Nucleus

Nuclear blebs are herniations of the nucleus that occur in diseased nuclei that cause nuclear rupture leading to cellular dysfunction. Chromatin and lamins are two of the major structural components of the nucleus that maintain its shape and function, but their relative roles in nuclear blebbing remain elusive. Lamin B is reported to be lost in blebs by qualitative data while quantitative studies reveal a spectrum of lamin B levels in nuclear blebs dependent on perturbation and cell type. Chromatin has been reported to be decreased or de-compacted in nuclear blebs, but again the data are not conclusive. To determine the composition of nuclear blebs, we compared the immunofluorescence intensity of lamin B and DNA in the main nucleus body and nuclear bleb across cell types and perturbations. Lamin B nuclear bleb levels varied drastically across MEF wild type and chromatin or lamins perturbations, HCT116 lamin B1-GFP imaging, and human disease model cells of progeria and prostate cancer. However, DNA concentration was consistently decreased to about half that of the main nucleus body across all measured conditions. Using Partial Wave Spectroscopic (PWS) microscopy to measure chromatin density in the nuclear bleb vs body we find similar results that DNA is consistently less dense in nuclear blebs. Thus, our data spanning many different cell types and perturbations supports that decreased DNA is a better marker of a nuclear bleb than lamin B levels that vary widely.

Nuclear blebs are herniations of the nucleus that occur in many human conditions including aging, heart disease, muscular dystrophy, and many cancers. Nuclear blebbing causes nuclear rupture and cellular dysfunction. However, understanding the formation, stability, and identification of nuclear blebs remains an ongoing challenge. Our previous studies reveal that nuclear blebs are best hallmarked by decreased DNA density. To determine if chromatin decompaction underlies decreased DNA density in nuclear blebs, we investigated the histone composition of nuclear blebs across multiple cell lines. Time lapse and immunofluorescence imaging revealed that global histone H2B and H3 levels are decreased in the nuclear bleb relative to the nuclear body. Next, we imaged histone modification states of euchromatin and heterochromatin, which respectively track decompact and compact states of chromatin. Overall, we find that nuclear blebs display variable histone modification state across cell lines, as euchromatin does not consistently enrich nor is heterochromatin consistently depleted. Nuclear blebs did consistently show active RNA Pol II initiation is enriched relative to elongation. Thus, we find that the local histone modification state is not an essential component of nuclear blebs while transcription initiation enrichment over elongation is reproducible across cell lines and conditions. Summary statementWe measured histones and their modification states in nuclear blebs. We find that chromatin state is variable while transcription initiation is consistently enriched relative to elongation in nuclear blebs.

Abnormal nuclear morphology is a hallmark of human diseases, including cancers and age-related disorders. Previously, maintenance of nuclear morphology and integrity was thought to be solely dependent on a force balance between nuclear mechanical resistance and actin antagonism. However, our recent work revealed that inhibiting RNA polymerase II suppresses nuclear blebbing independent of altering force balance, but the mechanism remains unknown. Through removing cell culture media serum and then adding it back, we can decrease and then restore transcriptional activity. Decreasing transcriptional activity decreases nuclear bleb formation, stability, and rupture while returning transcriptional activity restores nuclear blebbing. These modulations of transcriptional activity did not alter nuclear or actin mechanics. The mean square displacement (MSD) of chromatin domains labeled via transfected Cy3-dNTPs revealed that transcription activity regulates chromatin motion. To determine if increasing chromatin motion is a mechanism to increase nuclear blebbing, we used an established RAD51 inhibitor BO2. We verified BO2 increases chromatin domain motion which resulted in increased nuclear blebbing. We reveal the mechanism by which transcriptional activity drives nuclear blebbing is through chromatin motion. Thus, two hallmarks of human disease are directly linked via transcriptional activity and abnormal nuclear shape. Statement of SignificanceO_LINuclear blebs are hallmarks of disease progression that cause dysfunction, but how they are formed remains unanswered. C_LIO_LIWe find that chromatin motion generated by transcriptional activity is essential for both nuclear bleb formation and stability. This was independent of changes in nuclear stiffness or actin antagonism. C_LIO_LIThis finding provides a key advancement in our understanding of nuclear bleb formation. Furthermore, it reveals transcriptional activity as a novel contributor to nuclear blebbing in addition to the paradigm of nuclear shape determined as a force balance between nuclear resistance and actin antagonism. C_LI

