Showing papers in "Nucleus in 2018"
TL;DR: It is concluded that in aggregate the available evidence argues against stable loops and supports a model where TADs are dynamic structures that continually form and break throughout the cell cycle.
Abstract: Mammalian genomes are folded into spatial domains, which regulate gene expression by modulating enhancer-promoter contacts. Here, we review recent studies on the structure and function of Topologically Associating Domains (TADs) and chromatin loops. We discuss how loop extrusion models can explain TAD formation and evidence that TADs are formed by the ring-shaped protein complex, cohesin, and that TAD boundaries are established by the DNA-binding protein, CTCF. We discuss our recent genomic, biochemical and single-molecule imaging studies on CTCF and cohesin, which suggest that TADs and chromatin loops are dynamic structures. We highlight complementary polymer simulation studies and Hi-C studies employing acute depletion of CTCF and cohesin, which also support such a dynamic model. We discuss the limitations of each approach and conclude that in aggregate the available evidence argues against stable loops and supports a model where TADs are dynamic structures that continually form and break throughout the cell cycle.
188 citations
TL;DR: HGPS cells and animal preclinical models have provided insights into the molecular and cellular pathways that underlie the disease and have also highlighted possible mechanisms involved in normal aging.
Abstract: Hutchinson-Gilford progeria syndrome (HGPS) is a sporadic, autosomal dominant disorder characterized by premature and accelerated aging symptoms leading to death at the mean age of 14.6 years usual...
62 citations
TL;DR: Parts of nuclear lamin association with the genome are reviewed to argue towards a deregulation of large-scale and local spatial genome organization by a subset of lamin A mutations causing laminopathies.
Abstract: The nuclear lamina contributes to the regulation of gene expression and to chromatin organization. Mutations in A-type nuclear lamins cause laminopathies, some of which are associated with a loss of heterochromatin at the nuclear periphery. Until recently however, little if any information has been provided on where and how lamin A interacts with the genome and on how disease-causing lamin A mutations may rearrange genome conformation. Here, we review aspects of nuclear lamin association with the genome. We highlight recent evidence of reorganization of lamin A-chromatin interactions in cellular models of laminopathies, and implications on the 3-dimensional rearrangement of chromatin in these models, including patient cells. We discuss how a hot-spot lipodystrophic lamin A mutation alters chromatin conformation and epigenetic patterns at an anti-adipogenic locus, and conclude with remarks on links between lamin A, Polycomb and the pathophysiology of laminopathies. The recent findings presented here collectively argue towards a deregulation of large-scale and local spatial genome organization by a subset of lamin A mutations causing laminopathies.
46 citations
TL;DR: The function of NPCs as genome organizers and hubs for transcriptional regulation provides additional evidence that the compartmentalization of genes and transcriptional regulators within the nuclear space is an important mechanism of gene expression regulation.
Abstract: Nuclear pore complexes (NPCs), the channels connecting the nucleus with the cytoplasm, are the largest protein structures of the nuclear envelope. In addition to their role in regulating nucleocytoplasmic transport, increasing evidence shows that these multiprotein structures play central roles in the regulation of gene activity. In light of recent discoveries, NPCs are emerging as scaffolds that mediate the regulation of specific gene sets at the nuclear periphery. The function of NPCs as genome organizers and hubs for transcriptional regulation provides additional evidence that the compartmentalization of genes and transcriptional regulators within the nuclear space is an important mechanism of gene expression regulation.
43 citations
TL;DR: Recent studies showing that lamins play a role in DNA replication are summarized, and the emerging model that DNA damage and replication stress are “sensed” at the cytoplasm by proteins that normally survey this space in search of foreign nucleic acids is discussed.
Abstract: Mammalian nuclei are equipped with a framework of intermediate filaments that function as a karyoskeleton. This nuclear scaffold, formed primarily by lamins (A-type and B-type), maintains the spatial and functional organization of the genome and of sub-nuclear compartments. Over the past decade, a body of evidence has highlighted the significance of these structural nuclear proteins in the maintenance of nuclear architecture and mechanical stability, as well as genome function and integrity. The importance of these structures is now unquestioned given the wide range of degenerative diseases that stem from LMNA gene mutations, including muscular dystrophy disorders, peripheral neuropathies, lipodystrophies, and premature aging syndromes. Here, we review our knowledge about how alterations in nuclear lamins, either by mutation or reduced expression, impact cellular mechanisms that maintain genome integrity. Despite the fact that DNA replication is the major source of DNA damage and genomic instability in dividing cells, how alterations in lamins function impact replication remains minimally explored. We summarize recent studies showing that lamins play a role in DNA replication, and that the DNA damage that accumulates upon lamins dysfunction is elicited in part by deprotection of replication forks. We also discuss the emerging model that DNA damage and replication stress are "sensed" at the cytoplasm by proteins that normally survey this space in search of foreign nucleic acids. In turn, these cytosolic sensors activate innate immune responses, which are materializing as important players in aging and cancer, as well as in the response to cancer immunotherapy.
