Kaushik Sengupta

Lamins are nuclear intermediate filament proteins that maintain nuclear architecture through interactions with the chromatin. The A- and B-type lamins tether the genome at the peripheral lamina underlying the inner nuclear membrane in the form of heterochromatic lamina-associated domains (LADs). However, LADs associated with A-type lamins are confined not only to lamina but also to the nuclear core, thereby pointing to their distinct and multifarious roles in chromatin organisation and regulation. Previous studies involving lamin B1 depletion depicted the detachment of LADs from the nuclear periphery, accompanied by alteration of chromatin distribution while preserving the topologically associating domains (TADs) in structurally intact form. In this piece of work, we have shown, for the first time in HGSOC, the effects of lamin A/C knockdown on chromatin organisation, which was characterised by significant detethering of gene-poor chromatin from the periphery along with active (A) to inactive (B) compartment switching. This was associated with a rewiring of oncogenic super-enhancer elements corroborated by the differential gene expression profile. Overall analysis tipped the scale in favour of reduced cellular proliferation upon lamin A/C knockdown. This finding was strengthened by the observed proliferative potential of spheroids ex vivo and tumours in a mouse xenograft model.

Lamins are intermediate filament proteins of the nucleus that are present in nuclear lamina as well as nucleoplasm. They play diverse roles in maintaining the structure and rigidity of the nucleus as well as nuclear homeostasis. Lamins are of two main types—A and B. B-type lamins are expressed from the embryonic stage, whereas A-type lamins are expressed during cell differentiation. Both A- and B-type lamins form distinct but interacting networks that contribute to differential chromosome tethering and distribution within the nucleus. A- and B-type lamins maintain the euchromatin-to-heterochromatin ratio in health and disease. Interestingly, lamin A/B itself varies largely and distinctly in different types of cancer. Likewise, the lamina-associated domains of the chromatin network get significantly altered in the process of carcinogenesis. We have discussed here the differential expression of lamin proteins in different cancers, contributing to distinct genome organization, ultimately precipitating into diverse neoplastic transformation.

The filamentous nuclear lamina network is predominantly made up of lamin proteins, which play pivotal roles in maintaining nuclear architecture, chromatin structure, mechanotransduction and various other nuclear processes. Among the lamin proteins, A type is the principal mechanical component of the nucleus and controls gene expression via direct interactions with chromatin and chromatin modulator proteins. Mutations in the LMNA gene cause a plethora of diseases termed laminopathies, including various cardiac and muscular ailments, by disrupting nuclear integrity and mechanotransduction signalling. These mutations also alter chromatin organisation and epigenetic landscape, tantamount to compromised cellular homeostasis. In this chapter, we elaborate on lamin A’s molecular structure, assembly dynamics and its role in nuclear mechanotransduction and chromatin maintenance. Additionally, we highlighted the pathological consequences of lamin A dysfunction and discussed emerging approaches aimed at rationalising the cellular- and tissue-specific effects during laminopathies.

Lamins are intermediate filaments constituting the nuclear lamina which maintains the structural integrity of the nucleus and play a key role in the spatiotemporal genome organization. Mutations in lamin A/C have been associated with a plethora of diseases including dilated cardiomyopathy. In this study, we focused on lamin A mutants E161K and K97E which are widely reported in patients afflicted with dilated cardiomyopathy. We established that these mutations cause large scale disruption of the peripheral lamina and consequent heterochromatin (HC) organization, along with the formation of lamin A aggregates inside the nucleoplasm. Using coarse-grained polymer simulations, we explored the interplay between lamin, nuclear membrane, and chromatin showing that disruptions in these interactions can reproduce the experimental phenotypes. Simulations also predict altered positioning depending on HC content and altered lamin dynamics in E161K and K97E mutants. The predictions from the simulations were verified using 3D FISH to delineate the reorganization of chromosome territories in these mutants. Based on the disengagement of lamin A foci from HC, we subsequently investigated the dynamic properties of the mutant aggregates using advanced imaging techniques. Through this, we report that mutant lamin aggregates show internal rearrangement and external exchange at the mesoscopic-level reminiscent of liquid–liquid phase separation.

