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Phase separation forms membraneless compartments in the nuclei, including by establishing heterochromatin "domains" and repair foci. Pericentromeric heterochromatin mostly comprises repeated sequences prone to aberrant recombination, and "safe" homologous recombination (HR) repair of these sequences requires the movement of repair sites to the nuclear periphery before Rad51 recruitment and strand invasion. How this mobilization initiates is unknown, and the contribution of phase separation to these dynamics is unclear. Here, we show that Nup98 nucleoporin is recruited to heterochromatic repair sites before relocalization through Sec13 or Nup88 nucleoporins, and downstream from the Smc5/6 complex and SUMOylation. Remarkably, the phase separation properties of Nup98 are required and sufficient to mobilize repair sites and exclude Rad51, thus preventing aberrant recombination while promoting HR repair. Disrupting this pathway results in heterochromatin repair defects and widespread chromosome rearrangements, revealing a novel "off-pore" role for nucleoporins and phase separation in nuclear dynamics and genome integrity in a multicellular eukaryote., Competing Interests: Declaration of Interests The authors declare no competing interests.

Nuclear pore complexes are large multicomponent protein complexes that are embedded in the nuclear envelope, where they mediate nucleocytoplasmic transport. In addition to supporting transport, nuclear pore components, termed nucleoporins (Nups), can interact with chromatin and influence genome function. A subset of Nups can also localize to the nuclear interior and bind chromatin intranuclearly, providing an opportunity to investigate chromatin-associated functions of Nups outside of the transport context. This review focuses on the gene regulatory functions of such intranuclear Nups, with a particular emphasis on their identity as components of several chromatin regulatory complexes. Recent proteomic screens have identified Nups as interacting partners of active and repressive epigenetic machinery, architectural proteins, and DNA replication complexes, providing insight into molecular mechanisms via which Nups regulate gene expression programs. This review summarizes these interactions and discusses their potential functions in the broader framework of nuclear genome organization., (© 2023 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Cellular ability to mount an enhanced transcriptional response upon repeated exposure to external cues is termed transcriptional memory, which can be maintained epigenetically through cell divisions and can depend on a nuclear pore component Nup98. The majority of mechanistic knowledge on transcriptional memory has been derived from bulk molecular assays. To gain additional perspective on the mechanism and contribution of Nup98 to memory, we used single-molecule RNA FISH (smFISH) to examine the dynamics of transcription in Drosophila cells upon repeated exposure to the steroid hormone ecdysone. We combined smFISH with mathematical modeling and found that upon hormone exposure, cells rapidly activate a low-level transcriptional response, but simultaneously begin a slow transition into a specialized memory state characterized by a high rate of expression. Strikingly, our modeling predicted that this transition between non-memory and memory states is independent of the transcription stemming from initial activation. We confirmed this prediction experimentally by showing that inhibiting transcription during initial ecdysone exposure did not interfere with memory establishment. Together, our findings reveal that Nup98's role in transcriptional memory is to stabilize the forward rate of conversion from low to high expressing state, and that induced genes engage in two separate behaviors - transcription itself and the establishment of epigenetically propagated transcriptional memory., Competing Interests: PP, SL, MC No competing interests declared, (© 2022, Pascual-Garcia et al.)

