Positioning Heterochromatin at the Nuclear Periphery Suppresses Histone Turnover to Promote Epigenetic Inheritance.

In eukaryotes, heterochromatin is generally located at the nuclear periphery. This study investigates the biological significance of perinuclear positioning for heterochromatin maintenance and gene silencing. We identify the nuclear rim protein Amo1NUPL2 as a factor required for the propagation of heterochromatin at endogenous and ectopic sites in the fission yeast genome. Amo1 associates with the Rix1PELP1-containing RNA processing complex RIXC and with the histone chaperone complex FACT. RIXC, which binds to heterochromatin protein Swi6HP1 across silenced chromosomal domains and to surrounding boundary elements, connects heterochromatin with Amo1 at the nuclear periphery. In turn, the Amo1-enriched subdomain is critical for Swi6 association with FACT that precludes histone turnover to promote gene silencing and preserve epigenetic stability of heterochromatin. In addition to uncovering conserved factors required for perinuclear positioning of heterochromatin, these analyses elucidate a mechanism by which a peripheral subdomain enforces stable gene repression and maintains heterochromatin in a heritable manner.

CRISPR-Mediated Programmable 3D Genome Positioning and Nuclear Organization.

Programmable control of spatial genome organization is a powerful approach for studying how nuclear structure affects gene regulation and cellular function. Here, we develop a versatile CRISPR-genome organization (CRISPR-GO) system that can efficiently control the spatial positioning of genomic loci relative to specific nuclear compartments, including the nuclear periphery, Cajal bodies, and promyelocytic leukemia (PML) bodies. CRISPR-GO is chemically inducible and reversible, enabling interrogation of real-time dynamics of chromatin interactions with nuclear compartments in living cells. Inducible repositioning of genomic loci to the nuclear periphery allows for dissection of mitosis-dependent and -independent relocalization events and also for interrogation of the relationship between gene position and gene expression. CRISPR-GO mediates rapid de novo formation of Cajal bodies at desired chromatin loci and causes significant repression of endogenous gene expression over long distances (30-600 kb). The CRISPR-GO system offers a programmable platform to investigate large-scale spatial genome organization and function.

A nuclear architecture screen in Drosophila identifies Stonewall as a link between chromatin position at the nuclear periphery and germline stem cell fate.

The association of genomic loci to the nuclear periphery is proposed to facilitate cell type-specific gene repression and influence cell fate decisions. However, the interplay between gene position and expression remains incompletely understood, in part because the proteins that position genomic loci at the nuclear periphery remain unidentified. Here, we used an Oligopaint-based HiDRO screen targeting ∼1000 genes to discover novel regulators of nuclear architecture in Drosophila cells. We identified the heterochromatin-associated protein Stonewall (Stwl) as a factor promoting perinuclear chromatin positioning. In female germline stem cells (GSCs), Stwl binds and positions chromatin loci, including GSC differentiation genes, at the nuclear periphery. Strikingly, Stwl-dependent perinuclear positioning is associated with transcriptional repression, highlighting a likely mechanism for Stwl's known role in GSC maintenance and ovary homeostasis. Thus, our study identifies perinuclear anchors in Drosophila and demonstrates the importance of gene repression at the nuclear periphery for cell fate.

Calcineurin dephosphorylates topoisomerase IIß and regulates the formation of neuronal-activity-induced DNA breaks.

Neuronal activity induces topoisomerase IIß (Top2B) to generate DNA double-strand breaks (DSBs) within the promoters of neuronal early response genes (ERGs) and facilitate their transcription, and yet, the mechanisms that control Top2B-mediated DSB formation are unknown. Here, we report that stimulus-dependent calcium influx through NMDA receptors activates the phosphatase calcineurin to dephosphorylate Top2B at residues S1509 and S1511, which stimulates its DNA cleavage activity and induces it to form DSBs. Exposing mice to a fear conditioning paradigm also triggers Top2B dephosphorylation at S1509 and S1511 in the hippocampus, indicating that calcineurin also regulates Top2B-mediated DSB formation following physiological neuronal activity. Furthermore, calcineurin-Top2B interactions following neuronal activity and sites that incur activity-induced DSBs are preferentially localized at the nuclear periphery in neurons. Together, these results reveal how radial gene positioning and the compartmentalization of activity-dependent signaling govern the position and timing of activity-induced DSBs and regulate gene expression patterns in neurons.

Replication stress conferred by POT1 dysfunction promotes telomere relocalization to the nuclear pore.

