MINIREVIEW Gene silencing at the nuclear periphery Sigal Shaklai, Ninette Amariglio, Gideon Rechavi and Amos J. Simon Sheba Cancer Research Center and the Institute of Hematology, The Chaim Sheba Medical Center, Tel Hashomer and the Sackler School of Medicine, Tel Aviv University, Israel The nuclear lamina The nuclear envelope (NE), which separates the nucleus from the cytoplasm, consists of the outer (ONM) and inner (INM) nuclear membranes and nuclear pore com- plexes (NPCs). The ONM is continuous with the endo- plasmic reticulum (ER). The INM and ONM are separated by a lumenal space, but join at sites that are occupied by NPCs, which mediate bidirectional transport of macromolecules between the cytoplasm and the nucleus. The luminal space between the ONM and INM is crossed by giant protein complexes that bridge the NE and mechanically couple the cyto- skeleton to the nucleoskeleton (reviewed in [1]). In Keywords epigenetics; gene silencing; heterochromatin; histone modifications; LAP2; laminopathies; nuclear envelope; nuclear envelopathies; nuclear lamina; transcription Correspondence A. J. Simon, Sheba Cancer Research Center and the Institute of Hematology, The Chaim Sheba Medical Center, Tel Hashomer and the Sackler School of Medicine, Tel Aviv University, Israel Fax: 972 3 530 5351 Tel: 972 3 530 5814 E-mail: amos.simon@sheba.health.gov.il (Received 21 August 2006, revised 4 January 2007, accepted 8 January 2007) doi:10.1111/j.1742-4658.2007.05697.x The nuclear envelope (NE) is composed of inner and outer nuclear mem- branes (INM and ONM, respectively), nuclear pore complexes and an underlying mesh like supportive structure – the lamina. It has long been known that heterochromatin clusters at the nuclear periphery adjacent to the nuclear lamina, hinting that proteins of the lamina may participate in regulation of gene expression. Recent studies on the molecular mechanisms involved show that proteins of the nuclear envelope participate in regula- tion of transcription on several levels, from direct binding to transcription factors to induction of epigenetic histone modifications. Three INM pro- teins; lamin B receptor, lamina-associated polypeptide 2b and emerin, were shown to bind chromatin modifiers and ⁄ or transcriptional repressors indu- cing, at least in one case, histone deacetylation. Emerin and another INM protein, MAN1, have been linked to down-regulation of specific signaling pathways, the retino blastoma 1 ⁄ E2F MyoD and transforming growth fac- tor beta ⁄ bone morphogenic protein, respectively. Therefore, cumulative data suggests that proteins of the nuclear lamina regulate transcription by recruiting chromatin modifiers and transcription factors to the nuclear per- iphery. In this minireview we describe the recent literature concerning mechanisms of gene repression by proteins of the NE and suggest the hypothesis that the epigenetic ‘histone code’, dictating transcriptional repression, is ‘written’ in part, at the NE by its proteins. Finally, as aber- rant gene expression is one of the mechanisms speculated to underlie the newly discovered group of genetic diseases termed nuclear envelo- pathies ⁄ laminopathies, elucidating the repressive role of NE proteins is a major challenge to both researchers and clinicians. Abbreviations BAF, barrier-to-autointegration factor; EDMD, Emery–Dreifuss muscular dystrophy; GCL, germ cell less; HDAC, histone deacetylase; HP1, heterochromatin protein 1; IBSN, infantile bilateral striatal necrosis; INM, inner nuclear membrane; KASH, Klarsicht, ANC-1 and SYNE1 homology; LAP2b, lamina-associated polypeptide 2b; LBR, lamin B receptor; LEM domain, LAP2-emerin-MAN1 domain; ONM, outer nuclear membrane; NE, nuclear envelope; NES1, Nesprin-1; NES2g, Nesprin-2 giant; NPC, nuclear pore complexes; pRb, retinoblastoma protein; SREBP, sterol response element binding protein; SUN, S-phase arrest defective 1 and UNC-84 homology.; TGFb/BMP, transforming growth factor beta ⁄ bone morphogenic protein. FEBS Journal 274 (2007) 1383–1392 ª 2007 The Authors Journal compilation ª 2007 FEBS 1383 particular, SUN (S-phase arrest defective 1 and UNC- 84 homology) domain family of nuclear envelope pro- teins, such as Caenorhabditis elegans UNC-84 [2] and matefin ⁄ SUN-1 [3], interacts with various KASH [Klarsicht, actin-noncomplementing (ANC-1) and synap- tic nuclear envelope-1 (SYNE1) homology] domain partners, such as ANC-1 [4], UNC-83 [5], and ZYG-12 [6], to form SUN domain-dependent ‘bridges’ across the inner and outer nuclear membranes. In this network