Single molecule RNA fluorescence in situ hybridization (smRNA FISH) is a widely used method for examining cellular localization of RNA and assessing gene expression outputs. The Nuclear Pore Complex (NPC) is a nuclear macro-complex known to both mediate nucleocytoplasmic transport and influence transcription via interactions with chromatin. Consequently, depletion of NPC proteins can result in defects in either transcription or nuclear export of mRNA. To distinguish between these two different functions of NPC components, it is preferable to analyze transcription and mRNA export simultaneously or in the same cell. Here, we present a smRNA FISH protocol with downstream custom MATLAB image analysis for application in Drosophila larval salivary gland tissues. This method can detect both nuclear export and transcriptional phenotypes in the same cell and as a single assay, and can be adapted to many other cell types and organisms., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Dosage compensation in Drosophila melanogaster involves a 2-fold transcriptional upregulation of the male X chromosome, which relies on the X-chromosome-binding males-specific lethal (MSL) complex. However, how such 2-fold precision is accomplished remains unclear. Here, we show that a nuclear pore component, Mtor, is involved in setting the correct levels of transcription from the male X chromosome. Using larval tissues, we demonstrate that the depletion of Mtor results in selective upregulation at MSL targets of the male X, beyond the required 2-fold. Mtor and MSL components interact genetically, and depletion of Mtor can rescue the male lethality phenotype of MSL components. Using RNA fluorescence in situ hybridization (FISH) analysis and nascent transcript sequencing, we find that the effect of Mtor is not due to defects in mRNA export but occurs at the level of nascent transcription. These findings demonstrate a physiological role for Mtor in the process of dosage compensation, as a transcriptional attenuator of X chromosome gene expression., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The nuclear pore complex (NPC) is a massive nuclear envelope-embedded protein complex, the canonical function of which is to mediate selective nucleocytoplasmic transport. In addition to its transport function, the NPC has been shown to interact with the underlying chromatin and to influence both activating and repressive gene regulatory processes, contributing to the establishment and the epigenetic maintenance of cell identity. In this review, we discuss diverse gene regulatory functions of NPC components and emerging mechanisms underlying these functions, including roles in genome architecture, transcription complex assembly, chromatin remodeling, and coordination of transcription and mRNA export. These functional roles highlight the importance of the NPC as a nuclear scaffold directing genome organization and function., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Drosophila larval salivary gland polytene chromosome squashes have been used for decades to analyze genome-wide protein-binding patterns, transcriptional activation processes, and changes in chromatin structure at specific genetic loci. There have been many evolutions of the squashing protocol over the years, with sub-optimal reproducibility and low sample success rate as accepted caveats. However, low sample success rates are an obvious disadvantage when polytene chromosomes are used for more high-throughput approaches, such as genetic or antibody screens, or for experiments requiring high-quality chromosome structure preservation. Here we present an exceptionally reproducible squashing and fluorescence staining protocol, which generates high-quality fluorescence images on well-spread chromosomes. This is followed by our novel, semi-automated MATLAB analysis program used to determine correlations between fluorescence signals of interest at a single site on polytene chromosomes, in a pixel-by-pixel manner. In our case, we have used this approach to assess chromatin changes at genomic sites, ectopically targeted by nuclear pore proteins. The use of our analysis program increases the ability to make unbiased conclusions on changes in chromatin structure, or in protein recruitment to chromatin, regardless of sample variation in immunofluorescence staining. As it is simply based upon differences in fluorescence intensity at a defined location, the provided analysis program is not limited to analysis of polytene chromosome, and could be applied to many different contexts where correlation between fluorescent signals at any particular location is of interest., Competing Interests: Competing interestsAuthors declare no competing interests., (Copyright © 2020 The Authors; exclusive licensee Bio-protocol LLC.)

Spatial repositioning of genes in nuclear space has been extensively linked to regulation of gene expression, but the mechanisms behind this directed movement have remained uncertain. In this issue of Developmental Cell, Wang et al. (2020) describe a nuclear actin-myosin-based pathway driving the movement of activated genes to the nuclear periphery., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Interactions between the genome and the nuclear pore complex (NPC) have been implicated in multiple gene regulatory processes, but the underlying logic of these interactions remains poorly defined. Here, we report high-resolution chromatin binding maps of two core components of the NPC, Nup107 and Nup93, in Drosophila cells. Our investigation uncovered differential binding of these NPC subunits, where Nup107 preferentially targets active genes while Nup93 associates primarily with Polycomb-silenced regions. Comparison to Lamin-associated domains (LADs) revealed that NPC binding sites can be found within LADs, demonstrating a linear binding of the genome along the nuclear envelope. Importantly, we identified a functional role of Nup93 in silencing of Polycomb target genes and in spatial folding of Polycomb domains. Our findings lend to a model where different nuclear pores bind different types of chromatin via interactions with specific NPC sub-complexes, and a subset of Polycomb domains is stabilized by interactions with Nup93., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Nuclear pore complexes (NPCs) are canonically known to regulate nucleocytoplasmic transport. However, research efforts over the last decade have demonstrated that NPCs and their constituent nucleoporins (Nups) also interact with the genome and perform important roles in regulation of gene expression. It has become increasingly clear that many Nups execute these roles specifically through regulation of chromatin state, whether through interactions with histone modifiers and downstream changes in post-translational histone modifications, or through relationships with chromatin-remodeling proteins that can result in physical changes in nucleosome occupancy and chromatin compaction. This review focuses on these findings, highlighting the functional connection between NPCs/Nups and regulation of chromatin structure, and how this connection can manifest in regulation of transcription.

Nuclear pore complexes have emerged in recent years as chromatin-binding nuclear scaffolds, able to influence target gene expression. However, how nucleoporins (Nups) exert this control remains poorly understood. Here we show that ectopically tethering Drosophila Nups, especially Sec13, to chromatin is sufficient to induce chromatin decondensation. This decondensation is mediated through chromatin-remodeling complex PBAP, as PBAP is both robustly recruited by Sec13 and required for Sec13-induced decondensation. This phenomenon is not correlated with localization of the target locus to the nuclear periphery, but is correlated with robust recruitment of Nup Elys. Furthermore, we identified a biochemical interaction between endogenous Sec13 and Elys with PBAP, and a role for endogenous Elys in global as well as gene-specific chromatin decompaction. Together, these findings reveal a functional role and mechanism for specific nuclear pore components in promoting an open chromatin state., (© 2019 Kuhn et al.)

