Ectopic expression of meiotic cohesin generates chromosome instability in cancer cell line.

Many tumors express meiotic genes that could potentially drive somatic chromosome instability. While germline cohesin subunits SMC1B, STAG3, and REC8 are widely expressed in many cancers, messenger RNA and protein for RAD21L subunit are expressed at very low levels. To elucidate the potential of meiotic cohesins to contribute to genome instability, their expression was investigated in human cell lines, predominately in DLD-1. While the induction of the REC8 complex resulted in a mild mitotic phenotype, the expression of the RAD21L complex produced an arrested but viable cell pool, thus providing a source of DNA damage, mitotic chromosome missegregation, sporadic polyteny, and altered gene expression. We also found that genomic binding profiles of ectopically expressed meiotic cohesin complexes were reminiscent of their corresponding specific binding patterns in testis. Furthermore, meiotic cohesins were found to localize to the same sites as BORIS/CTCFL, rather than CTCF sites normally associated with the somatic cohesin complex. These findings highlight the existence of a germline epigenomic memory that is conserved in cells that normally do not express meiotic genes. Our results reveal a mechanism of action by unduly expressed meiotic cohesins that potentially links them to aneuploidy and chromosomal mutations in affected cells.

Distinct roles of nuclear basket proteins in directing the passage of mRNA through the nuclear pore.

The in vivo characterization of the exact copy number and the specific function of each composite protein within the nuclear pore complex (NPC) remains both desirable and challenging. Through the implementation of live-cell high-speed super-resolution single-molecule microscopy, we first quantified the native copies of nuclear basket (BSK) proteins (Nup153, Nup50, and Tpr) prior to knocking them down in a highly specific manner via an auxin-inducible degron strategy. Second, we determined the specific roles that BSK proteins play in the nuclear export kinetics of model messenger RNA (mRNA) substrates. Finally, the three-dimensional (3D) nuclear export routes of these mRNA substrates through native NPCs in the absence of specific BSK proteins were obtained and further validated via postlocalization computational simulations. We found that these BSK proteins possess the stoichiometric ratio of 1:1:1 and play distinct roles in the nuclear export of mRNAs within live cells. The absence of Tpr from the NPC predominantly reduces the probability of nuclear mRNAs entering the NPC for export. Complete depletion of Nup153 and Nup50 results in an mRNA nuclear export efficiency decrease of approximately four folds. mRNAs can gain their maximum successful export efficiency as the copy number of Nup153 increased from zero to only half the full complement natively within the NPC. Lastly, the absence of Tpr or Nup153 seems to alter the 3D export routes of mRNAs as they pass through the NPC. However, the removal of Nup50 alone has almost no impact upon mRNA export route and kinetics.

Human CCR4-NOT is a global regulator of gene expression and is a novel silencer of retrotransposon activation.

CCR4-NOT regulates multiple steps in gene regulation, including transcription, mRNA decay, protein ubiquitylation, and translation. It has been well studied in budding yeast; however, relatively less is known about its regulation and functions in mammals. To characterize the functions of the human CCR4-NOT complex, we developed a rapid auxin-induced degron system to deplete CNOT1 (the scaffold of the complex) and CNOT4 (E3 ubiquitin ligase) in cell culture. Transcriptome-wide measurements of gene-expression revealed that depleting CNOT1 changed several thousand transcripts, wherein most mRNAs were increased and resulted in a global decrease in mRNA decay rates. In contrast to what was observed in CNOT1-depleted cells, CNOT4 depletion only modestly changed RNA steady-state levels and, surprisingly, led to a global acceleration in mRNA decay. To further investigate the role of CCR4-NOT in transcription, we used transient transcriptome sequencing (TT-seq) to measure ongoing RNA synthesis. Depletion of either subunit resulted in increased RNA synthesis of several thousand genes. In contrast to most of the genome, a rapid reduction in the synthesis of KRAB-Zinc-Finger-proteins (KZNFs) genes, especially those clustered on chromosome 19, was observed. KZNFs are transcriptional repressors of retro-transposable elements (rTEs), and consistent with the decreased KZNFs expression, we observed a significant and rapid activation of rTEs, mainly Long interspersed Nuclear Elements (LINEs). Our data reveal that CCR4-NOT regulates gene expression and silences retrotransposons across the genome by maintaining KZNF expression. These data establish CCR4-NOT as a global regulator of gene expression, and we have identified a novel mammalian-specific function of the complex, the suppression of rTEs.

