Loss Of Emerin Research Articles

Attenuation of Wnt/β-catenin activity reverses enhanced generation of cardiomyocytes and cardiac defects caused by the loss of emerin.

Mutations in EMD, encoding emerin cause skeletal muscle and heart defects in patients with X-linked Emery-Dreifuss muscular dystrophy (X-EDMD) but the underlying mechanisms leading to cardiac defects are poorly understood. Here, we investigated the role of emerin in controlling cardiomyocyte proliferation and cardiac remodeling and explored its function in regulation of the Wnt/β-catenin pathway. We observed a remarkable increase of cardiomyocytes in emerin-null adult mice accompanied with decreased numbers of multinucleated cells. Depletion of emerin in mouse ES cell-derived cardiomyocytes by shRNA caused hyperactivation of Wnt/β-catenin signaling, increased proliferation and abrogated timely cardiac differentiation. Likewise, emerin-null mice exhibited increased Wnt/β-catenin signaling, cardiac dysfunction and perturbed hypertrophic remodeling following pressure overload. Pharmacological inhibition of β-catenin normalized proliferation and differentiation of ES cell-derived cardiomyocytes while inactivation of a single allele of β-catenin efficiently rescued cardiac dysfunction in emerin-null mice. We conclude that emerin constrains β-catenin signaling in the heart providing tight control of cardiomyocyte numbers. Enhanced Wnt/β-catenin signaling seems to contribute to cardiac defects observed in X-EDMD. Hence, therapeutic inhibition of Wnt/β-catenin signaling might be beneficial for X-EDMD patients.

Tyrosine phosphorylation of nuclear-membrane protein emerin by Src, Abl and other kinases

X-linked recessive Emery-Dreifuss muscular dystrophy (EDMD) is caused by loss of emerin, a nuclear-membrane protein with roles in nuclear architecture, gene regulation and signaling. Phosphoproteomic studies have identified 13 sites of tyrosine phosphorylation in emerin. We validated one study, confirming that emerin is hyper-tyrosine-phosphorylated in Her2-overexpressing cells. We discovered that non-receptor tyrosine kinases Src and Abl each phosphorylate emerin and a related protein, LAP2beta, directly. Src phosphorylated emerin specifically at Y59, Y74 and Y95; the corresponding triple Y-to-F (;FFF') mutation reduced tyrosine phosphorylation by approximately 70% in vitro and in vivo. Substitutions that removed a single hydroxyl moiety either decreased (Y19F, Y34, Y161F) or increased (Y4F) emerin binding to BAF in cells. Y19F, Y34F, Y161F and the FFF mutant also reduced recombinant emerin binding to BAF from HeLa lysates, demonstrating the involvement of both LEM-domain and distal phosphorylatable tyrosines in binding BAF. We conclude that emerin function is regulated by multiple tyrosine kinases, including Her2, Src and Abl, two of which (Her2, Src) regulate striated muscle. These findings suggest roles for emerin as a downstream effector and ;signal integrator' for tyrosine kinase signaling pathway(s) at the nuclear envelope.

Loss of GATA6 Leads to Nuclear Deformation and Aneuploidy in Ovarian Cancer

A prominent hallmark of most human cancer is aneuploidy, which is a result of the chromosomal instability of cancer cells and is thought to contribute to the initiation and progression of most carcinomas. The developmentally regulated GATA6 transcription factor is commonly lost in ovarian cancer, and the loss of its expression is closely associated with neoplastic transformation of the ovarian surface epithelium. In the present study, we found that reduction of GATA6 expression with small interfering RNA (siRNA) in human ovarian surface epithelial cells resulted in deformation of the nuclear envelope, failure of cytokinesis, and formation of polyploid and aneuploid cells. We further discovered that loss of the nuclear envelope protein emerin may mediate the consequences of GATA6 suppression. The nuclear phenotypes were reproduced by direct suppression of emerin with siRNA. Thus, we conclude that diminished expression of GATA6 leads to a compromised nuclear envelope that is causal for polyploidy and aneuploidy in ovarian tumorigenesis. The loss of emerin may be the basis of nuclear morphological deformation and subsequently the cause of aneuploidy in ovarian cancer cells.