of SUN–KASH interactions UNC-84 can bind either ANC-1, which binds actin, or UNC-83, which binds microtubules via an unidentified microtubule-dependent motor protein. Matefin ⁄ SUN-1 binds ZYG-12 dimers, which bind the microtubule-organizing centre. Human proteins SUN1 and SUN2 anchor Nesprin-2 (also known as syne-2 and NUANCE) giant (NES2g) at the ONM. NES2g and Nesprin-1 (also known as CPG2, syne-1, myne-1 and Enaptin) giant isoform (NES1g), each bind actin. Nesprin-3 (NES3) binds plectin, which links cytoplasmic intermediate filaments to actin (reviewed in [1,7,8]). These bridges physically connect the nucleus to component of the cytoskeleton. By ser- ving, both as mechanical adaptors and nuclear envelope receptors, it is proposed that SUN domain proteins con- nect cytoplasmic and nucleoplasmic activities [1]. At the nucleoplasm this complex is proposed to be bound by lamins [9]. Lining the nucleoplasmic side of the NE, and in close contact with it, is the nuclear lamina (reviewed in [10,10a]). It is a protein meshwork composed of lamins and a growing number of NE lamin binding pro- teins. The nuclear lamina is proposed to have essential roles in chromatin and NPCs architecture and organiza- tion [11–15], nuclear positioning [4,5], NE breakdown and reassembly during mitosis [16], DNA replication [17], RNA polymerase II-dependent gene expression [18], and transcriptional repression [19,20]. The number of lamin-binding INM proteins that have been identified in mammalian cells is growing rapidly [21]. The identifi- cation and analysis of these proteins are essential to understanding the diverse cellular functions attributed to the nuclear lamina. Lamins are type-V intermediate- filament proteins, which have a short N-terminal ‘head’ domain, a long a-helical coiled-coil ‘rod’ domain, and a globular ‘tail’ domain (reviewed in [10,10a]). In mam- malian cells there are two types of lamins: A- and B-types. Lamin A ⁄ C proteins are the alternatively spliced isoforms of LMNA gene. These lamins are expressed in a tissue-specific manner, disperse as soluble proteins during mitosis, and are probably incorporated into the nuclear lamina later than B-type lamins during postmitotic NE reassembly. B-type lamins are essential for cell viability and are expressed in all cells during development. During mitosis the B-type lamins are found in a membrane-bound form, attached to the dis- assembled inner nuclear membranes (and their associ- ating proteins), suggesting their complete cellular segregation from A-type lamins when the NE is disas- sembled [10a]. In mammalian cells lamins bind in vitro to many known INM proteins, including emerin, MAN1, lamin B receptor (LBR), lamina-associated polypeptides-1 and 2 (LAP1, LAP2) isoforms and Nes- prin-1a. In addition, lamins bind nucleoplasmic soluble proteins, such as the chromatin histones H2A and H2B dimers and barrier-to-autointegration factor (BAF), as well as LAP2a, Kruppel-like protein (MOK2), actin, retinoblastoma protein (RB), sterol response element binding protein (SREBP), components of RNA polym- erase II-dependent transcription complexes and DNA replication complexes [22,23]. Mutations in lamins and lamin-binding proteins cause a wide range of heritable or sporadic human diseases, which are collectively known as the ‘nuclear envelopathies’ or ‘laminopathies’ [24–26]. The majority of these disorders were linked to mutations in A-type lamins. However, mutations in four integral INM lamin binding proteins have also been implicated as a cause of ‘nuclear envelopathies’. Of the four proteins LBR, emerin, LAP2 and MAN1, the three latter share the conserved 40 amino acid chromatin binding LAP2-emerin-MAN1 (LEM) domain [27,28]. Heterochromatin and the nuclear periphery Various studies have established that a correlation exists between positioning of genes at the nuclear per- iphery and their silencing (reviewed in [29] and above). Gene poor chromosomes have been shown to be more peripherally configured than gene rich chromosomes [30–32] and transcriptionally silent genes are located at or translocated to the nuclear periphery upon silencing [33,34]. Additionally, several experiments in various model systems have shown that the translocation of chromatin regions to the nuclear periphery results in silencing of the genes in these regions. In Drosophila, insertion of the gypsy insulator into a DNA sequence caused translocation of that sequence to the nuclear periphery correlating with changes in gene expression [35]. In mammalian cells, the Ikaros