Identification and Cloning of an mRNA Coding for a Germ Cell-Specific A-Type Lamin in Mice

The nuclear lamins are karyoskeletal proteins which have important functions, such as maintaining nuclear envelope integrity and organizing high order nuclear structure during mitosis in higher eukaryotes. In somatic mammalian cells, the A-type and B-type lamins, composed of lamins A and C and lamins B1 and B2, are major components of the nuclear lamina. However, A-type lamins have as yet not been identified in germ cells and undifferentiated embryonic cells. Here we report the cloning of a new 52-kDa A-type lamin from mouse pachytene spermatocytes, termed lamin C2 because of its similarities with lamin C. It has a sequence identical to that of lamin C except that the N -terminal segment, containing the head and the α-helical coil 1A domains, is replaced with a short non-α-helical stretch of amino acids. In mice, lamin C2 was found to be specifically expressed in germ cells. This specific expression and unique structure suggests a role for lamin C2 in determining the organization of nuclear and chromosomal structures during spermatogenesis.     

Defects in lamin B1 expression or processing affect interphase chromosome position and gene expression

Radial organization of nuclei with peripheral gene-poor chromosomes and central gene-rich chromosomes is common and could depend on the nuclear boundary as a scaffold or position marker. To test this, we studied the role of the ubiquitous nuclear envelope (NE) component lamin B1 in NE stability, chromosome territory position, and gene expression. The stability of the lamin B1 lamina is dependent on lamin endoproteolysis (by Rce1) but not carboxymethylation (by Icmt), whereas lamin C lamina stability is not affected by the loss of full-length lamin B1 or its processing. Comparison of wild-type murine fibroblasts with fibroblasts lacking full-length lamin B1, or defective in CAAX processing, identified genes that depend on a stable processed lamin B1 lamina for normal expression. We also demonstrate that the position of mouse chromosome 18 but not 19 is dependent on such a stable nuclear lamina. The results implicate processed lamin B1 in the control of gene expression as well as chromosome position.     

Identification of a short nuclear lamin protein selectively expressed during meiotic stages of rat spermatogenesis

The nuclear lamina is a karyoskeletal structure located at the nuclear periphery and intimately associated with the inner nuclear membrane. It is composed of a multigene family of proteins, the lamins, which show a conspicuous cell type-specific expression pattern. The functional role of lamins has not been definitively established but available information indicates that they are involved in the organization of nuclear envelope and interphase chromatin. Spermatogenesis is characterized, among other features, by stage-specific changes in chromatin organization and function. These changes are accompanied by modifications in the organization and composition of the nuclear lamina. In previous experiments we have determined that rat spermatogenic cells express a lamin closely related, if not identical, to lamin B1 of somatic cells; whereas rat somatic lamins A, C, D and E were not detected. Considering that chromatin reorganizations during spermatogenesis may be directly or indirectly related to changes of the nuclear lamina we have decided to further investigate lamin expression during this process. Here we report on the identification of a 52 kDa protein of the rat which, according to immunocytochemical and biochemical data, appears to be a novel nuclear lamin. Using meiotic stage-specific markers, we have also demonstrated that this short lamin is selectively expressed during meiotic stages of spermatogenesis.     

A-type and B-type lamins initiate layer assembly at distinct areas of the nuclear envelope in living cells

To investigate nuclear lamina re-assembly in vivo, Drosophila A-type and B-type lamins were artificially expressed in Drosophila lamin Dm₀null mutant brain cells. Both exogenous lamin C (A-type) and Dm₀ (B-type) formed sub-layers at the nuclear periphery, and efficiently reverted the abnormal clustering of the NPC. Lamin C initially appeared where NPCs were clustered, and subsequently extended along the nuclear periphery accompanied by the recovery of the regular distribution of NPCs. In contrast, lamin Dm₀ did not show association with the clustered NPCs during lamina formation and NPC spacing recovered only after completion of a closed lamin Dm₀ layer. Further, when lamin Dm₀ and C were both expressed, they did not co-polymerize, initiating layer formation in separate regions. Thus, A and B-type lamins reveal differing properties during lamina assembly, with A-type having the primary role in organizing NPC distribution. This previously unknown complexity in the assembly of the nuclear lamina could be the basis for intricate nuclear envelope functions.     

