Early Polycomb-target deregulations in Hutchinson-Gilford Progeria Syndrome revealed by heterochromatin analysis
Endre SebestyénFabrizia MarulloFederica LuciniAndrea BianchiCristiano PetriniSara ValsoniIlaria OlivieriLaura AntonelliFrancesco GregorettiGennaro OlivaFrancesco FerrariChiara Lanzuolo
0
Citation
78
Reference
10
Related Paper
Abstract:
Abstract Hutchinson-Gilford progeria syndrome (HGPS) is characterized by the progressive accumulation of progerin, an aberrant form of Lamin A. This leads to chromatin structure disruption, in particular by interfering with Lamina Associated Domains. Although several cellular and molecular alterations have been characterized, it is still unclear how chromatin structural changes translate into premature senescence in HGPS. Moreover, early events in chromatin remodeling have not been detected so far. We developed a new high-throughput sequencing-based method, named SAMMY-seq, for genome-wide characterization of heterochromatin accessibility changes. Using SAMMY-seq, we detected early stage alterations of chromatin structure in HGPS primary fibroblasts. Of note, these structural changes do not disrupt the distribution of H3K9me3 but are associated with site-specific H3K27me3 variations and transcriptional dysregulation of Polycomb target genes. Our results show that SAMMY-seq represents a novel and sensitive tool to characterize heterochromatin alterations. Moreover, we found that the assembly of lamin associated domains is strictly connected to the correct Polycomb repression, rapidly lost in early HGPS pathogenesis.Keywords:
Progeria
Nuclear lamina
Polycomb-group proteins
Premature aging
Senescence
Envelope (radar)
Premature aging
Nuclear matrix
Translational Research
Cite
Citations (67)
The nuclear lamina is a network of structural filaments, the A and B type lamins, located at the nuclear envelope and throughout the nucleus. Lamin filaments provide the nucleus with mechanical stability and support many basic activities, including gene regulation. Mutations in LMNA , the gene encoding A type lamins, cause numerous human diseases, including the segmental premature aging disease Hutchinson–Gilford progeria syndrome (HGPS). Here we show that structural and mechanical properties of the lamina are altered in HGPS cells. We demonstrate by live-cell imaging and biochemical analysis that lamins A and C become trapped at the nuclear periphery in HGPS patient cells. Using micropipette aspiration, we show that the lamina in HGPS cells has a significantly reduced ability to rearrange under mechanical stress. Based on polarization microscopy results, we suggest that the lamins are disordered in the healthy nuclei, whereas the lamins in HGPS nuclei form orientationally ordered microdomains. The reduced deformability of the HGPS nuclear lamina possibly could be due to the inability of these orientationally ordered microdomains to dissipate mechanical stress. Surprisingly, intact HGPS cells exhibited a degree of resistance to acute mechanical stress similar to that of cells from healthy individuals. Thus, in contrast to the nuclear fragility seen in lmna null cells, the lamina network in HGPS cells has unique mechanical properties that might contribute to disease phenotypes by affecting responses to mechanical force and misregulation of mechanosensitive gene expression.
Progeria
LMNA
Nuclear lamina
Premature aging
Cite
Citations (374)
Lamins are intermediate filament proteins. They are structural components of the nuclear lamina, a filamentous meshwork beneath the inner nuclear membrane, which confers mechanical stability to the nucleus. Mutations in the human LMNA gene cause a wide range of diseases called laminopathies. Amongst these is the Hutchinson-Gilford progeria syndrome (HGPS), a rare premature aging disorder. One of the mutations causing HGPS is a heterozygous point mutation E145K within the central domain of the lamin A protein. The E145K mutation affects lamin filament assembly and induces profound changes in the composition and architecture of the patient cell nuclei. In vitro analyses of purified E145K lamin A reveal severe assembly defects into higher order lamin structures, indicating an abnormal lateral association of lamin protofilaments. Ex vivo expression of the wild-type and E145K lamin A in Xenopus oocytes showed influence of the mutant lamin A on the mechanical properties of the lamina as revealed by atomic force microscopy (AFM). Nuclear laminae made up of overexpressed E145K lamin A were stiffer than those harboring wild-type lamin A. In this work, mechanical properties of somatic cell nuclei were studied using AFM. In pilot experiments with HeLa cells AFM data acquisition and analysis was optimized. Next, the mechanical properties of the dermal fibroblasts of a four years old progeria patient bearing the E145K lamin A mutation were analyzed using AFM. The abnormal shape of nuclei expressing E145K lamin A and alterations in the cellular actin network were revealed by fluorescence microscopy. Lamina thickness was assessed by transmission electron microscopy. AFM probing of entire dermal fibroblasts revealed minor differences in the elastic moduli of nuclear and cytoplasmic cell regions. Thus, isolation of the nuclei was done to directly measure their mechanical properties by AFM. Isolated nuclei of the progeria patient (age 4) and the old person (age 61) were significantly stiffer than those of a young person (age 10). These results indicated that lamin A E145K alters the mechanical properties of the nuclei of the dermal fibroblasts obtained from a progeria patient. Thus, it was shown that the process of aging, be it natural or abnormal, increases the stiffness of nuclei.
