DNA replication in early mammalian embryos is patterned, predisposing lamina-associated regions to fragility.

DNA replication in differentiated cells follows a defined program, but when and how it is established during mammalian development is not known. Here we show using single-cell sequencing, that late replicating regions are established in association with the B compartment and the nuclear lamina from the first cell cycle after fertilization on both maternal and paternal genomes. Late replicating regions contain a relative paucity of active origins and few but long genes and low G/C content. In both bovine and mouse embryos, replication timing patterns are established prior to embryonic genome activation. Chromosome breaks, which form spontaneously in bovine embryos at sites concordant with human embryos, preferentially locate to late replicating regions. In mice, late replicating regions show enhanced fragility due to a sparsity of dormant origins that can be activated under conditions of replication stress. This pattern predisposes regions with long neuronal genes to fragility and genetic change prior to separation of soma and germ cell lineages. Our studies show that the segregation of early and late replicating regions is among the first layers of genome organization established after fertilization.

Progerin forms an abnormal meshwork and has a dominant-negative effect on the nuclear lamina.

Progerin, the protein that causes Hutchinson-Gilford progeria syndrome, triggers nuclear membrane (NM) ruptures and blebs, but the mechanisms are unclear. We suspected that the expression of progerin changes the overall structure of the nuclear lamina. High-resolution microscopy of smooth muscle cells (SMCs) revealed that lamin A and lamin B1 form independent meshworks with uniformly spaced openings (~0.085 µm2). The expression of progerin in SMCs resulted in the formation of an irregular meshwork with clusters of large openings (up to 1.4 µm2). The expression of progerin acted in a dominant-negative fashion to disrupt the morphology of the endogenous lamin B1 meshwork, triggering irregularities and large openings that closely resembled the irregularities and openings in the progerin meshwork. These abnormal meshworks were strongly associated with NM ruptures and blebs. Of note, the progerin meshwork was markedly abnormal in nuclear blebs that were deficient in lamin B1 (~50% of all blebs). That observation suggested that higher levels of lamin B1 expression might normalize the progerin meshwork and prevent NM ruptures and blebs. Indeed, increased lamin B1 expression reversed the morphological abnormalities in the progerin meshwork and markedly reduced the frequency of NM ruptures and blebs. Thus, progerin expression disrupts the overall structure of the nuclear lamina, but that effect-along with NM ruptures and blebs-can be abrogated by increased lamin B1 expression.

The probability of chromatin to be at the nuclear lamina has no systematic effect on its transcription level in fruit flies.

BACKGROUND: Multiple studies have demonstrated a negative correlation between gene expression and positioning of genes at the nuclear envelope (NE) lined by nuclear lamina, but the exact relationship remains unclear, especially in light of the highly stochastic, transient nature of the gene association with the NE. RESULTS: In this paper, we ask whether there is a causal, systematic, genome-wide relationship between the expression levels of the groups of genes in topologically associating domains (TADs) of Drosophila nuclei and the probabilities of TADs to be found at the NE. To investigate the nature of this possible relationship, we combine a coarse-grained dynamic model of the entire Drosophila nucleus with genome-wide gene expression data; we analyze the TAD averaged transcription levels of genes against the probabilities of individual TADs to be in contact with the NE in the control and lamins-depleted nuclei. Our findings demonstrate that, within the statistical error margin, the stochastic positioning of Drosophila melanogaster TADs at the NE does not, by itself, systematically affect the mean level of gene expression in these TADs, while the expected negative correlation is confirmed. The correlation is weak and disappears completely for TADs not containing lamina-associated domains (LADs) or TADs containing LADs, considered separately. Verifiable hypotheses regarding the underlying mechanism for the presence of the correlation without causality are discussed. These include the possibility that the epigenetic marks and affinity to the NE of a TAD are determined by various non-mutually exclusive mechanisms and remain relatively stable during interphase. CONCLUSIONS: At the level of TADs, the probability of chromatin being in contact with the nuclear envelope has no systematic, causal effect on the transcription level in Drosophila. The conclusion is reached by combining model-derived time-evolution of TAD locations within the nucleus with their experimental gene expression levels.

Interaction with B-type lamin reveals the function of Drosophila Keap1 xenobiotic response factor in nuclear architecture.

