The Interactive Fly

Zygotically transcribed genes

Genes regulating nucleo-cytoplasmic transport

  • Pre-assembled nuclear pores insert into the nuclear envelope during early development
  • Nuclear pores assemble from Nucleoporin condensates during oogenesis
  • A negative loop within the nuclear pore complex controls global chromatin organization
  • Core components of the nuclear pore bind distinct states of chromatin and contribute to Polycomb repression
    Nuclear pore complex and venes regulating nucleo-cytoplasmic transport



    Pre-assembled nuclear pores insert into the nuclear envelope during early development

    Nuclear pore complexes (NPCs) span the nuclear envelope (NE) and mediate nucleocytoplasmic transport. In metazoan oocytes and early embryos, NPCs reside not only within the NE, but also at some endoplasmic reticulum (ER) membrane sheets, termed annulate lamellae (AL). Although a role for AL as NPC storage pools has been discussed, it remains controversial whether and how they contribute to the NPC density at the NE. This study shows that AL insert into the NE as the ER feeds rapid nuclear expansion in Drosophila blastoderm embryos. NPCs within AL resemble pore scaffolds that mature only upon insertion into the NE. This paper delineates a topological model in which NE openings are critical for AL uptake that nevertheless occurs without compromising the permeability barrier of the NE. This unanticipated mode of pore insertion is developmentally regulated and operates prior to gastrulation (Hampoelz, 2016).

    In eukaryotes, the double membranous nuclear envelope (NE) encloses the nucleoplasm and separates it from the cytoplasm. The inner nuclear membrane (INM) provides contact with chromatin and the outer nuclear membrane (ONM) is continuous with the endoplasmic reticulum (ER). The two bilayers are fused at nuclear pore complexes (NPCs) that form aqueous channels through which regulated transport of macromolecules occurs. NPCs consist of multiple copies of ~30 different nucleoporins (Nups) that are organized into biochemically distinct sub-complexes. Two such modules, the inner ring complex (also called Nup93 complex) and the Y-complex (also called Nup107 complex) constitute the NPC scaffold that is symmetric across the NE plane. FG-Nups (containing phenylalanine-glycine rich intrinsically disordered protein domains) dock onto the scaffold. They constitute the permeability barrier and interact with translocating cargo complexes. Some of them (e.g., Nup214/88, Nup358 [RanBP2], and Nup153) introduce asymmetry by specifically binding to the cytoplasmic or nuclear face of the NPC, respectively (Hampoelz, 2016).

    Obviously, the sheer size and compositional complexity of NPCs renders its assembly and membrane insertion a very intricate task. Two distinct NPC assembly pathways that are temporally separated during the cell cycle have been described. First, during interphase, NPCs are assembled de novo onto an enclosed NE. Interphase assembly occurs ubiquitously throughout eukaryotes and strictly requires the fusion of the INM and ONM by a mechanism that is only partially understood. Second, no membrane fusion is required for NPC assembly at mitotic exit. This so-called postmitotic assembly mode is restricted to eukaryotes that disassemble their NPCs during mitosis into soluble sub-complexes after phosphorylation by mitotic kinases. In anaphase, de-phosphorylation of Nups is thought to trigger the ordered re-assembly onto the separated chromatids before or while membranes enclose daughter nuclei. Both insertion mechanisms rely on the stepwise recruitment of pre-assembled sub-complexes. An insertion of pre-assembled NPCs into the NE has not yet been described (Hampoelz, 2016).

    NPCs not only reside within the NE but are also found in stacked cytoplasmic membranes termed annulate lamellae (AL) that are a subdomain of the ER. Based on two-dimensional (2D) transmission electron micrographs these membrane stacks have been perceived as parallel membrane sheets decorated with NPCs (hereafter called AL-NPCs) that morphologically appear similar to their counterparts on the nuclear envelope (NE-NPCs). AL appear in some but not all transformed cell lines and are highly abundant in germ cells and early embryos throughout animal phyla, including Xenopus, Caenorhabditis elegans, sea urchin, Drosophila, and also humans. A role of AL as a storage compartment for maternally deposited Nups that can be made available for meiosis and the rapid cell cycles during early embryogenesis has been suggested but not experimentally proven. Despite these fundamental and long-standing pretensions the function of AL remains elusive and controversial, primarily for two reasons: (1) it has been difficult to conceive how the insertion of parallel stacked membrane sheets containing pre-assembled and possibly pre-oriented NPCs is topologically possible; and (2) direct experimental evidence for a contribution of AL-NPCs to the pool of NE-NPCs has never been obtained. On the contrary a previous study in Drosophila embryos has detected large soluble pools of transport channel Nups and concluded that NPC insertion likely proceeds from soluble cytosolic Nups (Hampoelz, 2016).

    This study addressed the function of AL in the physiological context of the Drosophila blastoderm embryo that is rich in AL, while it undergoes a series of 13 synchronized mitoses in a syncytium. Subsequently, the plasma membranes enclose the cortically aligned somatic nuclei in the extended 14th interphase, forming the first epithelial cell layer before the embryo initiates gastrulation. This occurs concomitantly with the broad onset of transcriptional activity on the zygotic genome, a major developmental transition present in all metazoan. In the syncytial blastoderm, cell-cycle progression is very rapid, with interphase durations of ~10 min during the early cell cycles. At least in mammalian cells, de novo NPC interphase assembly has been described to proceed with markedly slower kinetics. This led to a hypothesis that NPC assembly into a closed NE in Drosophila embryos might occur by a different, faster mechanism. By tracking NPCs in living embryos, this study demonstrates direct uptake of AL-NPCs into the NE, as the ER feeds nuclear expansion. A topological model was derived that explains how the INM becomes continuous with inserting membrane sheets from the ER. It is concluded that AL insertion to the NE is a previously unanticipated mode of NPC insertion that relies on pre-assembled, yet immature NPC scaffolds and operates prior to gastrulation (Hampoelz, 2016).