transcriptional regulator, which activates lymphocyte-specific expres- sion, was found to associate with transcriptionally inac- tive genes at centromeric loci [33]. Immunoglobulin loci in inactivated pro-T cells preferentially colocalized with lamin B at the nuclear periphery, while they were cen- trally configured and active in pro-B cells [36]. Simi- larly, dissociation of the transcriptional repressor Oct-1 from lamin B and the nuclear periphery was correlated Gene silencing at the nuclear periphery S. Shaklai et al. 1384 FEBS Journal 274 (2007) 1383–1392 ª 2007 The Authors Journal compilation ª 2007 FEBS with reduced inhibitory activity [37]. Several studies have made use of the lac-operator ⁄ repressor system [38] to demonstrate in vivo the ability of genetically engin- eered chromosome regions to undergo decondensation [39–41] and intranuclear repositioning [42–44] when activated by transcription factors or acidic activation domains. A clue to the mechanism by which intranu- clear translocation occurs comes from the work of Chu- ang and colleagues; they showed that migration of an interphase chromosome locus from the nuclear periph- ery to the nuclear center upon activation is disrupted by specific actin or nuclear myosin mutants [45]. While INM proteins in metazoans have been shown to func- tion as repressors of transcription, gene regulation at the nuclear periphery is probably a much more complex process. Recent studies in yeast suggest that proteins of the nuclear pore complex the nucleoporins (NUPS) function as inhibitors of gene repression or rather as activators of transcription. The nucleoporin Nup2p was shown to tether chromatin to the nuclear pore complex (NPC) blocking propagation of heterochromatin. Fur- thermore, interaction of Nup2p with numerous genes leads to their activation in what was coined the nucleo- pore to-gene-promoter interaction (Nup-PI) [46]. Simi- larly, transcriptionaly activated GAL1 genes are preferentially found at the nuclear periphery where they are linked to the NPC component Nup1 by SAGA interacting factors [47]. These studies support the notion that positioning of genes in the nuclear space correlates to their transcriptional activity, still leaving many unanswered questions as to the molecular mecha- nisms by which repositioning is transacted. Transcriptional repression by proteins of the nuclear envelope Peripherally located, transcriptionally silent chromatin has distinctive structural characteristics (at the DNA and chromatin levels) and has been shown to associate with proteins of the nuclear lamina. Whether these associations lead to the repressive chromatin pheno- type or are a result of it is still unknown. In a recent study in Drosophila melanogaster 500 genes interacting with the nuclear lamina protein B-type lamin (DmO, a Drosophila lamin), were identified and characterized [48]. In this study B-type lamin (DmO) was fused to the Escherichia coli enzyme DNA adenine methyl- transferase. The genomic DNA fragments that were methylated on their adenine residues were identified by cDNA microarray analysis. These genes displayed four main features: transcriptional inactivity, lack of ‘active’ histone marks, late replication timing and presence of long intergenic regions. Several large scale studies in mammalian tissues have also addressed the question of the components of nuclear envelope–chromatin associ- ated complexes. Schirmer et al. [21], in an attempt to identify new integral nuclear envelope proteins subjec- ted rat liver nuclear envelopes and cofractionated organelles to a subtractive proteomic analysis. Proteins remaining in the nuclear fractions included histones, chromatin associated proteins and transcription fac- tors. Georgatus and colleagues isolated mononucleo- somes attached to the LBR, from fractions of peripheral heterochromatin and demonstrated that they contain a distinct acetylatalion ⁄ methylation pat- tern befitting heterochromatin [49]. At least three INM proteins were shown to directly associate with chroma- tin modifiers and transcriptional repressors: 1. Lam- in B receptor (LBR) was found to associate with heterochromatin protein 1 (HP1) and histones H3 ⁄ H4 under deacetylating conditions [50]; 2. lamina-associ- ated polypeptide 2b (LAP2b) was shown by us to bind the transcriptional repressors germ cell less (GCL) [19] and histone deacetylase 3 (HDAC3) resulting in the latter case in deacetylation of histone H4 [20]; 3. Emerin was shown to associate with the death promoting fac- tor Btf [51], the