BackgroundDynamic chromatin behavior, which is related to chromatin accessibility, plays a critical role in various genome DNA functions such as RNA transcription and DNA replication/repair. Previous studies using highly synchronized cells showed that average local chromatin motion, captured by single-nucleosome imaging and tracking on a second time scale, remained almost constant throughout G1, S, and G2 phases in living human cells, although possible effects of prolonged drug treatments for cell-cycle synchronization could not be excluded. ResultsTo avoid possible effects of prolonged drug treatment, we combined single-nucleosome imaging with Fucci probes to visualize cell-cycle progression through G1, S, and G2. Using HeLa and HCT116 cells expressing H2B-HaloTag and Fucci probes, we found that local nucleosome motion remained similar on average throughout interphase, except for elevated motion in early G1. Transcription inhibition similarly increased nucleosome motion throughout interphase. Local nucleosome motion also increased following replication stress or DNA damage. ConclusionOur findings suggest that near-constant chromatin motion supports housekeeping functions under similar physical conditions during interphase. Our findings also suggest that cells can transiently change chromatin motion to perform ad hoc tasks in response to signals from inside and outside the cell, such as DNA damage.

The nuclear lamina is a vital structural component of eukaryotic cells, playing a pivotal role in both physiological processes, such as cell differentiation, and pathological conditions, including laminopathies and cancer metastasis. Lamina associated proteins, particularly lamins and nesprins, are integral to mechanosensing, chromatin organization, and gene regulation. However, their precise contributions to gene regulation remain incompletely understood. This study explores the functions of lamin A, LMNA, and SYNE2 in gene expression, with a particular focus on their influence on distal chromatin interactions and conformational changes. Using inducible shRNA knockdown, RNA-seq analysis, and dCas9-mediated live imaging of chromosomes, we demonstrate that lamin A affects RNA synthesis, LMNA governs chromatin spatial organization, and SYNE2 regulates chromatin modifications. Furthermore, both lamins and nesprins enhance telomere dynamics. These findings elucidate nuclear envelope-associated mechanisms in gene regulation, offering valuable insights into chromatin dynamics under both physiological and pathological contexts.

Abnormal nuclear blebbing occurs in many human diseases and causes nuclear rupture and dysfunction. Nuclear blebbing is caused by chromatin motion via RNA Pol II transcriptional activity and nuclear mechanical weakening. Camptothecin, a topoisomerase I inhibitor, rapidly suppresses nuclear blebbing within hours. We find that camptothecin does not decrease RNA Pol II phosphorylation, but does decrease newly synthesized RNA, likely by stalling RNA Pol II. However, camptothecin treatment suppresses nuclear blebbing more drastically than inhibition of transcription activity by alpha amanitin, suggesting a second mechanism of nuclear blebbing suppression. Dual micromanipulation nuclear force measures revealed camptothecin treatment increased chromatin-based nuclear stiffness but not lamin-based strain stiffening. Thus, inhibition of topoisomerase I via camptothecin drastically suppresses nuclear blebbing by both stalling RNA Pol II and increasing chromatin-based nuclear stiffness. Summary statementInhibition of topoisomerase I suppresses nuclear blebbing by stalling RNA Pol II activity and increasing chromatin-based nuclear spring constant.

Argonaute 1 (AGO1), a central component of the RNA interference (RNAi) pathway, has recently been implicated in nuclear functions, particularly in chromatin organization and gene regulation. However, the role of AGO1 in lncRNA-driven nuclear compartmentalization remains elusive. In this study, we uncover a novel function for AGO1 in the regulation of nuclear architecture through its interaction with the long non-coding RNA (lncRNA) NEAT1. NEAT1 lncRNA is required for the formation of paraspeckles, nuclear substructures involved in multiple cellular processes. We show that AGO1 physically interacts with NEAT1 and other key paraspeckle proteins (PSPs) and co-localizes with paraspeckles in the nucleus. AGO1 depletion disrupts expression of both NEAT1 isoforms, reduces the interaction of essential PSPs with NEAT1 lncRNA, and leads to impaired paraspeckle formation. Furthermore, depletion of NEAT1 results in the mis-localization of AGO1 from paraspeckles and alters active chromatin compartments. Together, our findings establish a new functional relationship between AGO1 and NEAT1 in nuclear compartmentalization and chromatin architecture.