BACKGROUND: The Keap1-Nrf2 pathway serves as a central regulator that mediates transcriptional responses to xenobiotic and oxidative stimuli. Recent studies have shown that Keap1 and Nrf2 can regulate transcripts beyond antioxidant and detoxifying genes, yet the underlying mechanisms remain unclear. Our research has uncovered that Drosophila Keap1 (dKeap1) and Nrf2 (CncC) proteins can control high-order chromatin structure, including heterochromatin. METHODS AND RESULTS: In this study, we identified the molecular interaction between dKeap1 and lamin Dm0, the Drosophila B-type lamin responsible for the architecture of nuclear lamina and chromatin. Ectopic expression of dKeap1 led to an ectopic localization of lamin to the intra-nuclear area, corelated with the spreading of the heterochromatin marker H3K9me2 into euchromatin regions. Additionally, mis-regulated dKeap1 disrupted the morphology of the nuclear lamina. Knocking down of dKeap1 partially rescued the lethality induced by lamin overexpression, suggesting their genetic interaction during development. CONCLUSIONS: The discovered dKeap1-lamin interaction suggests a novel role for the Keap1 oxidative/xenobiotic response factor in regulating chromatin architecture.

Widespread nuclear lamina injuries defeat proteostatic purposes of α-synuclein amyloid inclusions.

Biogenesis of inclusion bodies (IBs) facilitates protein quality control (PQC). Canonical aggresomes execute degradation of misfolded proteins while non-degradable amyloids sequester into insoluble protein deposits. Lewy bodies (LBs) are filamentous amyloid inclusions of α-synuclein, but PQC benefits and drawbacks associated with LB-like IBs remain underexplored. Here, we report that crosstalk between filamentous LB-like IBs and aggresome-like IBs of α-synuclein (Syn-aggresomes) buffer the load, aggregation state, and turnover of the amyloidogenic protein in mouse primary neurons and HEK293T cells. Filamentous LB-like IBs possess unorthodox PQC capacities of self-quarantining α-synuclein amyloids and being degradable upon receding fresh amyloidogenesis. Syn-aggresomes equilibrate biogenesis of filamentous LB-like IBs by facilitating spontaneous degradation of α-synuclein and conditional turnover of disintegrated α-synuclein amyloids. Thus, both types of IB primarily contribute to PQC. Incidentally, the overgrown perinuclear LB-like IBs become degenerative once these are misidentified by BICD2, a cargo-adapter for the cytosolic motor-protein dynein. Microscopy indicates that microtubules surrounding the perinuclear filamentous inclusions are also distorted, misbalancing the cytoskeleton-nucleoskeleton tension leading to widespread lamina injuries. Together, nucleocytoplasmic mixing, DNA damage, and deregulated transcription of stress chaperones defeat the proteostatic purposes of the filamentous amyloids of α-synuclein.

A high-content screen reveals new regulators of nuclear membrane stability.

Nuclear membrane rupture is a physiological response to multiple in vivo processes, such as cell migration, that can cause extensive genome instability and upregulate invasive and inflammatory pathways. However, the underlying molecular mechanisms of rupture are unclear and few regulators have been identified. In this study, we developed a reporter that is size excluded from re-compartmentalization following nuclear rupture events. This allows for robust detection of factors influencing nuclear integrity in fixed cells. We combined this with an automated image analysis pipeline in a high-content siRNA screen to identify new proteins that both increase and decrease nuclear rupture frequency in cancer cells. Pathway analysis identified an enrichment of nuclear membrane and ER factors in our hits and we demonstrate that one of these, the protein phosphatase CTDNEP1, is required for nuclear stability. Analysis of known rupture determinants, including an automated quantitative analysis of nuclear lamina gaps, are consistent with CTDNEP1 acting independently of actin and nuclear lamina organization. Our findings provide new insights into the molecular mechanism of nuclear rupture and define a highly adaptable program for rupture analysis that removes a substantial barrier to new discoveries in the field.

Lamins: The backbone of the nucleocytoskeleton interface.