splicing associated factor YT521-B [52] and similar to LAP2b, with the transcriptional repressor GCL [53]. Other components of the nuclear lamina shown to interact with transcriptional regula- tors include LAP2a, a nucleoplasmic LAP2 isoform, and lamin A ⁄ C [54–56]. LAP2a was shown to complex with lamin A ⁄ C and the retinoblastoma protein (pRb). Reduced levels of LAP2a or its aberrant localization caused mislocalization of pRb suggesting that LAP2a and lamin A ⁄ C serve as anchoring sites for this protein [55]. As mentioned, lamin A ⁄ C interacts with histones, components of the RNA II polymerase transcriptional complex (reviewed in [10]), SREBP [57] and dephos- phorylated pRb [55]. With regard to transcription, two INM LEM domain proteins, MAN1 and emerin, have been linked to specific pathways. MAN1 has been shown to antagonize TGFb ⁄ BMP signaling through binding to receptor-regulated Smads [sma (C. elegans) and mothers against decapentaplegic (DPP, Droso- phila) homologues], inhibiting downstream signaling and preventing normal ventralization in Xenopus laevis embryos [58–60,60a]. Emerin loss, responsible for X-linked Emery–Dreifuss muscular dystrophy, has been shown by two recent studies to result in deregula- tion of the Rb1 ⁄ E2F MyoD pathway involved in mus- cle regeneration [61,62]. Stewart and his colleagues evaluated regenerating muscle of emerin and lamin A null mice. In addition to Rb and MyoD, lack of emer- in resulted in up regulation of the transcriptional repression modifiers histone deacetylase 1 (HDAC1), S. Shaklai et al. Gene silencing at the nuclear periphery FEBS Journal 274 (2007) 1383–1392 ª 2007 The Authors Journal compilation ª 2007 FEBS 1385 histone methyl transferase Suv39H and HP1a in what was speculated to be a compensatory effect [61]. Both studies are in accordance with our previous findings [20,63], which link proteins of the nuclear envelope, such as the LAP2 family to epigenetic gene regulation. The epigenetic ‘histone code’ regulates transcription Histones undergo various types of post-translational modifications, including acetylation and methylation of lysines and arginines, phosphorylation of serines and threonines, ubiquitylation and sumoylation of lysines, as well as ribosylation. These reversible epigenetic modifications are executed by histone modifying enzymes, such as histone acetyl transferases and their antagonists histone deacetylases (HDACs), histone methyltransferases and their antagonists histone deme- thylases, histone kinases and their antagonists histone phosphatases and enzymes with sumoylation, ubiquity- lation and ribosylation activities. The epigenetic ‘his- tone code’ (or histone mark) is the pattern of these modifications. Its complexity results from the enor- mous number of combinations of modification type, number and sites on which they occur in each histone. For example, histone H3 can be acetylated on its lysine 9 (later on written as H3 K9 acetylation), phos- phorylated on the adjacent serine 10 residue and methylated on its lysine 27, individually or all at the same time. Further complexity results from the possi- bility of single lysine and arginine residues to undergo mono-, di- or tri- (in the case of lysine) methylation. The histone code influences the structure of the chro- matin fiber aiding or abating its ability to undergo transcription at that point. Site-specific combinations of histone modifications have been shown to correlate with transcriptional activation or repression. For example, the combination of H4 K8 acetylation, H3 K14 acetylation, and H3 S10 phosphorylation is often associated with transcriptional activation. Conversely, tri-methylation of H3 K9 and the lack of H3 and H4 acetylation correlate with transcriptional repression (reviewed in [64,65]). Evidence points to the concentra- tion of transcriptionally inactive heterochromatin, lacking histone acetylation at the nuclear periphery as opposed to acetylated, transcriptionaly competent euchro- matin at the nuclear interior [66]. Nuclear envelopathies ⁄ laminopathies and gene repression Mutations in proteins of the nuclear lamina have been shown to cause a wide array of genetic diseases termed nuclear envelopathies ⁄ laminopathies [24–26]. Although only few genes encoding nuclear lamina and pore complex proteins have been identified as causing these diseases the clinical manifestations are widely varied [10,67]. They encompass premature ageing syndromes, myopathies, neuropathies, lipodystrophies, dermopa- thies and varied combinations of