Ubiquitin ligase RNF123 Mediates Degradation of Heterochromatin Protein 1α and β in Lamin A/C Knock-Down Cells

Background

The nuclear lamina is a key determinant of nuclear architecture, integrity and functionality in metazoan nuclei. Mutations in the human lamin A gene lead to highly debilitating genetic diseases termed as laminopathies. Expression of lamin A mutations or reduction in levels of endogenous A-type lamins leads to nuclear defects such as abnormal nuclear morphology and disorganization of heterochromatin. This is accompanied by increased proteasomal degradation of certain nuclear proteins such as emerin, nesprin-1α, retinoblastoma protein and heterochromatin protein 1 (HP1). However, the pathways of proteasomal degradation have not been well characterized.

Methodology/Principal Findings

To investigate the mechanisms underlying the degradation of HP1 proteins upon lamin misexpression, we analyzed the effects of shRNA-mediated knock-down of lamins A and C in HeLa cells. Cells with reduced levels of expression of lamins A and C exhibited proteasomal degradation of HP1α and HP1β but not HP1γ. Since specific ubiquitin ligases are upregulated in lamin A/C knock-down cells, further studies were carried out with one of these ligases, RNF123, which has a putative HP1-binding motif. Ectopic expression of GFP-tagged RNF123 directly resulted in degradation of HP1α and HP1β. Mutational analysis showed that the canonical HP1-binding pentapeptide motif PXVXL in the N-terminus of RNF123 was required for binding to HP1 proteins and targeting them for degradation. The role of endogenous RNF123 in the degradation of HP1 isoforms was confirmed by RNF123 RNAi experiments. Furthermore, FRAP analysis suggested that HP1β was displaced from chromatin in laminopathic cells.

Conclusions/Significance

Our data support a role for RNF123 ubiquitin ligase in the degradation of HP1α and HP1β upon lamin A/C knock-down. Hence lamin misexpression can cause degradation of mislocalized proteins involved in key nuclear processes by induction of specific components of the ubiquitin-proteasome system.     

Both lamin A and lamin C mutations cause lamina instability as well as loss of internal nuclear lamin organization

We have applied the fluorescence loss of intensity after photobleaching (FLIP) technique to study the molecular dynamics and organization of nuclear lamin proteins in cell lines stably transfected with green fluorescent protein (GFP)-tagged A-type lamin cDNA. Normal lamin A and C proteins show abundant decoration of the inner layer of the nuclear membrane, the nuclear lamina, and a generally diffuse localization in the nuclear interior. Bleaching studies revealed that, while the GFP-tagged lamins in the lamina were virtually immobile, the intranuclear fraction of these molecules was partially mobile. Intranuclear lamin C was significantly more mobile than intranuclear lamina A. In search of a structural cause for the variety of inherited diseases caused by A-type lamin mutations, we have studied the molecular organization of GFP-tagged lamin A and lamin C mutants R453W and R386K, found in Emery-Dreifuss muscular dystrophy (EDMD), and lamin A and lamin C mutant R482W, found in patients with Dunnigan-type familial partial lipodystrophy (FPLD). In all mutants, a prominent increase in lamin mobility was observed, indicating loss of structural stability of lamin polymers, both at the perinuclear lamina and in the intranuclear lamin organization. While the lamin rod domain mutant showed overall increased mobility, the tail domain mutants showed mainly intranuclear destabilization, possibly as a result of loss of interaction with chromatin. Decreased stability of lamin mutant polymers was confirmed by flow cytometric analyses and immunoblotting of nuclear extracts. Our findings suggest a loss of function of A-type lamin mutant proteins in the organization of intranuclear chromatin and predict the loss of gene regulatory function in laminopathies.     

