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.

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.

A Glimpse into Chromatin Organization and Nuclear Lamina Contribution in Neuronal Differentiation.

During embryonic development, stem cells undergo the differentiation process so that they can specialize for different functions within the organism. Complex programs of gene transcription are crucial for this process to happen. Epigenetic modifications and the architecture of chromatin in the nucleus, through the formation of specific regions of active as well as inactive chromatin, allow the coordinated regulation of the genes for each cell fate. In this mini-review, we discuss the current knowledge regarding the regulation of three-dimensional chromatin structure during neuronal differentiation. We also focus on the role the nuclear lamina plays in neurogenesis to ensure the tethering of the chromatin to the nuclear envelope.

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.

Gene Regulatory Interactions at Lamina-Associated Domains.

The nuclear lamina provides a repressive chromatin environment at the nuclear periphery. However, whereas most genes in lamina-associated domains (LADs) are inactive, over ten percent reside in local euchromatic contexts and are expressed. How these genes are regulated and whether they are able to interact with regulatory elements remain unclear. Here, we integrate publicly available enhancer-capture Hi-C data with our own chromatin state and transcriptomic datasets to show that inferred enhancers of active genes in LADs are able to form connections with other enhancers within LADs and outside LADs. Fluorescence in situ hybridization analyses show proximity changes between differentially expressed genes in LADs and distant enhancers upon the induction of adipogenic differentiation. We also provide evidence of involvement of lamin A/C, but not lamin B1, in repressing genes at the border of an in-LAD active region within a topological domain. Our data favor a model where the spatial topology of chromatin at the nuclear lamina is compatible with gene expression in this dynamic nuclear compartment.

An atlas of lamina-associated chromatin across twelve human cell types reveals an intermediate chromatin subtype.

BACKGROUND: Association of chromatin with lamin proteins at the nuclear periphery has emerged as a potential mechanism to coordinate cell type-specific gene expression and maintain cellular identity via gene silencing. Unlike many histone modifications and chromatin-associated proteins, lamina-associated domains (LADs) are mapped genome-wide in relatively few genetically normal human cell types, which limits our understanding of the role peripheral chromatin plays in development and disease. RESULTS: To address this gap, we map LAMIN B1 occupancy across twelve human cell types encompassing pluripotent stem cells, intermediate progenitors, and differentiated cells from all three germ layers. Integrative analyses of this atlas with gene expression and repressive histone modification maps reveal that lamina-associated chromatin in all twelve cell types is organized into at least two subtypes defined by differences in LAMIN B1 occupancy, gene expression, chromatin accessibility, transposable elements, replication timing, and radial positioning. Imaging of fluorescently labeled DNA in single cells validates these subtypes and shows radial positioning of LADs with higher LAMIN B1 occupancy and heterochromatic histone modifications primarily embedded within the lamina. In contrast, the second subtype of lamina-associated chromatin is relatively gene dense, accessible, dynamic across development, and positioned adjacent to the lamina. Most genes gain or lose LAMIN B1 occupancy consistent with cell types along developmental trajectories; however, we also identify examples where the enhancer, but not the gene body and promoter, changes LAD state. CONCLUSIONS: Altogether, this atlas represents the largest resource to date for peripheral chromatin organization studies and reveals an intermediate chromatin subtype.

Chromatin Structure from Development to Ageing.

Nuclear structure influences genome architecture, which contributes to determine patterns of gene expression. Global changes in chromatin dynamics are essential during development and differentiation, and are one of the hallmarks of ageing. This chapter describes the molecular dynamics of chromatin structure that occur during development and ageing. In the first part, we introduce general information about the nuclear lamina, the chromatin structure, and the 3D organization of the genome. Next, we detail the molecular hallmarks found during development and ageing, including the role of DNA and histone modifications, 3D genome dynamics, and changes in the nuclear lamina. Within the chapter we discuss the implications that genome structure has on the mechanisms that drive development and ageing, and the physiological consequences when these mechanisms fail.

Local euchromatin enrichment in lamina-associated domains anticipates their repositioning in the adipogenic lineage.