disease manifestations [68]. The mutated genes underlying these disorders include lamin A ⁄ C, which is responsible for the auto- somal dominant form of Emery–Dreifuss muscular dystrophy (EDMD) and various other laminopathies, amongst them the Hutchison–Gilford progeria syn- drome (HGPS) (reviewed in [69]); emerin, an INM protein responsible for the X-linked cases of EDMD; MAN1, another INM protein responsible for three autosomal dominant diseases characterized by increased bone density and elevated TGFb-BMP expression [70]; mutated LBR results in Pelger–Hue ¨ t anomaly and Greenberg skeletal dysplasia, an autosomal ressesive chondrodystrophy and LAP2a which has recently shown to result in cardiomyopathy [71]. Two NPC pro- teins, ALADIN (also termed Adracalin or AAAS) and nup62, can be added to the expanding list of mutated nucler lamina proteins causing these diseases. Muta- tions in the WD-repeat ALADIN NPC protein cause the Triple A syndrome, a human autosomal recessive disorder characterized by an unusual array of tissue- specific defects [71a]. In collaboration with Mordechai Shohat and his colleagues we recently found that mutated nup62 causes autosomal recessive familial infantile bilateral striatal necrosis (IBSN) severe neuro- logical disorder [72], IBSN is characterized by symmet- rical degeneration of the caudate nucleus, putamen, and occasionally the globus pallidus, with little involve- ment of the rest of the brain. The question of how mutations in the same gene or group of genes, which are ubiquitously expressed, cause such a wide variety of tissue specific diseases has linked the laminopathies to the study of transcription regula- tion. Two major models attempt to explain how mutated lamins and NE proteins lead to the observed pathologies: the mechanical stress model and the gene expression model. The mechanical stress model suggests that nuclei that contain defective lamin or emerin pro- teins might be mechanically more fragile than their wildtype counterparts. This model relies on studies in C. elegans, D. melanogaster and mice, showing dra- matic defects in NE structure in nuclei that are deficient in lamins, and that this fragility could ultimately lead to nuclear damage and cell death [68]. The idea of enhanced nuclear fragility is particularly attractive as an explanation for the cardiac- and skeletal-muscle pathologies, as the forces that are generated during Gene silencing at the nuclear periphery S. Shaklai et al. 1386 FEBS Journal 274 (2007) 1383–1392 ª 2007 The Authors Journal compilation ª 2007 FEBS muscle contraction might potentially lead to preferen- tial breakage of nuclei that contain a defective nuclear lamina. Nuclei in noncontractile tissues might remain relatively unscathed, despite showing abnormal nuclear and NE organization. The second model, that of perturbed gene expression, is based on cumulative evidence showing involvement of nuclear lamina pro- teins in gene repression [29]. According to this model mutations in lamina proteins could promote diseases by compromising various gene regulatory pathways in different tissues. This model is supported by several lines of evidence: Primarily evidence to the association of transcriptional regulators with proteins of the nuc- lear lamina as described above, additionally morpholo- gical studies showing disrupted heterochromatin at the nuclear periphery of cells from laminopathy patients and finally impaired epigenetic histone modifications in lamin A mutated cells. Proteins of the nuclear lamina most probably exert their effect in several nonexclusive modes. One such example is the pRb protein which was shown to bind both lamin A and LAP2a [55,73]. pRb, besides being involved in inhibition of prolifer- ation, is important for skeletal muscle and adipose tis- sue differentiation, two tissues which are frequently affected in the ‘nuclear envelopathies’ [74]. In order to mediate at least some of its effects pRb recruits various histone modifying enzymes to its target promoters, amongst them HDAC1, 2 and 3. Interestingly, a pRb– HDAC3 complex was shown to be important for the regulation of adipocyte differentiation by peroxisome proliferator-activated receptor gamma [75]. Another example of the multifaceted effects of mutated lamina proteins are studies on cells from EDMD patients and lamin A knockout mice showing altered organization of heterochromatin at the nuclear periphery [76]. Simi- larly, light and electron microscopy analyses of HGPS fibroblasts reveal significant changes in nuclear