Transcriptional repression, apoptosis, human disease and the ...

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TRENDS in Biochemical Sciences Vol.26 No.1 January 2001

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Transcriptional repression, apoptosis, human disease and the functional evolution of the nuclear lamina Merav Cohen, Kenneth K. Lee, Katherine L.Wilson and Yosef Gruenbaum 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.

Merav Cohen Yosef Gruenbaum* Dept of Genetics,The Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel. *e-mail: [email protected] Kenneth K. Lee Katherine L.Wilson Dept of Cell Biology and Anatomy,The Johns Hopkins University School of Medicine, 725 N. Wolfe St, Baltimore, MD 21205, USA. All authors contributed equally to the article.

The main feature of eukaryotic cells is the nucleus, which enwraps the chromosomes and is the site of DNA replication, RNA transcription and processing, and ribosome assembly. The nuclear envelope (NE) is the boundary between the nucleus and cytoplasm. The NE is composed of the inner and outer nuclear membranes (INM and ONM, respectively), which are separated by a lumenal space continuous with the ER lumen. Communication between the nucleoplasm and cytoplasm takes place through pores in the nuclear envelope, where the inner and outer membranes join. Within these pores are nuclear pore complexes (NPCs), which mediate and regulate nuclear transport1. Underneath the INM is a meshwork of nuclear-specific intermediate filaments, termed the nuclear lamina, which includes lamin proteins plus a growing number of lamin-associated proteins2,3. Near the INM is the peripheral chromatin, a large proportion of which is heterochromatin (Fig. 1). Lamins are type V intermediate filament proteins. They range in size from 60 to 70 kDa and have a characteristic structure: a small N-terminal ‘head’, a 52-nm coiled-coil ‘rod’ and a globular Cterminal ‘tail’4. Lamins form α-helical coiled-coil dimers, which are the building blocks for further assembly. In vitro, lamin dimers associate to form head-to-tail polymers. The assembly pathway and final structure(s) of lamin filaments are poorly understood. Indeed, lamin filaments are probably unlike the 10-nm diameter cytoplasmic intermediate filaments, because no 10-nm filaments have been detected in electron micrograph cross-sections, and the lamina isolated from Xenopus oocyte nuclei forms an orthogonal network rather than linear filaments4. There is strong evidence that lamins are not restricted to the nuclear periphery but exist

throughout the nuclear interior5. It is not known if the peripheral and interior lamins form similar or different structures. However, given the growing number and types of lamin-binding proteins, some of these partners might influence the assembly or structural properties of lamins. The nuclear lamina provides structural support for chromosomes, and is required to maintain nuclear shape, space NPCs, replicate DNA and efficiently segregate chromosomes2,3. New functions for the lamina are discussed below. Metazoan evolution: a gradual increase in the complexity of nuclear lamina proteins

Investigators are identifying a growing number of integral and peripheral membrane proteins that associate with lamins (Fig. 2). Hence, the term ‘nuclear lamina’ is general, and includes both lamins and lamin-binding proteins. Vertebrates have three lamin genes. The LMNA gene encodes four alternatively spliced A-type lamins named A, C, A∆10 and C2. Two genes encode B-type lamins, LMNB1 (encoding lamin B1) and LMNB2 (encoding lamins B2 and B3)4. Several of these proteins are differentially expressed during development and differentiation, suggesting tissue-specific functions. In addition, vertebrates express many integral membrane proteins in the INM, including three isoforms of lamina-associated protein 1 (LAP1), at least five isoforms of LAP2, as well as emerin (mutations in which cause Emery–Dreifuss muscular dystrophy; EDMD), MAN1, lamin B receptor (LBR), nurim and probably UNC-84. A sixth isoform of LAP2, named LAP2α, lacks a transmembrane domain and is found throughout the nuclear interior during interphase. The LAP2 isoforms plus emerin and MAN1 are members of a family defined by a 43-residue ‘LEM’ domain near their N terminus6. There are two lamin genes in Drosophila (lamin Dm0 and lamin C), and one in Caenorhabditis elegans (Ref. 4). The number of lamins expressed in each organism fits a pattern in which more complex eukaryotes have greater lamin diversity. Eukaryotes are defined as more complex if they have more cells and more distinct cell types, tissues and organs. Thus, adult hermaphrodite C. elegans