    Collectively, the following scenario emerges from the data. AL are abundant in early Drosophila embryos and predominantly contribute to maintain the constant NE-NPC density in the expanding NE during interphase. The abundance of AL at the cortical nuclei layer thereby oscillates together with the progression of the consecutive interphases until the start of global transcription when AL disappear and the mode of NPC insertion changes. During each onset of early interphases, AL-NPCs are assembled similarly to NE-NPCs but since the combined nuclear surface of the two daughter nuclei is smaller as compared to the parental nucleus, they remain in the cytoplasm. As interphases progress, AL-NPCs feed into the pool of NE-NPCs alongside ER membranes that augment NE surface during rapid nuclear expansion. AL insertion is enabled by NE openings that might either persist from previous mitosis or form de novo by an unknown mechanism. Upon AL insertion, the NE permeability barrier remains unperturbed, likely because the NE openings are entirely surrounded by the ER network. The inserting NPCs comprise pre-assembled NPC scaffolds that recruit the full set of Nups only subsequent to insertion and only then establish transport competence (Hampoelz, 2016).

    Why do the expanding nuclei of the syncytial blastoderm maintain a constant number of NPCs per surface area despite their transcriptional inactivity? One might surmise that this is due to mechanical properties but also temporal constraints. The insertion of NPCs might be crucial to enable the massive influx of material into the nucleoplasm during nuclear expansion (volume increase). Indeed, the strained configuration of nuclei is reflected by their strong mechanical response (NE tumbling) upon disruption of the NE and permeability barrier after laser puncture. Second, the batch transfer of entire NPC scaffolds as inherent parts of membrane sheets overcomes the described kinetic constrains of interphase assembly in mammalian cells, that are not compatible with the short interphases in the Drosophila syncytium. Given the abundance of AL-NPCs and the reported high insertion rate of NPCs into the NE of Xenopus leavis oocytes it appears likely that similar mechanisms operate in vertebrates. It remains unclear how sufficient amounts of AL are generated to globally feed nuclear surface expansion over multiple cell cycles until the start of transcription. However, Nups are maternally provided and AL are abundant not only at the cortical layer of nuclei but also within the interior of the embryo. Therefore, a possibility that needs to be considered is that a source of AL-NPCs already generated during oogenesis feeds nuclear growth throughout the syncytial blastoderm (Hampoelz, 2016).

    In addition to their eminent role in transport, NE-NPCs organize the nuclear periphery by delineating zones of active euchromatin as compared to transcriptionally repressed heterochromatin in between pores. Crucial to this is that NPCs are laterally immobile within the NE, which was shown to depend on the nuclear lamina. Lamins are nuclear intermediate filament proteins and come in two major types: B-type Lamins are ubiquitous, while A-type Lamins are expressed exclusively when cells differentiate. Both proteins engage in distinct meshworks and also impact on NPC insertion rate. This work puts NPC organization and the mode of pore insertion into a developmental context. It is proposed that in Drosophila AL insertion is innate to earliest embryogenesis and diminishes when pores get laterally restricted and cluster at the NE. There are no A-type lamins expressed at that stage, and specifically expressed INM proteins could be crucial. Intriguingly, the formation of immobile pore clusters coincides with the transcriptional upregulation of hundreds of genes at zygotic induction, a developmental transition present in all metazoan that is accompanied by characteristic changes in chromatin signatures. This study revealed that the zygotically upregulated INM protein LBR, a developmentally controlled INM tether of peripheral heterochromatin, is sufficient to prematurely aggregate NPCs in blastoderm interphases, when artificially expressed earlier in embryogenesis. This also leads to larger AL likely because LBR counteracts AL insertion for which lateral NPC mobility is required. The data suggest a zygotically induced regulation that links pore insertion and organization, NE composition and ultimately also chromatin organization at the nuclear periphery. All of these events eventually contribute to the commitment of originally pluripotent somatic nuclei into distinct lineages (Hampoelz, 2016).

    Nuclear pores assemble from Nucleoporin condensates during oogenesis

    The molecular events that direct nuclear pore complex (NPC) assembly toward nuclear envelopes have been conceptualized in two pathways that occur during mitosis or interphase, respectively. In gametes and embryonic cells, NPCs also occur within stacked cytoplasmic membrane sheets, termed annulate lamellae (AL), which serve as NPC storage for early development. The mechanism of NPC biogenesis at cytoplasmic membranes remains unknown. This study shows that during Drosophila oogenesis, Nucleoporins condense into different precursor granules that interact and progress into NPCs. Nup358 is a key player that condenses into NPC assembly platforms while its mRNA localizes to their surface in a translation-dependent manner. In concert, Microtubule-dependent transport, the small GTPase Ran and nuclear transport receptors regulate NPC biogenesis in oocytes. This study has delineated a non-canonical NPC assembly mechanism that relies on Nucleoporin condensates and occurs away from the nucleus under conditions of cell cycle arrest (Hampoelz, 2019).