shape, including lobulation of the nuclear envelope, thickening of the nuclear lamina, loss of peripheral heterochroma- tin, and clustering of nuclear pores [77]. These struc- tural defects worsen as HGPS cells age in culture. The authors suggest that nuclear lamina defects in these cells are due to the disruption of lamin-related func- tions, ranging from the maintenance of nuclear shape to regulation of gene expression and DNA replication. Goldman and colleagues further analyzed the mecha- nisms responsible for the loss of heterochromatin in cells of HPGS patients [78]. For this purpose epigenetic marks regulating facultative and constitutive hetero- chromatin were examined. In cells originating from a female HGPS patient, the transcriptionally repressive histone H3 trimethylated on lysine 27 (H3 K27me3) marker of facultative heterochromatin, was lost on the inactive X chromosome (Xi). The methyltransferase responsible for this epigenetic modification, EZH2, was down-regulated. These alterations were detectable before the changes in nuclear shape, reported earlier [77]. Another transcriptionally repressive epigenetic mark, histone H3 trimethylated on lysine 9 (H3 K9me3) which marks pericentric constitutive hetero- chromatin, was down-regulated in these cells. This change correlated with an altered association of the H3K9me3 with HP1a and the calcinosis, Raynauds phenomenon, esophageal dysmotility, sclerodactyly, tel- engiectasia (CREST) antigen. In contrast to the decrea- ses in histone H3 methylation states an increase in trimethylation of histone H4 on lysine 20, an epigenetic mark for constitutive heterochromatin, was observed [78]. This study is the first to define specific alterations in histone lysine methylation as early events in disease pathology, suggesting that either mutated lamin A or general distortions of the nuclear lamina impair regula- tion of epigenetic modifications. HGPS has always been an appealing disease for researchers due to the possible implications for the study of ageing. Recently a direct link has been formed between heterochromatinization defects leading to HGPS, and ageing. Scaffidi and Misteli [79] showed that cell nuclei from old individuals acquire similar defects to those of HGPS patients, including changes in histone modifications and increased DNA damage. While cells from young indi- viduals (3–11 years old) showed robust staining of the transcriptional repressive heterochromatin marks HP1, LAP2s and Tri-Me-K9H3, a significant subpopulation of nuclei in cells from old individuals displayed reduced signals, similar to previous observations in HGPS cells [80]. These observations implicate lamin A and nuclear lamina-dependent epigenetic alterations as involved not only in nuclear envelopathies but also in the physiologi- cal process of aging. Summary The idea that genes are silenced at the nuclear periph- ery is not new. In the early 1960s Mirsky and col- leagues showed in electron micrographs of calf thymus nuclei the peripheral localization of condensed hetero- chromatic regions and the more centered localization of diffused euchromatic regions. RNA synthesis was more active in the diffused interior euchromatin than in the condensed peripheral heterochromatin [81]. The heterochromatic sex-chromatin body of Barr, which in female mammalian cells is composed of a segment of one X chromosome, was found by this group to carry unexpressed genes. The DNA of this inactivated X chromosome replicated later than that of other S. Shaklai et al. Gene silencing at the nuclear periphery FEBS Journal 274 (2007) 1383–1392 ª 2007 The Authors Journal compilation ª 2007 FEBS 1387 chromosomal segments [82]. Today we know that the inactivated X chromosome resides at the nuclear periphery and we use it as a compelling example of chromosome-wide, long-range epigenetic gene silencing in mammals (reviewed in [83]). Since the fundamental discoveries by the group of Mirsky the development of experimental tools, such as fluorescence in situ hybrid- ization combined with three-dimensional microscopy, to analyze chromosomes and proteins in living cells, together with complementary approaches that explore the computational biology, epigenetic modifications and gene expression profiling along the chromosomes, offer us today the possibility of visualizing ‘real time’ gene expression. We can follow the looping out or ‘jumping’ of loci from their gene repressed heterochro- matic territory at the nuclear periphery to more inter- nal gene active euchromatic territories for their