Yosef Gruenbaum

The nuclear lamina comes of age

Many nuclear proteins form lamin-dependent complexes, including LEM-domain proteins, nesprins and SUN-domain proteins. These complexes have roles in chromatin organization, gene regulation and signal transduction. Some link the nucleoskeleton to cytoskeletal structures, ensuring that the nucleus and centrosome assume appropriate intracellular positions. These complexes provide new insights into cell architecture, as well as a foundation for the understanding of the molecular mechanisms that underlie the human laminopathies — clinical disorders that range from Emery–Dreifuss muscular dystrophy to the accelerated ageing seen in Hutchinson–Gilford progeria syndrome.

Transcriptional repression, apoptosis, human disease and the functional evolution of the nuclear lamina.

The number and complexity of genes encoding nuclear lamina proteins has increased during metazoan evolution. Emerging evidence reveals that transcriptional repressors such as the retinoblastoma protein, and apoptotic regulators such as CED-4, have functional and dynamic interactions with the lamina. The discovery that mutations in nuclear lamina proteins cause heritable tissue-specific diseases, including Emery-Dreifuss muscular dystrophy, is prompting a fresh look at the nuclear lamina to devise models that can account for its diverse functions and dynamics, and to understand its enigmatic structure.

Methylation of CpG sequences in eukaryotic DNA.

An understanding of the function of DNA methylation in eukaryotes will require a clearer, more precise, knowledge of the distribution of methylated bases in the eukaryotic genome. Although it has been known for some time that 5-methylcytosine (m’Cyt) is the only methylated base in eukaryotes, little is known about the sequence specificity of this modification. One important fact which has emerged from base analyses is that >90% of the msCyt residues are found in the sequence CpG [ 11. It was therefore of interest to determine what fraction of this dinucleotide sequence is methylated. The answer to this question would not only contribute to our knowledge of the distribution of msCyt but would also shed light on the factors which make up the recognition signal for eukaryotic methylation. In an attempt to answer this question we have developed a new experimental procedure for detecting msCyt in CpG sequences. This dinucleotide is found to be highly methylated in animal cells and may be the major determinant in the placement of methyl groups on DNA.

The nuclear lamina and its functions in the nucleus.

The nuclear lamina is a structure near the inner nuclear membrane and the peripheral chromatin. It is composed of lamins, which are also present in the nuclear interior, and lamin-associated proteins. The increasing number of proteins that interact with lamins and the compound interactions between these proteins and chromatin-associated proteins make the nuclear lamina a highly complex but also a very exciting structure. The nuclear lamina is an essential component of metazoan cells. It is involved in most nuclear activities including DNA replication, RNA transcription, nuclear and chromatin organization, cell cycle regulation, cell development and differentiation, nuclear migration, and apoptosis. Specific mutations in nuclear lamina genes cause a wide range of heritable human diseases. These diseases include Emery-Dreifuss muscular dystrophy, limb girdle muscular dystrophy, dilated cardiomyopathy (DCM) with conduction system disease, familial partial lipodystrophy (FPLD), autosomal recessive axonal neuropathy (Charcot-Marie-Tooth disorder type 2, CMT2), mandibuloacral dysplasia (MAD), Hutchison Gilford Progeria syndrome (HGS), Greenberg Skeletal Dysplasia, and Pelger-Huet anomaly (PHA). Genetic analyses in Caenorhabditis elegans, Drosophila, and mice show new insights into the functions of the nuclear lamina, and recent structural analyses have begun to unravel the molecular structure and assembly of lamins and their associated proteins.