Methylated histones on mitotic chromosomes promote topoisomerase IIα function for high fidelity chromosome segregation.

DNA Topoisomerase IIα (TopoIIα) decatenates sister chromatids, allowing their segregation in mitosis. Without the TopoIIα Strand Passage Reaction (SPR), chromosome bridges and ultra-fine DNA bridges (UFBs) arise in anaphase. The TopoIIα C-terminal domain is dispensable for the SPR in vitro but essential for mitotic functions in vivo. Here, we present evidence that the Chromatin Tether (ChT) within the CTD interacts with specific methylated nucleosomes and is crucial for high-fidelity chromosome segregation. Mutation of individual αChT residues disrupts αChT-nucleosome interaction, induces loss of segregation fidelity and reduces association of TopoIIα with chromosomes. Specific methyltransferase inhibitors reducing histone H3 or H4 methylation decreased TopoIIα at centromeres and increased segregation errors. Methyltransferase inhibition did not further increase aberrant anaphases in the ChT mutants, indicating a functional connection. The evidence reveals novel cellular regulation whereby TopoIIα specifically interacts with methylated nucleosomes via the αChT to ensure high-fidelity chromosome segregation.

Influenza virus mRNAs encode determinants for nuclear export via the cellular TREX-2 complex.

Nuclear export of influenza A virus (IAV) mRNAs occurs through the nuclear pore complex (NPC). Using the Auxin-Induced Degron (AID) system to rapidly degrade proteins, we show that among the nucleoporins localized at the nucleoplasmic side of the NPC, TPR is the key nucleoporin required for nuclear export of influenza virus mRNAs. TPR recruits the TRanscription and EXport complex (TREX)-2 to the NPC for exporting a subset of cellular mRNAs. By degrading components of the TREX-2 complex (GANP, Germinal-center Associated Nuclear Protein; PCID2, PCI domain containing 2), we show that influenza mRNAs require the TREX-2 complex for nuclear export and replication. Furthermore, we found that cellular mRNAs whose export is dependent on GANP have a small number of exons, a high mean exon length, long 3' UTR, and low GC content. Some of these features are shared by influenza virus mRNAs. Additionally, we identified a 45 nucleotide RNA signal from influenza virus HA mRNA that is sufficient to mediate GANP-dependent mRNA export. Thus, we report a role for the TREX-2 complex in nuclear export of influenza mRNAs and identified RNA determinants associated with the TREX-2-dependent mRNA export.

Crm1 is a mitotic effector of Ran-GTP in somatic cells.

The Ran GTPase controls multiple cellular processes, including nuclear transport, mitotic checkpoints, spindle assembly and post-mitotic nuclear envelope reassembly. Here we examine the mitotic function of Crm1, the Ran-GTP-binding nuclear export receptor for leucine-rich cargo (bearing nuclear export sequence) and Snurportin-1 (ref. 3). We find that Crm1 localizes to kinetochores, and that Crm1 ternary complex assembly is essential for Ran-GTP-dependent recruitment of Ran GTPase-activating protein 1 (Ran-GAP1) and Ran-binding protein 2 (Ran-BP2) to kinetochores. We further show that Crm1 inhibition by leptomycin B disrupts mitotic progression and chromosome segregation. Analysis of spindles within leptomycin B-treated cells shows that their centromeres were under increased tension. In leptomycin B-treated cells, centromeres frequently associated with continuous microtubule bundles that spanned the centromeres, indicating that their kinetochores do not maintain discrete end-on attachments to single kinetochore fibres. Similar spindle defects were observed in temperature-sensitive Ran pathway mutants (tsBN2 cells). Taken together, our findings demonstrate that Crm1 and Ran-GTP are essential for Ran-BP2/Ran-GAP1 recruitment to kinetochores, for definition of kinetochore fibres and for chromosome segregation at anaphase. Thus, Crm1 is a critical Ran-GTP effector for mitotic spindle assembly and function in somatic cells.

