One-step purification of tag free and soluble lamin B1 from an E. coli bacterial expression system.

Lamin B1 is an intermediate filament protein that is a core component of the nuclear lamina. Structural studies and biochemical characterization of lamin B1 are severely hampered by the tendency of the protein to form inclusion bodies in E. coli bacterial expression systems. Therefore, the purity and consistency of the protein varies from batch to batch. In this work, we have purified a tag-free lamin B1 protein from a soluble fraction following bacterial expression. We also checked the functional properties of the purified as well as of the subsequently lyophilised protein. The current protocol helps to purify functional lamin B1 in a single step.

Scaling concepts in 'omics: Nuclear lamin-B scales with tumor growth and often predicts poor prognosis, unlike fibrosis.

Physicochemical principles such as stoichiometry and fractal assembly can give rise to characteristic scaling between components that potentially include coexpressed transcripts. For key structural factors within the nucleus and extracellular matrix, we discover specific gene-gene scaling exponents across many of the 32 tumor types in The Cancer Genome Atlas, and we demonstrate utility in predicting patient survival as well as scaling-informed machine learning (SIML). All tumors with adjacent tissue data show cancer-elevated proliferation genes, with some genes scaling with the nuclear filament LMNB1, including the transcription factor FOXM1 that we show directly regulates LMNB1 SIML shows that such regulated cancers cluster together with longer overall survival than dysregulated cancers, but high LMNB1 and FOXM1 in half of regulated cancers surprisingly predict poor survival, including for liver cancer. COL1A1 is also studied because it too increases in tumors, and a pan-cancer set of fibrosis genes shows substoichiometric scaling with COL1A1 but predicts patient outcome only for liver cancer-unexpectedly being prosurvival. Single-cell RNA-seq data show nontrivial scaling consistent with power laws from bulk RNA and protein analyses, and SIML segregates synthetic from contractile cancer fibroblasts. Our scaling approach thus yields fundamentals-based power laws relatable to survival, gene function, and experiments.

The prolyl-isomerase PIN1 is essential for nuclear Lamin-B structure and function and protects heterochromatin under mechanical stress.

Chromatin organization plays a crucial role in tissue homeostasis. Heterochromatin relaxation and consequent unscheduled mobilization of transposable elements (TEs) are emerging as key contributors of aging and aging-related pathologies, including Alzheimer's disease (AD) and cancer. However, the mechanisms governing heterochromatin maintenance or its relaxation in pathological conditions remain poorly understood. Here we show that PIN1, the only phosphorylation-specific cis/trans prolyl isomerase, whose loss is associated with premature aging and AD, is essential to preserve heterochromatin. We demonstrate that this PIN1 function is conserved from Drosophila to humans and prevents TE mobilization-dependent neurodegeneration and cognitive defects. Mechanistically, PIN1 maintains nuclear type-B Lamin structure and anchoring function for heterochromatin protein 1α (HP1α). This mechanism prevents nuclear envelope alterations and heterochromatin relaxation under mechanical stress, which is a key contributor to aging-related pathologies.

Increase in lamin B1 promotes telomere instability by disrupting the shelterin complex in human cells.

Telomere maintenance is essential to preserve genomic stability and involves telomere-specific proteins, DNA replication and repair proteins. Lamins are key components of the nuclear envelope and play numerous roles, including maintenance of the nuclear integrity, regulation of transcription, and DNA replication. Elevated levels of lamin B1, one of the major lamins, have been observed in some human pathologies and several cancers. Yet, the effect of lamin B1 dysregulation on telomere maintenance remains unknown. Here, we unveil that lamin B1 overexpression drives telomere instability through the disruption of the shelterin complex. Indeed, lamin B1 dysregulation leads to an increase in telomere dysfunction-induced foci, telomeric fusions and telomere losses in human cells. Telomere aberrations were preceded by mislocalizations of TRF2 and its binding partner RAP1. Interestingly, we identified new interactions between lamin B1 and these shelterin proteins, which are strongly enhanced at the nuclear periphery upon lamin B1 overexpression. Importantly, chromosomal fusions induced by lamin B1 in excess were rescued by TRF2 overexpression. These data indicated that lamin B1 overexpression triggers telomere instability through a mislocalization of TRF2. Altogether our results point to lamin B1 as a new interacting partner of TRF2, that is involved in telomere stability.