Nuclear lamins: key regulators of nuclear structure and activities

•  The lamin molecule ‐  Domain organization of lamins ‐  Lamins are divided to type A and type B ‐  Post‐translational processing of lamin molecules ‐  Lamin molecules in evolution •  The supramolecular assembly of lamins ‐  From lamin monomer to lamin dimer ‐  From dimers to filaments ‐  The roles of the different domains in the assembly of lamins ‐  Laminopathic mutations affect lamin filament assembly ‐  Lamin assembly in vivo •  Lamin‐binding proteins ‐  Lamins, chromatin and epigenesis ‐  Lamin binding to DNA ‐  Lamin binding to chromatin ‐  Lamins affect chromatin organization and epigenesis ‐  Lamins are involved in many nuclear functions ‐  Lamins determine the shape and stiffness of the nucleus ‐  Lamins and DNA replication ‐  Lamins in transcription and splicing •  Lamins and aging ‐  Lamins and laminopathies ‐  Mutations in lamins and their associated proteins causing ‘laminopathies’ ‐  Animal models for laminopathies ‐  Molecular models for laminopathies •  Lamins and stem cells ‐  The Notch pathway ‐  The Wnt/β‐catenin pathway ‐  Other pathways •  Lamins and cancer ‐  Lamins as biomarkers for cancer ‐  Lamins and cancer regulating pathways ‐  Lamins and cancer related aneuploidy •  Lamin and viruses

MAN1 and emerin have overlapping function(s) essential for chromosome segregation and cell division in Caenorhabditis elegans

Emerin and MAN1 are LEM domain-containing integral membrane proteins of the vertebrate nuclear envelope. The function of MAN1 is unknown, whereas emerin is known to interact with nuclear lamins, barrier-to-autointegration factor (BAF), nesprin-1α, and a transcription repressor. Mutations in emerin cause X-linked recessive Emery–Dreifuss muscular dystrophy. Emerin and MAN1 homologs are both conserved in Caenorhabditis elegans, but loss of Ce-emerin has no detectable phenotype. We therefore used C. elegans to test the hypothesis that Ce-MAN1 overlaps functionally with Ce-emerin. Supporting this model, Ce-MAN1 interacted directly with Ce-lamin and Ce-BAF in vitro and required Ce-lamin for its nuclear envelope localization. Interestingly, RNA interference-mediated removal of ≈90% of Ce-MAN1 was lethal to ≈15% of embryos. However, in the absence of Ce-emerin, ≈90% reduction of Ce-MAN1 was lethal to all embryos by the 100-cell stage, with a phenotype involving repeated cycles of anaphase chromosome bridging and cytokinesis [“cell untimely torn” (cut) phenotype]. Immunostaining showed that the anaphase-bridged chromatin specifically retained a mitosis-specific phosphohistone H3 epitope and failed to recruit detectable Ce-lamin or Ce-BAF. These findings show that LEM domain proteins are essential for cell division and that Ce-emerin and Ce-MAN1 share at least one and possibly multiple overlapping functions, which may be relevant to Emery–Dreifuss muscular dystrophy.

Lamins: Nuclear Intermediate Filament Proteins with Fundamental Functions in Nuclear Mechanics and Genome Regulation

Lamins are intermediate filament proteins that form a scaffold, termed nuclear lamina, at the nuclear periphery. A small fraction of lamins also localize throughout the nucleoplasm. Lamins bind to a growing number of nuclear protein complexes and are implicated in both nuclear and cytoskeletal organization, mechanical stability, chromatin organization, gene regulation, genome stability, differentiation, and tissue-specific functions. The lamin-based complexes and their specific functions also provide insights into possible disease mechanisms for human laminopathies, ranging from muscular dystrophy to accelerated aging, as observed in Hutchinson-Gilford progeria and atypical Werner syndromes.