Bub1 is essential for assembly of the functional inner centromere.

During mitosis, the inner centromeric region (ICR) recruits protein complexes that regulate sister chromatid cohesion, monitor tension, and modulate microtubule attachment. Biochemical pathways that govern formation of the inner centromere remain elusive. The kinetochore protein Bub1 was shown to promote assembly of the outer kinetochore components, such as BubR1 and CENP-F, on centromeres. Bub1 was also implicated in targeting of Shugoshin (Sgo) to the ICR. We show that Bub1 works as a master organizer of the ICR. Depletion of Bub1 from Xenopus laevis egg extract or from HeLa cells resulted in both destabilization and displacement of chromosomal passenger complex (CPC) from the ICR. Moreover, soluble Bub1 controls the binding of Sgo to chromatin, whereas the CPC restricts loading of Sgo specifically onto centromeres. We further provide evidence that Bub1 kinase activity is pivotal for recruitment of all of these components. Together, our findings demonstrate that Bub1 acts at multiple points to assure the correct kinetochore formation.

Analysis of Nucleoporin Function Using Inducible Degron Techniques.

Over the last decade, the use of auxin-inducible degrons (AID) to control the stability of target proteins has revolutionized the field of cell biology. AID-mediated degradation helps to overcome multiple hurdles that have been encountered in studying multisubunit protein complexes, like the nuclear pore complex (NPC), using classical biochemical and genetic methods. We have used the AID system for acute depletion of individual members of the NPC, called nucleoporins, in order to distinguish their roles both within established NPCs and during NPC assembly.Here, we describe a protocol for CRISPR/Cas9-mediated gene targeting of genes with the AID tag. As an example, we describe a step-by-step protocol for targeting of the NUP153 gene. We also provide recommendations for screening strategies and integration of the sequence encoding the Transport Inhibitor Response 1 (TIR1) protein, a E3-Ubiquitin ligase subunit necessary for AID-dependent protein degradation. In addition, we discuss applications of the NUP-AID system and functional assays for analysis of NUP-AID tagged cell lines.

Association of RanGAP to nuclear pore complex component, RanBP2/Nup358, is required for pupal development in Drosophila.

Ran's GTPase-activating protein (RanGAP) is tethered to the nuclear envelope (NE) in multicellular organisms. We investigated the consequences of RanGAP localization in human tissue culture cells and Drosophila. In tissue culture cells, disruption of RanGAP1 NE localization surprisingly has neither obvious impacts on viability nor nucleocytoplasmic transport of a model substrate. In Drosophila, we identified a region within nucleoporin dmRanBP2 required for direct tethering of dmRanGAP to the NE. A dmRanBP2 mutant lacking this region shows no apparent growth defects during larval stages but arrests at the early pupal stage. A direct fusion of dmRanGAP to the dmRanBP2 mutant rescues this arrest, indicating that dmRanGAP recruitment to dmRanBP2 per se is necessary for the pupal ecdysis sequence. Our results indicate that while the NE localization of RanGAP is widely conserved in multicellular organisms, the targeting mechanisms are not. Further, we find a requirement for this localization during pupal development.

RanBP1 controls the Ran pathway in mammalian cells through regulation of mitotic RCC1 dynamics.