Dysfunctional Connections Between the Nucleus and the Actin and Microtubule Networks in Laminopathic Models

Laminopathies encompass a wide array of human diseases associated to scattered mutations along LMNA, a single gene encoding A-type lamins. How such genetic alterations translate to cellular defects and generate such diverse disease phenotypes remains enigmatic. Recent work has identified nuclear envelope proteins—emerin and the linker of the nucleoskeleton and cytoskeleton (LINC) complex—which connect the nuclear lamina to the cytoskeleton. Here we quantitatively examine the composition of the nuclear envelope, as well as the architecture and functions of the cytoskeleton in cells derived from two laminopathic mouse models, including Hutchinson-Gilford progeria syndrome ( Lmna L530P/L530P) and Emery-Dreifuss muscular dystrophy ( Lmna −/−). Cells derived from the overtly aphenotypical model of X-linked Emery-Dreifuss muscular dystrophy ( Emd −/y) were also included. We find that the centrosome is detached from the nucleus, preventing centrosome polarization in cells under flow—defects that are mediated by the loss of emerin from the nuclear envelope. Moreover, while basal actin and focal adhesion structure are mildly affected, RhoA activation, cell-substratum adhesion, and cytoplasmic elasticity are greatly lowered, exclusively in laminopathic models in which the LINC complex is disrupted. These results indicate a new function for emerin in cell polarization and suggest that laminopathies are not directly associated with cells’ inability to polarize, but rather with cytoplasmic softening and weakened adhesion mediated by the disruption of the LINC complex across the nuclear envelope.

Disturbed nuclear orientation and cellular migration in A-type lamin deficient cells

The nuclear lamina and the cytoskeleton form an integrated structure that warrants proper mechanical functioning of cells. We have studied the correlation between structural alterations and migrational behaviour in fibroblasts with and without A-type lamins. We show that loss of A-type lamins causes loss of emerin and nesprin-3 from the nuclear envelope, concurring with a disturbance in the connection between the nucleus and the cytoskeleton in A-type lamin-deficient ( lmna −/−) cells. In these cells functional migration assays during in vitro wound healing revealed a delayed reorientation of the nucleus and the microtubule-organizing center during migration, as well as a loss of nuclear oscillatory rotation. These observations in fibroblasts isolated from lmna knockout mice were confirmed in a 3T3 cell line with stable reduction of lmna expression due to RNAi approach. Our results indicate that A-type lamins play a key role in maintaining directional movement governed by the cytoskeleton, and that the loss of these karyoskeletal proteins has important consequences for functioning of the cell as a mechanical entity.

Mechanical Properties of the Cell Nucleus and the Effect of Emerin Deficiency

Nuclear structure and mechanics are gaining recognition as important factors that affect gene expression, development, and differentiation in normal function and disease, yet the physical mechanisms that govern nuclear mechanical stability remain unclear. Here we examined the physical properties of the cell nucleus by imaging fluorescently labeled components of the inner nucleus (chromatin and nucleoli) and the nuclear envelope (lamins and membranes) in nuclei deformed by micropipette aspiration (confocal imaged microdeformation). We investigated nuclei, both isolated and in intact, living cells, and found that nuclear volume significantly decreased by 60–70% during aspiration. While nuclear membranes exhibited blebbing and fluid characteristics during aspiration, the nuclear lamina exhibited behavior of a solid-elastic shell. Under large deformations of GFP-lamin A-labeled nuclei, we observed a decay of fluorescence intensity into the tip of the deformed tongue that we interpreted in terms of nonlinear, two-dimensional elasticity theory. Here we applied this method to study nuclear envelope stability in disease and found that mouse embryo fibroblasts lacking the inner nuclear membrane protein, emerin, had a significantly decreased ratio of the area expansion to shear moduli ( K/ μ) compared to wild-type cells (2.1 ± 0.2 versus 5.1 ± 1.3). These data suggest that altered nuclear envelope elasticity caused by loss of emerin could contribute to increased nuclear fragility in Emery-Dreifuss muscular dystrophy patients with mutations in the emerin gene. Based on our experimental results and theoretical considerations, we present a model describing how the nucleus is stabilized in the pipette. Such a model is essential for interpreting the results of any micropipette study of the nucleus and porous materials in general.