Mutations in the telomere-binding protein POT1 are associated with solid tumors and leukemias. POT1 alterations cause rapid telomere elongation, ATR kinase activation, telomere fragility, and accelerated tumor development. Here, we define the impact of mutant POT1 alleles through complementary genetic and proteomic approaches based on CRISPR interference and biotin-based proximity labeling, respectively. These screens reveal that replication stress is a major vulnerability in cells expressing mutant POT1, which manifests as increased telomere mitotic DNA synthesis at telomeres. Our study also unveils a role for the nuclear pore complex in resolving replication defects at telomeres. Depletion of nuclear pore complex subunits in the context of POT1 dysfunction increases DNA damage signaling, telomere fragility and sister chromatid exchanges. Furthermore, we observed telomere repositioning to the nuclear periphery driven by nuclear F-actin polymerization in cells with POT1 mutations. In conclusion, our study establishes that relocalization of dysfunctional telomeres to the nuclear periphery is critical to preserve telomere repeat integrity.

DAXX safeguards heterochromatin formation in embryonic stem cells.

Genomes comprise a large fraction of repetitive sequences folded into constitutive heterochromatin, which protect genome integrity and cell identity. De novo formation of heterochromatin during preimplantation development is an essential step for preserving the ground-state of pluripotency and the self-renewal capacity of embryonic stem cells (ESCs). However, the molecular mechanisms responsible for the remodeling of constitutive heterochromatin are largely unknown. Here, we identify that DAXX, an H3.3 chaperone essential for the maintenance of mouse ESCs in the ground state, accumulates in pericentromeric regions independently of DNA methylation. DAXX recruits PML and SETDB1 to promote the formation of heterochromatin, forming foci that are hallmarks of ground-state ESCs. In the absence of DAXX or PML, the three-dimensional (3D) architecture and physical properties of pericentric and peripheral heterochromatin are disrupted, resulting in de-repression of major satellite DNA, transposable elements and genes associated with the nuclear lamina. Using epigenome editing tools, we observe that H3.3, and specifically H3.3K9 modification, directly contribute to maintaining pericentromeric chromatin conformation. Altogether, our data reveal that DAXX is crucial for the maintenance and 3D organization of the heterochromatin compartment and protects ESC viability.

Control of heterochromatin localization and silencing by the nuclear membrane protein Lem2.

Transcriptionally silent chromatin localizes to the nuclear periphery, which provides a special microenvironment for gene repression. A variety of nuclear membrane proteins interact with repressed chromatin, yet the functional role of these interactions remains poorly understood. Here, we show that, in Schizosaccharomyces pombe, the nuclear membrane protein Lem2 associates with chromatin and mediates silencing and heterochromatin localization. Unexpectedly, we found that these functions can be separated and assigned to different structural domains within Lem2, excluding a simple tethering mechanism. Chromatin association and tethering of centromeres to the periphery are mediated by the N-terminal LEM (LAP2-Emerin-MAN1) domain of Lem2, whereas telomere anchoring and heterochromatin silencing require exclusively its conserved C-terminal MSC (MAN1-Src1 C-terminal) domain. Particularly, silencing by Lem2 is epistatic with the Snf2/HDAC (histone deacetylase) repressor complex SHREC at telomeres, while its necessity can be bypassed by deleting Epe1, a JmjC protein with anti-silencing activity. Furthermore, we found that loss of Lem2 reduces heterochromatin association of SHREC, which is accompanied by increased binding of Epe1. This reveals a critical function of Lem2 in coordinating these antagonistic factors at heterochromatin. The distinct silencing and localization functions mediated by Lem2 suggest that these conserved LEM-containing proteins go beyond simple tethering to play active roles in perinuclear silencing.

Interactions of Chromatin with the Nuclear Lamina and Nuclear Pore Complexes.

Heterochromatin and euchromatin form different spatial compartments in the interphase nucleus, with heterochromatin being localized mainly at the nuclear periphery. The mechanisms responsible for peripheral localization of heterochromatin are still not fully understood. The nuclear lamina and nuclear pore complexes were obvious candidates for the role of heterochromatin binders. This review is focused on recent studies showing that heterochromatin interactions with the nuclear lamina and nuclear pore complexes maintain its peripheral localization. Differences in chromatin interactions with the nuclear envelope in cell populations and in individual cells are also discussed.

Histone methyltransferase activity programs nuclear peripheral genome positioning.