Beta-strand-mediated dimeric formation of the Ig-like domains of human lamin A/C and B1.

Lamins are nuclear intermediate filament proteins that play an essential role in maintaining the nuclear structure by forming a 3-D meshwork. Lamins consist of the N-terminal unstructured head, the coiled-coil rod domain, and the C-terminal tail, which is mostly unstructured except for the Ig-like domain. To date, the Ig-like domain has been characterized as a monomeric structure. Here, we determined the crystal structures of human lamin A/C, including the Ig-like domain and its N- and C-terminal flanking sequences. Interestingly, the structures showed a homodimer formed by beta-strand interactions between the N- and C-terminal flanking sequences. This interaction also provides a molecular implication for the creation of a 3-D meshwork between the 3.5-nm-thick filaments. Furthermore, we determined the crystal structure of the corresponding region of lamin B1. The structure showed a similar dimeric assembly, also formed by beta-strand interactions, albeit the intersubunit distance was much shorter. Since the Ig-like domain contains many genetic hotspots causing lamin-related diseases in lamin A/C, our findings will help understand the detailed assembly of lamins in a 3-D meshwork structure and lamin-related diseases at the molecular level.

Lamin B1 acetylation slows the G1 to S cell cycle transition through inhibition of DNA repair.

The integrity and regulation of the nuclear lamina is essential for nuclear organization and chromatin stability, with its dysregulation being linked to laminopathy diseases and cancer. Although numerous posttranslational modifications have been identified on lamins, few have been ascribed a regulatory function. Here, we establish that lamin B1 (LMNB1) acetylation at K134 is a molecular toggle that controls nuclear periphery stability, cell cycle progression, and DNA repair. LMNB1 acetylation prevents lamina disruption during herpesvirus type 1 (HSV-1) infection, thereby inhibiting virus production. We also demonstrate the broad impact of this site on laminar processes in uninfected cells. LMNB1 acetylation negatively regulates canonical nonhomologous end joining by impairing the recruitment of 53BP1 to damaged DNA. This defect causes a delay in DNA damage resolution and a persistent activation of the G1/S checkpoint. Altogether, we reveal LMNB1 acetylation as a mechanism for controlling DNA repair pathway choice and stabilizing the nuclear periphery.

A Rare Mutation in LMNB2 Associated with Lipodystrophy Drives Premature Cell Senescence.

Many proteins are causative for inherited partial lipodystrophies, including lamins, the essential constituents of the nuclear envelope scaffold called the lamina. By performing high throughput sequencing on a panel of genes involved in lipodystrophies, we identified a heterozygous mutation in LMNB2 gene (c.700C > T p.(Arg234Trp)) in a female patient presenting early onset type II diabetes, hypertriglyceridemia, and android fat distribution. This mutation is rare in the general population (frequency 0.013% in GnomAD) and was predicted pathogenic by a set of pathogenicity prediction software. Patient-derived fibroblasts showed nuclear shape abnormalities and premature senescence features, which are two typical cellular phenotypes associated with laminopathies. Moreover, we observed an atypical aggregation of lamin B2 in nucleoplasm, which co-distributes with emerin and lamin A/C, along with an abnormal distribution of lamin A/C at the nuclear envelope. Finally, reducing lamin B2 expression level by siRNA targeted toward LMNB2 transcripts resulted in decreased nuclear anomalies and senescence-associated beta-galactosidase, suggesting a role of the mutated protein in the occurrence of the observed cellular phenotype. Altogether, these results suggest that mutations in lamin B2 could produce premature senescence and partial lipodystrophy features as observed with certain mutants of lamin A/C.

