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Online ISSN: 1949-1042
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Aims and scope

Focuses on cell biology and nucleus structure, transport and dynamics, chromatin organization, subcellular organelles, DNA damage repair, RNA processing.

Recent publications
This paper provides a laboratory workflow for single-nucleus RNA-sequencing (snRNA-seq) including a protocol for gentle nuclei isolation from fresh frozen tumor biopsies, making it possible to analyze biobanked material. To develop this protocol, we used non-frozen and frozen human bladder tumors and cell lines. We tested different lysis buffers (IgePal and Nuclei EZ) and incubation times in combination with different approaches for tissue and cell dissection: sectioning, semi-automated dissociation, manual dissociation with pestles, and semi-automated dissociation combined with manual dissociation with pestles. Our results showed that a combination of IgePal lysis buffer, tissue dissection by sectioning, and short incubation time was the best conditions for gentle nuclei isolation applicable for snRNA-seq, and we found limited confounding transcriptomic changes based on the isolation procedure. This protocol makes it possible to analyze biobanked material from patients with well-described clinical and histopathological information and known clinical outcomes with snRNA-seq.
The establishment, maintenance and dynamic regulation of three-dimensional (3D) chromatin structures provide an important means for partitioning of genome into functionally distinctive domains, which helps to define specialized gene expression programs associated with developmental stages and cell types. Increasing evidence supports critical roles for intrinsically disordered regions (IDRs) harbored within transcription factors (TFs) and chromatin-modulatory proteins in inducing phase separation, a phenomenon of forming membrane-less condensates through partitioning of biomolecules. Such a process is also critically involved in the establishment of high-order chromatin structures and looping. IDR- and phase separation-driven 3D genome (re)organization often goes wrong in disease such as cancer. This review discusses about recent advances in understanding how phase separation of intrinsically disordered proteins (IDPs) modulates chromatin looping and gene expression.
Lamins A/C are nuclear intermediate filament proteins that are involved in diverse cellular mechanical and biochemical functions. Here, we report that recognition of Lamins A/C by a commonly used antibody (JOL-2) that binds the Lamin A/C Ig-fold and other antibodies targeting similar epitopes is highly dependent on cell density, even though Lamin A/Clevels do not change. We propose that the effect is caused by partial unfolding or masking of the C’E and/or EF loops of the Ig-fold in response to cell spreading. Surprisingly, JOL-2 antibody labeling was insensitive to disruption of cytoskeletal filaments or the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex. Furthermore, neither nuclear stiffness nor nucleo-cytoskeletal force transmission changed with cell density. These findings are important for the interpretation of immunofluorescence data for Lamin A/C and also raise the intriguing prospect that the conformational changes may play a role in Lamin A/C mediated cellular function.
Eukaryotic cells organize their genome within the nucleus with a double-layered membrane structure termed the nuclear envelope (NE) as the physical barrier. The NE not only shields the nuclear genome but also spatially separates transcription from translation. Proteins of the NE including nucleoskeleton proteins, inner nuclear membrane proteins, and nuclear pore complexes have been implicated in interacting with underlying genome and chromatin regulators to establish a higher-order chromatin architecture. Here, I summarize recent advances in the knowledge of NE proteins that are involved in chromatin organization, gene regulation, and coordination of transcription and mRNA export. These studies support an emerging view of plant NE as a central hub that contributes to chromatin organization and gene expression in response to various cellular and environmental cues.
The eukaryotic nucleus displays a variety of membraneless compartments with distinct biomolecular composition and specific cellular activities. Emerging evidence indicates that protein-based liquid–liquid phase separation (LLPS) plays an essential role in the formation and dynamic regulation of heterochromatin compartmentalization. This feature is especially conspicuous at the pericentric heterochromatin domains. In this review, we will describe our understanding of heterochromatin organization and LLPS. In addition, we will highlight the increasing importance of multivalent weak homo- and heteromolecular interactions in LLPS-mediated heterochromatin compartmentalization in the complex environment inside living cells.
The eukaryotic genome is organized in three dimensions within the nucleus. Transcriptionally active chromatin is spatially separated from silent heterochromatin, a large fraction of which is located at the nuclear periphery. However, the mechanisms by which chromatin is localized at the nuclear periphery remain poorly understood. Here we demonstrate that Proline Rich 14 (PRR14) protein organizes H3K9me3-modified heterochromatin at the nuclear lamina. We show that PRR14 dynamically associates with both the nuclear lamina and heterochromatin, and is able to reorganize heterochromatin in the nucleus of interphase cells independent of mitosis. We characterize two functional HP1-binding sites within PRR14 that contribute to its association with heterochromatin. We also demonstrate that PPR14 forms an anchoring surface for heterochromatin at the nuclear lamina where it interacts dynamically with HP1-associated chromatin. Our study proposes a model of dynamic heterochromatin organization at the nuclear lamina via the PRR14 tethering protein.
Enhancers are cis-regulatory elements that can stimulate gene expression from distance, and drive precise spatiotemporal gene expression profiles during development. Functional enhancers display specific features including an open chromatin conformation, Histone H3 lysine 27 acetylation, Histone H3 lysine 4 mono-methylation enrichment, and enhancer RNAs production. These features are modified upon developmental cues which impacts their activity. In this review, we describe the current state of knowledge about enhancer functions and the diverse chromatin signatures found on enhancers. We also discuss the dynamic changes of enhancer chromatin signatures, and their impact on lineage specific gene expression profiles, during development or cellular differentiation.
Autophagy has emerged as a key regulator of cell metabolism. Recently, we have demonstrated that autophagy is involved in RNA metabolism by regulating ribosomal RNA (rRNA) synthesis. We found that autophagy-deficient cells display much higher 47S precursor rRNA level, which is caused by the accumulation of SQSTM1/p62 (sequestosome 1) but not other autophagy receptors. Mechanistically, SQSTM1 accumulation potentiates the activation of MTOR (mechanistic target of rapamycin kinase) complex 1 (MTORC1) signaling, which facilitates the assembly of RNA polymerase I pre-initiation complex at ribosomal DNA (rDNA) promoter regions and leads to the activation of rDNA transcription. Finally, we showed that SQSTM1 accumulation is responsible for the increase in protein synthesis, cell growth and cell proliferation in autophagy-deficient cells. Taken together, our findings reveal a regulatory role of autophagy and autophagy receptor SQSTM1 in rRNA synthesis and may provide novel mechanisms for the hyperactivated rDNA transcription in autophagy-related human diseases. Abbreviations: 5-FUrd: 5-fluorouridine; LAP: MAP1LC3/LC3-associated phagocytosis; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; PIC: pre-initiation complex; POLR1: RNA polymerase I; POLR1A: RNA polymerase I subunit A; rDNA: ribosomal DNA; RRN3: RRN3 homolog, RNA polymerase I transcription factor; rRNA: ribosomal RNA; SQSTM1/p62: sequestosome 1; TP53INP2: tumor protein p53 inducible nuclear protein 2; UBTF: upstream binding transcription factor.
DFCC workflow. a) RNAP-Dendra2 stained nuclei of U2OS cells are imaged with a time interval of Δt by confocal microscopy. b) Flow fields between successive images are computed using Optical Flow. c) The spatial correlation in flow field direction (upper panel) and flow magnitude (lower panel) is computed over increasing space lags (averaged over the two spatial dimensions) and over accessible time lags (from blue to red). The spatial directional and magnitudinal correlation length, respectively, is obtained via regression to the Whittle-Matérn covariance model for every time lag (insets).
Spatial correlation of RNAP dynamics in the absence of serum, upon serum-stimulation and DRB treatment. a) Exemplary RNAP-Dendra2 stained nucleus. b) Directional and c) magnitudinal correlation length of RNAP over increasing time lag.
Spatial correlation of DNA and RNA Pol II dynamics for serum-starved, active, and stalled transcription. a) Superimposed directional correlation length for DNA and RNA Pol II in the absence of serum. b) Analogous for the magnitudinal correlation. c-d) Analogous for serum-stimulated cells. e-f) Analogous for DRB treatment in the presence of serum. While DNA dynamics become spatially correlated upon serum stimulation, RNA Pol II's directional and magnitudinal correlation decreases slightly. Upon stalling RNA Pol II at the initiation step by addition of DRB to the medium, RNA Pol II's directional correlation slightly increases, while the opposite trend is observed for DNA dynamics. In contrast, DRB treatment reduces the magnitudinal correlation length of both RNA Pol II and DNA.
Schematic representation of the observed spatially coherent motion of chromatin versus RNA Pol II in serum-starved, active, and promoter-paused states of transcription. Spatially coherent chromatin and RNA Pol II motion exhibit opposite trends upon transcription stimulation.
Gene transcription by RNA polymerase II (RNAPol II) is a tightly regulated process in the genomic, temporal, and spatial context. Recently, we have shown that chromatin exhibits spatially coherently moving regions over the entire nucleus, which is enhanced by transcription. Yet, it remains unclear how the mobility of RNA Pol II molecules is affected by transcription regulation and whether this response depends on the coordinated chromatin movement. We applied our Dense Flow reConstruction and Correlation method to analyze nucleus-wide coherent movements of RNA Pol II in living human cancer cells. We observe a spatially coherent movement of RNA Pol II molecules over (Formula presented.) 1 μm, which depends on transcriptional activity. Inducing transcription in quiescent cells decreased the coherent motion of RNA Pol II. We then quantify the spatial correlation length of RNA Pol II in the context of DNA motion. RNA Pol II and chromatin spatially coherent motions respond oppositely to transcriptional activities. Our study holds the potential of studying the chromatin environment in different nuclear processes. © 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
Dominant missense mutations in RanBP2/Nup358 cause Acute Necrotizing Encephalopathy (ANE), a pediatric disease where seemingly healthy individuals develop a cytokine storm that is restricted to the central nervous system in response to viral infection. Untreated, this condition leads to seizures, coma, long-term neurological damage and a high rate of mortality. The exact mechanism by which RanBP2 mutations contribute to the development of ANE remains elusive. In November 2021, a number of clinicians and basic scientists presented their work on this disease and on the interactions between RanBP2/Nup358, viral infections, the innate immune response and other cellular processes. © 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
Domain conservation and membrane orientation of DdSpastin. (a) Schematic of DdSpastin domains and membrane orientation by motif predictions using ELM 25. See text for further descriptions; PNS, perinuclear space. (b) Immunoprecipitation using GFP-Trap Agarose beads showing tubulin-binding of DdSpastin-GFP. Proteins in the supernatant (lysate; corresponding to ~10 6 cells) and the GFP-Trap eluate (corresponding to 1 × 10 7 cells) were separated by SDS-PAGE, and stained with Coomassie or evaluated by immunoblot staining with anti-β-tubulin; *, this particular band was analyzed by mass spectrometry resulting in a hit for α-tubulin (see table S1). (c) In vitro microtubule severing assay. Polymerized porcine brain tubulin and DdSpastin-GFP (green) were incubated with and without 1 mM ATP. The reaction mixture was fixed with formaldehyde on poly-L-lysine coated coverslips and stained with anti-α-tubulin (red). Green spots most likely represent DdSpastin-GFP clusters that have formed via hydrophobic interactions of the transmembrane domains. Bar, 5 µm. (d) Verification of membrane orientation using isolated nuclei from DdSpastin-GFP overexpression cells. Nuclei were fixed with and without Triton X-100 permeabilization. Merged images of three examples each and corresponding single channel images are shown. Bar, 2 μm.
Localization of DdSpastin-NEON (knock-in). Cells were fixed with glutaraldehyde and stained with DAPI(blue), and anti-α-tubulin (red). DdSpastin-NEON (green) accumulated at spindle Poles beginning in early telophase and in late telophase at the central spindle. The DdSpastin-NEON channel alone is shown below the merged images. A quantitative evaluation of all investigated cells is given in Table S2. Bar, 5 μm.
DdSpastin interactions and co-localizations. (a) Immunoblot of whole cell extracts of AX2 control cells, BirA*-DdSpastin cells and DdSpastin-BirA* cells stained with anti-BirA* antibodies. Fusion protein bands and BirA* with signal peptide are labeled with an asterisk. (b) BioID with nuclear extracts of DdSpastin-BirA* (lane 1-6) and negative control BirA* cells (lane 7). Western blots were stained with alkaline phosphate conjugated to the antibodies/protein stated on top. The interactors Src1 and Sun1 are labeled with red asterisks, DdSpastin-BirA* is labeled with a blue asterisk (lane 2). Lane 1 control w/o biotin incubation and lane 7 BirA* control show no specific bands at these positions. (c-h) Fluorescence microscopy of the strains stated in the figures. Cells were fixed with either glutaraldehyde (c-g) or methanol (h), and additionally labeled with DAPI (blue). Close-ups show co-localization (d, e, g, h). GFP-CHMP7 labeling is shown in a single channel because this protein has not been previously published (f). Bar, 5 μm.
Dictyostelium amoebae perform a semi-closed mitosis, in which the nuclear envelope is fenestrated at the insertion sites of the mitotic centrosomes and around the central spindle during karyokinesis. During late telophase the centrosome relocates to the cytoplasmic side of the nucleus, the central spindle disassembles and the nuclear fenestrae become closed. Our data indicate that Dictyostelium spastin (DdSpastin) is a microtubule-binding and severing type I membrane protein that plays a role in this process. Its mitotic localization is in agreement with a requirement for the removal of microtubules that would hinder closure of the fenestrae. Furthermore, DdSpastin interacts with the HeH/ LEM-family protein Src1 in BioID analyses as well as the inner nuclear membrane protein Sun1, and shows subcellular co-localizations with Src1, Sun1, the ESCRT component CHMP7 and the IST1-like protein filactin, suggesting that the principal pathway of mitotic nuclear envelope remodeling is conserved between animals and Dictyostelium amoebae.
