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. 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. This study 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, it was 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 this study 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, it was demonstrated 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 (Panda, 2014).

Innate immunity is an evolutionarily conserved mode of defense against invading pathogens. A major facet of innate immunity involves the recognition of pathogen-associated molecular patterns by pattern recognition receptors to initiate signaling pathways to induce antimicrobial gene expression. This system is robust and is the sole mode of protection against invading pathogens in insects and plants. The gene expression programs activated on pathogen detection are tightly orchestrated to regulate downstream immune responses. The best-characterized example is the lipopolysaccharide (LPS)-dependent gene expression program. This response is divided into two stages; within minutes, a rapid primary response independent of new protein synthesis is initiated, which instructs the downstream translation-dependent secondary response. Many primary response genes have active chromatin marks and features of transcriptional pausing, including high occupancy of the initiating form of RNA polymerase II (RNAPII), S5 phosphorylated (S5P), along with negative elongation factor complex (NELF) and DRB Sensitivity- Inducing Factor complex (DSIF), which prevent transcriptional elongation. Paused RNAPII can be activated by the positive transcription elongation factor b (P-TEFb) in a stimulus-dependent manner, which phosphorylates NELF, DSIF, and RNAPII to release the pause and promote transcriptional elongation and thus the production of mature mRNAs. Indeed, a large number of LPS-induced primary response genes are controlled at the level of pausing including the classical gene TNF-α. Furthermore, this is conserved in Drosophila as the LPS-inducible homolog of TNF-α (Eiger) is also regulated by pausing. Furthermore, depletion of the pausing factor NELF reduced RNAPII occupancy on the promoters of LPS-stimulated genes in Drosophila (Panda, 2014).

Although many signaling pathways that regulate antibacterial and antifungal gene expression programs have been well characterized in insects, a understanding of antiviral gene expression programs is less clear. It was recently found that viral infection can lead to a rapid antiviral gene expression program, and that one-half of these virus-inducible genes are regulated at the level of transcriptional pausing (Xu, 2012). That study also found that NELF is required for RNAPII occupancy at these pausing-regulated genes. These data suggest a conserved role for this mode of gene regulation in the control of antiviral gene expression; however, whether there are specific factors required to promote high RNAPII occupancy at these promoters or to promote the future activation at particular loci remains unclear (Panda, 2014).

Nucleoporins (Nups), first identified for their role in nuclear-cytoplasmic transport, have recently been found to have roles outside of the nuclear pore. Initially, a subset of Nups was found to be mobile, able to move off and on the pore. The intranuclear accumulation of one such Nup, Nup98, is linked to ongoing nuclear transcription and chemical inhibition of RNA polymerase II was shown to abrogate its intranuclear mobility. Moreover, Nup98 was subsequently found to directly control gene expression of a subset of developmentally regulated genes. Nup98 is recruited to these loci during developmental transcriptional activation, and this association is required for the expression of such genes, particularly for the rapid induction of hormoneactivated developmental gene targets. It was recently shown that Nup98 is similarly involved in the transcriptional regulation of IFN-γ-induced gene expression, suggesting that the transcriptional roles of Nups may be involved in immunity (Panda, 2014).

Based on these findings, and given the fact that many developmental and immune genes are regulated by transcriptional pausing, it is hypothesized that Nup98 also may regulate virus-induced antiviral genes. Nup98 was found to play a broadly antiviral role in insects; cells or adult flies depleted of Nup98 are more susceptible to infection against a panel of disparate RNA viruses. This includes human arboviruses from diverse families of viruses: Sindbis virus (SINV), an alphavirus; vesicular stomatitis virus (VSV), a rhabdovirus; and West Nile virus (Kunjin strain), a flavivirus. Interestingly, this study found that in the experimental system that was used, transient depletion of Nup98 did not affect general nuclear pore function; nuclear import of NFκB and nuclear export of mRNAs and proteins were intact. This led us to the discovery of a role for Nup98 in promoting antiviral gene expression in Drosophila. Through transcriptional profiling, it was found that 36 genes were virus-induced and Nup98-dependent. Importantly, a subset of Nup98-dependent virus-induced genes were antiviral themselves, suggesting that Nup98 directly regulates expression of these antiviral genes. Moreover, single-molecule RNA fluorescent in situ hybridization (FISH) revealed reduced levels of virus-induced mRNAs, but not their localization, again supporting a role in the direct regulation of gene expression at the level of transcription. These Nup98- dependent virus-induced genes are translation-independent and regulated by the pausing-release factor P-TEFb. Mechanistically, Nup98 was found to binds to the promoter of these antiviral genes and positively regulates the levels of active RNAPII at these loci at the basal state. Taken together, the data suggest that Nup98 binds to these loci and facilitates the engagement of RNAPII; subsequent virus challenge signals P-TEFb to activate transcription at these loci, inducing antiviral gene expression. These findings demonstrate a previously unidentified requirement for Nup98 in antiviral defense via direct transcriptional initiation of antiviral genes and its coordination with transcriptional pausing to restrict viral infection (Panda, 2014).