Heterochromatin is a membraneless compartment within the cell nucleus. In recent years, a controversy arose on whether heterochromatin organization is driven by liquid-liquid phase separation or not. While many heterochromatin proteins were shown to undergo liquid-liquid phase separation in vitro, other studies reported that this does not happen in cells. Here, we tested the ability of heterochromatin proteins to generate heterochromatin barrier compartments in cells. We found that, while several proteins (H1.0, H1.4, HP1alpha, HP1beta, Mbd1, Mbd2 and MeCP2) form barrier compartments in mouse and/or human cells this differs between cell types. In addition, not all compartments in the same cell form barriers. We established and experimentally validated a model that predicted the ability to form barrier compartments is dependent on the protein accumulation in heterochromatin followed by the competition between compartments for the nucleoplasm pool of the protein and resulted in larger size for the barrier compartments. These findings resolve the existing controversy and rationalize how in cells heterochromatin compartments form and compete to establish dynamic barriers to the entry and exit of its components. HighlightsHeterochromatin barrier formation differs between proteins, cell lines and heterochromatin compartments within the cell. Barrier formation depends on heterochromatin anchors, including ligands and other scaffolds. Barrier compartments are defined by their larger size and higher protein enrichment. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/729812v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@a63021org.highwire.dtl.DTLVardef@a20362org.highwire.dtl.DTLVardef@8c2390org.highwire.dtl.DTLVardef@72dde3_HPS_FORMAT_FIGEXP M_FIG C_FIG

The nuclear pore complex (NPC), composed of proteins termed nucleoporins (Nups), intercalates the nuclear envelope, and is primarily involved in protein trafficking and mRNA export. At the nuclear basket, Nups have been associated with chromatin organization and postulated to function as transcriptional hubs, working in tandem with mRNA export machinery. However, little is known about the intermediate process of RNA splicing at the NPC. Here, we used BioID to screen for interactors of basket-Nups Nup153 and TPR and discovered the enrichment of splicing proteins across all spliceosome complexes (E, A, B, B*, P). The peripheral nature of the interaction between Nup153 and selected splicing components was confirmed by in-situ proximity ligation assay and STED microscopy. The presence of splicing components at the NPC, reduced upon splicing inhibition, is partly dependent on Nup153 and functionally correlated to the splicing of Nup153-bound genes. Assessed by DamID, Nup153-bound genes ([~]500) are characterized by multiple long introns with lower-than-average GC content. Positioned at the periphery but distinct from the neighbouring lamina-associated domain (LADs) in chromatin signatures and expression levels, these genes showed Nup153-dependent splicing defect, suggesting that splicing occurs at the NPC. Altogether, our data substantiates the gene gating theory bringing transcription and export, now accompanied by speckle-distant splicing events, at the level of the NPC.

Abnormalities in nuclear morphology are an important diagnostic tool to determine malignancy in cancer cells and are characterised by nuclear blebbing and deformations. Nuclear shape is mostly maintained by a dense protein meshwork of lamins, consisting of 4 lamin subtypes, of which the individual contribution to nuclear shape maintenance remains elusive. In this study, we decouple the roles of lamin A, C, and B1 across cancer cell lines with varying malignant potential (HeLa, HT1080, and MDA-MB-231). Using single-cell correlation analysis, we directly link reduced lamin A/C, and not lamin B1, expression levels to nuclear deformability. We found that the nuclear shape of the more malignant MDA-MB-231 cells is approximately 4-fold more sensitive to lamin A/C than HeLa and HT1080 cells. Biochemical analyses reveal cell-type-specific variation in lamin A/C interactions and homodimer formation that correlates with nuclear shape deformations. In contrast to healthy mouse embryonic fibroblast cells, malignant cells exhibit reduced dimerisation, which correlates with nuclear deformability. As such, our study links, for the first time, the lamin A/C dimerisation state to nuclear abnormalities, thereby providing new avenues for investigating cancer progression.