Single-Cell DamID to Capture Contacts Between DNA and the Nuclear Lamina in Individual Mammalian Cells.

The organization of DNA within the eukaryotic nucleus is important for cellular processes such as regulation of gene expression and repair of DNA damage. To comprehend cell-to-cell variation within a complex system, systematic analysis of individual cells is necessary. While many tools exist to capture DNA conformation and chromatin context, these methods generally require large populations of cells for sufficient output. Here we describe single-cell DamID, a technique to capture contacts between DNA and a given protein of interest. By fusing the bacterial methyltransferase Dam to nuclear lamina protein lamin B1, genomic regions in contact with the nuclear periphery can be mapped. Single-cell DamID generates contact maps with sufficient throughput and resolution to reliably identify patterns of similarity as well as variation in nuclear organization of interphase chromosomes.

Replication dynamics of individual loci in single living cells reveal changes in the degree of replication stochasticity through S phase.

Eukaryotic genomes are replicated under the control of a highly sophisticated program during the restricted time period corresponding to S phase. The most widely used replication timing assays, which are performed on populations of millions of cells, suggest that most of the genome is synchronously replicated on homologous chromosomes. We investigated the stochastic nature of this temporal program, by comparing the precise replication times of allelic loci within single vertebrate cells progressing through S phase at six loci replicated from very early to very late. We show that replication timing is strictly controlled for the three loci replicated in the first half of S phase. Out of the three loci replicated in the second part of S phase, two present a significantly more stochastic pattern. Surprisingly, we find that the locus replicated at the very end of S phase, presents stochasticity similar to those replicated in early S phase. We suggest that the richness of loci in efficient origins of replication, which decreases from early- to late-replicating regions, and the strength of interaction with the nuclear lamina may underlie the variation of timing control during S phase.

Concentric organization of A- and B-type lamins predicts their distinct roles in the spatial organization and stability of the nuclear lamina.

The nuclear lamina is an intermediate filament meshwork adjacent to the inner nuclear membrane (INM) that plays a critical role in maintaining nuclear shape and regulating gene expression through chromatin interactions. Studies have demonstrated that A- and B-type lamins, the filamentous proteins that make up the nuclear lamina, form independent but interacting networks. However, whether these lamin subtypes exhibit a distinct spatial organization or whether their organization has any functional consequences is unknown. Using stochastic optical reconstruction microscopy (STORM) our studies reveal that lamin B1 and lamin A/C form concentric but overlapping networks, with lamin B1 forming the outer concentric ring located adjacent to the INM. The more peripheral localization of lamin B1 is mediated by its carboxyl-terminal farnesyl group. Lamin B1 localization is also curvature- and strain-dependent, while the localization of lamin A/C is not. We also show that lamin B1's outer-facing localization stabilizes nuclear shape by restraining outward protrusions of the lamin A/C network. These two findings, that lamin B1 forms an outer concentric ring and that its localization is energy-dependent, are significant as they suggest a distinct model for the nuclear lamina-one that is able to predict its behavior and clarifies the distinct roles of individual nuclear lamin proteins and the consequences of their perturbation.

Optimization of DamID for use in primary cultures of mouse hepatocytes.

DamID, a method to identify DNA associating with a particular protein, was originally developed for use in immortalized tissue culture lines. The power of this technique has led to its adaptation for a number of additional systems. Here we report adaptations for its use in primary cells isolated from rodents with emphasis on the challenges this presents. Specifically, we present several modifications that allow the method to be performed in mouse acutely isolated primary hepatocytes while seemingly maintaining tissue genome architecture. We also describe the downstream bioinformatic analysis necessary to identify LADs and discuss some of the parameters and their effects with regards to the sensitivity of the method.

Lamin B1 mapping reveals the existence of dynamic and functional euchromatin lamin B1 domains.