42 citations
TL;DR: It is demonstrated that chromatin and the lamina provide distinct mechanical contributions to nuclear mechanical response, and several new exciting possibilities for understanding nuclear morphology, organization, and mechanics are suggested.
Abstract: The cell nucleus houses, protects, and arranges the genome within the cell. Therefore, nuclear mechanics and morphology are important for dictating gene regulation, and these properties are perturbed in many human diseases, such as cancers and progerias. The field of nuclear mechanics has long been dominated by studies of the nuclear lamina, the intermediate filament shell residing just beneath the nuclear membrane. However, a growing body of work shows that chromatin and chromatin-related factors within the nucleus are an essential part of the mechanical response of the cell nucleus to forces. Recently, our group demonstrated that chromatin and the lamina provide distinct mechanical contributions to nuclear mechanical response. The lamina is indeed important for robust response to large, whole-nucleus stresses, but chromatin dominates the short-extension response. These findings offer a clarifying perspective on varied nuclear mechanics measurements and observations, and they suggest several new exciting possibilities for understanding nuclear morphology, organization, and mechanics.
40 citations
TL;DR: The phenotypes generated by mutations affecting genes directly acting on DNA function, as mutations in the helicases WRN and BLM or in the polymerase polδ, share many of the traits of progeroid laminopathies and support the hypothesis of a concerted implication of DNA function and lamins in aging.
Abstract: Progeroid syndromes induced by mutations in lamin A or in its interactors - named progeroid laminopathies - are model systems for the dissection of the molecular pathways causing physiological and premature aging. A large amount of data, based mainly on the Hutchinson Gilford Progeria syndrome (HGPS), one of the best characterized progeroid laminopathy, has highlighted the role of lamins in multiple DNA activities, including replication, repair, chromatin organization and telomere function. On the other hand, the phenotypes generated by mutations affecting genes directly acting on DNA function, as mutations in the helicases WRN and BLM or in the polymerase polδ, share many of the traits of progeroid laminopathies. These evidences support the hypothesis of a concerted implication of DNA function and lamins in aging. We focus here on these aspects to contribute to the comprehension of the driving forces acting in progeroid syndromes and premature aging.
40 citations
TL;DR: It is suggested that there is no motor, and thermal motion within the nucleus drives extrusion, and diffusive extrusion can in principle generate loops of the size observed in the data.
Abstract: Chromatin loop extrusion is a popular model for the formation of CTCF loops and topological domains. Recent HiC data have revealed a strong bias in favour of a particular arrangement of the CTCF bi...
36 citations
TL;DR: Clinical aspects of EDMD in context to causative genes, genotype-phenotype correlation and its emplacement within phenotypic spectrum of skeletal muscle diseases associated with envelopathies are presented.
Abstract: Emery-Dreifuss muscular dystrophy (EDMD), clinically characterized by scapulo-humero-peroneal muscle atrophy and weakness, multi-joint contractures with spine rigidity and cardiomyopathy with conduction defects, is associated with structural/functional defect of genes that encode the proteins of nuclear envelope, including lamin A and several lamin-interacting proteins. This paper presents clinical aspects of EDMD in context to causative genes, genotype-phenotype correlation and its emplacement within phenotypic spectrum of skeletal muscle diseases associated with envelopathies.
33 citations
TL;DR: The goal of this manuscript is to combine the most recent evidences in an updated review of cardiac laminopathies associated with mutations in the LMNA gene to show the state-of-the-art of such a complex disease group.