Lmo7 is an emerin-binding protein that regulates the transcription of emerin and many other muscle-relevant genes

X-linked Emery-Dreifuss muscular dystrophy (X-EDMD) is inherited through mutations in emerin, a nuclear membrane protein. Emerin has proposed roles in nuclear architecture and gene regulation, but direct molecular links to disease were unknown. We report that Lim-domain only 7 (Lmo7) binds emerin directly with 125 nM affinity; the C-terminal half of human Lmo7 (hLmo7C) was sufficient to bind emerin in vitro. Lmo7 appeared relevant to EDMD because a deletion that removes Lmo7 (plus eight exons of a neighboring gene) in mice causes dystrophic muscles [Semenova, E., Wang, X., Jablonski, M.M., Levorse, J. and Tilghman, S.M. (2003) An engineered 800 kilobase deletion of Uchl3 and Lmo7 on mouse chromosome 14 causes defects in viability, postnatal growth and degeneration of muscle and retina. Hum. Mol. Genet., 12, 1301-1312]. Lmo7 localizes in the nucleus, cytoplasm and cell surface, particularly adhesion junctions [Ooshio, T., Irie, K., Morimoto, K., Fukuhara, A., Imai, T. and Takai, Y. (2004) Involvement of LMO7 in the association of two cell-cell adhesion molecules, nectin and E-cadherin, through afadin and alpha-actinin in epithelial cells. J. Biol. Chem., 279, 31365-31373]. Our data suggest endogenous Lmo7 is a nucleocytoplasmic shuttling protein, and might also localize at focal adhesions in HeLa cells. Two key results show that Lmo7 regulates emerin gene expression: rat Lmo7 isoforms directly activated a luciferase reporter gene in vivo, and emerin mRNA expression decreased 93% in Lmo7-downregulated HeLa cells. Thus, Lmo7 not only binds emerin protein but is also required for emerin gene transcription. Microarray analysis of Lmo7-downregulated HeLa cells identified over 4200 misregulated genes, including 46 genes important for muscle or heart. Misregulation of 11 genes, including four (CREBBP, NAP1L1, LAP2, RBL2) known to be misregulated in X-EDMD patients and emerin-null mice [Bakay, M., Wang, Z., Melcon, G., Schiltz, L., Xuan, J., Zhao, P., Sartorelli, V., Seo, J., Pegoraro, E., Angelini, C. et al. (2006) Nuclear envelope dystrophies show a transcriptional fingerprint suggesting disruption of Rb-MyoD pathways in muscle regeneration. Brain, 129, 996-1013; Melcon, G., Kozlov, S., Cutler, D.A., Sullivan, T., Hernandez, L., Zhao, P., Mitchell, S., Nader, G., Bakay, M., Rottman, J.N. et al. (2006) Loss of emerin at the nuclear envelope disrupts the Rb1/E2F and MyoD pathways during muscle regeneration. Hum. Mol. Genet., 15, 637-651] was confirmed by real-time PCR. Overexpression of wild-type emerin, but not emerin mutant P183H (which causes EDMD and selectively disrupts binding to Lmo7), decreased the expression of CREBBP, NAP1L1 and LAP2, suggesting Lmo7 activity is both EDMD-relevant and inhibited by direct binding to emerin. We conclude that Lmo7 positively regulates many EDMD-relevant genes (including emerin), and is feedback-regulated by binding to emerin.

