InteractiveFly: GeneBrief

Nucleoporin 93kD-1: Biological Overview | References

Gene name - Nucleoporin 93kD-1

Synonyms - Nup93

Cytological map position - 12C6-12C6

Function - nuclear pore complex

Keywords - nuclear pore complex - scaffold nucleoporin considered important for the overall integrity of the nuclear pore complex - functions in supporting Smad nuclear import - a core component of the inner ring sub-complex - associates primarily with Polycomb-silenced regions - Nup93 recruits Nup62 to suppress chromatin tethering by Nup155

Symbol - Nup93-1

FlyBase ID: FBgn0027537

Genetic map position - chrX:13,822,826-13,825,910

Classification - Nup93

Cellular location - nuclear envelope

NCBI link: EntrezGene, Nucleotide, Protein
Nup93-1 orthologs: Biolitmine

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 (reviewed in van Steensel, 2017). 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 (Lapetina, 2017; Van de Vosse, 2013). 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).

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 (Kalverda, 2010). 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 (Busayavalasa, 2012). 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).

Specific nucleoporin requirement for Smad nuclear translocation

Cytoplasm-to-nucleus translocation of Smad is a fundamental step in transforming growth factor beta (TGF-beta) signal transduction. This study identified a subset of nucleoporins that, in conjunction with Moleskin (Msk, Drosophila Imp7/8), specifically mediate activation-induced nuclear translocation of MAD (Drosophila Smad1) but not the constitutive import of proteins harboring a classic nuclear localization signal (cNLS) or the spontaneous nuclear import of Medea (Drosophila Smad4). Surprisingly, many of these nucleoporins, including Sec13, Nup75, Nup93, and Nup205, are scaffold nucleoporins considered important for the overall integrity of the nuclear pore complex (NPC) but not known to have cargo-specific functions. The roles of these nucleoporins in supporting Smad nuclear import are separate from their previously assigned functions in NPC assembly. Furthermore, novel pathway-specific functions of Sec13 and Nup93 were uncovered; both Sec13 and Nup93 are able to preferentially interact with the phosphorylated/activated form of MAD, and Nup93 acts to recruit the importin Msk to the nuclear periphery. These findings, together with the observation that Sec13 and Nup93 could interact directly with Msk, suggest their direct involvement in the nuclear import of MAD. Thus, this study has delineated the nucleoporin requirement of MAD nuclear import, reflecting a unique trans-NPC mechanism (Chen, 2010).

This study identified a distinct nucleoporin cohort, including both non-FG nucleoporins and FG-nucleoporins, that represents a unique trans-NPC mechanism for signal-activated MAD. Such specificity in nucleoporin utilization may reflect different demands of constitutive and signal-induced nuclear import events. Most unexpectedly, several non-FG nucleoporins, including Sec13, Nup93, Nup75, and Nup205, appear to act in concert with Msk to selectively transport MAD, but not the cNLS-cargo or basal-state Medea, into the nucleus. This is the first indication that beyond their involvement in the general assembly of the NPC, non-FG nucleoporins could play discrete roles in specific nuclear transport pathways. This study further identified the distinct functions served by two non-FG scaffold nucleoporins, Sec13 and Nup93, that are critical and specific for the nuclear import of MAD. These findings suggest a novel functional interplay between the MAD nuclear import machinery and the NPC (Chen, 2010).

Sec13 is part of the Nup107-160 complex, and Nup93 is part of the Nup53-93 complex; both are scaffolds of the NPC. It is emphasized that these findings are not in conflict with the established roles of Sec13 and Nup93 in general NPC assembly but broaden the functions of these non-FG nucleoporins to specific nuclear import pathways. It was somewhat surprising that depletion of Nup75 and Sec13 had little impact on MAb414 staining and nuclear envelope permeability, in contrast to the more severe phenotypes exhibited by the knockdown of other components in the Nup107-160 complex (i.e., nup145, nup107, and nup160). It was hardly possible to detect Sec13 after RNAi, so the lack of impact on MAb414 staining could not be attributed to incomplete depletion of Sec13. Therefore, these observations suggest that knocking down individual components of the Nup107-160 complex could lead to different phenotypes regarding MAD nuclear import, the MAb414 staining pattern, and the permeability of the NPC, arguing that each nucleoporin in the Nup107-160 complex serves distinct functions (Chen, 2010).

