Nuclear Pore Complex Proteins Research Articles

Interactions of nuclear transport factors and surface-conjugated FG nucleoporins: Insights and limitations.

Protein-protein interactions are central to biological processes. In vitro methods to examine protein-protein interactions are generally categorized into two classes: in-solution and surface-based methods. Here, using the multivalent interactions between nucleocytoplasmic transport factors and intrinsically disordered FG repeat containing nuclear pore complex proteins as a model system, we examined the utility of three surface-based methods: atomic force microscopy, quartz crystal microbalance with dissipation, and surface plasmon resonance. Although results were comparable to those of previous reports, the apparent effect of mass transport limitations was demonstrated. Additional experiments with a loss-of-interaction FG repeat mutant variant demonstrated that the binding events that take place on surfaces can be unexpectedly complex, suggesting particular care must be exercised in interpretation of such data.

The Role of Transcription Factors and Nuclear Pore Proteins in Controlling the Spatial Organization of the Yeast Genome

Loss of nuclear pore complex (NPC) proteins, transcription factors (TFs), histone modification enzymes, Mediator, and factors involved in mRNA export disrupts the physical interaction of chromosomal sites with NPCs. Conditional inactivation and ectopic tethering experiments support a direct role for the TFs Gcn4 and Nup2 in mediating interaction with the NPC but suggest an indirect role for factors involved in mRNA export or transcription. A conserved "positioning domain" within Gcn4 controls interaction with the NPC and inter-chromosomal clustering and promotes transcription of target genes. Such a function may be quite common; a comprehensive screen reveals that tethering of most yeast TFs is sufficient to promote targeting to the NPC. While some TFs require Nup100, others do not, suggesting two distinct targeting mechanisms. These results highlight an important and underappreciated function of TFs in controlling the spatial organization of the yeast genome through interaction with the NPC.

The nucleoporin ELYS regulates nuclear size by controlling NPC number and nuclear import capacity.

How intracellular organelles acquire their characteristic sizes is a fundamental question in cell biology. Given stereotypical changes in nuclear size in cancer, it is important to understand the mechanisms that control nuclear size in human cells. Using a high-throughput imaging RNAi screen, we identify and mechanistically characterize ELYS, a nucleoporin required for post-mitotic nuclear pore complex (NPC) assembly, as a determinant of nuclear size in mammalian cells. ELYS knockdown results in small nuclei, reduced nuclear lamin B2 localization, lower NPC density, and decreased nuclear import. Increasing nuclear import by importin α overexpression rescues nuclear size and lamin B2 import, while inhibiting importin α/β-mediated nuclear import decreases nuclear size. Conversely, ELYS overexpression increases nuclear size, enriches nuclear lamin B2 at the nuclear periphery, and elevates NPC density and nuclear import. Consistent with these observations, knockdown or inhibition of exportin 1 increases nuclear size. Thus, we identify ELYS as a novel positive effector of mammalian nuclear size and propose that nuclear size is sensitive to NPC density and nuclear import capacity.

Nicotiana benthamiana exportin 1 is required for elicitor-induced phytoalexin production, cell death induction, and resistance against potato late blight pathogen Phytophthora infestans

The oomycete Phytophthora infestans is the causal agent of potato late blight, one of the most devastating pathogens for potato. To investigate the plant mechanisms for resistance against P. infestans, the Solanaceae model plant Nicotiana benthamiana was employed in this study. Previously, we reported that NbNup75, a nuclear pore complex protein, is required for the resistance against P. infestans, suggesting that nuclear-pore-mediated transport is involved in the induction of defense responses. In this study, we investigated the role of N. benthamiana exportin 1 NbXpo1 in disease resistance. Mammalian and yeast exportin 1 proteins are known as a regulator for nuclear-pore-mediated export of proteins and RNAs. NbXpo1-silenced N. benthamiana showed minor growth defects and significantly decreased resistance to P. infestans. Gene silencing of NbXpo1 compromised defense responses induced by the elicitor INF1 (a secretory protein of P. infestans), such as production of the phytoalexin capsidiol and induction of cell death. In NbXpo1-silenced plants, the genes for capsidiol biosynthesis, NbEAS, and defense-related MAP kinase, NbWIPK, were significantly downregulated, while genes encoding the plant defensin, NbPDF, and anti-microbial protein thionin, NbTHI, were upregulated. These results indicate that N. benthamiana Xpo1 is involved in activating a specific group of defense-related genes.