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absent in C. elegans, including LBR, nurim and Atype lamins. Not surprisingly, some lamina proteins might be unique to vertebrates. For example, neither Drosophila nor C. elegans encode obvious orthologs for LAP1 or LAP2. Metazoan nuclear lamina proteins are absent from yeast, and probably plants

Fig. 1. Schematic view of the nuclear envelope, lamina and chromatin. Shown are the inner and outer membranes of the nuclear envelope, with their enclosed lumen. Also shown are lamin filaments and selected nuclear envelope proteins including lamina-associated protein 1 (LAP1), emerin, LAP2β, MAN1, UNC-84, lamin B receptor (LBR), nurim and otefin. Otefin is a peripheral membrane protein that binds lamins; the other depicted proteins are anchored in the inner membrane via one or more transmembrane domains. Most nuclear envelope proteins bind specifically to either A-type or B-type lamins, which are depicted hypothetically as heteropolymers by different shades of orange. Question marks indicate proteins whose binding properties are unknown. The distinctly shaped knobs on A- and B-type lamins represent specific binding sites for histones and other proteins, such as transcriptional regulators (not shown; see Fig. 5). The peripheral lamina is proposed to tether proteins that regulate gene expression or modify chromatin structure. Chromatin located near the nuclear envelope is depicted as structurally condensed, to indicate that it is mostly transcriptionally inactive. Lamins are also found in the interior of the nucleus, along with the α isoform of LAP2. The structure of the ‘internal lamina’ is not understood, and is therefore hypothetically depicted as filaments that are thin, disconnected and crossbridged. We have used artistic license to depict direct connections between the peripheral and interior lamin filaments, and to suggest alternative structures for lamin filaments.

nematodes, which consist of 959 body cells, are defined as less complex than adult Drosophila fruit flies, which have ten times as many cells and can form wings, legs, eyes, sensory bristles and other specialized structures. The nearly complete genome sequences for C. elegans and Drosophila melanogaster allowed a search for homologs of vertebrate INM proteins in ‘lower’ eukaryotes (Fig. 2). C. elegans has three LEM-domain genes, including homologs of vertebrate emerin and MAN1 (Ref. 7). A homolog for lem-3 has not yet been identified in vertebrates. UNC-84, an abundant INM protein in C. elegans, is required for nuclear migration during development8. Homologs of unc-84 are present in Drosophila and humans, but nothing is known about their localization or function. The Drosophila genome has at least six LEM-domain genes, three of which are probably homologs of MAN1, emerin and lem-3. The other Drosophila LEM-domain proteins are otefin, an otefin-like protein and a novel protein with four putative transmembrane domains (Fig. 2). Several INM proteins present in vertebrates and Drosophila are http://tibs.trends.com

The yeast Saccharomyces cerevisiae contains no lamin genes9, and lacks all other INM proteins known in multicellular eukaryotes. This complete lack of nuclear lamina proteins in S. cerevisiae contrasts with the conservation of NPC proteins in yeast10, and suggests that metazoan nuclear lamina proteins provide functions unique to multicellular animals. What about the other multicellular eukaryotes, plants? DNA sequence information is available in GenBank for large fractions of the Arabidopsis genome and many plant cDNAs. Despite previous reports, lamina proteins appear to be absent from plants. Conversely, plant proteins such as MAF1, which localizes to the plant NE, lack homologs in animals11. These results suggest that NE proteins and functions evolved separately in plants and animals. Thus, several unique INM proteins first appeared in metazoans and increased in number and complexity during evolution. Metazoan nuclear lamina proteins first appeared probably around the transition between closed and open mitosis (see below). Increase in the efficiency and extent of nuclear envelope disassembly during animal evolution

S. cerevisiae has a closed mitosis wherein the NE remains intact12. During closed mitosis, tubulin proteins are imported to allow mitotic spindles to assemble inside the nucleus. By contrast, the mammalian NE undergoes ‘open’ mitosis, in which the nuclear lamina and NPCs reversibly disassemble, the nuclear membranes merge into the ER and nuclear proteins are released into the cytoplasm. During late anaphase and telophase this process is reversed; nuclear membranes reassociate with chromatin, NPCs assemble and nuclear proteins including lamins are imported back into the nucleus13. In vertebrates, the disassembly of the NE defines the transition between prophase and prometaphase12. Based on their extent and timing of NE breakdown, C. elegans and Drosophila embryos are intermediate between yeast and vertebrates (Fig. 3). For example, in C. elegans, the nuclear membranes and lamina remain intact except at spindle poles until after the metaphase–anaphase transition, and completely disassemble only during mid-late anaphase7. In Drosophila early embryos, NPCs disassemble during prophase, similar to vertebrates. However,

