James M. Holaska

Nesprin‐1α self‐associates and binds directly to emerin and lamin A in vitro

Nesprin‐1α is a spectrin repeat (SR)‐containing, transmembrane protein of the inner nuclear membrane, and is highly expressed in muscle cells. A yeast two‐hybrid screen for nesprin‐1α‐interacting proteins showed that nesprin‐1α interacted with itself. Blot overlay experiments revealed that nesprin‐1αs third SR binds the fifth SR. The carboxy‐terminal half of nesprin‐1α directly bound lamin A, a nuclear intermediate filament protein. Biochemical analysis demonstrated that nesprin‐1α dimers bind directly to the nucleoplasmic domain of emerin, an inner nuclear membrane protein, with an affinity of 4 nM. Binding was optimal for full nucleoplasmic dimers of nesprin‐1α, since nesprin fragments SR1‐5 and SR5‐7 bound emerin as monomers with affinities of 53 nM and 250 mM, respectively. We propose that membrane‐anchored nesprin‐1α antiparallel dimers interact with both emerin and lamin A to provide scaffolding at the inner nuclear membrane.

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 ∼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.

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.

Disruption of nesprin-1 produces an Emery Dreifuss muscular dystrophy-like phenotype in mice

Mutations in the gene encoding the inner nuclear membrane proteins lamins A and C produce cardiac and skeletal muscle dysfunction referred to as Emery Dreifuss muscular dystrophy. Lamins A and C participate in the LINC complex that, along with the nesprin and SUN proteins, LInk the Nucleoskeleton with the Cytoskeleton. Nesprins 1 and 2 are giant spectrin-repeat containing proteins that have large and small forms. The nesprins contain a transmembrane anchor that tethers to the nuclear membrane followed by a short domain that resides within the lumen between the inner and outer nuclear membrane. Nesprins luminal domain binds directly to SUN proteins. We generated mice where the C-terminus of nesprin-1 was deleted. This strategy produced a protein lacking the transmembrane and luminal domains that together are referred to as the KASH domain. Mice homozygous for this mutation exhibit lethality with approximately half dying at or near birth from respiratory failure. Surviving mice display hindlimb weakness and an abnormal gait. With increasing age, kyphoscoliosis, muscle pathology and cardiac conduction defects develop. The protein components of the LINC complex, including mutant nesprin-1alpha, lamin A/C and SUN2, are localized at the nuclear membrane in this model. However, the LINC components do not normally associate since coimmunoprecipitation experiments with SUN2 and nesprin reveal that mutant nesprin-1 protein no longer interacts with SUN2. These findings demonstrate the role of the LINC complex, and nesprin-1, in neuromuscular and cardiac disease.

The nuclear envelope, lamins and nuclear assembly.

The nuclear lamina is composed of both A- and B-type lamins and lamin-binding proteins. Many lamin-binding proteins are integral proteins of the inner nuclear membrane. Lamins and inner nuclear membrane proteins are important for a variety of cell functions, including nuclear assembly, replication, transcription, and nuclear integrity. Recent advances in the field in the past year include the identification of a family of spectrin-repeat-containing inner nuclear membrane proteins and other novel inner-membrane proteins, and the discovery of a nuclear membrane fusion complex. There is also growing evidence that A- and B-type lamins and their binding partners have distinct roles during nuclear assembly and interphase.

Nesprin-1 mutations in human and murine cardiomyopathy

Mutations in LMNA, the gene encoding the nuclear membrane proteins, lamins A and C, produce cardiac and muscle disease. In the heart, these autosomal dominant LMNA mutations lead to cardiomyopathy frequently associated with cardiac conduction system disease. Herein, we describe a patient with the R374H missense variant in nesprin-1alpha, a protein that binds lamin A/C. This individual developed dilated cardiomyopathy requiring cardiac transplantation. Fibroblasts from this individual had increased expression of nesprin-1alpha and lamins A and C, indicating changes in the nuclear membrane complex. We characterized mice lacking the carboxy-terminus of nesprin-1 since this model expresses nesprin-1 without its carboxy-terminal KASH domain. These Delta/DeltaKASH mice have a normally assembled but dysfunctional nuclear membrane complex and provide a model for nesprin-1 mutations. We found that Delta/DeltaKASH mice develop cardiomyopathy with associated cardiac conduction system disease. Older mutant animals were found to have elongated P wave duration, elevated atrial and ventricular effective refractory periods indicating conduction defects in the myocardium, and reduced fractional shortening. Cardiomyocyte nuclei were found to be elongated with reduced heterochromatin in the Delta/DeltaKASH hearts. These findings mirror what has been described from lamin A/C gene mutations and reinforce the importance of an intact nuclear membrane complex for a normally functioning heart.