Spatial organization of the genome in the nucleus plays a critical role in development and regulation of transcription. A genomic region that resides at the nuclear periphery is part of the chromatin layer marked with histone H3 lysine 9 dimethyl (H3K9me2), but chromatin reorganization during cell differentiation can cause movement in and out of this nuclear compartment with patterns specific for individual cell fates. Here we describe a CRISPR-based system that allows visualization coupled with forced spatial relocalization of a target genomic locus in live cells. We demonstrate that a specified locus can be tethered to the nuclear periphery through direct binding to a dCas9-Lap2ß fusion protein at the nuclear membrane, or via targeting of a histone methyltransferase (HMT), G9a fused to dCas9, that promotes H3K9me2 labeling and localization to the nuclear periphery. The enzymatic activity of the HMT is sufficient to promote this repositioning, while disruption of the catalytic activity abolishes the localization effect. We further demonstrate that dCas9-G9a-mediated localization to the nuclear periphery is independent of nuclear actin polymerization. Our data suggest a function for epigenetic histone modifying enzymes in spatial chromatin organization and provide a system for tracking and labeling targeted genomic regions in live cells.

KMT1 family methyltransferases regulate heterochromatin-nuclear periphery tethering via histone and non-histone protein methylation.

Euchromatic histone methyltransferases (EHMTs), members of the KMT1 family, methylate histone and non-histone proteins. Here, we uncover a novel role for EHMTs in regulating heterochromatin anchorage to the nuclear periphery (NP) via non-histone methylation. We show that EHMTs methylate and stabilize LaminB1 (LMNB1), which associates with the H3K9me2-marked peripheral heterochromatin. Loss of LMNB1 methylation or EHMTs abrogates heterochromatin anchorage at the NP We further demonstrate that the loss of EHMTs induces many hallmarks of aging including global reduction of H3K27methyl marks and altered nuclear morphology. Consistent with this, we observe a gradual depletion of EHMTs, which correlates with loss of methylated LMNB1 and peripheral heterochromatin in aging human fibroblasts. Restoration of EHMT expression reverts peripheral heterochromatin defects in aged cells. Collectively, our work elucidates a new mechanism by which EHMTs regulate heterochromatin domain organization and reveals their impact on fundamental changes associated with the intrinsic aging process.

Tidying-up the plant nuclear space: domains, functions, and dynamics.

Understanding how the packaging of chromatin in the nucleus is regulated and organized to guide complex cellular and developmental programmes, as well as responses to environmental cues is a major question in biology. Technological advances have allowed remarkable progress within this field over the last years. However, we still know very little about how the 3D genome organization within the cell nucleus contributes to the regulation of gene expression. The nuclear space is compartmentalized in several domains such as the nucleolus, chromocentres, telomeres, protein bodies, and the nuclear periphery without the presence of a membrane around these domains. The role of these domains and their possible impact on nuclear activities is currently under intense investigation. In this review, we discuss new data from research in plants that clarify functional links between the organization of different nuclear domains and plant genome function with an emphasis on the potential of this organization for gene regulation.

A decade of understanding spatio-temporal regulation of DNA repair by the nuclear architecture.

The nucleus is a hub for gene expression and is a highly organized entity. The nucleoplasm is heterogeneous, owing to the preferential localization of specific metabolic factors, which lead to the definition of nuclear compartments or bodies. The genome is organized into chromosome territories, as well as heterochromatin and euchromatin domains. Recent observations have indicated that nuclear organization is important for maintaining genomic stability. For example, nuclear organization has been implicated in stabilizing damaged DNA, repair-pathway choice, and in preventing chromosomal rearrangements. Over the past decade, several studies have revealed that dynamic changes in the nuclear architecture are important during double-strand break repair. Stemming from work in yeast, relocation of a damaged site prior to repair appears to be at least partially conserved in multicellular eukaryotes. In this review, we will discuss genome and nucleoplasm architecture, particularly the importance of the nuclear periphery in genome stability. We will also discuss how the site of relocation regulates repair-pathway choice.

Measuring Inaccessible Chromatin Genome-Wide Using Protect-seq.

Chromatin accessibility has been an immensely powerful metric for identifying and understanding regulatory elements in the genome. Many important regulatory elements, such as enhancers and transcriptional start sites, are characterized by "open" or nucleosome-free regions. Understanding the areas of the genome that are not considered open chromatin has been more difficult. Protect-seq is a genomics technique that aims to identify inaccessible chromatin associated with the nuclear periphery. These regions are enriched for histone modifications associated with transcriptional repression and correlate with loci identified by other techniques measuring heterochromatin and peripheral localization. Here, we discuss the protocol and best practices to perform Protect-seq.

A nuclear architecture screen in Drosophila identifies Stonewall as a link between chromatin position at the nuclear periphery and germline stem cell fate.