The Ran GTPase plays critical roles in multiple cellular processes including interphase nucleocytoplasmic transport and mitotic spindle assembly. During mitosis in mammalian cells, GTP-bound Ran (Ran-GTP) is concentrated near mitotic chromatin while GDP-bound Ran (Ran-GDP) is more abundant distal to chromosomes. This pattern spatially controls spindle formation because Ran-GTP locally releases spindle assembly factors (SAFs), such as Hepatoma Up-Regulated Protein (HURP), from inhibitory interactions near chromosomes. Regulator of Chromatin Condensation 1 (RCC1) is Ran's chromatin-bound exchange factor, and RanBP1 is a conserved Ran-GTP-binding protein that has been implicated as a mitotic regulator of RCC1 in embryonic systems. Here, we show that RanBP1 controls mitotic RCC1 dynamics in human somatic tissue culture cells. In addition, we observed the re-localization of HURP in metaphase cells after RanBP1 degradation, consistent with the idea that altered RCC1 dynamics functionally modulate SAF activities. Together, our findings reveal an important mitotic role for RanBP1 in human somatic cells, controlling the spatial distribution and magnitude of mitotic Ran-GTP production and thereby ensuring the accurate execution of Ran-dependent mitotic events. ABBREVIATIONS: AID: Auxin-induced degron; FLIP: Fluorescence loss in photobleaching; FRAP: Fluorescence recovery after photobleaching; GDP: guanosine diphosphate; GTP: guanosine triphosphate; HURP: Hepatoma Up-Regulated Protein; NE: nuclear envelope; NEBD: Nuclear Envelope Breakdown; RanBP1: Ran-binding protein 1; RanGAP1: Ran GTPase-Activating Protein 1; RCC1: Regulator of Chromatin Condensation 1; RRR complex: RCC1/Ran/RanBP1 heterotrimeric complex; SAF: Spindle Assembly Factor; TIR1: Transport Inhibitor Response 1 protein; XEE: Xenopus egg extract.

PICH regulates the abundance and localization of SUMOylated proteins on mitotic chromosomes.

Proper chromosome segregation is essential for faithful cell division and if not maintained results in defective cell function caused by the abnormal distribution of genetic information. Polo-like kinase 1-interacting checkpoint helicase (PICH) is a DNA translocase essential for chromosome bridge resolution during mitosis. Its function in resolving chromosome bridges requires both DNA translocase activity and ability to bind chromosomal proteins modified by the small ubiquitin-like modifier (SUMO). However, it is unclear how these activities cooperate to resolve chromosome bridges. Here, we show that PICH specifically disperses SUMO2/3 foci on mitotic chromosomes. This PICH function is apparent toward SUMOylated topoisomerase IIα (TopoIIα) after inhibition of TopoIIα by ICRF-193. Conditional depletion of PICH using the auxin-inducible degron (AID) system resulted in the retention of SUMO2/3-modified chromosomal proteins, including TopoIIα, indicating that PICH functions to reduce the association of these proteins with chromosomes. Replacement of PICH with its translocase-deficient mutants led to increased SUMO2/3 foci on chromosomes, suggesting that the reduction of SUMO2/3 foci requires the remodeling activity of PICH. In vitro assays showed that PICH specifically attenuates SUMOylated TopoIIα activity using its SUMO-binding ability. Taking the results together, we propose a novel function of PICH in remodeling SUMOylated proteins to ensure faithful chromosome segregation.

IRBIT Directs Differentiation of Intestinal Stem Cell Progeny to Maintain Tissue Homeostasis.

The maintenance of the intestinal epithelium is ensured by the controlled proliferation of intestinal stem cells (ISCs) and differentiation of their progeny into various cell types, including enterocytes (ECs) that both mediate nutrient absorption and provide a barrier against pathogens. The signals that regulate transition of proliferative ISCs into differentiated ECs are not fully understood. IRBIT is an evolutionarily conserved protein that regulates ribonucleotide reductase (RNR), an enzyme critical for the generation of DNA precursors. Here, we show that IRBIT expression in ISC progeny within the Drosophila midgut epithelium cells regulates their differentiation via suppression of RNR activity. Disruption of this IRBIT-RNR regulatory circuit causes a premature loss of intestinal tissue integrity. Furthermore, age-related dysplasia can be reversed by suppression of RNR activity in ISC progeny. Collectively, our findings demonstrate a role of the IRBIT-RNR pathway in gut homeostasis.

Nucleoporin TPR is an integral component of the TREX-2 mRNA export pathway.