The challenging question ahead is how these non-FG nucleoporins mediate the nuclear import of MAD. Interestingly, Sec13 has been shown to dynamically transit between the cytoplasm and the nucleus, and endogenous Sec13 is partitioned among NPC, the intranuclear space, and the endoplasmic reticulum (ER). With theobservation that Sec13 preferentially interacts with phosphorylated/activated MAD, it is possible that Sec13 could act as an active trafficker rather than as a stationary component of the NPC to mediate the nuclear import of MAD. Whether phosphorylated MAD reaches the NPC via random diffusion or is guided by particular factors remains an open question, and it will be interesting to investigate whether Sec13 might be involved (Chen, 2010).

Msk has a characteristic nuclear rim localization pattern that is shown in this study to be important for its ability to transport MAD into the nucleus. Two of the nucleoporins that are required for MAD nuclear import, Nup93 and Nup358, appear to be responsible for targeting Msk to the nuclear periphery. Deletion of the C-terminal region of Msk disrupted its nuclear rim distribution and also significantly weakened the Msk-Nup93 interaction. It is unclear whether the same C-terminal deletion of Msk would affect the Msk-Nup358 interaction as well. Thus, the question remains, between Nup93 and Nup358, which one is more directly responsible for recruiting Msk to the nuclear periphery. Interestingly, Impβ is also concentrated to the nuclear periphery, like Msk, but such localization has been shown to depend on Nup153 instead of Nup93 and Nup358. Therefore, different importins are apparently recruited to the NPC through distinct nucleoporins, another direct indication that various nuclear import pathways operate through different modes of interaction with the NPC (Chen, 2010).

As exemplified by Sec13, Nup93, and Nup358, the nucleoporins implicated in MAD nuclear import serve distinct functions at different stages of the import process. One appealing model is that Msk is positioned by Nup93 and Nup358 to the vicinity of NPC, and perhaps Sec13 engages phosphorylated MAD and, through its own trafficking ability, delivers MAD to Msk, which completes the translocation across the NPC. While the data clearly suggest that Sec13 and Nup93 play roles distinct from those of the other components of the Nup107-160 or Nup53-93 complex, it is not suggested that they function in isolation from the other nucleoporins. Nor is it possible at this point to rule out a possible requirement for other nucleoporins in the nuclear import of MAD. Nevertheless, the direct physical interaction between Sec13/Nup93 and MAD or Msk, as well as the very selective impact of Sec13 and Nup93 RNAi on the nuclear import of MAD, but not other cargoes, is consistent with the interpretation that Sec13 and Nup93 are directly involved in the nuclear import of MAD (Chen, 2010).

Thisr genetic dissection of the Smad nuclear import pathway has important implications for the model of NPC structure and function. The findings in this study depart from the current dogma that puts only FG-nucleoporins at the center of the NPC-importin interplay. The diversity in trans-NPC routes and the pathway-specific involvement of non-FG nucleoporins need to be incorporated into models of NPC function in nuclear transport. It is increasingly clear that there are multiple distinct routes through the NPC that are taken by different importin/cargo complexes. The question, then, is how the NPC can accommodate these different passages. X-ray crystal structure analysis and electron microscopy have suggested that the Nup107-160 complex assumes a Y-shaped topography, raising speculations that such a porous assembly may leave room for additional trans-NPC passages besides the central tunnel, which is densely populated by FG-nucleoporins. One could also speculate that maybe the NPC can assume different configurations upon receiving different importin/cargo complexes to enable the translocation process (Chen, 2010).

Functions of Nup93 orthologs in other species

HOXA repression is mediated by nucleoporin Nup93 assisted by its interactors Nup188 and Nup205