The mode of mitosis is dramatically modified by deletion of a single nuclear pore complex gene in Aspergillus nidulans

Nuclear pore complex (NPC) proteins (Nups) play multiple roles during mitosis. In this study we expand these roles and reveal that in Aspergillus nidulans, compromising the core Nup84-120 subcomplex of the NPC modifies the mitotic behavior of the nuclear envelope (NE). In wildtype cells, the NE undergoes simultaneous double pinching events to separate daughter nuclei during mitotic exit, whereas in Nup84-120 complex mutants, only one restriction of the NE is observed. Investigating the basis for this modified behavior of the NE in Nup deleted cells uncovered previously unrealized roles for core Nups in mitotic exit. During wildtype anaphase, the NE surrounds the two separating daughter DNA masses which typically flank the central nucleolus, to form three distinct nuclear compartments. In contrast, deletion of core Nups frequently results in early nucleolar eviction from the mitotic nucleus, in turn causing an uncharacteristic dumbbell-shaped NE morphology of anaphase nuclei with a nuclear membrane bridge connecting the two forming G1 nuclei. Importantly, the absence of the nucleolus between the separating daughter nuclei during anaphase delays chromosome segregation and progression into G1 as nuclei remain connected by chromatin bridges. Proteins localizing to late segregating chromosome arms are observed between forming daughter nuclei, and the mitotic spindle fails to resolve in a timely manner. These chromatin bridges are occupied by the Aurora kinase until nuclei have fully separated, suggesting involvement of Aurora in monitoring mitotic spindle and nuclear membrane resolution during mitotic exit. Our findings thus reveal a novel requirement for core Nups in mediating nucleolar positioning during mitosis, which dictates the pattern of NE fissions during karyokinesis and facilitates normal chromosome segregation. The findings additionally demonstrate that the mode of mitosis can be dramatically modified by deletion of a single NPC gene and reveals surprising fluidity in mitotic mechanisms.

Correction to: Biallelic Variants in the Nuclear Pore Complex Protein NUP93 Are Associated with Non-progressive Congenital Ataxia.

The original version of this article unfortunately contained mistake in Fig.3 image.

Extensive Reduction of the Nuclear Pore Complex in Nucleomorphs

The nuclear pore complex (NPC) is a large macromolecular assembly situated within the pores of the nuclear envelope. Through interactions between its subcomplexes and import proteins, the NPC mediates the transport of molecules into and out of the nucleus and facilitates dynamic chromatin regulation and gene expression. Accordingly, the NPC constitutes a highly integrated nuclear component that is ubiquitous and conserved among eukaryotes. Potential exceptions to this are nucleomorphs: Highly reduced, relict nuclei that were derived from green and red algae following their endosymbiotic integration into two lineages, the chlorarachniophytes and the cryptophyceans. A previous investigation failed to identify NPC genes in nucleomorph genomes suggesting that these genes have either been relocated to the host nucleus or lost. Here, we sought to investigate the composition of the NPC in nucleomorphs by using genomic and transcriptomic data to identify and phylogenetically classify NPC proteins in nucleomorph-containing algae. Although we found NPC proteins in all examined lineages, most of those found in chlorarachniophytes and cryptophyceans were single copy, host-related proteins that lacked signal peptides. Two exceptions were Nup98 and Rae1, which had clear nucleomorph-derived homologs. However, these proteins alone are likely insufficient to structure a canonical NPC and previous reports revealed that Nup98 and Rae1 have other nuclear functions. Ultimately, these data indicate that nucleomorphs represent eukaryotic nuclei without a canonical NPC, raising fundamental questions about their structure and function.

