Chromosome Capture Research Articles

The effect of taxol microinjection on the microtubular structure in polar body formation of starfish oocytes

In starfish oocytes, microtubules (MTs) form a spindle, which plays an important role in contributing to the selective loss of chromosomes and centrosomes to the polar bodies (PBs) during meiosis. When Taxol was locally injected near the germinal vesicle (GV) or the mitotic apparatus during meiosis I, PB formation was inhibited as mentioned below. In the oocytes, which were injected with Taxol after spindle formation, the spindle became large, and then the volume of the first PB also increased more than that of the control. In contrast, in the oocytes injected with Taxol before the spindle formation, chromosome capture and alignment were inhibited. These oocytes did not form PB, but only a bulge at the cell cortex was occasionally observed. Moreover, in the oocytes injected with Taxol before GV breakdown, the chromosomes did not gather in one place, and then two asters were observed at distant positions from the cell cortex. These results suggested that MTs lost not only the ability to obtain the bipolar attachment of chromosomes by Taxol injection but also the aster closer to the cell cortex lost its interaction with the cell cortex of the animal pole.

Pharmacogene regulatory elements: from discovery to applications.

Regulatory elements play an important role in the variability of individual responses to drug treatment. This has been established through studies on three classes of elements that regulate RNA and protein abundance: promoters, enhancers and microRNAs. Each of these elements, and genetic variants within them, are being characterized at an exponential pace by next-generation sequencing (NGS) technologies. In this review, we outline examples of how each class of element affects drug response via regulation of drug targets, transporters and enzymes. We also discuss the impact of NGS technologies such as chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA sequencing (RNA-Seq), and the ramifications of new techniques such as high-throughput chromosome capture (Hi-C), chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) and massively parallel reporter assays (MPRA). NGS approaches are generating data faster than they can be analyzed, and new methods will be required to prioritize laboratory results before they are ready for the clinic. However, there is no doubt that these approaches will bring about a systems-level understanding of the interplay between genetic variants and drug response. An understanding of the importance of regulatory variants in pharmacogenomics will facilitate the identification of responders versus non-responders, the prevention of adverse effects and the optimization of therapies for individual patients.

15q11.2–13.3 chromatin analysis reveals epigenetic regulation of CHRNA7 with deficiencies in Rett and autism brain

Copy number variations (CNVs) within human 15q11.2-13.3 show reduced penetrance and variable expressivity in a range of neurologic disorders. Therefore, characterizing 15q11.2-13.3 chromatin structure is important for understanding the regulation of this locus during normal neuronal development. Deletion of the Prader-Willi imprinting center (PWS-IC) within 15q11.2-13.3 disrupts long-range imprinted gene expression resulting in Prader-Willi syndrome. Previous results establish that MeCP2 binds to the PWS-IC and is required for optimal expression of distal GABRB3 and UBE3A. To examine the hypothesis that MeCP2 facilitates 15q11.2-13.3 transcription by linking the PWS-IC with distant elements, chromosome capture conformation on chip (4C) analysis was performed in human SH-SY5Y neuroblastoma cells. SH-SY5Y neurons had 2.84-fold fewer 15q11.2-13.3 PWS-IC chromatin interactions than undifferentiated SH-SY5Y neuroblasts, revealing developmental chromatin de-condensation of the locus. Out of 68 PWS-IC interactions with15q11.2-13.3 identified by 4C analysis and 62 15q11.2-13.3 MeCP2-binding sites identified by previous ChIP-chip studies, only five sites showed overlap. Remarkably, two of these overlapping PWS-IC- and MeCP2-bound sites mapped to sites flanking CHRNA7 (cholinergic receptor nicotinic alpha 7) encoding the cholinergic receptor, nicotinic, alpha 7. PWS-IC interaction with CHRNA7 in neurons was independently confirmed by fluorescent in situ hybridization analysis. Subsequent quantitative transcriptional analyses of frontal cortex from Rett syndrome and autism patients revealed significantly reduced CHRNA7 expression compared with controls. Together, these results suggest that transcription of CHRNA7 is modulated by chromatin interactions with the PWS-IC. Thus, loss of long-range chromatin interactions within 15q11.2-13.3 may contribute to multiple human neurodevelopmental disorders.