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Fig. 2. Proposed orthologs to lamina proteins in Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster and vertebrates. The first number shows the number of genes; the number of splicing isoforms is in parentheses. Brown indicates that no ortholog was found; light yellow indicates the presence of a single gene; yellow indicates multiple genes or multiple isoforms. Question marks indicate genes not yet found in the still-incomplete vertebrate databases. Data was obtained from the GenBank database. The S. cerevisiae protein Sad1 contains a region of homology to UNC-84, but is positioned on the outer nuclear membrane and ER, and was excluded from this figure15.

until mid–late anaphase the nuclear membranes (and a fraction of the lamina) remain intact and are supplemented by a temporary second layer of membranes14. Thus metazoan evolution might have been accompanied by an increase in the ability of different NE components to disassemble early in mitosis. The correlation between NE complexity and the enhanced extent and efficiency of NE disassembly at mitosis is intriguing. Open mitosis creates new problems because mechanisms are needed to (i) ensure that all chromosomes end up in a single nucleus; (ii) reassemble the NE and interior architecture; and (iii) re-establish the interphase organization of chromatin and subnuclear organelles. Furthermore, lamin filaments, no matter how useful during interphase, might interfere with chromosome segregation during mitosis13. Thus, open mitosis might have obligatorily co-evolved with nuclear lamina proteins. Having more lamina proteins probably conferred a selective advantage to metazoan creatures, perhaps related to improvements in chromatin organization, or improved nuclear signaling or gene expression. In addition, open mitosis exposes the chromatin to cytosolic proteins, which might provide new means to regulate the cell cycle through access to cytosolic replication licensing factors15. Furthermore, the process of nuclear assembly itself might provide new mechanisms for regulating chromatin structure during development and differentiation. Roles for nuclear lamina in apoptosis

Apoptosis16, or programmed cell death, can regulate cell number, sculpt tissues during development and http://tibs.trends.com

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eliminate damaged cells. During apoptosis, nuclei undergo specific morphological changes, including proteolytic cleavage of the nuclear lamina, clustering of NPCs, detachment of chromatin from the NE and DNA cleavage. In C. elegans, the caspase CED-3 executes apoptosis by cleaving target proteins. CED-3 is activated by the APAF1 homolog, CED-4. However, CED-4 is normally prevented from activating CED-3 by the BCL2 homolog, CED-9. Cell death is triggered when the BH3-domain protein, EGL-1, binds and inhibits CED-9 (Ref. 17). In normal C. elegans cells, antibodies against CED-9 and CED-4 revealed that both proteins are localized to the mitochondria. However, when apoptosis is triggered, either by EGL-1 activation or CED-9 destruction, CED-4 rapidly translocates to the envelope (Fig. 4). This translocation does not depend on CED-3, suggesting that CED-4 translocates to the NE before caspase activation18. Although the exact location of CED-4 in the NE is unknown, it appears to overlap the lamina18. This result indicates that the lamina provides an attachment site for apoptotic signaling machinery. Both A- and B-type lamins, LAP2α and LAP2β are early targets for caspase degradation, before detectable DNA cleavage or chromatin condensation occurs19,20. Other INM proteins, including LBR, are targeted later21. Lamins are cleaved in the α-helical rod domain, probably by caspase 6 (Ref. 22). LAP2β is cleaved by caspase 3 (Ref. 21). Cleaved fragments of lamins and LAP2β remain associated with the NE but are not known to play further roles. Apoptotic nuclei resemble nuclei in lamindeficient cells, which have clustered NPCs, detached chromatin and odd shapes23–25. Thus, lamin destruction probably causes these same changes during apoptosis. Direct evidence for the importance of lamins in apoptosis was found by expressing an uncleavable mutant form of lamin in cultured cells22; although caspases were activated, chromatin failed to condense and DNA cleavage was delayed. This result suggests that lamin degradation facilitates nuclease activation during apoptosis. It would be interesting to follow apoptosis in C. elegans cells that express uncleavable lamin, and to determine whether CED-4 can translocate to a lamin-depleted NE. Heterochromatin tends to associate with the INM

