation approach for the isolation of MAR DNA and other fractions and further developed heavy stable isotope-diluted ultrahigh-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) for the specific quantification of epigenetic DNA modifications in the subchromatin structures. By this approach, we showed for the first time that the content of a DNA demethylation intermediate, 5-hydroxymethylcytosine (5hmdC), in MARs decreased significantly in four tested cell lines compared to the contents in genomic DNA. In particular, the content of DNA 5hmdC in the MARs of 293T cell lines decreased the most at approximately 41.09%. Together, our findings implicate that MAR DNA is less sensitive than genomic DNA to DNA demethylation.
- Published
- 2021
3. 1H, 13C and 15N resonance assignments and solution structures of the two RRM domains of Matrin-3
- Author
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Shigeyuki Yokoyama, Yutaka Muto, Fahu He, Kanako Kuwasako, Mikako Shirouzu, Peter Güntert, Takashi Nagata, Akiko Tanaka, Kengo Tsuda, Mari Takahashi, Takanori Kigawa, Masayuki Takizawa, Naohiro Kobayashi, and Satoru Watanabe
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Zinc finger ,RNA recognition motif ,Chemistry ,RNA ,medicine.disease ,Nuclear matrix ,Biochemistry ,Cell biology ,chemistry.chemical_compound ,Structural Biology ,RNA splicing ,Transcriptional regulation ,medicine ,Amyotrophic lateral sclerosis ,DNA - Abstract
Matrin-3 is a multifunctional protein that binds to both DNA and RNA. Its DNA-binding activity is linked to the formation of the nuclear matrix and transcriptional regulation, while its RNA-binding activity is linked to mRNA metabolism including splicing, transport, stabilization, and degradation. Correspondingly, Matrin-3 has two zinc finger domains for DNA binding and two consecutive RNA recognition motif (RRM) domains for RNA binding. Matrin-3 has been reported to cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) when its disordered region contains pathogenic mutations. Simultaneously, it has been shown that the RNA-binding activity of Matrin-3 mediated by its RRM domains, affects the formation of insoluble cytoplasmic granules, which are related to the pathogenic mechanism of ALS/FTD. Thus, the effect of the RRM domains on the phase separation of condensed protein/RNA mixtures has to be clarified for a comprehensive understanding of ALS/FTD. Here, we report the 1H, 15N, and 13C resonance assignments of the two RNA binding domains and their solution structures. The resonance assignments and the solution structures obtained in this work will contribute to the elucidation of the molecular basis of Matrin-3 in the pathogenic mechanism of ALS and/or FTD.
- Published
- 2021
4. Domain Model of Eukaryotic Genome Organization: From DNA Loops Fixed on the Nuclear Matrix to TADs
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Sergey V. Razin, Irina V. Zhegalova, and Omar L. Kantidze
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Genome ,Biophysics ,Eukaryota ,Nuclear Matrix ,General Medicine ,DNA ,Geriatrics and Gerontology ,Biochemistry ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,Chromatin - Abstract
The article reviews the development of ideas on the domain organization of eukaryotic genome, with special attention on the studies of DNA loops anchored to the nuclear matrix and their role in the emergence of the modern model of eukaryotic genome spatial organization. Critical analysis of results demonstrating that topologically associated chromatin domains are structural-functional blocks of the genome supports the notion that these blocks are fundamentally different from domains whose existence was proposed by the domain hypothesis of eukaryotic genome organization formulated in the 1980s. Based on the discussed evidence, it is concluded that the model postulating that eukaryotic genome is built from uniformly organized structural-functional blocks has proven to be untenable.
- Published
- 2022
5. Differential incorporation of SUN-domain proteins into LINC complexes is coupled to gene expression.
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May, Christopher K. and Carroll, Christopher W.
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NUCLEAR matrix , *CYTOSKELETON , *HELA cells , *GENETIC regulation , *GENE expression - Abstract
LInkers of Nucleoskeleton and Cytoskeleton (LINC) complexes, composed of SUN and KASH-domain proteins, span the nuclear envelope and physically connect the nuclear interior to cytoskeletal elements. Most human cells contain two SUN proteins, Sun1 and Sun2, and several KASH-proteins suggesting that multiple functionally distinct LINC complexes co-exist in the nuclear envelope. We show here, however, that while Sun1 and Sun2 in HeLa cells are each able to bind KASH-domains, Sun1 is more efficiently incorporated into LINC complexes under normal growth conditions. Furthermore, the balance of Sun1 and Sun2 incorporated into LINC complexes is cell type-specific and is correlated with SRF/Mkl1-dependent gene expression. In addition, we found that Sun1 has a LINC complex-independent role in transcriptional control, possibly by regulating the SRF/Mkl1 pathway. Together, these data reveal novel insights into the mechanisms of LINC complex regulation and demonstrate that Sun1 modulates gene expression independently of its incorporation into LINC complexes. [ABSTRACT FROM AUTHOR]
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- 2018
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6. Nucleoskeleton proteins for nuclear dynamics
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Kei Miyamoto and Masahiko Harata
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Nuclear Envelope ,Cellular differentiation ,Biology ,Biochemistry ,03 medical and health sciences ,0302 clinical medicine ,medicine ,Transcriptional regulation ,Animals ,Humans ,Nuclear Matrix ,Cytoskeleton ,Molecular Biology ,Actin ,030304 developmental biology ,Cell Nucleus ,0303 health sciences ,Nuclear Proteins ,Cell Differentiation ,General Medicine ,Actins ,Lamins ,Chromatin ,Cell biology ,Cell nucleus ,medicine.anatomical_structure ,Gene Expression Regulation ,Nucleus ,030217 neurology & neurosurgery ,Lamin - Abstract
The eukaryotic nucleus shows organized structures of chromosomes, transcriptional components and their associated proteins. It has been believed that such a dense nuclear environment prevents the formation of a cytoskeleton-like network of protein filaments. However, accumulating evidence suggests that the cell nucleus also possesses structural filamentous components to support nuclear organization and compartments, which are referred to as nucleoskeleton proteins. Nucleoskeleton proteins including lamins and actin influence nuclear dynamics including transcriptional regulation, chromatin organization and DNA damage responses. Furthermore, these nucleoskeleton proteins play a pivotal role in cellular differentiation and animal development. In this commentary, we discuss how nucleoskeleton-based regulatory mechanisms orchestrate nuclear dynamics.
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- 2021
7. The dynamics of the nuclear environment and their impact on gene function
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Marco Biggiogera, Claudio Casali, L. Zannino, and Stella Siciliani
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Cell Nucleus ,Dynamics (mechanics) ,Cell ,Nuclear Proteins ,General Medicine ,Biology ,Cell cycle ,Biochemistry ,Chromatin ,Cell Compartmentation ,Cell nucleus ,Phenotype ,medicine.anatomical_structure ,Gene Expression Regulation ,medicine ,Animals ,Humans ,Nuclear Matrix ,Molecular Biology ,Nucleus ,Neuroscience ,Gene ,Function (biology) - Abstract
In the last decades, it has become increasingly clear how the modulation of spatial organization of chromatin over time and through the cell cycle is closely connected to gene function regulation. Different physicochemical stimuli contribute to the realization of specific transcriptional programs and finally to a specific cellular phenotype. In this review, we aim to describe the current knowledge about the dynamics regulating the movements and the interactions of molecules within the nucleus and their impact on gene functions. In particular, taking into account that these forces exert their effect in a nuclear environment characterized by a high concentration of molecules, we will discuss the role of proteins and structures that regulate these movements and transduce physicochemical signals acting on the cell to the nucleus.
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- 2020
8. Combining Proximity Labeling and Cross-Linking Mass Spectrometry for Proteomic Dissection of Nuclear Envelope Interactome
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Ming-Jou Chien, Kurt Yun Mou, Cheng-Hao Liu, Fu An Li, You-Chiun Chang, and Yu-Hsiang Cheng
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Proteomics ,0301 basic medicine ,General method ,030102 biochemistry & molecular biology ,Nuclear Envelope ,Chemistry ,Dissection ,Proteins ,General Chemistry ,Mass spectrometry ,Nuclear matrix ,Biochemistry ,Interactome ,Mass Spectrometry ,03 medical and health sciences ,Cross-Linking Reagents ,030104 developmental biology ,Biophysics ,Humans ,Inner membrane ,Protein network ,Envelope (waves) - Abstract
Proximity labeling (PL) and chemical cross-linking (XL) mass spectrometry are two powerful methods to dissect protein-protein interactions (PPIs) in cells. Although PL typically captures neighboring proteins within a range of 10-20 nm of a single bait protein, chemical XL defines direct protein-protein contacts within 1 nm in a systemic manner. Here, we develop a new method, named PL/XL-MS, to harness the advantages of both PL and XL. PL/XL-MS can enrich a subcellular compartment by PL and simultaneously identify PPIs of multiple proteins from XL data. We applied PL/XL-MS to dissect the human nuclear envelope interactome. PL/XL-MS successfully enriched the nuclear envelope proteins and identified most known inner nuclear membrane proteins. By searching the cross-linked peptides, we successfully observed 109 literature-curated PPIs of 14 nuclear envelope proteins. Based on the homoprotein XL data, we experimentally characterized a nuclear matrix protein, Matrin-3, and observed its preferential localization near the nuclear envelope. PL/XL-MS is a simple and general method for studying protein networks in a subproteome of interest.
- Published
- 2020
9. Short-term hypoxia triggers ROS and SAFB mediated nuclear matrix and mRNA splicing remodeling
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Chrysa Taze, Sotiria Drakouli, Martina Samiotaki, George Panayotou, George Simos, Eleni Georgatsou, and Ilias Mylonis
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Vascular Endothelial Growth Factor A ,Organic Chemistry ,Clinical Biochemistry ,Matrix Attachment Region Binding Proteins ,Protein Serine-Threonine Kinases ,Hypoxia-Inducible Factor 1, alpha Subunit ,Biochemistry ,Cell Hypoxia ,Nuclear Matrix-Associated Proteins ,Receptors, Estrogen ,Humans ,Nuclear Matrix ,RNA, Messenger ,Reactive Oxygen Species ,Hypoxia - Abstract
The cellular response to hypoxia, in addition to HIF-dependent transcriptional reprogramming, also involves less characterized transcription-independent processes, such as alternative splicing of the VEGFA transcript leading to the production of the proangiogenic VEGF form. We now show that this event depends on reorganization of the splicing machinery, triggered after short-term hypoxia by ROS production and intranuclear redistribution of the nucleoskeletal proteins SAFB1/2. Exposure to low oxygen causes fast dissociation of SAFB1/2 from the nuclear matrix, which is reversible, inhibited by antioxidant treatment, and also observed under normoxia when the mitochondrial electron transport chain is blocked. This is accompanied by altered interactions between SAFB1/2 and the splicing machinery, translocation of kinase SRPK1 to the cytoplasm, and dephosphorylation of RS-splicing factors. Depletion of SAFB1/2 under normoxia phenocopies the hypoxic and ROS-mediated switch in VEGF mRNA splicing. These data suggest that ROS-dependent remodeling of the nuclear architecture can promote production of splicing variants that facilitate adaptation to hypoxia.
