Laminopathies: A consequence of mechanical stress or gene expression disturbances?

Transcription

1 BIOLOGY AND HEALTH Journal website: Narrative review Laminopathies: A consequence of mechanical stress or gene expression disturbances? Alicia Jonker Alicia Jonker I Maastricht University Faculty of Health, Medicine and Life Sciences (FHML) Bachelor GW BGZ Date: Tutor: Jos Broers Correspondence: alicia.jonker@student.maastrichtuniversity.nl Abstract Laminopathies are fourteen rare, distinct diseases associated with mutations in lamins, which are nuclear membrane proteins. These laminopathies include muscular laminopathies, lipodystrophies, and developmental-progeroid syndromes. The aim of this review is to give an overview of the two main hypotheses which have been suggested in order to explain the different phenotypes occurring in laminopathies, based on several in vivo and in vitro studies. These mechanisms include the mechanical stress hypothesis, which states that laminopathies occur as a consequence of increased nuclear fragility and impaired ability to respond to mechanical stress, and the gene expression hypothesis, which states that mutations in lamins or its product result in disturbed chromatin dynamics and an aberrant gene expression. Concludingly, this review argues that these mechanisms probably interact amongst each other in causing laminopathies, which is explained by the mechanosignalling hypothesis. Introduction Laminopathies include fourteen, phenotypically diverse diseases, caused by mutations in lamins (1). There are three main groups of diseases, namely muscular laminopathies, lipodystrophies, and developmentalprogeroid syndromes (2). Lamins are classified as type V intermediate filament and are the principal component of the lamina, underneath the inner nuclear membrane (INM) (3-5). In mammals, the different isoforms are A- and B-type lamins, according to their sequence homologies. B-type lamins are expressed at the earliest stages of development and later on, while the expression of the soluble A-type lamins seems related to cell differentiation (6). Mutations in A-type lamins result in laminopathies such as Hutchinson-Gilford Progeria (HGPS), Emery-Dreiffus Muscular Dystrophy (EDMD), Lamb-Girdle Muscular Dystrophy (LGMD), Familial Partial Lipodystrophy (FPLD), Acro-Mandibular Dysplasia (MAD), Charcot- Marie-Tooth type B (CMT2B), and Restrictive Dermopathy (RD). Lamin B mutations are less frequent, 1

2 but are present in adult-onset demyelinating leukodystrophy (ADLD) (2, 7). Premature aging syndromes, or progeroid syndromes such as HGPS, are characterized by clinical features of accelerated aging, such as hair loss, skin tightness, cardiovascular diseases, osteoporosis, lipodystrophy, and reduced lifespan (2). When evaluating the mechanisms behind the development of laminopathies, there are two main hypotheses to be distinguished. The mechanical stress hypothesis shortly states that changes in viscoelastic behaviour of the nucleus related to mutations of A-type lamins are responsible for the phenotypes of the different laminopathies (8). Especially tissues susceptible to stress, such as striated muscle, are affected (9). The structural stability of the nucleus must be maintained in order to protect genome integrity (10). The gene expression hypothesis on the other hand, states that laminopathies are mainly caused by the disturbed chromatin dynamics, and therefore aberrant gene expression, through mutations in lamin A or associated products (2). These different hypotheses stem directly from the different functions of lamins, namely maintenance of nuclear shape, nuclear positioning and genome organization (11). This review aims to give a general overview of the background and evidence for each of these mechanisms, and in which way they can contribute to the different phenotypes in laminopathies. First, the general structure and properties of lamins will be discussed, in order to better understand the functions of lamins. Afterwards, the mechanism of the mechanical stress hypothesis will shortly be reviewed, followed by evidence for this hypothesis in the form of in vitro and in vivo studies. This mechanism will be coupled to the pathology of some laminopathies. The same structure will be used to explain the gene expression hypothesis. To finalize, a conclusion will be given on which hypothesis seems to fit the pathology of laminopathies best. 1. Structure and properties of lamins Lamins have different structural roles, including maintenance of nuclear shape, rigidity and resistance to mechanical stress (1). In order to understand the structural roles of lamins, and therefore the pathologies associated with mutations, first the structure and properties of lamins need to be understood. In mammals, there are two A-type lamins derived from the LMNA gene, which is located on chromosome 1, namely lamin A and lamin C. Both lamins A and C have a large number of binding partners, including other nuclear lamins, INM proteins, chromatin-associated proteins and DNA binding proteins (12). Lamin B1 and lamin B2 are derived from LMNB1 on chromosome 5 and LMNB2 on chromosome 19, respectively (13, 14). All lamins consist of a long central alpha helical rod domain, flanked by an N terminal head and a C terminal tail domain (fig. 1). The nuclear localization signal (NLS) is situated in the grossly unstructured C terminal (15), as are the immunoglobulin fold (Ig-fold) (16, 17) and the CaaX box at the termination (18). Both lamins are transcribed as precursor proteins that undergo cysteine farnesylation at the CaaX box, which is the attachment of a hydrophobic group in order to attach the protein to the membrane. A-type lamins do not retain the farnesyl group due to a second cleavage step by, but B-type lamins are permanently farnesylated (8). The lamins also differ in their expression patterns: B-type lamins are expressed in most embryonic stem cells (19), while lamin A is expressed later, namely during differentiation (20). Lamin B proteins are very essential during animal development, which explains why mutations in lamin B appear lethal during foetal development or within minutes after birth (19, 21, 22). However, mutations in the LMNA gene only result in growth retardation after birth (23, 24): the so-called laminopathies. 2