In progeria cells and cells in intact beating embryonic chick hearts, high Gaussian curvature favors nuclear rupture, even when myosin stress is inhibited. A. Mesenchymal stem/progenitor cells from patients with progeria have a defective nuclear lamina, which increases nuclear rupture in standard 2D culture. The nuclear bleb or scar shows abundant lamin-A but loss of lamin-B (yellow arrows). Myosin-II inhibitor blebbistatin reduces stress on the nucleus, but blebs/scars persist. iPSC = induced pluripotent stem cell. Images: dashed circles and lines trace the local curvature; scale = 5 µm. B. Rupture frequency, as indicated by % progeria cells with blebs/scars, is highest at the nuclear pole and decreases by ~half after blebbistatin (blebb.) treatment. NT = non-treated; 594 cells; � 242 cells per condition. C. Lower: For 6 non-treated and 6 blebb.-treated progeria cells (randomly selected), curvature was measured at ~20 locations around the nuclear perimeter (inset image, scale = 5 µm). Plots show the curvature distributions. Upper: For progeria cells from panel B with blebs/
Migration through constricting pores causes frequent nuclear rupture; multiple such ruptures at distinct sites within a single nucleus seem to be independent events that depend strongly on pore curvature. A. Nuclei rupture as cells migrate through constricting pores from the Top to the Bottom of a Transwell. Images: nuclear blebs and scars (yellow arrows) in migrated U2OS human bone cancer cells on the Bottom of a 3 µm pore membrane. Some nuclei show multi-site rupture, with 2 or 3+ blebs/scars. B. Distributions of bleb/scar number and location among U2OS cells that have migrated through pores of varying diameter D pore . � 90 cells per D pore . C. Rupture frequency during migration, as indicated by % migrated U2OS cells with blebs/scars, increases with pore curvature. This trend holds overall (black diamonds) as well as among migrated cells with 1 (dark gray), 2 (light gray), and 3+ (light blue) rupture sites. Filled points are measured values from panel B. Unfilled are estimates of multi-site rupture frequency, obtained by treating each rupture as an independent event with probability equal to the measured % of 1-bleb/scar
Nuclear entry into a constriction causes instantaneous dilution of lamin-B at the leading tip of the nucleus, while lamin-A requires a critical strain rate to flow. A. Lamin-A and lamin-B networks are predicted to have viscous and elastic responses, respectively, to applied stress. B. Cells were detached, treated with latrunculin to depolymerize the actin cytoskeleton and pulled under controlled pressure ∆P into micropipettes. C. U2OS cells were transfected with lamin-B-GFP and mCherry-cGAS. (i) Representative images of cell aspiration into a low-curvature or a high-curvature pipette. Arrow head = nuclear envelope rupture, indicated by nuclear accumulation of cGAS. Scale = 5 µm. (ii) For 17 cells pulled at a slow or fast rate into pipettes of varying curvature, lamin-B intensity at the nuclear tip was measured at t = 15 s. Plots show distributions of these intensities for all conditions. Based on mean µ � SEM σ values from Xia, Pfeifer 2019; � 3 cells per condition. D. A549 cells with gene-edited RFP-lamin-B were transfected with GFP-lamin-A and aspirated at varying rates into pipettes of fixed
Nuclear rupture has long been associated with deficits or defects in lamins, with recent results also indicating a role for actomyosin stress, but key physical determinants of rupture remain unclear. Here, lamin-B filaments stably interact with the nuclear membrane at sites of low Gaussian curvature yet dilute at high curvature to favor rupture, whereas lamin-A depletion requires high strain-rates. Live-cell imaging of lamin-B1 gene-edited cancer cells is complemented by fixed-cell imaging of rupture in: iPS-derived progeria patients cells, cells within beating chick embryo hearts, and cancer cells with multi-site rupture after migration through small pores. Data fit a model of stiff filaments that detach from a curved surface.Rupture is modestly suppressed by inhibiting myosin-II and by hypotonic stress, which slow the strain-rates. Lamin-A dilution and rupture probability indeed increase above a threshold rate of nuclear pulling. Curvature-sensing mechanisms of proteins at plasma membranes, including Piezo1, might thus apply at nuclear membranes. Summary statement: High nuclear curvature drives lamina dilution and nuclear envelope rupture even when myosin stress is inhibited. Stiff filaments generally dilute from sites of high Gaussian curvature, providing mathematical fits of experiments.
Flow diagram elucidating the main experimental steps of in situ NuMat preparation.
Visualization of in situ NuMat. D. melanogaster embryos at early stages of development (0-2 hr) were used to prepare nuclear matrices in situ. A. Visualization by TEM. Images obtained by TEM of resinless sections of embryos carrying intact nuclei, in situ NuMat and RNase A treated in situ NuMat. The fine filaments seen in NuMat, are lost upon RNase A treatment, leaving large gaps in the nuclear structure and leading to collapse of nuclei. B. Visualization by confocal microscopy. Unextracted embryo and embryo with in situ NuMat, were immuno-stained with anti-Lamin Dm0 and DAPI and imaged by confocal microscopy. In unextracted embryos, Lamin Dm0 appears as a ring at the nuclear periphery of intact nuclei. After in situ NuMat preparation, no DAPI staining is observed in the nucleus, as chromatin has been digested and extracted out. Lamin Dm0 staining can now be seen in the nuclear interior as well. C. STED visualization of Lamin Dm0 stained in situ NuMat. D. Remnants of nucleolus remain associated with in situ NuMat. Confocal images of unextracted embryo and embryo with in situ NuMat, were immuno-stained with anti-Fibrillarin, antiLamin Dm0 and DAPI. Loss of DAPI staining indicates extraction of chromatin. In situ NuMat shows prominent staining with fibrillarin indicating remnants of nucleolus remain associated with the nuclear substructure. E. In situ NuMat prepared without crosslinking, without stabilization or with over-crosslinking. In situ NuMat is efficiently prepared as evident by absence of DAPI staining, even when the embryos are not crosslinked or are not stabilized. The circular morphology of the nuclei remains intact, but internal lamin staining is not visible. Over-crosslinking results in clumps of DNA that remains unextracted as visualized by DAPI staining. Internal lamin staining is also not sufficiently revealed. All of the confocal image were acquired using a Leica SP8 confocal microscope. The whole embryo images were taken with 20X objective and the higher magnification images were taken with 63X objective. Images were processed using LAS X software from Leica.
In situ NuMat prepared with early D. melanogaster embryo with a mitotic wave. In situ NuMat prepared with early syncytial embryos, captures a snapshot of an embryo with nuclei at different stages of mitosis. Immuno-staining with anti-Lamin Dm0 and anti-BEAF 32 reveals the dynamics of these nuclear proteins at different mitotic stages. A subset of BEAF 32 stays associated with mitotic nuclei even when the nuclear envelope (defined by Lamin Dm0) is dissolved.
In situ NuMat preparation protocol can be used in conjunction with fly genetics. A. Fly cross scheme to generate a fly line carrying tagged isoforms of BEAF 32 in the same fly. B. Immuno-staining of unextracted/in situ NuMat prepared salivary glands with anti-Lamin Dm0, anti-Myc and anti-FLAG antibodies. Myc-tagged BEAF 32A and FLAG-tagged 32B, colocalize on several bands of the polytene chromosome in the salivary gland nuclei. After in situ NuMat preparation, 32A gets extracted out and 32B remains associated with NuMat.
The study of nuclear matrix (NuMat) over the last 40 years has been limited to either isolated nuclei from tissues or cells grown in culture. Here, we provide a protocol for NuMat preparation in intact Drosophila melanogaster embryos and its use in dissecting the components of nuclear architecture. The protocol does not require isolation of nuclei and therefore maintains the three-dimensional milieu of an intact embryo, which is biologically more relevant compared to cells in culture. One of the advantages of this protocol is that only a small number of embryos are required. The protocol has been extended to larval tissues like salivary glands with little modification. Taken together, it becomes possible to carry out such studies in parallel to genetic experiments using mutant/transgenic flies. This protocol, therefore, opens the powerful field of fly genetics to cell biology in the study of nuclear architecture. Summary: Nuclear Matrix is a biochemically defined entity and a basic component of the nuclear architecture. Here we present a protocol to isolate and visualize Nuclear Matrix in situ in the Drosophila melanogaster and its potential applications.
Nuclear Speckles exhibit changes in morphology upon stress. (a) Immunofluorescence for speckle marker Sc35 and Tubulin indicating changes in NS upon Etoposide and Actinomycin D treatment in fixed cells. (b) Representative confocal images for Sc35 Immunofluorescence under Control, Actinomycin D and Etoposide treatment. (c) IF analysis of NS number in maximum-intensity Z projections of Sc35 immunostaining after Control, Etoposide and Actinomycin D treatment. Quantification is depicted as the frequency distribution of NS number/cell. N = 3, n = 40. Error bars represent SD. (d) Frequency distribution of NS volume as measured by volumetric analysis of maximum-intensity Z projections of Sc35 immunostaining. N = 3, n = 40. Error bars represent SD. (e) Schematic describing purification of nuclei for immunostaining. (f) Representative images for immunostaining of Sc35 and γH2aX for purified nuclei upon Etoposide and Actinomycin D treatment. (g) Frequency distribution of Nuclear speckle number per nuclei as measured by the fluorescence intensity of Sc35. N = 3, n = 40.Error bars represent SD. (h) Frequency distribution of NS volume measured by volumetric analysis of Sc35 staining of purified nuclei. N = 3, n = 40. Error bars represent SD.
DNase 1 nicking leads to the collapse of NS-morphology. (a) Schematic for isolation of nuclei and DNase 1 treatment. (b) IF images for Tubulin (green) and Sc35 (red) staining in whole cells and purified nuclei. (c) Representative images of NS morphologies (Sc35 staining) observed upon DNase 1 treatment. (d) Quantification of different NS morphologies observed upon increasing concentrations of DNase 1. Error bars represent SD. (e) Representative images of Sc35 Immunostaining of Nuclei treated with DNase 1(2 U) over 10, 20, and 30 minutes of incubation. (f) Quantification of different NS morphologies observed upon DNase 1 treatment time course. Error bars represent SD.
NS changes upon DNase 1 treatment are consistent over different physiological states. (a) Representative images for nuclei purified post Treatment of cells with Etoposide and Actinomycin D, stained for Sc35 and Nucleolar marker Nucleolin. (b) Representative IF images for nuclei purified after Etoposide and Actinomycin D treatment and subsequent DNase 1 (2 U) treatment. The Red channel marks NS staining and the green channel shows an absence of any Tubulin staining. (c) Nucleolin(green) and Sc35 (red) staining in DNase 1(2 U) treated nuclei post Etoposide and Actinomycin D treatment. (d) Percentage of NS morphologies observed post-DNase 1 treatment of purified nuclei from Etoposide and Actinomycin D treated cells. Error bars represent Standard deviation, N = 3, n = 50. e) Poly A mRNA FISH using FAM oligo dT for control and DNase 1 treated nuclei.
SR proteins exhibit strong chromatin association. (a) Schematic of Nuclear fractionation, sequential salt elution and Western blotting. (b) Western blots for supernatants released from chromatin after sequential salt elution. Consecutive lanes represent Control and Etoposide (10 uM, 4 hours) treated samples for each fraction. SR proteins (probed as pSRSF or SRSF) elute at higher salt concentrations compared to transcription factor Stat3. Histone H3 shows a stronger binding with maximum elution over 300 mM NaCl. Tubulin is used for indicating the purity of nuclear fractions. (c) Western blot for salt-based elution of the supernatants released and the ultimate chromatin pellet probed for SR proteins, Histone H3 and Tubulin. The blot shows clear enrichment of Histone H3 in the pellet and maximum elution of SR proteins at 600 mM NaCl. (d) Actinomycin D treatment (5 μg/ml) shows no alteration in chromatin association of SR proteins compared to DMSO control, as indicated by pSRSF Western blots. (e) Western blots for pSRSF proteins post MNase treatment followed by differential salt elution, probed for transcription factor Stat 3, chromatin remodeler HDAC1, and splicing factor hnRNPA1 showing weak chromatin association and H3 and SR proteins showing stronger association. (f) Western blot of supernatant released after MNase treatment of 30, 45, and 60 minutes and the remaining pellet.
Model: In unperturbed conditions nuclear speckles (NS) and chromatin are closely interacting as indicated by the strong association of NS constituent SR proteins with chromatin. Such an interaction might act as a 'tether' for NS. Perturbations like limited DNase 1 nicking of chromatin leads to 'untethering' of NS from chromatin which results in destabilization of NS into isotropic distribution or aggregation of constituent proteins as observed by the immunostaining.
Nuclear Speckles (NS) are phase-separated condensates of protein and RNA whose components dynamically coordinate RNA transcription, splicing, transport and DNA repair. NS, probed largely by imaging studies, remained historically well known as Interchromatin Granule Clusters, and biochemical properties, especially their association with Chromatin have been largely unexplored. In this study, we tested whether NS exhibit any stable association with chromatin and show that limited DNAse-1 nicking of chromatin leads to the collapse of NS into isotropic distribution or aggregates of constituent proteins without affecting other nuclear structures. Further biochemical probing revealed that NS proteins were tightly associated with chromatin, extractable only by high-salt treatment just like histone proteins. NS were also co-released with solubilised mono-dinucleosomal chromatin fraction following the MNase digestion of chromatin. We propose a model that NS-chromatin constitutes a “putative stable association” whose coupling might be subject to the combined regulation from both chromatin and NS changes. Abbreviations: NS: Nuclear speckles; DSB: double strand breaks; PTM: posttranslational modifications; DDR: DNA damage repair; RBP-RNA binding proteins; TAD: topologically associated domains; LCR: low complexity regions; IDR: intrinsically disordered regions.
DNA Supercoiling in vivo (a) Twin-supercoiled domain model explaining the generation of dynamic positive supercoils ahead and negative supercoils behind of the protein complex that translocates along the DNA double helix [15]. Although this supercoiling regulates the variety of DNA transactions, excessive DNA supercoils will halt the further progression of translocating complex if not properly resolved. (b) Methods for detection of DNA supercoiling in vivo. Top panel: DNA supercoiling have been most frequently probed with psoralen. Psoralen freely crosses cellular membranes, intercalates between DNA bases and forms crosslinks between the two strands when exposed to UV light [127]. It has a different preference for relaxed, positively supercoiled, and negatively supercoiled DNA (blue curve). Taking advantage of this psoralen property, supercoiled DNA have been mapped in bacteria, yeast, Drosophila, and human cells [66,69,119,145]. Recently developed GapR-seq assay is based on the ability of the bacterial protein GapR to preferentially recognize overtwisted DNA (green curve). Chromatin immunoprecipitation of GapR combined with high-throughput sequencing was used to generate maps of positive supercoiling in bacteria and yeast [97]. Detection of topoisomerase activity sites (Middle panel) and non-B DNA structures (Bottom panel) are also powerful methods to predict DNA supercoiling in vivo [71,132,146]. There has been considerable concordance between the studies supporting the main prophecies of the twin-supercoiled domain model: negative torsional stress accumulated at the upstream promoter region of the active genes, while positive torsional stress accrues in a transcription-dependent manner in gene bodies and downstream to the 3' ends of genes. -Sc (negatively supercoiled DNA); R (relaxed DNA); +Sc (positively supercoiled DNA). Blue triangle (Non-B DNA).