Loss of emerin at the nuclear envelope disrupts the Rb1/E2F and MyoD pathways during muscle regeneration

Emery-Dreifuss muscular dystrophy (EDMD1) is caused by mutations in either the X-linked gene emerin (EMD) or the autosomal lamin A/C (LMNA) gene. Here, we describe the derivation of mice lacking emerin in an attempt to derive a mouse model for EDMD1. Although mice lacking emerin show no overt pathology, muscle regeneration in these mice revealed defects. A bioinformatic array analysis of regenerating Emd null muscle revealed abnormalities in cell-cycle parameters and delayed myogenic differentiation, which were associated with perturbations to transcriptional pathways regulated by the retinoblastoma (Rb1) and MyoD genes. Temporal activation of MyoD transcriptional targets was significantly delayed, whereas targets of the Rb1/E2F transcriptional repressor complex remained inappropriately active. The inappropriate modulation of Rb1/MyoD transcriptional targets was associated with up-regulation of Rb1, MyoD and their co-activators/repressors transcripts, suggesting a compensatory effort to overcome a molecular block to differentiation at the myoblast/myotube transition during regeneration. This compensation appeared to be effective for MyoD transcriptional targets, although was less effective for Rb1 targets. Analysis of Rb1 phosphorylation states showed prolonged hyper-phosphorylation at key developmental stages in Emd null myogenic cells, both in vivo and in vitro. We also analyzed the same pathways in Lmna null muscle, which shows extensive dystrophy. Surprisingly, Lmna null muscle did not show the same perturbations to Rb- and MyoD-dependent pathways. We did observe increased transcriptional expression of Lap2alpha and delayed expression of Rb1, which may regulate alternative transcriptional pathways in the Lmna null myoblasts. We suggest that the dominant LMNA mutations seen in many clinically disparate laminopathies may similarly alter Rb function, with regard to either the timing of exit from the cell cycle or terminal differentiation programs or both.

Emerin Caps the Pointed End of Actin Filaments: Evidence for an Actin Cortical Network at the Nuclear Inner Membrane

X-linked Emery-Dreifuss muscular dystrophy is caused by loss of emerin, a LEM-domain protein of the nuclear inner membrane. To better understand emerin function, we used affinity chromatography to purify emerin-binding proteins from nuclear extracts of HeLa cells. Complexes that included actin, αII-spectrin and additional proteins, bound specifically to emerin. Actin polymerization assays in the presence or absence of gelsolin or capping protein showed that emerin binds and stabilizes the pointed end of actin filaments, increasing the actin polymerization rate 4- to 12-fold. We propose that emerin contributes to the formation of an actin-based cortical network at the nuclear inner membrane, conceptually analogous to the actin cortical network at the plasma membrane. Thus, in addition to disrupting transcription factors that bind emerin, loss of emerin may destabilize nuclear envelope architecture by weakening a nuclear actin network.

Transcriptional Repressor Germ Cell-less (GCL) and Barrier to Autointegration Factor (BAF) Compete for Binding to Emerin in Vitro

Emerin belongs to the "LEM domain" family of nuclear proteins, which contain a characteristic approximately 40-residue LEM motif. The LEM domain mediates direct binding to barrier to autointegration factor (BAF), a conserved 10-kDa chromatin protein essential for embryogenesis in Caenorhabditis elegans. In mammalian cells, BAF recruits emerin to chromatin during nuclear assembly. BAF also mediates chromatin decondensation during nuclear assembly. The LEM domain and central region of emerin are essential for binding to BAF and lamin A, respectively. However, two other conserved regions of emerin lacked ascribed functions, suggesting that emerin could have additional partners. We discovered that these "unascribed" domains of emerin mediate direct binding to a transcriptional repressor, germ cell-less (GCL). GCL co-immunoprecipitates with emerin from HeLa cells. We determined the binding affinities of emerin for GCL, BAF, and lamin A and analyzed their oligomeric interactions. We showed that emerin forms stable complexes with either lamin A plus GCL or lamin A plus BAF. Importantly, BAF competed with GCL for binding to emerin in vitro, predicting that emerin can form at least two distinct types of complexes in vivo. Loss of emerin causes Emery-Dreifuss muscular dystrophy, a tissue-specific inherited disease that affects skeletal muscles, major tendons, and the cardiac conduction system. Although GCL alone cannot explain the disease mechanism, our results strongly support gene expression models for Emery-Dreifuss muscular dystrophy by showing that emerin binds directly to a transcriptional repressor, GCL, and by suggesting that emerin-repressor complexes might be regulated by BAF. Biochemical roles for emerin in gene expression are discussed.