3 2. The mechanical stress hypothesis 2.1 The mechanism Lamins self-assemble into higher order oligomeric structures via dimer formation (25-27). The head, rod and tail domains play active roles in this assembly (26, 28). A- and B-type lamins form separate networks in the nucleus, which interact amongst themselves (29). However, the exact mechanisms behind the polymerization of the nuclear lamins to form the final laminar structure are still poorly understood. Based on studies on lamin assembly (30), it has been proposed that B-type lamins form the base, especially B1, on which the laminar superstructure, composed of lamin A amongst others, is assembled (8). The nuclear envelope consists of three intimately linked substructures: namely the nuclear membranes, the nuclear lamina and the nuclear pore complexes (NPCs). The outer nuclear membrane (ONM) is continuous with the endoplasmic reticulum. The NPCs allow connections between the ONM and inner nuclear membrane (INM) and are important in nucleocytoplasmic transport. The INM consists of lamins and nuclear envelope transmembrane proteins (NETs): a specific set of proteins, of which more than 90 have been identified, but only some fully characterized (31, 32). The lamina can be seen as a mechanoresponsive element of the cell as it transmits mechanical cues from the ECM. This mechanoresponsive property of the lamina is important in the mechanical stress hypothesis. Lamins give elasticity to the lamina and are important in shock absorbing (33, 34). Therefore, the lamins regulate mechanical behaviour of the nucleus. Viscoelasticity of the nucleus depends entirely on A-type lamins, where B-type lamins are responsible for resisting nuclear deformation in response to force (35). Stiffness of a tissue is largely dependent on the relative expression ratio of A- and B-type lamins: soft tissues, such as the brain, exhibit a low lamin A:lamin B ratio, where harder load-bearing tissues, such as muscles, bone, and cartilage, exhibit a high ratio (36). Since lamin A mutations are involved in diseases linked to mechanical force transmission of muscles, the effect lamin A has on viscoelasticity of the nucleus is an important aim of future research. The way a nucleus is built can be compared to the tensegrity architecture, as described by R. Buckminster Fuller (37). In this model, structures gain integrity and stability through a pervasive tensional force, rather than through continuous compression. The nucleus is a socalled self-stabilizing tensegrity structure, in which compression elements do not touch, but are suspended by connections to a continuous series of tension elements, which can be compared to cables (fig. 2). Thus, structural stability depends on continuous tension: a tensile prestress generated by the contractile actomyosin filaments and extracellular tethering sites to the ECM and to other cells (38-41). Since the structural members are connected by elements that transmit tensional forces throughout the whole tensegrity structure, application of a local force results in an integrated structural response. 3

4 The linker of nucleoskeleton and cytoskeleton (LINC) complex physically connects the nuclear lamina to the cytoskeleton (fig. 3). The complex is comprised of SUN (Sad1 & UNC-84) domain proteins spanning the INM (42) and is connected to nesprins (Syne, Myne, and NUANCE) at the ONM (43-45). Actin, microtubules and cytoplasmic intermediate filaments are also connected to the different isoforms of nesprin (46, 47). Therefore, the LINC complex is essential in sensing of physical forces and translating them into biochemical and biological responses (43, 44, 48), and maintaining proper shape and structure of the nucleus in response to mechanical forces (49, 50). LINC complexes and A-type lamins are also crucial in nucleo-skeletal coupling and nuclear positioning (51, 52). Mutations in LMNA result in disruption of the mechanotransduction pathway, through defects in the nucleocytoskeletal coupling (53). In mammalian cells, this is the effect of improper localized components of the LINC complex (54, 55) and disturbed interaction of various downstream signalling molecules with A-type lamins (56). Integrity of the nuclear-cytoskeletal linkages is especially important in muscle function (57-60). So far, in all LMNA (9, 61) and nesprin mutations (59, 62, 63) causing striated muscle disease, the nesprin/sun/lamin interactions are disturbed and the nucleo-cytoskeletal linkages are dysfunctional (9, 36, 59, 60, 62, 64). Concluding, according to the mechanical stress hypothesis, laminopathies occur when LMNA mutations, or mutations in genes coding for A-type lamin associated proteins, result in an increased fragility or deformations of the nuclei and a disturbed ability of the nucleus to cope with mechanical stress. This explains why especially load bearing tissues, such as striated muscle and bone, are affected in laminopathies. 