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- 2022
10. Cloning and Characterization of a Human Genomic Sequence that Alleviates Repeat-Induced Gene Silencing.
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Fukuma, Miki, Ganmyo, Yuto, Miura, Osamu, Ohyama, Takashi, and Shimizu, Noriaki
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NUCLEOTIDE sequence , *GENE silencing , *MOLECULAR cloning , *PLASMIDS , *NUCLEAR matrix - Abstract
Plasmids bearing a mammalian replication initiation region (IR) and a nuclear matrix attachment region (MAR) are spontaneously amplified in transfected mammalian cells, and such amplification generates chromosomal homogeneously staining regions (HSRs) or extrachromosomal double minutes (DMs). This method provides a novel, efficient, and rapid way to establish cells that stably produce high levels of recombinant proteins. However, because IR/MAR plasmids are amplified as repeats, they are frequently targeted by repeat-induced gene silencing (RIGS), which silences a variety of repeated sequences in transgenes and the genome. To address this problem, we developed a novel screening system using the IR/MAR plasmid to isolate human genome sequences that alleviate RIGS. The screen identified a 3,271 bp sequence (B-3-31) that elevated transgene expression without affecting the amplification process. Neither non-B structure (i.e., the inverted repeats or bending) nor known epigenetic modifier elements such as MARs, insulators, UCOEs, or STARs could explain the anti-silencing activity of B-3-31. Instead, the activity was distributed throughout the entire B-3-31 sequence, which was extremely A/T-rich and CpG-poor. Because B-3-31 effectively and reproducibly alleviated RIGS of repeated genes, it could be used to increase recombinant protein production. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
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11. Centromere Protein (CENP)-W Interacts with Heterogeneous Nuclear Ribonucleoprotein (hnRNP) U and May Contribute to Kinetochore-Microtubule Attachment in Mitotic Cells.
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Chun, Younghwa, Kim, Raehyung, and Lee, Soojin
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CENTROMERE , *HETEROGENOUS nucleation , *NUCLEOPROTEINS , *KINETOCHORE , *MITOSIS - Abstract
Background: Recent studies have shown that heterogeneous nuclear ribonucleoprotein U (hnRNP U), a component of the hnRNP complex, contributes to stabilize the kinetochore-microtubule interaction during mitosis. CENP-W was identified as an inner centromere component that plays crucial roles in the formation of a functional kinetochore complex. Results: We report that hnRNP U interacts with CENP-W, and the interaction between hnRNP U and CENP-W mutually increased each other’s protein stability by inhibiting the proteasome-mediated degradation. Further, their co-localization was observed chiefly in the nuclear matrix region and at the microtubule-kinetochore interface during interphase and mitosis, respectively. Both microtubule-stabilizing and microtubule-destabilizing agents significantly decreased the protein stability of CENP-W. Furthermore, loss of microtubules and defects in microtubule organization were observed in CENP-W-depleted cells. Conclusion: Our data imply that CENP-W plays an important role in the attachment and interaction between microtubules and kinetochore during mitosis. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
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12. Nuclear lipids: key signaling effectors in the nervous system and other tissues1
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Robert W. Ledeen and Gusheng Wu
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nuclear envelope ,nuclear chromatin ,inner nuclear membrane ,outer nuclear membrane ,nuclear matrix ,nuclear phospholipids ,Biochemistry ,QD415-436 - Abstract
Lipids have long been recognized as quantitatively minor components of the nucleus, where they were initially thought to have little functional importance; but they now command growing interest, with recognition of their diverse signaling and modulating properties in that organelle. This applies to the lipid-poor compartments of the nucleoplasm as well as the relatively lipid-rich nuclear envelope. Phosphoglycerides and sphingomyelin, as the predominant lipids, have attracted the most interest among researchers, but some of the less-abundant lipids such as gangliosides, sphingosine, and sphingosine phosphate are now becoming recognized as functionally important nuclear constituents. Among recent advances in this emerging field are detailed findings on the metabolic enzymes that synthesize and catabolize nuclear lipids; the fact that these are localized primarily within the nucleus itself indicates considerable autonomy with respect to lipid metabolism. Current studies suggest several key processes involving RNA and DNA reactivity that are dependent on these lipid-initiated events. Neural cell nuclei have been the subject of such investigations, with results that closely parallel the more numerous studies on nuclei of extraneural cells.This review attempts to outline some of the major findings on nuclear lipids of diverse cell types; results with nonneural nuclei will hopefully provide useful guideposts to further studies of neural systems.
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- 2004
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13. Biochemical deconstruction and reconstruction of Nuclear Matrix reveals the layers of nuclear organization (Updated February 21, 2023).
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NUCLEAR matrix ,NUCLEAR membranes ,DECONSTRUCTION ,IN situ processing (Mining) - Published
- 2023
14. Biochemical deconstruction and reconstruction of the Nuclear Matrix reveal the layers of nuclear organization.
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NUCLEAR matrix ,NUCLEAR membranes ,DECONSTRUCTION ,IN situ processing (Mining) - Published
- 2023
15. Gene editing of PKLR gene in human hematopoietic progenitors through 5' and 3' UTR modified TALEN mRNA
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Quintana-Bustamante, O., Fañanas-Baquero, S., Orman, I., Torres, Raul, Duchateau, P., Poirot, L., Gouble, A., Bueren, J.A., Segovia, J.C., and Universitat Autònoma de Barcelona
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Genome engineering ,0301 basic medicine ,Hydrolases ,Genetic enhancement ,Artificial Gene Amplification and Extension ,Engineering and technology ,Synthetic genome editing ,Biochemistry ,Polymerase Chain Reaction ,Mice ,0302 clinical medicine ,Genome editing ,Nucleic Acids ,Synthetic bioengineering ,3' Untranslated Regions ,Cells, Cultured ,Gene Editing ,Transcription activator-like effector nuclease ,Multidisciplinary ,Database and informatics methods ,Sequence analysis ,Enzymes ,Cell biology ,TALENs ,030220 oncology & carcinogenesis ,Medicine ,Cellular Structures and Organelles ,Stem cell ,Research Article ,Nucleases ,Bioinformatics ,Science ,Pyruvate Kinase ,Bioengineering ,Nucleofection ,Biology ,Research and Analysis Methods ,03 medical and health sciences ,Transcription Activator-Like Effector Nucleases ,DNA-binding proteins ,Genetics ,Animals ,Humans ,Nuclear Matrix ,Molecular Biology Techniques ,Molecular Biology ,Gene ,Synthetic biology ,DNA sequence analysis ,Cell Nucleus ,Biology and life sciences ,Synthetic genomics ,HEK 293 cells ,Proteins ,Cell Biology ,DNA ,Hematopoietic Stem Cells ,Nested Polymerase Chain Reaction ,030104 developmental biology ,HEK293 Cells ,Genetic Loci ,Enzymology ,Homologous recombination ,5' Untranslated Regions - Abstract
TheauthorswouldliketothankMiguelA.MartinforthecarefulmaintenanceofNSGmice,andRebecaSa ́nchezandOmairaAlberquillafortheirtechnicalassistanceinflowcytometry.TheauthorsalsothankFundacio ́n Botı ́n forpromotingtranslationalresearchattheHemato-poieticInnovativeTherapiesDivisionoftheCIEMAT Pyruvate Kinase Deficiency (PKD) is a rare erythroid metabolic disease caused by mutations in the PKLR gene, which encodes the erythroid specific Pyruvate Kinase enzyme. Erythrocytes from PKD patients show an energetic imbalance and are susceptible to hemolysis. Gene editing of hematopoietic stem cells (HSCs) would provide a therapeutic benefit and improve safety of gene therapy approaches to treat PKD patients. In previous studies, we established a gene editing protocol that corrected the PKD phenotype of PKD-iPSC lines through a TALEN mediated homologous recombination strategy. With the goal of moving toward more clinically relevant stem cells, we aim at editing the PKLR gene in primary human hematopoietic progenitors and hematopoietic stem cells (HPSCs). After nucleofection of the gene editing tools and selection with puromycin, up to 96% colony forming units showed precise integration. However, a low yield of gene edited HPSCs was associated to the procedure. To reduce toxicity while increasing efficacy, we worked on i) optimizing gene editing tools and ii) defining optimal expansion and selection times. Different versions of specific nucleases (TALEN and CRISPR-Cas9) were compared. TALEN mRNAs with 5' and 3' added motifs to increase RNA stability were the most efficient nucleases to obtain high gene editing frequency and low toxicity. Shortening ex vivo manipulation did not reduce the efficiency of homologous recombination and preserved the hematopoietic progenitor potential of the nucleofected HPSCs. Lastly, a very low level of gene edited HPSCs were detected after engraftment in immunodeficient (NSG) mice. Overall, we showed that gene editing of the PKLR gene in HPSCs is feasible, although further improvements must to be done before the clinical use of the gene editing to correct PKD. Sí
- Published
- 2021
16. Measuring nuclear calcium and actin assembly in living cells
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Robert Grosse, Bing Zhao, Mahira Safaralizade, and Ronja Fuderer
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Nuclear Localization Signals ,chemistry.chemical_element ,Calcium ,Biochemistry ,Actin-Related Protein 2-3 Complex ,Mice ,medicine ,Animals ,Humans ,Nuclear Matrix ,Calcium Signaling ,Molecular Biology ,Actin ,Calcium signaling ,Cell Nucleus ,General Medicine ,Actins ,Chromatin ,Cell nucleus ,Actin Cytoskeleton ,medicine.anatomical_structure ,HEK293 Cells ,chemistry ,GCaMP ,Biophysics ,NIH 3T3 Cells ,Nucleus ,Nuclear localization sequence - Abstract
Nuclear calcium signalling has emerged as a critical mechanism regulating processes like chromatin organization and gene expression. Recently, we have shown that nuclear calcium elevation triggers rapid and transient actin filament assembly inside the nucleus. Here, we constructed and employed a nuclear-specific calcium sensor based upon the new generation of genetically encoded probes jGCaMP7f. By fusing a nuclear localization signal to jGCaMP7f, we achieved highly efficient nuclear-specific targeting. Comparing the jGCaMP7f-NLS probe with the previous GCaMP6f-NLS calcium sensor showed clearly that jGCaMP7f-NLS is more sensitive and reverses significantly quicker thereby reflecting rapid nuclear calcium transients in a closely physiological manner. We further confirm that nuclear calcium transients precede nuclear actin polymerization by several seconds. Our data show that calcium-triggered nuclear actin assembly in fibroblasts is independent of the actin nucleating Arp2/3 complex. Together, jGCaMP7f-NLS represents an easy to use, reliable and highly sensitive nuclear calcium sensor that allows to tightly interrogate real-time, spatiotemporal calcium signalling and calcium-elicited effects in the nucleus of living cells.