Nup2 performs diverse interphase functions in Aspergillus nidulans.

The nuclear pore complex (NPC) protein Nup2 plays interphase nuclear transport roles and in Aspergillus nidulans also functions to bridge NPCs at mitotic chromatin for their faithful coinheritance to daughter G1 nuclei. In this study, we further investigate the interphase functions of Nup2 in A. nidulans. Although Nup2 is not required for nuclear import of all nuclear proteins after mitosis, it is required for normal G1 nuclear accumulation of the NPC nuclear basket–associated components Mad2 and Mlp1 as well as the THO complex protein Tho2. Targeting of Mlp1 to nuclei partially rescues the interphase delay seen in nup2 mutants indicating that some of the interphase defects in Nup2-deleted cells are due to Mlp1 mislocalization. Among the inner nuclear membrane proteins, Nup2 affects the localization of Ima1, orthologues of which are involved in nuclear movement. Interestingly, nup2 mutant G1 nuclei also exhibit an abnormally long period of extensive to-and-fro movement immediately after mitosis in a manner dependent on the microtubule cytoskeleton. This indicates that Nup2 is required to limit the transient postmitotic nuclear migration typical of many filamentous fungi. The findings reveal that Nup2 is a multifunctional protein that performs diverse functions during both interphase and mitosis in A. nidulans.

The karyosphere capsule in Rana temporaria oocytes contains structural and DNA-binding proteins

ABSTRACTDuring the last stages of oogenesis, oocyte chromosomes condense and come close together, forming the so-called karyosphere. Karyosphere formation is accompanied by an essential decrease in transcriptional activity. In the grass frog Rana temporaria, the karyosphere is surrounded by an extrachromosomal capsule that separates the chromosomes from the rest of the nucleoplasm. The karyosphere capsule (KC) of R. temporaria has been investigated in detail at the ultrastructural level, but its protein composition remained largely unknown. We demonstrate here that nuclear actin, especially F-actin, as well as lamins A/C and B are the most abundant proteins of the KC. Key proteins of nuclear pore complexes, such as Nup93 and Nup35, are also detectable in the KC. New antibodies recognizing the telomere-binding protein TRF2 allowed us to localize TRF2 in nuclear speckles. We also found that the R. temporaria KC contains some proteins involved in chromatin remodeling, including topoisomerase II and ATRX. Thus, we believe that KC isolates the chromosomes from the rest of the nucleoplasm during the final period of oocyte growth (late diplotene) and represents a specialized oocyte nuclear compartment to store a variety of factors involved in nuclear metabolism that can be used in future early development.Abbreviations: BrUTP: 5-bromouridine 5’-triphosphate; CytD: cytochalasin D; IGCs: interchromatin granule clasters; IgG: immunoglobulin G; KC: karyosphere capsule; Mw: molecular weight; NE: nuclear envelope; PBS: phosphate buffered saline; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; Topo II: topoisomerase II

Toward the "unravelling" of HIV: Host cell factors involved in HIV-1 core uncoating.