Pushing the (nuclear) envelope into meiosis

A recent study shows that a short isoform of a mammalian nuclear lamin is important for homologous chromosome interactions during meiotic prophase in mice.Meiosis is the specialized cell division process required for sexual reproduction. As cells enter meiotic prophase, a relatively long period preceding the two chromosome divisions, nuclei and chromosomes undergo remodeling to promote interactions between homologous chromosomes. Each chromosome must find and identify its unique partner within the volume of the nucleus, a process that obviously involves large-scale chromosome movements.Over 100 years ago, cytological analysis of meiotic cells revealed a unique chromosome configuration termed the meiotic ''bouquet'', in which chromosome ends seem to be attached to the nuclear periphery, frequently in a tight cluster. The presence of the bouquet was found to coincide with the stage during which homologous chromosomes undergo pairing and synapsis. This was the first indication that interactions between the chromosomes and the nuclear envelope might be important for meiotic pairing. More recent analysis in diverse model systems has revealed that the bouquet is a consequence of interactions between chromosomes and cytoskeletal elements - microtubules or actin cables - via a protein bridge that spans the nuclear envelope. A study recently published in PLOS Genetics [1] has shed further light on the role of the nuclear lamina in meiotic progression by studying the role of a meiosis-specific isoform of a nuclear lamin protein.In metazoans the nuclear envelope is fortified by the nuclear lamina, a meshwork of intermediate filament proteins (lamins) and associated proteins that underlies the inner nuclear membrane. The lamina confers structural rigidity to nuclei and also interacts with a wide variety of nucleoplasmic, transmembrane and chromosome-associated proteins. The composition of the lamina in metazoans shows tissue-specific variability and developmental regulation. Most differentiated mammalian cells express both A-type lamins (lamins A and C, which are generated by alternative splicing of the LMNA gene) and B-type lamins (encoded by two different genes), whereas some invertebrates express only a single lamin protein. Stem cells typically lack A-type lamins, which are also dispensable for early development in mice.Among the nuclear envelope components that interact with lamins are LINC (linker of nucleoskeleton and cytoskeleton) complexes. These versatile networks involve a pair of SUN/KASH proteins that bridge both membranes of the nuclear envelope. SUN domain proteins traverse the inner membrane, with their amino termini projecting into the nucleus and their SUN domains in the lumen between the two membranes. Their partners have membrane-spanning regions adjacent to their carboxy-terminal KASH domains, short peptides that bind to the SUN domains. Using a variety of interaction modules, LINC complexes create connections between nuclear structures such as the lamina or chromosomes and cytoskeletal elements such as actin filaments or microtubules. Throughout the eukaryotes, they have essential roles in diverse processes, including the positioning and migration of nuclei within cells and anchorage of centrosomes to the nuclear envelope. During meiosis, specific LINC complexes are recruited to interact with chromosomes through the expression of meiosis-specific proteins that bind to telomeres or, less frequently, to other specialized loci [2]. These connections, probably in conjunction with meiosis-specific modifications to the cytoskeleton and motor proteins, lead to large-scale chromosome motions that facilitate homologous chromosome pairing. These movements involve dramatic motion of the LINC proteins within the nuclear membrane, sometimes involving movements of up to several micrometers that occur within a few seconds [3]. This stands in sharp contrast to the behavior of some of the same protein complexes in somatic or premeiotic cells, in which they show highly constrained motion and minimal turnover [3].In the new PLOS Genetics study [1], groups led by Manfred Alsheimer and Ricardo Benavente, both of the University of Würzburg, have now engineered a disruption of an exon in the mouse LMNA gene that is specific to the meiotic isoform lamin C2 to generate C2-deficient mice (C2^-/- mice). These collaborators have previously provided important insights into the regulation and functions of cell-type specific lamin isoforms, particularly during meiosis. Using antibodies, they characterized the lamin isoforms present in rat spermatocytes [4]. Immunolocalization revealed that a truncated isoform of lamin C (lamin C2) was localized in a patchy pattern along the nuclear envelope, along with a short B-type lamin (lamin B3) [4]. Because these short isoforms lack domains implicated in interactions between lamin subunits, they and others