- Published
- 2020
17. The Cultural Divide: Exponential Growth in Classical 2D and Metabolic Equilibrium in 3D Environments.
- Author
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Wrzesinski, Krzysztof, Rogowska-Wrzesinska, Adelina, Kanlaya, Rattiyaporn, Borkowski, Kamil, Schwämmle, Veit, Dai, Jie, Joensen, Kira Eyd, Wojdyla, Katarzyna, Carvalho, Vasco Botelho, and Fey, Stephen J.
- Subjects
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CELL metabolism , *CELL culture , *PROTEOMICS , *GLYCOLYSIS , *PENTOSE phosphate pathway , *OXIDATIVE phosphorylation , *UREA synthesis - Abstract
Introduction: Cellular metabolism can be considered to have two extremes: one is characterized by exponential growth (in 2D cultures) and the other by a dynamic equilibrium (in 3D cultures). We have analyzed the proteome and cellular architecture at these two extremes and found that they are dramatically different. Results: Structurally, actin organization is changed, microtubules are increased and keratins 8 and 18 decreased. Metabolically, glycolysis, fatty acid metabolism and the pentose phosphate shunt are increased while TCA cycle and oxidative phosphorylation is unchanged. Enzymes involved in cholesterol and urea synthesis are increased consistent with the attainment of cholesterol and urea production rates seen in vivo. DNA repair enzymes are increased even though cells are predominantly in Go. Transport around the cell – along the microtubules, through the nuclear pore and in various types of vesicles has been prioritized. There are numerous coherent changes in transcription, splicing, translation, protein folding and degradation. The amount of individual proteins within complexes is shown to be highly coordinated. Typically subunits which initiate a particular function are present in increased amounts compared to other subunits of the same complex. Summary: We have previously demonstrated that cells at dynamic equilibrium can match the physiological performance of cells in tissues in vivo. Here we describe the multitude of protein changes necessary to achieve this performance. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
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18. Colon Cancer-Specific Antigen-2 May Be Used as a Detecting and Prognostic Marker in Colorectal Cancer: A Preliminary Observation.
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Xue, Gang, Wang, Xiaojuan, Yang, Yong, Liu, Degui, Cheng, Ying, Zhou, Jun, and Cao, Yongkuan
- Subjects
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ANTIGENS , *BIOMARKERS , *COLON cancer prognosis , *BLOOD serum analysis , *COLON cancer treatment , *NUCLEAR matrix - Abstract
Background: A specific and sensitive serum marker for colorectal cancer (CRC) detection and surveillance is central to effective treatment. It was preliminarily reported that some nuclear matrix proteins may be served as a specific blood based marker for colon cancer. The objective of this study is to evaluate the value of serum CCSA-2 detection in diagnosis, prognostic estimation and surveillance for CRC. Method: Serum CCSA-2 protein was measured in 181 various patient populations and 20 healthy donors before surgery. For 106 CRC patients, it was also measured on day 7 after surgery. Among them, 49 CRC patients' CCSA-2 protein were measured during the follow-up period according to NCCN Guideline. Results: The serum CCSA-2 concentration in CRC patients was significantly higher than which in other patients and healthy individuals. Serum CCSA-2, at the cut-off point of 64.10 ng/mL, had a sensitivity of 98.10% and a specificity of 97.90% in separating CRC populations from all other individuals. The CCSA-2 assay was significantly more sensitive than CEA and CA19-9 assay in CRC detection. After surgery, the serum CCSA-2 level of CRC patients declined significantly, but it rebounded to a high level when recurrences occurred. The pre-operative serum CCSA-2 level in patients who had a relapse within the follow-up period was significantly higher than which in patients without relapse. Conclusions: Serum CCSA-2 not only may be a potential biomarker using in screening and surveillance of CRC, but also may be an independent prognostic marker for CRC patients. Further clinical trials need to be performed in a larger population of patients to ulteriorly confirm these results. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
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19. A dynamic actin-dependent nucleoskeleton and cell identity
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Nadine Hosny El Said, Piergiorgio Percipalle, Syed Raza Mahmood, and Tomas Venit
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Nuclear gene ,Transcription, Genetic ,Cellular differentiation ,Biology ,Biochemistry ,03 medical and health sciences ,0302 clinical medicine ,Animals ,Humans ,Nuclear Matrix ,Cytoskeleton ,Molecular Biology ,Gene ,Actin ,030304 developmental biology ,Ribonucleoprotein ,Cell Nucleus ,0303 health sciences ,Genome ,RNA ,Cell Differentiation ,General Medicine ,DNA-Directed RNA Polymerases ,Actins ,Chromatin ,Cell biology ,Cytoskeletal Proteins ,Genes, Mitochondrial ,Gene Expression Regulation ,030217 neurology & neurosurgery - Abstract
Actin is an essential regulator of cellular functions. In the eukaryotic cell nucleus, actin regulates chromatin as a bona fide component of chromatin remodelling complexes, it associates with nuclear RNA polymerases to regulate transcription and is involved in co-transcriptional assembly of nascent RNAs into ribonucleoprotein complexes. Actin dynamics are, therefore, emerging as a major regulatory factor affecting diverse cellular processes. Importantly, the involvement of actin dynamics in nuclear functions is redefining the concept of nucleoskeleton from a rigid scaffold to a dynamic entity that is likely linked to the three-dimensional organization of the nuclear genome. In this review, we discuss how nuclear actin, by regulating chromatin structure through phase separation may contribute to the architecture of the nuclear genome during cell differentiation and facilitate the expression of specific gene programs. We focus specifically on mitochondrial genes and how their dysregulation in the absence of actin raises important questions about the role of cytoskeletal proteins in regulating chromatin structure. The discovery of a novel pool of mitochondrial actin that serves as ‘mitoskeleton’ to facilitate organization of mtDNA supports a general role for actin in genome architecture and a possible function of distinct actin pools in the communication between nucleus and mitochondria.
- Published
- 2020
20. Visualization of endogenous nuclear F-actin in mouse embryos reveals abnormal actin assembly after somatic cell nuclear transfer
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Shunya Ihashi, Taiki Shindo, Junko Tomikawa, Yuko Sakamoto, Kei Miyamoto, and Tomomi Okuno
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Male ,Nuclear Transfer Techniques ,Somatic cell ,Phalloidin ,Phalloidine ,Zygote ,Green Fluorescent Proteins ,macromolecular substances ,Microfilament ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,Mice ,0302 clinical medicine ,medicine ,Animals ,Humans ,Nuclear Matrix ,Paraformaldehyde ,Molecular Biology ,Actin ,030304 developmental biology ,Cell Nucleus ,0303 health sciences ,Pronucleus ,Staining and Labeling ,Nuclear Proteins ,General Medicine ,Actins ,Cell biology ,medicine.anatomical_structure ,chemistry ,Somatic cell nuclear transfer ,Female ,Nucleus ,030217 neurology & neurosurgery - Abstract
Actin in the nucleus, referred to as nuclear actin, is involved in a variety of nuclear events. Nuclear actin is present as a globular (G-actin) and filamentous form (F-actin), and dynamic assembly/disassembly of nuclear actin profoundly affects nuclear functions. However, it is still challenging to observe endogenous nuclear F-actin. Here, we present a condition to visualize endogenous nuclear F-actin of mouse zygotes using different fixation methods. Zygotes fixed with paraformaldehyde and treated with fluorescently conjugated phalloidin show both short and long actin filaments in their pronuclei. Short nuclear actin filaments are characteristic of phalloidin staining, rather than the consequence of severing actin filaments by the fixation process, since long nuclear actin filaments probed with the nuclear actin chromobody are not disassembled into short filaments after fixation with paraformaldehyde. Furthermore, we find that nuclear actin assembly is impaired after somatic cell nuclear transfer (SCNT), suggesting abnormal nucleoskeleton structures in SCNT embryos. Taken together, our presented method for visualizing nuclear F-actin with phalloidin can be used to observe the states of nuclear actin assembly, and revealed improper reprogramming of actin nucleoskeleton structures in cloned mouse embryos.
- Published
- 2020
21. Neuron-enriched phosphatase and actin regulator 3 (Phactr3)/ nuclear scaffold-associated PP1-inhibiting protein (Scapinin) regulates dendritic morphology via its protein phosphatase 1-binding domain
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Tomoaki Miyata, Masaaki Tsuda, Shizuku Shoji, Mamoru Fukuchi, Maki Kaito, Yuta Ishibashi, Daisuke Ihara, Tetsuya Yamada, Yamato Hida, Toshihisa Ohtsuka, Miho Mizukoshi, Akiko Tabuchi, Keietsu Kikuchi, Mitsuru Ishikawa, Marisa Kaneda, and Tomoyuki Hakamata
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0301 basic medicine ,Male ,Phosphatase ,Biophysics ,macromolecular substances ,Biochemistry ,Rats, Sprague-Dawley ,03 medical and health sciences ,0302 clinical medicine ,Nuclear Matrix-Associated Proteins ,Protein Domains ,Protein Phosphatase 1 ,medicine ,Animals ,Nuclear Matrix ,RNA, Messenger ,Axon ,Molecular Biology ,Actin ,Cerebral Cortex ,Chemistry ,Microfilament Proteins ,Wild type ,Nuclear Proteins ,Protein phosphatase 1 ,Cell Biology ,Dendrites ,Cell biology ,Up-Regulation ,Mice, Inbred C57BL ,030104 developmental biology ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,Mutation ,Female ,Neuron ,Postsynaptic density ,Binding domain ,Protein Binding ,Subcellular Fractions - Abstract
Phosphatase and actin regulator 3/nuclear scaffold-associated protein phosphatase 1-inhibiting protein (Phactr3/Scapinin) is an actin- and protein phosphatase 1 (PP1)-binding protein known to negatively regulate axon elongation. In this study, we examined the expression pattern of Phactr3/Scapinin in several tissues and investigated the effect of Phactr3/Scapinin on dendritic morphology of cortical neurons. Results showed that Phactr3/Scapinin expression was up-regulated in the developing brain and enriched in neurons and in the postsynaptic density fraction, but not in astrocytes. Overexpression of wild type or mutant Phactr3/Scapinin, which lacked actin-binding activity, resulted in increased dendritic complexity and percentage of spines with a mushroom or stubby shape, as well as a decrease in spine density. However, overexpression of mutant Phactr3/Scapinin that lacked PP1-binding activity did not. Taken together, these findings suggest that Phactr3/Scapinin expression is neuronal and might contribute to synaptic formation via distinct actin- and PP1-binding domains involved in dendritic and axonal morphology, respectively.