Nuclear pore complexes (NPCs) are important for cellular functions beyond nucleocytoplasmic trafficking, including genome organization and gene expression. This multi-faceted nature and the slow turnover of NPC components complicates investigations of how individual nucleoporins act in these diverse processes. To address this question, we apply an Auxin-Induced Degron (AID) system to distinguish roles of basket nucleoporins NUP153, NUP50 and TPR. Acute depletion of TPR causes rapid and pronounced changes in transcriptomic profiles. These changes are dissimilar to shifts observed after loss of NUP153 or NUP50, but closely related to changes caused by depletion of mRNA export receptor NXF1 or the GANP subunit of the TRanscription-EXport-2 (TREX-2) mRNA export complex. Moreover, TPR depletion disrupts association of TREX-2 subunits (GANP, PCID2, ENY2) to NPCs and results in abnormal RNA transcription and export. Our findings demonstrate a unique and pivotal role of TPR in gene expression through TREX-2- and/or NXF1-dependent mRNA turnover.

The Ran GTPase regulates kinetochore function.

The Ran GTPase is required for nuclear assembly, nuclear transport, spindle assembly, and mitotic regulation. While the first three processes are relatively well understood, details of Ran's role in mitotic progression remain obscure. We have found that elevated levels of Ran's exchange factor (RCC1) abrogate the spindle assembly checkpoint in Xenopus egg extracts, restore APC/C activity, and disrupt the kinetochore localization of checkpoint regulators, including Mad2, CENP-E, Bub1, and Bub3. Depletion of Ran's GTPase activating protein (RanGAP1) and its accessory factor (RanBP1) similarly abrogates checkpoint arrest. By contrast, the addition of RanGAP1 and RanBP1 to extracts with exogenous RCC1 restores the spindle checkpoint. Together, these observations suggest that the spindle checkpoint is directly responsive to Ran-GTP levels. Finally, we observe a clear wave of RCC1 association to mitotic chromosomes at the metaphase-anaphase transition in normal cycling extracts, suggesting that this mechanism has an important role in unperturbed cell cycles.

SUMO-2/3 regulates topoisomerase II in mitosis.

We have analyzed the abundance of SUMO-conjugated species during the cell cycle in Xenopus egg extracts. The predominant SUMO conjugation products associated with mitotic chromosomes arose from SUMO conjugation of topoisomerase II. Topoisomerase II was modified exclusively by SUMO-2/3 during mitosis under normal circumstances, although we observed conjugation of topoisomerase II to SUMO-1 in extracts with exogenous SUMO-1 protein. Inhibition of SUMO modification by a dominant-negative mutant of the SUMO-conjugating enzyme Ubc9 (dnUbc9) did not detectably alter topoisomerase II activity, but it did increase the amount of unmodified topoisomerase II retained on mitotic chromosomes after high salt washing. dnUbc9 did not disrupt the assembly of condensed mitotic chromosomes or block progression of extracts through mitosis, but it did block the dissociation of sister chromatids at the metaphase-anaphase transition. Together, our results suggest that SUMO conjugation is important for chromosome segregation in metazoan systems, and that mobilization of topoisomerase II from mitotic chromatin may be a key target of this modification.

The Nup107-160 nucleoporin complex is required for correct bipolar spindle assembly.

The Nup107-160 complex is a critical subunit of the nuclear pore. This complex localizes to kinetochores in mitotic mammalian cells, where its function is unknown. To examine Nup107-160 complex recruitment to kinetochores, we stained human cells with antisera to four complex components. Each antibody stained not only kinetochores but also prometaphase spindle poles and proximal spindle fibers, mirroring the dual prometaphase localization of the spindle checkpoint proteins Mad1, Mad2, Bub3, and Cdc20. Indeed, expanded crescents of the Nup107-160 complex encircled unattached kinetochores, similar to the hyperaccumulation observed of dynamic outer kinetochore checkpoint proteins and motors at unattached kinetochores. In mitotic Xenopus egg extracts, the Nup107-160 complex localized throughout reconstituted spindles. When the Nup107-160 complex was depleted from extracts, the spindle checkpoint remained intact, but spindle assembly was rendered strikingly defective. Microtubule nucleation around sperm centrosomes seemed normal, but the microtubules quickly disassembled, leaving largely unattached sperm chromatin. Notably, Ran-GTP caused normal assembly of microtubule asters in depleted extracts, indicating that this defect was upstream of Ran or independent of it. We conclude that the Nup107-160 complex is dynamic in mitosis and that it promotes spindle assembly in a manner that is distinct from its functions at interphase nuclear pores.