proposed that these proteins might form a more flexible network. This idea was supported by experiments in which meiosis-specific lamin C2 was ectopically expressed in fibroblasts and found to be more mobile within the nuclear envelope than full-length lamin C [5]. Expression of lamin C2 also resulted in aberrant localization of Sun1 in these cells. The collaborators also demonstrated that spermatogenesis was disrupted in Lmna^-/- mice, although oocyte meiosis was not obviously perturbed [6]. Although defects in meiosis-specific processes were observed in the knockout mice, it was not possible to rule out an indirect effect of lamin depletion in somatic cells on meiosis in spermatocytes, prior to the new study.An important feature of the new research [1] is that the C2^-/- mice show normal expression of all other A-type lamins. The C2^-/- males recapitulate the meiotic failure seen in Lmna^-/- mice. Nevertheless, their chromosomes frequently fail to synapse and they engage in heterologous associations or show aberrant telomere-telomere interactions; all of these defects are rare in wild-type spermatocytes. As a result of extensive apoptosis and failure of sperm maturation, the males are completely infertile. However, females are fertile, despite some evidence for pairing defects in C2^-/- oocytes.These sex-specific differences in the effects of lamin C2 loss are somewhat surprising. They could in part reflect differential implementation of meiotic checkpoints, which cull defective spermatocytes more ruthlessly than oocytes [7]. However, analysis of homologous pairing and synapsis in the C2^-/- mutant mice also revealed more severe defects in males. Both male and female mice lacking Sun1 protein are completely sterile and show synaptic failure during meiotic prophase [8]. This suggests that LINC-mediated chromosome dynamics are essential for homolog interactions during meiosis in both sexes. The milder defects caused by loss of lamin C2 in both male and female meiosis suggest that it has a less direct role in mediating chromosome movement than Sun1. This is consistent with the idea that expression of short lamin isoforms during meiosis acts primarily to increase the mobility of proteins within the nuclear envelope, relative to somatic cells. It seems likely that the dynamics of pairing, synapsis and recombination differ dramatically between spermatocytes, which are produced continually during the adult life of the male, and oocytes, which undergo meiotic prophase during fetal development. Such differences might render male meiosis more sensitive to changes in nuclear envelope organization or dynamics.The modifications made to the mouse nuclear envelope during meiosis are likely to be conserved in concept, if not in detail, in other taxa. As mentioned above, the isoforms and expression patterns of lamin proteins have diverged rapidly among the metazoa, as have the structures and functions of LINC complexes. For example, amphibians lack lamin C (and lamin C2), suggesting that its meiotic role in mammals is a recent innovation. Furthermore, the mouse Sun1 protein has a C2H2 zinc finger lacking in primate orthologs, which might suggest that it has evolved a distinct way to connect with meiotic chromosomes. It is thus not currently clear which aspects of meiotic lamina remodeling in mice can be extrapolated to other species.In Caenorhabditis elegans, meiotic chromosome dynamics are probably mediated by post-translational modification of the amino-terminal (nucleoplasmic) domain of sun-1 [9]. It is not yet known how this modification contributes to the function of the meiotic LINC complex. Direct observation has indicated that the motion of LINC complexes within the nuclear envelope becomes much less constrained as cells enter meiosis [3]. Phosphorylation of sun-1 may weaken interactions between the LINC complexes and the lamina to increase their mobility within the nuclear envelope, and/or promote interactions between LINC complexes to create high load-bearing aggregates of these proteins necessary to drive chromosome movement. It is not currently known whether the lamina itself is modified in C. elegans meiotic nuclei, but it is easy to imagine that phosphorylation could also be used to tweak protein-protein interactions within the lamina to optimize its properties during meiosis and other specialized cellular processes. It is likely that metazoans have evolved a wide range of mechanisms to modify their nuclear envelopes to meet the special demands of meiotic prophase.Homologous chromosome pairing remains one of the most mysterious aspects of meiosis. This new work in mice [1] adds an important piece of the puzzle by illuminating how the nuclear lamina can be modified to facilitate meiotic chromosome dynamics. To understand this process will clearly require looking beyond the chromosomes, and even beyond the nucleus, to the cellular networks connected by LINC complexes.     