2.2 Evidence from previous research Multiple studies have been done on tissue samples of laminopathy patients and LMNA knockdown cells, in order to study the structure and behaviour of the nucleus. Besides these in vitro studies, there is also in vivo evidence from studies on animals, mainly mice In vitro studies Single molecule force spectroscopy has been used to study the unfolding of lamin A protein domains in response to externally applied force in different LMNA mutations (65, 66). It appeared that a mutation causing autosomal dominant EDMD, namely R453W, resulted in an altered mechanoresistance of the Ig domain itself, which had consequences for the lamin A-emerin 4

5 interaction and thus the mechanotransduction properties (66). A study on fibroblasts of 16 different disease-causing lamin A variants found that all affected chromosome orientation, but only the mutations that cause muscle disease affect nuclear movement and positioning (9). The assembly of individual LINC complexes does not seem to be affected by these muscle disease causing lamin A/C mutations, but the ability of LINC complexes to resist forces exerted by moving actin cables does appear to be impaired in cells lacking lamin A/C. This is probably due to lamin A/C s anchoring function of the transmembrane actin-associated nuclear (TAN) line, which couples the nucleus to moving actin cables, enabling forces to be exerted on the nucleus (8). Tamiello et al. (2013) studied nuclear fragility in cultured dermal fibroblasts from a compound progeroid syndrome patient in different growth substrate stiffness conditions. They found that malformations and nucleus ruptures occur when cells grow on substrates with a stiffness higher than 10 kpa (67). Moreover, a study using electron microscopy on human dermal fibroblast cultures from HGPS cells and LMNA knockdown cells showed non-lethal ruptures of the nuclear envelope even without application of external force (68). Collectively, these studies clearly demonstrate the increased nuclear fragility of LMNA mutant cells, both in the presence and absence of external mechanical forces. If the fragility of nuclei is already increased in vitro, the consequences of living on a laminopathic cell may be even more detrimental. Tissue samples from patients with Delated Cardiomyopathy (DCM), Emery Dreiffus Muscular Dystrophy (EDMD), Hutchinson-Gilford Progeria Syndrome (HGPS), and Familial Partial Lipodystrophy (FPLD) have shown mechanically compromised nuclei, which are often deformed and lacking integrity (9, 69). Electron microscopic analyses of biopsy samples obtained from heart muscle tissue in DCM patients showed abnormally elongated nuclei, reduced nuclear stiffness (9, 53, 69, 70), increased nuclear fragility and discontinuity in nuclear lamina (71, 72). The fibroblasts of HGPS patients, in contrast, showed stiffened nuclei (73, 74). Fidzianska et al. (2008) used immunohistochemical and ultrastructural analysis to study cardiomyocytes from an end-stage DCM patient with a missense point mutation in the LMNA gene. They found the nuclear shape to be altered in thirty percent of the cardiomyocyte nuclei, and, more intriguingly, the appearance of sarcoplasmic organelles within the nuclear matrix of well enveloped nuclei (75), a phenomenon which has not been observed in laminopathic striated muscle cells before. Although this study supports the mechanical hypothesis, they also found chromatin disorganization in approximately one third of the nuclei, suggesting a role of the other hypothesis in occurrence of DCM as well. It is also to be noted that this study only used cells from one patient, so it is hard to say whether the observations would be similar in other DCM cases. A study with a greater power is the study of Gupta et al. (2010), where 25 unrelated DCM patient samples were studied for cardiomyocyte nuclear abnormalities. In eight of those 25 patients, major nuclear abnormalities were detected (72). They also demonstrated that a large deletion of LMNA resulted in reduced protein levels in the nuclear envelope, leading to cardiomyocyte nuclear envelope disruption and thus leading to the pathogenesis of DCM. This finding is confirmed by a study on striated muscle from a patient with EDMD with electron microscopy. This study revealed defects in the nuclear membrane as well, with extrusions of chromatin in the endothelial cells (76), which would explain the EDMD phenotype of muscle wasting. However, this last study also used only one patient s cells. These studies on laminopathic cells commonly show the appearance of nuclear deformations, comparable to the deformations found in LMNA knock down cells. These findings aid to the assumption that laminopathies are 5