Nuclear genome architecture relies on interactions between the genome and various nuclear scaffolds. One such a nuclear scaffold is the nuclear pore complex (NPC), which in addition to its nuclear transport function, can interact with underlying chromatin. In particular, NPCs have been recently reported to associate with a number of enhancers and superenhancers in metazoan genomes, and select NPC components have been shown to promote the formation of specific genomic loops. Here, we provide a brief overview of current models of enhancer function, and discuss recent evidence that NPCs bind enhancers and contribute to topological genome organization. We also examine possible models of how gene and enhancer targeting to NPCs may contribute to tissue-specific genome architecture and expression programs, including the possibility that NPCs may promote phase separation of transcriptional compartments., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Nuclear pore complex components (Nups) have been implicated in transcriptional regulation, yet what regulatory steps are controlled by metazoan Nups remains unclear. We identified the presence of multiple Nups at promoters, enhancers, and insulators in the Drosophila genome. In line with this binding, we uncovered a functional role for Nup98 in mediating enhancer-promoter looping at ecdysone-inducible genes. These genes were found to be stably associated with nuclear pores before and after activation. Although changing levels of Nup98 disrupted enhancer-promoter contacts, it did not affect ongoing transcription but instead compromised subsequent transcriptional activation or transcriptional memory. In support of the enhancer-looping role, we found Nup98 to gain and retain physical interactions with architectural proteins upon stimulation with ecdysone. Together, our data identify Nups as a class of architectural proteins for enhancers and supports a model in which animal genomes use the nuclear pore as an organizing scaffold for inducible poised genes., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Knowing the configuration of the nuclear pore is essential for appreciating the underlying mechanisms of nucleo-cytoplasmic communication. Now, Fernandez-Martinez et al. present a high-resolution structure of the cytoplasmic nuclear pore-mRNA export holo-complex, challenging our textbook depiction of this massive membrane-embedded complex., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The eukaryotic cell nucleus houses an organism's genome and is the location within the cell where all signaling induced and development-driven gene expression programs are ultimately specified. The genome is enclosed and separated from the cytoplasm by the nuclear envelope (NE), a double-lipid membrane bilayer, which contains a large variety of trans-membrane and associated protein complexes. In recent years, research regarding multiple aspects of the cell nucleus points to a highly dynamic and coordinated concert of efforts between chromatin and the NE in regulation of gene expression. Details of how this concert is orchestrated and how it directs cell differentiation and disease are coming to light at a rapid pace. Here we review existing and emerging concepts of how interactions between the genome and the NE may contribute to tissue specific gene expression programs to determine cell fate.

The nuclear pore complex is a transport channel embedded in the nuclear envelope and made up of 30 different components termed nucleoporins (Nups). In addition to their classical role in transport, a subset of Nups has a conserved role in the regulation of transcription via direct binding to chromatin. The molecular details of this function remain obscure, and it is unknown how metazoan Nups are recruited to their chromatin locations or what transcription steps they regulate. Here, we demonstrate genome-wide and physical association between Nup98 and histone-modifying complexes MBD-R2/NSL [corrected] and Trx/MLL. Importantly, we identify a requirement for MBD-R2 in recruitment of Nup98 to many of its genomic target sites. Consistent with its interaction with the Trx/MLL complex, Nup98 is shown to be necessary for Hox gene expression in developing fly tissues. These findings introduce roles of Nup98 in epigenetic regulation that may underlie the basis of oncogenicity of Nup98 fusions in leukemia.

In response to infection, the innate immune system rapidly activates an elaborate and tightly orchestrated gene expression program to induce critical antimicrobial genes. While many key players in this program have been identified in disparate biological systems, it is clear that there are additional uncharacterized mechanisms at play. Our previous studies revealed that a rapidly-induced antiviral gene expression program is active against disparate human arthropod-borne viruses in Drosophila. Moreover, one-half of this program is regulated at the level of transcriptional pausing. Here we found that Nup98, a virus-induced gene, was antiviral against a panel of viruses both in cells and adult flies since its depletion significantly enhanced viral infection. Mechanistically, we found that Nup98 promotes antiviral gene expression in Drosophila at the level of transcription. Expression profiling revealed that the virus-induced activation of 36 genes was abrogated upon loss of Nup98; and we found that a subset of these Nup98-dependent genes were antiviral. These Nup98-dependent virus-induced genes are Cdk9-dependent and translation-independent suggesting that these are rapidly induced primary response genes. Biochemically, we demonstrate that Nup98 is directly bound to the promoters of virus-induced genes, and that it promotes occupancy of the initiating form of RNA polymerase II at these promoters, which are rapidly induced on viral infection to restrict human arboviruses in insects.