The nuclear pore complex (NPC) is a large multi-protein structure that enables movement of macromolecules, such as mRNA and proteins, between the nucleoplasm and cytoplasm. There has been great interest in how the physical state of the NPC can influence nuclear-cytoplasmic transport. The hypothesis that the NPC may be mechanosensitive is supported by prior reports showing that the diameter of the NPC increases with nuclear envelope stretch as well as increased ECM stiffness. We therefore sought to develop a biosensor-based approach to determine if the NPC experiences mechanical tension. Using a previously developed FRET-force biosensor, known as TSmod, we developed a gp210 tension sensor. gp210 is a transmembrane nucleoporin, which may serve to anchor the NPC into the nuclear envelope. Using a CRISPR knock-in strategy, we developed a HeLa cell line which expresses the gp210 tension sensor at endogenous levels. Using this sensor, we observed that gp210 forces increase in response to osmotically induced nuclear swelling. Cell attachment, ECM stiffness, the nuclear LINC complex, chromatin condensation, and actomyosin contractility were all observed to influence gp210 forces. Surprisingly, gp210 forces were increased with chromatin relaxation and myosin light chain kinase inhibition, indicating that NPC forces may be differentially regulated from forces on the LINC complex. Our data support a hypothesis where nuclear strain, rather than cytoskeletal forces, is the predominant source for NPC forces. Our studies demonstrate that NPC proteins do experience mechanical tension. We anticipate that the gp210 force sensor will be of use for future studies of NPC mechanobiology.

Despite their importance, how linker histone H1s interact in chromatin and especially how the highly positively charged and intrinsically disordered H1 C-terminal domain (CTD) binds and stabilizes nucleosomes and higher-order chromatin structures remains unclear. Using single-molecule FRET we found that about half of the H1 CTDs in H1-nucleosome complexes exhibit well-defined FRET values indicative of distinct, static conformations, while the remainder of the population exhibits dynamically changing values, similar to that observed for H1 in the absence of nucleosomes. We also find that the first 30 residues of the CTD participate in relatively localized interactions with the first [~]20 bp of linker DNA, and that two separate regions in the CTD contribute to H1-dependent organization of linker DNA, consistent with some non-random CTD-linker DNA interactions. Finally, our data show that acetylation mimetics within the histone H3 tail induce decondensation and enhanced dynamics of the nucleosome-bound H1 CTD. (148 words)

The RNA helicase UPF1 is best known for its key role in mRNA surveillance but has been implicated in additional cellular processes both in the nucleus and in the cytoplasm. In human cells, the vast majority of UPF1 resides in the cytoplasm and only small amounts can be detected in the nucleus at steady state. It was previously shown that its export from the nucleus to the cytoplasm is Crm1-dependent, yet neither the nuclear export signal (NES) nor the nuclear localization signal (NLS) has been identified. Here, we provide evidence for a noncanonical NLS in UPF1, map the NES to amino acids 89-105 and show that L103 and F105 are essential for UPF1s export to the cytoplasm. Examination of additional UPF1 mutants revealed that a functional helicase domain but not the association with RNA is crucial for the shuttling capacity of UPF1.

This article has been withdrawn by bioRxiv owing to a technical error that created a duplicate posting of this manuscript. Please see doi: https://doi.org/10.1101/2020.06.24.168757 to access the preprint.