Chromatin mechanics and gene expression (a) The pre-initiation complex formation often involves the recruitment of chromatin remodeling complexes and histone acetyltransferases on the promoter. Core histone rearrangement and/or acetylation release negative supercoils previously constrained by the nucleosomes. Negative supercoiling increases affinity of TFs to promoter DNA, helps recruitment of transcription machinery and assist promoter DNA melting. (b) Nucleosome destabilization in the gene body is a mechanism to achieve high elongation efficiency. Positive supercoiling in front of transcribing Pol II propagates faster than the rate of elongation. The resulting torsional stress weakens the contacts between DNA and core histone by promoting H2A/H2B dimer eviction from the nucleosomes. Chromatin responds to DNA supercoiling by confinement of gene domain. This confined state of chromatin enhances the frequency of interaction among distal transcription regulators and Pol II.
Enhancer-Promoter (e-p) communication There are at least three models by which an enhancer-promoter communication is established (a). The classical model (i) where transcription factors bind within their target enhancer and promoter form a stable complex between enhancer and promoter to stabilize the chromatin loop. In 'kiss-and-run' model (II), only transient physical contact between enhancer and promoter is required to regulate promoter activity. In proximity model (III), the enhancer communicates with the target promoter in a distance-dependent manner through the high local concentrations of transcription factors established by 'hub' or 'condensate' formation. Enhancer (red rectangle) is located far from the promoter (green rectangle) and may not communicate in a linear scale (B, Top panel). Bidirectional transcription at the enhancer region induces positive torsional stress resulting in confinement of region between enhancer and promoter (B, Middle panel). Enhanced spatial exploration of chromatin fiber promotes establishing functional E-P communication. Upon activation of the targeted promoter, the enhancer transcription is no longer required (B, Bottom panel). For clarity, transcription factors and Pol II complex have been omitted.
TADs formation Schematic showing active transcription supports TAD formation in DNA supercoiling dependent fashion.
The compaction of linear DNA into micrometer-sized nuclear boundaries involves the establishment of specific three-dimensional (3D) DNA structures complexed with histone proteins that form chromatin. The resulting structures modulate essential nuclear processes such as transcription, replication, and repair to facilitate or impede their multi-step progression and these contribute to dynamic modification of the 3D-genome organization. It is generally accepted that protein–protein and protein–DNA interactions form the basis of 3D-genome organization. However, the constant generation of mechanical forces, torques, and other stresses produced by various proteins translocating along DNA could be playing a larger role in genome organization than currently appreciated. Clearly, a thorough understanding of the mechanical determinants imposed by DNA transactions on the 3D organization of the genome is required. We provide here an overview of our current knowledge and highlight the importance of DNA and chromatin mechanics in gene expression.
Loss of UIPs results in altered nuclear shape and nuclear import. a, b) Wt and strains harboring indicated deletions were examined for nuclear morphology and distribution of nuclear pores using GFP-Esc1 (a) and GFP-Nup49 (b) plasmids respectively. The maximum intensity projection (MIP) of representative cells is shown. DAPI staining is used to define the nucleus. The abnormality in the nuclear membrane and pore complex distribution is shown by yellow arrows and arrowheads respectively. Scale-2μm . c) Quantification of defects is shown in the bar graph as the fraction of cell population showing abnormality (n>300, 3 independent experiments, error bars indicate SEM). d)The circularity index of the nuclear envelope for the indicated strains is shown. 25-30 cells picked randomly were used for measurement. The horizontal line shows the mean of the distribution.
List of strains used in the study.
List of plasmids used in the study.
A double membrane bilayer perforated by nuclear pore complexes (NPCs) governs the shape of the nucleus, the prominent distinguishing organelle of a eukaryotic cell. Despite the absence of lamins in yeasts, the nuclear morphology is stably maintained and shape changes occur in a regulated fashion. In a quest to identify factors that contribute to regulation of nuclear shape and function in Saccharomyces cerevisiae, we used a fluorescence imaging based approach. Here we report the identification of a novel protein, Uip4p, that is required for regulation of nuclear morphology. Loss of Uip4 compromises NPC function and loss of nuclear envelope (NE) integrity. Our localization studies show that Uip4 localizes to the NE and endoplasmic reticulum (ER) network. Furthermore, we demonstrate that the localization and expression of Uip4 is regulated during growth, which is crucial for NPC distribution.
Cellular senescence provokes a dramatic alteration of chromatin organization and gene expression profile of proinflammatory factors, thereby contributing to various age-related pathologies via the senescence-associated secretory phenotype (SASP). Chromatin organization and global gene expression are maintained through the CCCTC-binding factor (CTCF). However, the molecular mechanism underlying CTCF regulation and its association with SASP gene expression remains to be fully elucidated. A recent study by our team showed that noncoding RNA (ncRNA) derived from normally silenced pericentromeric repetitive sequences directly impair the DNA binding of CTCF. This CTCF disturbance increases the accessibility of chromatin at the loci of SASP genes and caused the transcription of inflammatory factors. This mechanism may promote malignant transformation.
Purified MeCP2 forms liquid-like droplets in physiologically crowding environments. (A) Analysis of human MeCP2 protein sequence. Top: Schematic overview of human MeCP2 structure. NTD: N-terminal domain; MBD: methyl binding domain; ID: intervening domain; NID: N-CoR interacting domain; CTD: C-terminal domain; TRD: transcriptional repression domain. Bottom black line: PONDR prediction ( of MeCP2 ordered/disordered regions, >0.5 is considered disordered. Bottom gray line: protein charge, >0 means positively charged ( /emboss/charge). The Isoelectric point (PI) of MeCP2 is predicted 10.56 using INNOVAGEN ( Amino acid labeling is according to human MeCP2 isoform 1.(B) Validation of MeCP2 purity. The MeCP2 and GFP-MeCP2 proteins were expressed in bacteria by IPTG induction, purified using chitin beads and eluted by DTT. The final protein concentrations were measured by Pierce™ 660 nm Protein Assay Reagent. 2 µg and 10 µg purified protein were then used for SDS polyacrylamide gel electrophoresis and tris borate EDTA polyacrylamide gel electrophoresis, respectively. Left: SDS polyacrylamide gel electrophoresis of purified human MeCP2 and GFP-MeCP2 followed by Coomassie staining. 2 µg each lane. Right: tris borate EDTA polyacrylamide gel
Validation and calibration of a cellular system mimicking in vivo MeCP2 physiological behavior. (A) Scheme of the experiment: C2C12 myoblast (mb-) were transfected with a plasmid encoding for MeCP2-GFP. After 20 h, transfected cells (mb+) were sorted into two categories, low and high expressing, according to the GFP intensity using a Fluorescence-activated cell sorting (FACS).(B) Immunofluorescence staining showing MeCP2 levels in mouse myoblasts before and after transfection of MeCP2-GFP. Scale bars = 5 µm. Boxplots show the MeCP2 heterochromatin mean intensity and the mean heterochromatin cluster area of untransfected, low and high MeCP2 expressing myoblasts of three independent replicates (***p < 0.001, Wilcoxon test).(C) Quantification of total MeCP2 in mouse myoblasts. The concentration of MeCP2-GFP standard was determined by SDS-PAGE and coomassie staining in comparison to a BSA standard series. The MeCP2 standard was used to quantify the MeCP2 protein level in untransfected, low and high expressing FACS sorted mouse myoblasts by Western blot against MeCP2 for three independent replicates (average values ± standard deviation). Full gels and Blots are shown in Fig. S3.(D) Scheme of the calculation of the nuclear volume based on thresholded 0.2 µm z-stacks.(E) Scheme of the cell segmentation and the calculations
DNA promotes the liquid-liquid phase separation of MeCP2. The synthesized DNA was labeled with DRAQ5. The in vitro phase separation assay at different conditions was done by incubation at room temperature for 45 min. Then, the mixtures were transferred to chambers made of double-sided tapes and sealed with coverslips. The fluorescent and DIC images were taken using the Nikon Eclipse TiE2 microscope. MeCP2: 3 µM, NaCl: 150 mM, no PEG.(A) Fluorescent images of MeCP2 droplets in the presence of DRAQ5 labeled DNA with different concentration and length. Scale bar = 10 µm.(B) Quantification of size, area and number of droplets from (A). The red channel was applied for droplet segmentation by bandpass filter and threshold based on the mean intensity in/out droplets. Droplets with size >0.1 µm2 were considered and droplet parameters were measured.≥3 images were taken for each condition. The droplets number and sum droplet area per image and mean droplet area were plotted with mean ± SD (standard deviation).
Cytosine methylation restricts droplet growth in vitro and in vivo. (A) Fluorescent images showing the MeCP2 phase property in the presence of methylated and unmethylated DNA. The synthesized 800 bp DNA was labeled with DRAQ5. 1% GFP-MeCP2 was mixed with 99% untagged MeCP2. The in vitro phase separation assay at different conditions was done by incubation at room temperature for 45 min. Then, the mixtures were transferred to chambers made of double-sided tapes and sealed with coverslips. The fluorescent and DIC images were taken using the Nikon Eclipse TiE2 microscope. MeCP2 and DNA concentrations (conc.) are as mentioned. NaCl: 150 mM, no PEG. Representative images of the GFP channel are shown. Scale bar = 10 µm.(B) Graphs showing the influence of mCpG on the LLPS properties of MeCP2 based on droplet size, sum area and number from (C). Droplets were segmented by a bandpass filter and thresholded based on the mean GFP intensities in/out droplets. The droplet parameters were measured and plotted. conc.: concentration.(C) DNA methylation detection. 1 µl of 10 µM 42 bp and 20 bp DNA with and without CpG methylation was treated with HpaII and MspI respectively for 2 h ar 37°C
DNA and methyl-DNA addition to MeCP2 LLPS emulates the three mobility populations observed in heterochromatin in cells. (A) 10 µM MeCP2 containing 1% GFP-MeCP2 was mixed with of 5 ng/µl 800 bp (methyl-)DNA with or without crowding agents (5% PEG 8000) in a buffer containing 150 mM NaCl and incubated 45-60 min at room temperature before imaging. GFP-MeCP2 tracks were obtained using TrackMate, sorting out tracks with less than 4 spots and analyzed in SMTracker. Square displacement cumulative distribution of the steps is shown as described in Figure 2. A curve containing the square displacement analysis of the tracks located in heterochromatin in myoblasts with low MeCP2 from Figure 4 has been added for comparison.(B) Mouse embryonic fibroblast proficient (MEF-P) or deficient (MEF-PM) in DNA methylation were transfected with pEG-MeCP2 and pMaSatmRFP or pMaSat-miRFP703 using AMAXA transfection and seeded on glass slides in DMEM 15% FBS at 37°C, 5% CO 2 for 16-20 h. During image acquisition, cells were maintained in PBS. MeCP2 tracks were obtained in TrackMate, sorting out the tracks containing less than 5 spots and analyzed using SMTracker. Square displacement curves and best fit model for MeCP2 is shown as described in Figure 2.(C) Effect of (methyl)-DNA in MeCP2 populations fixed in Gaussian-mixture model with the diffusion coefficient obtained for live cells (fast: 0.520; slow: 0.095; static: 0.023). Last column belongs to the sorted tracks in low MeCP2 myoblast for the heterochromatin compartment as shown in Figure 4.(D) Comparison of the population variation with the same diffusion coefficients forcing three population model in Gaussian-mixture model in cells deficient (MEF-PM) or proficient (MEF-P) in 5mC maintenance.(E) C2C12 myoblasts were transfected with of pEG-MeCP2R111G and pMaSat-mRFP using AMAXA transfection and seeded on glass slides in DMEM 20% FBS at 37°C, 5% CO 2 . During acquisition, cells were maintained in PBS. MeCP2 tracks were obtained in TrackMate, sorting out the tracks containing less than 5 spots and analyzed using SMTracker. Square displacement curves and best fit model for MeCP2 and MaSat is shown as described in Figure 2. The data from Figure 4(a) corresponding to low levels of MeCP2 has been added for reference. The residuals showed the 2-population model as the simplest fitting model according to Bayesian Information Criterion for the R111G mutant.(F) Exemplary cell for MeCP2 R111G mutant, which showed no enrichment in heterochromatin neither in the pre-bleach image nor in the track overview. Scale bar = 2 µm.(G) MeCP2 wt versus R111G comparison of the population variation with the same diffusion coefficients forcing a three population model in the Gaussianmixture model. The third population is completely absent in the R111G mutant.5mC: DNA with cytosine methylation.
Heterochromatin is the highly compacted form of chromatin with various condensation levels hallmarked by high DNA methylation. MeCP2 is mostly known as a DNA methylation reader but has also been reported as a heterochromatin organizer. Here, we combine liquid–liquid phase separation (LLPS) analysis and single-molecule tracking with quantification of local MeCP2 concentrations in vitro and in vivo to explore the mechanism of MeCP2-driven heterochromatin organization and dynamics. We show that MeCP2 alone forms liquid-like spherical droplets via multivalent electrostatic interactions and with isotropic mobility. Crowded environments and DNA promote MeCP2 LLPS and slow down MeCP2 mobility. DNA methylation, however, restricts the growth of heterochromatin compartments correlating with immobilization of MeCP2. Furthermore, MeCP2 self-interaction is required for LLPS and is disrupted by Rett syndrome mutations. In summary, we are able to model the heterochromatin compartmentalization as well as MeCP2 concentration and heterogeneous motion in the minimal in vitro system. © 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
Cryo-ET analysis of the C. elegans lamin filaments. (a). A schematic view of the Ce-lamin protein , indicating the different domains of the protein. The intrinsically disordered head domain (blue), is composed of 48 amino acids. The first 14 aa are shown as a black line, followed by 35 conserved aa which are found in other organisms. The coiled-coil rod domain (large green box) is composed of the canonical helix segments and their linkers. Helix 1A (35 aa), helix 1B (134 aa) and helix 2 (129 aa) are shown. The tail domain (yellow) is composed of an Ig-like domain (112 aa) flanked by unfolded protein stretches (black lines). The positions of the nuclear localization sequence (vertical gray stripe) and the CaaX motif (vertical light gray stripe) are indicated. (b). The filaments are detected in an x-y slice through a cryo-electron tomogram, 3 nm in thickness. Arrowheads point to some of the globular domains seen along the filaments. Scale bar 100 nm. (c). Structural class averages of in silico segmented filaments, extracted from cryo-tomograms with a box size of 70 2 nm 2 . The filaments appears two protofilaments (horizontaly oriented) that are often bent. This appearance indicates the flexibility of the filaments. Additional densities, presumably Iglike domains, are often seen (arrowheads). (d). In silico reconstituted filaments, from the structural class averages shown in c., indicate the flexibility and interactions between the protofilaments.