BACKGROUND: Interactions of chromatin with the nuclear lamina via lamina-associated domains (LADs) confer structural stability to the genome. The dynamics of positioning of LADs during differentiation, and how LADs impinge on developmental gene expression, remains, however, elusive. RESULTS: We examined changes in the association of lamin B1 with the genome in the first 72 h of differentiation of adipose stem cells into adipocytes. We demonstrate a repositioning of entire stand-alone LADs and of LAD edges as a prominent nuclear structural feature of early adipogenesis. Whereas adipogenic genes are released from LADs, LADs sequester downregulated or repressed genes irrelevant for the adipose lineage. However, LAD repositioning only partly concurs with gene expression changes. Differentially expressed genes in LADs, including LADs conserved throughout differentiation, reside in local euchromatic and lamin-depleted sub-domains. In these sub-domains, pre-differentiation histone modification profiles correlate with the LAD versus inter-LAD outcome of these genes during adipogenic commitment. Lastly, we link differentially expressed genes in LADs to short-range enhancers which overall co-partition with these genes in LADs versus inter-LADs during differentiation. CONCLUSIONS: We conclude that LADs are predictable structural features of adipose nuclear architecture that restrain non-adipogenic genes in a repressive environment.

The nuclear lamina binds the EBV genome during latency and regulates viral gene expression.

The Epstein Barr virus (EBV) infects almost 95% of the population worldwide. While typically asymptomatic, EBV latent infection is associated with several malignancies of epithelial and lymphoid origin in immunocompromised individuals. In latently infected cells, the EBV genome persists as a chromatinized episome that expresses a limited set of viral genes in different patterns, referred to as latency types, which coincide with varying stages of infection and various malignancies. We have previously demonstrated that latency types correlate with differences in the composition and structure of the EBV episome. Several cellular factors, including the nuclear lamina, regulate chromatin composition and architecture. While the interaction of the viral genome with the nuclear lamina has been studied in the context of EBV lytic reactivation, the role of the nuclear lamina in controlling EBV latency has not been investigated. Here, we report that the nuclear lamina is an essential epigenetic regulator of the EBV episome. We observed that in B cells, EBV infection affects the composition of the nuclear lamina by inducing the expression of lamin A/C, but only in EBV+ cells expressing the Type III latency program. Using ChIP-Seq, we determined that lamin B1 and lamin A/C bind the EBV genome, and their binding correlates with deposition of the histone repressive mark H3K9me2. By RNA-Seq, we observed that knock-out of lamin A/C in B cells alters EBV gene expression. Our data indicate that the interaction between lamins and the EBV episome contributes to the epigenetic control of viral gene expression during latency, suggesting a restrictive function of the nuclear lamina as part of the host response against viral DNA entry into the nucleus.

CTCF supports preferentially short lamina-associated domains.

More than one third of the mammalian genome is in a close association with the nuclear lamina, thus these genomic regions were termed lamina-associated domains (LADs). This association is fundamental for many aspects of chromatin biology including transcription, replication, and DNA damage repair. LADs association with the nuclear envelope is thought to be dependent on two major mechanisms: The first mechanism is the interaction between nuclear membrane proteins such as LBR with heterochromatin modifications that are enriched in LADs chromatin. The second mechanism is based on proteins that bind the borders of the LADs and support the association of the LADs with the nuclear envelope. Two factors were suggested to support the second mechanism: CCCTC-binding factor (CTCF) and YY1 based on their enriched binding to LADs borders. However, this mechanism has not been proven yet at a whole genome level. Here, to test if CTCF supports the LADs landscape, we generated melanoma cells with a partial loss of function (pLoF) of CTCF by the CRISPR-Cas9 system and determined the LADs landscape by lamin B ChIP-seq analysis. We found that under regular growth conditions, CTCF pLoF led to modest changes in the LADs landscape that included an increase in the signal of 2% of the LADs and a decrease in the signal of 8% of the LADs. However, CTCF importance for the LADs landscape was much higher upon induction of a chromatin stress. We induced chromatin stress by inhibiting RNA polymerase II, an intervention that is known to alter chromatin compaction and supercoiling. Notably, only in CTCF pLoF cells, the chromatin stress led to the dissociation of 7% of the LADs from the lamina. The CTCF-dependent LADs had almost three times shorter median length than the non-affected LADs, were enriched in CTCF binding at their borders, and were higher in their facultative-status (cell-type specific). Thus, it appears that CTCF is a key factor in facilitating the association of short facultative LADs with the nuclear lamina upon chromatin stress.

PNET2 is a component of the plant nuclear lamina and is required for proper genome organization and activity.

The interaction between chromatin and the nuclear lamina (NL) is intrinsically important to the establishment of three-dimensional chromatin architecture and spatiotemporal regulation of gene expression. However, critical regulators involved in this process are poorly understood in plants. Here, we report that Arabidopsis PNET2 and its two homologs are bona fide inner nuclear membrane proteins and integral components of the NL. PNET2s physically interact with the plant nucleoskeleton and engage nucleosome-enriched chromatin at the nuclear periphery. Loss of all three PNET2s leads to severely disrupted growth and development, concomitant activation of abiotic and biotic stress responses, and ultimate lethality in Arabidopsis. The pent2 triple mutant also displays drastic transcriptome changes accompanied by a globally altered chromatin architecture revealed by HiC analysis. Our study identified PNET2 as an inner nuclear membrane (INM) component of the NL, which associates with chromatin and play a critical role in orchestrating gene expression and chromatin organization in plants.

