Beyond A and B Compartments: how major nuclear locales define nuclear genome organization and function

141st ENMC International Workshop Inaugural Meeting of the EURO-Laminopathies Project Nuclear Envelope-linked Rare Human Diseases: From Molecular Pathophysiology towards Clinical Applications 10–12 March 2006, Naarden, The Netherlands

Lamin A/C (LMNA), a key component of the nuclear envelope, is essential for maintaining nuclear integrity and genome organization [W. Xie et al., Curr. Biol. 26, 2651-2658 (2016)]. While LMNA dysregulation has been implicated in genomic instability across cancer and aging, the underlying mechanisms remain poorly understood [S. Graziano et al., Nucleus 9, 258-275 (2018)]. Here, we define a mechanistic role for LMNA in preserving genome stability in small-cell lung cancer (SCLC), a malignancy marked by extreme genomic instability [N. Takahashi et al., Cancer Res. Commun. 2, 503-517 (2022)]. LMNA depletion promotes R-loop accumulation, transcription-replication conflicts, replication stress, DNA breaks, and micronuclei formation. Mechanistically, LMNA deficiency disrupts nuclear pore complex organization, specifically reducing phenylalanine-glycine (FG)-nucleoporin incorporation, resulting in impaired RNA export and nuclear retention of RNA. LMNA expression is repressed by EZH2 and reexpressed during SCLC differentiation from neuroendocrine (NE) to non-NE states, and low LMNA levels correlate with poor clinical outcomes. These findings establish LMNA as a key regulator of nuclear transport and genome integrity, linking nuclear architecture to SCLC progression and therapeutic vulnerability.

Rationale: Mechanical forces are transduced to nuclear responses via the linkers of the nucleoskeleton and cytoskeleton (LINC) complex, which couples the cytoskeleton to the nuclear lamina and associated chromatin. While disruption of the LINC complex can cause cardiomyopathy, the relevant interactions that bridge the nucleoskeleton to cytoskeleton are poorly understood in the cardiomyocyte, where cytoskeletal organization is unique. Furthermore, while microtubules and desmin intermediate filaments associate closely with cardiomyocyte nuclei, the importance of these interactions is unknown. Objective: Here, we sought to determine how cytoskeletal interactions with the LINC complex regulate nuclear homeostasis in the cardiomyocyte. Methods and Results: To this end, we acutely disrupted the LINC complex, microtubules, actin, and intermediate filaments and assessed the consequences on nuclear morphology and genome organization in rat ventricular cardiomyocytes via a combination of super-resolution imaging, biophysical, and genomic approaches. We find that a balance of dynamic microtubules and desmin intermediate filaments is required to maintain nuclear shape and the fidelity of the nuclear envelope and lamina. Upon depletion of desmin (or nesprin [nuclear envelope spectrin repeat protein]-3, its binding partner in the LINC complex), polymerizing microtubules collapse the nucleus and drive infolding of the nuclear membrane. This results in DNA damage, a loss of genome organization, and broad transcriptional changes. The collapse in nuclear integrity is concomitant with compromised contractile function and may contribute to the pathophysiological changes observed in desmin-related myopathies. Conclusions: Disrupting the tethering of desmin to the nucleus results in a loss of nuclear homeostasis and rapid alterations to cardiomyocyte function. Our data suggest that a balance of forces imposed by intermediate filaments and microtubules is required to maintain nuclear structure and genome organization in the cardiomyocyte.

Lamins are nuclear intermediate filament proteins. They provide mechanical stability, organize chromatin and regulate transcription, replication, nuclear assembly and nuclear positioning. Recent studies provide new insights into the role of lamins in development, differentiation and tissue response to mechanical, reactive oxygen species and thermal stresses. These studies also propose the existence of separate filament networks for A- and B-type lamins and identify new roles for the different networks. Furthermore, they show changes in lamin composition in different cell types, propose explanations for the more than 14 distinct human diseases caused by lamin A and lamin C mutations and propose a role for lamin B1 in these diseases.

Lamin A/C (LMNA), a key component of the nuclear envelope, is essential for maintaining nuclear integrity and genome organization [1]. While LMNA dysregulation has been implicated in genomic instability across cancer and aging, the underlying mechanisms remain poorly understood [2]. Here, we investigate LMNA’s role in small-cell lung cancer (SCLC), a highly aggressive malignancy characterized by extreme genomic instability [3, 4]. We demonstrate that LMNA depletion promotes R-loop accumulation, transcription-replication conflicts, replication stress, DNA breaks, and micronuclei formation. Mechanistically, LMNA loss disrupts nuclear pore complex distribution, reducing phenylalanine-glycine (FG)-nucleoporin incorporation and impairing RNA export efficiency. Furthermore, we show that LMNA expression is epigenetically repressed by EZH2 during SCLC differentiation from neuroendocrine (NE) to non-NE states. Clinically, low LMNA levels correlate with significantly worse survival in SCLC patients. These findings uncover a novel role for LMNA in safeguarding genome integrity and shaping tumor heterogeneity, with broad implications for cancer and aging.