- Published
- 2020
22. Identification of evolutionarily conserved nuclear matrix proteins and their prokaryotic origins
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Rahul Sureka and Rakesh Mishra
- Subjects
biology ,Endosymbiosis ,General Chemistry ,Computational biology ,Mitochondrion ,biology.organism_classification ,Nuclear matrix ,Biochemistry ,Chloroplast ,Evolution, Molecular ,Drosophila melanogaster ,Eukaryotic Cells ,Nuclear Matrix-Associated Proteins ,Prokaryotic Cells ,Evolutionary biology ,Homo sapiens ,Organelle ,RNA splicing ,Melanogaster ,Animals ,Nuclear Matrix ,Endomembrane system ,Phylogeny ,Cellular localization - Abstract
Compared to prokaryotic cells, a typical eukaryotic cell is much more complex along with its endomembrane system and membrane-bound organelles. Although the endosymbiosis theories convincingly explain the evolution of membrane-bound organelles such as mitochondria and chloroplasts, very little is understood about the evolutionary origins of the nucleus, the defining feature of eukaryotes. Most studies on nuclear evolution have not been able to take into consideration the underlying structural framework of the nucleus, attributed to the nuclear matrix (NuMat), a ribonucleoproteinaceous structure. This can largely be attributed to the lack of annotation of its core components. Since NuMat has been shown to provide a structural platform for facilitating a variety of nuclear functions such as replication, transcription, and splicing, it is important to identify its protein components to better understand these processes. In this study, we address this issue using the developing embryos of Drosophila melanogaster and Danio rerio and identify 362 core NuMat proteins that are conserved between the two organisms. We further compare our results with publicly available Mus musculus NuMat dataset and Homo sapiens cellular localization dataset to define the core homologous NuMat proteins consisting of 252 proteins. We find that of them, 86 protein groups have originated from pre-existing proteins in prokaryotes. While 36 were conserved across all eukaryotic supergroups, 14 new proteins evolved before the evolution of the last eukaryotic common ancestor and together, these 50 proteins out of the 252 core conserved NuMat proteins are conserved across all eukaryotes, indicating their indispensable nature for nuclear function for over 1.5 billion years of eukaryotic history. Our analysis paves the way to understand the evolution of the complex internal nuclear architecture and its functions.
- Published
- 2019
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23. Intranuclear Mobility of Estrogen Receptor: Implication for Transcriptional Regulation
- Author
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Takashi Hashimoto, Mitsuhiro Kawata, and Ken-ichi Matsuda
- Subjects
0301 basic medicine ,Histology ,Physiology ,Estrogen receptor ,Review ,Biochemistry ,Pathology and Forensic Medicine ,03 medical and health sciences ,0302 clinical medicine ,Transcription (biology) ,Live cell imaging ,Transcriptional regulation ,Transcription factor ,Gene ,Chemistry ,Scaffold attachment factor B ,living imaging ,Cell Biology ,SAFB ,Nuclear matrix ,Cell biology ,030104 developmental biology ,nuclear matrix ,FRAP ,030217 neurology & neurosurgery ,estrogen receptor - Abstract
The estrogen receptor (ER) is a ligand-dependent transcription factor that has two subtypes: ERα and ERβ. ERs regulate transcription of estrogen-responsive genes through interactions with multiple intranuclear components, such as cofactors and the nuclear matrix. Live cell imaging using fluorescent protein-labeled ERs has revealed that ligand-activated ERs are highly mobile in the nucleus, with transient association with the DNA and nuclear matrix. Scaffold attachment factor B (SAFB) 1 and its paralogue, SAFB2, are nuclear matrix-binding proteins that negatively modulate ERα-mediated transcription. Expression of SAFB1 and SAFB2 reduces the mobility of ERα in the presence of ligand. This regulatory machinery is emerging as an epigenetic-like mechanism that alters transcriptional activity through control of intranuclear molecular mobility.
- Published
- 2018
24. Linking nuclear matrix–localized PIAS1 to chromatin SUMOylation via direct binding of histones H3 and H2A.Z
- Author
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Jiwen Li, Jiemin Wong, Wei Wei, Qingqing Guan, Jing Luo, Huifang Zhang, Yunpeng Zhang, Xiang Xu, Jialun Li, Zhaosu Chen, and Lujian Liao
- Subjects
Transcription, Genetic ,SUMO protein ,DSP, dithiobis succinimidyl propionate ,PIAS1 ,Biochemistry ,Histones ,Protein Domains ,Transcription (biology) ,histone SUMOylation ,Humans ,Protein inhibitor of activated STAT ,Molecular Biology ,biology ,co-IP, coimmunoprecipitation ,Chemistry ,SUMO, small ubiquitin-related modifier ,Sumoylation ,Cell Biology ,Nuclear matrix ,Protein Inhibitors of Activated STAT ,Chromatin ,Ubiquitin ligase ,Cell biology ,ChIP, chromatin immunoprecipitation ,HEK293 Cells ,Histone ,SUMO ,nuclear matrix ,Small Ubiquitin-Related Modifier Proteins ,biology.protein ,Function (biology) ,Research Article ,HeLa Cells - Abstract
As a conserved posttranslational modification, SUMOylation has been shown to play important roles in chromatin-related biological processes including transcription. However, how the SUMOylation machinery associates with chromatin is not clear. Here, we present evidence that multiple SUMOylation machinery components, including SUMO E1 proteins SAE1 and SAE2 and the PIAS (protein inhibitor of activated STAT) family SUMO E3 ligases, are primarily associated with the nuclear matrix rather than with chromatin. We show using nuclease digestion that all PIAS family proteins maintain nuclear matrix association in the absence of chromatin. Of importance, we identify multiple histones including H3 and H2A.Z as directly interacting with PIAS1 and demonstrate that this interaction requires the PIAS1 SAP (SAF-A/B, Acinus, and PIAS) domain. We demonstrate that PIAS1 promotes SUMOylation of histones H3 and H2B in both a SAP domain- and an E3 ligase activity-dependent manner. Furthermore, we show that PIAS1 binds to heat shock-induced genes and represses their expression and that this function also requires the SAP domain. Altogether, our study reveals for the first time the nuclear matrix as the compartment most enriched in SUMO E1 and PIAS family E3 ligases. Our finding that PIAS1 interacts directly with histone proteins also suggests a molecular mechanism as to how nuclear matrix-associated PIAS1 is able to regulate transcription and other chromatin-related processes.
- Published
- 2021
25. Nucleocytoplasmic Shuttling of the Zinc Finger Protein EZI Is Mediated by lmportin-7-dependent Nuclear Import and CRM1 -independent Export Mechanisms.
- Author
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Saijou, Eiko, Itoh, Tohru, Kyung-Woon Kim, Iemura, Shun-ichiro, Natsume, Tohru, and Miyajima, Atsushi
- Subjects
- *
CELL membranes , *NUCLEAR matrix , *MOLECULAR biology , *EUKARYOTIC cells , *BIOCHEMICAL genetics , *BIOORGANIC chemistry , *BIOCHEMISTRY - Abstract
Nucleocytoplasmic translocation constitutes a foundation for nuclear proteins to exert their proper functions and hence for various biological reactions to occur normally in eukaryotic cells. We reported previously that EZI/Zfp467, a 12 zinc finger motif-containing protein, localizes predominantly in the nucleus, yet the underlying mechanism still remains elusive. Here we constructed a series of mutant forms of EZI and examined their subcellular localization. The results delineated a non-canonical nuclear localization signal in the region covering the 9th to the 12th zinc fingers, which was necessary for nuclear accumulation of EZI as well as sufficient to confer nuclear localizing ability to a heterologous protein. We also found that the N-terminal domain of EZI is necessary for its nuclear export, the process of which was not sensitive to the CRM1 inhibitor leptomycin B. An interaction proteomics approach and the following co-immunoprecipitation experiments identified the nuclear import receptor importin-7 as a molecule that associated with EZI and, importantly, short interfering RNA-mediated knock- down of importin-7 expression completely abrogated nuclear accumulation of EZI. Taken together, these results identify EZI as a novel cargo protein for importin-7 and demonstrate a nucleocytoplasmic shuttling mechanism that is mediated by importin-7-dependent nuclear localization and CRM1-independent nuclear export. [ABSTRACT FROM AUTHOR]
- Published
- 2007
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26. Cooperation of ERK and SCFSkp2 for MKP-1 Destruction Provides a Positive Feedback Regulation of Proliferating Signaling.
- Author
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Yun-wei Lin and Jia-ling Yang
- Subjects
- *
MITOGEN-activated protein kinases , *PROTEIN kinases , *NUCLEAR matrix , *PROTEOLYSIS , *PROTEIN metabolism , *PROTEOLYTIC enzymes , *PROTEOMICS , *BIOCHEMISTRY - Abstract
The dual-specificity MAPK phosphatase MKP-1/CL100/DUSP1 is an inducible nuclear protein controlled by p44/42 MAPK (ERK1/2) in a negative feedback mechanism to inhibit kinase activity. Here, we report on the molecular basis for a novel positive feed-back mechanism to sustain ERK activation by triggering MKP-1 proteolysis. Active ERK2 docking to the DEF motif (FXFP, residues 339–342) of N-terminally truncated MKP-1 in vitro initiated phosphorylation at the Ser296/Ser323 domain, which was not affected by substituting Ala for Ser at Ser359/Ser364. The DEF and Ser296/Ser323 sites were essential for ubiquitin-mediated MKP-1 proteolysis stimulated by MKK1-ERK signaling in H293 cells, whereas the N-terminal domain and Ser359/Ser364 sites were dispensable. ERK activation by serum increased the endogenous level of ubiquitinated phosphor-Ser296 MKP-1 and the degradation of MKP-1. Intriguingly, active ERK-promoted phospho-Ser296 MKP-1 bound to SCFSkp2 ubiquitin ligase in vivo and in vitro. Forced expression of Skp2 enhanced MKP-1 polyubiquitinatinn and proteolysis upon ERK activation, whereas depletion of endogenous Skp2 suppressed such events. The kinetics of ERK signaling stimulated by serum correlated with the endogenous MKP-1 degradation rate in a Skp2-dependent manner. Thus, MKP-1 proteolysis can be achieved via ERK and SCFSkp2 cooperation, thereby sustaining ERK activation. [ABSTRACT FROM AUTHOR]
- Published
- 2006
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27. DNA Length Modulates the Affinity of Fragments of Genomic DNA for the Nuclear Matrix In Vitro
- Author
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David García-Vilchis and Armando Aranda-Anzaldo
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Male ,0301 basic medicine ,HMG-box ,DNA polymerase ,Base pair ,Biochemistry ,03 medical and health sciences ,0302 clinical medicine ,Animals ,Nuclear Matrix ,Rats, Wistar ,Molecular Biology ,chemistry.chemical_classification ,DNA ligase ,DNA clamp ,biology ,DNA replication ,DNA ,Cell Biology ,Matrix Attachment Regions ,Molecular biology ,Rats ,030104 developmental biology ,chemistry ,030220 oncology & carcinogenesis ,Hepatocytes ,biology.protein ,Biophysics ,DNA supercoil ,In vitro recombination - Abstract
Classical observations have shown that during the interphase the chromosomal DNA of metazoans is organized in supercoiled loops attached to a compartment known as the nuclear matrix (NM). Fragments of chromosomal DNA able to bind the isolated NM in vitro are known as matrix associated/attachment/addressed regions or MARs. No specific consensus sequence or motif has been found that may constitute a universal, defining feature of MARs. On the other hand, high-salt resistant DNA-NM interactions in situ define true DNA loop anchorage regions or LARs, that might correspond to a subset of the potential MARs but are not necessarily identical to MARs characterized in vitro, since there are several examples of MARs able to bind the NM in vitro but which are not actually bound to the NM in situ. In the present work we assayed the capacity of two LARs, as well as of shorter fragments within such LARs, for binding to the NM in vitro. Paradoxically the isolated (≈2 kb) LARs cannot bind to the NM in vitro while their shorter (≈300 pb) sub-fragments and other non-related but equally short DNA fragments, bind to the NM in a high-salt resistant fashion. Our results suggest that the ability of a given DNA fragment for binding to the NM in vitro primarily depends on the length of the fragment, suggesting that binding to the NM is modulated by the local topology of the DNA fragment in suspension that it is known to depend on the DNA length. J. Cell. Biochem. 118: 4487-4497, 2017. © 2017 Wiley Periodicals, Inc.