6 caused by mutations in A-type lamins and the subsequent disruptions in nuclear lamina integrity In vivo studies Fibroblasts derived from LMNA null mice were found to have nuclei with highly irregular shapes (77). In this study, point mutations were introduced into the A-type lamin DNA by two-stage PCR. Four of the mutations had been linked to DCM, EDMD and FPLD. It appeared that one of the mutations responsible for DCM, namely L85R, resulted in an incompetent lamin C mutant in terms of assembly into the lamina. However, the mutation did not cause mislocalization of the lamina to the nucleoplasm, which is possibly the result of a hydrophobic interaction of the farnesyl lipid tail with protein or lipid components of the INM. The EDMD mutation, L530P, has only a marginal effect on the localization of lamin A to the nuclear periphery, because the mutation resides within the nonhelical C-terminal domain of A-type lamins. Therefore, the lamina organization remains quite normal. It does however seem possible that those mutations that fail to dramatically perturb lamin A assembly may interfere with lamin C behaviour (77, 78). All LMNA mutations associated with DCM and EDMD appeared to be able to alter the nuclear envelope and nuclear architecture (77). 2.3 Pathology Lamin A networks deviate from lamin B1 in their unique viscoelastic response as compared to strain hardening behaviour of lamin B1, which is to be explained by their distinct higher order associations (79). Dilated Cardiomyopathy is caused by mutations E161K and R190W (80-82), resulting in a fragile and poorly elastic matrix, which is unable to bear shear strain deformation (35). This explains the abnormally elongated nuclei with impaired elasticity as seen in lamin A mutations. Nuclear envelope blebbing, elongated nuclei, lamina thickening, and overall fragility of the lamina are different types of nuclear deformation which have been observed in the different laminopathies (6). 6 Dilated cardiomyopathy, associated with more than 120 different LMNA mutations and mutations of other nuclear envelope proteins (83-85), is characterized by progressive thinning of the ventricular wall and weakened cardiac contractility. Parts of the disease pathogenesis are impaired mechanotransduction (86) and nuclear envelope fragility (87), associated with low lamin A/C levels. DCM and Emery-Dreiffus Muscular Dystrophy (EDMD) have certain clinical features in common, which is why the suggestion was made that DCM and EDMD represent a single disease entity with variable expression of symptoms that are perhaps modified by additional genetic factors (77, 78, 88). The same may be true of limb girdle dystrophy with atrioventricular conduction disturbances. All LMNA mutations mapped in this disorder compromise lamin A or C assembly, or both (89). The increased nuclear fragility as a result of compromised nuclear envelope integrity could explain some of the aspects of DCM and EDMD phenotypes, for instance an elevated incidence of mechanical damage to cardiac and skeletal muscle nuclei, which may be unable to withstand forces generated during repeated contraction (90). Because muscle cells are unaffected in other laminopathies, such as Familial Partial Lipodystrophy (FPLD), nuclear fragility cannot be the universal mechanism to account for all diseases. FPLD is characterized by a loss of subcutaneous fat from the extremities and from the trunk, and an accumulation of excess fat in the face, neck, back and visceral abdomen (91). The mechanical stress hypothesis does not seem sufficient to explain the mechanisms behind these types of laminopathies, since adipose tissue is not exposed to mechanical stress in the way that striated and cardiac myocytes are. In the premature aging disorder Hutchinson-Gilford progeria syndrome (HGPS), a farnesylated mutant product of the LMNA gene, namely progerin, causes the nucleus to be more frail and less fluid (73). This supports the notion that farnesylated lamins are more

7 tightly anchored to the membrane and less soluble (92). However, the nuclear envelope being more fragile does not explain the features of genetic instability and p53- dependent premature senescence (93). The gene expression hypothesis might be more suited to explain lipodystrophy- and progeria-related laminopathies. 3. Gene expression hypothesis 3.1 The mechanism All the so far investigated laminopathies have defects in chromatin organization and/or dynamics, which suggests a loss of chromatin functionality as a major pathogenetic mechanism (94-100). Chromatin dynamics is the whole of heterochromatin maintenance and compartmentalization of chromosome domains, DNA damage response and genome stability, chromatin conformational changes before and after mitosis, gene silencing and transcriptional activation, and chromatin remodelling at specific promoters (2). Multiple proteins are involved, such as DNA binding factors, enzymes involved in histone marks deposition or reading, nucleosome sliding, eviction or replacement, molecules regulating DNA methylation (12), epigenetic enzymes, DNA repair factors, heterochromatin proteins, ATPases, and nuclear actin (2). Among lamins and INM proteins, many bind directly to chromatin or recruit intermediate factors. The interaction of lamins with naked DNA happens in a non sequence-specific manner (101, 102). Lamins have three main functions in chromatin regulation, which are (1) modulation and maintenance of the heterochromatin domains (12, 103), (2) recruitment of the DNA damage response machinery (104, 105), and (3) transcription factor binding. Heterochromatin regulation is regulated through diverse chromatin-binding factors, such as histone methyltransferase Suv39H1, histones H2, H3 and H4 (106), heterochromatin proteins HP1alpha (107) and - beta, bridging proteins such as LAP2alpha and BAF, and nucleosomal proteins. HP1 interaction with lamins is essential for heterochromatin anchorage at the end of mitosis (107). The complex interaction between lamins 7 and chromatin also involves nuclear membrane proteins, such as lamin B receptor, LAP2beta (103), emerin (108), BAF (109), and SUN proteins (110, 111), which also interact with chromatin partners (106, 112, 113). Prelamin A is essential in heterochromatin anchorage, through binding heterochromatin protein HP1, LAP2alpha, and BAF (114). However, accumulation of prelamin A leads to reorganization of heterochromatin domains in both physiological (109, 114, 115) and pathological (94-96, 116, 117) circumstances. Lamina-Associated chromatin Domains (LADs) are highly conserved gene-poor chromosome domains associated with lamins (118) which include about 40% of the human genome (119). Perinucleolar heterochromatin is defined as Nucleolus-Associated Domains (NADs) (120). Both NADs and LADs are mobile, can be relocated upon certain stimuli (120), and are aimed at maintaining a cell-type specific default setting in nuclei (120). Location of genes in the perinucleolar area seems to be mostly dependent on BAF and lamin A (121). Insulator sequences, such as CTCF (120) and D4Z4 (122), flank the LADs and NADs and anchor the chromatin domains at the nuclear periphery similar to lamin A (122). The role of lamins in the recruitment of the DNA damage response is mainly exerted in recruiting 53BP1 in the nucleus and localizing 53BP1 foci in cells subjective to oxidative stress, in which both lamin A (123) and prelamin A (114) are important. DNA is mostly repaired by non-homologous end joining. Lamin B1 anchors proteins, such as PCNA, which are involved in nucleotide excision repair (124). However, recruitment of PCNA relies on lamin A Ig-fold interaction (125). Lamin B1 loss as a result of UV damage causes disruption of DNA repair proteins (124), indicating that genome integrity relies on both lamin A and lamin B1. The final function of lamins in chromatin dynamics is transcription factor binding at the nuclear periphery. Prelamin A binds SREBP1, Sp1, and Oct-1 ( ) and lamin A and C anchor prb, Mok2, and cfos (129, 130). Transcription factor Oct-1 can be recruited to the

8 nuclear envelope by lamin B1 (131, 132). These interactions are mostly inhibitory on gene transcription (126, 133, 134). Lamin A contributes to the formation of heterochromatin domains through influencing histone dynamics, where the absence of lamin A/C induces hypermobility of histones and loss of heterochromatin, which impairs differentiation marker expression (135). This explains why non-functional lamins, such as progerin, alter chromatin dynamics in fibroblasts ( ). 3.2 Research Recent studies have demonstrated the physiological importance of lamins in rearranging chromatin during the initial steps of development. Studies have been conducted in vitro and in vivo In vitro Abnormal chromatin organization as a result of lamin mutations was first observed in Emery-Dreifuss muscular dystrophy (EDMD) muscle fibres (99), through electron microscopy study of muscle biopsies from EDMD patients. Sabatelli et al. (2001) found nuclear alterations in about 10% of the muscle fibres, namely peripheral heterochromatin loss or detachment from the nuclear envelope and interchromatin texture alterations, (99). Similar findings were done in EDMD fibroblasts (139) and mature muscle fibres (98, 99), where a detachment of peripheral heterochromatin from the nuclear lamina and focal loss of heterochromatin domains were observed. Subcutaneous adipose tissue from seven FPLD patients and ten healthy control participants was obtained and studied by Araujo-Vilar et al. (2009) and they found peripheral heterochromatin defects and nuclear fibrous dense lamina in adipocytes of the FPLD patients (140). This finding was associated with prelamin A accumulation, which reduced the expression of several genes involved in adipogenesis. The balance between proliferation and differentiation in adipocytes may be 8 disturbed as a result of prelamin A accumulation, leading to a less efficient tissue generation. Early ultrastructural analyses on Hutchinson-Gilford progeria (HGPS) and Mandibuloacral dysplasia (MADA) cells confirmed these findings, in observing a complete loss of peripheral heterochromatin and dispersed euchromatin (94, 96, 116). Three cell lines from HGPS patients showed nuclear envelope-nuclear lamina abnormalities and loss of peripheral heterochromatin, which was more pronounced with increased progerin concentrations (94). When this farnesylated protein was destabilized through farnesyl-transferase inhibitor (mevinolin) treatment, the amount of progerin was slightly reduced and heterochromatin areas were restored (94). The progerin level lowering effects of this treatment were even more pronounced in combined treatment with trichostatin A. This suggests that morpho-functional defects in HGPS nuclei are directly related to progerin accumulation. Goldman et al. (2004) had similar findings, however their study population only existed out of one patient (116). In a study on fibroblasts from three MADA patients and three control subjects, who were age and sex matched, the prelamin A levels were shown to be increased in MADA cells (96). There were also alterations in peripheral heterochromatin shown, which appeared to be correlated with the age of the patient In vivo Research conducted on the morphology and transcriptional activity of myonuclei from LMNA-null mice found that there were fewer myonuclei in these knockout mice, with half of these nuclei showing abnormalities (141). There also appeared to be a lack of coordinated transcription in myonuclei lacking A-type lamins, as examined by assaying the transcriptional activity through acetylated histone H3 and PABPN1 levels. The abnormally shaped myonuclei were accumulated at the myotendinous junction, of which the structure was also perturbed, in terms of disorganised sarcomeres, reduced interdigitation with the tendon, and lipid and collaged deposition. The mice