Abstract: Cardiac laminopathies, associated with mutations in the LMNA gene, encompass a wide spectrum of clinical manifestations, involving electrical and mechanical alterations of cardiomyocytes. Thus, dilated cardiomyopathy, bradyarrhythmias and atrial or ventricular tachyarrhythmias may occur in a number of combined phenotypes. Nowadays, some attempt has been made to identify clinical predictors for the most life-threatening complications of LMNA-associated heart disease, i.e. sudden cardiac death and end-stage heart failure. The goal of this manuscript is to combine the most recent evidences in an updated review to show the state-of-the-art of such a complex disease group. This is supposed to be the starting point to collect more data and design new ad hoc studies to identify clinically useful predictors to stratify risk in mutation carriers, including probands and their asymptomatic relatives.
33 citations
TL;DR: The evidence on how the stem cell nucleus responds and adapts to physical forces is reviewed, and a perspective on how this mechano-adaptation may function to drive and enforce stem cell differentiation is provided.
Abstract: Exogenous mechanical forces are transmitted through the cell and to the nucleus, initiating mechanotransductive signaling cascades with profound effects on cellular function and stem cell fate. A growing body of evidence has shown that the force sensing and force-responsive elements of the nucleus adapt to these mechanotransductive events, tuning their response to future mechanical input. The mechanisms underlying this "mechano-adaptation" are only just beginning to be elucidated, and it remains poorly understood how these components act and adapt in tandem to drive stem cell differentiation. Here, we review the evidence on how the stem cell nucleus responds and adapts to physical forces, and provide a perspective on how this mechano-adaptation may function to drive and enforce stem cell differentiation.
TL;DR: It is suggested that these previously overlooked classes of CTCF binding regions may have different roles in regulating diverse chromatin-based phenomena, and may impact the understanding of heritable epigenetic regulation in cancer cells and normal germ cells.
Abstract: CCCTC-binding factor (CTCF) is a conserved, essential regulator of chromatin architecture containing a unique array of 11 zinc fingers (ZFs). Gene duplication and sequence divergence during early amniote evolution generated the CTCF paralog Brother Of the Regulator of Imprinted Sites (BORIS), which has a DNA binding specificity identical to that of CTCF but divergent N- and C-termini. While healthy somatic tissues express only CTCF, CTCF and BORIS are normally co-expressed in meiotic and post-meiotic germ cells, and aberrant activation of BORIS occurs in tumors and some cancer cell lines. This has led to a model in which CTCF and BORIS compete for binding to some but not all genomic target sites; however, regulation of CTCF and BORIS genomic co-occupancy is not well understood. We recently addressed this issue, finding evidence for two major classes of CTCF target sequences, some of which contain single CTCF target sites (1xCTSes) and others containing two adjacent CTCF motifs (2xCTSes). The functional and chromatin structural features of 2xCTSes are distinct from those of 1xCTS-containing regions bound by a CTCF monomer. We suggest that these previously overlooked classes of CTCF binding regions may have different roles in regulating diverse chromatin-based phenomena, and may impact our understanding of heritable epigenetic regulation in cancer cells and normal germ cells.
TL;DR: Fine-excision alignment mass spectrometry (FEA-MS) is developed to quantify progerin and its phosphorylation levels in patient iPS cells differentiated to mesenchymal stem cells (MSCs), suggesting a differential mechano-responsiveness might best explain the stiff tissue defects in Progeria.
Abstract: Interphase phosphorylation of lamin-A,C depends dynamically on a cell's microenvironment, including the stiffness of extracellular matrix. However, phosphorylation dynamics is poorly understood for...
Journal Article•
TL;DR: Having encouraged by the linguistic term in decision models, a method of multi attribute group decision making is proposed that amalgamates the idea of picture fuzzy sets and linguistic term sets to discourse the situations where the real-life problems fail to express in numerical form.
Abstract: Having encouraged by the linguistic term in decision models, it is proposed a method of multi attribute group decision making. This amalgamates the idea of picture fuzzy sets and linguistic term sets to discourse the situations where the real-life problems fail to express in numerical form. Firstly, it is introduced the concept of picture fuzzy linguistic number and comparison rules for ranking the alternatives are discussed. Further the aggregation operators based on picture fuzzy linguistic information are introduced. Finally, it is introduced a technique to obtain satisfactory results about real-life complex problems, and it is given a descriptive example to discuss the reliability and effectiveness of the suggested technique by using group decision criteria. Subject classification: 03E72
TL;DR: Data show that TGF β2 contributes to the pathogenesis of Emery-Dreifuss Muscular Dystrophy type 2 and Limb-Girdle muscular dystrophy 1B and can be considered a potential biomarker of those diseases.
