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.

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.

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.

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.

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.

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.

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.

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.

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.

RanBP1 governs spindle assembly by defining mitotic Ran-GTP production.

Accurate control of the Ras-related nuclear protein (Ran) GTPase cycle depends on the regulated activity of regulator of chromosome condensation 1 (RCC1), Ran's nucleotide exchange factor. RanBP1 has been characterized as a coactivator of the Ran GTPase-activating protein RanGAP1. RanBP1 can also form a stable complex with Ran and RCC1, although the dynamics and function of this complex remain poorly understood. Here, we show that formation of the heterotrimeric RCC1/Ran/RanBP1 complex in M phase Xenopus egg extracts controls both RCC1's enzymatic activity and partitioning between the chromatin-bound and soluble pools of RCC1. This mechanism is critical for spatial control of Ran-guanosine triphosphate (GTP) gradients that guide mitotic spindle assembly. Moreover, phosphorylation of RanBP1 drives changes in the dynamics of chromatin-bound RCC1 pools at the metaphase-anaphase transition. Our findings reveal an important mitotic role for RanBP1, controlling the spatial distribution and magnitude of mitotic Ran-GTP production and thereby ensuring accurate execution of Ran-dependent mitotic events.

Enzyme regulation. IRBIT is a novel regulator of ribonucleotide reductase in higher eukaryotes.

Ribonucleotide reductase (RNR) supplies the balanced pools of deoxynucleotide triphosphates (dNTPs) necessary for DNA replication and maintenance of genomic integrity. RNR is subject to allosteric regulatory mechanisms in all eukaryotes, as well as to control by small protein inhibitors Sml1p and Spd1p in budding and fission yeast, respectively. Here, we show that the metazoan protein IRBIT forms a deoxyadenosine triphosphate (dATP)-dependent complex with RNR, which stabilizes dATP in the activity site of RNR and thus inhibits the enzyme. Formation of the RNR-IRBIT complex is regulated through phosphorylation of IRBIT, and ablation of IRBIT expression in HeLa cells causes imbalanced dNTP pools and altered cell cycle progression. We demonstrate a mechanism for RNR regulation in higher eukaryotes that acts by enhancing allosteric RNR inhibition by dATP.

SUMOylation of Psmd1 controls Adrm1 interaction with the proteasome.

SUMOylation is the covalent conjugation of SUMO polypeptides to cellular target proteins. Psmd1 is a subunit of the proteasomal 19S regulatory particle that acts as a docking site for Adrm1, another proteasome subunit that recruits ubiquitinated substrates for proteolysis. Here, we show that the SUMO deconjugating enzyme xSENP1 specifically interacts with Psmd1 and that disruption of xSENP1 targeting delays mitotic exit. Psmd1 becomes SUMOylated through the action of the SUMO E3 enzyme PIASy. We mapped SUMOylation sites within Psmd1 and found that SUMOylation of a critical lysine immediately adjacent to the Adrm1-binding domain regulates the association of Adrm1 with Psmd1. Together, our findings suggest that the interaction of Psmd1 with Adrm1 is controlled by SUMOylation in a manner that may alter proteasome composition and function. These findings demonstrate a mechanism for regulation of ubiquitin-mediated protein degradation by ubiquitin-like proteins of the SUMO family.

Xenopus HJURP and condensin II are required for CENP-A assembly.

Centromeric protein A (CENP-A) is the epigenetic mark of centromeres. CENP-A replenishment is necessary in each cell cycle to compensate for the dilution associated to DNA replication, but how this is achieved mechanistically is largely unknown. We have developed an assay using Xenopus egg extracts that can recapitulate the spatial and temporal specificity of CENP-A deposition observed in human cells, providing us with a robust in vitro system amenable to molecular dissection. Here we show that this deposition depends on Xenopus Holliday junction-recognizing protein (xHJURP), a member of the HJURP/Scm3 family recently identified in yeast and human cells, further supporting the essential role of these chaperones in CENP-A loading. Despite little sequence homology, human HJURP can substitute for xHJURP. We also report that condensin II, but not condensin I, is required for CENP-A assembly and contributes to retention of centromeric CENP-A nucleosomes both in mitosis and interphase. We propose that the chromatin structure imposed by condensin II at centromeres enables CENP-A incorporation initiated by xHJURP.

The SUMO protease SENP6 is essential for inner kinetochore assembly.

We have analyzed the mitotic function of SENP6, a small ubiquitin-like modifier (SUMO) protease that disassembles conjugated SUMO-2/3 chains. Cells lacking SENP6 showed defects in spindle assembly and metaphase chromosome congression. Analysis of kinetochore composition in these cells revealed that a subset of proteins became undetectable on inner kinetochores after SENP6 depletion, particularly the CENP-H/I/K complex, whereas other changes in kinetochore composition mimicked defects previously reported to result from CENP-H/I/K depletion. We further found that CENP-I is degraded through the action of RNF4, a ubiquitin ligase which targets polysumoylated proteins for proteasomal degradation, and that SENP6 stabilizes CENP-I by antagonizing RNF4. Together, these findings reveal a novel mechanism whereby the finely balanced activities of SENP6 and RNF4 control vertebrate kinetochore assembly through SUMO-targeted destabilization of inner plate components.

The Nup107-160 complex and gamma-TuRC regulate microtubule polymerization at kinetochores.

The metazoan nuclear pore complex (NPC) disassembles during mitosis, and many of its constituents distribute onto spindles and kinetochores, including the Nup107-160 sub-complex. We have found that Nup107-160 interacts with the gamma-tubulin ring complex (gamma-TuRC), an essential and conserved microtubule nucleator, and recruits gamma-TuRC to unattached kinetochores. The unattached kinetochores nucleate microtubules in a manner that is regulated by Ran GTPase; such microtubules contribute to the formation of kinetochore fibres (k-fibres), microtubule bundles connecting kinetochores to spindle poles. Our data indicate that Nup107-160 and gamma-TuRC act cooperatively to promote spindle assembly through microtubule nucleation at kinetochores: HeLa cells lacking Nup107-160 or gamma-TuRC were profoundly deficient in kinetochore-associated microtubule nucleation. Moreover, co-precipitated Nup107-160- gamma-TuRC complexes nucleated microtubule formation in assays using purified tubulin. Although Ran did not regulate microtubule nucleation by gamma-TuRC alone, Nup107-160-gamma-TuRC complexes required Ran-GTP for microtubule nucleation. Collectively, our observations show that Nup107-160 promotes spindle assembly through Ran-GTP-regulated nucleation of microtubules by gamma-TuRC at kinetochores, and reveal a relationship between nucleoporins and the microtubule cytoskeleton.