9 demonstrated severely affected muscle contraction within weeks of birth, with subsequent decreased force generation. This study provides evidence for the importance of A-type lamins in correct myonuclear function, and suggests the role of lamin mutations in the occurrence of EDMD, namely myotendinous junction disorganization and consequential reduction in force generation and muscle wasting. In both mouse (141) and human (111, 142) laminopathic muscle fibres, the myonuclei appear elongated. The gene regulation hypothesis would explain these elongated nuclei as multiple nuclei being clustered, due to failure to properly position during muscle stem cell differentiation and regeneration (111). In EDMD, this would lead to altered regulation of myonuclear domains (the cytoplasmic regions regulated by each myonucleus), as the altered sarcomere structure which has been observed in LMNA knockout mice suggests (141). Several defects in transcriptional regulation in EDMD muscle have been demonstrated, such as altered histone H3 trimethylated at lysine 27 (H3K27) and phosphorylated RNA polymerase II (143) and defective acetylation of histone H3 on lysine 9 (141). In a C. elegans model of EDMD1, where emerin expression was lacking, showed a downregulation of histone deacetylases (144), which suggests that enzymes involved in epigenetic modification of histones could play a role in EDMD pathogenesis. Solovei at al. conducted a study on mice with lamin B receptor (Lbr) and lamin A (LMNA) mutations, using histological study and transcriptome analysis, in order to investigate the mechanisms regulating chromatin distribution (103). It appeared that the absence of both LBR and lamin A/C leads to a loss of peripheral heterochromatin and an inversion of architecture, with heterochromatin facing the nuclear interior. They found that there seems to be a hierarchical function of nuclear envelope components in regulating heterochromatin domains. Expression of LBR is sufficient to retain heterochromatin at the nuclear periphery, but lamin A/C expression needs LBR or other LMNA partner expression to ensure anchorage. These studies collectively show the importance of lamins in the regulation of heterochromatin domains and, consequently, regulation of gene transcription. If mutations in lamins occur, the gene transcription is altered, which could explain the different phenotypes as seen in laminopathies. 3.3 Pathology It appears to be quite hard to find a consistent relationship between mutation location on the LMNA gene and its subsequent effect, because mutations causing muscular dystrophies appear to be spread all along the gene (145). However, the majority of lipopdystrophy-causing mutations are located at codon R482, on the C-terminal globular domain, which is highly conserved in human A-type lamin genes (26). The vast majority of mutations causing progeria affect splicing (13). The position of the mutation on the LMNA gene relative to the Nuclear Localization Signal (NLS) seems to play a significant role in the type of laminopathy induced. Mutations at the N-terminal side of the NLS are more likely to cause cardiomyopathy and muscle atrophy, where mutations at the C-terminal side more likely result in progeroid symptoms (146) Muscular dystrophies The reason why EDMD apparently only affects striated muscle is not completely elucidated, but there are several suggestions based on reported data (2). First, muscle represents the only tissue where prelamin A increases during normal differentiation (111, 147). Because prelamin A regulates large scale heterochromatin organization (115, 148), the altered prelamin A modulation occurring in EDMD2 (111) would affect heterochromatin dynamics related to differentiation-linked positioning of chromatin domains and specific gene expression (143, 147). Second, lamin point mutations in EDMD2 have been shown to impair reorganization of heterochromatic arrays during 9

10 muscle-specific promoter activation (149), such as the myogenin promoter (150). This implies that lamin mutations also affect gene expression at specific sites. Third, emerin seems to have an upstream role with respect to lamins in regulation of muscle and neuronal genes, as an experiment in C. elegans using DamID technology has shown (144). A lack of emerin shifted these sequences to lamin association, which would explain the much lower frequency of emerin mutations causing EDMD1 as compared to the huge number of pathogenetic LMNA mutations (144). Finally, several studies suggest that the interaction of lamin A/C and emerin with their binding partners, such as SUN1, SUN2, BAF, and LAP2alpha, could also be important in the effects of lamin mutations on chromatin, specifically in muscle (2, 109, 111, 147, 148). These findings suggest that deregulation of chromatin dynamics and gene expression, as a result of altered lamin-mediated chromatin remodelling, may be important in the pathogenesis of EDMD Lipodystrophies Lipodystrophic phenotypes, which are characterized by a diminished subcutaneous adipose tissue in the extremities and an adipose accumulation on the face and neck at the onset of puberty (151), all share the common feature of prelamin A accumulation at diverse levels (152). For familial partial