Viral Mimicry of Cdc2/Cyclin-Dependent Kinase 1 Mediates Disruption of Nuclear Lamina during Human Cytomegalovirus Nuclear Egress

The nuclear lamina is a major obstacle encountered by herpesvirus nucleocapsids in their passage from the nucleus to the cytoplasm (nuclear egress). We found that the human cytomegalovirus (HCMV)-encoded protein kinase UL97, which is required for efficient nuclear egress, phosphorylates the nuclear lamina component lamin A/C in vitro on sites targeted by Cdc2/cyclin-dependent kinase 1, the enzyme that is responsible for breaking down the nuclear lamina during mitosis. Quantitative mass spectrometry analyses, comparing lamin A/C isolated from cells infected with viruses either expressing or lacking UL97 activity, revealed UL97-dependent phosphorylation of lamin A/C on the serine at residue 22 (Ser²²). Transient treatment of HCMV-infected cells with maribavir, an inhibitor of UL97 kinase activity, reduced lamin A/C phosphorylation by approximately 50%, consistent with UL97 directly phosphorylating lamin A/C during HCMV replication. Phosphorylation of lamin A/C during viral replication was accompanied by changes in the shape of the nucleus, as well as thinning, invaginations, and discrete breaks in the nuclear lamina, all of which required UL97 activity. As Ser²² is a phosphorylation site of particularly strong relevance for lamin A/C disassembly, our data support a model wherein viral mimicry of a mitotic host cell kinase activity promotes nuclear egress while accommodating viral arrest of the cell cycle.     

Disruption of Nuclear Lamin Organization Alters the Distribution of Replication Factors and Inhibits DNA Synthesis

The nuclear lamina is a fibrous structure that lies at the interface between the nuclear envelope and the nucleoplasm. The major proteins comprising the lamina, the nuclear lamins, are also found in foci in the nucleoplasm, distinct from the peripheral lamina. The nuclear lamins have been associated with a number of processes in the nucleus, including DNA replication. To further characterize the specific role of lamins in DNA replication, we have used a truncated human lamin as a dominant negative mutant to perturb lamin organization. This protein disrupts the lamin organization of nuclei when microinjected into mammalian cells and also disrupts the lamin organization of in vitro assembled nuclei when added to Xenopus laevis interphase egg extracts. In both cases, the lamina appears to be completely absent, and instead the endogenous lamins and the mutant lamin protein are found in nucleoplasmic aggregates. Coincident with the disruption of lamin organization, there is a dramatic reduction in DNA replication. As a consequence of this disruption, the distributions of PCNA and the large subunit of the RFC complex, proteins required for the elongation phase of DNA replication, are altered such that they are found within the intranucleoplasmic lamin aggregates. In contrast, the distribution of XMCM3, XORC2, and DNA polymerase α, proteins required for the initiation stage of DNA replication, remains unaltered. The data presented demonstrate that the nuclear lamins may be required for the elongation phase of DNA replication.     

Increased plasticity of the nuclear envelope and hypermobility of telomeres due to the loss of A–type lamins

Background

The nuclear lamina provides structural support to the nucleus and has a central role in defining nuclear organization. Defects in its filamentous constituents, the lamins, lead to a class of diseases collectively referred to as laminopathies. On the cellular level, lamin mutations affect the physical integrity of nuclei and nucleo-cytoskeletal interactions, resulting in increased susceptibility to mechanical stress and altered gene expression.

Methods

In this study we quantitatively compared nuclear deformation and chromatin mobility in fibroblasts from a homozygous nonsense LMNA mutation patient and a Hutchinson–Gilford progeria syndrome patient with wild type dermal fibroblasts, based on the visualization of mCitrine labeled telomere-binding protein TRF2 with light-economical imaging techniques and cytometric analyses.

Results

Without application of external forces, we found that the absence of functional lamin A/C leads to increased nuclear plasticity on the hour and minute time scale but also to increased intranuclear mobility down to the second time scale. In contrast, progeria cells show overall reduced nuclear dynamics. Experimental manipulation (farnesyltransferase inhibition or lamin A/C silencing) confirmed that these changes in mobility are caused by abnormal or reduced lamin A/C expression.

Conclusions

These observations demonstrate that A-type lamins affect both nuclear membrane and telomere dynamics.

General significance

Because of the pivotal role of dynamics in nuclear function, these differences likely contribute to or represent novel mechanisms in laminopathy development.     