Abstract: Among rare diseases caused by mutations in LMNA gene, Emery-Dreifuss Muscular Dystrophy type 2 and Limb-Girdle muscular Dystrophy 1B are characterized by muscle weakness and wasting, joint contractures, cardiomyopathy with conduction system disorders. Circulating biomarkers for these pathologies have not been identified. Here, we analyzed the secretome of a cohort of patients affected by these muscular laminopathies in the attempt to identify a common signature. Multiplex cytokine assay showed that transforming growth factor beta 2 (TGF β2) and interleukin 17 serum levels are consistently elevated in the vast majority of examined patients, while interleukin 6 and basic fibroblast growth factor are altered in subgroups of patients. Levels of TGF β2 are also increased in fibroblast and myoblast cultures established from patient biopsies as well as in serum from mice bearing the H222P Lmna mutation causing Emery-Dreifuss Muscular Dystrophy in humans. Both patient serum and fibroblast conditioned media activated a TGF β2-dependent fibrogenic program in normal human myoblasts and tenocytes and inhibited myoblast differentiation. Consistent with these results, a TGF β2 neutralizing antibody avoided fibrogenic marker activation and myogenesis impairment. Cell intrinsic TGF β2-dependent mechanisms were also determined in laminopathic cells, where TGF β2 activated AKT/mTOR phosphorylation. These data show that TGF β2 contributes to the pathogenesis of Emery-Dreifuss Muscular Dystrophy type 2 and Limb-Girdle muscular Dystrophy 1B and can be considered a potential biomarker of those diseases. Further, the evidence of TGF β2 pathogenetic effects in tenocytes provides the first mechanistic insight into occurrence of joint contractures in muscular laminopathies.
TL;DR: Novel cellular models recapitulating adipogenic developmental pathways provide scalable tools for disease modeling and therapeutic screening and could initiate metabolic inflexibility of adipose tissue extracellular matrix and TGF-beta signaling.
Abstract: Mutations in LMNA, encoding A-type lamins, are responsible for laminopathies including muscular dystrophies, lipodystrophies, and premature ageing syndromes. LMNA mutations have been shown to alter nuclear structure and stiffness, binding to partners at the nuclear envelope or within the nucleoplasm, gene expression and/or prelamin A maturation. LMNA-associated lipodystrophic features, combining generalized or partial fat atrophy and metabolic alterations associated with insulin resistance, could result from altered adipocyte differentiation or from altered fat structure. Recent studies shed some light on how pathogenic A-type lamin variants could trigger lipodystrophy, metabolic complications, and precocious cardiovascular events. Alterations in adipose tissue extracellular matrix and TGF-beta signaling could initiate metabolic inflexibility. Premature senescence of vascular cells could contribute to cardiovascular complications. In affected families, metabolic alterations occur at an earlier age across generations, which could result from epigenetic deregulation induced by LMNA mutations. Novel cellular models recapitulating adipogenic developmental pathways provide scalable tools for disease modeling and therapeutic screening.
TL;DR: The main clinical features of these syndromes, their associated comorbidities and the clues for the differential diagnosis with other lipodystrophic disorders are summarized.
Abstract: The nuclear lamina is a complex reticular structure that covers the inner face of the nucleus membrane in metazoan cells. It is mainly formed by intermediate filaments called lamins, and exerts essential functions to maintain the cellular viability. Lamin A/C provides mechanical steadiness to the nucleus and regulates genetic machinery. Laminopathies are tissue-specific or systemic disorders caused by variants in LMNA gene (primary laminopathies) or in other genes encoding proteins which are playing some role in prelamin A maturation or in lamin A/C function (secondary laminopathies). Those disorders in which adipose tissue is affected are called laminopathic lipodystrophies and include type 2 familial partial lipodystrophy and certain premature aging syndromes. This work summarizes the main clinical features of these syndromes, their associated comorbidities and the clues for the differential diagnosis with other lipodystrophic disorders.
TL;DR: The current understanding of nuclear removal mechanisms in these three cell types are discussed and how recent studies into nuclear degradation in keratinocytes could contribute to a wider understanding of these molecular mechanisms in both health and pathology are expanded upon.