Early HIV-1 replication requires fusion of viral and cellular membranes that release a fullerene-shaped viral core structure, formed by a lattice of primarily hexameric capsid (CA) rings, into the cellular cytoplasm. The core contains positive-polarity single-strand viral genomic RNA and enzymes that synthesize viral double-strand DNA that is integrated into a host chromosome. Only HIV-1 that release intact cores into the cytoplasm are infectious [1], and disassembly of the core occurs by a regulated process called uncoating. For the purpose of this article, we define uncoating as any dissociation of CA from the viral core. However, the precise mechanism, timing, and location of uncoating is contentious. Recent live-cell single-particle tracking of infectious HIV-1 cores has provided unprecedented insight into uncoating kinetics [2]. One such study found that core integrity, measured by “leakiness” of a core-trapped fluorescent marker, was lost in the cytoplasm approximately 30 min after fusion (after first-strand transfer of reverse transcription) and that this was required for productive infection [3]. However, any such loss of core integrity must not allow innate cellular sensors, such as cyclic guanosine monophosphate–adenosine monophosphate synthase (cGAS), to identify and restrict retroviral DNA in the cytoplasm [4]. Another recent study found that a small amount of CA dissociates gradually from the core post-entry before the remaining core structure docks at the nuclear pore complex (NPC), leading to accelerated CA loss, nuclear entry, integration of viral DNA into host DNA, and productive infection [5]. As these two studies measure different events and are not mutually exclusive, it is our view, consistent with that expressed in the review by Francis and Melikyan [2], that HIV-1 uncoating may begin by the loss of core integrity and gradual CA dissociation in the cytoplasm, followed by CA-dependent nuclear docking and accelerated CA loss at the NPC. An alternative model of uncoating is that an intact core arrives at the NPC before uncoating [1]. Here, we discuss the host cell factors subverted by HIV-1 to regulate ordered uncoating in cells (Fig 1), with the caveat that the spatiotemporal specifics of where and when host factors interact with the core and affect CA uncoating requires further investigation. Open in a separate window Fig 1 Host cell factors regulate HIV-1 uncoating. The HIV-1 virion, which contains the CypA, ERK2, and eEF1A host proteins that regulate uncoating, binds and fuses with the host cell membrane, and the core is released into the cytoplasm. PDZD8 and CypA binds CA to stabilize cores to promote infection; however, infection is inhibited if CypA associates with MxB, in which MxB oligomers bind CA to hyperstabilize cores. Cellular eEF1A interacts with HIV-1 RT and facilitates uncoating. Dia1 and Dia2 bind CA–NC complexes to facilitate uncoating likely by localized microtubules stabilization. Dynein interacts with the core via the BICD2 adapter protein or direct interaction with IN, and kinesin-1 interacts with the core via the FEZ1 (phosphorylated by MARK2) adapter protein. This is important for transport of the replication complexes through the cytoplasm to MTOCs at the nuclear periphery as well as uncoating. Pin1 binds CA phosphorylated by MEK1/2 activated ERK2 during virion maturation to facilitate uncoating. MELK also phosphorylates CA to promote uncoating. HIV-1 RT binds eEF1A to stabilize the RTC. The NPC proteins Nup358 and Nup153 help import the PIC, and Nup358 is relocalized to the cytoplasm by KIF5B, while Nup153 prolongs CA association with PICs in the nucleus. TNPO3 localizes CPSF6 to the nucleus to prevent CPSF6-dependent hyperstable cores, and TNPO3 may also help complete CA dissociation from replication complexes in the nucleus. This figure does not attempt to illustrate the spatiotemporal specifics of where and when host factors interact with the core and regulate CA uncoating. Green text: promotes uncoating for optimal kinetics. Orange text: delays uncoating for optimal kinetics. Red text: causes hyperstable cores and inhibits infection. Only viral and host proteins relevant in uncoating and discussed in this article are included in the figure. CA, capsid; CPSF6, cleavage and polyadenylation specificity factor 6; CypA, cyclophilin A; BICD2, bicaudal D2; Dia1/2, diaphanous-related formins 1 and 2; eEF1A, eukaryotic translation elongation factor 1A; ERK2, extracellular signal-regulated kinase 2; FEZ1, fasciculation and elongation factor zeta 1; IN, integrase; KIF5B, kinesin-1 heavy chain; NC, nucleocapsid; NPC, nuclear pore complex; Nup, nucleoporin; MARK2, microtubule affinity-regulating kinase 2; MEK1/2, mitogen-activated protein 1 and 2; MELK, maternal embryonic leucine zipper kinase; MTOC, microtubule-organizing centers; MxB, myxovirus resistance protein B; PDZD8, PDZ domain-containing 8 protein; PIC, pre-integration complex; RT, reverse transcriptase; RTC, reverse transcription complexes; TNPO3, transportin 3.

Nucleoporin 54 contributes to homologous recombination repair and post-replicative DNA integrity.

