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.

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.

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.

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.

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.

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.

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.

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.

Eukaryotic cell function depends on the physical separation of nucleoplasmic and cytoplasmic components by the nuclear envelope (NE). Molecular communication between the two compartments involves active, signal-mediated trafficking, a function that is exclusively performed by nuclear pore complexes (NPCs). The individual NPC components and the mechanisms that are involved in nuclear trafficking are well documented and have become textbook knowledge. However, in addition to their roles as nuclear gatekeepers, NPC components—nucleoporins—have been shown to have critical roles in chromatin organization and gene regulation. These findings have sparked new enthusiasm to study the roles of this multiprotein complex in nuclear organization and explore novel functions that in some cases appear to go beyond a role in transport. Here, we discuss our present view of NPC biogenesis, which is tightly linked to proper cell cycle progression and cell differentiation. In addition, we summarize new data suggesting that NPCs represent dynamic hubs for the integration of gene regulation and nuclear transport processes.

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.