lipodystrophy (FPLD2), two main pathogenic mechanisms have been described, involving altered chromatin dynamics. The first mechanism relates the altered peripheral chromatin organization to accumulated prelamin A and BAF in FPLD2 cells (100, 126, 153). Prelamin A triggers BAF recruitment to the nuclear envelope, as does its mutated form R482Q, as observed in FPLD2 cases (153). The second mechanism involves an altered transcription factor import in the nuclei due to prelamin A: impaired SREBP1 translocation and SREBP1-mediated transactivation of adipocyte-specific genes (126, 140) for example. Also, Oct-1 activity is impaired due to prelamin A accumulation (154), which has been shown to block the autophagic process: a mechanism involved in white adipose tissue differentiation (155, 156). Thus, lipodystrophy-linked LMNA mutations could affect chromatin activity by altering transcription factor and/or transcriptional regulators targeting and activity. Mutated lamin A in FPLD2 has been shown to alter adipocyte gene expression, including PPARG2, RB1, CCND3, and LPL in thigh but not in abdomen subcutaneous adipose tissue (140). To complicate matters further, there are two distinct and opposite clinical phenotypes within the same lamin A mutations, namely lipoatrophy and adipose tissue hypertrophy (2) Progeroid and developmental laminopathies In progeroid and developmental laminopathies, prelamin A or progerin accumulation elicits complete loss of heterochromatin in the nucleus in most cases (94, 96, 117). This is explained by the ability of the farnesylated prelamin A form to increase nuclear size, elicit nuclear envelope misfolding and heterochromatin dispersion (115, 152). Prelamin A/progerin interaction with the transcription factor SIRT1 possibly causes different chromatin epigenetic modifications (157, 158). But there are also findings which suggest the interplay of prelamin A/progerin and HDACs (94, 157) and/or DNA methyltransferases (94, 115) is involved in pathogenetic mechanisms. Cell cycle progression and population doublings may influence lamin-mediated epigenetic modifications (94, 96, 159). Most likely, lamin A and prelamin A forms contribute to the default setting of chromatin in cells by locking in a particular transcriptional status, and therefore accumulation of prelamin A, as in restrictive dermopathy (RD) or mandibuloacral dysplasia with type B lipodystrophy (MADB), or mutated prelamin A forms, as in other progeroid laminopathies, impairs fundamental function of the lamin network, which causes unordered localization of chromatin domains and loss or increase of some markers, depending on stochastic events (2). The notion that inhibition of enzymes involved in heterochromatin organization improves the phenotype in progeria models (94, 159) explains that the active enzymes are probably entrapped by prelamin 10

11 A/progerin. Inhibition of these mutated proteins may therefore lead to restoration of heterochromatin dynamics. HGPS and (Mandibuloacral dysplasia) MADA nuclei are more susceptible to DNA damage, even though prelamin A accumulation per se does not necessarily induce genomic instability (114, 160), because the recruitment of DNA repair factors is impaired (161). Furthermore, a rare progeroid disorder involving major bones is caused when BAF, a master regulator of the interplay between progerin and chromatin (109) which binds both emerin and DNA, is mutated (162). One mechanism that has been proposed as a cause of altered chromatin dynamics in HGPS is a disruption of functions of some replication and repair factors because of progerin accumulation, which causes mislocalization of xeroderma pigmentosum group A (XPA) protein to the replication forks and replication fork stalling and arresting cell-cycle progression (163). This would explain both the altered DNA damage repair and the prolonged S-phase in progeroid laminopathies (164). One can conclude that a certain rate of nuclear lamina constituents is apparently required for proper chromatin modulation and that a disruption in this condition causes cell cycle arrest and senescence linked to the relocation and activation/inactivation of specific genes (165, 166). Upregulation of lamin B1 and SUN1 and recruitment of BAF and LAP2alpha to the nuclear periphery are part of the cellular response to the prelamin A/progerin accumulation, but it is unclear whether this worsens the cellular phenotype or attenuates deleterious effects on chromatin (2). the nucleus to cope with mechanical stress. Examples of these deformations are nuclear envelope blebbing, elongated nuclei, lamina thickening, and overall fragility of the lamina (6). Such structural abnormalities have been observed in the nuclei of laminopathic cells (6, 9, 53, 67-77, 87), as has an impaired viscoelasticity (35, 79). The occurrence of defects in the ability of cells to cope with mechanical stress may explain the deformed nuclei in load-bearing tissues, such as striated and cardiac muscle and bone (8, 9, 86, 90). These deformations in nuclei per se may not fully explain the different phenotypes in laminopathies, especially with regard to lipodystrophies and progeroid and developmental laminopathies. This is where the gene expression hypothesis comes in. According to the gene expression hypothesis, there are at least four modes of lamin action in chromatin regulation which are disturbed in laminopathies, namely (1) transcription factor retention at the nuclear lamina (94, , 167), (2) altered interplay between mutated lamins and