The nuclear pore complex (NPC) machinery is emerging as an important determinant in the maintenance of genome integrity and sensitivity to DNA double-strand break (DSB)-inducing agents, such as ionising radiation (IR). In this study, using a high-throughput siRNA screen, we identified the central channel NPC protein Nup54, and concomitantly its molecular partners Nup62 and Nup58, as novel factors implicated in radiosensitivity. Nup54 depletion caused an increase in cell death by mitotic catastrophe after IR, and specifically enhanced both the duration of the G2 arrest and the radiosensitivity of cells that contained replicated DNA at the time of IR exposure. Nup54-depleted cells also exhibited increased formation of chromosome aberrations arisen from replicated DNA. Interestingly, we found that Nup54 is epistatic with the homologous recombination (HR) factor Rad51. Moreover, using specific DNA damage repair reporters, we observed a decreased HR repair activity upon Nup54 knockdown. In agreement with a role in HR repair, we also demonstrated a decreased formation of HR-linked DNA synthesis foci and sister chromatid exchanges after IR in cells depleted of Nup54. Our study reveals a novel role for Nup54 in the response to IR and the maintenance of HR-mediated genome integrity.

NUP155 insufficiency recalibrates a pluripotent transcriptome with network remodeling of a cardiogenic signaling module

BackgroundAtrial fibrillation is a cardiac disease driven by numerous idiopathic etiologies. NUP155 is a nuclear pore complex protein that has been identified as a clinical driver of atrial fibrillation, yet the precise mechanism is unknown. The present study employs a systems biology algorithm to identify effects of NUP155 disruption on cardiogenicity in a model of stem cell-derived differentiation.MethodsEmbryonic stem (ES) cell lines (n = 5) with truncated NUP155 were cultured in parallel with wild type (WT) ES cells (n = 5), and then harvested for RNAseq. Samples were run on an Illumina HiSeq 2000. Reads were analyzed using Strand NGS, Cytoscape, DAVID and Ingenuity Pathways Analysis to deconvolute the NUP155-disrupted transcriptome. Network topological analysis identified key features that controlled framework architecture and functional enrichment.ResultsIn NUP155 truncated ES cells, significant expression changes were detected in 326 genes compared to WT. These genes segregated into clusters that enriched for specific gene ontologies. Deconvolution of the collective framework into discrete sub-networks identified a module with the highest score that enriched for Cardiovascular System Development, and revealed NTRK1/TRKA and SRSF2/SC35 as critical hubs within this cardiogenic module.ConclusionsThe strategy of pluripotent transcriptome deconvolution used in the current study identified a novel association of NUP155 with potential drivers of arrhythmogenic AF. Here, NUP155 regulates cardioplasticity of a sub-network embedded within a larger framework of genome integrity, and exemplifies how transcriptome cardiogenicity in an embryonic stem cell genome is recalibrated by nucleoporin dysfunction.

The yeast Ty1 retrotransposon requires components of the nuclear pore complex for transcription and genomic integration.

Nuclear pore complexes (NPCs) orchestrate cargo between the cytoplasm and nucleus and regulate chromatin organization. NPC proteins, or nucleoporins (Nups), are required for human immunodeficiency virus type 1 (HIV-1) gene expression and genomic integration of viral DNA. We utilize the Ty1 retrotransposon of Saccharomyces cerevisiae (S. cerevisiae) to study retroviral integration because retrotransposons are the progenitors of retroviruses and have conserved integrase (IN) enzymes. Ty1-IN targets Ty1 elements into the genome upstream of RNA polymerase (Pol) III transcribed genes such as transfer RNA (tRNA) genes. Evidence that S. cerevisiae tRNA genes are recruited to NPCs prompted our investigation of a functional role for the NPC in Ty1 targeting into the genome. We find that Ty1 mobility is reduced in multiple Nup mutants that cannot be accounted for by defects in Ty1 gene expression, cDNA production or Ty1-IN nuclear entry. Instead, we find that Ty1 insertion upstream of tRNA genes is impaired. We also identify Nup mutants with wild type Ty1 mobility but impaired Ty1 targeting. The NPC nuclear basket, which interacts with chromatin, is required for both Ty1 expression and nucleosome targeting. Deletion of components of the NPC nuclear basket causes mis-targeting of Ty1 elements to the ends of chromosomes.