The incorporation of variant histone H3.3 into the genome is tightly linked with transcriptional activity, yet its precise regulatory mechanisms remain elusive. Traditional methods like Chromatin Immunoprecipitation offer static views of H3.3 distribution, lacking dynamic insights. Here, using the SNAP tag system, we employed Fluorescence Recovery After Photobleaching (FRAP) and live-cell imaging to investigate H3.3 mobility and decay kinetics in live mouse embryonic fibroblast cells. Our focus on interferon-induced transcriptional activation revealed rapid H3.3 exchange, indicative of its transcriptional regulatory role. Transcription inhibition hindered H3.3 mobility, emphasizing its involvement in transcription. Additionally, we probed into turnover dynamics(decay) of H3.1-SNAP and H3.3-SNAP variants, uncovering differential decay rates influenced by transcriptional activity and histone modifiers such as NSD2 and HIRA. Live-cell imaging showed faster decay of H3.3 compared to H3.1, further exacerbated upon NSD2/HIRA loss. Notably, HIRA and NSD2, regulators of H3.3 dynamics, proved crucial for both H3.3 mobility and decay, underscoring their pivotal role. These findings deepen our understanding of epigenetic regulation, emphasizing the dynamic nature of histone turnover in cellular function and its implications for disease pathogenesis. Taken together, this study sheds light on the dynamic behavior of H3.3 and its regulatory mechanisms, providing valuable insights into epigenetic regulation in cellular processes and disease contexts.

Among flowering plants, genome size varies remarkably, by >2200-fold, and this variation depends on the loss and gain of non-coding DNA sequences that form distinct heterochromatin complexes during interphase. In plants with giant genomes, most chromatin remains condensed during interphase, forming a dense network of heterochromatin threads called interphase chromonemata. Using super-resolution light and electron microscopy, we studied the ultrastructure of chromonemata during and after replication in root meristem nuclei of Nigella damascena L. During S-phase, heterochromatin undergoes transient decondensation locally at DNA replication sites. Due to the abundance of heterochromatin, the replication leads to a robust disassembly of the chromonema meshwork and a general reorganization of the nuclear morphology visible even by conventional light microscopy. After replication, heterochromatin recondenses, restoring the chromonema structure. Thus, we show that heterochromatin replication in interphase nuclei of giant-genome plants induces a global nuclear reorganization.

The linear DNA sequence of mammalian chromosomes is organized in large blocks of DNA with similar sequence properties, producing a pattern of dark and light staining bands on mitotic chromosomes. Cytogenetic banding is essentially invariant between people and cell-types and thus may be assumed unrelated to genome regulation. We investigate whether large blocks of Alu-rich R-bands and L1-rich G-bands provide a framework upon which functional genome architecture is built. We examine two models of large-scale chromatin condensation: X-chromosome inactivation and formation of senescence-associated heterochromatin foci (SAHFs). XIST RNA triggers gene silencing but also formation of the condensed Barr Body (BB), thought to reflect cumulative gene silencing. However, we find Alu-rich regions are depleted from the L1-rich BB, supporting it is a dense core but not the entire chromosome. Alu-rich bands are also gene-rich, affirming our earlier findings that genes localize at the outer periphery of the BB. SAHFs similarly form within each territory by coalescence of syntenic L1 regions depleted for highly Alu-rich DNA. Analysis of senescent cell Hi-C data also shows large contiguous blocks of G-band and R-band DNA remodel as a segmental unit. Entire dark-bands gain distal intrachromosomal interactions as L1-rich regions form the SAHF. Most striking is that sharp Alu peaks within R-bands resist these changes in condensation. We further show that Chr19, which is exceptionally Alu rich, fails to form a SAHF. Collective results show regulation of genome architecture corresponding to large blocks of DNA and demonstrate resistance of segments with high Alu to chromosome condensation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=179 HEIGHT=200 SRC="FIGDIR/small/574742v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@5ac4b9org.highwire.dtl.DTLVardef@1298a63org.highwire.dtl.DTLVardef@3c8b01org.highwire.dtl.DTLVardef@c9f5f4_HPS_FORMAT_FIGEXP M_FIG C_FIG

Transcription of the majority of eukaryotic genes is accompanied by splicing, a process that depends on the assembly of the spliceosome on introns. The timing of spliceosome assembly varies significantly between introns, transcripts, genes and species. While quick co-transcriptional intron removal has been demonstrated for many mammalian genes, most splicing events do not occur immediately after intron synthesis. In this study, we utilized the highly expressed Tg gene, which forms exceptionally long transcription loops (Leidescher et al., 2022), providing a convenient model for studying splicing dynamics using advanced light microscopy. Our single-cell oligopainting-based analysis revealed a delay in splicing several tens of kilobases downstream of a transcribed intron, a finding further supported by standard cell population analyses. We speculate that this phenomenon is due to the abnormally high transcription rate of the Tg gene, which may lead to a localized deficiency in splicing factors and, consequently, delayed spliceosome assembly on thousands of nascent transcripts decorating the gene. Additionally, we found that, in contrast to short introns (<10 kb), long Tg intron (>50 kb) is spliced promptly, providing further support for the idea that intron length may modulate splicing speed.