The nuclear pore complex (NPC) mediates nuclear transport of RNA and proteins into and out of the nucleus. Certain nucleoporins have additional functions in chromatin organization and transcription regulation. Nup93 is a scaffold nucleoporin at the nuclear pore complex which is associated with human chromosomes 5, 7 and 16 and with the promoters of the HOXA gene as revealed by ChIP-on-chip studies using tiling microarrays for these chromosomes. However, the functional consequences of the association of Nup93 with HOXA is unknown. This study examined the association of Nup93 with the HOXA gene cluster and its consequences on HOXA gene expression in diploid colorectal cancer cells (DLD1). Nup93 showed a specific enrichment ~1 Kb upstream of the transcription start site of each of the HOXA1, HOXA3 and HOXA5 promoters, respectively. Furthermore, the association of Nup93 with HOXA was assisted by its interacting partners Nup188 and Nup205. The depletion of the Nup93 sub-complex significantly upregulated HOXA gene expression levels. However, expression levels of a control gene locus (GLCCI1) on human chromosome 7 were unaffected. Three-dimensional fluorescence in situ hybridization (3D-FISH) analyses revealed that the depletion of the Nup93 sub-complex (but not Nup98) disengages the HOXA gene locus from the nuclear periphery, suggesting a potential role for Nup93 in tethering and repressing the HOXA gene cluster. Consistently, Nup93 knockdown increased active histone marks (H3K9ac), decreased repressive histone marks (H3K27me3) on the HOXA1 promoter and increased transcription elongation marks (H3K36me3) within the HOXA1 gene. Moreover, the combined depletion of Nup93 and CTCF (a known organizer of HOXA gene cluster) but not Nup93 alone, significantly increased GLCCI1 gene expression levels. Taken together, this suggests a novel role for Nup93 and its interactors in repressing the HOXA gene cluster. This study reveals that the nucleoporin Nup93 assisted by its interactors Nup188 and Nup205 mediates the repression of HOXA gene expression (Labade, 2016).

The regulated expression of the HOX family of transcription factors is required during early development and differentiation, whereas its untimely expression in differentiated cells is associated with disease. HOX gene expression is also maintained in differentiated cells such as human skin fibroblasts in a manner that retains their tissue-specific origin. ChIP-chip studies using tiling microarrays revealed an association of Nup93 with human chromosomes 5, 7 and 16. Nup93 was enriched on the HOXA sub-cluster of human chromosome 7. This study shows that Nup93 associates with the HOXA gene cluster in a manner dependent on its interactors Nup188 and Nup205. Furthermore, depletion of these nucleoporins showed a significant increase in the expression levels of HOXA genes. This was consistent with a disengagement of the HOXA gene locus from the nuclear periphery in Nup93-/Nup188-/Nup205-depleted cells. In addition, the upregulation of HOXA genes upon Nup93 depletion was associated with an increase in active histone marks, reduced inactive marks and enrichment of a transcription elongation mark (Labade, 2016).

Several studies across organisms have consistently shown an association of the mobile nucleoporins such as Nup98, Nup50 and Nup153 with chromatin in addition to regulating nuclear transport. This study corroborated previous findings of Brown (2008) and shows that Nup93 indeed associates with the promoters of HOXA1, HOXA3 and HOXA5 and represses HOXA gene expression. It is conceivable that the repressive mechanism of Nup93 could potentially extend to other HOX gene clusters such as the HOXB, HOXC and HOXD, respectively. Chromatin conformation capture assays (such as 5C) have shown that the silenced HOXA gene cluster adopts a folded loop structure in a human myeloid leukemia cell line THP1. It is speculated that nucleoporins such as Nup93 may further modulate the local three-dimensional organization of the topologically associated domains (TADs) within the HOX gene cluster. These studies provide evidence to the growing body of literature which reinforce the role of nucleoporins in regulating chromatin organization and gene expression. Gene expression regulation is typically accompanied by an altered occupancy of active and inactive histone marks on gene promoters. The active state of the HOXA gene cluster is marked by active histone marks such as H3K9ac and H3K4me3, while the inactive state shows an enrichment of inactive marks such as H3K9me3 and H3K27me3. For instance, histone deacetylases and PRC2 complex proteins modify levels of active and inactive histone marks on the HOXA gene cluster in NT2/D1 embryonal carcinoma cells. Interestingly, a recent Dam-ID study showed that Nup93 associates with chromatin at the nuclear periphery (Ibarra, 2016). Considering the localization of the HOXA gene cluster on the gene poor chromosome 7 territory, proximal to the nuclear periphery, this study found a sequestration of the HOXA gene cluster to the nuclear periphery potentially mediated by the Nup93 sub-complex but not Nup98. It is surmised that the depletion of Nup93, Nup188 or Nup205 and their reduced stability, enhances the accessibility of the HOXA gene cluster to transcriptional activators and epigenetic modulators that could facilitate their untimely expression of HOXA genes-the physiological ramifications of which remain unclear (Labade, 2016).