Evolutionary conservation and cell type specificity of nuclear compartments in the brain.

In the cell nucleus, radial genome organization relative to the nuclear lamina exhibits both cell type-specific variability and evolutionarily conserved distribution. A recent study by Ahanger, Delgado et al. explores the in vivo spatial distribution of the genome in the brain and identifies conserved genomic localization across mammalian species that are correlated with gene expression states.

Aberrant nuclear lamina contributes to the malignancy of human gliomas.

Glioma is the most common type of tumor in the central nervous system, accounting for around 80% of all malignant brain tumors. Previous studies showed a significant association between nuclear morphology and the malignant progress of gliomas. By virtue of integrated proteomics and genomics analyses as well as experimental validations, we identify three nuclear lamin genes (LMNA, LMNB1, and LMNB2) that are significantly upregulated in glioma tissues compared with normal brain tissues. We show that elevated expressions of LMNB1, LMNB2, and LMNA in glioma cells are highly associated with the rapid progression of the disease and the knockdown of LMNB1, LMNB2, and LMNA dramatically suppresses glioma progression in both in vitro and in vivo mouse models. Moreover, the repression of glioma cell growth by lamin knockdown is mediated by the pRb-mediated G1-S inhibition. On the contrary, overexpression of lamins in normal human astrocytes dramatically induced nuclear morphological aberrations and accelerated cell growth. Together, our multi-omics-based analysis has revealed a previously unrecognized role of lamin genes in gliomagenesis, providing a strong support for the key link between aberrant tumor nuclear shape and the survival of glioma patients. Based on these findings, lamins are proposed to be potential oncogene targets for therapeutic treatments of brain tumors.

Checkpoint activation drives global gene expression changes in Drosophila nuclear lamina mutants.

The nuclear lamina (NL) lines the inner nuclear membrane. This extensive protein network organizes chromatin and contributes to the regulation of transcription, DNA replication, and repair. Lap2-emerin-MAN1 domain (LEM-D) proteins are key members of the NL, representing proteins that connect the NL to the genome through shared interactions with the chromatin-binding protein Barrier-to-Autointegration Factor (BAF). Functions of the LEM-D protein emerin and BAF are essential during Drosophila melanogaster oogenesis. Indeed, loss of either emerin or BAF blocks germ cell development and causes loss of germline stem cells, defects linked to the deformation of NL structure, and non-canonical activation of Checkpoint kinase 2 (Chk2). Here, we investigate the contributions of emerin and BAF to gene expression in the ovary. Profiling RNAs from emerin and baf mutant ovaries revealed that nearly all baf misregulated genes were shared with emerin mutants, defining a set of NL-regulated genes. Strikingly, loss of Chk2 restored the expression of most NL-regulated genes, identifying a large class of Chk2-dependent genes (CDGs). Nonetheless, some genes remained misexpressed upon Chk2 loss, identifying a smaller class of emerin-dependent genes (EDGs). Properties of EDGs suggest a shared role for emerin and BAF in the repression of developmental genes. Properties of CDGs demonstrate that Chk2 activation drives global misexpression of genes in the emerin and baf mutant backgrounds. Notably, CDGs were found upregulated in lamin-B mutant backgrounds. These observations predict that Chk2 activation might have a general role in gene expression changes found in NL-associated diseases, such as laminopathies.

The Nuclear Lamina.

Lamins interact with a host of nuclear membrane proteins, transcription factors, chromatin regulators, signaling molecules, splicing factors, and even chromatin itself to form a nuclear subcompartment, the nuclear lamina, that is involved in a variety of cellular processes such as the governance of nuclear integrity, nuclear positioning, mitosis, DNA repair, DNA replication, splicing, signaling, mechanotransduction and -sensation, transcriptional regulation, and genome organization. Lamins are the primary scaffold for this nuclear subcompartment, but interactions with lamin-associated peptides in the inner nuclear membrane are self-reinforcing and mutually required. Lamins also interact, directly and indirectly, with peripheral heterochromatin domains called lamina-associated domains (LADs) and help to regulate dynamic 3D genome organization and expression of developmentally regulated genes.

Inherent genomic properties underlie the epigenomic heterogeneity of human induced pluripotent stem cells.