Constitutive heterochromatin is transcriptionally repressed; it is also highly condensed, late replicating, rich in repetitive sequences, does not recombine during meiosis and has regular nucleosome spacing. Repressive chromatin structure can spread to genes near heterochromatin26. A comparison between four model organisms – S. cerevisiae, C. elegans, D. melanogaster and mouse – shows that the amount of constitutive heterochromatin increases from less to more complex

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Fig. 3. Increase in the extent and timing of nuclear envelope (NE) disassembly during animal evolution. Abbreviations: +, indicates the component is mostly intact; +/–, partial NE disassembly and release to the cytoplasm; +/– – , almost complete disassembly; –, complete disassembly; NPC, nuclear pore complex. For details see Refs 7,12,14,31.

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organisms. Heterochromatin in S. cerevisiae is limited to telomeres and silent mating type loci26. C. elegans has condensed chromatin at its periphery (e.g. see electron micrographs in Ref. 27) and tandem repeats, a hallmark of constitutive heterochromatin, accounting for ~2.7% of the genome28. The Drosophila genome contains an estimated 30% heterochromatin, including most of the Y chromosome. About 40% of human chromosome 22 is repetitive noncoding DNA (Ref. 29). Cytological studies show that a large proportion of condensed chromatin, including centromeres and telomeres, borders the INM (Ref. 30). The positioning of inactive DNA is not random, because the inactive X chromosome in female mammalian cells is located near the NE, whereas the active X chromosome extends into the nuclear interior31. Even in S. cerevisiae, INM proximity helps to silence partially silenced genes: when the DNAbinding domain of the yeast transcription factor Gal4 was fused to a transmembrane protein, Gal4 became localized to the nuclear periphery, as did target genes containing the Gal4 operator. Furthermore, these genes became completely http://tibs.trends.com

silenced, in a Sir (silent information regulatory protein)-dependent manner32. Many nuclear lamina proteins interact directly with chromosomal proteins and might thereby affect chromatin structure at the nuclear periphery. For example, LBR interacts with the heterochromatin-specific protein HP1 (Ref. 33), and LEM-domain protein LAP2β interacts with barrier-to-autointegration factor (BAF)34, a DNAbinding protein that inhibits autointegration of retroviral DNA (Ref. 35). Lamins themselves can bind specific histones36. Young arrest (YA) is a Drosophila protein with developmentally regulated expression that is required for the transition from meiosis to mitosis; YA binds to both lamin B and chromatin37. A key question for the future is whether interactions between NE proteins and chromatin directly modulate the higher-order structure of chromatin. Nuclear lamina and transcription

A growing number of transcription factors, most of which are repressors, localize at the nuclear periphery. Oct-1, a transcription factor containing a

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Fig. 4. Proposed translocation of apoptotic regulatory protein CED-4 to the nuclear envelope in Caenorhabditis elegans (Ref. 18). CED-9 (green), which is homologous to mammalian BCL2, is located on mitochondrial membranes in normal cells. CED-9 binds CED-4 (red; homologous to mammalian APAF1), and prevents CED-4 from activating apoptosis. EGL-1 (blue) initiates the death signal by releasing CED-4. CED-4 then translocates to the nuclear lamina, where it is proposed to bind an unidentified lamina protein6 (X, yellow).

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(GCL) protein, which in Drosophila is required to establish the germ cell lineage during development41. The mouse GCL is active in Drosophila (Ref. 42), where it localizes primarily at the NE. Mouse GCL protein interacts with DP protein, which heterodimerizes with transcription factor E2F and regulates the cell cycle43. Intriguingly, mouse GCL was independently identified in a two-hybrid screen by its interaction with the β-specific region of the INM protein LAP2β; in addition, GCL co-localizes with LAP2β at the nuclear lamina (A. Simon, pers. commun.). Figure 5 shows a model in which these pair-wise interactions (e.g. between Rb and lamin A) occur simultaneously to repress a given target gene. However, it is equally possible that Rb and LAP2β form separate complexes. The nuclear periphery is also used for tethering transcription activators. For example, at low glucose levels, the insulin activator IPF-1/PDX-1 is localized to the nuclear periphery. Elevated glucose causes a rapid translocation of IPF-1/PDX-1 to the nucleoplasm where it stimulates the transcription of the insulin gene44. Oct-1, Rb, GCL and IPF-1/PDX-1 provide strong support for the hypothesis that nuclear lamina proteins play active roles in transcriptional repression and chromatin structure. Further support for this idea comes from the Drosophila Su(HW) and Mod(mdg4) proteins, which establish chromatin boundaries that regulate homeotic gene expression45. Immunostaining of Drosophila cells showed that Su(HW)–Mod(mdg4) complexes are restricted to only 10–20 spots, most of which are located near the nuclear lamina45.