Abstract: Eukaryotic nuclei are essential organelles, storing the majority of the cellular DNA, comprising the site of most DNA and RNA synthesis, controlling gene expression and therefore regulating cellular function. The majority of mammalian cells retain their nucleus throughout their lifetime, however, in three mammalian tissues the nucleus is entirely removed and its removal is essential for cell function. Lens fibre cells, erythroblasts and epidermal keratinocytes all lose their nucleus in the terminal differentiation pathways of these cell types. However, relatively little is known about the pathways that lead to complete nuclear removal and about how these pathways are regulated. In this review, we aim to discuss the current understanding of nuclear removal mechanisms in these three cell types and expand upon how recent studies into nuclear degradation in keratinocytes, an easily accessible experimental model, could contribute to a wider understanding of these molecular mechanisms in both health and pathology.
TL;DR: The calculation of genome structures provides a useful tool for assessing the quality of single-cell Hi-C data because it requires a self-consistent network of interactions, relating to the underlying 3D conformation, with few errors, as well as sufficient longer-range cis- and trans-chromosomal contacts.
Abstract: Single-cell chromosome conformation capture approaches are revealing the extent of cell-to-cell variability in the organization and packaging of genomes. These single-cell methods, unlike their mul...
TL;DR: It is shown that the PIAS4-mediated SUMOylation regulates the exchange of H2A.Z-2 at DNA damage sites, and the SUMoylation of H 2A.
Abstract: Histone exchange and histone post-translational modifications play important roles in the regulation of DNA metabolism, by re-organizing the chromatin configuration. We previously demonstrated that the histone variant H2A.Z-2 is rapidly exchanged at damaged sites after DNA double strand break induction in human cells. In yeast, the small ubiquitin-like modifier (SUMO) modification of H2A.Z is involved in the DNA damage response. However, whether the SUMO modification regulates the exchange of human H2A.Z-2 at DNA damage sites remains unclear. Here, we show that H2A.Z-2 is SUMOylated in a damage-dependent manner, and the SUMOylation of H2A.Z-2 is suppressed by the depletion of the SUMO E3 ligase, PIAS4. Moreover, PIAS4 depletion represses the incorporation and eviction of H2A.Z-2 at damaged sites. These findings demonstrate that the PIAS4-mediated SUMOylation regulates the exchange of H2A.Z-2 at DNA damage sites.
Journal Article•
TL;DR: The basic set theoretic operations of BPVHFSs are defined and their related results are studied and their fitness is verified using principle of mathematical induction.
Abstract: In this manuscript, a novel structure of bipolar-valued hesitant fuzzy set (BPVHFS) is proposed as a generalization of fuzzy set (FS). The basic set theoretic operations of BPVHFSs are defined and their related results are studied. Based on the basic operations, some aggregation operators for BPVHFSs are developed and their fitness is verified using principle of mathematical induction. The multiattribute decision making (MADM) is established in the framework of BPVHFSs and a numerical example is provided to illustrate the process. The article ended with some concluding remarks along with some future directions.
TL;DR: A state-of-the-art portrait of the mRNA quality control research is presented and the current hypotheses proposed for dynamics of the cooperation between the involved components and how it leads to their shared goal: mRNAquality control prior to export into the cytoplasm are discussed.
Abstract: Despite extensive research on how mRNAs are quality controlled prior to export into the cytoplasm, the exact underlying mechanisms are still under debate. Specifically, it is unclear how quality control proteins at the entry of the nuclear pore complex (NPC) distinguish normal and aberrant mRNAs. While some of the involved components are suggested to act as switches and recruit different factors to normal versus aberrant mRNAs, some experimental and computational evidence suggests that the combined effect of the regulated stochastic interactions between the involved components could potentially achieve an efficient quality control of mRNAs. In this review, we present a state-of-the-art portrait of the mRNA quality control research and discuss the current hypotheses proposed for dynamics of the cooperation between the involved components and how it leads to their shared goal: mRNA quality control prior to export into the cytoplasm.
TL;DR: This review focuses on mammalian K DM2A and KDM2B, the only two lysine demethylases whose genes have been described to produce also an alternative isoform lacking the N-terminal demethylase domain, which might represent a more common evolutionarily conserved regulatory mechanism.
Abstract: Aberrant levels of histone modifications lead to chromatin malfunctioning and consequently to various developmental defects and human diseases. Therefore, the proteins bearing the ability to modify histones have been extensively studied and the molecular mechanisms of their action are now fairly well understood. However, little attention has been paid to naturally occurring alternative isoforms of chromatin modifying proteins and to their biological roles. In this review, we focus on mammalian KDM2A and KDM2B, the only two lysine demethylases whose genes have been described to produce also an alternative isoform lacking the N-terminal demethylase domain. These short KDM2A/B-SF isoforms arise through alternative promoter usage and seem to play important roles in development and disease. We hypothesise about the biological significance of these alternative isoforms, which might represent a more common evolutionarily conserved regulatory mechanism.