The classically described role for Nups is within the nuclear pore complex, with specific functions in the transport of macromolecules in and out of the nucleus; however, recent studies have found both on-pore and off-pore roles for a subset of Nups. Using two independent assays, this study found no defect in nuclear export or import on depletion of Nup98 or several other nuclear pore proteins. This finding is consistent with two recent studies that found no role for Nup98 in nuclear transport when Nup98 was compromised (Light, 2013; Sabri, 2007). In addition, using single-molecule RNA FISH, this study detected no defect in nuclear export of B52 or CG9008 mRNA on depletion of Nup98. Nup98 may regulate the transport of additional antiviral genes, but nevertheless the results suggest that a subset of antiviral genes is regulated by Nup98 at the transcriptional level. Altogether, this suggests an off-pore transport-independent role for Nup98 in antiviral defense (Panda, 2014).

Viral infection leads to the rapid induction of an antiviral transcriptional response. In metazoans, RNAPII recruitment and activation are known to regulate signal-dependent gene transcription; however, the factors involved in recruiting and stabilizing RNAPII at the promoter in a context and gene-specific manner in response to diverse stimuli, including viral infection, are unclear. This study has demonstrated that Nup98 depletion reduces the level of RNAPII S5 phosphorylated (S5P) at the promoters of virally induced genes and, consequently, the level of transcripts. This suggests that Nup98 either promotes recruitment of RNAPII or maintains the initiating form of RNAPII at the promoters to facilitate transcription. Along with identifying this Nup98-dependent pathway in antiviral defense, the findings also shed light on the mechanistic involvement of Nups in transcription. Although several studies have identified a functional role for Nups in transcriptional activation, the detailed mechanism behind this role has not been fully deciphered. The current results suggest a specific step in the transcriptional process, RNAPII S5 activity at the target gene promoter, which is regulated by Nup98. It was recently shown that transcriptional pausing regulates one-half of the virus-induced genes in Drosophila, which is significantly enriched compared with the genome as a whole (Xu, 2012; Panda, 2014 and references therein).

Transcriptionally paused genes have high occupancy of RNAPII S5P, are primary response genes, and are dependent on P-TEFb for their induction. This study found that the seven virus-induced Nup98-dependent genes that were validated are primary response genes; there are high levels of RNAPII S5P at the promoters basally, and they are dependent on P-TEFb for their induction. This suggests that Nup98 maintains active RNAPII at these genes, keeping them poised for future activation by PTEFb. In the absence of Nup98, along with the loss of RNAPII S5P, these antiviral genes are no longer efficiently induced, resulting in elevated levels of viral infection. This model is consistent with the recent results demonstrating recruitment of Nup98 to the promoter of developmentally regulated genes independent of transcription elongation, because Nup98 recruitment is insensitive to flavopiridol treatment, and that many developmental genes are known to be regulated at the level of pausing. Consistent with the hypothesis that Nup98 is a specific transcription activator, it also has been demonstrated that Nup98 interacts with histone-modifying enzymes CBP/p300 and histone deacetylases (Kasper, 1999; Bai, 2006) (Panda, 2014).

Given that Nup98 regulates gene expression of developmental genes in mammals and Drosophila and this study observed regulation of antiviral genes in Drosophila, it is suggested that Nup98 may have functions in regulating cell intrinsic antiviral gene expression in mammalian systems. Indeed, it has long been recognized that Nup98 is involved in antiviral defense (Enninga, 2002, Satterly, 2007; Chen, 2010; von Kobbe, 2000). Nup98 depletion reduces the nuclear export of specific immune regulated genes, including IFN-stimulated genes (Satterly, 2007). Furthermore, viral infection was enhanced in Nup98-deficient MEFs (Satterly, 2007). These antiviral phenotypes were ascribed to transport defects; however, it has not been explored whether Nup98 also directly regulates the induction of antiviral primary response genes. Interestingly, a recent study found that Nup98 impacts the regulation of IFN-γ-responsive genes (Light, 2013). In this study, Nup98 was required to set the reactivation state of IFN-γ-responsive genes without affecting the initial activation (Light, 2013). Mechanistically, the study found that Nup98 binds the promoter of IFN-γ-responsive genes and is required for the maintenance of histone H3K4 dimethylation during transcriptional memory (Light, 2013). Whether Nup98 affects the transcriptional memory of immune-regulated genes in Drosophila remains to be seen (Panda, 2014).