    Nuclear pore complexes (NPCs) bridge the nuclear envelope (NE) and mediate nucleocytoplasmic exchange. They are giant assemblies of about 110 MDa in animals with an elaborate structure and composition. About 30 different genes encode for NPC components, termed nucleoporins (Nups). Those are subclassified into scaffold Nups that assemble into a cylindrical architecture with a ~50 nm wide central channel; and intrinsically disordered phenylalanine-glycine rich FG-Nups that line this channel. Scaffold Nups assemble into the so-called Y and inner ring complexes that form the outer and inner rings, respectively (see The Nuclear Pore Complex as a Flexible and Dynamic Gate). FG-Nups (containing phenylalanine-glycine repeats) have the capacity to phase separate in vitro. In vivo, they establish a unique biophysical milieu within the central channel that is impermeable to inert molecules. FG-Nups transiently interact with nuclear transport receptors (NTRs, also called importins, exportins, or karyopherins) that form complexes with cargo and cross the permeability barrier. Transport directionality across the NE is ensured by the small GTPase Ran. RCC1, the RanGTP exchange factor (RanGEF) is chromatin associated and maintains a high RanGTP concentration in the nucleus. The Ran GTPase activating protein (RanGAP) binds to Nup358 (also called RanBP2) at the cytoplasmic face of the NPC and ensures high RanGDP levels in the cytosol. Although RanGTP displaces cargo from import complexes in the nucleoplasm, GTP hydrolysis disassembles export complexes once they arrive at the cytoplasmic face. Nup358 is absent from lower eukaryotes but essential in animals and involved in active nuclear transport, cell cycle progression, malignant transformation, and viral infection (Hampoelz, 2019).

    NPC assembly is an intricate process. In multicellular organisms, two assembly pathways were described. First, the relatively rapid assembly of NPCs from pre-existing building blocks concomitantly with nuclear envelope (NE) reformation at the end of open mitosis is referred to as 'post-mitotic' assembly. This pathway is spatially directed to chromatin by the Nup Elys. Temporal control is provided by cell-cycle-dependent kinases and phosphatases. Second, interphase assembly is a relatively slow process that generates NPCs from scratch in order to double their number for the next mitosis. It proceeds from inside out through the NE and requires the active nuclear import of Nups. Here, Nup153 spatially directs the Y complex to the inner nuclear membrane (Vollmer, 2015; Hampoelz, 2019 and references therein).

    Little is known about the early steps of NPC assembly that occur prior to membrane association. FG-Nups serve as a velcro for scaffold Nups. They, however, have a considerable aggregation propensity in isolation that has to be controlled during NPC biogenesis in vivo. Non-NPC-associated Nups are chaperoned by importin β. RanGTP dissociates importin β complexes and thereby releases Nups for interphase and post-mitotic NPC assembly. Likewise, RanGTP induces NPC assembly in vitro, but the in vivo relevance of this finding remains to be tested (Hampoelz, 2019).

    In multicellular organisms, nuclear pores also reside in stacked membrane sheets of the endoplasmic reticulum (ER), termed annulate lamellae (AL). Those are particularly prominent in gametes and embryos of a multitude of species including Drosophila. In early fly embryos, AL insert into the NE in order to supply the rapidly growing nuclei with additional membranes and NPCs (Hampoelz, 2016). AL are therefore thought to be maternally provided NPC storage pools. How AL assemble in the absence of a nuclear compartment, which spatially coordinates the process in case of the two previously characterized pathways, remains elusive. This study has investigated AL-NPC biogenesis in vivo during Drosophila melanogaster oogenesis. AL-NPC biogenesis was shown to be vastly abundant during oogenesis. It depends on the condensation of Nups into compositionally different granules that are transported along microtubules (MTs) and regulated by Nup358 in concert with Ran and NTRs. This NPC biogenesis is mechanistically distinct from both canonical NPC assembly pathways and progresses away from chromatin. It is proposed that instead, Nup358 condensates fulfill the role of spatially directing NPC biogenesis, in the absence of a bona fide nuclear compartment (Hampoelz, 2019).

    Little has been known about the biogenesis of AL and the spatial cues that allow NPC formation away from the nuclear compartment. This work addresses these questions during Drosophila oogenesis and suggests a third, non-canonical NPC assembly mechanism. Already in nurse cells, Nup358 condenses into large granules (see A Model for NPC Biogenesis beyond the Nuclear Compartment). Condensation might be fostered by local translation of nup358 transcripts that enrich at the surface of Nup358 granules in a translation dependent manner. In nurse cells, AL biogenesis is suppressed, and only limited NPC assembly is observed within Nup358 granules. This could be due to the available amount or configuration of ER membranes or because high cytoplasmic concentrations of RanGDP promote the formation of Importin-Nup complexes that prevent other Nups from condensation. Nup358 granules become assembly platforms for AL-NPCs once they travel through ring canals into the ooplasm. Scaffold and FG-Nups condense into oocyte specific granules, possibly facilitated by elevated levels of RanGTP that dissociates the respective Nups from Importin. In the oocyte, NPC precursor granule interactions are promoted by MT dependent granule dynamics. Upon interaction, granules transfer material and assemble Nups onto available ER membrane, ultimately leading to the formation of larger stacks with multiple membrane sheets. Those are inherited to the embryo where they supplement dividing nuclei with NPCs throughout early embryogenesis (Hampoelz, 2019).

    The phase-separating properties of FG-Nups have been subject to extensive research in vitro. This study provides evidence for condensation of Nups in vivo. Several properties, namely the coalescence of Nup358 granules, the transfer of material between granules, the high molecular mobility within granules, and the contact shapes observed upon granule interactions, are hallmarks of biomolecular condensates. Such condensates are defined as 'non-membranous organelles'. Although AL inherently contain stacked membrane sheets, they retain at least some characteristics of a phase separated condensate such as a milieu that is distinct from the surrounding cytoplasm. These findings underline the importance of phase separation at membranes that was also observed in other biological systems (Hampoelz, 2019).