transcription [34,45,84,85]. However, still little is known about the molecular mechanisms responsible for nuclear lamina-dependent gene regulation. In recent years great advancement has been achieved in understanding the role of the nuclear lamina ⁄ envel- ope in regulation of transcription. A major boost to the subject arrived from the unexpected finding that mutations in nuclear lamina ⁄ envelope proteins are the cause of a large family of diseases with varied expres- sion. Involvement of INM proteins in transcription occurs on different levels. By binding transcription fac- tors they inhibit gene expression on the basic linear DNA level and by binding chromatin modifiers they influence gene expression at the epigenetic level. Our model (Fig. 1) suggests that under certain, yet unknown, physiological conditions, NE proteins, such as LAP2b and LBR are triggered to modify the chro- matin in their vicinity in order to induce gene silencing there. The transition from nucleoplasm decondensed gene active euchromatin to condensed gene silenced heterochromatin requires the creation of transcrip- tional repressive environment at the nuclear periphery. This can be achieved by forming repressive complexes by the NE proteins as illustrated in Fig. 1. By serving as docking sites at the INM, LAP2b, LBR, and poss- ibly other INM proteins, can anchor DNA, chromatin and chromatin modifiers in order to execute reversible epigenetic modifications on DNA, histone tails and transcription factors. We propose that the outcome of these energy-free or energy-dependent enzymatic reactions is the remodeling of the INM-attached chro- matin, such that specific loci and genes are transcrip- tionally inhibited. Our model, based on LAP2b and Fig. 1. Gene silencing at the nuclear periphery. The ONM is a continuation of the endoplasmic reticulum. It joins the INM at the NPCs. Lamins A ⁄ C (black line) and B (red line) are shown as filaments at the nuclear periphery and across the nucleoplasm (lamin A ⁄ C). Associa- tions of the INM proteins LAP2b, LBR, emerin and MAN1 with lamins, chromatin and their specific partners are shown: LAP2b binds BAF, HDAC3, GCL and HA95; LBR binds HP1, histones H3 and H4; MAN1 binds BAF and GCL, emerin binds BAF and GCL competitively, YT521-B and actin. The question marks indicate, yet unidentified, LAP2b-associating proteins catalyzing gene silencing through epigenetic modifications. The two chromatin states, of gene-active unwrapped euchromatin at the nucleoplasm, and of gene-silenced condensed heterochromatin at the vicinity of the INM are circled. In the latter state, epigenetic modifications on histones and DNA are illustrated. The intranuclear complex containing LAP2a, lamin A, Rb and BAF proteins is shown. C, cytosol; NL, nuclear lamina; NP, nucleoplasm; NPC, nuc- lear pore complex; ONM, outer nuclear membrane; INM, inner nuclear membrane; H-Chr (GR), heterochromatin (gene repression); Ec-Chr (GA), euchromatin (gene activation); Me, methylation; deAC, deacetylation; Ri, ribosylation; Ub, ubiquitination; P, phosphorylation. Gene silencing at the nuclear periphery S. Shaklai et al. 1388 FEBS Journal 274 (2007) 1383–1392 ª 2007 The Authors Journal compilation ª 2007 FEBS LBR studies, suggests that two collaborative under- acetylated chromatin complexes are formed at and anchored to the NE. In one complex, LAP2b recruits the enzyme (HDAC3) while in the other complex LBR recruits the substrates (histones H3 ⁄ H4) [20,50]. In both cases, acetylation conditions, alleviated the LAP2b ⁄ HDAC3 dependent transcriptional repression [20] or dissociated the LBR–HP1–histones repressive complex [50]. The proposed concept places proteins of the nuclear lamina as high hierarchical transcriptional regulators. This may have implications in the study of cancer dis- eases, where a strong link was established in recent years between gene inactivation and tumorigenesis, mainly in hematological malignancies [63], and NE ⁄ lamina associated diseases and ageing in which perturbed gene regulation and peripheral heterochro- matin formation have been shown to exist [79,86]. Acknowledgements Our research is supported by the Israel Science Fund grant no. 804. We thank ‘PA’AMEI TIKVA’ founda- tion for their support of our research. GR holds the Djerasi Chair for Oncology (Sackler School of Medi- cine, Tel Aviv University, Israel). References 1 Tzur YB, Wilson KL & Gruenbaum Y (2006) SUN- domain proteins. ‘Velcro’ That Links Nucleoskeleton Cytoskeleton. 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