The association of genomic loci to the nuclear periphery is proposed to facilitate cell-type specific gene repression and influence cell fate decisions. However, the interplay between gene position and expression remains incompletely understood, in part because the proteins that position genomic loci at the nuclear periphery remain unidentified. Here, we used an Oligopaint-based HiDRO screen targeting ~1000 genes to discover novel regulators of nuclear architecture in Drosophila cells. We identified the heterochromatin-associated protein, Stonewall (Stwl), as a factor promoting perinuclear chromatin positioning. In female germline stem cells (GSCs), Stwl binds and positions chromatin loci, including GSC differentiation genes, at the nuclear periphery. Strikingly, Stwl-dependent perinuclear positioning is associated with transcriptional repression, highlighting a likely mechanism for Stwl's known role in GSC maintenance and ovary homeostasis. Thus, our study identifies perinuclear anchors in Drosophila and demonstrates the importance of gene repression at the nuclear periphery for cell fate.

Characterization of a Plant Nuclear Matrix Constituent Protein in Liverwort.

The nuclear lamina (NL) is a complex network of nuclear lamins and lamina-associated nuclear membrane proteins, which scaffold the nucleus to maintain structural integrity. In animals, type V intermediate filaments are the main constituents of NL. Plant genomes do not encode any homologs of these intermediate filaments, yet plant nuclei contain lamina-like structures that are present in their nuclei. In Arabidopsis thaliana, CROWDED NUCLEI (CRWN), which are required for maintaining structural integrity of the nucleus and specific perinuclear chromatin anchoring, are strong candidates for plant lamin proteins. Recent studies revealed additional roles of Arabidopsis Nuclear Matrix Constituent Proteins (NMCPs) in modulating plants' response to pathogen and abiotic stresses. However, detailed analyses of Arabidopsis NMCP activities are challenging due to the presence of multiple homologs and their functional redundancy. In this study, we investigated the sole NMCP gene in the liverwort Marchantia polymorpha (MpNMCP). We found that MpNMCP proteins preferentially were localized to the nuclear periphery. Using CRISPR/Cas9 techniques, we generated an MpNMCP loss-of-function mutant, which displayed reduced growth rate and curly thallus lobes. At an organelle level, MpNMCP mutants did not show any alteration in nuclear morphology. Transcriptome analyses indicated that MpNMCP was involved in regulating biotic and abiotic stress responses. Additionally, a highly repetitive genomic region on the male sex chromosome, which was preferentially tethered at the nuclear periphery in wild-type thalli, decondensed in the MpNMCP mutants and located in the nuclear interior. This perinuclear chromatin anchoring, however, was not directly controlled by MpNMCP. Altogether, our results unveiled that NMCP in plants have conserved functions in modulating stress responses.

CRISPR-Cas-Mediated Tethering Recruits the Yeast HMR Mating-Type Locus to the Nuclear Periphery but Fails to Silence Gene Expression.

To investigate the relationship between genome structure and function, we have developed a programmable CRISPR-Cas system for nuclear peripheral recruitment in yeast. We benchmarked this system at the HMR and GAL2 loci, both of which are well-characterized model systems for localization to the nuclear periphery. Using microscopy and gene silencing assays, we demonstrate that CRISPR-Cas-mediated tethering can recruit the HMR locus but does not detectably silence reporter gene expression. A previously reported Gal4-mediated tethering system does silence gene expression, and we demonstrate that the silencing effect has an unexpected dependence on the properties of the protein tether. The CRISPR-Cas system was unable to recruit GAL2 to the nuclear periphery. Our results reveal potential challenges for synthetic genome structure perturbations and suggest that distinct functional effects can arise from subtle structural differences in how genes are recruited to the periphery.

Transcription-independent TFIIIC-bound sites cluster near heterochromatin boundaries within lamina-associated domains in C. elegans.

BACKGROUND: Chromatin organization is central to precise control of gene expression. In various eukaryotic species, domains of pervasive cis-chromatin interactions demarcate functional domains of the genomes. In nematode Caenorhabditis elegans, however, pervasive chromatin contact domains are limited to the dosage-compensated sex chromosome, leaving the principle of C. elegans chromatin organization unclear. Transcription factor III C (TFIIIC) is a basal transcription factor complex for RNA polymerase III, and is implicated in chromatin organization. TFIIIC binding without RNA polymerase III co-occupancy, referred to as extra-TFIIIC binding, has been implicated in insulating active and inactive chromatin domains in yeasts, flies, and mammalian cells. Whether extra-TFIIIC sites are present and contribute to chromatin organization in C. elegans remains unknown. RESULTS: We identified 504 TFIIIC-bound sites absent of RNA polymerase III and TATA-binding protein co-occupancy characteristic of extra-TFIIIC sites in C. elegans embryos. Extra-TFIIIC sites constituted half of all identified TFIIIC binding sites in the genome. Extra-TFIIIC sites formed dense clusters in cis. The clusters of extra-TFIIIC sites were highly over-represented within the distal arm domains of the autosomes that presented a high level of heterochromatin-associated histone H3K9 trimethylation (H3K9me3). Furthermore, extra-TFIIIC clusters were embedded in the lamina-associated domains. Despite the heterochromatin environment of extra-TFIIIC sites, the individual clusters of extra-TFIIIC sites were devoid of and resided near the individual H3K9me3-marked regions. CONCLUSION: Clusters of extra-TFIIIC sites were pervasive in the arm domains of C. elegans autosomes, near the outer boundaries of H3K9me3-marked regions. Given the reported activity of extra-TFIIIC sites in heterochromatin insulation in yeasts, our observation raised the possibility that TFIIIC may also demarcate heterochromatin in C. elegans.