Progeria
LMNA
Nuclear lamina
Premature aging
Cite
Citations (0)
LMNA
Progeria
Nuclear lamina
Premature aging
Cite
Citations (13)
More than 20 mutations in the gene encoding A-type lamins (LMNA) cause progeria, a rare premature aging disorder. The major pathognomonic hallmarks of progeria cells are seen as nuclear deformations or blebs that are related to the redistribution of A- and B-type lamins within the nuclear lamina. However, the functional significance of these progeria-associated blebs remains unknown. We have carried out an analysis of the structural and functional consequences of progeria-associated nuclear blebs in dermal fibroblasts from a progeria patient carrying a rare point mutation p.S143F (C428T) in lamin A/C. These blebs form microdomains that are devoid of major structural components of the nuclear envelope (NE)/lamina including B-type lamins and nuclear pore complexes (NPCs) and are enriched in A-type lamins. Using laser capture microdissection and comparative genomic hybridization (CGH) analyses, we show that, while these domains are devoid of centromeric heterochromatin and gene-poor regions of chromosomes, they are enriched in gene-rich chromosomal regions. The active form of RNA polymerase II is also greatly enriched in blebs as well as nascent RNA but the nuclear co-activator SKIP is significantly reduced in blebs compared to other transcription factors. Our results suggest that the p.S143F progeria mutation has a severe impact not only on the structure of the lamina but also on the organization of interphase chromatin domains and transcription. These structural defects are likely to contribute to gene expression changes reported in progeria and other types of laminopathies.
Progeria
LMNA
Nuclear lamina
Premature aging
Cite
Citations (38)
The A‐and B‐type nuclear lamins (LA/C, LB1 and LB2) form separate but interconnecting networks within the nuclear lamina and nucleoplasm. Hundreds of mutations in the LMNA gene cause a wide range of diseases including the premature aging disease, Hutchinson‐Gilford Progeria Syndrome (HGPS). The nuclei of HGPS patients’ cells exhibit a wide range of changes in the architecture of their nuclei, including the formation of LA/C rich nuclear envelope blebs, a loss of peripheral heterochromatin, alterations in epigenetic modifications of chromatin, and changes in the RNA pol II transcriptional machinery. Recently we have focused our attention on the mechanisms responsible for the premature senescenece of cells derived from HGPS patients. The results demonstrate that the nuclear lamins are critical factors in the proliferation and senescence of both normal diploid and HGPS cells. Supported by the NCI and the Progeria Research Foundation.
Progeria
LMNA
Nuclear lamina
Premature aging
Nucleoplasm
Euchromatin
Cite
Citations (0)
Mutations in the human lamin A gene (LMNA) cause a wide range of diseases (laminopathies). Among these is the Hutchinson-Gilford progeria syndrome (HGPS), a rare premature aging disease. Most HGPS patients carry a silent point mutation, which activates a cryptic splice site resulting in the expression of a permanently isoprenylated and truncated lamin AΔ50/progerin. Another type of mutant lamin A namely, E145K-lamin A, also causes HGPS. E145K-lamin A induces profound changes in the nuclear architecture of patient cells as well as after expression in cultured cells. The E145K mutation is located in the α-helical central domain of lamin A, which is involved in lamin filament assembly. In vitro analyses of purified E145K-lamin A have revealed severe assembly defects into higher order lamin structures, which indicates an abnormal lateral association of protofilaments. To analyze how the altered assembly observed in vitro might influence the mechanics of a nuclear lamina formed by E145K-lamin A, mutant and wild type lamin A were ectopically expressed in amphibian oocytes. Both types form a lamina consisting of multi-layered sheets of filaments at the inner side of the nuclear envelope. The mechanical properties of isolated nuclei were measured by atomic force microscopy (AFM). From the resulting force curves, the stiffness of the lamina was estimated. The thickness of the resulting lamin A layer was then measured by TEM. The two parameters allowed us to estimate the elastic modulus (Young's modulus) of the lamina. Lamin A sheets made from E145K filaments have a higher Young's modulus compared to wild type filaments, i.e. the E145K-lamin A sheets are more rigid than wild type laminae of comparable thickness.