The nuclear lamina (NL) is a crucial component of the inner nuclear membrane (INM) and consists of lamin filaments and associated proteins. Lamins are type V intermediate filament proteins essential for maintaining the integrity and mechanical properties of the nucleus. In human cells, 'B-type' lamins (lamin B1 and lamin B2) are ubiquitously expressed, while 'A-type' lamins (lamin A, lamin C, and minor isoforms) are expressed in a tissue- and development-specific manner. Lamins homopolymerize to form filaments that localize primarily near the INM, but A-type lamins also localize to and function in the nucleoplasm. Lamins play central roles in the assembly, structure, positioning, and mechanics of the nucleus, modulating cell signaling and influencing development, differentiation, and other activities. This review highlights recent findings on the structure and regulation of lamin filaments, providing insights into their multifaceted functions, including their role as "mechanosensors", delving into the emerging significance of lamin filaments as vital links between cytoskeletal and nuclear structures, chromatin organization, and the genome.

Intermediate filaments in the heart: The dynamic duo of desmin and lamins orchestrates mechanical force transmission.

The intermediate filament (IF) cytoskeleton supports cellular structural integrity, particularly in response to mechanical stress. The most abundant IF proteins in mature cardiomyocytes are desmin and lamins. The desmin network tethers the contractile apparatus and organelles to the nuclear envelope and the sarcolemma, while lamins, as components of the nuclear lamina, provide structural stability to the nucleus and the genome. Mutations in desmin or A-type lamins typically result in cardiomyopathies and recent studies emphasized the synergistic roles of desmin and lamins in the maintenance of nuclear integrity in cardiac myocytes. Here we explore the emerging roles of the interdependent relationship between desmin and lamins in providing resilience to nuclear structure while transducing extracellular mechanical cues into the nucleus.

Choreography of lamina-associated domains: structure meets dynamics.

Lamina-associated domains are large regions of heterochromatin positioned at the nuclear periphery. These domains have been implicated in gene repression, especially in the context of development. In mammals, LAD organization is dependent on nuclear lamins, inner nuclear membrane proteins, and chromatin state. In addition, chromatin readers and modifier proteins have been implicated in this organization, potentially serving as molecular tethers that interact with both nuclear envelope proteins and chromatin. More recent studies have focused on teasing apart the rules that govern dynamic LAD organization and how LAD organization, in turn, relates to gene regulation and overall 3D genome organization. This review highlights recent studies in mammalian cells uncovering factors that instruct the choreography of LAD organization, re-organization, and dynamics at the nuclear lamina, including LAD dynamics in interphase and through mitotic exit, when LAD organization is re-established, as well as intra-LAD subdomain variations.

Lamins as structural nuclear elements through evolution.

Lamins are nuclear intermediate filament proteins with important, well-established roles in humans and other vertebrates. Lamins interact with DNA and numerous proteins at the nuclear envelope to determine the mechanical properties of the nucleus, coordinate chromatin organization, and modulate gene expression. Many of these functions are conserved in the lamin homologs found in basal metazoan organisms, including Drosophila and Caenorhabditis elegans. Lamin homologs have also been recently identified in non-metazoans, like the amoeba Dictyostelium discoideum, yet how these proteins compare functionally to the metazoan isoforms is only beginning to emerge. A better understanding of these distantly related lamins is not only valuable for a more complete picture of eukaryotic evolution, but may also provide new insights into the function of vertebrate lamins.

Scaffold, mechanics and functions of nuclear lamins.

Nuclear lamins are type-V intermediate filaments that are involved in many nuclear processes. In mammals, A- and B-type lamins assemble into separate physical meshwork underneath the inner nuclear membrane, the nuclear lamina, with some residual fraction localized within the nucleoplasm. Lamins are the major part of the nucleoskeleton, providing mechanical strength and flexibility to protect the genome and allow nuclear deformability, while also contributing to gene regulation via interactions with chromatin. While lamins are the evolutionary ancestors of all intermediate filament family proteins, their ultimate filamentous assembly is markedly different from their cytoplasmic counterparts. Interestingly, hundreds of genetic mutations in the lamina proteins have been causally linked with a broad range of human pathologies, termed laminopathies. These include muscular, neurological and metabolic disorders, as well as premature aging diseases. Recent technological advances have contributed to resolving the filamentous structure of lamins and the corresponding lamina organization. In this review, we revisit the multiscale lamin organization and discuss its implications on nuclear mechanics and chromatin organization within lamina-associated domains.

Deviating from the norm: Nuclear organisation in trypanosomes.