Lamins (A/C and B) are major constituents of the nuclear lamina (NL). Structurally conserved lamina-associated domains (LADs) are formed by genomic regions that contact the NL. Lamins are also found in the nucleoplasm, with a yet unknown function. Here we map the genome-wide localization of lamin B1 in an euchromatin-enriched fraction of the mouse genome and follow its dynamics during the epithelial-to-mesenchymal transition (EMT). Lamin B1 associates with actively expressed and open euchromatin regions, forming dynamic euchromatin lamin B1-associated domains (eLADs) of about 0.3 Mb. Hi-C data link eLADs to the 3D organization of the mouse genome during EMT and correlate lamin B1 enrichment at topologically associating domain (TAD) borders with increased border strength. Having reduced levels of lamin B1 alters the EMT transcriptional signature and compromises the acquisition of mesenchymal traits. Thus, during EMT, the process of genome reorganization in mouse involves dynamic changes in eLADs.

E3 ubiquitin ligase HECW2 targets PCNA and lamin B1.

Lamins constitute the major architectural proteins of the nuclear lamina that help in maintaining nuclear organization. Mutations in lamins are associated with diverse degenerative diseases, collectively termed laminopathies. HECW2, a HECT-type E3 ubiquitin ligase, is transcriptionally upregulated in HeLa cells expressing Emery-Dreifuss muscular dystrophy-causing-lamin A mutants. However, the role of HECW2 upregulation in mediating downstream effects in lamin mutant-expressing cells was previously unexplored. Here, we show that HECW2 interacts with two lamin A-binding proteins, proliferating cell nuclear antigen (PCNA), via a canonical PCNA-interacting protein (PIP) motif, and lamin B1. HECW2 mediates their ubiquitination and targets them for proteasomal degradation. Cells expressing lamin A mutants G232E and Q294P, in which HECW2 is upregulated, show increased proteasomal degradation of PCNA and lamin B1 most likely mediated by HECW2. Our findings establish HECW2 as an E3 ubiquitin ligase for PCNA and lamin B1 which regulates their levels in laminopathic cells. We also found that HECW2 interacts with wild-type lamin A and ubiquitinates it and this interaction is reduced in case of lamin mutants G232E and Q294P. Our findings suggest that interplay among HECW2, lamin A, PCNA, and lamin B1 determines their respective homeostatic levels in the cell and dysregulation of these interactions may contribute to the pathogenicity of laminopathies.

DCM associated LMNA mutations cause distortions in lamina structure and assembly.

BACKGROUND: A and B-type lamins are integral scaffolding components of the nuclear lamina which impart rigidity and shape to all metazoan nuclei. Over 450 mutations in A-type lamins are associated with 16 human diseases including dilated cardiomyopathy (DCM). Here, we show that DCM mutants perturb the self-association of lamin A (LA) and it's binding with lamin B1 (LB1). METHODS: We used confocal and superresolution microscopy (NSIM) to study the effect of LA mutants on the nuclear lamina. We further used circular dichroism, fluorescence spectroscopy and isothermal titration calorimetry (ITC) to probe the structural modulations, self-association and heteropolymeric association of mutant LA. RESULTS: Transfection of mutants in cultured cell lines result in the formation of nuclear aggregates of varied size and distribution. Endogenous LB1 is sequestered into these aggregates. This is consistent with the ten-fold increase in association constant of the mutant proteins compared to the wild type. These mutants exhibit differential heterotypic interaction with LB1, along with significant secondary and tertiary structural alterations of the interacting proteins. Thermodynamic studies demonstrate that the mutants bind to LB1 with different stoichiometry, affinity and energetics. CONCLUSIONS: In this report we show that increased self-association propensity of mutant LA modulates the LA-LB1 interaction and precludes the formation of an otherwise uniform laminar network. GENERAL SIGNIFICANCE: Our results might highlight the role of homotypic and heterotypic interactions of LA in the pathogenesis of DCM and hence laminopathies in the broader sense.