- Published
- 2017
28. The Set of Structural DNA-Nuclear Matrix Interactions in Neurons Is Cell-Type Specific and Rather Independent of Functional Constraints
- Author
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Juan Carlos Rivera-Mulia, Armando Aranda-Anzaldo, and Evangelina Silva-Santiago
- Subjects
0301 basic medicine ,Cell Biology ,Nuclear matrix ,Biochemistry ,Cell biology ,Chromatin ,Nuclear DNA ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,medicine.anatomical_structure ,chemistry ,medicine ,Nucleoid ,DNA supercoil ,Scaffold/matrix attachment region ,Molecular Biology ,Nucleus ,DNA - Abstract
In metazoans, nuclear DNA is organized during the interphase in negatively supercoiled loops anchored to a compartment or substructure known as the nuclear matrix. The interactions between DNA and the nuclear matrix (NM) are of higher affinity than those between DNA and chromatin proteins since the last ones do not resist the procedures for extracting the NM. The structural interactions DNA-NM constitute a set of topological relationships that define a nuclear higher order structure (NHOS) although there are further higher order levels of organization within the nucleus. So far, the evidence derived from studies with primary hepatocytes and naive B lymphocytes indicates that the NHOS is cell-type specific at the local and at the large-scale level, and so it has been suggested that such NHOS is primary determined by structural and thermodynamic constraints. We carried out a comparative characterization of the NHOS of postmitotic cortical neurons with that of hepatocytes and naive B lymphocytes. Our results indicate that the NHOS of neurons is completely different at the large scale and at the local level from that one observed in hepatocytes or in naive B lymphocytes, confirming on the one hand that the set of structural DNA-NM interactions is cell-type specific and supporting, on the other hand the notion that structural constraints that impinge on chromosomal DNA and the NM are more important for determining this NHOS than functional constraints related to replication and/or transcription. J. Cell. Biochem. 118: 2151-2160, 2017. © 2016 Wiley Periodicals, Inc.
- Published
- 2017
29. Nuclear-Cytoplasmic Shuttling of the Oncogenic Mouse UNP/USP4 Deubiquitylating Enzyme.
- Author
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Soboleva, Tatiana A., Jans, David A., Johnson-Saliba, Melanie, and Baker, Rohan T.
- Subjects
- *
ENZYMES , *RETINOBLASTOMA , *TUMOR suppressor proteins , *NUCLEAR matrix , *PROTEINS , *BIOCHEMISTRY - Abstract
The oncogenic deubiquitylating enzyme (DUB) Unp/Usp4, which binds to the retinoblastoma family of tumor suppressor proteins, was originally described as a nuclear protein. However, more recent studies have shown it to be cytoplasmic. In addition, analysis of its subcellular localization has been complicated by the existence of the paralog Usp15. In this study, we resolved this controversy by investigating the localization of exogenously expressed Usp4 (using red fluorescent protein-Usp4) and of endogenous Usp4 (using highly specific antibodies that can distinguish Usp4 from Usp15). We found that by inhibiting nuclear export with leptomycin B, both exogenous and endogenous Usp4 accumulate in the nucleus. Further, using a Rev-green fluorescent protein-based export assay, we confirmed the existence of a nuclear export signal (133VEVYLLELKL142) in Usp4. In addition, a functional nuclear import signal (766QPQKKKK772) was also identified, which was specifically recognized by importin α/β. Finally, we show that the equilibrium of Usp4 subcellular localization varies between different cell types. Usp4 is thus the first DUB reported to have nucleocytoplasmic shuttling properties. The implications of this shuttling for its function as a DUB are discussed. [ABSTRACT FROM AUTHOR]
- Published
- 2005
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30. KIN17 encodes an RNA-binding protein and is expressed during mouse spermatogenesis.
- Author
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Pinon-Lataillade, Ghislaine, Masson, Christel, Bernardino-Sgherri, Jacqueline, Henriot, Véronique, Mauffrey, Philippe, Frobert, Yveline, Araneda, Silvia, and Angulo, Jaime F.
- Subjects
- *
SERTOLI cells , *SPERMATOGENESIS , *DNA , *NUCLEAR matrix , *METABOLISM , *BIOCHEMISTRY - Abstract
Genotoxic agents deform DNA structure thus eliciting a complex genetic response allowing recovery and cell survival. The Kin17 gene is up-regulated during this response. This gene encodes a conserved nuclear protein that shares a DNA-binding domain with the bacterial RecA protein. The KIN17 protein binds DNA and displays enhanced expression levels in proliferating cultured cells, suggesting a role in nuclear metabolism. We investigated this by studying the expression profile of KIN17 protein during mouse spermatogenesis. As expected, the expression level of Kin17 is higher in proliferating than in differentiated cells. KIN17 is selectively extracted from this tissue by detergents and a fraction was tightly associated with the nuclear matrix. Germinal cells ubiquitously express Kin17 and the protein is located mainly in the nucleus except in elongated spermatids where cytoplasmic staining is also observed. Sertoli and germ cells that are no longer mitotically active express KIN17, suggesting a general role in all testicular cell types. In adult testis a significant proportion of KIN17 co-purifies with polyadenylated RNA. KIN17 directly binds RNA, preferentially poly(G) and poly(U) homopolymers. These results together with the identification of KIN17 as a component of the human spliceosome indicate that this protein may participate in RNA processing. [ABSTRACT FROM AUTHOR]
- Published
- 2004
- Full Text
- View/download PDF
31. Structure-Function Analysis of the Estrogen Receptor α Corepressor Scalfford Attchement Factor-B1.
- Author
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Townson, Steven M., Kang, Kaiyan, Lee, Adrian V., and Oesterreich, Steffi
- Subjects
- *
ESTROGEN , *ESTROGEN receptors , *NUCLEAR matrix , *CELL nuclei , *CHROMATIN , *RNA , *GENETIC transcription , *HISTONE deacetylase , *YEAST , *PROTEINS , *BIOCHEMISTRY - Abstract
Scaffold attachment factor-B1 (SAFB1) is a nuclear matrix protein that has been proposed to couple chromatin structure, transcription, and RNA processing. We have previously shown that SAFB1 can repress estrogen receptor (ERα)-mediated transactivation. Here we present a structure-function study showing that transactivation is mediated via an intrinsic and transferable C-terminal repression domain (RD). A similar C-terminal RD was found in the family member SAFB2. Removal of the RD from SAFB1 resulted in a dominant-negative SAFB1 protein that increased ligand-dependent and -independent ERα activity. SAFBIRD-mediated repression was partly blocked by histone deacetylase inhibitors; however, no histone deacetylase inhibitors were identified in a yeast two-hybrid screen using the RD as bait. Instead, SAFBIRD was found to interact with TAFII68, a member of the basal transcription machinery. We propose a model in which SAFB1 represses ERα activity via indirect association with histone deacetylation and interaction with the basal transcription machinery. [ABSTRACT FROM AUTHOR]
- Published
- 2004
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- View/download PDF
32. Werner Syndrome Protein Contains Three Structure-specific DNA Binding Domains.
- Author
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von Kobbe, Cayetano, Thomäs, Nikolas H., Czyzewski, Bryan K., Pavletich, Nikola P., and Bohr, Vilhelm A.
- Subjects
- *
WERNER'S syndrome , *GENETIC mutation , *NUCLEAR matrix , *DNA helicases , *PROTEIN binding , *BIOCHEMISTRY - Abstract
Werner syndrome (WS) is a premature aging syndrome caused by mutations in the WS gene (WRN) and a deficiency in the function of the Werner protein (WRN). WRN is a multifunctional nuclear protein that catalyzes three DNA-dependent reactions: a 3'-5'-exonuclease, an ATPase, and a 3'-5'-helicase. Deficiency in WRN results in a cellular phenotype of genomic instability. The biochemical characteristics of WRN and the cellular phenotype of WRN mutants suggest that WRN plays an important role in DNA metabolic pathways such as recombination, transcription, replication, and repair. The catalytic activities of WRN have been extensively studied and are fairly well understood. However, much less is known about the domain-specific interactions between WRN and its DNA substrates. This study identifies and characterizes three distinct WRN DNA binding domains using recombinant truncated fragments of WRN and five DNA substrates (long forked duplex, blunt-ended duplex, single-stranded DNA, 5'-overhang duplex, and Holliday junction). Substrate-specific DNA binding activity was detected in three domains, one Nterminal and two different C-terminal WRN fragments (RecQ conserved domain and helicase RNase D conserved domain-containing domains). The substrate specificity of each DNA binding domain may indicate that each protein domain has a distinct biological function. The importance of these results is discussed with respect to proposed roles for WRN in distinct DNA metabolic pathways. [ABSTRACT FROM AUTHOR]
- Published
- 2003
- Full Text
- View/download PDF
33. Analysis of quinolinequinone reactivity, cytotoxicity, and anti-HIV-1 properties
- Author
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Ayna Alfadhli, Andrew Mack, Eric Barklis, Sam Berk, Logan Harper, and Christopher Ritchie
- Subjects
0301 basic medicine ,Anti-HIV Agents ,Clinical Biochemistry ,Pharmaceutical Science ,Microbial Sensitivity Tests ,01 natural sciences ,Biochemistry ,Article ,Virus ,Structure-Activity Relationship ,03 medical and health sciences ,chemistry.chemical_compound ,Drug Discovery ,Humans ,Structure–activity relationship ,Nuclear Matrix ,Cytotoxicity ,Molecular Biology ,Cells, Cultured ,Cell Proliferation ,Viral matrix protein ,Dose-Response Relationship, Drug ,Molecular Structure ,010405 organic chemistry ,Cell growth ,Chemistry ,Organic Chemistry ,RNA ,Glutathione ,In vitro ,0104 chemical sciences ,HEK293 Cells ,030104 developmental biology ,Chemical engineering ,HIV-1 ,Quinolines ,RNA, Viral ,Molecular Medicine - Abstract
We have analyzed a set of quinolinequinones with respect to their reactivities, cytotoxicities, and anti-HIV-1 properties. Most of the quinolinequinones were reactive with glutathione, and several acted as sulfhydryl crosslinking agents. Quinolinequinones inhibited binding of the HIV-1 matrix protein to RNA to varying degrees, and several quinolinequinones showed the capacity to crosslink HIV-1 matrix proteins in vitro, and HIV-1 structural proteins in virus particles. Cytotoxicity assays yielded quinolinequinone CC50 values in the low micromolar range, reducing the potential therapeutic value of these compounds. However, one compound, 6,7-dichloro-5,8-quinolinequinone potently inactivated HIV-1, suggesting that quinolinequinones may prove useful in the preparation of inactivated virus vaccines or for other virucidal purposes.