The absence of detectable methylated bases in Drosophila melanogaster DNA

Most of the eukaryotic organisms are methylated at specific cytosine residues in their DNA. For more than 2 decades efforts have been made to answer the question of whether the DNA of the fruit fly Drosophila melanogaster is methylated, but the results were inconclusive. The answer to this question is now attracting special interest in light of the fact that in recent years substantial evidence has accumulated, suggesting a correlation between vertebrate DNA methylation and gene expression [1]. Drosophila in particular is an interesting organism in this respect as it goes through severabdefined developmental stages and its genome organization has been extensively investigated. Here, a variety of highly sensitive methods have been used to analyze methylated bases in Drosophila melanogaster DNA. 5-Methyl-cytosine, which is the common methylated base in DNA of eukaryotic organisms, could not be detected in any of the developmental stages of this organism. There is no indication for other modifications of this DNA as well. The welldefined clonally inherited patterns of methylation of the genetic material in mammals [1] and the absence of such methylation patterns in Drosophila DNA suggested here, bring into focus the longstanding enigma of the biological role played by methylation patterns. 2.1. Preparation of DNA DNA of Drosophila melanogaster embryos, larvae, pupae and adults was prepared from isolated nuclei. The nuclei were lysed in 1 mM Tris (pH 8) and after centrifugation at 10 000 x g the chromatin pellet was suspended in a lysis mixture containing 0.5% (w/v) sodium lauryl sulphate; 2.5 mM EDTA; 0.5 M NaC1; 10 mM Tris (pH 8) and 100 ~g proteinase K/ml (Merck Co.). The mixture was incubated for 2 h at 37°C and RNase treated (300/~g pancreatic RNase/ml for l h at 37°C). Phenol extraction was followed by chloroform:isoamyl alcohol (24: 1, v/v) extraction and ethanol precipitation. The DNA was hydrolyzed to free bases for analysis as in [2].

Meiotic Chromosome Homology Search Involves Modifications of the Nuclear Envelope Protein Matefin/SUN-1

Genome haploidization during meiosis depends on recognition and association of parental homologous chromosomes. The C. elegans SUN/KASH domain proteins Matefin/SUN-1 and ZYG-12 have a conserved role in this process. They bridge the nuclear envelope, connecting the cytoplasm and the nucleoplasm to transmit forces that allow chromosome movement and homolog pairing and prevent nonhomologous synapsis. Here, we show that Matefin/SUN-1 forms rapidly moving aggregates at putative chromosomal attachment sites in the meiotic transition zone (TZ). We analyzed requirements for aggregate formation and identified multiple phosphotarget residues in the nucleoplasmic domain of Matefin/SUN-1. These CHK-2 dependent phosphorylations occur in leptotene/zygotene, diminish during pachytene and are involved in pairing. Mimicking phosphorylation causes an extended TZ and univalents at diakinesis. Our data suggest that the properties of the nuclear envelope are altered during the time window when homologs are sorted and Matefin/SUN-1 aggregates form, thereby controling the movement, homologous pairing and interhomolog recombination of chromosomes.

The supramolecular organization of the C. elegans nuclear lamin filament.

Nuclear lamins are involved in most nuclear activities and are essential for retaining the mechano-elastic properties of the nucleus. They are nuclear intermediate filament (IF) proteins forming a distinct meshwork-like layer adhering to the inner nuclear membrane, called the nuclear lamina. Here, we present for the first time, the three-dimensional supramolecular organization of lamin 10 nm filaments and paracrystalline fibres. We show that Caenorhabditis elegans nuclear lamin forms 10 nm IF-like filaments, which are distinct from their cytoplasmic counterparts. The IF-like lamin filaments are composed of three and four tetrameric protofilaments, each of which contains two partially staggered anti-parallel head-to-tail polymers. The beaded appearance of the lamin filaments stems from paired globular tail domains, which are spaced regularly, alternating between 21 nm and 27 nm. A mutation in an evolutionarily conserved residue that causes Hutchison-Gilford progeria syndrome in humans alters the supramolecular structure of the lamin filaments. On the basis of our structural analysis, we propose an assembly pathway that yields the observed 10 nm IF-like lamin filaments and paracrystalline fibres. These results serve also as a platform for understanding the effect of laminopathic mutations on lamin supramolecular organization.