The nucleus, a highly organized and dynamic organelle, plays a crucial role in regulating cellular processes. During cell differentiation, profound changes occur in gene expression, chromatin organization, and nuclear morphology. This review explores the intricate relationship between nuclear architecture and cellular function, focusing on the roles of the nuclear lamina, nuclear pore complexes (NPCs), sub-nuclear bodies, and the nuclear scaffold. These components collectively maintain nuclear integrity, organize chromatin, and interact with key regulatory factors. The dynamic remodeling of chromatin, its interactions with nuclear structures, and epigenetic modifications work in concert to modulate gene accessibility and ensure precise spatiotemporal control of gene expression. The nuclear lamina stabilizes nuclear shape and is associated with inactive chromatin regions, while NPCs facilitate selective transport. Sub-nuclear bodies contribute to genome organization and gene regulation, often by influencing RNA processing. The nuclear scaffold provides structural support, impacting 3D genome organization, which is crucial for proper gene expression during differentiation. This review underscores the significance of nuclear architecture in regulating gene expression and guiding cell differentiation. Further investigation into nuclear structure and 3D genome organization will deepen our understanding of the mechanisms governing cell fate determination.

Karyotype engineering reveals spatio-temporal control of replication firing and gene contacts

Nuclear architecture is the organization of the genome within a cell nucleus with respect to different nuclear landmarks such as the nuclear lamina, nuclear matrix or nucleoli. Recently, nuclear architecture has emerged as a major regulator of gene expression in mammalian cells. However, studies connecting nuclear architecture with gene expression are largely population-averaged and do not report on the heterogeneity in genome organization or gene expression within a population. In this report we present a method for combining 3D DNA fluorescence in situ hybridization (FISH) with single-molecule RNA FISH (smFISH) and immunofluorescence to study nuclear architecture-dependent gene regulation on a cell-by-cell basis. We further combine our method with imaging-based cell cycle staging to correlate nuclear architecture with gene expression across the cell cycle. We present this in the context of the cyclin-A2 (CCNA2) gene, which has known cell cycle-dependent expression. We show that, across the cell cycle, the expression of a CCNA2 gene copy is stochastic and depends neither on its sub-nuclear position - which usually lies close to nuclear lamina - nor on the expression from other copies of the gene.This article has an associated First Person interview with the first author of the paper.

The nucleus is a complex organelle that hosts the genome and is essential for vital processes like DNA replication, DNA repair, transcription, and splicing. The genome is non-randomly organized in the three-dimensional space of the nucleus. This functional sub-compartmentalization was thought to be organized on the framework of nuclear matrix (NuMat), a non-chromatin scaffold that functions as a substratum for various molecular processes of the nucleus. More recently, nuclear bodies or membrane-less subcompartments of the nucleus are thought to arise due to phase separation of chromatin, RNA, and proteins. The nuclear architecture is an amalgamation of the relative organization of chromatin, epigenetic landscape, the nuclear bodies, and the nucleoskeleton in the three-dimensional space of the nucleus. During mitosis, the nucleus undergoes drastic changes in morphology to the degree that it ceases to exist as such; various nuclear components, including the envelope that defines the nucleus, disintegrate, and the chromatin acquires mitosis-specific epigenetic marks and condenses to form chromosome. Upon mitotic exit, chromosomes are decondensed, re-establish hierarchical genome organization, and regain epigenetic and transcriptional status similar to that of the mother cell. How this mitotic memory is inherited during cell division remains a puzzle. NuMat components that are a part of the mitotic chromosome in the form of mitotic chromosome scaffold (MiCS) could potentially be the seeds that guide the relative re-establishment of the epigenome, chromosome territories, and the nuclear bodies. Here, we synthesize the advances towards understanding cellular memory of nuclear architecture across mitosis and propose a hypothesis that a subset of NuMat proteome essential for nucleation of various nuclear bodies are retained in MiCS to serve as seeds of mitotic memory, thus ensuring the daughter cells re-establish the complex status of nuclear architecture similar to that of the mother cells, thereby maintaining the pre-mitotic transcriptional status.