Human induced pluripotent stem cells (hiPSCs) show variable differentiation potential due to their epigenomic heterogeneity, whose extent/attributes remain unclear, except for well-studied elements/chromosomes such as imprints and the X chromosomes. Here, we show that seven hiPSC lines with variable germline potential exhibit substantial epigenomic heterogeneity, despite their uniform transcriptomes. Nearly a quarter of autosomal regions bear potentially differential chromatin modifications, with promoters/CpG islands for H3K27me3/H2AK119ub1 and evolutionarily young retrotransposons for H3K4me3. We identify 145 large autosomal blocks (≥100 kb) with differential H3K9me3 enrichment, many of which are lamina-associated domains (LADs) in somatic but not in embryonic stem cells. A majority of these epigenomic heterogeneities are independent of genetic variations. We identify an X chromosome state with chromosome-wide H3K9me3 that stably prevents X chromosome erosion. Importantly, the germline potential of female hiPSCs correlates with X chromosome inactivation. We propose that inherent genomic properties, including CpG density, transposons, and LADs, engender epigenomic heterogeneity in hiPSCs.

Lamin C is required to establish genome organization after mitosis.

BACKGROUND: The dynamic 3D organization of the genome is central to gene regulation and development. The nuclear lamina influences genome organization through the tethering of lamina-associated domains (LADs) to the nuclear periphery. Evidence suggests that lamins A and C are the predominant lamins involved in the peripheral association of LADs, potentially serving different roles. RESULTS: Here, we examine chromosome architecture in mouse cells in which lamin A or lamin C are downregulated. We find that lamin C, and not lamin A, is required for the 3D organization of LADs and overall chromosome organization. Striking differences in localization are present as cells exit mitosis and persist through early G1 and are linked to differential phosphorylation. Whereas lamin A associates with the nascent nuclear envelope (NE) during telophase, lamin C remains in the interior, surrounding globular LAD aggregates enriched on euchromatic regions. Lamin C association with the NE is delayed until several hours into G1 and correlates temporally and spatially with the post-mitotic NE association of LADs. Post-mitotic LAD association with the NE, and global 3D genome organization, is perturbed only in cells depleted of lamin C, and not lamin A. CONCLUSIONS: Lamin C regulates LAD dynamics during exit from mitosis and is a key regulator of genome organization in mammalian cells. This reveals an unexpectedly central role for lamin C in genome organization, including inter-chromosomal LAD-LAD segregation and LAD scaffolding at the NE, raising intriguing questions about the individual and overlapping roles of lamin A/C in cellular function and disease.

Small-Molecule Therapeutic Perspectives for the Treatment of Progeria.

Hutchinson-Gilford progeria syndrome (HGPS), or progeria, is an extremely rare disorder that belongs to the class of laminopathies, diseases characterized by alterations in the genes that encode for the lamin proteins or for their associated interacting proteins. In particular, progeria is caused by a point mutation in the gene that codifies for the lamin A gene. This mutation ultimately leads to the biosynthesis of a mutated version of lamin A called progerin, which accumulates abnormally in the nuclear lamina. This accumulation elicits several alterations at the nuclear, cellular, and tissue levels that are phenotypically reflected in a systemic disorder with important alterations, mainly in the cardiovascular system, bones, skin, and overall growth, which results in premature death at an average age of 14.5 years. In 2020, lonafarnib became the first (and only) FDA approved drug for treating progeria. In this context, the present review focuses on the different therapeutic strategies currently under development, with special attention to the new small molecules described in recent years, which may represent the upcoming first-in-class drugs with new mechanisms of action endowed with effectiveness not only to treat but also to cure progeria.

A hub-and-spoke nuclear lamina architecture in trypanosomes.

The nuclear lamina supports many functions, including maintaining nuclear structure and gene expression control, and correct spatio-temporal assembly is vital to meet these activities. Recently, multiple lamina systems have been described that, despite independent evolutionary origins, share analogous functions. In trypanosomatids the two known lamina proteins, NUP-1 and NUP-2, have molecular masses of 450 and 170 kDa, respectively, which demands a distinct architecture from the ∼60 kDa lamin-based system of metazoa and other lineages. To uncover organizational principles for the trypanosome lamina we generated NUP-1 deletion mutants to identify domains and their arrangements responsible for oligomerization. We found that both the N- and C-termini act as interaction hubs, and that perturbation of these interactions impacts additional components of the lamina and nuclear envelope. Furthermore, the assembly of NUP-1 terminal domains suggests intrinsic organizational capacity. Remarkably, there is little impact on silencing of telomeric variant surface glycoprotein genes. We suggest that both terminal domains of NUP-1 have roles in assembling the trypanosome lamina and propose a novel architecture based on a hub-and-spoke configuration.