Helix 1A restricts lateral assembly of C. elegans lamin filaments, while helix 1B and 2 are indispensable. (a). Schematic illustration of the Ce-lamin and the deletion mutations (red) that were used. No filaments were detected by negative stained electron microscopy analysis of Ce-lamin Δcoil 1B and Δcoil 2. (b). A cryo-EM image of the Ce-lamin Δcoil 1A filaments (arrows). C. Structural class averages indicate several protofilaments and the repeating density clouds (oriented vertically). Horizontally oriented filaments are decorated by vertical cloud densities, presumably the Ig-like fold domains. (d). Reconstituted filaments from the Ce-lamin Δcoil 1A cryo-EM analysis, showing an alternating pattern of 28 ± 2 nm and 16 ± 2 nm (these distances indicated in the figure). Scale bar 50 nm, indicated for C and D.
The assembly of C. elegans lamin filaments in the presence and absence of helix 1A. (a). Lamin dimers assemble into headto-tail polymers, which laterally assemble in an antiparallel fashion into protofilaments. In vitro assembled Ce-lamin is further assembled into ~8-10 nm thick filaments. Helix 1A in green, helices 1B and 2 in gray, flexible tail in pink and Ig-like domain in red. (b). In the absence of helix 1A the assembly continues to form ~20 nm thick filaments in which slight change in the repeat was observed.
Lamins are the major constituent of the nuclear lamina, a protein meshwork underlying the inner nuclear membrane. Nuclear lamins are type V intermediate filaments that assemble into ~3.5 nm thick filaments. To date, only the conditions for the in vitro assembly of Caenorhabditis elegans lamin (Ce-lamin) are known. Here, we investigated the assembly of Ce-lamin filaments by cryo-electron microscopy and tomography. We show that Ce-lamin is composed of ~3.5 nm protofilaments that further interact in vitro and are often seen as 6–8 nm thick filaments. We show that the assembly of lamin filaments is undisturbed by the removal of flexible domains, that is, the intrinsically unstructured head and tail domains. In contrast, much of the coiled-coil domains are scaffold elements that are essential for filament assembly. Moreover, our results suggest that Ce-lamin helix 1A has a minor scaffolding role but is important to the lateral assembly regulation of lamin protofilaments.
Scheme of DFCC analysis pipeline. A) 2D live nucleus imaging of RNA Pol II (RPb1-Dendra2) of U2OS cell line. Images are imaged by spinning disk microscopy with a time interval of Δt (200 ms). B) The raw images were pre-processed by testing the lateral drift and applying de-noising step. C) Horn-Schunck Optical Flow algorithm was applied to estimate the flow fields for each pixel over the entire nucleus. D) displacement fields output of successive two images that are estimated by Optical Flow algorithm. E) The vector`s angles were defined on a linear scale. The difference between the angles of the two vectors ~ V 1 and ~ V 2 in approximately the negative x-direction is 2π À 2 2 , with 2 small. Yet, because of the periodicity of 2π, the change can also be defined to 2 2 . F) The spatial correlation function for the displacement field direction (upper panel) and magnitude (lower panel) is computed over increasing space lags and over accessible time lags (from golden to blue). Whittle-Matérn covariance model for every time lag was applied to calculate the spatial correlation length for both direction and magnitude (insets).
Activation of transcription results in coordinated movement of chromatin over a range of micrometers. To investigate how transcriptional regulation affects the mobility of RNA Pol II molecules and whether this movement response depends on the coordinated movement of chromatin, we used our Dense Flow reConstruction and Correlation (DFCC) method. Using DFCC, we studies the nucleus-wide coherent movements of RNA Pol II in the context of DNA in humancancer cells. This study showed the dependance of coherent movements of RNA Pol II molecules (above 1 µm) on transcriptional activity. Here, we share the dataset of this study, includes nucleus-wide live imaging and analysis of DNA and RNA polymerase II in different transcription states, and the code for teh analysis. Our dataset may provide researchers interested in the long-range organization of chromatin in living cell images with the ability to link the structural genomic compartment to dynamic information. .
Protein structure of common lamin outlined in [33] (a) and mutations of the LMNA gene seen in various cancer studies of multiple cancer tissue origins depicted on NCI's GDC [81] (b).
Lamin levels within a single tumor mass can differ [44], which likely depend on the surrounding ECM, but could it also correspond to ease of access to resources? Darker staining corresponds to a higher lamin concentration, and in (a) the core tumor has significantly higher lamin than the periphery. (b) and (c) are zoom-in regions of different subsets of the cancer population notated: Be (benign), and aggressive tumors, Ca GP 3 (low-grade Gleason Pattern tumors) and Ca GP 4/5 (high-grade Gleason Pattern tumors). See [44] for how they define each tumor grade.
Positive (a) and negative (b) modulation from baseline (see control) for three different prostate cancer lines: LNCaP, DU145, and PC3 [44]. An upregulation of lamin shows a higher proliferation rate for each cell line, whereas a knockdown shows a decrease. This proliferation rate could coincide with an increase in transport through the nuclear envelope, allowing for a greater amount of nuclear 'activity', including protein, transcription, and other transport to facilitate fast growth.
Two different molecular weight trafficking rates in yeast cells of containing different FG Nup mutations. An individual mutation type is normalized to the wild-type yeast to see transport differences [66]. Interestingly, it is possible for certain mutations to show a 'preference' to one molecular weight over another. Might this happen in human cells with Nup mutations or structural changes?
Nuclear lamins and transport are intrinsically linked, but their relationship is yet to be fully unraveled. A multitude of complex, coupled interactions between lamins and nucleoporins (Nups), which mediate active transport into and out of the nucleus, combined with well documented dysregulation of lamins in many cancers, suggests that lamins and nuclear transport may play a pivotal role in carcinogenesis and the preservation of cancer. Changes of function related to lamin/Nup activity can principally lead to DNA damage, further increasing the genetic diversity within a tumor, which could lead to the reduction the effectiveness of antineoplastic treatments. This review discusses and synthesizes different connections of lamins to nuclear transport and offers a number of outlook questions, the answers to which could reveal a new perspective on the connection of lamins to molecular transport of cancer therapeutics, in addition to their established role in nuclear mechanics.
Nucleus, chromatin, and chromosome organization studies heavily rely on fluorescence microscopy imaging to elucidate the distribution and abundance of structural and regulatory components. Three-dimensional (3D) image stacks are a source of quantitative data on signal intensity level and distribution and on the type and shape of distribution patterns in space. Their analysis can lead to novel insights that are otherwise missed in qualitative-only analyses. Quantitative image analysis requires specific software and workflows for image rendering, processing, segmentation, setting measurement points and reference frames and exporting target data before further numerical processing and plotting. These tasks often call for the development of customized computational scripts and require an expertise that is not broadly available to the community of experimental biologists. Yet, the increasing accessibility of high- and super-resolution imaging methods fuels the demand for user-friendly image analysis workflows. Here, we provide a compendium of strategies developed by participants of a training school from the COST action INDEPTH to analyze the spatial distribution of nuclear and chromosomal signals from 3D image stacks, acquired by diffraction-limited confocal microscopy and super-resolution microscopy methods (SIM and STED). While the examples make use of one specific commercial software package, the workflows can easily be adapted to concurrent commercial and open-source software. The aim is to encourage biologists lacking custom-script-based expertise to venture into quantitative image analysis and to better exploit the discovery potential of their images. Abbreviations: 3D FISH: three-dimensional fluorescence in situ hybridization; 3D: three-dimensional; ASY1: ASYNAPTIC 1; CC: chromocenters; CO: Crossover; DAPI: 4',6-diamidino-2-phenylindole; DMC1: DNA MEIOTIC RECOMBINASE 1; DSB: Double-Strand Break; FISH: fluorescence in situ hybridization; GFP: GREEN FLUORESCENT PROTEIN; HEI10: HUMAN ENHANCER OF INVASION 10; NCO: Non-Crossover; NE: Nuclear Envelope; Oligo-FISH: oligonucleotide fluorescence in situ hybridization; RNPII: RNA Polymerase II; SC: Synaptonemal Complex; SIM: structured illumination microscopy; ZMM (ZIP: MSH4: MSH5 and MER3 proteins); ZYP1: ZIPPER-LIKE PROTEIN 1.
Models of accessibility control mechanisms by chromatin. (a) Steric occlusion at binding sites by nucleosomes or oligo-nucleosome contacts can prevent productive binding interactions and reduce the effective concentration of a TF or polymerase (blue circles) in a genomic region. If all binding sites are obscured, the protein is not concentrated in the genomic region, even though diffusion may be unaffected. (b) Liquid-liquid phase separation of chromatin and associated proteins can prevent proteins from entering three-dimensional regions of the nucleus (compartments) based on the proteins' chemical properties such as charge. (c) If chromatin is crosslinked into a gel, it would exclude proteins larger than the pore size of the gel regardless of their chemical properties. (d) Volume exclusion due to crowding can reduce the concentration of soluble protein in a manner that depends more weakly on size than a gel (c).
Access to DNA is a prerequisite to the execution of essential cellular processes that include transcription, replication, chromosomal segregation, and DNA repair. How the proteins that regulate these processes function in the context of chromatin and its dynamic architectures is an intensive field of study. Over the past decade, genome-wide assays and new imaging approaches have enabled a greater understanding of how access to the genome is regulated by nucleosomes and associated proteins. Additional mechanisms that may control DNA accessibility in vivo include chromatin compaction and phase separation – processes that are beginning to be understood. Here, we review the ongoing development of accessibility measurements, we summarize the different molecular and structural mechanisms that shape the accessibility landscape, and we detail the many important biological functions that are linked to chromatin accessibility.
A schematic of a canonical kinetochore. The main structure of the kinetochore consists of constitutive centromere-associated network (CCAN), which includes five subgroups (CENP-L-N, CENP-H-I-K-M, CENP-O-P-Q-R-U, CENP-T-W-S-X and CENP-C), and the KMN (Knl1, Mis12, and Ndc80 complexes) network. Kinetochore position is specified by CENP-A-containing nucleosomes, upon which CCAN assembles. CCAN recruits KMN, which directly binds microtubules, during mitosis. Two independent pathways, CENP-C and CENP-T, link KMN to CCAN.
Phosphorylation-mediated competitive exclusion between Ccp1 and Ndc80 at the N-terminus of CENP-T regulates the recruitment of KMN. The Ccp1-binding domain of CENP-T is localized adjacent to the Ndc80-binding domain at the N-terminal region of CENP-T. When cells enter mitosis, the Ccp1-binding domain of CENP-T is phosphorylated by the Cdk1 kinase. Phosphorylation of the Ccp1-binding domain dissociates Ccp1 from CENP-T, allowing Ndc80C to bind to the Ndc80-binding domain. Ndc80C then directly interacts with microtubules to facilitate chromosome segregation. When cells exit from mitosis, the Ccp1-binding domain is dephosphorylated, which recruits Ccp1. Reassociation of Ccp1 with the Ccp1-binding domain blocks the binding of Ndc80C to CENP-T during interphase. P: phosphorylation.
The kinetochore is a large proteinaceous structure assembled on the centromeres of chromosomes. The complex machinery links chromosomes to the mitotic spindle and is essential for accurate chromosome segregation during cell division. The kinetochore is composed of two submodules: the inner and outer kinetochore. The inner kinetochore is assembled on centromeric chromatin and persists with centromeres throughout the cell cycle. The outer kinetochore attaches microtubules to the inner kinetochore, and assembles only during mitosis. The review focuses on recent advances in our understanding of the mechanisms governing the proper assembly of the outer kinetochore during mitosis and highlights open questions for future investigation.
Fluorescence labeling technologies and their benefits and drawbacks.
The nucleus, central to cellular activity, relies on both direct mechanical input as well as its molecular transducers to sense external stimuli and respond by regulating intra-nuclear chromatin organization that determines cell function and fate. In mesenchymal stem cells of musculoskeletal tissues, changes in nuclear structures are emerging as a key modulator of their differentiation and proliferation programs. In this review we will first introduce the structural elements of the nucleoskeleton and discuss the current literature on how nuclear structure and signaling are altered in relation to environmental and tissue level mechanical cues. We will focus on state-of-the-art techniques to apply mechanical force and methods to measure nuclear mechanics in conjunction with DNA, RNA, and protein visualization in living cells. Ultimately, combining real-time nuclear deformations and chromatin dynamics can be a powerful tool to study mechanisms of how forces affect the dynamics of genome function.
Schematic representation of key parameters in modeling of plant nuclear architecture. (a) Nuclear shape determines the available space for the genome. (b) Chromosome territories provide major attachments sites for chromosomes and determine broad chromosome architecture. Exemplar schematic of the A. thaliana rosette chromosome configuration presented. (c) Domains and DNA loops provide local chromosome environments for genomic areas with shared features. (d) Chromatin modifications (red and gray flags) determine characteristics of individual monomers in chromosomal polymer chains.
3D modeling of local chromosome conformation in A. thaliana. Left, 2D Capture Hi-C interaction maps of a 170 kb region in the A. thaliana genome that contains a gene cluster of co-expressed neighboring genes. Right, 3D modeling of the same 170 kb region using TADbit. In green, gene cluster. In (a), the gene cluster is silenced. In (b), the gene cluster is expressed. Adapted from Nützmann et al. [59].