    Several lines of evidence, namely 1,6-hexanediol treatment, depletion of BicD and embargoed, and the interference with the Ran nucleotide status suggest that condensation of Nups into NPC precursor granules is critical for AL biogenesis. It is further underlined by the fact that Colchicine treatment counteracts the RanQ69L phenotype by reducing the number of MT-promoted granule interactions. Condensation concentrates NPC constituents in a constrained volume within the large ooplasm and might prevent unspecific interactions of soluble Nups. MT dynamics enhances interactions of otherwise unmixed, compositionally heterogeneous NPC precursor condensates and is a prerequisite for NPC assembly from condensed granules. It also prevents unwanted fusion and relaxation of compositionally homogeneous condensates of the same type. Facilitated interactions of granules could be of particular importance in the highly viscous ooplasm, where cytoskeleton-induced streaming is critical for the efficient distribution of various components (Hampoelz, 2019).

    The two canonical NPC assembly pathways rely on the stepwise and orchestrated assembly of soluble Nups or subcomplexes onto either anaphase chromatin or the NE surface during interphase, respectively. However, these spatial cues are absent in the ooplasm and alternative mechanisms to locally concentrate assembly modules must be important. It is believed that the condensation of Nups replaces the canonical cues, in line with previous work that had shown functions of natively unfolded FG-Nups to stabilize each other but also NPC scaffold components during yeast NPC assembly. Controlled interactions and material transfer between condensates might account for specific steps of assembly and even provide a certain order, although this concept remains to be further tested (Hampoelz, 2019).

    The data strongly suggest Nup358 granules as assembly platforms, where NPCs are seeded onto ER membranes. Nup358 has no reported role in initiating the assembly process in both previously described pathways (Weberruss, 2016). On the contrary, during interphase Nup358 assembles rather late onto the NPC scaffold. Although such information is not available for post-mitotic assembly, its mitotic localization to kinetochores could indicate an early role for Nup358. Indeed, Nup358 is of structural importance for the pore scaffold, given that its loss destabilizes the outer Y complex at the cytoplasmic ring at NE-NPCs. It is thus conceivable that Nup358 could not only stabilize but also recruit scaffold components onto membranes. Post-mitotic and interphase NPC assembly are initiated by two distinct Nups, Elys and Nup153, respectively. Elys localizes the Y complex onto anaphase chromatin and is dispensable for interphase assembly but also for AL formation, given that its depletion induces AL. In contrast, Nup153 seeds NPCs during interphase assembly onto the inner nuclear membrane, and it has been suggested to have a similar role at the ER during AL biogenesis. This study, however, found that Nup153 is absent from AL in oocytes(Hampoelz, 2019).

    Despite all molecular and conceptual differences, the common driving force for NPC biogenesis at and beyond the nucleus is Ran that coordinates the availability of Nups for assembly by dissociating them from NTRs. Nup358 binds RanGAP and directly links the NPC to Ran activity. At the NE this is eminent to ensure a sharp Ran gradient and thus efficient nucleocytoplasmic transport. This study shows that this interaction is preserved beyond nuclei, because RanGAP strongly enriches at Nup358 granules in a Nup358-dependent manner. One might speculate that within the RanGTP milieu of the ooplasm, RanGAP induces a local Ran gradient at Nup358 granules that drives NPC biogenesis; conceptually similar to the nuclear compartment for interphase or postmitotic NPC assembly (Figure 7C and 7C'). Thereby, the observed progressive dilution of Nup358 and RanGAP at Nup358 granules in the oocyte could be important to drive their progression into AL. It might be caused by ooplasmic RanGTP that favors complex formation between Crm1 and Nup358. Although this would be consistent with the observed embargoed gene silencing phenotype, the enhanced condensation of Nup358 upon global induction of RanGTP argues for an alternative interpretation: Nup358 functionally interacts with both, Importins and Crm1 under specific conditions. It is thus not clear how exactly it is being chaperoned. The phenotype observed under embargoed gene silencing conditions might be indirectly caused by disturbance of the spatial distribution of Ran and NTRs across nurse cells and oocytes, as indicated by the variety in phenotype across individuals. Yet it stresses the importance of spatially controlled Nup condensation to assemble AL-NPCs. In any scenario, NTR-mediated de-condensation of Nup358 and the consequent reduction of local RanGAP activity would regulate the progression of NPC biogenesis by determining the availability of soluble, 'assembly prone' Nups and the degree of mixing at granule interfaces (Hampoelz, 2019).

    Various aspects of AL-NPC biogenesis are markedly different from both canonical NPC assembly pathways. During oogenesis, Nup condensation, local translation and MT dependent dynamics interplay with Ran activity in order to faithfully assemble AL in oocytes. They are inherited to the embryo where this pool of ready-made NPCs supplements nuclei during the rapid interphases of the blastoderm stage. Because AL are present in a plethora of species, similar mechanisms are likely to operate throughout animals (Hampoelz, 2019).

    A negative loop within the nuclear pore complex controls global chromatin organization

    The nuclear pore complex (NPC) tethers chromatin to create an environment for gene regulation, but little is known about how this activity is regulated to avoid excessive tethering of the genome. Tethering specific genomic loci to the NPC appears to contribute to transcriptional activation. Also, the NPC has been further implicated in creating a repressive environment or retaining genes at the periphery after repression, possibly contributing to epigenetic transcriptional memory. This paper proposes a negative regulatory loop within the NPC controlling the chromatin attachment state, in which Nup155 and Nup93 recruit Nup62 to suppress chromatin tethering by Nup155. Depletion of Nup62 severely disrupts chromatin distribution in the nuclei of female germlines and somatic cells, which can be reversed by codepleting Nup155. See a model for the chromatin attachment state controlled by an internal regulatory circuit in the NPC. Thus, this universal regulatory system within the NPC is crucial to control large-scale chromatin organization in the nucleus (Breuer, 2015).