At first glance the nucleus is a highly conserved organelle. Overall nuclear morphology, the octagonal nuclear pore complex, the presence of peripheral heterochromatin and the nuclear envelope appear near constant features right down to the ultrastructural level. New work is revealing significant compositional divergence within these nuclear structures and their associated functions, likely reflecting adaptations and distinct mechanisms between eukaryotic lineages and especially the trypanosomatids. While many examples of mechanistic divergence currently lack obvious functional interpretations, these studies underscore the malleability of nuclear architecture. I will discuss some recent findings highlighting these facets within trypanosomes, together with the underlying evolutionary framework and make a call for the exploration of nuclear function in non-canonical experimental organisms.

The nuclear lamina is required for proper development and nuclear shape distortion in tomato.

The nuclear lamina in plant cells is composed of plant-specific proteins, including nuclear matrix constituent proteins (NMCPs), which have been postulated to be functional analogs of lamin proteins that provide structural integrity to the organelle and help stabilize the three-dimensional organization of the genome. Using genomic editing, we generated alleles for the three genes encoding NMCPs in cultivated tomato (Solanum lycopersicum) to determine if the consequences of perturbing the nuclear lamina in this crop species were similar to or distinct from those observed in the model Arabidopsis thaliana. Loss of the sole NMCP2-class protein was lethal in tomato but is tolerated in Arabidopsis. Moreover, depletion of NMCP1-type nuclear lamina proteins leads to distinct developmental phenotypes in tomato, including leaf morphology defects and reduced root growth rate (in nmcp1b mutants), compared with cognate mutants in Arabidopsis. These findings suggest that the nuclear lamina interfaces with different developmental and signaling pathways in tomato compared with Arabidopsis. At the subcellular level, however, tomato nmcp mutants resembled their Arabidopsis counterparts in displaying smaller and more spherical nuclei in differentiated cells. This result argues that the plant nuclear lamina facilitates nuclear shape distortion in response to forces exerted on the organelle within the cell.

Nuclear proteostasis imbalance in laminopathy-associated premature aging diseases.

Laminopathies are a group of rare genetic disorders with heterogeneous clinical phenotypes such as premature aging, cardiomyopathy, lipodystrophy, muscular dystrophy, microcephaly, epilepsy, and so on. The cellular phenomena associated with laminopathy invariably show disruption of nucleoskeleton of lamina due to deregulated expression, localization, function, and interaction of mutant lamin proteins. Impaired spatial and temporal tethering of lamin proteins to the lamina or nucleoplasmic aggregation of lamins are the primary molecular events that can trigger nuclear proteotoxicity by modulating differential protein-protein interactions, sequestering quality control proteins, and initiating a cascade of abnormal post-translational modifications. Clearly, laminopathic cells exhibit moderate to high nuclear proteotoxicity, raising the question of whether an imbalance in nuclear proteostasis is involved in laminopathic diseases, particularly in diseases of early aging such as HGPS and laminopathy-associated premature aging. Here, we review nuclear proteostasis and its deregulation in the context of lamin proteins and laminopathies.

The plant nuclear lamina disassembles to regulate genome folding in stress conditions.

The nuclear lamina is a complex network of nuclear lamins and lamin-associated nuclear membrane proteins, which scaffold the nucleus to maintain structural integrity. In Arabidopsis thaliana, nuclear matrix constituent proteins (NMCPs) are essential components of the nuclear lamina and are required to maintain the structural integrity of the nucleus and specific perinuclear chromatin anchoring. At the nuclear periphery, suppressed chromatin overlapping with repetitive sequences and inactive protein-coding genes are enriched. At a chromosomal level, plant chromatin organization in interphase nuclei is flexible and responds to various developmental cues and environmental stimuli. On the basis of these observations in Arabidopsis, and given the role of NMCP genes (CRWN1 and CRWN4) in organizing chromatin positioning at the nuclear periphery, one can expect considerable changes in chromatin-nuclear lamina interactions when the global chromatin organization patterns are being altered in plants. Here we report the highly flexible nature of the plant nuclear lamina, which disassembles substantially under various stress conditions. Focusing on heat stress, we reveal that chromatin domains, initially tethered to the nuclear envelope, remain largely associated with CRWN1 and become scattered in the inner nuclear space. By investigating the three-dimensional chromatin contact network, we further reveal that CRWN1 proteins play a structural role in shaping the changes in genome folding under heat stress. Also, CRWN1 acts as a negative transcriptional coregulator to modulate the shift of the plant transcriptome profile in response to heat stress.

The secret life of chromatin tethers.