Non-farnesylated B-type lamin can tether chromatin inside the nucleus and its chromatin interaction requires the Ig-fold region.

Lamins are thought to direct heterochromatin to the nuclear lamina (NL); however, this function of lamin has not been clearly demonstrated in vivo. To address this, we analyzed polytene chromosome morphology when artificial lamin variants were expressed in Drosophila endoreplicating cells. We found that the CaaX-motif-deleted B-type lamin Dm0, but not A-type lamin C, was able to form a nuclear envelope-independent layer that was closely associated with chromatin. Other nuclear envelope proteins were not detected in this "ectopic lamina," and the associated chromatin showed a repressive histone modification maker but not a permissive histone modification marker nor RNA polymerase II proteins. Furthermore, deletion of the C-terminal lamin-Ig-fold domain prevents chromatin association with this ectopic lamina. Thus, non-farnesylated B-type lamin Dm0 can form an ectopic lamina and induce changes to chromatin structure and status inside the interphase nucleus.

Delivering Single-Walled Carbon Nanotubes to the Nucleus Using Engineered Nuclear Protein Domains.

Single-walled carbon nanotubes (SWCNTs) have great potential for cell-based therapies due to their unique intrinsic optical and physical characteristics. Consequently, broad classes of dispersants have been identified that individually suspend SWCNTs in water and cell media in addition to reducing nanotube toxicity to cells. Unambiguous control and verification of the localization and distribution of SWCNTs within cells, particularly to the nucleus, is needed to advance subcellular technologies utilizing nanotubes. Here we report delivery of SWCNTs to the nucleus by noncovalently attaching the tail domain of the nuclear protein lamin B1 (LB1), which we engineer from the full-length LMNB1 cDNA. More than half of this low molecular weight globular protein is intrinsically disordered but has an immunoglobulin-fold composed of a central hydrophobic core, which is highly suitable for associating with SWCNTs, stably suspending SWCNTs in water and cell media. In addition, LB1 has an exposed nuclear localization sequence to promote active nuclear import of SWCNTs. These SWCNTs-LB1 dispersions in water and cell media display near-infrared (NIR) absorption spectra with sharp van Hove peaks and an NIR fluorescence spectra, suggesting that LB1 individually disperses nanotubes. The dispersing capability of SWCNTs by LB1 is similar to that by albumin proteins. The SWCNTs-LB1 dispersions with concentrations ≥150 µg/mL (≥30 µg/mL) in water (cell media) remain stable for ≥75 days (≥3 days) at 4 °C (37 °C). Further, molecular dynamics modeling of association of LB1 with SWCNTs reveal that the exposure of the nuclear localization sequence is independent of LB1 binding conformation. Measurements from confocal Raman spectroscopy and microscopy, NIR fluorescence imaging of SWCNTs, and fluorescence lifetime imaging microscopy show that millions of these SWCNTs-LB1 complexes enter HeLa cells, localize to the nucleus of cells, and interact with DNA. We postulate that the modification of native cellular proteins as noncovalent dispersing agents to provide specific transport will open new possibilities to utilize both SWCNT and protein properties for multifunctional subcellular targeting applications. Specifically, nuclear targeting could allow delivery of anticancer therapies, genetic treatments, or DNA to the nucleus.

Molecular Events in Lamin B1 Homopolymerization: A Biophysical Characterization.

Lamin B1 is one of the major constituents of the nuclear lamina, a filamentous network underlying the nucleoplasmic side of the inner nuclear membrane. Homopolymerization of lamin B1, coupled to the homotypic and heterotypic association of other lamin types, is central to building the higher order network pattern inside the nucleus. This in turn maintains the mechanical and functional integrity of the lamina. We have characterized the molecular basis of the self-association of lamin B1 using spectroscopic and calorimetric methods. We report that concentration dependent lamin B1 oligomerization involves significant alterations in secondary and tertiary structures of the protein resulting in fairly observable compaction in size. Comparison of the energetics of the homotypic association of lamin B1 with that of lamin A reported earlier led to the finding that lamin A oligomers had higher thermodynamic stability. This leads us to conjecture that lamin B1 has less stress bearing ability compared to lamin A.