50 ways to build a spindle: the complexity of microtubule generation during mitosis

The accurate segregation of duplicated chromosomes, essential for the development and viability of a eukaryotic organism, requires the formation of a robust microtubule (MT)-based spindle apparatus. Entry into mitosis or meiosis precipitates a cascade of signalling events which result in the activation of pathways responsible for a dramatic reorganisation of the MT cytoskeleton: through changes in the properties of MT-associated proteins, local concentrations of free tubulin dimer and through enhanced MT nucleation. The latter is generally thought to be driven by localisation and activation of γ-tubulin-containing complexes (γ-TuSC and γ-TuRC) at specific subcellular locations. For example, upon entering mitosis, animal cells concentrate γ-tubulin at centrosomes to tenfold the normal level during interphase, resulting in an aster-driven search and capture of chromosomes and bipolar mitotic spindle formation. Thus, in these cells, centrosomes have traditionally been perceived as the primary microtubule organising centre during spindle formation. However, studies in meiotic cells, plants and cell-free extracts have revealed the existence of complementary mechanisms of spindle formation, mitotic chromatin, kinetochores and nucleation from existing MTs or the cytoplasm can all contribute to a bipolar spindle apparatus. Here, we outline the individual known mechanisms responsible for spindle formation and formulate ideas regarding the relationship between them in assembling a functional spindle apparatus.

A First-Passage-Time Theory for Search and Capture of Chromosomes by Microtubules in Mitosis

The mitotic spindle is an important intermediate structure in eukaryotic cell division, in which each of a pair of duplicated chromosomes is attached through microtubules to centrosomal bodies located close to the two poles of the dividing cell. Several mechanisms are at work toward the formation of the spindle, one of which is the 'capture' of chromosome pairs, held together by kinetochores, by randomly searching microtubules. Although the entire cell cycle can be up to 24 hours long, the mitotic phase typically takes only less than an hour. How does the cell keep the duration of mitosis within this limit? Previous theoretical studies have suggested that the chromosome search and capture is optimized by tuning the microtubule dynamic parameters to minimize the search time. In this paper, we examine this conjecture. We compute the mean search time for a single target by microtubules from a single nucleating site, using a systematic and rigorous theoretical approach, for arbitrary kinetic parameters. The result is extended to multiple targets and nucleating sites by physical arguments. Estimates of mitotic time scales are then obtained for different cells using experimental data. In yeast and mammalian cells, the observed changes in microtubule kinetics between interphase and mitosis are beneficial in reducing the search time. In Xenopus extracts, by contrast, the opposite effect is observed, in agreement with the current understanding that large cells use additional mechanisms to regulate the duration of the mitotic phase.

Microtubule Stabilization Triggers the Plus-End Accumulation of Kif18A/kinesin-8

The precise control of spindle microtubule (MT) dynamics is essential for chromosome capture and alignment. Kif18A/kinesin-8, an essential regulator of kinetochore MT dynamics, accumulates at its plus-ends in metaphase but not prometaphase cells. The underlying mechanism of time-dependent and kinetochore MT-specific plus-end accumulation of Kif18A is unknown. Here, we examined the factors required for the MT plus-end accumulation of Kif18A. In Eg5 inhibitor-treated cells, Kif18A localized along the MTs in the monopolar spindle and rarely accumulated at their plus-ends, indicating that MT-kinetochore association was not sufficient to induce Kif18A accumulation. In contrast, taxol treatment triggered the rapid MT plus-end accumulation of Kif18A regardless of kinetochore association. Furthermore, Aurora B inhibitor-induced stabilization of the plus-ends of kinetochore MTs promoted the plus-end accumulation of Kif18A. In the absence of Kif18A, treatment with taxol but not Eg5 inhibitor causes highly elongated mitotic MTs, suggesting the importance of plus-end accumulation for the MT length-controlling activity of Kif18A. Taken together, we propose that there is a mutual regulation of kinetochore MT plus-end dynamics and Kif18A accumulation, which may contribute to the highly regulated and ordered changes in kinetochore MT dynamics during chromosome congression and oscillation.