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Nuclear lamina and genetic diseases Fig. 5. A speculative model for the involvement of the nuclear lamina in transcriptional repression. Transcriptional repressor retinoblastoma (Rb) associates with the nuclear lamina when it is active as a repressor, whereas hyperphosphorylated Rb is not associated with the lamina. Rb represses genes required for entry into S-phase by binding to E2F–DP heterodimers, and recruiting histone deacetylases (not depicted).The model is based on binding observed between pairs of proteins in vitro: lamins A/C –Rb, Rb–E2F, E2F–DP, DP–GCL (germ cell-less), GCL–LAP2β , LAP2β–BAF, and LAP2β–lamin B (see text for details). It is not yet known which, if any, of these putative interactions occur in vivo in the context of gene regulation. Nuclear lamina filaments are depicted generically as a orange rod, with round knobs to represent binding sites specific for A-type lamins, and triangular knobs to represent specific binding sites on B-type lamins.

POU domain that represses the aging-associated collagenase gene, co-localizes with lamin B (Ref. 38). In aging cells, the departure of Oct-1 from the NE coincides with increased collagenase activity; both events are reverted in immortalized cells38. This result suggests that Oct-1 is active as a repressor only when it is located at the NE. The retinoblastoma (Rb) protein binds transcription factor E2F and represses transcription by recruiting histone deacetylase39. The active (hypophosphorylated) form of Rb co-localizes with lamins A/C at the nuclear periphery in vivo and binds lamins A/C in vitro40. Thus, transcriptional repression by Rb correlates with its lamin-binding activity (Fig. 5). A third example is the germ-cell-less http://tibs.trends.com

Interactions between the nuclear lamina and transcription factors might explain how mutations in nuclear lamina proteins cause inherited diseases. For example, the X-linked form of Emery–Dreifuss muscular dystrophy (EDMD) is caused by mutations in the emerin gene46, whereas autosomal–dominant EDMD is caused by mutations in LMNA (Ref. 47; Fig. 6). Other mutations in LMNA cause cardiomyopathy and lipodystrophy47. These diseases are proposed to result from defects in gene expression, due to loss of specific attachment sites on the nuclear lamina needed to establish or maintain particular patterns of gene expression48. In this model, a missense mutation that disrupts the binding of just one factor to lamin filaments would cause a limited phenotype, whereas complete loss of LMNA would disrupt all proteins that depend on A-type lamin filaments. This prediction is supported by the severe combined phenotype seen in LMNAknockout mice49, which have defects in muscle, fat and possibly bone (B. Burke, pers. commun.), consistent with defects in tissue mesenchymal stem cells48.

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Fig. 6. A schematic map of mutations in human lamin A that cause three distinct inherited diseases. Shown is the lamin A protein (Ref. 50). The N-terminal head, coiled-coil rod and C-terminal tail domains are shown in different shades of blue. Mutations clinically associated with inherited dilated cardiomyopathy type 1A are shown in red.The mutations associated with autosomal–recessive and autosomal–dominant Emery–Dreifuss muscular dystrophy (EDMD) are shown in green and blue, respectively. Mutations associated with Dunnington-type partial lipodystrophy are shown in brown (mutation data taken from Ref. 47). More than half of the mutated residues that are associated with human diseases are identical in the lamin A gene in Xenopus and lamin A genes in hydra9. Numbers indicate exons, which are drawn approximately to scale according to the number of amino acid residues encoded. Mutations associated with sporadic (noninherited) disease are omitted.The clustering of mutations for dilated cardiomyopathy 1A and Dunnington-type lipodystrophy to distinct regions of the lamin protein indicates that these regions are used as attachment sites for specific binding partners relevant to each disease.