    Cytological study of the chromatin attachment state to the nuclear envelope is experimentally challenging, as chromatin usually occupies the entire nucleus. However, meiotic chromatin becomes fully detached from the nuclear envelope and compacted into a spherical structure, the karyosome, after recombination in Drosophila oocytes. Chromatin detachment and karyosome formation are crucial to make a single spindle and allow subsequent chromosome segregation and are conserved features also seen in mammalian oocytes. By taking advantage of this unique nuclear organization in oocytes, this study sought factors required for chromatin detachment from the nuclear envelope by individually knocking down various nuclear proteins in the female germline (the oocyte and nurse cells) by RNAi (Breuer, 2015).

    Strikingly, the depletion of either of two nuclear pore proteins, Nup62 or Nup93, led to disruption of the compact karyosome morphology, while the depletion of several other pore proteins did not. The chromatin shifted near the nuclear periphery, resulting in strong (Nup62) or partial (Nup93) overlap with a nuclear pore marker in the oocytes in comparison with the control RNAi, which was confirmed by superresolution microscopy. Nup93 is a linker scaffold protein known to be required for the recruitment of Nup62, one of the central channel proteins containing FG repeats (Sachdev, 2012). It was confirmed that the defect is not an off-target effect by rescue experiments using RNAi-resistant transgenes. In addition, similar karyosome defects were observed in female sterile Nup62 mutants. In nurse cells (polytenized germline cells that support oocyte growth), chromatin also distributed irregularly and more toward the nuclear periphery after RNAi of these genes. This demonstrates a general role for both genes in global chromatin organization rather than being restricted to oocytes (Breuer, 2015).

    To identify the cause of the karyosome defect upon Nup62 or Nup93 RNAi, the structural integrity and transport function of NPC was tested. RNAi of Nup62 or Nup93 did not disrupt the overall structural integrity of the NPC, as judged by the localization of FG-containing subunits and the core scaffold subunit Nup107. The active import function of the NPC showed small differences as assessed by fluorescence recovery after photobleaching (FRAP) of GFP fused with a nuclear localization signal (NLS). There was a significant increase in the nuclear size of early oocytes, which may be caused by a reduced ability of the nuclear pore to act as a diffusion barrier (Breuer, 2015).

    Next, a relationship was examined with the meiotic recombination checkpoint, which is known to disrupt karyosome formation in the presence of unrepaired double-strand breaks (DSBs) in oocytes. Inactivation of the checkpoint did not suppress the karyosome defects of Nup62 or Nup93 RNAi, demonstrating that the defect is independent of the meiotic recombination checkpoint in oocytes (Breuer, 2015).

    Considering the above results, it was hypothesized that chromatin is excessively anchored to the NPC in RNAi of Nup62 or Nup93. If this was the case, it was predicted that chromatin specifically interacting with the NPC must be preferentially accumulated at the nuclear periphery rather than random chromatin. In order to test this, previously identified genomic loci bound to another nuclear pore component, Nup98, were used in Drosophila S2 culture cells. Nup98 has two distinct populations -- one at nuclear pores and the other in the nucleoplasm -- that bind distinct genomic loci in S2 cells. Nurse cells were subjected to fluorescence in situ hybridization (FISH) using individual probes corresponding to genomic loci known to be associated with Nup98 within the NPC or located in the nucleoplasm in S2 cells and were costained with a DNA dye. A proportion was measured of the total DNA signals in the nuclear periphery region, defined by a distance from the nuclear lamina of <10% of the nuclear radius, which occupies ~20% of the nuclear area. In control RNAi, ~16%-17% of the total DNA (propidium iodide or DAPI signal) was located in the nuclear periphery region. For all genomic loci (three NPC-bound and four nucleoplasmic), 17%-25% of the signal foci were found in the nuclear periphery. This indicates that there is no preference for periphery locations of the total DNA or of these specific genomic regions in wild-type nurse cells. When Nup93 was knocked down, there was a small increase (from 16%-17% to 20%-24%) in the total DNA that occupies the nuclear periphery (Nup93 RNAi was used, since it gives a milder phenotype than Nup62 RNAi). Strikingly, a strong, consistent redistribution of all NPC-bound genomic loci to the periphery (from 17%-25% to >40%) was observed, whereas the nucleoplasmic loci showed smaller variable changes. The increases for NPC-bound loci were significantly higher than the increases for both total DNA and the nucleoplasmic loci, supporting the hypothesis that depletion of Nup62 or Nup93 results in an excessive attachment of specific chromatin regions to the NPC (Breuer, 2015).

    The results suggest that Nup62 and Nup93 suppress the interaction between chromatin and another NPC subunit. If this is the case, codepletion of this hypothetical NPC subunit that mediates chromatin attachment to the nuclear pore should restore detachment of chromatin in Nup62- or Nup93-depleted oocytes. Several NPC subunits have previously been shown to have chromatin-binding activity, including Nup155, Nup50, and ELYS/Mel-28. Flies expressing two shRNAs were generated: one for Nup62 and the other for each of the aforementioned chromatin-binding NPC subunits, the non-chromatin-bound Nup160, or a control. Codepletion of Nup155 specifically restored normal karyosome morphology and detachment from the nuclear periphery in Nup62-depleted oocytes. Furthermore, in nurse cells, simultaneous RNAi of Nup155 also restored normal chromatin distribution caused by Nup62 RNAi. Crucially, codepletion of Nup155 did not rescue the larger nuclear size in Nup62-depleted oocytes. This demonstrates that Nup62's function on chromatin organization is independent of its function on nuclear size maintenance, which may reflect its function as a diffusion barrier (Breuer, 2015).