Chromosomes are the carriers of inheritable traits and define cell function and development. This is not only based on the linear DNA sequence of chromosomes but also on the additional molecular information they are associated with, including the transcription machinery, histone modifications, and their three-dimensional folding. The synergistic application of experimental approaches and computer simulations has helped to unveil how these organizational layers of the genome interplay in various organisms. However, such multidisciplinary approaches are still rarely explored in the plant kingdom. Here, we provide an overview of our current knowledge on plant 3D genome organization and review recent efforts to integrate cutting-edge experiments from microscopy and next-generation sequencing approaches with theoretical models. Building on these recent approaches, we propose possible avenues to extend the application of theoretical modeling in the characterization of the 3D genome organization in plants.
Time-course analysis of NE assembly. (a-d) Representative fluorescence images of the NE assembly assay at each time point. Nuclei were stained with Hoechst 33342 (magenta), and the S12 fraction was stained with ER-targeted GFP (green). White arrows (d) indicate bleb-like structures. Bars, 5 µm. (e-h) 3D volumetric rendering images of NE assembly at each time point. Nuclei were stained with Hoechst 33342 (magenta), and the S12 fraction was stained with ER-targeted GFP (green). Red arrows (f) indicate membranes associated with chromatin. Bars, 2 µm. (i, j) Representative fluorescence images of the NE assembly assay at each time point. Nuclei were stained with Hoechst 33342 (magenta), and the nuclear envelop was stained with anti-Nup43 antibody (cyan). Bars, 5 µm.
GTP-and ATP-dependent NE assembly. (a-c) Representative fluorescence images of the NE assembly assay in the presence of nucleotides, as indicated in each panel. Nuclei were stained with Hoechst 33342 (magenta), and the S12 fraction was stained with ER-targeted GFP (green). Bars, 5 µm. (d) The percentage of nuclei with NE closure. Each box is bounded by the lower and upper quartiles, the central bar represents the median, the whiskers indicate minimum and maximum values, and beeswarm plots indicate individual data points (technical replicates). The numbers of individual biological experiments are n > 20.
The coordinated regulation of the nucelar envelope (NE) reassembly during cell division is an essential event. However, there is little information on the molecular components involved in NE assembly in plant cells. Here we developed an in vitro assay of NE assembly using tobacco BY-2 cultured cells. To start the NE assembly reaction, the demembranated nuclei and the S12 fraction (cytosol and microsomes) were mixed in the presence of GTP and ATP nucleotides. Time–course analysis indicated that tubule structures were extended from the microsomal vesicles that accumulated on the demembranated nuclei, and finally sealed the NE. Immunofluorescence confirmed that the assembled membrane contains a component of nuclear pore complex. The efficiency of the NE assembly is significantly inhibited by GTPγS that suppresses membrane fusion. This in-vitro assay system may elucidate the role of specific proteins and provide important insights into the molecular machinery of NE assembly in plant cells.
HP1β differs from HP1α and HP1γ in that it contains an acidic linker domain. (a) Comparison of order/disorder prediction of HP1 homologs by the PONDR algorithm, a website tool ( VLXT scores are shown on the y-axis, amino acid positions are shown on the x-axis. (b) Ion exchange chromatography analysis of HP1 proteins. 50 µg of HP1 proteins were diluted in 500 µL buffer B (20 mM Tris-HCl, pH 8.0) and loaded on a 1 mL Resource Q column and analyzed by using a Äkta Pure FPLC system. (c) Net charge distribution per residue (NCPR) of HP1 proteins (CIDER, Negatively charged amino acids are marked in red, positively charged amino acids in blue. The pI of IDR-H in HP1 proteins is indicated.
HP1β cannot self-phase separate because of its acidic linker domain. (a) DIC images of HP1 droplets at 4°C in a buffer containing 20 mM HEPES pH 7.2, 75 mM KCl and 1 mM DTT using the 63x objective of a DeltaVision Personal Microscope (scale bar: 10 µm). Protein concentrations are as indicated. N.D.: not done. (b) Phase separation of engineered HP1β at 170 µM and 4°C with four amino acid substitutions in the IDR-H changing it from acidic to basic (HP1β RKRK). A zoomed-in image is shown with the same magnification as in (a). Scale bar: 10 µm.
HP1β can form phase-separated droplets in the presence of histones. (a) Illustration of isolating mononucleosomes by MNase treatment (left). (b) Mononucleosome solution was incubated with or without 30 µg of HP1β at 4°C in a buffer containing 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 3 mM CaCl 2 , 0.1% NP-40 and 1.5 mM EDTA. Phase-separated droplets were pelleted by centrifugation. Proteins in the supernatant (S) and phase-separated droplets (P) were separated and visualized by coomassie blue SDS-PAGE gels and western blotting with an anti-H3 antibody. (c-e) HP1 phase separation in the presence of histones isolated by acid-extraction from HEK293T cells in a buffer of 20 mM HEPES pH 7.2, 75 mM KCl and 1 mM DTT. 50 µM of HP1 homologs were incubated with 50 µM of histones (scale bar: 10 µm) (c). 3 to 100 µM of HP1β was incubated with 100 µM of histones. HP1β phase-separated droplets were separated and visualized as above (d). 50 µM of HP1β was incubated with 50 µM of histones in a buffer with NaCl concentrations ranging from 50 to 800 mM. Proteins in the P and S fractions were analyzed as above (e). (f) Phase diagram of HP1β with protein and salt concentration as order parameters. Phase separation was scored by the presence or absence of droplets in the sample.
Trimethylation of K9 of histone H3 (H3K9me3) and histone dimerization are required for HP1β phase separation. (a) HP1β protein from 3 to 100 µM was incubated with 100 µM of histones at 4°C in a buffer containing 20 mM HEPES pH 7.2, 75 mM KCl and 1 mM DTT. HP1β phase-separated droplets were separated by spin down. Proteins in P and S fractions were analyzed by SDS-PAGE gels and visualized western blot with anti-H3K9me3 antibody. (b) Representative DIC images show HP1β phase separation assay outcome in the presence of histones or histone H3 peptide (aa 1-20) carrying H3K9me3. 25 µM of HP1β was incubated with either 25 µM core histones or H3K9me3 peptide (aa 1-20). (c) Analysis of histones by size exclusion chromatography (SEC). 250 µg of histones were diluted in a buffer of 20 mM Tris-HCl, 300 mM NaCl, pH 7.4 and separated on an equilibrated Superdex 200 Increase 10/300 GL column. For size comparison a protein marker mix including carbonic anhydrase (29 kDa), bovine serum albumin (66 kDa), alcohol dehydrogenase (150 kDa), beta-amylase (200 kDa), apoferritin (443 kDa), and thyroglobulin (669 kDa) was analyzed under identical conditions. (d) Histone H3 peptide (aa 1-20) carrying H3K9me3, or H3K9me1 or H3K9ac was incubated with 25 µM of HP1β and histones. Proteins in S and P fractions were analyzed and visualized by coomassie stained SDS-PAGE gels and H3 peptides by fluorescent imaging.
HP1β phase separation contributes to heterochromatin formation in vivo. (a) Illustration of the binding of H3K9me3 and the CD domain of HP1β. The amino acids, tyrosine (Y) 21, tryptophan (W) 42 and phenylalanine (F) 45, form an aromatic cage for H3K9me3 peptide that is abolished by the replacement of K41W42 with alanine (A) [15]. (b and c) Schematic representations show the CRISPR/Cas9 gene-editing strategy used to generate MIN tagged HP1β mESCs. The donor harbors the MIN tag sequence (attP) and homology arms to the genomic sequence 55 and 33 of the translational start site. The targeting region was amplified with primers as indicated and assessed by Sanger sequencing. (d) Schematic representation shows the strategy to generate GFP-HP1β WT and KW mESC lines with Bxb1 mediated recombination. (e) Gel electrophoresis of the multiplex PCR for validation of GFP-HP1β mESCs with primers as indicated in (d). 343 bp and 259 bp sequences were amplified from E14 and GFP-HP1β cells, respectively. (f) Representative images of GFP-HP1β WT and KW mESCs stained with an anti-H3K9me3 antibody. Scale bar: 5 µm. See overview images in Figure S10. (g) FRAP quantification of GFP-HP1β WT and GFP-HP1β KW. Curves show average GFP signal relative to the fluorescence signal prior to bleaching (WT, n = 20 and KW_C (chromocenter), n = 6 and KW_D (diffuse), n = 6). The areas used for FRAP are indicated by circles in (f).
Liquid-liquid phase separation (LLPS) mediated formation of membraneless organelles has been proposed to coordinate biological processes in space and time. Previously, the formation of phase-separated droplets was described as a unique property of HP1α. Here, we demonstrate that the positive net charge of the intrinsically disordered hinge region (IDR-H) of HP1 proteins is critical for phase separation and that the exchange of four acidic amino acids is sufficient to confer LLPS properties to HP1β. Surprisingly, the addition of mono-nucleosomes promoted H3K9me3-dependent LLPS of HP1β which could be specifically disrupted with methylated but not acetylated H3K9 peptides. HP1β mutants defective in H3K9me3 binding were less efficient in phase separationin vitro and failed to accumulate at heterochromatin in vivo. We propose that multivalent interactions of HP1β with H3K9me3-modified nucleosomes via its chromodomain and dimerization via its chromoshadow domain enable phase separation and contribute to the formation of heterochromatin compartments in vivo.
Behavior of a polymer pinned by one or two anchors to a surface NP without attraction potential. This experiment models the space explored (distance to NP) by a chromatin polymer anchored at one or both ends to a surface representing the nuclear lamina devoid of any attraction strength toward the polymer. (a) Mean distance from bead center to NP along the polymer, with bead 1 as the sole anchor. Gray shades represent increasing polymer stiffness L P (legend bottom right), from flexible (L P = 5 nm) to near-rigid (L P = 200 nm). (b) Mean distance from bead center to NP along a polymer anchored at both ends (beads 1 and 12) to NP, with a Euclidian distance between them of (i) d E = 50 nm (relaxed polymer) and (ii) d E = 300 nm (stretched polymer). Data for other d E values are shown in Figure S1a. In (a) and (b), black lines represent the theoretical rigid approximation (see Supplemental information 2); gray shades represent increasing L P ; lines connect data points for L P = 5 nm for clearer visualization of the trends. (c) Distance from bead center to NP as a function of Euclidian distance d E between anchors, for increasing polymer stiffness L P (gray shades) and at indicated bead position i along the chain (top). Given approximations of L P as a function of chromatin compaction (Supplemental information 1), our simulations model chromatin behavior at the nuclear periphery for euchromatic domains (L P = 5 and 50 nm) and heterochromatic domains (L P = 100 and 200 nm).
Polymer configurations at a surface NP fitted with an attraction potential toward the chromatin polymer. This experiment tests the effect of introducing a variable attraction force in the nuclear lamina on the conformation of chromatin at the lamina (in the form of tail, trains or loops) and on the size of these configurations (number of polymer beads). (a,b) Number of beads in tailtrain-loop configurations as a function of attraction strength (x axis) and polymer stiffness (L P ; gray, red and blue shades; legend) in simulations with (a) one anchor point and (b) two anchor points and indicated d E between them. (c) Definition of polymer adsorption/desorption regimes at NP from the proportion of beads in a tail-train-loop configuration (F tail/train/loop = <F train > -<F other >), with: adsorption when F tail/train/loop > 50% (red), adsorption-desorption transitions when 0 < F tail/train/loop ≤ 50% (yellow), and desorption when F tail/train/loop ≤ 0 (green). Numbers are F tail/train/loop in percent of the modeled structures. We refer to Supplementary information 1 to relate chromatin polymer stiffness (L P ) to chromatin compaction in the nucleus. While low persistence length (L P = 5-50 nm; (semi)-flexible polymer) characterizes euchromatin, elevated persistence lengths (L P = 100-200 nm; semi-flexible to rigid polymer) are properties of heterochromatin. We infer that regardless of the number of anchors, euchromatin is more prone than heterochromatin to stochastic associations with the lamina (modeled as adsorption-desorption regimes) even when it is stretched.
Interactions of chromatin with the nuclear lamina imposes a radial genome distribution important for nuclear functions. How physical properties of chromatin affect these interactions is unclear. We used polymer simulations to model how physical parameters of chromatin affect its interaction with the lamina. Impact of polymer stiffness is greater than stretching on its configurations at the lamina; these are manifested as trains describing extended interactions, and loops describing desorbed regions . Conferring an attraction potential leads to persistent interaction and adsorption-desorption regimes manifested by fluctuations between trains and loops. These are modulated by polymer stiffness and stretching, with a dominant impact of stiffness on resulting structural configurations. We infer that flexible euchromatin is more prone to stochastic interactions with lamins than rigid heterochromatin characterizing constitutive LADs. Our models provide insights on the physical properties of chromatin as a polymer which affect the dynamics and patterns of interactions with the nuclear lamina.
Figure S4: Simulated Hi-C data. (A) Simulated Hi-C matrices for loop changes (See Supplementary Table S2). The original Hi-C matrix is shown on the left, with matrices that are increasingly more divergent from left to right. Green circles indicate where the matrix has been perturbed to change the chromatin loop structure. (B) Simulated Hi-C matrices for compartment changes (See Supplementary Table S2). The original Hi-C matrix is shown on the left, with matrices that are increasingly more divergent from left to right. Green arrows indicate where the matrix has been perturbed to change the chromatin compartment structure.
Data on genome organization and output over time, or the 4D Nucleome (4DN), require synthesis for meaningful interpretation. Development of tools for the efficient integration of these data is needed, especially for the time dimension. We present the “4DNvestigator”, a user-friendly network based toolbox for the analysis of time series genome-wide genome structure (Hi-C) and gene expression (RNA-seq) data. Additionally, we provide methods to quantify network entropy, tensor entropy, and statistically significant changes in time series Hi-C data at different genomic scales. Availability:
Distribution of nuclear envelope proteins from metazoa across eukaryotes. Coulson plot demonstrating presence or absence of NE proteins across the eukaryotes. Filled circles indicate proteins identified, open circles indicate proteins for which orthologs were not found. Rows are predicted proteins and columns are organisms. Supergroups are colourised using the same system as in Figure 2. Three groups are recognized: Group A; highly conserved across Eukaryotic supergroups, Group B; originated in an amorphean ancestor of Metazoa.