Conclusions

The nuclear lamina cannot be viewed merely as the ‘nuts and bolts’ of nuclear structure. Although the

References 1 Görlich, D. and Kutay, U. (1999) Transport between the cell nucleus and the cytoplasm. Annu. Rev. Cell Dev. Biol. 15, 607–660 2 Goldberg, M. et al. (1999) The nuclear lamina: molecular organization and interaction with chromatin. Crit. Rev. Eukaryotic Gene Expr. 9, 285–293 3 Gotzmann, J. and Foisner, R. (1999) Lamins and lamin-binding proteins in functional chromatin organization. Crit. Rev. Eukaryotic Gene Expr. 9, 257–265 4 Stuurman, N. et al. (1998) Nuclear lamins: their structure, assembly, and interactions J. Struct. Biol. 122, 42–66 5 Moir, R.D. et al. (2000) Disruption on nuclear lamin organization blocks the elongation phase of DNA replication. J. Cell Biol. 149, 1179–1192 6 Lin, F. et al. (2000) MAN1, an inner nuclear membrane protein that shares the LEM domain with lamina-associated polypeptide 2 and emerin. J. Biol. Chem. 275, 4840–4847 7 Lee, K.K. et al. (2000) C. elegans nuclear envelope proteins emerin, MAN1, lamin, and nucleoporins reveal unique timing of nuclear envelope breakdown during mitosis. Mol. Biol. Cell 11, 3089–3099

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EDMD Acknowledgements We thank biomedical illustrator M. Linkinhoker for rendering Figs 1 and 5; H. Cedar, C. Machamer, A. Simon, D. Shumaker and J. Liu for useful discussions and comments on the manuscript; and A. Simon for sharing results before publication.This work was supported by grants from the USA–Israel Binational Science Foundation (BSF), the Israel Science Foundation (ISF) #125/98 and the German–Israel Foundation (GIF) #1-573036.13 (toY.G.), and by grants from the W.W. Smith CharitableTrust and NIH-RO1GM48646 (to K.L.W.).

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lamina is certainly an essential structural element in the nucleus, the increasing evidence for human ‘laminopathy’ diseases suggests that some laminassociated proteins have highly specialized, and in some cases possibly tissue-specific, functions. Recent studies suggest that activities such as transcription repression, growth control and apoptotic signaling each depend on the dynamic assembly of lamin-based structures. These findings join previous work showing that the lamina is essential for DNA replication. The evolutionary view suggests that metazoan evolution has been accompanied by an expansion in the number and ‘flavors’ of nuclear lamins and lamin-binding proteins. Thus, more lamin-associated proteins will probably emerge, including new and known proteins whose functions are finally linked to a 3D context within the nucleus.

8 Malone, C. et al. (1999) UNC-84 localizes to the nuclear envelope and is required for nuclear migration and anchoring during C. elegans development. Development 126, 3171–3181 9 Erber, A. et al. (1998) Molecular phylogeny of metazoan intermediate filament proteins. J. Mol. Evol. 47, 751–762 10 Rout, M.P. et al. (2000) The yeast nuclear pore complex: composition, architecture, and transport mechanism. J. Cell Biol. 148, 635–651 11 Gindullis, F. and Meier, I. (1999) Matrix attachment region binding protein MFP1 is localized in discrete domains at the nuclear envelope. Plant Cell 11, 1117–1128 12 Gerace, L. and Burke, B. (1988) Functional organization of the nuclear envelope. Annu. Rev. Cell Biol. 4, 335–374 13 Gant, T.M. and Wilson, K.L. (1997) Nuclear assembly. Annu. Rev. Cell Dev. Biol. 13, 669–695 14 Paddy, M.R. et al. (1996) Time-resolved, in vivo studies of mitotic spindle formation and nuclear lamina breakdown in Drosophila early embryos. J. Cell Sci. 109, 591–607 15 Madine, M.A. et al. (2000) The roles of the MCM, ORC, and Cdc6 proteins in determining the replication competence of chromatin in quiescent cells. J. Struct. Biol. 129, 198–210