    It was also found that single depletion of Nup155 led to a large reduction of Nup62 (one of the FG-containing subunits) from the nuclear envelope and its accumulation in the cytoplasm. However, it did not significantly reduce the total amount of the FG-containing subunits at the nuclear envelope in both Nup155 and Nup62/Nup155 double RNAi. This demonstrates that Nup155 is required for Nup62 recruitment, and the apparent rescue of the Nup62 depletion defect by Nup155 codepletion is not due to a loss of integrity or a reduced number of nuclear pores. Taken together, the results suggest a negative regulatory loop in which Nup155 recruits Nup62 to the nuclear pores, and, in turn, Nup62 suppresses chromatin anchoring by Nup155 (Breuer, 2015).

    A potential negative regulatory circuit was uncovered within the NPC that controls the chromatin attachment state to the nuclear pores in the oocytes and nurse cells. Therefore, attempts were made to test whether a common regulatory system also controls chromatin organization in somatic cells. Using the Drosophila S2 cell line, Nup62 or Nup155 were depleted individually and simultaneously by RNAi. Control RNAi cells showed a relatively even distribution of chromatin within the nucleus except for a dense region that corresponds to heterochromatin. In contrast, Nup62 RNAi resulted in an uneven distribution of chromatin within the nucleus. To quantify this, the area that chromatin occupies relative to the nuclear area was measured. The cells depleted of Nup62 showed a significant decrease in chromatin occupancy compared with a control RNAi. Strikingly, double depletion of Nup62 and Nup155 showed a chromatin occupancy similar to the control. This rescue was reversed by RNAi-resistant full-length Nup155 but not by resistant Nup155 lacking the chromatin-binding region. No significant change in chromatin occupancy was observed upon Nup155 depletion alone. This demonstrated the presence of a common negative loop within the NPC that controls the global chromatin distribution between female germline cells and somatic cells (Breuer, 2015).

    Recent reports described the role of the NPC to tether chromatin and thus create an environment for gene regulation. While recruitment mechanisms for specific genes have been described, very little is known about whether or how this tethering is regulated. This study makes two major conceptual advances in understanding of global chromatin organization, especially the critical role and regulation of the NPC-mediated tethering. First, it highlights a far greater role of the NPC in large-scale chromatin organization than previously anticipated. Second, it points to a universal regulatory circuit inside the NPC that controls the attachment state of chromatin to the nuclear pore. This consists of a negative regulatory loop in which chromatin-binding Nup155 recruits the central channel protein Nup62, which in turn suppresses chromatin binding. As nuclear pore components associate with the genome to positively or negatively influence gene expression, this regulatory loop might be part of a wider network for the NPC to control gene expression, depending on the cellular and developmental context. Although a genuine and direct regulatory role of this loop has yet to be demonstrated, its intrinsic capacity supplies the NPC with a key mechanism to globally or locally organize the metazoan genome. On the other hand, any change or imbalance in this regulatory network might have dramatic effects for the nuclear architecture and, concomitantly, the expression profile of the cell. This may have a significant medical implication, as nuclear pore components not only are known to deteriorate with age but are also affected in several tissue-specific human diseases (Breuer, 2015).

    Core components of the nuclear pore bind distinct states of chromatin and contribute to Polycomb repression

    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. This study reports high-resolution chromatin binding maps of two core components of the NPC, Nup107 and Nup93, in Drosophila cells. This 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, this study identified a functional role of Nup93 in silencing of Polycomb target genes and in spatial folding of Polycomb domains. These 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 (Gozalo, 2019).

    Spatial architecture in the nucleus is set up by interactions between the genome and protein components of nuclear macro-complexes and scaffolds. The most prominent nuclear scaffold is the nuclear envelope (NE), which consists of a double membrane interspersed by a variety of trans-membrane and closely associated proteins. Chromatin re-organization and gene re-positioning during cellular differentiation involves losing or gaining interactions between the genome and the NE, and such rearrangements can influence gene expression programs. For instance, the nuclear lamina, which is a filamentous protein network underlying the NE, has been extensively implicated in setting up tissue-specific genome organization by sequestering genes destined for silencing. Genome-wide mapping of Lamin-associated domains (LADs), as well as related functional studies, have led to the current view of the nuclear lamina as a compartment for stable gene repression. Another major component of the NE is the nuclear pore complex (NPC), which consists of multiple copies of approximately 30 different proteins termed nucleoporins (Nups) and is responsible for selective nucleo-cytoplasmic transport. In addition to transport-related functions, NPCs and individual Nups are also involved in genome organization and gene regulation through physical interactions with the genome. Yet unlike the nuclear lamina, the functional relationship between NPCs and genome regulation appears to be considerably more varied and remains less understood (Gozalo, 2019).