Lamins are nuclear intermediate filaments constituting the nuclear lamina which maintains the structural integrity of the nucleus and play a key role in the spatial organisation of the genome. Mutations in the lamin protein have been associated with diverse diseases collectively known as laminopathies. In this study, we focused on two lamin A mutants - E161K and K97E - associated with dilated cardiomyopathy (DCM). Through confocal imaging, we established that these mutations cause large scale disruption of the peripheral lamin and consequent heterochromatin organisation, along with the formation of lamin aggregates inside the nucleoplasm. Using coarse-grained polymer simulations, we uncovered the role of lamin-lamin, lamin-membrane and lamin-chromatin interactions in maintaining wild-type lamin and chromatin organisation and showed that disruptions in these interactions can reproduce the experimental observations in the lamin mutants. These predictions were verified using 3D-FISH experiments to quantify the reorganisation of chromosome territories in these mutants. Using advanced imaging methods, we characterised the dynamical properties of the lamin aggregates in the mutants to show for the first time a liquid-like state of the lamin aggregates through a liquid-liquid phase separation. The altered lamin and chromatin interactions in the mutants thus manifest as liquid-like aggregates in the nucleoplasm leading to disruption of the spatial organisation of the genome in these laminopathy-associated mutants.

Background Information: Lamins are type V intermediate filament proteins underlying the inner nuclear membrane which provide structural rigidity to the nucleus, tether the chromosomes, maintain nuclear homeostasis, and remain dynamically associated with developmentally regulated regions of the genome. A large number of mutations particularly in the LMNA gene encoding lamin A/C results in a wide array of human diseases, collectively termed as laminopathies. Dilated Cardiomyopathy (DCM) is one such laminopathic cardiovascular disease which is associated with systolic dysfunction of left or both ventricles leading to cardiac arrhythmia which ultimately culminates into myocardial infarction.Results: In this work, we have unraveled the epigenetic landscape to address the regulation of gene expression in mouse myoblast cell line in the context of the missense mutation LMNA 289A<G (Lys97Glu) that is found in DCM‐afflicted patient with severe symptoms. Significant changes in H3‐specific epigenetic modifications indicated a dysregulation in transcription machinery which was investigated by RNA sequencing analysis. The major pathways involved in IL‐17 signaling, cellular response to interferon‐beta and gamma, cytokine production, and related pathways are found to be downregulated. Analysis of the promoter sequences of the genes in the abovementioned pathways led us to the master regulator NF‐κB and its regulatory network.Conclusions: We report here for the first time that there is a significant downregulation of the NF‐κB pathway, which has been implicated in cardio‐protection elsewhere.Significance: This provides a new pathophysiological explanation that correlates an LMNA mutation and dilated cardiomyopathy.

Extensive research for the last two decades has significantly contributed to understanding the roles of lamins in the maintenance of nuclear architecture and genome organization which is drastically modified in neoplasia. It must be emphasized that alteration in lamin A/C expression and distribution is a consistent event during tumorigenesis of almost all tissues of human bodies. One of the important signatures of a cancer cell is its inability to repair DNA damage which befalls several genomic events that transform the cells to be sensitive to chemotherapeutic agents. This genomic and chromosomal instability is the most common feature found in cases of high-grade ovarian serous carcinoma. Here, we report elevated levels of lamins in OVCAR3 cells (high-grade ovarian serous carcinoma cell line) in comparison to IOSE (immortalised ovarian surface epithelial cells) and, consequently, altered damage repair machinery in OVCAR3. We have analysed the changes in global gene expression as a sequel to DNA damage induced by etoposide in ovarian carcinoma where lamin A is particularly elevated in expression and reported some differentially expressed genes associated with pathways conferring cellular proliferation and chemoresistance. We hereby establish the role of elevated lamin A in neoplastic transformation in the context of high-grade ovarian serous cancer through a combination of HR and NHEJ mechanisms.

Changes in the microdynamics landscape of Ig fold R453W modulate the mobility and assembly of full length lamin A.