Compensation for chronic oxidative stress in ALADIN null mice.

ABSTRACTMutations in the AAAS gene coding for the nuclear pore complex protein ALADIN lead to the autosomal recessive disorder triple A syndrome. Triple A patients present with a characteristic phenotype including alacrima, achalasia and adrenal insufficiency. Patient fibroblasts show increased levels of oxidative stress, and several in vitro studies have demonstrated that the nucleoporin ALADIN is involved in both the cellular oxidative stress response and adrenal steroidogenesis. It is known that ALADIN knock-out mice lack a phenotype resembling human triple A syndrome. The objective of this study was to determine whether the application of chronic oxidative stress by ingestion of paraquat would generate a triple A-like phenotype in ALADIN null mice. Adult male mice were fed either a paraquat (0.25 g/kg diet) or control diet for 11 days. After application of chronic oxidative stress, ALADIN knock-out mice presented with an unexpected compensated glutathione metabolism, but lacked a phenotype resembling human triple A syndrome. We did not observe increased levels of oxidative stress and alterations in adrenal steroidogenesis in mice depleted for ALADIN. This study stresses the species-specific role of the nucleoporin ALADIN, which in mice involves a novel compensatory mechanism for regulating the cellular glutathione redox response.

Tau Protein Disrupts Nucleocytoplasmic Transport in Alzheimerrs Disease

Tau protein, which normally functions to stabilize microtubules, is the major constituent of neurofibrillary tangles in Alzheimer’s disease (AD). The mechanism underlying tau-associated neural damage remains unclear. We now show that tau can interact with nuclear pore complex (NPC) constituents and affect their structural and functional integrity. Pathological tau leads to dissociation of nuclear pore complex proteins, and impairs nuclear export and import in vitro, in tau overexpressing transgenic mouse models, and in human AD tissue. Moreover, a nuclear pore component, Nup98, surprisingly colocalizes with neurofibrillary tangles in neuronal soma, and both in vivo and in vitro directly interacts with tau to greatly facilitate its aggregation. These data support the hypothesis that tau directly interacts with nuclear pore complex constituents, leading to their mislocalization and to disruption of nuclear pore function, raising the possibility that nuclear pore dysfunction contributes to tau-induced neurotoxicity in Alzheimer’s disease.

CDK1 and PLK1 coordinate the disassembly and reassembly of the nuclear envelope in vertebrate mitosis.

Micronuclei (MN) arise from chromosomes or fragments that fail to be incorporated into the primary nucleus after cell division. These structures are a major source of genetic instability caused by DNA repair and replication defects coupled to aberrant Nuclear Envelope (NE). These problems ultimately lead to a spectrum of chromosome rearrangements called chromothripsis, a phenomenon that is a hallmark of several cancers. Despite its importance, the molecular mechanism at the origin of this instability is still not understood. Here we show that lagging chromatin, although it can efficiently assemble Lamin A/C, always fails to recruit Nuclear Pore Complexes (NPCs) proteins and that Polo-Like Kinase (PLK1) negatively regulates NPC assembly. We also provide evidence for the requirement of PLK1 activity for the disassembly of NPCs, but not Lamina A/C, at mitotic entry. Altogether this study reveals the existence of independent regulatory pathways for Lamin A/C and NPC reorganization during mitosis where Lamin A/C targeting to the chromatin is controlled by CDK1 activity (a clock-based model) while the NPC loading is also spatially monitored by PLK1.

A new role for the nuclear basket network.

Our view of the nuclear pore complexes (NPCs) as gateways between the nuclear and cytoplasmic compartments has been largely expanded in recent years. NPCs have now demonstrated roles in genome regulation and maintenance from single cells to multicellular organisms. Both NPC proteins as well as components of the NPC basket act as dynamic scaffolds for silencing factors, and chromatin and cell cycle regulators. Components of the NPC basket also couple mRNA production and export, and prevent the exit of unprocessed mRNAs from the nucleus. Our recent work describes a novel function of the fission yeast nuclear basket component - the translocated promoter region (TPR) nucleoporin Alm1 - in proper localization of the proteasome to the nuclear envelope. Here we discuss how regulation of proteasome localization to the nuclear envelope by Alm1 is key to maintain kinetochores homeostasis and proper chromosome segregation.