The nuclear envelope plays an essential role in organizing the genome inside of the nucleus. The inner nuclear membrane is coated with a meshwork of filamentous lamin proteins that provide a surface to organize a variety of cellular processes. A subset of nuclear lamina- and membrane-associated proteins functions as anchors to hold transcriptionally silent heterochromatin at the nuclear periphery. While most chromatin tethers are integral membrane proteins, a limited number are lamina-bound. One example is the mammalian proline-rich 14 (PRR14) protein. PRR14 is a recently characterized protein with unique function that is different from other known chromatin tethers. Here, we review our current understanding of PRR14 structure and function in organizing heterochromatin at the nuclear periphery.

The Interplay between Oxidative Stress and the Nuclear Lamina Contributes to Laminopathies and Age-Related Diseases.

Oxidative stress is a physiological condition that arises when there is an imbalance between the production of reactive oxygen species (ROS) and the ability of cells to neutralize them. ROS can damage cellular macromolecules, including lipids, proteins, and DNA, leading to cellular senescence and physiological aging. The nuclear lamina (NL) is a meshwork of intermediate filaments that provides structural support to the nucleus and plays crucial roles in various nuclear functions, such as DNA replication and transcription. Emerging evidence suggests that oxidative stress disrupts the integrity and function of the NL, leading to dysregulation of gene expression, DNA damage, and cellular senescence. This review highlights the current understanding of the interplay between oxidative stress and the NL, along with its implications for human health. Specifically, elucidation of the mechanisms underlying the interplay between oxidative stress and the NL is essential for the development of effective treatments for laminopathies and age-related diseases.

The nuclear lamina couples mechanical forces to cell fate in the preimplantation embryo via actin organization.

During preimplantation development, contractile forces generated at the apical cortex segregate cells into inner and outer positions of the embryo, establishing the inner cell mass (ICM) and trophectoderm. To which extent these forces influence ICM-trophectoderm fate remains unresolved. Here, we found that the nuclear lamina is coupled to the cortex via an F-actin meshwork in mouse and human embryos. Actomyosin contractility increases during development, upregulating Lamin-A levels, but upon internalization cells lose their apical cortex and downregulate Lamin-A. Low Lamin-A shifts the localization of actin nucleators from nucleus to cytoplasm increasing cytoplasmic F-actin abundance. This results in stabilization of Amot, Yap phosphorylation and acquisition of ICM over trophectoderm fate. By contrast, in outer cells, Lamin-A levels increase with contractility. This prevents Yap phosphorylation enabling Cdx2 to specify the trophectoderm. Thus, forces transmitted to the nuclear lamina control actin organization to differentially regulate the factors specifying lineage identity.

Differential contributions of nuclear lamina association and genome compartmentalization to gene regulation.

Chromatin regions that interact with the nuclear lamina are often heterochromatic, repressed in gene expression, and in the spatial B compartment. However, exceptions to this trend allow us to examine the relative impact of lamin association and spatial compartment on gene regulation. Here, we compared lamin association, gene expression, Hi-C, and histone mark datasets from cell lines representing different states of differentiation across different cell-type lineages. With these data, we compare, for example, gene expression differences when a B compartment region is associated with the nuclear lamina in one cell type but not in another. In general, we observed an additive rather than redundant effect of lamin association and compartment status. But, whether compartment status or lamin association had a dominant influence on gene expression varied by cell type. Finally, we identified how compartment and lamin association influence the likelihood of gene induction or repression in response to physicochemical treatment.

InterLINCing Chromatin Organization and Mechanobiology in Laminopathies.

PURPOSE OF REVIEW: In this review, we explore the chromatin-related consequences of laminopathy-linked mutations through the lens of mechanotransduction. RECENT FINDINGS: Multiple studies have highlighted the role of the nuclear lamina in maintaining the integrity of the nucleus. The lamina also has a critical role in 3D genome organization. Mutations in lamina proteins associated with various laminopathies result in the loss of organization of DNA at the nuclear periphery. However, it remains unclear if or how these two aspects of lamin function are connected. Recent data suggests that unlinking the cytoskeleton from the nuclear lamina may be beneficial to slow progress of deleterious phenotypes observed in laminopathies. In this review, we highlight emerging data that suggest interlinked chromatin- and mechanical biology-related pathways are interconnected in the pathogenesis of laminopathies.