Frequency of gene ontology annotation for nuclear envelope proteins from the metazoan cohort. bar chart showing frequency of GO annotation of Biological process of highly conserved proteins across supergroups (Group A), proteins present across Amorphea (Group B) and proteins restricted to Metazoa (Group C) from .Figure 3
Distribution of trypanosomatid nuclear envelope proteins across eukaryotes. Coulson plot demonstrating presence or absence of trypanosome NE orthologs across the eukaryotes. filled circles indicate proteins identified, open circles indicate proteins for which orthologs were not found. rows are predicted proteins and columns are organisms. supergroups are colourised using the same system as Figure 2. Two groups are recognized: Group A; scattered distribution across taxa and Group C; restricted to kinetoplastids. TriTrypDB [49] accession IDs are shown on the left.
Eukaryotic cells arose over 1.5 billion years ago, with the endomembrane system a central feature, facilitating evolution of specialised intracellular compartments. Endomembranes include the nuclear envelope (NE) that divides the cytoplasm from the nucleoplasm. The NE possesses universal features, specifically a double lipid bilayer membrane, nuclear pore complexes (NPCs), and continuity with the endoplasmic reticulum, indicating a common evolutionary origin. However, the levels of specialisation between eukaryotic lineages remains unclear, despite clear evidence for distinct mechanisms underpinning various nuclear activities. Several distinct modes of molecular evolution facilitate organellar diversification and include gene loss (sculpting), replacement/repurposing (backfilling), paralog expansion and emergence of novel genes in specific lineages. To understand mechanisms that apply to the NE, we exploited previously described proteome datasets of purified nuclear envelopes from model systems for comparative analysis. We find enrichment of core nuclear functions amongst the most widely conserved proteins, which account for a small fraction of the total, while the largest cohorts are likely lineage-specific. This, together with consideration of additional published studies, suggests that, despite a common origin, the NE has evolved as a highly diverse organelle with significant lineage-specific functionality.
Evaluation of a 3D gift-wrapping method of segmentation. (a) Example of a nucleus raw slice (left) after Otsu-modified segmentation (middle) and gift-wrapping segmentation (right). Artefactual indentation at the nuclear border (arrow #1); Nucleolus border (arrow #2).(b) Comparison of Otsu and gift-wrapping methods using standardized microspheres. Microsphere volume of 1, 2.5 and 4 µm diameter (n = 28, 24, and 15, respectively) were computed by the Otsu (green triangle) and gift-wrapping (gray square) methods and compared to theoretical volumes (red circle) (Supplemental table 2).(c) Comparison of nuclear volumes after segmentation of plant nuclei by the Otsu or gift-wrapping methods. Nuclei were split into two categories: guard cells (n = 375) and pavement cells (n = 127) (Supplemental table 4) were segmented by the two methods and volumes of the segmented nuclei were computed by NucleusJ 2.0. Modified Otsu method (gray); giftwrapping (green). Student t-test P-value: *** <0.0001, * = 0.0046.(d) Principal component analysis of morphology parameters (Flatness, Elongation, New surface area and Volume) obtained after segmentation by the gift-wrapping (left) or Otsu (right) methods of the same nuclei as in Figure 2c. Guard cell nuclei (GC, black) and pavement cell nuclei (PC, orange).
NucleusJ2.0 analysis of the k4c1c4 mutant with 1altered nuclear morphology and chromatin organization. Nuclear morphology parameters were computed by the gift-wrapping segmentation method using an initial dataset of WT (n = 663) and k4c1c4 mutant (n = 881) nuclei described in Supplemental table 4.(a) Nuclear morphology parameters computed by NucleusJ 2.0 on GC and PC cells: nuclear volume (µm 3 ), flatness, elongation and surface area (µm 2 ). WT (n = 502) and k4c1c4 mutant (n = 672) (Supplemental table 4). Student t-test P-value: * <0.01, ** <0.001 and *** <0.0001.(b) Chromatin organization parameters computed by NucleusJ 2.0: number of chromocentres (NbCc), Mean of chromocentre volume (VCc Mean, µm 3 ), total volume of chromocentres (VCc Total, µm 3 ), and distance between chromocentre barycenter and the nuclear envelope (d(CcBarycentre/NE), µm). WT (n = 186) and k4c1c4 mutant (n = 81) (Supplemental table 6). Student t-test P-value: ns > 0.01, ** <0.001 and *** <0.0001.(c) and d) Principal component analysis using morphological parameters (n = 1544; Supplemental table 4) and chromatin organization parameters (n = 267; Supplemental table 6).(e) Typical images chosen with parameters close to the median values of morphological parameters and chromatin organization parameters. Z-projection of raw nuclei. Scale Bar 2 µm. GC: guard cell, PC: pavement cell.
Analysis of aspect and position of 180pb and 5S rDNA repeats revealed by 3D-DNA FISH using NucleusJ 2.0. NucleusJ 2.0 parameters applied to A) 180bp signals and B) 5S rDNA. Parameters: number of DNA FISH signals, mean volume of FISH signal (µm 3 ), total volume of FISH signal (µm 3 ), distance between FISH signal border and the nuclear envelope (d(FISH signal Border/ NE), µm) and distance between FISH signal barycenter and the nuclear envelope (d(Barycenter of FISH signal/NE), µm) (Supplemental tables 7-8). Student t-test P-value: ns >0.01, * <0.01, ** <0.001 and *** <0.0001. C) Typical 3D DNA FISH Z-projection of pavement cell nuclei of WT (n = 65 for 180bp and n = 32 for 5S) and D) k4c1c4 mutant (n = 95 for 180bp and n = 48 for 5S) (Supplemental tables 7-8). From left to right: Hoechst (DNA, blue), 55 TYE 563 LNA probe (180bp, purple), CY5 PCR probe (5S, green) and merge. Scale Bar 2 µm.
OMERO-FSU Datasets.
NucleusJ 1.0, an ImageJ plugin, is a useful tool to analyze nuclear morphology and chromatin organization in plant and animal cells. NucleusJ 2.0 is a new release of NucleusJ, in which image processing is achieved more quickly using a command-lineuser interface. Starting with large collection of 3D nuclei, segmentation can be performed by the previously developed Otsu-modified method or by a new 3D gift-wrapping method, taking better account of nuclear indentations and unstained nucleoli. These two complementary methods are compared for their accuracy by using three types of datasets available to the community at . Finally, NucleusJ 2.0 was evaluated using original plant genetic material by assessing its efficiency on nuclei stained with DNA dyes or after 3D-DNA Fluorescence in situ hybridization. With these improvements, NucleusJ 2.0 permits the generation of large user-curated datasets that will be useful for software benchmarking or to train convolution neural networks.
The functional organization of the plant nuclear envelope has recently gained increasing attention due to new connections made between the proteins of the nuclear periphery and important plant biological processes. Better understood from animal research, nuclear envelope and nuclear peripheral proteins are known to play roles in nuclear morphology, subcellular nuclear anchoring and movement, chromatin tethering and the mechanical interplay between nucleoplasm and cytoplasm. However, precisely how these roles translate to functionality in a broader biological context is often not well understood. Plants have their own set of nuclear envelope-associated proteins that sometimes overlap with their animal counterparts, but are more often plant-unique, suggesting that functionalities evolved after the split of Opisthokonta and Streptophyta about one billion years ago. During the past few years, significant progress has been made in discovering broader biological roles of plant nuclear envelope proteins, increasing the number of known plant nuclear envelope proteins, and connecting known proteins to chromatin organization, gene expression, and the regulation of nuclear calcium. Interactions of viruses with the plant nuclear envelope are another emerging theme. Here, we survey these recent advances and propose directions for the future development of this still relatively new, yet rapidly advancing field.
Number of Paralogues of LINC complex and associated components in monocot and dicot lineages.
Schematic representation summarizing key functions of nuclear periphery components in plants.
Schematic comparison of nuclear periphery components for monocot and dicot crops and the model species Arabidopsis thaliana.
In this review, we explore recent advances in knowledge of the structure and dynamics of the plant nuclear envelope. As a paradigm, we focused our attention on the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, a structurally conserved bridging complex comprising SUN domain proteins in the inner nuclear membrane and KASH domain proteins in the outer nuclear membrane. Studies have revealed that this bridging complex has multiple functions with structural roles in positioning the nucleus within the cell, conveying signals across the membrane and organizing chromatin in the 3D nuclear space with impact on gene transcription. We also provide an up-to-date survey in nuclear dynamics research achieved so far in the model plant Arabidopsis thaliana that highlights its potential impact on several key plant functions such as growth, seed maturation and germination, reproduction and response to biotic and abiotic stress. Finally, we bring evidences that most of the constituents of the LINC Complex and associated components are, with some specificities, conserved in monocot and dicot crop species and are displaying very similar functions to those described for Arabidopsis. This leads us to suggest that a better knowledge of this system and a better account of its potential applications will in the future enhance the resilience and productivity of crop plants. © 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
Lamin polymerization and phosphorylation. (a) Schematic representation of lamin polymerization. Lamins form dimers through rod domain interactions. Lamin dimers associate longitudinally into polar head-to-tail polymers. (b) Distribution of phosphorylation sites in Lamin A/C. Phosphorylation sites are stratified by the cell-cycle phase in which the residue is reported to be phosphorylated.
Regulators and functions of lamin phosphorylation. (a) Kinases and phosphatases known to regulate lamin phosphorylation are shown. Phosphorylation of lamins could cause depolymerization and localization to the nuclear interior. (b) Phosphorylated Lamin A/C bind to enhancers of active genes in the nuclear interior.
Decades of studies have established that nuclear lamin polymers form the nuclear lamina, a protein meshwork that supports the nuclear envelope structure and tethers heterochromatin to the nuclear periphery. Much less is known about unpolymerized nuclear lamins in the nuclear interior, some of which are now known to undergo specific phosphorylation. A recent finding that phosphorylated lamins bind gene enhancer regions offers a new hypothesis that lamin phosphorylation may influence transcriptional regulation in the nuclear interior. In this review, we discuss the regulation, localization, and functions of phosphorylated lamins. We summarize kinases that phosphorylate lamins in a variety of biological contexts. Our discussion extends to laminopathies, a spectrum of degenerative disorders caused by lamin gene mutations, such as cardiomyopathies and progeria. We compare the prevailing hypothesis for laminopathy pathogenesis based on lamins’ function at the nuclear lamina with an emerging hypothesis based on phosphorylated lamins’ function in the nuclear interior.
Lamins interact with the nuclear membrane and chromatin but the precise players and mechanisms of these interactions are unknown. Here, we tested whether the removal of the CaaX motif from Lamin B disrupts its attachment to the nuclear membrane and affects chromatin distribution. We used Drosophila melanogaster LamA25 homozygous mutants that lack the CaaX box. We found that the mutant Lamin B was not confined to the nuclear periphery but was distributed throughout the nuclear interior, colocalizing with chromosomes in salivary gland and proventriculus. The peripheral position of Lamin C, nuclear pore complex (NPC), heterochromatin protein 1a (HP1a), H3K9me2- and H3K27me3-associated chromatin remained intact. The fluorescence intensity of the DAPI-stained peripheral chromatin significantly decreased and that of the central chromatin significantly increased in the proventriculus nuclei of the mutantflies compared to wild-type. However, the mutation had little effect on chromatin radial distribution inside highly polytenized salivary gland nuclei.
The reduction of nuclear F-actin in progerinexpressing cells. (a) Human dermal fibroblast (HDF) cells containing GFP-lamin A or GFP-progerin cDNA regulated by a Doxinducible promoter were transfected with nAC-mCherry, and the cells cultured with or without Dox for 96 hr. Typical mCherry images corresponding to the nuclei with F-actin (w/ F-actin) and to nuclei without F-actin (w/o F-actin) are shown. The numbers under panels indicate the percentage of cells possessing nuclear F-actin in each cell group. Scale bar, 2 μm. (b) Relative frequency of nuclei with nuclear F-actin. The mean value of lamin A-Dox(+) was defined as 1.0. Data shown are mean ± standard error measurement (SEM) of six independent experiments. For the quantification, 735 cells (lamin A-Dox(+)) and 771 cells (progerin-Dox(+)) were analyzed. (c) Relative transcriptional activity of the TCF/LEF promoter measured by luciferase-assay. The mean measured value in lamin A-Dox(-) was assigned as 1.0. Data shown are mean ± SEM of four independent experiments. (d) The relative expression level of TCF-1 in control and progerin-expressing cells. The expression level was measured by qRT-PCR and was normalized with respect to that of GAPDH gene. The expression level of TCF-1 in lamin A-Dox(-) was assigned as 1.0. Data shown are mean ± SEM of four independent experiments. n.s., not significant; *, P < 0.05; **, P < 0.01.
Indices of nuclear actin in progerin-expressing cells. (a) The relative expression level of ACTB gene in progerin-expressing cells. The expression level was measured by qRT-PCR and was normalized with respect to that of GAPDH gene. The expression level of ACTB in progerin-Dox(-) cells was assigned as 1.0. Data shown are mean ± SEM of three independent experiments. (b) Images of progerin-expressing cells stained by DAPI and DNase I conjugated with Alexa Fluor 594 (DNase I). Scale bar, 10 μm. (c) The relative amount of nuclear G-actin was defined as the ratio of nuclear to the cytoplasmic signal intensity of DNase I-staining of progerinexpressing cells. Data are shown as a box plot. The mean value of progerin-Dox(-) cells was assigned as 1.0. Error bars indicate standard deviation (SD) of three independent experiments. For quantification, 88 cells (progerin-Dox(-)) and 87 cells (progerin-Dox (+)) were analyzed. More than 20 cells were analyzed in each independent experiment. (d) The relative amount of nuclear mCherry-NLS-actin was defined as the ratio of nuclear to the cytoplasmic signal intensity of mCherry-fluorescence in progerin-expressing cells and shown as in C. Error bars indicate SD of three independent experiments. For quantification, 63 cells and 83 cells were analyzed. More than 20 cells were analyzed in each independent experiment. (e) Relative frequency of nuclei possessing nuclear F-actin in progerin-expressing cells treated with FTI. The mean value of progerin-expressing cells without FTI treatment was defined as 1.0. Data shown are mean ± SEM of three (4.0 μM FTI) and four (0, 0.5, 1.0, and 2.0 μM FTI) independent experiments. For quantification, 459 cells (0 μM FTI), 429 cells (0.5 μM FTI), 533 cells (1.0 μM FTI), 506 cells (2.0 μM FTI), and 335 cells (4.0 μM FTI) were analyzed. n.s., not significant.