Nucleoporins regulate nuclear import and export of mRNA, RNA and proteins. In addition, an increasing number of evidences implicate nucleoporins in gene regulation. Furthermore, the composition of the nuclear pore complex (NPC) is variable across cells types, which interestingly has limited effect on nuclear transport. In embryonic stem cells, Nup210 is absent but is specifically incorporated into the NPC during differentiation. Dam-ID studies reveal that Nup153 regulates expression of cell identity genes independent of its role in nuclear transport. Nup98 is involved in both nucleocytoplasmic transport and gene regulation , since Nup98 interacts with the mRNA export factor Rae1 and regulates mRNA export. Nup98 associates with developmentally active genes such as GRIK1, ERBB4, NRG1 and DCC and regulates their expression levels during differentiation. The Nup98-HOXA9 fusion protein associates with and inappropriately activates the HOX gene cluster in mouse embryonic stem cells in a manner dependent on the Crm1 protein. Interestingly, Nup98 depletion in DLD1 cells did not alter either the spatial localization or the expression levels of the HOXA gene, notwithstanding its impact on nuclear transport. This suggests an independent role for Nup98 in regulating nuclear transport but not HOXA gene expression. However, in cells depleted of Nup93, Nup188 or Nup205, nuclear export was relatively unaffected although nuclear import was reduced. Taken together, these findings implicate nucleoporins such as Nup93, Nup188 and Nup205 as modulators of chromatin organization in addition to their nuclear transport functions (Labade, 2016).

The mechanisms by which core nucleoporins associate with DNA are unclear. More importantly, several findings suggest that nucleoporins are involved in chromatin remodeling owing to their association with chromatin modifiers such as the SAGA complex, HDACs, RSC complex, SUMO proteases, SENP1, SENP2 and MSL complex. Chromatin remodeling complexes such as the SAGA complex-a transcriptional activator, associates with the nuclear pore complex and activates HXK1, INO1 and GAL genes when recruited to the NPC. Nup2, Nup60, Nic96, Nup116, Mlp1 and Mlp2 are enriched on transcriptionally active regions in S. cerevisiae. Furthermore, ARP6 links the active housekeeping gene RPP1A, involved in ribosome biogenesis to the nuclear pore complex. Nup170p represses ribosomal biogenesis genes and genes on the sub-telomeric region. Nup120 and Nup133 also core nucleoporins repress SUC2 gene expression in yeast. Interestingly, Nup93 tethers and regulates the expression of cell identity genes, predominantly localized at the nuclear periphery. The tethering of HOXA gene cluster to the nuclear periphery and its repression by the Nup93 sub-complex adds to the repertoire of nucleoporin-mediated gene repression events. Analyses of protein-protein interaction networks using BIOGRID of human Nup93 shows that Nup93 interacts with chromatin modifiers such as HDAC11, HDAC9, HDAC5 and PCR2 complex proteins -- EED and Suz12. It is conceivable that Nup93 and its interactors associate with transcriptional repressors in repressing the HOXA gene cluster, although no direct association was detected between Nup93 and the chromatin repressive complex (PRC2). ChIP-mass spectrometric approaches may identify putative interactors of Nup93 involved in chromatin organization (Labade, 2016).

Regulation of HOXA gene expression is essential during early development, since the aberrant expression of HOX genes leads to developmental defects. Active HOX genes cluster into transcriptionally active domains as shown using chromatin conformation capture analyses in mouse embryonic tissues. Similarly, HOXA gene regulation is also important in adult tissues since their aberrant expression is associated with various cancers. Furthermore, CTCF is an important regulator of the 3D organization and silencing of the HOXA gene cluster. Notably, CTCF is associated closer to the 5' region of the HOX gene cluster, in a manner that does not overlap with Nup93 binding sites, suggesting the complementary and potentially independent roles of Nup93 and CTCF in the maintenance of HOXA gene repression. However, it is unclear if CTCF silences HOXA gene cluster in differentiated cells by recruiting regulatory proteins such as PRC2. This study showed that Nup93 depletion in DLD1 cells did not alter the levels of CTCF or PRC2 complex proteins-EZH2, Suz12 and EED. It is surmised that CTCF or PRC2 proteins may have altered chromatin occupancy in the absence of Nup93 sub-complex, which remains to be elucidated by ChIP-sequencing of CTCF or PRC2 complex proteins in the absence of Nup93. This is further consistent with the significant upregulation of the GLCCI1 gene in cells depleted of both Nup93 and CTCF as compared CTCF-depleted cells (Labade, 2016).