Progeria
LMNA
Nuclear lamina
Premature aging
Cite
Citations (34)
Hutchinson–Gilford progeria syndrome (HGPS) is an extremely rare premature aging disease presenting many features resembling the normal aging process. HGPS patients die before the age of 20 years due to cardiovascular problems and heart failure. HGPS is linked to mutations in the LMNA gene encoding the intermediate filament protein lamin A. Lamin A is a major component of the nuclear lamina, a scaffold structure at the nuclear envelope that defines mechanochemical properties of the nucleus and is involved in chromatin organization and epigenetic regulation. Lamin A is also present in the nuclear interior where it fulfills lamina-independent functions in cell signaling and gene regulation. The most common LMNA mutation linked to HGPS leads to mis-splicing of the LMNA mRNA and produces a mutant lamin A protein called progerin that tightly associates with the inner nuclear membrane and affects the dynamic properties of lamins. Progerin expression impairs many important cellular processes providing insight into potential disease mechanisms. These include changes in mechanosignaling, altered chromatin organization and impaired genome stability, and changes in signaling pathways, leading to impaired regulation of adult stem cells, defective extracellular matrix production and premature cell senescence. In this review, we discuss these pathways and their potential contribution to the disease pathologies as well as therapeutic approaches used in preclinical and clinical tests.
LMNA
Progeria
Nuclear lamina
Premature aging
Cite
Citations (100)
Abstract The lamina is a filamentous meshwork beneath the inner nuclear membrane that confers mechanical stability to nuclei. The E145K mutation in lamin A causes Hutchinson‐Gilford progeria syndrome (HGPS). It affects lamin filament assembly and induces profound changes in the nuclear architecture. Expression of wild‐type and E145K lamin A in Xenopus oocytes followed by atomic force microscopy (AFM) probing of isolated oocyte nuclei has shown significant changes in the mechanical properties of the lamina. Nuclei of oocytes expressing E145K lamin A are stiffer than those expressing wild‐type lamin A. Here we present mechanical measurements by AFM on dermal fibroblasts obtained from a 4‐year‐old progeria patient bearing the E145K lamin A mutation and compared it to fibroblasts obtained from 2 healthy donors of 10 and 61 years of age, respectively. The abnormal shape of nuclei expressing E145K lamin A was analyzed by fluorescence microscopy. Lamina thickness was measured using electron micrographs. Fluorescence microscopy showed alterations in the actin network of progeria cells. AFM probing of whole dermal fibroblasts did not demonstrate significant differences in the elastic moduli of nuclear and cytoplasmic cell regions. In contrast, AFM measurements of isolated nuclei showed that nuclei of progeria and old person's cells are significantly stiffer than those of the young person, indicating that the process of aging, be it natural or abnormal, increases nuclear stiffness. Our results corroborate AFM data obtained using Xenopus oocyte nuclei and prove that the presence of E145K lamin A abnormally increases nuclear stiffness.
Progeria
Nuclear lamina
Premature aging
Nuclear membrane
LMNA
Cite
Citations (25)
Hutchinson–Gilford progeria syndrome (HGPS) is a dominant autosomal premature aging syndrome caused by the expression of a truncated prelamin A designated progerin (Pgn). A-type and B-type lamins are intermediate filament proteins that polymerize to form the nuclear lamina network apposed to the inner nuclear membrane of vertebrate somatic cells. It is not known if in vivo both type of lamins assemble independently or co-assemble. The blebbing and disorganization of the nuclear envelope and adjacent heterochromatin in cells from patients with HGPS is a hallmark of the disease, and the ex vivo reversal of this phenotype is considered important for the development of therapeutic strategies. Here, we investigated the alterations in the lamina structure that may underlie the disorganization caused in nuclei by Pgn expression. We studied the polymerization of enhanced green fluorescent protein- and red fluorescent protein-tagged wild-type and mutated lamins in the nuclear envelope of living cells by measuring fluorescence resonance energy transfer (FRET) that occurs between the two fluorophores when tagged lamins interact. Using time domain fluorescence lifetime imaging microscopy that allows a quantitative analysis of FRET signals, we show that wild-type lamins A and B1 polymerize in distinct homopolymers that further interact in the lamina. In contrast, expressed Pgn co-assembles with lamin B1 and lamin A to form a mixed heteropolymer in which A-type and B-type lamin segregation is lost. We propose that such structural lamina alterations may be part of the primary mechanisms leading to HGPS, possibly by impairing functions specific for each lamin type such as nuclear membrane biogenesis, signal transduction, nuclear compartmentalization and gene regulation.
Progeria
Nuclear lamina
LMNA
Premature aging
Cite
Citations (114)