- Published
- 2016
34. TGF-β induces PML SUMOylation, degradation and PML nuclear body disruption
- Author
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Faten El-Asmi, Laurent Dianoux, Bouchra El-Mchichi, Mohamed Ali Maroui, Mounira K. Chelbi-Alix, Toxicité environnementale, cibles thérapeutiques, signalisation cellulaire (T3S - UMR_S 1124), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP)
- Subjects
0301 basic medicine ,viruses ,[SDV]Life Sciences [q-bio] ,Immunology ,SUMO protein ,Apoptosis ,Protein degradation ,Promyelocytic Leukemia Protein ,Caspase 8 ,Biochemistry ,03 medical and health sciences ,0302 clinical medicine ,Interferon ,Transforming Growth Factor beta ,Cell Line, Tumor ,medicine ,Immunology and Allergy ,Humans ,Nuclear Matrix ,Molecular Biology ,Cell Nucleus ,Nucleoplasm ,Chemistry ,Alternative splicing ,virus diseases ,Interferon-alpha ,Sumoylation ,Hematology ,Nuclear matrix ,Cell biology ,Enzyme Activation ,030104 developmental biology ,HEK293 Cells ,030220 oncology & carcinogenesis ,Proteolysis ,Small Ubiquitin-Related Modifier Proteins ,Ectopic expression ,medicine.drug ,Protein Binding - Abstract
ProMyelocytic Leukemia (PML) protein is essential for the formation of nuclear matrix-associated organelles named PML nuclear bodies (NBs) that act as a platform for post-translational modifications and protein degradation. PML NBs harbor transiently and permanently localized proteins and are associated with the regulation of several cellular functions including apoptosis. There are seven PML isoforms, six nuclear (PMLI-VI) and one cytoplasmic (PMLVII), which are encoded by a single gene via alternative RNA splicing. It has been reported that murine PML-null primary cells are resistant to TGF-β-induced apoptosis and that cytoplasmic PML is an essential activator of TGF-β signaling. The role and the fate of interferon (IFN)-enhanced PML NBs in response to TGF-β have not been investigated. Here we show that IFNα potentiated TGF-β-mediated apoptosis in human cells. IFNα or ectopic expression of PMLIV, but not of PMLIII, enhanced TGF-β-induced caspase 8 activation. In response to TGF-β, both PMLIII and PMLIV were conjugated to SUMO and shifted from the nucleoplasm to the nuclear matrix, however only PMLIV, via its specific C-terminal region, interacted with caspase 8 and recruited it within PML NBs. This process was followed by a caspase-dependent PML degradation and PML NB disruption. Taken together, these findings highlight the role of PML NBs in the enhancement by IFN of TGF-β-induced apoptosis and caspase 8 activation.
- Published
- 2019
35. Optogenetics reveals Cdc42 local activation by scaffold-mediated positive feedback and Ras GTPase
- Author
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Vincent Vincenzetti, Laura Merlini, Iker Lamas, Sophie G. Martin, and Aleksandar Vjestica
- Subjects
0301 basic medicine ,Scaffold protein ,Molecular biology ,Hydrolases ,Cell Polarity/genetics ,Feedback, Physiological/physiology ,Nuclear Matrix/physiology ,Optogenetics ,Organisms, Genetically Modified ,Schizosaccharomyces/genetics ,Schizosaccharomyces/metabolism ,Schizosaccharomyces pombe Proteins/genetics ,Schizosaccharomyces pombe Proteins/metabolism ,cdc42 GTP-Binding Protein/genetics ,cdc42 GTP-Binding Protein/metabolism ,ras Proteins/physiology ,Cell Membranes ,CDC42 ,GTPase ,Biochemistry ,Signaling Molecules ,0302 clinical medicine ,Cell Signaling ,Cell polarity ,Guanine Nucleotide Exchange Factors ,Small GTPase ,Biology (General) ,cdc42 GTP-Binding Protein ,Feedback, Physiological ,0303 health sciences ,Brain Mapping ,biology ,Chemistry ,Effector ,General Neuroscience ,Mechanisms of Signal Transduction ,Cell Polarity ,3. Good health ,Cell biology ,Enzymes ,Bioassays and Physiological Analysis ,Guanine nucleotide exchange factor ,Cellular Structures and Organelles ,biological phenomena, cell phenomena, and immunity ,General Agricultural and Biological Sciences ,Research Article ,Signal Transduction ,Feedback Regulation ,Imaging Techniques ,QH301-705.5 ,DNA construction ,Research and Analysis Methods ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Fluorescence Imaging ,Schizosaccharomyces ,Nuclear Matrix ,030304 developmental biology ,Positive feedback ,General Immunology and Microbiology ,Biology and Life Sciences ,Membrane Proteins ,Proteins ,Cell Biology ,Neurophysiological Analysis ,biology.organism_classification ,Guanosine Triphosphatase ,030104 developmental biology ,Molecular biology techniques ,Schizosaccharomyces pombe ,Plasmid Construction ,Enzymology ,ras Proteins ,Schizosaccharomyces pombe Proteins ,030217 neurology & neurosurgery ,Neuroscience - Abstract
Local activity of the small GTPase Cdc42 is critical for cell polarization. Whereas scaffold-mediated positive feedback was proposed to break symmetry of budding yeast cells and produce a single zone of Cdc42 activity, the existence of similar regulation has not been probed in other organisms. Here, we address this problem using rod-shaped cells of fission yeast Schizosaccharomyces pombe, which exhibit zones of active Cdc42-GTP at both cell poles. We implemented the CRY2-CIB1 optogenetic system for acute light-dependent protein recruitment to the plasma membrane, which allowed to directly demonstrate positive feedback. Indeed, optogenetic recruitment of constitutively active Cdc42 leads to co-recruitment of the guanine nucleotide exchange factor (GEF) Scd1 and endogenous Cdc42, in a manner dependent on the scaffold protein Scd2. We show that Scd2 function is dispensable when the positive feedback operates through an engineered interaction between the GEF and a Cdc42 effector, the p21-activated kinase 1 (Pak1). Remarkably, this rewired positive feedback confers viability and allows cells to form 2 zones of active Cdc42 even when otherwise essential Cdc42 activators are lacking. These cells further revealed that the small GTPase Ras1 plays a role in both localizing the GEF Scd1 and promoting its activity, which potentiates the positive feedback. We conclude that scaffold-mediated positive feedback, gated by Ras activity, confers robust polarization for rod-shape formation., The small GTPase Cdc42 is a key regulator of cell polarization. This study uses optogenetic and genetic strategies to show that Cdc42 is under positive feedback regulation potentiated by Ras GTPase activity.
- Published
- 2019
- Full Text
- View/download PDF
36. The epicenter of chromosomal fragility of Fra14A2, the mouse ortholog of human FRA3B common fragile site, is largely attached to the nuclear matrix in lymphocytes but not in other cell types that do not express such a fragility
- Author
-
Armando Aranda-Anzaldo and Rolando Guadarrama‐Ponce
- Subjects
0301 basic medicine ,Male ,Cell type ,Biology ,Biochemistry ,Chromosomes ,law.invention ,03 medical and health sciences ,Mice ,0302 clinical medicine ,FHIT ,law ,Animals ,Nuclear Matrix ,Lymphocytes ,Molecular Biology ,Gene ,Metaphase ,Cells, Cultured ,Cell Proliferation ,Chromosomal fragile site ,Chromosome Fragile Sites ,Chromosome Fragility ,DNA replication ,Cell Biology ,Nuclear matrix ,Cell biology ,Acid Anhydride Hydrolases ,Neoplasm Proteins ,030104 developmental biology ,030220 oncology & carcinogenesis ,Hepatocytes ,Suppressor - Abstract
Common fragile sites (CFSs) correspond to chromosomal regions susceptible to present breaks, discontinuities or constrictions in metaphase chromosomes from cells subjected to replication stress. They are considered as genomic regions intrinsically difficult to replicate and they are evolutionary conserved at least in mammals. However, the recent discovery that CFSs are cell-type specific indicates that DNA sequence by itself cannot account for CFS instability. Nevertheless, the large gene FHIT that includes FRA3B, the most highly expressed CFS in human lymphocytes, is commonly deleted in a variety of tumors suggesting a tumor suppressor role for its product. Here, we report that the epicenter of fragility of Fra14A2/Fhit, the mouse ortholog of human FRA3B/FHIT that like its human counterpart is the most highly expressed CFS in mouse lymphocytes, is largely attached to the nuclear matrix compartment in naive B lymphocytes but not in primary hepatocytes or cortical neurons that do not express such a CFS. Our results suggest a structural explanation for the difficult-to-replicate nature of such a region and so for its common fragility in lymphocytes, that is independent of the possible tumor suppressor role of the gene harboring such CFS.