A yeast‐based recombinogenic targeting toolset for transgenic analysis of human disease genes

Transgenic mouse models are valuable resources for analyzing functions of genes involved in human diseases. Mouse models provide critical insights into biological processes, including in vivo visualization of vasculature critical to our understanding of the immune system. Generating transgenic mice requires the capture and modification of large-insert DNAs representing genes of interest. We have developed a methodology using a yeast-bacterial shuttle vector, pClasper, that enables the capture and modification of bacterial artificial chromosomes (BAC)-sized DNA inserts. Numerous improvements and technical advances in the original pClasper vector have allowed greater flexibility and utility in this system. Examples of such pClasper mediated gene modifications include: Claspette-mediated capture of large-insert genomic fragments from BACs-human polycystic kidney disease-1 (PKD1); modification of pClasperA clones by the RareGap method-PKD1 mutations; Claspette-mediated modification of pClasper clones-mouse albumin-1 gene; and, of most relevance to our interest in lymph node vasculature-Claspimer-mediated modification of pClasper clones-high endothelial venule and lymphatic vessel genes. Mice that have been generated with these methods include mice with fluorescent high endothelial venules.

Rare Deletions at 16p13.11 Predispose to a Diverse Spectrum of Sporadic Epilepsy Syndromes

Deletions at 16p13.11 are associated with schizophrenia, mental retardation, and most recently idiopathic generalized epilepsy. To evaluate the role of 16p13.11 deletions, as well as other structural variation, in epilepsy disorders, we used genome-wide screens to identify copy number variation in 3812 patients with a diverse spectrum of epilepsy syndromes and in 1299 neurologically-normal controls. Large deletions (> 100 kb) at 16p13.11 were observed in 23 patients, whereas no control had a deletion greater than 16 kb. Patients, even those with identically sized 16p13.11 deletions, presented with highly variable epilepsy phenotypes. For a subset of patients with a 16p13.11 deletion, we show a consistent reduction of expression for included genes, suggesting that haploinsufficiency might contribute to pathogenicity. We also investigated another possible mechanism of pathogenicity by using hybridization-based capture and next-generation sequencing of the homologous chromosome for ten 16p13.11-deletion patients to look for unmasked recessive mutations. Follow-up genotyping of suggestive polymorphisms failed to identify any convincing recessive-acting mutations in the homologous interval corresponding to the deletion. The observation that two of the 16p13.11 deletions were larger than 2 Mb in size led us to screen for other large deletions. We found 12 additional genomic regions harboring deletions > 2 Mb in epilepsy patients, and none in controls. Additional evaluation is needed to characterize the role of these exceedingly large, non-locus-specific deletions in epilepsy. Collectively, these data implicate 16p13.11 and possibly other large deletions as risk factors for a wide range of epilepsy disorders, and they appear to point toward haploinsufficiency as a contributor to the pathogenicity of deletions.

Mitotic force generators and chromosome segregation.

The mitotic spindle uses dynamic microtubules and mitotic motors to generate the pico-Newton scale forces that are needed to drive the mitotic movements that underlie chromosome capture, alignment and segregation. Here, we consider the biophysical and molecular basis of force-generation for chromosome movements in the spindle, and, with reference to the Drosophila embryo mitotic spindle, we briefly discuss how mathematical modeling can complement experimental analysis to illuminate the mechanisms of chromosome-to-pole motility during anaphase A and spindle elongation during anaphase B.

Kinetochores Generate Microtubules with Distal Plus Ends: Their Roles and Limited Lifetime in Mitosis

SummaryIn early mitosis, microtubules can be generated at kinetochores as well as at spindle poles. However, the role and regulation of kinetochore-derived microtubules have been unclear. In general, metaphase spindle microtubules are oriented such that their plus ends bind to kinetochores. However, we now have evidence that, during early mitosis in budding yeast, microtubules are generated at kinetochores with distal plus ends. These kinetochore-derived microtubules interact along their length with microtubules that extend from a spindle pole, facilitating kinetochore loading onto the lateral surface of spindle pole microtubules. Once kinetochores are loaded, microtubules are no longer generated at kinetochores, and those that remain disappear rapidly and do not contribute to the metaphase spindle. Stu2 (the ortholog of vertebrate XMAP215/ch-TOG) localizes to kinetochores and plays a central role in regulating kinetochore-derived microtubules. Our work provides insight into microtubule generation at kinetochores and the mechanisms that facilitate initial kinetochore interaction with spindle pole microtubules.