16 Thompson, E.B. (1998) Apoptosis. Annu. Rev. Physiol. 60, 525–533 17 Hengartner, M.O. (1999) Programmed cell death in the nematode C. elegans. Recent Prog. Horm. Res. 54, 213–222 18 Chen, F. et al. (2000) Translocation of C. elegans CED-4 to nuclear membranes during programmed cell death. Science 287, 1485–1489 19 Lazebnik, Y.A. et al. (1993) Nuclear events of apoptosis in vitro in cell-free mitotic extracts: a model system for analysis of the active phase of apoptosis. J. Cell Biol. 123, 7–22 20 Gotzmann, J. et al. (2000) Caspase-mediated cleavage of the chromosome-binding domain of lamina-associated polypeptide 2α. J. Cell Sci. 113, 3769–3780 21 Buendia, B. et al. (1999) Caspase-dependent proteolysis of integral and peripheral proteins of nuclear membranes and nuclear pore complex proteins during apoptosis. J. Cell Sci. 112, 1743–1753 22 Rao, L. et al. (1996) Lamin proteolysis facilitates nuclear events during apoptosis. J. Cell Biol. 135, 1441–1455 23 Lenz-Bohme, B. et al. (1997) Insertional mutation of the Drosophila nuclear lamin dm(0) gene

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results in defective nuclear envelopes, clustering of nuclear pore complexes, and accumulation of annulate lamellae. J. Cell Biol. 137, 1001–1016 Harel, A. et al. (1998) Structural organization and biological roles of the nuclear lamina. In Textbook of Gene Therapy and Molecular Biology: From Basic Mechanism to Clinical Applications (Vol. 1) (Boulikas, T., ed.), pp. 529–542, Gene Therapy Press Liu, J. et al. (2000) Essential roles for Caenorhabditis elegans lamin gene in nuclear organization, cell cycle progression, and spatial organization of nuclear pore complexes. Mol. Biol. Cell 11, 3937–3947 Cockell, M. and Gasser, S.M. (1999) Nuclear compartments and gene regulation. Curr. Opin. Genet. Dev. 9, 199–205 White, J.G. et al. (1976) The structure of the ventral nerve cord of Caenorhabditis elegans. Philos. Trans. R. Soc. London Ser. B 275, 327–348 Wilson, R.K. (1999) How the worm was won. The C. elegans genome sequencing project. Trends Genet. 15, 51–58 Dunham, I. et al. (1999) The DNA sequence of human chromosome 22. Nature 402, 489–495 Qumsiyeh, M.B. (1999) Structure and function of the nucleus: anatomy and physiology of chromatin. Cell Mol. Life Sci. 55, 1129–1140 Kay, R.R. and Johnston, R.I. (1973) The nuclear envelope: current problems of structure and of function. Sub-Cell. Biochem. 2, 127–166 Andrulis, E.D. et al. (1998) Perinuclear localization of chromatin facilitates transcriptional silencing. Nature 394, 592–595 Ye, Q.A. et al. (1997) Domain-specific interactions


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Unwinding the ‘Gordian knot’ of helicase action Panos Soultanas and Dale B.Wigley Helicases are enzymes involved in every aspect of nucleic acid metabolism. Recent structural and biochemical evidence is beginning to provide details of their molecular mechanism of action. Crystal structures of helicases have revealed an underlying common structural fold. However, although there are many similarities between the mechanisms of different classes of helicase, not all aspects of the helicase activity are the same in all members of this enzyme family.

Panos Soultanas School of Chemistry, University of Nottingham, University Park, Nottingham, UK NG7 2RD. Dale B.Wigley* ICRF Clare Hall Laboratories, Blanche Lane, South Mimms, Herts, UK EN6 3LD. *e-mail: [email protected]

The sequence of the bases in the DNA of each organism contains the genetic information that determines its characteristics. However, this genetic information is locked within a double helix formed by two interacting and antiparallel DNA strands. Specific hydrogen-bonded pairs are formed between the bases of the interacting strands. Gaining access to the genetic information is of crucial importance for the replication and repair of this information as well as to translate it into proteins. For RNA, there is a need to remove

unwanted secondary structures and to dissociate RNA–DNA heteroduplexes. Therefore, a diverse class of enzymes has evolved whose functions are to allow access to the genetic information. These are known as ‘helicases’. Since the discovery of the first helicase from Escherichia coli nearly a quarter of a century ago1, a large number of these enzymes have been identified and characterized in many organisms. Primary-structure comparisons have identified several conserved regions of amino acid sequence homology thought to be characteristic of helicases (so-called ‘helicase signature motifs’) and have resulted in the classification of these proteins into distinct superfamilies based upon the extent of homology of their primary sequences2. Superfamilies I and II represent the largest and most closely related groups of helicases. They

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