The cell nucleus. A study in Burgundy.

Wilhelm Bernhard's revolutionary microscopy techniques helped him put forward the hypothesis of specialized compartmentalization of the nucleus. He also described for the first time the nuclear bodies and peri-chromatin fibrils, and demonstrated that these granules contain an RNA component. The tradition of biennial workshops, named after this great scientist, continues, and this year it took place in the heart of Burgundy, in Dijon, France (May 20-24, 2019, organized by INSERM UMR1231, UBFC), where well-fed participants emphasized the importance of viewing the cell nucleus as a hub of specialized colloidal compartments that orchestrate replication, transcription and nuclear transport.

RCC1 regulates inner centromeric composition in a Ran-independent fashion.

RCC1 associates to chromatin dynamically within mitosis and catalyzes Ran-GTP production. Exogenous RCC1 disrupts kinetochore structure in Xenopus egg extracts (XEEs), but the molecular basis of this disruption remains unknown. We have investigated this question, utilizing replicated chromosomes that possess paired sister kinetochores. We find that exogenous RCC1 evicts a specific subset of inner KT proteins including Shugoshin-1 (Sgo1) and the chromosome passenger complex (CPC). We generated RCC1 mutants that separate its enzymatic activity and chromatin binding. Strikingly, Sgo1 and CPC eviction depended only on RCC1's chromatin affinity but not its capacity to produce Ran-GTP. RCC1 similarly released Sgo1 and CPC from synthetic kinetochores assembled on CENP-A nucleosome arrays. Together, our findings indicate RCC1 regulates kinetochores at the metaphase-anaphase transition through Ran-GTP-independent displacement of Sgo1 and CPC.

Quantitative assessment of chromosome instability induced through chemical disruption of mitotic progression.

Most solid tumors are aneuploid, carrying an abnormal number of chromosomes, and they frequently missegregate whole chromosomes in a phenomenon termed chromosome instability (CIN). While CIN can be provoked through disruption of numerous mitotic pathways, it is not clear which of these mechanisms are most critical, or whether alternative mechanisms could also contribute significantly in vivo. One difficulty in determining the relative importance of candidate CIN regulators has been the lack of a straightforward, quantitative assay for CIN in live human cells: While gross mitotic abnormalities can be detected visually, moderate levels of CIN may not be obvious, and are thus problematic to measure. To address this issue, we have developed the first Human Artificial Chromosome (HAC)-based quantitative live-cell assay for mitotic chromosome segregation in human cells. We have produced U2OS-Phoenix cells carrying the alphoid(tetO)-HAC encoding copies of eGFP fused to the destruction box (DB) of anaphase promoting complex/cyclosome (APC/C) substrate hSecurin and sequences encoding the tetracycline repressor fused to mCherry (TetR-mCherry). Upon HAC missegregation, daughter cells that do not obtain a copy of the HAC are GFP negative in the subsequent interphase. The HAC can also be monitored live following the TetR-mCherry signal. U2OS-Phoenix cells show low inherent levels of CIN, which can be enhanced by agents that target mitotic progression through distinct mechanisms. This assay allows direct detection of CIN induced by clinically important agents without conspicuous mitotic defects, allowing us to score increased levels of CIN that fall below the threshold required for discernable morphological disruption.

Phosphorylation of Xenopus p31(comet) potentiates mitotic checkpoint exit.

p31(comet) plays an important role in spindle assembly checkpoint (SAC) silencing. However, how p31(comet)'s activity is regulated remains unclear. Here we show that the timing of M-phase exit in Xenopus egg extracts (XEEs) depends upon SAC activity, even under conditions that are permissive for spindle assembly. p31(comet) antagonizes the SAC, promoting XEE progression into anaphase after spindles are fully formed. We further show that mitotic p31(comet) phosphorylation by Inhibitor of nuclear factor κ-B kinase-ß (IKK-ß) enhances this role in SAC silencing. Together, our findings implicate IKK-ß in the control of anaphase timing in XEE through p31(comet) activation and SAC downregulation.