- Published
- 2019
37. Matrin3 promotes homologous recombinational repair by regulation of RAD51
- Author
-
Atsuhiko Fukuto, Aiko Kinomura, Satoshi Tashiro, Jiying Sun, Lin Shi, and Yasunori Horikoshi
- Subjects
0303 health sciences ,DNA repair ,DNA damage ,RAD51 ,RNA ,General Medicine ,Biology ,Nuclear matrix ,Biochemistry ,Cell biology ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,chemistry ,030220 oncology & carcinogenesis ,Homologous chromosome ,Homologous recombination ,Molecular Biology ,DNA ,030304 developmental biology - Abstract
Matrin3 is a highly conserved inner nuclear matrix protein involved in multiple stages of RNA metabolism. Although Matrin3 may also play a role in DNA repair, its precise roles have remained unclear. In this study, we showed that the depletion of Matrin3 led to decreased homologous recombination (HR) efficiency and increased radiation sensitivity of cells. Matrin3-depleted cells showed impaired DNA damage-dependent focus formation of RAD51, a key protein in HR. These findings suggest that Matrin3 promotes HR by regulating RAD51.
- Published
- 2019
38. Localization of Arg-X proteolysis in the supramolecular structures of cell nuclei during the induction of growth in mature wheat germs
- Author
-
Ruslan Ivanov and Gulnar Hamidovna Vafina
- Subjects
0106 biological sciences ,0301 basic medicine ,Nucleoplasm ,biology ,medicine.diagnostic_test ,Proteolysis ,Cell ,Plant Science ,Cleavage (embryo) ,Nuclear matrix ,01 natural sciences ,Protamine ,Chromatin ,03 medical and health sciences ,030104 developmental biology ,medicine.anatomical_structure ,Histone ,Biochemistry ,medicine ,biology.protein ,Biophysics ,Agronomy and Crop Science ,010606 plant biology & botany - Abstract
Proteolysis is a form of the biological control providing fast physiological response to changing conditions of an environment. The goal of the present work was to analyze the localization of the Arg-X proteolysis in complexes of histones and non-histone proteins isolated from supra-molecular structures (nucleoplasm, chromatin, the nuclear matrix) of cell nuclei of mature wheat germs during induction of growth. Cell nuclei were isolated from germs, cleared, and then nucleoplasm, chromatin, nuclear matrix were extracted by increasing ionic strength of solution. From isolated supra-molecular structures, non-histone proteins were separated from histones by using ion exchange chromatography. The Arg-X proteolytic activity was assessed by cleavage of Arg-X bonds in the arginine-enriched protein protamine in all nuclear fractions. It was shown that in the coleoptile, which grows by cell elongation, Arg-X proteolysis activity was at the level of nuclear matrix structures, namely the linker histone HI and core histones, and in mesocotyls, Arg-X proteolysis activity was found in the core histones of the fraction of chromatin tightly bound to the nuclear matrix. A possible role of Arg-X proteolysis in the fine regulation of morphogenetic mechanisms occurring during seed germination is suggested.
- Published
- 2016
39. The Arabidopsis CROWDED NUCLEI genes regulate seed germination by modulating degradation of ABI5 protein
- Author
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Wenming Zhao, Ni Zhan, Chunmei Guan, Jianru Zuo, Yan Liang, Jian Feng, and Bo Ren
- Subjects
0301 basic medicine ,biology ,Wild type ,food and beverages ,Plant Science ,Protein degradation ,biology.organism_classification ,Nuclear matrix ,Biochemistry ,General Biochemistry, Genetics and Molecular Biology ,Cell biology ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,chemistry ,Seedling ,Germination ,Arabidopsis ,Botany ,Abscisic acid ,Transcription factor - Abstract
In Arabidopsis, the phytohormone abscisic acid (ABA) plays a vital role in inhibiting seed germination and in post-germination seedling establishment. In the ABA signaling pathway, ABI5, a basic Leu zipper transcription factor, has important functions in the regulation of seed germination. ABI5 protein localizes in nuclear bodies, along with AFP, COP1, and SIZ1, and was degraded through the 26S proteasome pathway. However, the mechanisms of ABI5 nuclear body formation and ABI5 protein degradation remain obscure. In this study, we found that the Arabidopsis CROWDED NUCLEI (CRWN) proteins, predicted nuclear matrix proteins essential for maintenance of nuclear morphology, also participate in ABA-controlled seed germination by regulating the degradation of ABI5 protein. During seed germination, the crwn mutants are hypersensitive to ABA and have higher levels of ABI5 protein compared to wild type. Genetic analysis suggested that CRWNs act upstream of ABI5. The observation that CRWN3 colocalizes with ABI5 in nuclear bodies indicates that CRWNs might participate in ABI5 protein degradation in nuclear bodies. Moreover, we revealed that the extreme C-terminal of CRWN3 protein is necessary for its function in the response to ABA in germination. Our results suggested important roles of CRWNs in ABI5 nuclear body organization and ABI5 protein degradation during seed germination.
- Published
- 2015
40. Tight DNA-protein complexes isolated from barley seedlings are rich in potential guanine quadruplex sequences
- Author
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Ilva Trapina, Tatjana Sjakste, Marion S. Röder, Elina Leonova, Rudolfs Petrovs, and Nikolajs Sjakste
- Subjects
Circular dichroism ,Guanine ,lcsh:Medicine ,Plant Science ,GC-rich DNA ,G-quadruplex ,Deproteinisation-resistant DNA-protein complexes ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,chemistry.chemical_compound ,Barley ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,Oligonucleotide ,Chemistry ,General Neuroscience ,lcsh:R ,030302 biochemistry & molecular biology ,Structural gene ,Cell Biology ,Genomics ,General Medicine ,Nuclear matrix ,G-quadruplexes ,Chromatin ,Biochemistry ,General Agricultural and Biological Sciences ,DNA - Abstract
Background The concept of chromatin domains attached to the nuclear matrix is being revisited, with nucleus described as a set of topologically associating domains. The significance of the tightly bound to DNA proteins (TBP), a protein group that remains attached to DNA after its deproteinization should be also revisited, as the existence of these interactions is in good agreement with the concept of the topologically associating domain. The work aimed to characterize the DNA component of TBP isolated from barley seedlings. Methods The tight DNA-protein complexes from the first leaves, coleoptiles, and roots of barley seedlings were isolated by purification with chromatography on nitrocellulose or exhaustive digestion of DNA with DNase I. Cloning and transformation were performed using pMOSBBlue Blunt Ended Cloning Kit. Inserts were amplified by PCR, and sequencing was performed on the MegaBace 1000 Sequencing System. The BLAST search was performed using sequence databases at NCBI, CR-EST, and TREP and Ensembl Plants databases. Comparison to MAR/SAR sequences was performed using http://smartdb.bioinf.med.uni-goettingen.de/cgi-bin/SMARtDB/smar.cgi database. The prediction of G quadruplexes (GQ) was performed with the aid of R-studio library pqsfinder. CD spectra were recorded on a Chirascan CS/3D spectrometer. Results Although the barley genome is AT-rich (43% of GC pairs), most DNA fragments associated with TBP were GC-rich (up to 70% in some fractions). Both fractionation procedures yielded a high proportion of CT-motif sequences presented predominantly by the 16-bp CC(TCTCCC)2 TC fragment present in clones derived from the TBP-bound DNA and absent in free DNA. BLAST analysis revealed alignment with different barley repeats. Some clones, however, aligned with both nuclear and chloroplast structural genes. Alignments with MAR/SAR motifs were very few. The analysis produced by the pqsfinder program revealed numerous potential quadruplex-forming sites in the TBP-bound sequences. A set of oligonucleotides containing sites of possible GQs were designed and ordered. Three of them represented the minus strand of the CT-repeat. Two were derived from sequences of two clones of nitrocellulose retained fraction from leaves and contained GC-rich motifs different from the CT motif. Circular dichroism spectroscopy revealed profound changes in spectra when oligonucleotides were incubated with 100 mM KCl. There was either an increase of positive band in the area of 260 nm or the formation of a positive band at 290 nm. In the former case, changes are typical for parallel G-quadruplexes and, in the latter, 3 + 1 structures. Discussion The G-quadruplexes anchor proteins are probably involved in the maintenance of the topologically associated domain structure.
- Published
- 2020
41. Differential Detergent Lysis of Cellular Fractions for Immunoprecipitation
- Author
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Thomas O. Kohl and James A. DeCaprio
- Subjects
0301 basic medicine ,Lysis ,Octoxynol ,Immunoprecipitation ,Detergents ,Digitonin ,Cell Fractionation ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,chemistry.chemical_compound ,Cytosol ,0302 clinical medicine ,Animals ,Humans ,Cells, Cultured ,Cellular compartment ,Cell Nucleus ,Nucleoplasm ,Chemistry ,Cell Membrane ,Extraction (chemistry) ,Membrane Proteins ,Nuclear Proteins ,Proteins ,Nuclear matrix ,030104 developmental biology ,Biochemistry ,Cytoplasm ,030215 immunology - Abstract
Differential detergent fractionation of cells is a rapid method for extraction of cytoplasmic and nuclear proteins in preparation of an immunoprecipitation. This method can be applied for use of adherent or suspension cells and can significantly reduce nonspecific background in an immunoprecipitation by separation of cellular compartments into individual fractions. The lysis of cells by differential detergents permits the rapid extraction of proteins from the cytoplasm (digitonin), the cytoplasmic membranes, and organelles (Triton X-100), and nucleoplasm (Tween/DOC), facilitated through the use of distinct extraction buffers. Cytoplasmic and nuclear matrix proteins as well as DNA are left behind during the detergent-based extraction.
- Published
- 2020
42. In vitro binding of nuclear proteins to the barley plastocyanin gene promoter region.
- Author
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Nielsen, Peter S. and Gausing, Kirsten
- Subjects
- *
NUCLEAR matrix , *BARLEY , *PLASTOCYANIN , *PROMOTERS (Genetics) , *PHOTOSYNTHESIS , *BIOCHEMISTRY - Abstract
Discusses the in vitro binding of nuclear proteins to the barley plastocyanin gene promoter region. Role of plastocyanin in photosynthesis; Mapping of DNA sites in the plastocyanin gene that mediate binding of barley nuclear proteins; Characteristics of three specific binding sites.
- Published
- 1993
- Full Text
- View/download PDF
43. The nucleoskeleton and the topology of transcription.
- Author
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Cook, Peter R.