The C-terminal domain of Nup93 is essential for assembly of the structural backbone of nuclear pore complexes

Nuclear pore complexes (NPCs) are large macromolecular assemblies that control all transport across the nuclear envelope. They are formed by about 30 nucleoporins (Nups), which can be roughly categorized into those forming the structural skeleton of the pore and those creating the central channel and thus providing the transport and gating properties of the NPC. This study shows that the conserved nucleoporin Nup93 is essential for NPC assembly and connects both portions of the NPC. Although the C-terminal domain of the protein is necessary and sufficient for the assembly of a minimal structural backbone, full-length Nup93 is required for the additional recruitment of the Nup62 complex and the establishment of transport-competent NPCs (Sachdev, 2012).

Nucleoporin-mediated regulation of cell identity genes

The organization of the genome in the three-dimensional space of the nucleus is coupled with cell type-specific gene expression. However, how nuclear architecture influences transcription that governs cell identity remains unknown. This study shows that nuclear pore complex (NPC) components Nup93 and Nup153 bind superenhancers (SE), regulatory structures that drive the expression of key genes that specify cell identity. Nucleoporin-associated SEs localize preferentially to the nuclear periphery, and absence of Nup153 and Nup93 results in dramatic transcriptional changes of SE-associated genes. These results reveal a crucial role of NPC components in the regulation of cell type-specifying genes and highlight nuclear architecture as a regulatory layer of genome functions in cell fate (Ibarra, 2016).


Search PubMed for articles about Drosophila

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

Brown, C. R., Kennedy, C. J., Delmar, V. A., Forbes, D. J. and Silver, P. A. (2008). Global histone acetylation induces functional genomic reorganization at mammalian nuclear pore complexes. Genes Dev 22(5): 627-639. PubMed ID: 18316479

Busayavalasa, K., Chen, X., Farrants, A. K., Wagner, N. and Sabri, N. (2012). The Nup155-mediated organisation of inner nuclear membrane proteins is independent of Nup155 anchoring to the metazoan nuclear pore complex. J Cell Sci 125(Pt 18): 4214-4218. PubMed ID: 22718353

Chen, X. and Xu, L. (2010). Specific nucleoporin requirement for Smad nuclear translocation. Mol. Cell. Biol. 30(16): 4022-34. PubMed Citation: 20547758

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

Ibarra, A., Benner, C., Tyagi, S., Cool, J. and Hetzer, M. W. (2016). Nucleoporin-mediated regulation of cell identity genes. Genes Dev 30(20): 2253-2258. PubMed ID: 27807035

Iglesias, N., Paulo, J. A., Tatarakis, A., Wang, X., Edwards, A. L., Bhanu, N. V., Garcia, B. A., Haas, W., Gygi, S. P. and Moazed, D. (2020). Native Chromatin Proteomics Reveals a Role for Specific Nucleoporins in Heterochromatin Organization and Maintenance. Mol Cell 77(1): 51-66 e58. PubMed ID: 31784357

Kalverda, B. and Fornerod, M. (2010). Characterization of genome-nucleoporin interactions in Drosophila links chromatin insulators to the nuclear pore complex. Cell Cycle 9(24): 4812-4817. PubMed ID: 21150273

Labade, A. S., Karmodiya, K. and Sengupta, K. (2016). HOXA repression is mediated by nucleoporin Nup93 assisted by its interactors Nup188 and Nup205. Epigenetics Chromatin 9: 54. PubMed ID: 27980680

Lapetina, D. L., Ptak, C., Roesner, U. K. and Wozniak, R. W. (2017). Yeast silencing factor Sir4 and a subset of nucleoporins form a complex distinct from nuclear pore complexes. J Cell Biol 216(10): 3145-3159. PubMed ID: 28883038

Sachdev, R., Sieverding, C., Flotenmeyer, M. and Antonin, W. (2012). The C-terminal domain of Nup93 is essential for assembly of the structural backbone of nuclear pore complexes. Mol Biol Cell 23(4): 740-749. PubMed ID: 22171326

Van de Vosse, D. W., Wan, Y., Lapetina, D. L., Chen, W. M., Chiang, J. H., Aitchison, J. D. and Wozniak, R. W. (2013). A role for the nucleoporin Nup170p in chromatin structure and gene silencing. Cell 152(5): 969-983. PubMed ID: 23452847

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

Biological Overview

date revised: 28 February 2020

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