Changes at the nuclear lamina alter binding of pioneer factor Foxa2 in aged liver

Increasing evidence suggests that regulation of heterochromatin at the nuclear envelope underlies metabolic disease susceptibility and age‐dependent metabolic changes, but the mechanism is unknown. Here, we profile lamina‐associated domains (LADs) using lamin B1 ChIP‐Seq in young and old hepatocytes and find that, although lamin B1 resides at a large fraction of domains at both ages, a third of lamin B1‐associated regions are bound exclusively at each age in vivo. Regions occupied by lamin B1 solely in young livers are enriched for the forkhead motif, bound by Foxa pioneer factors. We also show that Foxa2 binds more sites in Zmpste24 mutant mice, a progeroid laminopathy model, similar to increased Foxa2 occupancy in old livers. Aged and Zmpste24‐deficient livers share several features, including nuclear lamina abnormalities, increased Foxa2 binding, de‐repression of PPAR‐ and LXR‐dependent gene expression, and fatty liver. In old livers, additional Foxa2 binding is correlated to loss of lamin B1 and heterochromatin (H3K9me3 occupancy) at these loci. Our observations suggest that changes at the nuclear lamina are linked to altered Foxa2 binding, enabling opening of chromatin and de‐repression of genes encoding lipid synthesis and storage targets that contribute to etiology of hepatic steatosis.     

A carboxyl-terminal interaction of lamin B1 is dependent on the CAAX endoprotease Rce1 and carboxymethylation

The mammalian nuclear lamina protein lamin B1 is posttranslationally modified by farnesylation, endoproteolysis, and carboxymethylation at a carboxyl-terminal CAAX motif. In this work, we demonstrate that the CAAX endoprotease Rce1 is required for lamin B1 endoproteolysis, demonstrate an independent pool of proteolyzed but nonmethylated lamin B1, as well as fully processed lamin B1, in interphase nuclei, and show a role for methylation in the organization of lamin B1 into domains of the nuclear lamina. Deficiency in the endoproteolysis or methylation of lamin B1 results in loss of integrity and deformity of the nuclear lamina. These data show that the organization of the nuclear envelope and lamina is dependent on a mechanism involving the methylation of lamin B1, and they identify a potential mechanism of laminopathy involving a B-type lamin.     

Dynamic properties of nuclear lamins: lamin B is associated with sites of DNA replication

The nuclear lamins form a fibrous structure, the nuclear lamina, at the periphery of the nucleus. Recent results suggest that lamins are also present as foci or spots in the nucleoplasm at various times during interphase of the cell cycle (Goldman, A. E., R. D. Moir, M. Montag- Lowy, M. Stewart, and R. D. Goldman. 1992. J. Cell Biol. 104:725-732; Bridger, J. M., I. R. Kill, M. O'Farrell, and C. J. Hutchison. 1993. J. Cell Sci. 104:297-306). In this report we demonstrate that during mid- late S-phase, nuclear foci detected with lamin B antibodies are coincident with sites of DNA replication as detected by the colocalization of sites of incorporation of bromodeoxyuridine (BrDU) or proliferating cell nuclear antigen (PCNA). The relationship between lamin B and BrDU is not maintained in the following G1 stage of the cell cycle. Furthermore, the nuclear staining patterns seen with antibodies directed against lamins A and C in mid-late S-phase do not coalign with the lamin B/BrDU-containing structures. These results imply that there is a role for lamin B in the organization of replicating chromatin during S phase.     

Towards understanding lamin gene regulation

The lamins are components of the nuclear lamina, which forms a fibrous meshwork lining the inner nuclear membrane. Lamina-membrane interactions play a crucial role during nuclear disassembly and reassembly at mitosis, whereas lamina-chromatin association has been proposed to be essential for chromatin organization. The composition of the lamina changes considerably during embryonic development and cell differentiation. Recent studies have provided insights into the regulation of the lamin genes.     