Complementation of HGPS phenotypes by an artificial increase of nuclear F-actin. (a) Frequency of nuclei possessing nuclear F-actin in progerin-expressing cells. The progerinexpressing cells were transfected with a control solution (mock), EYFP-actin (actin), or EYFP-NLS-actin (NLS-actin) together with nAC-mCherry. The mean value of cells transfected with a mock plasmid was defined as 1.0. Data shown are mean ± SEM of four independent experiments. For quantification, 418 cells (mock), 408 cells (actin), and 446 cells (NLS-actin) were analyzed. (b) The nucleus of progerin-expressing cells expressing mCherry-actin, mCherry-NLS-actin, or mCherry-NLS-G13R actin. Images are shown of GFP-progerin, mCherry-actin derivatives, and DAPI-staining. The image of DAPI was merged with GFP and mCherry signals (right panels). Scale bar, 2 μm. (c) The NII value of progerin-Dox(+) cells expressing mCherry-actin (actin), mCherry-NLS-actin (NLS-actin), or mCherry-NLS-G13R actin (NLS-G13R actin). Data shown are mean ± SEM of three independent experiments. For quantification, 526 cells (no transfection, progerin-Dox(-)), 317 cells (no transfection, progerin-Dox(+)), 310 cells (actin), 312 cells (NLS-actin), and 300 cells (NLS-G13R actin) were analyzed. (d) Relative luciferase activity of the TCF/LEF promoter analyzed by luciferase-assay in progerin-Dox(-) and progerin-Dox(+) cells. The cells were transfected with a plasmid expressing mCherry (mock) or mCherry-NLS-actin (NLS-actin). The value in progerin-Dox(-) cells expressing mCherry was assigned as 1.0. Data shown are mean ± SEM of three independent experiments. n.s., not significant; *, P < 0.05; **, P < 0.01.
Complementation of nuclear irregularity of progerinexpressing cells by treatment with jasplakinolide. (a) The progerin-expressing cells were treated with 10 nM or 50 nM jasplakinolide for 24 hours, and then stained with DAPI. DAPI and GFP-progerin images of the cells are compared with those of cells not treated with jasplakinolide (w/o Jasp). Scale bar, 10 μm. (b) The NII values of progerin-Dox(-) cells without jasplakinolide treatment (w/o Jasp) and of progerin-Dox(+) cells with or without jasplakinolide treatment (w/o Jasp, 10 nM, 20 nM or 50 nM) were compared. The NII value was determined in each cell group. Error bars indicate SEM of three independent experiments. For quantification, 526 cells (w/o Jasp, progerin-Dox(-)), 317 cells (w/o Jasp, progerin-Dox(+)), 453 cells (Jasp 10 nM), 630 cells (Jasp 20 nM), and 596 cells (Jasp 50 nM) were analyzed in each experiment. (c) The relative expression level of TCF-1 in progerin-expressing cells treated with jasplakinolide. The expression level was measured by qRT-PCR and was normalized with respect to that of GAPDH gene. The expression level of TCF-1 in progerin-Dox(+) without jasplakinolide treatment was assigned as 1.0. Data shown are mean ± SEM of three independent experiments. n.s., not significant; *, P<0.05; **, P < 0.01; ****, P < 0.0001.
Effects of inducing artificial nuclear F-actin in DNA damage repair and cell senescence. (a) γH2AX foci in progerinexpressing cells expressing mCherry-actin, mCherry-NLS-actin, or mCherry-NLS-G13R actin were detected by fluorescence microscopy. Representative images of γH2AX and GFPprogerin are shown. Scale bar, 2 μm. (b) The number of γH2AX foci per nucleus in progerin-Dox(-) and progerin-Dox (+) cells. The latter cells expressing mCherry-actin (actin), mCherry-NLS-actin (NLS-actin), or mCherry-NLS-G13R actin (NLS-G13R actin) were also analyzed. Data shown are mean ± SEM of three independent experiments, and 100 cells were analyzed in each experiment. (c) The relative signal intensity of SA-β-gal (arbitrary units) in progerin-expressing cells with or without the expression of mCherry-NLS-actin. The relative intensity of progerin-expressing cells without mCherry-NLSactin was assigned as 1.0. Data shown are mean ± SEM of three independent experiments. n.s., not significant; *, P < 0.05; ****, P < 0.0001.
Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disorder caused by a mutation of lamin A, which contributes to nuclear architecture and the spatial organization of chromatin in the nucleus. The expression of a lamin A mutant, named progerin, leads to functional and structural disruption of nuclear organization. Since progerin lacks a part of the actin-binding site of lamin A, we hypothesized that nuclear actin dynamics and function are altered in HGPS cells. Nuclear F-actin is required for the organization of nuclear shape, transcriptional regulation, DNA damage repair, and activation of Wnt/β-catenin signaling. Here we show that the expression of progerin decreases nuclear F-actin and impairs F-actin-regulated transcription. When nuclear F-actin levels are increased by overexpression of nuclear-targeted actin or by using jasplakinolide, a compound that stabilizes F-actin, the irregularity of nuclear shape and defects in gene expression can be reversed. These observations provide evidence for a novel relationship between nuclear actin and the etiology of HGPS.
Schematic representation of the NE and of its components. The NE is composed of two phospholipid bilayers (INM and ONM), separated by a perinuclear space. The ONM is an extension of the ER and is directly connected with the INM at NPCs. Both the ONM and INM contain a set of proteins, including the LEM domain proteins, as LAP2, emerin, and MAN1. The LINC complex puts in contact the lumen of the NE with the cytoskeleton and is formed by the SUN and KASH proteins as nesprin. Chromatin is at the nuclear periphery via bridging elements as BAF.
The nuclear envelope compartmentalizes chromatin in eukaryotic cells. The main nuclear envelope components are lamins that associate with a panoply of factors, including the LEM domain proteins. The nuclear envelope of mammalian cells opens up during cell division. It is reassembled and associated with chromatin at the end of mitosis when telomeres tether to the nuclear periphery. Lamins, LEM domain proteins, and DNA binding factors, as BAF, contribute to the reorganization of chromatin. In this context, an emerging role is that of the ESCRT complex, a machinery operating in multiple membrane assembly pathways, including nuclear envelope reformation. Research in this area is unraveling how, mechanistically, ESCRTs link to nuclear envelope associated factors as LEM domain proteins. Importantly, ESCRTs work also during interphase for repairing nuclear envelope ruptures. Altogether the advances in this field are giving new clues for the interpretation of diseases implicating nuclear envelope fragility, as laminopathies and cancer. Abbreviations na, not analyzed; ko, knockout; kd, knockdown; NE, nuclear envelope; LEM, LAP2-emerin-MAN1 (LEM)-domain containing proteins; LINC, linker of nucleoskeleton and cytoskeleton complexes; Cyt, cytoplasm; Chr, chromatin; MB, midbody; End, endosomes; Tel, telomeres; INM, inner nuclear membrane; NP, nucleoplasm; NPC, Nuclear Pore Complex; ER, Endoplasmic Reticulum; SPB, spindle pole body.
Punctate inclusions in progerin-expressing nuclei. Confocal images of HUVECs labeled via immunocytochemistry for endogenous LA/C, overexpressed LA and HA-tagged progerin. Control and LA cells show uniform equitorial labeling with come wrinkles due to actin fibers whereas progerin-expressing nuclei show punctate inclusions (arrows). The z-resolution for the lamin channel (488 nm) was chosen at 1.0 μm, so folds and puncta of the nuclear face may appear in the same confocal frame as the midline edge.
Confocal fluorescence microscopy confocal sections for cells patterned on lines. Fixed HUVECs were stained for Lamin (control) or HA (Progeria), and all cells were also stained for actin (phallodin) and DNA (Hoechst 33342). (a) Lamin A/C (control) stained with a lamin A/C antibody. (b) Control cell stained for actin to check the orientation of folds against the filament structures. (c) Lamin A control cells with Hoescht staining for DNA. (d) Merge of the lamin and actin channels shows nuclear alignment with the stripes and lamin folds coincident with the actin filaments. (e) Progerin-expressing cells stained with anti-HA to label HA-progerin express more wrinkles. (f) Progerinexpressing cells stained for actin to show the orientation of folds against the filament structures. (g) Progerin-expressing cells with Hoescht 33342 staining for DNA. (h) Merge of the lamin and actin shows lamin folds distinct from actin filaments. For both conditions the z-resolution for the lamin channel (488 nm) was chosen at 3.5 μm, actin channel (561 nm) 1.9 μm and DNA channel (405 nm) 1.3 μm.
Formation of wrinkles for cells under one-dimensional confinement. (a) Length of deformations or wrinkles for control, exogenous lamin A or HA-progerin expressing endothelial cells cultured on 20 µm diameter stripes. (b) On 20 µm diameter stripes, wrinkles in control cells and exogenous lamin A expressing cells (+ lamin A) primarily align with the stripe axis whereas HA-progerinexpressing cells do not show preferred orientation. (c) On 20 µm diameter stripes, treatment with latrunculin A and fixation at different time points shows an exponential decay. (d) Fits of exponential decay shows the differential decay constants for control and exogenous lamin A versus HA-progerin cells. Fits same for 4 points as 2 points. 30-50 cells per condition considered. * indicates statistically similar p > 0.05; ** indicates 0.001 < p < 0.05; (c and d) no * indicates statistically different with p < 0.001 using unpaired Students t-test.
Model of nuclear lamina under force. (a) The nuclear lamina for control cells experiences a thinning of membrane and dilation of lamin A network. (b) The nuclear lamina for progerin-expressing cells experience high stress and buckle at the aggregates irrespective of force application. Wrinkles then emanate from the aggregate space. (c) In control cells cytoskeletal forces are balanced through the nuclear lamina and are propagated from one side of the nucleus to the other. (d) With wrinkles or defects in progerin-expressing cells forces may be disrupted along the lamina.
The nuclear lamina is a meshwork of intermediate filament proteins, and lamin A is the primary mechanical protein. An altered splicing of lamin A, known as progerin, causes the disease Hutchinson-Gilford progeria syndrome. Progerin-expressing cells have altered nuclear shapes and stiffened nuclear lamina with microaggregates of progerin. Here, progerin microaggregate inclusions in the lamina are shown to lead to cellular and multicellular dysfunction. We show with Comsol simulations that stiffened inclusions causes redistribution of normally homogeneous forces, and this redistribution is dependent on the stiffness difference and relatively independent of inclusion size. We also show mechanotransmission changes associated with progerin expression in cells under confinement and cells under external forces. Endothelial cells expressing progerin do not align properly with patterning. Fibroblasts expressing progerin do not align properly to applied cyclic force. Combined, these studies show that altered nuclear lamina mechanics and microstructure impacts cytoskeletal force transmission through the cell.
Crm1 inhibits spindle assembly. Recombinant Crm1 (c-g) was added to mitotic extract supplemented with rhodamine-labeled tubulin and compared to GST (a) and Transportin controls (b). Typical images for each reaction are shown. GST (A) addition did not interfere with the production of strong bipolar spindles, while 15 μM Transportin (B) strongly inhibited bipolar spindle formation. Low concentrations (1-2 μM) of Crm1 (c and d) had little effect on bipolar spindle formation. However, increasing concentrations (8-10 μM) Crm1 had an increasingly deleterious effect on bipolar spindle formation (e and f). Bipolar spindle assembly was completely inhibited by 15 μM Crm1 (g).
A model for Exportin action in nuclear pore assembly. The exportins Crm1 and Exportin-t are proposed to regulate nuclear pore assembly in vitro by binding to critical Nup subunits in regions distant from the chromatin and preventing the subunits from assembling in those distant loci. In the model this inhibition is released around chromatin in response to the locally high concentration of RanGTP that is produced by the chromatin-bound Ran-GEF, RCC1. Because nuclear membrane assembly has not yet sealed off the genome in these early stages of nuclear assembly, RanGTP produced by RCC1 is able to locally leave the forming nucleus and release nearby soluble Nup subunits from exportin inhibition. This frees the Nup subunits to assemble into complete nuclear pore complexes in the forming nuclear membranes (green). A similar model has been proposed for the importins Importin β and Transportin (Trn) in their regulation of nuclear pore assembly during the cell cycle [41,47]. Exportin-5 (not shown) could act similarly to Crm1 and Exportin-t in the mechanism shown here.
Xenopus egg extracts are a powerful in vitro tool for studying complex biological processes, including nuclear reconstitution, nuclear membrane and pore assembly, and spindle assembly. Extracts have been further used to demonstrate a moonlighting regulatory role for nuclear import receptors or importins on these cell cycle assembly events. Here we show that exportins can also play a role in these events. Addition of Crm1, Exportin-t, or Exportin-5 decreased nuclear pore assembly in vitro. RanQ69L-GTP, a constitutively active form of RanGTP, ameliorated inhibition. Both Crm1 and Exportin-t inhibited fusion of nuclear membranes, again counteracted by RanQ69L-GTP. In mitotic extracts, Crm1 and Exportin-t negatively impacted spindle assembly. Pulldowns from the extracts using Crm1- or Exportin-t-beads revealed nucleoporins known to be essential for both nuclear pore and spindle assembly, with RanQ69L-GTP decreasing a subset of these target interactions. This study suggests a model where exportins, like importins, can regulate major mitotic assembly events.
Overview of cohesin, CTCF, and loop extrusion. (a) Overview of mammalian cohesin and some of its regulatory proteins. (b) Overview of CTCF with N-terminal, 11 Zinc Fingers, and C-terminal domains. (c) Simplified sketch of cohesin-mediated loop extrusion and the convergent CTCF rule. (d) Summary of key parameters constraining loop extrusion models in mouse embryonic stem cells (mESCs) [65] and human HeLa cells [83], with mESC residence times taken from [70]. * These are cohesin G1 residence times (both STAG1 and STAG2), but after these studies were published it was found that STAG2-cohesin binds DNA substantially more dynamically than STAG1-cohesin [38], suggesting that putative loop extruding G1 cohesins have at least two residence times. ** Estimated from [83] (~180,000 and ~120,000 CTCF proteins and sites per HeLa cell) with added assumption that 45% of CTCF proteins are bound to cognate sites (~45%, i.e. mean of mESC and U2OS in [70]). *** 305,900 is the mean of the LC-MS and FCS estimates reported in [83]. **** Cohesin density is estimated from ~159,437 dynamically bound (~13.7 min residence time) cohesin proteins (SCC1-mEGFP) in G1 and the reported HeLa genome sizes 7.9 Gb, both taken from [83]. It is important to note that these are genomic averages: e.g. CTCF residence time is for an average site (some sites will have slower and faster binding), cohesin density may not be uniform throughout the genome, and since the two in vitro cohesin loop extrusion papers disagreed on whether cohesin is monomeric [23] or dimeric [21], densities for both monomeric [1] and dimeric [2] are shown.