The nuclear envelope protein emerin binds directly to histone deacetylase 3 (HDAC3) and activates HDAC3 activity

Background: Emerin regulates the expression of a large number of genes. Results: Emerin binds HDAC3, mediates its nuclear envelope localization, and activates HDAC3 activity. Conclusion: Decreased HDAC3 activity may contribute to changes in genomic organization seen in emerin-null cells. Significance: These studies uncovered a putative mechanism for initiating and maintaining repressed genes at the nuclear periphery. Organization of the genome is critical for maintaining cell-specific gene expression, ensuring proper cell function. It is well established that the nuclear lamina preferentially associates with repressed chromatin. However, the molecular mechanisms underlying repressive chromatin formation and maintenance at the nuclear lamina remain poorly understood. Here we show that emerin binds directly to HDAC3, the catalytic subunit of the nuclear co-repressor (NCoR) complex, and recruits HDAC3 to the nuclear periphery. Emerin binding stimulated the catalytic activity of HDAC3, and emerin-null cells exhibit increased H4K5 acetylation, which is the preferred target of the NCoR complex. Emerin-null cells exhibit an epigenetic signature similar to that seen in HDAC3-null cells. Emerin-null cells also had significantly less HDAC3 at the nuclear lamina. Collectively, these data support a model whereby emerin facilitates repressive chromatin formation at the nuclear periphery by increasing the catalytic activity of HDAC3.

Emerin and the Nuclear Lamina in Muscle and Cardiac Disease

The human genome is contained within the nucleus and is separated from the cytoplasm by the nuclear envelope. Mutations in the nuclear envelope proteins emerin and lamin A cause a number of diseases including premature aging syndromes, muscular dystrophy, and cardiomyopathy. Emerin and lamin A are implicated in regulating muscle- and heart-specific gene expression and nuclear architecture. For example, lamin A regulates the expression and localization of gap junction and intercalated disc components. Additionally, emerin and lamin A are also required to maintain nuclear envelope integrity. Demonstrating the importance of maintaining nuclear integrity in heart disease, atrioventricular node cells lacking lamin A exhibit increased nuclear deformation and apoptosis. This review highlights the present understanding of lamin A and emerin function in regulating nuclear architecture, gene expression, and cell signaling and discusses putative mechanisms for how specific mutations in lamin A and emerin cause cardiac- or muscle-specific disease.

Emerin and histone deacetylase 3 (HDAC3) cooperatively regulate expression and nuclear positions of MyoD, Myf5, and Pax7 genes during myogenesis

The spatial organization of chromatin is critical in establishing cell-type dependent gene expression programs. The inner nuclear membrane protein emerin has been implicated in regulating global chromatin architecture. We show emerin associates with genomic loci of muscle differentiation promoting factors in murine myogenic progenitors, including Myf5 and MyoD. Prior to their transcriptional activation Myf5 and MyoD loci localized to the nuclear lamina in proliferating progenitors and moved to the nucleoplasm upon transcriptional activation during differentiation. The Pax7 locus, which is transcribed in proliferating progenitors, localized to the nucleoplasm and Pax7 moved to the nuclear lamina upon repression during differentiation. Localization of Myf5, MyoD, and Pax7 to the nuclear lamina and proper temporal expression of these genes required emerin and HDAC3. Interestingly, activation of HDAC3 catalytic activity rescued both Myf5 localization to the nuclear lamina and its expression. Collectively, these data support a model whereby emerin facilitates repressive chromatin formation at the nuclear lamina by activating the catalytic activity of HDAC3 to regulate the coordinated spatiotemporal expression of myogenic differentiation genes.

Emerin in health and disease.

Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the genes encoding emerin, lamins A and C and FHL1. Additional EDMD-like syndromes are caused by mutations in nesprins and LUMA. This review will specifically focus on emerin function and the current thinking for how loss or mutations in emerin cause EDMD. Emerin is a well-conserved, ubiquitously expressed protein of the inner nuclear membrane. Emerin has been shown to have diverse functions, including the regulation of gene expression, cell signaling, nuclear structure and chromatin architecture. This review will focus on the relationships between these functions and the EDMD disease phenotype. Additionally it will highlight open questions concerning emerins roles in cell and nuclear biology and disease.