We investigated the role of LMNA in adipose tissue by developing a novel mouse model of lipodystrophy. Transgenic mice were generated that express the LMNA mutation that causes familial partial lipodystrophy of the Dunnigan type (FPLD2). The phenotype observed in FPLD-transgenic mice resembles many of the features of human FPLD2, including lack of fat accumulation, insulin resistance, and enlarged, fatty liver. Similar to the human disease, FPLD-transgenic mice appear to develop normally, but after several weeks they are unable to accumulate fat to the same extent as their wild-type littermates. One poorly understood aspect of lipodystrophies is the mechanism of fat loss. To this end, we have examined the effects of the FPLD2 mutation on fat cell function. Contrary to the current literature, which suggests FPLD2 results in a loss of fat, we found that the key mechanism contributing to the lack of fat accumulation involves not a loss, but an apparent inability of the adipose tissue to renew itself. Specifically, preadipocytes are unable to differentiate into mature and fully functional adipocytes. These findings provide insights not only for the treatment of lipodystrophies, but also for the study of adipogenesis, obesity, and insulin resistance.

Nuclear bodies: multifunctional companions of the genome

1. Early Embryonic Cell Fate Decisions in the Mouse Yojiro Yamanaka and Amy Ralston Abstract Introduction Lineage Establishment and the Pre?stem Cell Program: Formation of the Blastocyst Lineage Maintenance and the Stem Cell Program: Beyond the Blastocyst The Second Lineage Decision: Subdividing the ICM Cell Signaling Regulates PE/EPI Specification Establishment and Modulation of Pluripotency in the EPI Lineage Conclusion 2. Nuclear Architecture in Stem Cells Kelly J. Morris, Mita Chotalia and Ana Pombo Abstract Introduction Functional Compartmentalization of the ES Cell Nucleus Stem Cell Features of Other Nucleoplasmic Subcompartments Chromatin Features Characteristic of ES Cell Nuclei Conclusion 3. Epigenetic Regulation of Pluripotency Eleni M. Tomazou and Alexander Meissner Abstract Introduction Epigenetic Modifications The Epigenome of ES Cells Conclusion 4. Autosomal Lyonization of Replication Domains During Early Mammalian Development Ichiro Hiratani and David M. Gilbert Abstract Introduction Replication Timing Program: An Elusive Measure of Genome Organization An Evolutionarily Conserved Epigenetic Fingerprint Replication Timing as a Quantitative Index of 3?Dimensional Genome Organization Replication Timing Reveals An Epigenetic Transition: Autosomal Lyonization at the Epiblast Stage Replication Timing and Cellular Reprogramming: Further Support for Autosomal Lyonization Maintenance and Alteration of Replication Timing Program and Its Potential Roles Conclusion 5. PRESERVATION OF GENOMIC INTEGRITY IN MOUSE EMBRYONIC STEM CELLS Peter J. Stambrook and Elisia D. Tichy Abstract Introduction and Historical Perspective Mutation Frequencies in Somatic Cells Protection of the Mouse ES Cell Genome Conclusion 6. Transcriptional Regulation in Embryonic Stem Cells Jian?Chien Dominic Heng and Huck?Hui Ng Abstract Introduction Embryonic Stem Cells as a Model to Study Transcriptional Regulation Transcription Factors Governing ESC Pluripotency TranscriptionalRegulatory Network Technologies for Dissecting the Transcriptional Regulatory Network The Core Transcriptional Regulatory Network: Oct4, Sox2 and Nanog Expanded Transcriptional Regulatory Network Enhanceosomes: Transcription Factor Complex Integration of Signaling Pathways to Transcriptional Network Interface Between Transcriptional and Epigenetic Regulation Conclusion 7. ALTERNATIVE SPLICING IN STEM CELL SELF?RENEWAL AND DIFERENTIATION David A. Nelles and Gene W. Yeo Abstract Introduction Introduction to Alternative Splicing Alternative Splicing of Genes Implicated in Stemness and Differentiation Genome?Wide Methods to Identify and Detect Alternative Splicing Events Regulation of Alternative Splicing by RNA Binding Proteins Conclusion and Perspectives 8. MicroRNA Regulation of Embryonic Stem Cell Self?Renewal and Differentiation Collin Melton and Robert Blelloch Abstract Introduction: The Self?Renewal Program Embryonic Stem Cells miRNA Biogenesis and Function ESCC miRNAs Promote Self?Renewal miRNAs Induced during ESC Differentiation Suppress the Self?Renewal Program Regulatory Networks Controlling miRNA Expression miRNAs Can Promote or Inhibit Dedifferentiation to iPS Cells miRNAs in Somatic Stem Cells miRNAs in Cancer Cells Conclusion 9. TELOMERES AND TELOMERASE IN ADULT STEM CELLS and PLURIPOTENT EMBRYONIC STEM CELLS Rosa M. Marion and Maria A. Blasco Abstract Introduction Regulation of Telomeres and Telomerase Role of Telomeres and Telomerase in Adult SC Compartments Telomeres and Telomerase Regulation During Reprogramming by SCNT Telomeres and Telomerase Regulation During iPS Cell Generation Telomerase Activation is Essential for the Good Quality of the Resulting iPS Cells Regulation of Telomere Reprogramming Conclusion 10. X Chromosome Inactivation and Embryonic Stem Cells Tahsin Stefan Barakat and Joost Gribnau Abstract Introduction Cis Acting Factors in XCI Trans Acting Factors in XCI Counting and Choice Silencing and Maintenance of