TL;DR: An improved knowledge of the complex pathophysiological pathways involved in cardiolaminopathies and in the determinants of their progression to more severe forms will help to improve clinical management and to better target pharmacological and non-pharmacological treatments.
Abstract: Lamin A/C gene mutations can be associated with cardiac diseases, usually referred to as ‘cardiolaminopathies’ characterized by arrhythmic disorders and/or left ventricular or biventricular dysfunc...
TL;DR: This work highlights work that addresses how the dynamic organization of the chromatin fiber affects the repair of damaged DNA and how, conversely, DNA damage and repair affect the structure and dynamics of chromatin in the budding yeast nucleus.
Abstract: Maintaining the integrity of the genome in the face of DNA damage is crucial to ensure the survival of the cell and normal development. DNA lesions and repair occur in the context of the chromatin fiber, whose 3D organization and movements in the restricted volume of the nucleus are under intense scrutiny. Here, we highlight work from our and other labs that addresses how the dynamic organization of the chromatin fiber affects the repair of damaged DNA and how, conversely, DNA damage and repair affect the structure and dynamics of chromatin in the budding yeast nucleus.
TL;DR: Genotypes such as KL-257, KL-236, Surbhi, Bahgsu and Himani can be used as diverse parents in breeding programmes for generating improved lines in context to oil content and can be use in future breeding and improvement programmes in this crop.
Abstract: Flax is an important commercial crop yielding oil, fibre and medicinal products. Present study reports the genetic diversity analysis in a collection of 28 genotypes of flax, including 9 exotic lines. Randomly amplified polymorphic DNA and inter-simple sequence repeat markers were used to study the genetic diversity. The characterized genotypes showed high level of genetic variation with respect to oil and fibre contents of genotypes. Altogether, 27 markers amplified 130 fragments with an average of 4.8 fragments and mean polymorphism information content and marker index values of 0.385 and 1.90 respectively. Jaccards coefficient based dendrogram grouped all genotypes into three groups and separated genotypes based on their oil and fibre contents. STRUCTURE analysis showed two genetic stocks for all the analyzed genotypes and recorded higher admixture in indigenous germplasm. Overall, the results showed that the characterized genotypes were highly diverse genetically and can be used in future breeding and improvement programmes in this crop. Specifically, genotypes such as KL-257, KL-236, Surbhi, Bahgsu and Himani can be used as diverse parents in breeding programmes for generating improved lines in context to oil content.
TL;DR: Live imaging and Fluorescence Recovery After Photobleaching of labelled H2B, showed that the depletion of Lamin B1, Fibrillarin (FBL) or Nucleostemin (GNL3), enhances H 2B-ECFP mobility in the nucleolus.
Abstract: Eukaryotic cells have 2 to 3 discrete nucleoli required for ribosome synthesis. Nucleoli are phase separated nuclear sub-organelles. Here we examined the role of nuclear Lamins and nucleolar factors in modulating the compartmentalization and dynamics of histone 2B (H2B-ECFP) in the nucleolus. Live imaging and Fluorescence Recovery After Photobleaching (FRAP) of labelled H2B, showed that the depletion of Lamin B1, Fibrillarin (FBL) or Nucleostemin (GNL3), enhances H2B-ECFP mobility in the nucleolus. Furthermore, Nucleolin knockdown significantly decreases H2B-ECFP compartmentalization in the nucleolus, while H2B-ECFP residence and mobility in the nucleolus was prolonged upon Nucleolin overexpression. Co-expression of N-terminal and RNA binding domain (RBD) deletion mutants of Nucleolin or inhibiting 45S rRNA synthesis reduces the sequestration of H2B-ECFP in the nucleolus. Taken together, these studies reveal a crucial role of Nucleolin-rRNA complex in modulating the compartmentalization, stability and dynamics of H2B within the nucleolus.
TL;DR: An increase in nuclear localization of MVI upon cell stimulation, and identification of potential nuclear localization (NLS) and nuclear export (NES) signals within MVI heavy chain are shown to be functional as the MVI nuclear presence was affected by the inhibitors of nuclear import and export.