Functional compartmentalization of the genome relies on interactions between genomic regions and various nuclear scaffolds and macro-complexes. The Nuclear Pore Complex (NPC) is a large nuclear envelope-embedded protein complex, which creates a highly regulated transport channel between the nucleus and the cytoplasm. In addition to its central role in transport, the NPC has been linked to genome compartmentalization via binding to specific regions of the genome and association with gene regulatory machinery. Although originally proposed to preferentially associate with active genes, the NPC has now been implicated in both gene activating and gene silencing processes. Here, we review recent findings that highlight the roles of various components of the NPC in transcriptional activation, transcriptional memory, heterochromatin formation, post-transcriptional gene silencing and RNA processing. Together, these findings suggest that the nuclear pore is utilized as a regulatory platform for a number of distinct gene expression processes and further point to its central role in setting up particular expression environments on the genomic template., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Nuclear pore complexes have recently been shown to play roles in gene activation; however their potential involvement in metazoan transcription remains unclear. Here we show that the nucleoporins Sec13, Nup98, and Nup88, as well as a group of FG-repeat nucleoporins, bind to the Drosophila genome at functionally distinct loci that often do not represent nuclear envelope contact sites. Whereas Nup88 localizes to silent loci, Sec13, Nup98, and a subset of FG-repeat nucleoporins bind to developmentally regulated genes undergoing transcription induction. Strikingly, RNAi-mediated knockdown of intranuclear Sec13 and Nup98 specifically inhibits transcription of their target genes and prevents efficient reactivation of transcription after heat shock, suggesting an essential role of NPC components in regulating complex gene expression programs of multicellular organisms., (2010 Elsevier Inc. All rights reserved.)

Nuclear-pore complexes (NPCs) are large protein channels that span the nuclear envelope (NE), which is a double membrane that encloses the nuclear genome of eukaryotes. Each of the typically 2,000-4,000 pores in the NE of vertebrate cells is composed of multiple copies of 30 different proteins known as nucleoporins. The evolutionarily conserved NPC proteins have the well-characterized function of mediating the transport of molecules between the nucleoplasm and the cytoplasm. Mutations in nucleoporins are often linked to specific developmental defects and disease, and the resulting phenotypes are usually interpreted as the consequences of perturbed nuclear transport activity. However, recent evidence suggests that NPCs have additional functions in chromatin organization and gene regulation, some of which might be independent of nuclear transport. Here, we review the transport-dependent and transport-independent roles of NPCs in the regulation of nuclear function and gene expression.

Chromatin insulators have been implicated in the establishment of independent gene expression domains and in the nuclear organization of chromatin. Post-translational modification of proteins by Small Ubiquitin-like Modifier (SUMO) has been reported to regulate their activity and subnuclear localization. We present evidence suggesting that two protein components of the gypsy chromatin insulator of Dorsophila melanogaster, Mod(mdg4)2.2 and CP190, are sumoylated, and that SUMO is associated with a subset of genomic insulator sites. Disruption of the SUMO conjugation pathway improves the enhancer-blocking function of a partially active insulator, indicating that SUMO modification acts to regulate negatively the activity of the gypsy insulator. Sumoylation does not affect the ability of CP190 and Mod(mdg4)2.2 to bind chromatin, but instead appears to regulate the nuclear organization of gypsy insulator complexes. The results suggest that long-range interactions of insulator proteins are inhibited by sumoylation and that the establishment of chromatin domains can be regulated by SUMO conjugation.

Chromatin insulators are gene regulatory elements implicated in the establishment of independent chromatin domains. The gypsy insulator of D. melanogaster confers its activity through a protein complex that consists of three known components, Su(Hw), Mod(mdg4)2.2, and CP190. We have identified a factor, Drosophila Topoisomerase I-interacting RS protein (dTopors) that interacts with the insulator protein complex and is required for gypsy insulator function. In the absence of Mod(mdg4)2.2, nuclear clustering of insulator complexes is disrupted and insulator activity is compromised. Overexpression of dTopors in the mod(mdg4)2.2 null mutant rescues insulator activity and restores the formation of nuclear insulator bodies. dTopors associates with the nuclear lamina, and mutations in lamin disrupt dTopors localization as well as nuclear organization and activity of the gypsy insulator. Thus, dTopors appears to be involved in the establishment of chromatin organization through its ability to mediate the association of insulator complexes with a fixed nuclear substrate.

Functional compartmentalization of eukaryotic genomes is thought to be necessary for the proper regulation of gene expression. Chromatin insulators or boundary elements have been implicated in the establishment of this compartmentalization, as they may be involved in creating independent chromatin domains. Recent advances in understanding the mechanisms of insulator function suggest a role for boundary elements in determining transcriptional identity of chromatin and in organizing chromatin into structural compartments within the nucleus. Insulators may thus be involved in setting up topological chromatin domains associated with particular transcriptional states.