    Given the close proximity of nuclear pores to the underlying chromatin, it is not surprising that multiple studies have now identified binding of Nups to subsets of genes and regulatory elements in a number of species. Many of these studies have reported preferential association of particular Nups with actively transcribing genes or re-localization of genes to the NPCs during activation. These findings have led to the predominant view of the NPC as a nuclear compartment for active processes, functionally opposed to those of the nuclear lamina. However, at least in metazoan systems, this view is confounded by the reported intranuclear presence of Nups that have been classified as dynamic. The ~30 conserved Nups that comprise the NPC can be either dynamic, meaning they are able to come on and off the NPC during interphase, or stable, meaning they are core components of the NE-embedded NPC for the majority of the cell cycle. Currently, many of the reported contacts between active genes and Nups have been described for dynamic Nups, such as Nup98, Nup153, and Nup62, and can frequently occur in the nucleoplasm. Consequently, it is unclear whether genomic binding to actual NPCs is functionally distinct from intranuclear Nup binding (Gozalo, 2019).

    Genomic binding to actual NPCs can be determined by mapping chromatin-binding patterns of stable Nups, which are components of the outer-ring Nup107-Nup160 and the inner-ring Nup93-Nup205 sub-complexes. Interestingly, previous studies that profiled chromatin binding of stable Nups did not identify enrichment for transcribing loci and reported prevalence of repressive chromatin. Similarly, DamID profiling of Nup98, artificially tethered to the NPC and thus used as a marker for actual NPC binding, showed no enrichment for active genes and instead exhibited high incidence of motifs for the architectural protein Su(Hw) in Drosophila cells. These studies conflict with the simplified view of the NPC as a scaffold for gene activation and highlight the complexity of NPC-genome interactions. It should be noted that the majority of the NPC genome-binding datasets, mentioned above, were produced using either the DamID technique, which tends to generate wide binding peaks, or the lower-resolution ChIP-chip approach, and thus may have given an incomplete picture of the locations and functions of NPC-genome contacts (Gozalo, 2019).

    One hypothesis, which can explain this dichotomy of both active and silent regions at the NPC, is that individual stable Nups bind distinct regions of the genome and regulate distinct chromatin-associated processes. This study set out to explore this hypothesis by generating precise binding maps of stable Nups, using an optimized ChIP-seq approach. ChIP-seq maps revealed that Nup107, a core component of the outer ring sub-complex, and Nup93, a core component of the inner ring sub-complex, bind highly non-overlapping regions of the genome. Specifically, while Nup107 preferentially targets active promoters, as has been reported for other Nups, Nup93 associates primarily with silenced regions bound by Polycomb group (PcG) proteins. PcG proteins are conserved regulators of epigenetically maintained gene repression, which often bind the genome in long Polycomb (Pc) domains. In agreement with its binding pattern, this study found that Nup93 plays a functional role in the silencing and long-range interactions of Pc targets. Together, the results emphasize the concept that different sub-complexes of the nuclear pore interact with and influence distinct chromatin states, revealing a complex landscape of NE-genome interactions (Gozalo, 2019).

    The results provide high-resolution chromatin binding maps of stable NPC components and offer a resource for future comparisons to a variety of genomic features. These maps and analysis contribute several insights into the nuclear organization field. First, it was found that representative members of the two core sub-complexes of the NPC, Nup107 and Nup93, bind to active and silenced regions, respectively. This differential binding helps explain the variability in previous conclusions on NPC-genome contacts and extends understanding of how NPC-genome contacts shape three-dimensional genome architecture. ChIP-seq maps of Nup107 are consistent with the predominant view of the NPC as a place for targeting active genes and suggest that this function is carried out primarily through associations of the genome with the outer-ring NPC sub-complex. Although currently it is not possible to definitively prove that all identified binding peaks of Nup107 and Nup93 represent NPC binding, comparison to LADs and immunofluorescence localization analysis, as well as DNA FISH analysis of select loci, suggest that a large proportion of these binding peaks represent regions present at the nuclear periphery, at actual NPCs (Gozalo, 2019).

    Second, the findings describe a functional connection between Nup93, a conserved subunit of the inner-ring NPC sub-complex, and Polycomb complexes, which are key regulators of developmental gene silencing. ChIP-seq map of Nup93 demonstrates preferential targeting of Nup93 to a large subset of PcG domains, particularly those that exhibit the highest level of Pc binding. Importantly, it was found that lowering levels of Nup93 leads to de-repression of Pc targets in both cultured cells and fly tissues. These findings suggest that (1) a core NPC subunit is involved in the epigenetic maintenance of silencing via its chromatin binding role; and (2) a subclass of particularly stable PcG chromatin domains are targeted to the nuclear periphery, where they require Nup93 for optimal silencing. The function of the Nup93 sub-complex in gene repression appears to be highly conserved, as strikingly, the S. pombe homolog of Nup93 was found by Moazed and colleagues to be required for silencing and nuclear clustering of heterochromatin (Iglesias, 2019). Nup93 has also been previously shown to be required for the repression of the HoxA gene cluster in mammalian cells (Labade, 2016). This is in line with previous findings that another subunit of this sub-complex, Nup155, associates with histone deacetylases in mammalian cells and targets repressed heterochromatin in yeast. The role of Nup93 in PcG silencing is also potentially related to the previously reported link of Nup153 to PcG-mediated repression in mouse ES cells. In further support of this notion, several Nups have been previously identified in a genome-wide imaging screen for factors that affect nuclear distribution of PcG proteins (Gozalo, 2019).