3D point scanning super-resolution microscopy via polarization modulation.

We report a new approach to achieving super-resolution in point-scanning microscopy through polarization modulation for the first time, to the best of our knowledge. By modulating linearly polarized incident light, the emission extent of fluorescent dyes changes periodically, adding sparsity in each recording, which contributes to the super-resolution reconstruction. To recover the super-resolution result, a sparse penalty-based deconvolution method is implemented onto the polarization-modulated dataset subsequently. By simply inserting a vortex half-wave retarder into a typical confocal microscope, we obtain the super-resolution experimental results of both nuclear pore complex proteins and tubulins in vero cells, which evidence a sub-diffraction resolution of λ/5. In addition, three-dimensional (3D) super-resolution on spatial distributed single molecules is simulated, where the significant resolution improvement in both lateral and axial directions further confirms its capacity in 3D imaging applications. Considering no constraint on fluorescence dyes and easy implementation in a point-scanning microscope, we envision that the polarization-modulated confocal microscope would be a helpful alternative in biological imaging.

Yeast silencing factor Sir4 and a subset of nucleoporins form a complex distinct from nuclear pore complexes.

Interactions occurring at the nuclear envelope (NE)-chromatin interface influence both NE structure and chromatin organization. Insights into the functions of NE-chromatin interactions have come from the study of yeast subtelomeric chromatin and its association with the NE, including the identification of various proteins necessary for tethering subtelomeric chromatin to the NE and the silencing of resident genes. Here we show that four of these proteins-the silencing factor Sir4, NE-associated Esc1, the SUMO E3 ligase Siz2, and the nuclear pore complex (NPC) protein Nup170-physically and functionally interact with one another and a subset of NPC components (nucleoporins or Nups). Importantly, this group of Nups is largely restricted to members of the inner and outer NPC rings, but it lacks numerous others including cytoplasmically and nucleoplasmically positioned Nups. We propose that this Sir4-associated Nup complex is distinct from holo-NPCs and that it plays a role in subtelomeric chromatin organization and NE tethering.

Epigenetic Transcriptional Memory of GAL Genes Depends on Growth in Glucose and the Tup1 Transcription Factor in Saccharomyces cerevisiae.

Previously expressed inducible genes can remain poised for faster reactivation for multiple cell divisions, a conserved phenomenon called epigenetic transcriptional memory. The GAL genes in Saccharomyces cerevisiae show faster reactivation for up to seven generations after being repressed. During memory, previously produced Gal1 protein enhances the rate of reactivation of GAL1, GAL10, GAL2, and GAL7 These genes also interact with the nuclear pore complex (NPC) and localize to the nuclear periphery both when active and during memory. Peripheral localization of GAL1 during memory requires the Gal1 protein, a memory-specific cis-acting element in the promoter, and the NPC protein Nup100 However, unlike other examples of transcriptional memory, the interaction with NPC is not required for faster GAL gene reactivation. Rather, downstream of Gal1, the Tup1 transcription factor and growth in glucose promote GAL transcriptional memory. Cells only show signs of memory and only benefit from memory when growing in glucose. Tup1 promotes memory-specific chromatin changes at the GAL1 promoter: incorporation of histone variant H2A.Z and dimethylation of histone H3, lysine 4. Tup1 and H2A.Z function downstream of Gal1 to promote binding of a preinitiation form of RNA Polymerase II at the GAL1 promoter, poising the gene for faster reactivation. This mechanism allows cells to integrate a previous experience (growth in galactose, reflected by Gal1 levels) with current conditions (growth in glucose, potentially through Tup1 function) to overcome repression and to poise critical GAL genes for future reactivation.