Distinct functional domains in emerin bind lamin A and DNA-bridging protein BAF.

Loss of emerin, a lamin-binding nuclear membrane protein, causes Emery-Dreifuss muscular dystrophy. We analyzed 13 site-directed mutations, and four disease-causing mutations that do not disrupt emerin stability or localization. We show that emerin binds directly to barrier-to-autointegration factor (BAF), a DNA-bridging protein, and that this binding to BAF requires conserved residues in the LEM-motif of emerin. Emerin has two distinct functional domains: the LEM-domain at the N-terminus, which mediates binding to BAF, and a second functional domain in the central region, which mediates binding to lamin A. Disease mutation Delta95-99 mapped to the lamin-binding domain and disrupted lamin A binding in vitro. Two other disease-linked residues, Ser54 and Pro183, mapped outside the BAF and lamin-binding domains, suggesting that emerin may have additional functional domains relevant to disease. The disease-linked emerin proteins all remained active for binding to BAF, both in vitro and in vivo, suggesting that disease can result from the loss of specific molecular interactions between emerin and either lamin A or putative novel partner(s). The demonstration that emerin binds directly to BAF, coupled to similar results for LAP2, provides proof in principle that all LEM-domain nuclear proteins can interact with BAF, with interesting implications for chromatin attachment to the nuclear envelope.

Properties of lamin A mutants found in Emery-Dreifuss muscular dystrophy, cardiomyopathy and Dunnigan-type partial lipodystrophy.

Autosomal dominant Emery-Dreifuss muscular dystrophy is caused by mutations in the LMNA gene, which encodes lamin A and lamin C. Mutations in this gene also give rise to limb girdle muscular dystrophy type 1B, dilated cardiomyopathy with atrioventricular conduction defect and Dunnigan-type partial lipodystrophy. The properties of the mutant lamins that cause muscular dystrophy, lipodystrophy and dilated cardiomyopathy are not known. We transfected C2C12 myoblasts with cDNA encoding wild-type lamin A and 15 mutant forms found in patients affected by these diseases. Immunofluorescence microscopy showed that four mutants, N195K, E358K, M371K and R386K, could have a dramatically aberrant localization, with decreased nuclear rim staining and formation of intranuclear foci. The distributions of endogenous lamin A/C, lamin B1 and lamin B2 were also altered in cells expressing these four mutants and three of them caused a loss of emerin from the nuclear envelope. In the yeast two-hybrid assay, the 15 lamin A mutants studied interacted with themselves and with wild-type lamin A and lamin B1. Pulse-chase experiments showed no decrease in the stability of several representative lamin A mutants compared with wild-type. These results indicate that some lamin A mutants causing disease can be aberrantly localized, partially disrupt the endogenous lamina and alter emerin localization, whereas others localize normally in transfected cells.

Solution structure of the constant region of nuclear envelope protein LAP2 reveals two LEM-domain structures: one binds BAF and the other binds DNA.

The nuclear envelope proteins LAP2, emerin and MAN1 share a conserved approximately 40-residue 'LEM' motif. Loss of emerin causes Emery-Dreifuss muscular dystrophy. We have solved the solution NMR structure of the constant region of human LAP2 (residues 1-168). Human LAP2(1-168) has two structurally independent, non-interacting domains located at residues 1-50 ('LAP2-N') and residues 111-152 (LEM-domain), connected by an approximately 60-residue flexible linker. The two domains are structurally homologous, comprising a helical turn followed by two helices connected by an 11-12-residue loop. This motif is shared by subdomains of T4 endonuclease VII and transcription factor rho, despite negligible (< or =15%) sequence identity. NMR chemical shift mapping demonstrated that the LEM-domain binds BAF (barrier-to-autointegration factor), whereas LAP2-N binds DNA. Both binding surfaces comprise helix 1, the N-terminus of helix 2 and the inter-helical loop. Binding selectivity is determined by the nature of the surface residues in these binding sites, which are predominantly positively charged for LAP2-N and hydrophobic for the LEM-domain. Thus, LEM and LEM-like motifs form a common structure that evolution has customized for binding to BAF or DNA.