A mechanistic role for nuclear function of testis-specific actin related proteins (ARPs) is proposed here through contributions of ARP subunit swapping in canonical chromatin regulatory complexes. This is significant to our understanding of both mechanisms controlling regulation of spermiogenesis, and the expanding functional roles of the ARPs in cell biology. Among these roles, actins and ARPs are pivotal not only in cytoskeletal regulation, but also in intranuclear chromatin organization, influencing gene regulation and nucleosome remodeling. This study focuses on two testis-specific ARPs, ACTL7A and ACTL7B, exploring their intranuclear activities and broader implications utilizing combined in vivo, in vitro, and in silico approaches. ACTL7A and ACTL7B, previously associated with structural roles, are hypothesized here to serve in chromatin regulation during germline development. This study confirms the intranuclear presence of ACTL7B in spermatocytes and round spermatids, revealing a potential role in intranuclear processes, and identifies a putative nuclear localization sequence conserved across mammalian ACTL7B, indicating a potentially unique mode of nuclear transport which differs from conventional actin. Ablation of ACTL7B leads to varied transcriptional changes reported here. Additionally, in the absence of ACTL7A or ACTL7B there is a loss of intranuclear localization of HDAC1 and HDAC3, which are known regulators of epigenetic associated acetylation changes that in turn regulate gene expression. Thus, these HDACs are implicated as contributors to the aberrant gene expression observed in the KO mouse testis transcriptomic analysis. Furthermore, this study employed and confirmed the accuracy of in silico models to predict ARP interactions with Helicase-SANT-associated (HSA) domains, uncovering putative roles for testis-specific ARPs in nucleosome remodeling complexes. In these models, ACTL7A and ACTL7B were found capable of binding to INO80 and SWI/SNF nucleosome remodeler family members in a manner akin to nuclear actin and ACTL6A. These models thus implicate germline-specific ARP subunit swapping within chromatin regulatory complexes as a potential regulatory mechanism for chromatin and associated molecular machinery adaptations in nuclear reorganizations required during spermiogenesis. These results hold implications for male fertility and epigenetic programing in the male-germline that warrant significant future investigation. In summary, this study reveals that ACTL7A and ACTL7B play intranuclear gene regulation roles in male gametogenesis, adding to the multifaceted roles identified also spanning structural, acrosomal, and flagellar stability. ACTL7A and ACTL7B unique nuclear transport, impact on HDAC nuclear associations, impact on transcriptional processes, and proposed mechanism for involvement in nucleosome remodeling complexes supported by AI facilitated in silico modeling contribute to a more comprehensive understanding of the indispensable functions of ARPs broadly in cell biology, and specifically in male fertility.

The interplay between cells and their surrounding microenvironment drives multiple cellular functions, including migration, proliferation, and cell fate transitions. The nucleus is a mechanosensitive organelle that adapts external mechanical and biochemical signals provided by the environment into nuclear changes with functional consequences for cell biology. However, the morphological and functional changes of the nucleus induced by 3D extracellular signals remain unclear. Here, we demonstrated that cells derived from 3D conditions conserve changes from cell confinement and show an aberrant nuclear morphology and localization of lamin B1, even in the absence of cellular confinement. We found that actin polymerization and protein kinase C (PKC) activity mediate the abnormal distribution of lamin B1 in 3D conditions-derived cells. These cells present altered chromatin compaction, gene transcription and cellular functions such as cell viability and migration. By combining biomechanical techniques and single-nucleus analysis, we have determined that the nucleus from 3D conditions-derived cells shows a different mechanical behavior and biophysical signature than the nucleus from control cells. Together, our work substantiates novel insights into how the extracellular environment alters the cell biology by promoting permanent changes in the chromatin, morphology, lamin B1 distribution, and the mechanical response of the nucleus.