- Subjects
- *
NUCLEAR matrix , *GENETIC transcription , *NONHISTONE chromosomal proteins , *CELL nuclei , *SALT , *BIOCHEMISTRY - Abstract
Transcription is conventionally believed to occur by passage of a mobile polymerase along a fixed template. Evidence for this model is derived almost entirely from material prepared using hypotonic salt concentrations. Studies on subnuclear structures isolated using hypertonic conditions, and more recently using conditions closer to the physiological, suggest an alternative. Transcription occurs as the template moves past a polymerase attached to a nucleoskeleton; this skeleton is the active site of transcription. Evidence for the two models is summarised. Much of it is consistent with the polymerase being attached and not freely diffusible. Some consequences of such a model are discussed. [ABSTRACT FROM AUTHOR]
- Published
- 1989
- Full Text
- View/download PDF
44. Modification of nuclear matrix proteins by ADP-ribosylation.
- Author
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Wesierska-Gadek, Józefa and Sauermann, Georg
- Subjects
- *
NUCLEAR matrix , *ADENOSINE diphosphate , *RIBOSE , *BIOCHEMISTRY , *BIOSYNTHESIS , *PROTEINS - Abstract
Nuclear matrices were isolated by treatment of isolated HeLa cell nuclei with high DNase I, pancreatic RNase and salt concentrations. ADP-ribosylated nuclear matrix proteins were identified by electrophoresis, blotting and autoradiography. In one experimental approach nuclear matrix proteins were labeled by exposure of permeabilized cells to the labeled precursor [32P]NAD. Alternatively, the cellular proteins were prelabeled with [35S]methionine and the ADP-ribosylated nuclear matrix proteins separated by aminophenyl boronate column chromatography. By both methods bands of modified proteins, though with differing intensities, were detected at 41, 43, 46, 51, 60, 64, 69, 73, 116, 140, 220 and 300 kDa. Approximately 2% of the total nuclear ADP-ribosyltransferase activity, but only 0.07% of the nuclear DNA, was tightly associated with the isolated nuclear matrix. The matrix-associated enzyme catalyzes the incorporation of [32P]ADP-ribose into acid-insoluble products of molecular mass 116 kDa and above, in a 3-aminobenzamide-inhibited, time-dependent reaction. The possible function of ADP-ribosylation of nuclear matrix proteins and of the attachment of ADP-ribosyltransferase to the nuclear matrix in the regulation of matrix-associated biochemical processes is discussed. [ABSTRACT FROM AUTHOR]
- Published
- 1985
- Full Text
- View/download PDF
45. Chinese Hamster Nuclear Proteins.
- Author
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Peters, Keith E., Okada, Tadashi A., and Comings, David E.
- Subjects
- *
HAMSTERS , *MURIDAE , *NUCLEAR matrix , *NONHISTONE chromosomal proteins , *CELL nuclei , *BIOCHEMISTRY , *MEDICAL sciences - Abstract
A comparison, by two-dimensional gel electrophoresis, of total interphase nuclear, metaphase chromosomal and nuclear matrix proteins from Chinese hamster V-79 cells was undertaken to examine the distribution of these proteins during mitosis. We have found a number of differences among these populations, although the two-dimensional gel patterns are generally similar. The most striking observation is that a loose cluster of six inter-phase nuclear polypeptides, with isoelectric points in urea between 5,7 and 6.7 and molecular masses ranging from 53 to 75 kDa, is greatly enriched in chromosome preparations. Each of these species is prominent also in the nuclear matrix. Preliminary evidence suggests that one of these polypeptides is the intermediate filament protein, vimentin. In addition, two major polypeptides of interphase nuclear preparations, a basic 94-kDa species and an approximately 65-kDa species, are absent from chromosomes. The latter polypeptide is the nuclear pore-lamina complex lamin B. Actin is present in all of these fractions, but tubulin has not been observed. hnRNP particle polypeptides are major components of the nuclear matrix, but are markedly reduced in metaphase chromosomes. The intermediate and basic 65 -75'kDa nuclear matrix polypeptides we have previously demonstrated to be major components of rat liver nuclear matrix, are reduced in Chinese hamster matrix preparations and at least one of these species, a minor, basic, 68-kDa polypeptide, is missing entirely from metaphase chromosomes. These results are discussed in relation to nuclear and chromosome structure and the possibility of contamination of nuclear protein preparations from cultured cell lines with intermediate filaments. [ABSTRACT FROM AUTHOR]
- Published
- 1982
- Full Text
- View/download PDF
46. The novel SAR-binding domain of scaffold attachment factor A (SAF-A) is a target in apoptotic nuclear breakdown.
- Author
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Göhring, Frank, Schwab, Birgit L., Nicotera, Pierluigi, Leist, Marcel, and Fackelmayer, Frank O.
- Subjects
- *
APOPTOSIS , *NUCLEAR matrix , *BIOCHEMISTRY , *PROTEOLYSIS , *PROTEIN metabolism , *MOLECULAR genetics - Abstract
The scaffold attachment factor A (SAF-A) is an abundant component of the nuclear scaffold and of chromatin, and also occurs in heterogeneous nuclear ribonucleoprotein (hnRNP) complexes. Evidence from previous experiments had suggested that SAF-A most likely has at least two different functions, being involved both in nuclear architecture and RNA metabolism. We now show that the protein has a novel scaffold-associated region (SAR)-specific bipartite DNA-binding domain which is independent from the previously identified RNA-binding domain, the RGG box. During apoptosis, but not during necrosis, SAF-A is cleaved in a caspase-dependent way. Cleavage occurs within the bipartite DNA-binding domain, resulting in a loss of DNA-binding activity and a concomitant detachment of SAF-A from nuclear structural sites. On the other hand, cleavage does not compromise the association of SAF-A with hnRNP complexes, indicating that the function of SAF-A in RNA metabolism is not affected in apoptosis. Our results suggest that detachment of SAF-A from SARs, caused by apoptotic proteolysis of its DNA-binding domain, is linked to the formation of oligonucleosomal-sized DNA fragments and could therefore contribute to nuclear breakdown in apoptotic cells. [ABSTRACT FROM AUTHOR]
- Published
- 1997
- Full Text
- View/download PDF
47. Nonhistone Proteins and Nuclear Matrix Structures
- Author
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A. Oscar Pogo
- Subjects
Non-histone protein ,Biochemistry ,Chemistry ,Nuclear matrix - Published
- 2018
48. Advances in the computational and molecular understanding of the prostate cancer cell nucleus
- Author
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Neil M. Carleton, George Lee, Robert W. Veltri, and Anant Madabhushi
- Subjects
0301 basic medicine ,Biochemical recurrence ,Male ,Cell Nucleus Shape ,Computer science ,Prostate cancer cell ,Context (language use) ,Computational biology ,Biochemistry ,Genomic Instability ,Article ,Epigenesis, Genetic ,Machine Learning ,03 medical and health sciences ,Prostate cancer ,0302 clinical medicine ,Image Interpretation, Computer-Assisted ,medicine ,Tumor Microenvironment ,Humans ,Molecular Biology ,Prostatic Neoplasms ,Cell Biology ,Nuclear matrix ,medicine.disease ,Prognosis ,Chromatin ,Cell nucleus ,030104 developmental biology ,medicine.anatomical_structure ,Cell Transformation, Neoplastic ,030220 oncology & carcinogenesis ,Cell Nucleus Size ,Identification (biology) ,Nucleus - Abstract
Nuclear alterations are a hallmark of many types of cancers, including prostate cancer (PCa). Recent evidence shows that subvisual changes, ones that may not be visually perceptible to a pathologist, to the nucleus and its ultrastructural components can precede visual histopathological recognition of cancer. Alterations to nuclear features such as nuclear size and shape, texture, and spatial architecture reflect the complex molecular level changes that occur during oncogenesis. Quantitative nuclear morphometry, a field that uses computational approaches to identify and quantify malignancy-induced nuclear changes, can enable a detailed and objective analysis of the prostate cancer cell nucleus. Recent advances in machine learning-based approaches can now automatically mine data related to these changes to aid in diagnostic decision-making and prediction of PCa prognoses. In this review, we use prostate cancer as a case study to connect the molecular level mechanisms that underlie these nuclear changes to the machine learning computational approaches, bridging the gap between the clinical and computational understanding of PCa. First, we discuss recent developments to our understanding of molecular events that drive nuclear alterations in the context of prostate cancer: the role of the nuclear matrix and lamina in size and shape changes, the role of three-dimensional chromatin organization and epigenetic modifications in textural changes, and role the tumor microenvironment in altering nuclear spatial topology. We then discuss the advances in the applications of machine learning algorithms to automatically segment nuclei in prostate histopathological images, extract nuclear features to aid in diagnostic decision-making, and predict potential outcomes such as biochemical recurrence and survival. Lastly, we discuss the challenges and opportunities associated with translation of the quantitative nuclear morphometry methodology into the clinical space. Ultimately, accurate identification and quantification of nuclear alterations can contribute to the field of nucleomics and has applications for computationally driven precision oncologic patient care.
- Published
- 2018
49. Promyelocytic Leukemia Protein (PML) Requirement for Interferon-induced Global Cellular SUMOylation
- Author
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Mohamed Ali Maroui, Frédéric Lamoliatte, Francis P. McManus, Pierre Thibault, Ghizlane Maarifi, and Mounira K. Chelbi-Alix
- Subjects
0301 basic medicine ,Gene isoform ,viruses ,SUMO protein ,Promyelocytic Leukemia Protein ,Proteomics ,Biochemistry ,Analytical Chemistry ,03 medical and health sciences ,Promyelocytic leukemia protein ,0302 clinical medicine ,Interferon ,medicine ,Humans ,Molecular Biology ,biology ,Chemistry ,Research ,Alternative splicing ,virus diseases ,Interferon-alpha ,Sumoylation ,Nuclear matrix ,Ubiquitin ligase ,Cell biology ,030104 developmental biology ,HEK293 Cells ,030220 oncology & carcinogenesis ,biology.protein ,medicine.drug - Abstract
We report that interferon (IFN) α treatment at short and long periods increases the global cellular SUMOylation and requires the presence of the SUMO E3 ligase promyelocytic leukemia protein (PML), the organizer of PML nuclear bodies (NBs). Several PML isoforms (PMLI-PMLVII) derived from a single PML gene by alternative splicing, share the same N-terminal region but differ in their C-terminal sequences. Introducing each of the human PML isoform in PML-negative cells revealed that enhanced SUMOylation in response to IFN is orchestrated by PMLIII and PMLIV. Large-scale proteomics experiments enabled the identification of 558 SUMO sites on 389 proteins, of which 172 sites showed differential regulation upon IFNα stimulation, including K49 from UBC9, the sole SUMO E2 protein. Furthermore, IFNα induces PML-dependent UBC9 transfer to the nuclear matrix where it colocalizes with PML within the NBs and enhances cellular SUMOylation levels. Our results demonstrate that SUMOylated UBC9 and PML are key players for IFN-increased cellular SUMOylation.
- Published
- 2018
50. Computational interaction study of scaffold/ matrix attachment regions (S/MARS) with nuclear matrix Protein in human chromosome 3 Contactin 4(CNTN4) gene
- Author
-
Suresh Kumar
- Subjects
Scaffold ,Chromosome 3 ,Chemistry ,Cell Biology ,Mars Exploration Program ,Nuclear matrix ,Scaffold/matrix attachment region ,Molecular Biology ,Biochemistry ,Gene ,Biotechnology ,Cell biology - Published
- 2018
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