Comparative genomics has recently provided unprecedented insights into the biology and evolution of the fungal lineage. In the postgenomics era, a major research interest focuses now on detailing the functions of fungal genomes, i.e. how genomic information manifests into complex phenotypes. Emerging evidence across diverse eukaryotes has revealed that the organization of DNA within the nucleus is critically important. Here, we discuss the current knowledge on the fungal genome organization, from the association of chromosomes within the nucleus to topological structures at individual genes and the genetic factors required for this hierarchical organization. Chromosome conformation capture followed by high-throughput sequencing (Hi-C) has elucidated how fungal genomes are globally organized in Rabl configuration, in which centromere or telomere bundles are associated with opposite faces of the nuclear envelope. Further, fungal genomes are regionally organized into topologically associated domain-like (TAD-like) chromatin structures. We discuss how chromatin organization impacts the proper function of DNA-templated processes across the fungal genome. Nevertheless, this view is limited to a few fungal taxa given the paucity of fungal Hi-C experiments. We advocate for exploring genome organization across diverse fungal lineages to ensure the future understanding of the impact of nuclear organization on fungal genome function.

Alterations to mechanical forces have been long-appreciated to be sufficient to drive cardiac pathophysiological remodeling; however, the mechanism by which cardiomyocytes sense and transduce mechanical stressors remains poorly understood. In part, mechanical forces can be transduced to transcriptional responses by direct strain transmission to the nucleus via the Linkers of the Nucleo- and Cytoskeleton (LINC) complex, which couples the cytoskeleton to the nuclear lamina and DNA. While LINC complex mutations are known to cause cardiomyopathy, cytoskeletal-LINC interactions are understudied in the cardiomyocyte. To probe these interactions, we acutely disrupted the LINC complex as well as microtubules, actin, and intermediate filaments and assessed the consequences on baseline nuclear homeostasis in the cardiomyocyte. Our result show that a balance of microtubules and desmin intermediate filaments is required to maintain nuclear shape and the fidelity of the nuclear envelope and lamina. Upon acute depletion of desmin (or Nesprin-3, its binding partner in the LINC complex), microtubules drive infolding of the nuclear membrane. This results in DNA damage, a loss of genome organization, and broad transcriptional changes. Desmin knockdown also causes compromised excitation-contraction coupling and contractile function. Together, our data suggest that a balance of forces imposed by intermediate filaments and microtubules is required to maintain nuclear structure and genome organization in the cardiomyocyte, and that this process is important for maintaining proper myocyte health and function.

The nuclear genome is spatially organized into a three-dimensional (3D) architecture by physical association of large chromosomal domains with subnuclear compartments including the nuclear lamina at the radial periphery and nuclear speckles within the nucleoplasm1–5. However, how spatial genome architecture regulates human brain development has been overlooked owing to technical limitations. Here, we generate high-resolution maps of genomic interactions with the lamina and speckles in cells of the neurogenic lineage isolated from midgestational human cortex, uncovering an intimate association between subnuclear genome compartmentalization, chromatin state and transcription. During cortical neurogenesis, spatial genome organization is extensively remodeled, relocating hundreds of neuronal genes from the lamina to speckles including key neurodevelopmental genes bivalent for H3K27me3 and H3K4me3. At the lamina, bivalent genes have exceptionally low expression, and relocation to speckles enhances resolution of bivalent chromatin to H3K4me3 and increases transcription >7-fold. We further demonstrate that proximity to the nuclear periphery – not the presence of H3K27me3 – is the dominant factor in maintaining the lowly expressed, poised state of bivalent genes embedded in the lamina. In addition to uncovering a critical role of subnuclear genome compartmentalization in neurogenic transcriptional regulation, our results establish a new paradigm in which knowing the spatial location of a gene is necessary to understanding its epigenomic regulation.