One-step vs. Multi-step CTCF-cohesin interaction mechanisms. (a) One-step CTCF-cohesin mechanism. If 1-step mechanism, it is not clear why cohesin could not extrude past the C-terminal domain of CTCF to interact with the N-terminal domain on the other side. (b) One-step CTCF-cohesin mechanism with directionally sensitive domains. For a one-step mechanism to work, the N-terminal CTCF domain and cohesin would both have to exhibit a directional sensitivity as illustrated. (c) Multi-step CTCFcohesin mechanism. For an N-terminal encounter, pausing is eventually followed by stabilization. For a C-terminal encounter, pausing is not followed by stabilization, so cohesin eventually extrudes past or dissociates. (d) Instead of a one-step mechanism, a multi-step mechanism would involve transient pausing of cohesin next to CTCF (1), followed by stabilization of cohesin only from the N-terminal side of CTCF, through either direct protein-protein interaction (2), CTCF 'turning OFF' the cohesin motor (ATPase) perhaps mediated via PDS5A/B and/or ESCO1 (3), or through CTCF preventing WAPL-mediated release of cohesin from chromatin by CTCF binding to the same RAD21/STAG2 interface as WAPL does (4). It is important to note both that these mechanisms are not mutually exclusive, and that many other mechanisms could contribute.
Distinct classes of chromatin loops. (a-b) Micro-C maps and CTCF and Cohesin (Smc1a) ChIP-Seq shown for wt-CTCF mESCs and ΔRBR i -CTCF mESCs, illustrating Type 1 RBR i -dependent loops that can be explained by loss of CTCF/cohesin binding (a) and Type 2 RBR i -dependent loops that cannot be explained by loss of CTCF/cohesin binding (b). (c) Sketch of a role for CTCF clustering in blocking cohesin extrusion. Figures 4a-c are partially reproduced and edited from [36] with permission.
CTCF-mediated loops can be disrupted with only modest effects on TADs/insulation. (a-b) Hi-C contact matrices at 10 kb resolution of the HOXA locus in wt-CTCF and Y226A/F228A-CTCF HAP1 cells from [40]. Figures 5 is partially reproduced and edited from [40] with permission.
Mammalian genome structure is closely linked to function. At the scale of kilobases to megabases, CTCF and cohesin organize the genome into chromatin loops. Mechanistically, cohesin is proposed to extrude chromatin loops bidirectionally until it encounters occupied CTCF DNA-binding sites. Curiously, loops form predominantly between CTCF binding sites in a convergent orientation. How CTCF interacts with and blocks cohesin extrusion in an orientation-specific manner has remained a mechanistic mystery. Here, we review recent papers that have shed light on these processes and suggest a multi-step interaction between CTCF and cohesin. This interaction may first involve a pausing step, where CTCF halts cohesin extrusion, followed by a stabilization step of the CTCF-cohesin complex, resulting in a chromatin loop. Finally, we discuss our own recent studies on an internal RNA-Binding Region (RBRi) in CTCF to elucidate its role in regulating CTCF clustering, target search mechanisms and chromatin loop formation and future challenges.
Structure of SATB1 protein and its potential post-translational modifications. Along the x-axis representing the amino acid positions in SATB1 protein, are depicted its important structural features and domains. ULD -ubiquitin-like domain, CUTL -CUT-like domain, CUT1 and CUT2 domains, EP -the peptide encoded by the predicted extra exon of the long SATB1 isoform, Qcompositional bias represented by a poly-Q domain and a stretch of prolines, HD -homeodomain. In the two upper segments separated by the dashed lines, there are potential murine and human post-translational modifications, extracted from the PhosphoSitePlus® database [44]. The abundance of gray lollipop visualizations in the chart indicate a number of sites that could potentially be phosphorylated and thus hold a great potential to regulate valency of SATB1 and affect its biophysical and/or regulatory properties.
Satb1 expression in relation to early T cell development. Color of depicted cell types indicates relative Satb1 gene expression. The background color symbolizes distinct body compartments harboring the depicted cell types. Satb1 is predominantly expressed during the double positive stage of T cell development, indicating a potential role of SATB1 at this stage. Important master regulators and key events of the T cell development are also depicted to better understand the processes and the genes which SATB1 could regulate. ESC -embryonic stem cell, HSC -hematopoietic stem cell, MPP -multipotent precursor, LMPPlymphoid-primed multipotent precursor, CLP -common lymphoid precursor 'A' type, ETP -early T cell precursor, DN -CD4CD8 double negative T cell, ISP -immature single-positive T cell, DP -CD4CD8 double positive T cell, CD4 -CD4 single positive T cell, CD8 -CD8 single positive T cell.
SATB1 in developing T cells mainly localizes to interchromatin regions and to the euchromatin/heterochromatin boundary. This localization pattern surrounding central heterochromatin, typical for T cells, was originally described as a cage-like structure. Super-resolution images indicate that the interconnected cage may instead correspond to individual scattered spheres and tendrils. Here we suggest that SATB1 exists in multiple variants such as the predicted isoforms of variable length (see Figure 1). Additionally, SATB1 is post-translationally modified including phosphorylation, methylation, sumoylation, yielding multiple SATB1 variants with potentially distinct functions. A hypothesis is that some SATB1 variants may be responsible for purely the structural organization of the nucleus (brown dots) in cooperation with the previously recognized elements of the nuclear matrix, such as the meshwork of lncRNAs. Other variants may have either repressive (red dots) or activatory (green dots) roles, depending on the chromatin modifying enzymes that SATB1 recruits (see Figure 5). On top of that, SATB1 possibly organizes the genome keeping certain regions looped out from heterochromatin. Another variant of SATB1 can further loop out genes and regulatory elements and bring them to transcriptional condensates via its IDR targeting or by other mechanisms to ensure another mode of positive transcriptional regulation.
Proposed model on SATB1's diverse modes of action and how their deregulation may result to disease. Different variants of SATB1 introduced in Figures 1 and 3 can define quite diverse interactomes localized within the cell nucleus. Consequently, different chromatin modifying or remodeling complexes are recruited by different SATB1 variants to the regulated genes. Chromatin accessibility is modified and transcription activation or repression occurs. Moreover, SATB1 mediates long-range promoter-enhancer communication and ultimately regulates chromatin organization. In its absence, the interactome and/or the loopscape structure of the genome is altered resulting in a modified transcriptome. Here we demonstrate how transcription of two genes is controlled either positively or negatively by two distinct SATB1 variants. This transcription state is deregulated in the Satb1 conditional knockout mice, ultimately leading to a disease such as autoimmunity. A link between deregulated SATB1 protein and altered chromatin landscape and/or genome organization in human autoimmune diseases has not been thoroughly studied yet.
The regulatory circuits that define developmental decisions of thymocytes are still incompletely resolved. SATB1 protein is predominantly expressed at the CD4⁺CD8⁺cell stage exerting its broad transcription regulation potential with both activatory and repressive roles. A series of post-translational modifications and the presence of potential SATB1 protein isoforms indicate the complexity of its regulatory potential. The most apparent mechanism of its involvement in gene expression regulation is via the orchestration of long-range chromatin loops between genes and their regulatory elements. Multiple SATB1 perturbations in mice uncovered a link to autoimmune diseases while clinical investigations on cancer research uncovered that SATB1 has a promoting role in several types of cancer and can be used as a prognostic biomarker. SATB1 is a multivalent tissue-specific factor with a broad and yet undetermined regulatory potential. Future investigations on this protein could further uncover T cell-specific regulatory pathways and link them to (patho)physiology.
Schematic diagram of chromatin loop-cluster stabilization via lamin meshwork inside the nucleoplasm.
Active movements of chromatin loops inside the nucleoplasm.
Nuclear lamins form an elastic meshwork underlying the inner nuclear membrane and provide mechanical rigidity to the nucleus and maintain shape. Lamins also maintain chromosome positioning and play important roles in several nuclear processes like replication, DNA damage repair, transcription, and epigenetic modifications. LMNA mutations affect cardiac tissue, muscle tissues, adipose tissues to precipitate several diseases collectively termed as laminopathies. However, the rationale behind LMNA mutations and laminopathies continues to elude scientists. During interphase, several chromosomes form inter/intrachromosomal contacts inside nucleoplasm and several chromosomal loops also stretch out to make a ‘loop-cluster’ which are key players to regulate gene expressions. In this perspective, we have proposed that the lamin network in tandem with nuclear actin and myosin provide mechanical rigidity to the chromosomal contacts and facilitate loop-clusters movements. LMNA mutations thus might perturb the landscape of chromosomal contacts or loop-clusters positioning which can impair gene expression profile.
Timeline of measurements and modeling of chromatin structures.
Timeline of measurements and concepts of chromatin functions.
Sizes of structural chromatin units measured with different methods.
Sizes of functional chromatin units measured with different methods.
Decades of investigation on genomic DNA have brought us deeper insights into its organization within the nucleus and its metabolic mechanisms. This was fueled by the parallel development of experimental techniques and has stimulated model building to simulate genome conformation in agreement with the experimental data. Here, we will discuss our recent discoveries on the chromatin units of DNA replication and DNA damage response. We will highlight their remarkable structural similarities and how both revealed themselves as clusters of nanofocal structures each on the hundred thousand base pair size range corresponding well with chromatin loop sizes. We propose that the function of these two global genomic processes is determined by the loop level organization of chromatin structure with structure dictating function. Abbreviations: 3D-SIM: 3D-structured illumination microscopy; 3C: chromosome conformation capture; DDR: DNA damage response; FISH: fluorescent in situ hybridization; Hi-C: high conformation capture; HiP-HoP: highly predictive heteromorphic polymer model; IOD: inter-origin distance; LAD: lamina associated domain; STED: stimulated emission depletion microscopy; STORM: stochastic optical reconstruction microscopy; SBS: strings and binders switch model; TAD: topologically associated domain
KEGG pathways significantly enriched in the differentially expressed genes between beta-actin WT and KO MEFs.
Cell fate-related genes are differentially expressed between beta-actin WT and KO MEFs. Genes differentially expressed (Padj<0.05, fold change >2) were subject to Gene Ontology (GO) enrichment analysis using DAVID Bioinformatics Resources. The expression level of genes associated with (a) Blood vessel development, (b) Neuron differentiation and (c) Positive regulation of fat cell differentiation are shown.
Speculative model describing the involvement of nuclear β-actin in epigenetic regulation during neuronal reprogramming.
In the eukaryotic cell nucleus, cytoskeletal proteins are emerging as essential players in nuclear function. In particular, actin regulates chromatin as part of ATP-dependent chromatin remodeling complexes, it modulates transcription and it is incorporated into nascent ribonucleoprotein complexes, accompanying them from the site of transcription to polyribosomes. The nuclear actin pool is undistinguishable from the cytoplasmic one in terms of its ability to undergo polymerization and it has also been implicated in the dynamics of chromatin, regulating heterochromatin segregation at the nuclear lamina and maintaining heterochromatin levels in the nuclear interiors. One of the next frontiers is, therefore, to determine a possible involvement of nuclear actin in the functional architecture of the cell nucleus by regulating the hierarchical organization of chromatin and, thus, genome organization. Here, we discuss the repertoire of these potential actin functions and how they are likely to play a role in the context of cellular differentiation.
Dynamics of the NE during the normal cell-cycle. (a) Interphase NE and ER organization. The interphase ER is continuous the NE and forms an interconnected network of membrane sheets and tubules. (b) In metaphase, the NE is absorbed into the mitotic ER, which is largely excluded from the spindle (purple). (c) Similarly, the mitotic ER remains largely shielded from the anaphase spindle during chromosome segregation. (d-e) (also see section 3 for details) In telophase, segregated chromosome masses recruit membranes to reform the NE. The chromosome regions in contact with the spindle assemble the core NE (thick red lines), whereas the chromosome peripheral regions assemble the non-core NE with NPCs (thick dark green lines). The core membranes abutting the central spindle are termed the 'inner core'; the core membranes abutting the spindle pole and its microtubules are termed the 'outer core'. (d) Two hypothetical models for the delivery of (core) membranes into the anaphase/telophase spindle (see section 5 for details): 1. core membrane delivery by direct ER tubule infiltration (red arrows); 2. core membrane delivery by extension of the nascent NE from chromosome periphery/non-core domain (green arrows). (f) (see section 3) In the subsequent interphase, the core NE initially lacking NPCs forms pore-free islands, which progressively assemble NPCs through an inside-out mechanism (purple arrows).
The nuclear envelope (NE) is composed of two lipid bilayer membranes that enclose the eukaryotic genome. In interphase, the NE is perforated by thousands of nuclear pore complexes (NPCs), which allow transport in and out of the nucleus. During mitosis in metazoans, the NE is broken down and then reassembled in a manner that enables proper chromosome segregation and the formation of a single nucleus in each daughter cell. Defects in coordinating NE reformation and chromosome segregation can cause aberrant nuclear architecture. This includes the formation of micronuclei, which can trigger a catastrophic mutational process commonly observed in cancers called chromothripsis. Here, we discuss the current understanding of the coordination of NE reformation with chromosome segregation during mitotic exit in metazoans. We review differing models in the field and highlight recent work suggesting that normal NE reformation and chromosome segregation are physically linked through the timing of mitotic spindle disassembly.
Bromodomain AAA+ ATPases (ATPases associated with diverse cellular activities) are emerging as oncogenic proteins and compelling targets for anticancer therapies. However, structural and biochemical insight into these machines is missing. A recent study by Cho et al. reports the first cryo-EM structure of a bromodomain AAA+ ATPase and provides first insights into the functions of this putative histone chaperone. © 2020, © 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
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Heinrich Leonhardt
  • Ludwig-Maximilians-University of Munich
Eric C Schirmer
  • The University of Edinburgh
Katherine L Wilson
  • Johns Hopkins University
Almouzni Almouzni
  • French National Centre for Scientific Research
Danièle Hernandez-Verdun
  • Paris Diderot University