Plant lamin-like proteins mediate chromatin tethering at the nuclear periphery.

BACKGROUND: The nuclear envelope not only serves as a physical barrier separating nuclear content from the cytoplasm but also plays critical roles in modulating the three-dimensional organization of genomic DNA. For both plants and animals, the nuclear periphery is a functional compartment enriched with heterochromatin. To date, how plants manage to selectively tether chromatin at the nuclear periphery is unclear. RESULTS: By conducting dual-color fluorescence in situ hybridization experiments on 2C nuclei, we show that in Arabidopsis thaliana, specific chromatin positioning at the nuclear periphery requires plant lamin-like proteins CROWDED NUCLEI 1 (CRWN1), CRWN4, and DNA methylation in CHG and CHH contexts. With chromosome painting and Hi-C analyses, we show global attenuation of spatial chromatin compartmentalization and chromatin positioning patterns at the nuclear periphery in both the crwn1 and crwn4 mutants. Furthermore, ChIP-seq analysis indicates that CRWN1 directly interacts with chromatin domains localized at the nuclear periphery, which mainly contains non-accessible chromatin. CONCLUSIONS: In summary, we conclude that CRWN1 is a key component of the lamina-chromatin network in plants. It is functionally equivalent to animal lamins, playing critical roles in modulating patterns of chromatin positioning at the nuclear periphery.

Emerin anchors Msx1 and its protein partners at the nuclear periphery to inhibit myogenesis.

BACKGROUND: Previous studies have shown that in myogenic precursors, the homeoprotein Msx1 and its protein partners, histone methyltransferases and repressive histone marks, tend to be enriched on target myogenic regulatory genes at the nuclear periphery. The nuclear periphery localization of Msx1 and its protein partners is required for Msx1's function of preventing myogenic precursors from pre-maturation through repressing target myogenic regulatory genes. However, the mechanisms underlying the maintenance of Msx1 and its protein partners' nuclear periphery localization are unknown. RESULTS: We show that an inner nuclear membrane protein, Emerin, performs as an anchor settled at the inner nuclear membrane to keep Msx1 and its protein partners Ezh2, H3K27me3 enriching at the nuclear periphery, and participates in inhibition of myogenesis mediated by Msx1. Msx1 interacts with Emerin both in C2C12 myoblasts and mouse developing limbs, which is the prerequisite for Emerin mediating the precise location of Msx1, Ezh2, and H3K27me3. The deficiency of Emerin in C2C12 myoblasts disturbs the nuclear periphery localization of Msx1, Ezh2, and H3K27me3, directly indicating Emerin functioning as an anchor. Furthermore, Emerin cooperates with Msx1 to repress target myogenic regulatory genes, and assists Msx1 with inhibition of myogenesis. CONCLUSIONS: Emerin cooperates with Msx1 to inhibit myogenesis through maintaining the nuclear periphery localization of Msx1 and Msx1's protein partners.

The Nuclear Lamina as an Organizer of Chromosome Architecture.

The nuclear lamina (NL) is a meshwork of lamins and lamin-associated proteins adjoining the inner side of the nuclear envelope. In early embryonic cells, the NL mainly suppresses background transcription, whereas, in differentiated cell types, its disruption affects gene expression more severely. Normally, the NL serves as a backbone for multiple chromatin anchoring sites, thus shaping the spatial organization of chromosomes in the interphase nucleus. However, upon cell senescence, aging, or in some types of terminally differentiated cells and lamin-associated diseases, the loss of NL-chromatin tethering causes drastic alterations in chromosome architecture. Here, we provide an overview of the recent advances in the field of NL-chromatin interactions, focusing on their impact on chromatin positioning, compaction, repression, and spatial organization.