Abstract: Myosin VI (MVI) is a unique actin-based motor protein moving towards the minus end of actin filaments, in the opposite direction than other known myosins. Besides well described functions of MVI in endocytosis and maintenance of Golgi apparatus, there are few reports showing its involvement in transcription. We previously demonstrated that in neurosecretory PC12 cells MVI was present in the cytoplasm and nucleus, and its depletion caused substantial inhibition of cell migration and proliferation. Here, we show an increase in nuclear localization of MVI upon cell stimulation, and identification of potential nuclear localization (NLS) and nuclear export (NES) signals within MVI heavy chain. These signals seem to be functional as the MVI nuclear presence was affected by the inhibitors of nuclear import (ivermectin) and export (leptomycin B). In nuclei of stimulated cells, MVI colocalized with active RNA polymerase II, BrUTP-containing transcription sites and transcription factor SP1 as well as SC35 and PML proteins, markers of nuclear speckles and PML bodies, respectively. Mass spectrometry analysis of samples of a GST-pull-down assay with the MVI tail domain as a "bait" identified several new potential MVI binding partners. Among them are proteins involved in transcription and post-transcriptional processes. We confirmed interaction of MVI with heterogeneous nuclear ribonucleoprotein U (hnRNPU) and nucleolin, proteins involved in pre-mRNA binding and transport, and nucleolar function, respectively. Our data provide an insight into mechanisms of involvement of MVI in nuclear processes via interaction with nuclear proteins and support a notion for important role(s) for MVI in gene expression.
TL;DR: It is proposed that Nup98 functions to regulate DHX9 activity within the nucleoplasm, which may represent a paradigm for understanding how N up98, and possibly other FG-Nup proteins, could direct the diverse cellular activities of multiple DBPs.
Abstract: The nucleoporin Nup98 has been linked to the regulation of transcription and RNA metabolism, 1-3 but the mechanisms by which Nup98 contributes to these processes remains largely undefined. Recently, we uncovered interactions between Nup98 and several DExH/D-box proteins (DBPs), a protein family well-known for modulating gene expression and RNA metabolism. 4-6 Analysis of Nup98 and one of these DBPs, DHX9, showed that they directly interact, their association is facilitated by RNA, and Nup98 binding stimulates DHX9 ATPase activity. 7 Furthermore, these proteins were dependent on one another for their proper association with a subset of gene loci to control transcription and modulate mRNA splicing. 7 On the basis of these observations, we proposed that Nup98 functions to regulate DHX9 activity within the nucleoplasm. 7 Since Nup98 is associated with several DBPs, regulation of DHX9 by Nup98 may represent a paradigm for understanding how Nup98, and possibly other FG-Nup proteins, could direct the diverse cellular activities of multiple DBPs.
TL;DR: The results recognize innate immunity as a player in LMNA-RM pathogenesis and Modulation of TLR7/9 signaling pathways and decrease of macrophage-mediated inflammation might be potential therapeutic strategies in LM NA-RM management.
Abstract: Laminopathies are a heterogeneous group of diseases, caused by mutations in lamin A/C proteins. The most common laminopathy (LMNA-related myopathies, LMNA-RM) affects skeletal and cardiac muscles; muscle histopathology is variable, ranging from mild unspecific changes to dystrophic features, sometimes with inflammatory evidence. Whether the genetic defect might activate innate immune components, leading to chronic inflammation, myofiber necrosis and fibrosis, is still unknown. By qPCR, a significant up-regulation of Toll-like receptor (TLR) 7 and 9 transcripts was found in LMNA-RM compared to other myopathic and non-myopathic muscles. A marked TLR7/9 staining was observed on LMNA-RM blood vessels and muscle fibers and, when present, on infiltrating cells, mainly macrophages, scattered in the tissue or localized close to degenerated muscle fibers and connective tissue. Our results recognize innate immunity as a player in LMNA-RM pathogenesis. Modulation of TLR7/9 signaling pathways and decrease of macrophage-mediated inflammation might be potential therapeutic strategies in LMNA-RM management. Abbreviations DMD, Duchenne muscular dystrophy; EDMD2, Emery-Dreifuss muscular dystrophy type 2; FSHD, facio-scapulo-humeral muscular dystrophy; LGMD1B, limb-girdle muscular dystrophy type 1B; LMNA-CMD, LMNA-related congenital muscular dystrophy; LMNA-RM, LMNA-related myopathies; sIBM, sporadic inclusion body myositis; TLR, Toll-like receptor.