Association of lamin A/C with muscle gene-specific promoters in myoblasts

The A-type and B-type lamins form a filamentous meshwork underneath the inner nuclear membrane called the nuclear lamina, which is an important component of nuclear architecture in metazoan cells. The lamina interacts with large, mostly repressive chromatin domains at the nuclear periphery. In addition, genome–lamina interactions also involve dynamic association of lamin A/C with gene promoters in adipocytes. Mutations in the human lamin A gene cause a spectrum of hereditary diseases called the laminopathies which affect muscle, cardiac and adipose tissues. Since most mutations in lamin A/C affect skeletal muscle, we investigated lamin–chromatin interactions at promoters of muscle specific genes in both muscle and non-muscle cell lines by ChIP-qPCR. We observed that lamin A/C was specifically associated with promoter regions of muscle genes in myoblasts but not in fibroblasts. Lamin A/C dissociated from the promoter regions of the differentiation specific MyoD, myogenin and muscle creatine kinase genes when myoblasts were induced to differentiate. In the promoter regions of the myogenin and MyoD genes, the binding of lamin A/C in myoblasts inversely correlated with the active histone mark, H3K4me3. Lamin A/C binding on muscle genes was reduced and differentiation potential was enhanced on treatment of myoblasts with a histone deacetylase inhibitor. These findings suggest a role for lamina–chromatin interactions in muscle differentiation and have important implications for the pathological mechanisms of striated muscle associated laminopathies.     

Non-farnesylated B-type lamin can tether chromatin inside the nucleus and its chromatin interaction requires the Ig-fold region

Lamins are thought to direct heterochromatin to the nuclear lamina (NL); however, this function of lamin has not been clearly demonstrated in vivo. To address this, we analyzed polytene chromosome morphology when artificial lamin variants were expressed in Drosophila endoreplicating cells. We found that the CaaX-motif-deleted B-type lamin Dm₀, but not A-type lamin C, was able to form a nuclear envelope-independent layer that was closely associated with chromatin. Other nuclear envelope proteins were not detected in this “ectopic lamina,” and the associated chromatin showed a repressive histone modification maker but not a permissive histone modification marker nor RNA polymerase II proteins. Furthermore, deletion of the C-terminal lamin-Ig-fold domain prevents chromatin association with this ectopic lamina. Thus, non-farnesylated B-type lamin Dm₀ can form an ectopic lamina and induce changes to chromatin structure and status inside the interphase nucleus.     

p34cdc2 acts as a lamin kinase in fission yeast

The nuclear lamina is an intermediate filament network that underlies the nuclear membrane in higher eukaryotic cells. During mitosis in higher eukaryotes, nuclear lamins are phosphorylated by a mitosis-specific kinase and this induces disassembly of the lamina structure. Recently, p34cdc2 protein kinase purified from starfish has been shown to induce phosphorylation of lamin proteins and disassembly of the nuclear lamina when incubated with isolated chick nuclei suggesting that p34cdc2 is likely to be the mitotic lamin kinase (Peter, M., J. Nakagawa, M. Dorée, J.C. Labbe, and E.A. Nigg. 1990b. Cell. 45:145-153). To confirm and extend these studies using genetic techniques, we have investigated the role of p34cdc2 in lamin phosphorylation in the fission yeast. As fission yeast lamins have not been identified, we have introduced a cDNA encoding the chicken lamin B2 protein into fission yeast. We report here that the chicken lamin B2 protein expressed in fission yeast is assembled into a structure that associates with the nucleus during interphase and becomes dispersed throughout the cytoplasm when cells enter mitosis. Mitotic reorganization correlates with phosphorylation of the chicken lamin B2 protein by a mitosis-specific yeast lamin kinase with similarities to the mitotic lamin kinase of higher eukaryotes. We show that a lamin kinase activity can be detected in cell-free yeast extracts and in p34cdc2 immunoprecipitates prepared from yeast cells arrested in mitosis. The fission yeast lamin kinase activity is temperature sensitive in extracts and immunoprecipitates prepared from strains bearing temperature-sensitive mutations in the cdc2 gene. These results in conjunction with the previously reported biochemical studies strongly suggest that disassembly of the nuclear lamina at mitosis in higher eukaryotic cells is a consequence of direct phosphorylation of nuclear lamins by p34cdc2.