Nuclear pore complex components (Nups) have been implicated in transcriptional regulation, yet what regulatory steps are controlled by metazoan Nups remains unclear. This study identified the presence of multiple Nups at promoters, enhancers, and insulators in the Drosophila genome. In line with this binding, a functional role for Nup98 was uncovered 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, Nup98 was found to gain and retain physical interactions with architectural proteins upon stimulation with ecdysone. Together, these 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 (Pascual-Garcia, 2017).

Together, the results identify a specific transcriptional function for Nup98 in enhancer looping and reveal Nups as a class of architectural proteins that in addition to cohesins, CTCF, and Mediator components can promote enhancer-promoter interactions. Interestingly, the NPC has been recently shown to associate with the Mediator complex and to depend on a specific Mediator subunit, Med31, for binding the GAL1 gene in yeast. The current genome-wide analysis and identification of physical interactions with architectural proteins further suggest that Nups may contribute to other topological contacts, such as those of insulators. This notion is further supported by previous links of Nups to insulators in both yeast and fly cells (Pascual-Garcia, 2017).

Somewhat surprisingly, this study found that similarly to depletion of Nup98, Nup98 overexpression appears to disrupt the enhancer-promoter contact and transcriptional memory. This is likely due to the involvement of Nup98 in self-interactions, which can lead to aberrant oligomerization and mis-localized chromatin binding of Nup98 that prevent its participation in productive contacts. The biophysical properties of the intrinsically disordered FG domains of Nup98 involve the ability to polymerize and phase-separate. Given recent implications of intrinsically disordered and phase-separating proteins in transcriptional regulation and nuclear structure, it will be particularly interesting to determine whether these properties of the FG domains underlie the identified functions of Nup98 in enhancer-promoter looping or in epigenetic memory of transcription (Pascual-Garcia, 2017).

The implications of these findings are potentially far reaching in the context of developmental gene regulation. Establishment and maintenance of appropriate enhancer-promoter contacts may be a general mechanism by which Nups contribute to regulation of gene expression during cell differentiation. Mutations in multiple Nups lead to defects in differentiation of specific cell lineages in the fly, zebrafish, and mouse models. The identified roles of the NPC in genome architecture may represent one of the mechanisms by which Nups contribute to tissue-specific processes. In addition to ecdysone-inducible genes, chromatin binding analysis identified Hox genes as a recurrent target of NPC binding, previous work has identified a physical association between Nup98 and an epigenetic regulator of Hox gene activity, Trithorax (Trx). Trx and its mammalian homologs are HMTs responsible for methylation of H3 lysine K4, which is in line with the previously demonstrated dependence of promoter-associated H3K4 di-methylation levels on Nup98 in yeast and human cells. It remains to be determined how enhancer-promoter looping is integrated with H3K4 di-methylation in transcriptional memory, but an intriguing possibility is the potential role of Nup98-stabilized enhancer-promoter contacts in Trx-mediated epigenetic regulation during development (Pascual-Garcia, 2017).

The results also provide an example of uncoupling activation-induced enhancer-promoter contacts from transcriptional activation. A common model for how enhancers function proposes that enhancers loop over to contact their target promoters to drive transcriptional activation, implying a direct cause to effect relationship from looping to transcription. But the current data suggest that this relationship is not always this linear and can involve stabilization of enhancer-promoter loops that are not required for ongoing transcription. The increased enhancer-promoter interaction described in this study appears to function as part of a memory system through a period of repression (Pascual-Garcia, 2017).

Furthermore, genome-wide analysis of Nup binding revealed that nuclear pores target both active and silenced loci in similar proportions. PcG-bound regions, marked by the H3K27Me3 mark, appear to be common targets of the stable NPCs, which is supported by a previous report of Nup153 playing a role in PcG-mediated gene silencing. On the basis of the rest of the current findings, it is proposed that a specific subset of genes associates with NPCs independently of expression, for the purposes of stabilizing possible or future activation-induced long-range contacts. Although the transcriptional memory function of Nup98 is not limited to NPC targets and has been shown to apply to intranuclear genes, it is possible that NPC binding may be particularly beneficial for genes with complex regulatory landscapes, such as ecdysone-inducible or Hox genes, where pre-scaffolding and epigenetic maintenance of enhancer-promoter architecture is most critical (Pascual-Garcia, 2017).