    Thus, a proposed model envisions that certain nuclear pores may interact with active chromatin via the Nup107 sub-complex, while other nuclear pores may associate with silent chromatin via the Nup93 sub-complex. This model is based on the ability to ChIP-seq distinct regions of the genome with different structural NPC components, and it is further supported by biochemical interaction data and the specificity of the functional effect of Nup93. The proposed model is consistent with previous findings in yeast, which reported the binding of a component of the Nup93 sub-complex to silent chromatin and the possible existence of this sub-complex as a type of an independent nuclear pore-related complex, present at the nuclear periphery. In this context, it remains to be determined whether some of the Nup93-PcG interactions similarly occur as an independent complex or if they are normally part of actual NPCs and whether the observed functional effect of Nup93 on PcG silencing always takes place at the NPC (Gozalo, 2019).

    Interestingly, it was found that the Nup93-targeted PcG domains tend to preferentially interact with each other in nuclear space. It is intriguing that other Nups, such as Nup98 and Mlp1/2, have been previously shown to facilitate long-range contacts of transcribing genes, such as enhancer-promoter and 5'-3' loops. It appears that stabilization of long-range contacts, either at active or silent genes, may be generally promoted by NPC binding, but the nature of contacts depends on the particular Nup involved. Based on the combined results, it is hypothesized that Nup93 binding may promote stabilization of PcG domains that are destined to be highly repressed. This stabilization may involve promoting long-range interactions between Pc sites, as well as possibly helping sequester PcG domains into specific nuclear compartments, away from gene activity. The findings also suggest that in the case of PcG silencing, long-range interactions are more functionally involved in gene repression than localization to the nuclear periphery is, since de-repression of PcG targets is consistently associated with loss of long-range interactions (Gozalo, 2019).

    Furthermore, the results suggest that some of the previously defined LADs are in fact interrupted or flanked with NPC-associated chromatin. In this manner, it appears that at least a fraction of mapped LADs may be complex, containing Nup-targeted sub-environments. These conclusions are also supported by the recent refined analysis of mammalian LADs, which revealed LAD interruptions that contain marks of active chromatin, termed 'Disruption in Peripheral signal' (DiPs). If such DiPs are biologically meaningful, the data would suggest that some such DiPs may be NPC-bound areas of the genome, characterized by functions distinct from the surrounding LADs. An intriguing conjecture is that positioning genes at NPCs within LADs may facilitate ready switching of transcriptional states, such that genes can shift between adjacent active and silent states, depending on incoming signals (Gozalo, 2019).

    Finally, this analysis demonstrated widespread genomic binding by a non-stable Nup Elys, which is currently the only Nup with a known direct chromatin binding activity (Zierhut, 2014). Interestingly, Nup107 is almost exclusively found at Elys binding sites. It is tempting to speculate that Elys serves as a chromatin tethering Nup for the Nup107 sub-complex components in the interphase genome, much like it has been demonstrated to do post-mitotically, during NPC assembly. The reproducibility of Elys-Nup107 binding patterns further invokes the possibility that post-mitotic targeting of the Elys/Nup107 sub-complex to chromatin occurs at specific sites in the genome and as such, may participate in the correct re-establishment of chromatin states and nuclear architecture after mitosis. On the other hand, Nup93 similarly shares a large fraction of its binding sites with Elys, suggesting that Elys may carry a similar function in targeting the inner ring sub-complex to chromatin. Presently it remains unclear how this specificity of Nup93 versus Nup107 genome targeting may be established. But together, the findings support the model where different subunits of the NPC have evolved unique functions in chromatin regulation. Individual Nups appear to be able to facilitate either activating or repressive processes and to assist nuclear organization of chromatin domains and key proteins complexes (Gozalo, 2019).


    References

    Breuer, M. and Ohkura, H. (2015). A negative loop within the nuclear pore complex controls global chromatin organization. Genes Dev 29: 1789-1794. PubMed ID: 26341556

    Gozalo, A., Duke, A., Lan, Y., Pascual-Garcia, P., Talamas, J. A., Nguyen, S. C., Shah, P. P., Jain, R., Joyce, E. F. and Capelson, M. (2019). Core components of the nuclear pore bind distinct states of chromatin and contribute to Polycomb repression. Mol Cell. PubMed ID: 31784359

    Hampoelz, B., Mackmull, M. T., Machado, P., Ronchi, P., Bui, K. H., Schieber, N., Santarella-Mellwig, R., Necakov, A., Andres-Pons, A., Philippe, J. M., Lecuit, T., Schwab, Y. and Beck, M. (2016). Pre-assembled nuclear pores insert into the nuclear envelope during early development. Cell 166 (3): 664-678. PubMed ID: 27397507

    Hampoelz, B., Schwarz, A., Ronchi, P., Bragulat-Teixidor, H., Tischer, C., Gaspar, I., Ephrussi, A., Schwab, Y. and Beck, M. (2019). Nuclear pores assemble from Nucleoporin condensates during oogenesis. Cell 179(3): 671-686. PubMed ID: 31626769

    Vollmer, B., Lorenz, M., Moreno-Andres, D., Bodenhofer, M., De Magistris, P., Astrinidis, S. A., Schooley, A., Flotenmeyer, M., Leptihn, S. and Antonin, W. (2015). Nup153 recruits the Nup107-160 complex to the inner nuclear membrane for interphasic nuclear pore complex assembly. Dev Cell 33(6): 717-728. PubMed ID: 26051542

    Weberruss, M. and Antonin, W. (2016). Perforating the nuclear boundary - how nuclear pore complexes assemble. J Cell Sci 129(24): 4439-4447. PubMed ID: 27856507

    Zierhut, C., Jenness, C., Kimura, H. and Funabiki, H. (2014). Nucleosomal regulation of chromatin composition and nuclear assembly revealed by histone depletion. Nat Struct Mol Biol 21(7): 617-625. PubMed ID: 24952593


    Zygotically transcribed genes

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