The tail domain of lamin B1 is more strongly modulated by divalent cations than lamin A.

The nucleoskeleton contains mainly nuclear intermediate filaments made of lamin proteins. Lamins provide nuclear structure and also play a role in various nuclear processes including signal transduction, transcription regulation and chromatin organization. The disparate functions of lamins may be related to the intrinsic disorder of the tail domains, which allows for altered and promiscuous binding. Here, we show modulation of lamin tail domain structures in the presence of divalent cations. We utilize changes in fluorescence of tryptophan residues within the Ig-fold flanked by disordered regions to experimentally measure protein thermodynamics. Using spectroscopy experiments and molecular dynamics simulations, we show that the tail domain of lamin B1 shows enhanced association with both Ca(2+) and Mg(2+) compared to the tail domain of lamin A. Binding curves show a similar KD between protein and ion (250-300 µM) for both proteins with both ions. However, we observe a maximum binding of ions to lamin B1 tail domain which is 2-3 times greater than that for lamin A tail domain by both experiment and simulation. Using simulations, we show that divalent ion association alters the Ig-fold by pinning flanking regions. With cells in culture, we observe altered lamin B1 organization in the presence of excess Mg(2+) more so than for lamin A. We suggest that the differential sensitivity to divalent cations contributes to the vastly different functionalities and binding of the 2 proteins.

Nuclear lamin stiffness is a barrier to 3D migration, but softness can limit survival.

Cell migration through solid tissue often involves large contortions of the nucleus, but biological significance is largely unclear. The nucleoskeletal protein lamin-A varies both within and between cell types and was shown here to contribute to cell sorting and survival in migration through constraining micropores. Lamin-A proved rate-limiting in 3D migration of diverse human cells that ranged from glioma and adenocarcinoma lines to primary mesenchymal stem cells (MSCs). Stoichiometry of A- to B-type lamins established an activation barrier, with high lamin-A:B producing extruded nuclear shapes after migration. Because the juxtaposed A and B polymer assemblies respectively conferred viscous and elastic stiffness to the nucleus, subpopulations with different A:B levels sorted in 3D migration. However, net migration was also biphasic in lamin-A, as wild-type lamin-A levels protected against stress-induced death, whereas deep knockdown caused broad defects in stress resistance. In vivo xenografts proved consistent with A:B-based cell sorting, and intermediate A:B-enhanced tumor growth. Lamins thus impede 3D migration but also promote survival against migration-induced stresses.

A Bayesian changepoint analysis of ChIP-Seq data of Lamin B.

The spatial organisation of the chromosomes in the nucleus is influenced by chromatin regions binding to the nucleic lamina, i.e., the inner part of the nucleic envelope. To investigate the architecture of chromosomes in the interphase nucleus, it is thus of high interest to detect such chromatin segments. This goal can be achieved by considering the fibrous protein Lamin B as a surrogate, since regions of high abundance of Lamin B can indicate chromatin segments attached to the nucleic lamina. We analyse ChIP-Seq (Chromatin-Immunoprecipitation Sequencing) data from an experiment that is designed to record Lamin B abundance. We introduce a Bayesian segmentation procedure in which a Markov Chain Monte Carlo (MCMC) algorithm is used for inference about the desired segmentation. The procedure is based on a Bayesian hierarchical model. Inference allows the distinction between regions of high versus low levels of Lamin B, and therefore, gives an insight into the binding of the chromatin to the nucleic envelope. An implementation of this approach is available in the statistical software environment R. This article is part of a special issue entitled: Computational proteomics in the post-identification era. Guest Editors: Martin Eisenacher and Christian Stephan.