LADs (12, 94, 96, 98, 99, , , 116, , 139, 140, 168), (3) specific effects at promoters exerted by lamin-dependent alterations of epigenetic molecules (2, 12, 141, 143, 144), and (4) impaired tethering of DNA damage repair factors, mostly in progeroid laminopathies (104, 105, 114, , 161, 163, 164). Defects in these different effects of lamins on chromatin organization explain some of the phenotypes, such as senescence of cells in progeroid diseases (93, 94, 96, 117, 151, 164, 165) and lipoatrophy and adipose tissue hypertrophy in lipodystrophies (2, 151). Conclusion After studying the different mechanisms explaining the occurrence of laminopathies, one can conclude that both hypotheses are probably at interplay in the pathology of laminopathies. According to the mechanical stress hypothesis, laminopathies occur when mutations in A-type lamins or A-type lamin associated proteins result in an increased fragility or deformation of the nucleus and a disturbed ability of 11 LMNA mutations leading to lipodystrophies can be explained by two of the mechanisms within the gene expression hypothesis. They could affect chromatin activity by altering transcription factor and/or transcriptional regulators targeting and activity through prelamin A accumulation (126, 140, ), and through altered peripheral chromatin organization (100, 126, 152). The gene expression theory also partly explains the phenotypes of progeroid and development

12 laminopathies. Prelamin A and progeroid accumulation lead to a complete loss of heterochromatin in most laminopathies (94, 96, 115, 117, 152). There also appears to be an altered interaction between progerin/prelamin A and transcription factors, leading to epigenetic modifications (157, 158). Furthermore, the accumulation of progerin likely disrupts the function of some replication and repair factors, causing replication forks stalling and arresting cell-cycle progression, and subsequently leading to the phenotype of senescence in HGPS (163, 164). However, the gene expression hypothesis is likely supported by the mechanical stress hypothesis. Abnormal gene expression may be impaired further when nuclei are deformed due the cell s inabilities to adapt to mechanical stress. The two hypotheses are combined in the so-called mechanosignalling hypothesis (169), which involves mechanosensing. Mechanosensing is the ability of cells and nuclei to sense and respond to mechanical forces of varying magnitude, direction and frequency (41, 170) and happens at the level of the ECM. A tensional force is generated on the cell, which is sensed and processed by the cytoskeleton in the form of both biochemical signals and mechanical forces terminating in the nucleus and initiating effector responses (171). Matrix stiffness and nuclear tension induce conformational changes in lamin coiled-coil dimers through tension-inhibited phosphorylation: unstressed lamin A/C is degraded under low-stress conditions and stabilized under high-stress conditions (92, 172). Chromatin itself can also sense and respond to mechanical forces exerted on or by the cell s cytoskeleton (92, 173). There is little research on the exact link between impaired adaptations to mechanical stress and altered gene expression. Therefore, this is an interesting future field of research. When the links between the mechanical stress hypothesis and the gene expression hypothesis are better understood, the different phenotypes of laminopathies may be better understood as well, which could support the development of medicines to treat these diseases. Literature 1. Gruenbaum Y, Foisner R. Lamins: nuclear intermediate filament proteins with fundamental functions in nuclear mechanics and genome regulation. Annu Rev Biochem. 2015;84: Camozzi D, Capanni C, Cenni V, Mattioli E, Columbaro M, Squarzoni S, et al. Diverse lamindependent mechanisms interact to control chromatin dynamics. Focus on laminopathies. Nucleus. 2014;5(5): Aebi U, Cohn J, Buhle L, Gerace L. The nuclear lamina is a meshwork of intermediate-type filaments. Nature. 1986;323(6088): Fisher DZ, Chaudhary N, Blobel G. cdna sequencing of nuclear lamins A and C reveals primary and secondary structural homology to intermediate filament proteins. Proc Natl Acad Sci U S A. 1986;83(17): Goldman AE, Maul G, Steinert PM, Yang HY, Goldman RD. Keratin-like proteins that coisolate with intermediate filaments of BHK-21 cells are nuclear lamins. Proc Natl Acad Sci U S A. 1986;83(11): Dechat T, Pfleghaar K, Sengupta K, Shimi T, Shumaker DK, Solimando L, et al. Nuclear lamins: major factors in the structural organization and function of the nucleus and chromatin. Genes Dev. 2008;22(7): Burke B, Stewart CL. Functional architecture of the cell's nucleus in development, aging, and disease. Curr Top Dev Biol. 2014;109: Dutta S, Bhattacharyya M, Sengupta K. Implications and Assessment of the Elastic Behavior of Lamins in Laminopathies. Cells. 2016;5(4). 9. Folker ES, Ostlund C, Luxton GW, Worman HJ, Gundersen GG. Lamin A variants that cause striated muscle disease are defective in anchoring transmembrane actin-associated nuclear lines for nuclear movement. Proc Natl Acad Sci U S A. 2011;108(1): Shimamoto Y, Tamura S, Masumoto H, Maeshima K. Nucleosome-nucleosome interactions via histone tails and linker DNA regulate nuclear rigidity. Mol Biol Cell. 2017;28(11): Shimi T, Kittisopikul M, Tran J, Goldman AE, Adam SA, Zheng Y, et al. Structural organization of nuclear lamins A, C, B1, and B2 revealed by 12

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