Irc15 Is a Microtubule-Associated Protein that Regulates Microtubule Dynamics in Saccharomyces cerevisiae

Microtubules are polymers composed of alpha-beta tubulin heterodimers that assemble into microtubules. Microtubules are dynamic structures that have periods of both growth and shrinkage by addition and removal of subunits from the polymer. Microtubules stochastically switch between periods of growth and shrinkage, termed dynamic instability. Dynamic instability is coupled to the GTPase activity of the beta-tubulin subunit of the tubulin heterodimer. Microtubule dynamics are regulated by microtubule-associated proteins (MAPs) that interact with microtubules to regulate dynamic instability. MAPs in budding yeast have been identified that bind microtubule ends (Bim1), that stabilize microtubule structures (Stu2), that bundle microtubules by forming cross-bridges (Ase1), and that interact with microtubules at the kinetochore (Cin8, Kar3, Kip3). IRC15 was previously identified in four different genetic screens for mutants affecting chromosome transmission or repair [11-14]. Here we present evidence that Irc15 is a microtubule-associated protein, localizing to microtubules in vivo and binding to purified microtubules in vitro. Irc15 regulates microtubule dynamics in vivo and loss of IRC15 function leads to delayed mitotic progression, resulting from failure to establish tension between sister kinetochores.

Genetics and Epigenetics of the PU.1 Upstream Regulatory Element: AML1 Binding Sites Are Critical for Leuekmia Induced by the AML/ETO9a Fusion Oncogene

The level of expression of the transcription factor PU.1 is a critical determinant of lineage commitment in normal hematopoiesis, and dysregulation of PU.1 leads to development of leukemia. In mice with targeted disruption of the PU.1 upstream regulatory element (URE), expression of PU.1 is decreased to 20% of wild type levels and results in development of acute myeloid leukemia (AML). These data suggests that tightly regulated PU.1 expression is important to maintain normal hematopoiesis and prevent leukemogenesis. Previously, we reported that AML1 (RUNX1) regulated PU.1 expression. Here we demonstrate that AMLl regulates PU.1 through 3 AML1 binding sites in the URE. Mice with targeted mutations in the 3 AML1 binding sites have decreased PU.1 expression in multiple hematopoietic lineages at multiple different developmental stages. Conditional targeting of AML1 in transgenic mice in which the URE homology region 2 (H2, containing all 3 AML1 binding sites) is used to drive expression of a reporter decreased reporter gene expression, suggesting that AML1 regulates PU.1 through these 3 sites in URE homology region 2. Using a second mouse model with a targeted mutation in the PU.1 binding site in the PU.1 URE (which is flanked by the 3 AML1 sites), we demonstrated that PU.1 indeed autoregulates itself through the URE. These results demonstrated that AML1 regulates PU.1 through the 3 AML1 sites in the URE. However, while low levels of PU.1 lead to leukemia, we have not observed frank leukemia development in AML1 conditional knockouts or in mice with targeted disruption of the 3 AML1 sites in the PU.1 URE. We hypothesized that this might be the case because disruption of AML1 or the AML1 sites reduces PU.1 levels to about 40% of wild type, but not as great as that found in PU.1 URE knockouts, which do progress to AML (20% of wild type). We hypothesized that downregulation of PU.1 as a result of binding of AML1/ETO fusion proteins to the URE might result in further reductions of PU.1 expression, and contribute to leukemogenesis. Therefore, we predicted that development of leukemia might be delayed in mice with mutations in the PU.1 URE AML1 DNA binding sites, and this was indeed the case in a modle using a retrovirus expressing the AML1/ETO9a form. We further explored the effect of AML1 and PU.1 binding on chromatin strucutre using chromatin immunoprecipitation (Chip) in the AML1 and PU.1 site URE knockin models, and found that AML1 is involved in H3K4me3 and H3/H4 acetylation of histone tails in the PU.1 URE, while PU.1 is involved in H3/H4 acetylation but not H3K4me3; H3K4 methylation and H3 acetylation decreased in AML1 sites mutant knockin mice and H3 acetylation decreased in PU.1 site mutant knockin mice. Mutation of the AML1 site in mice not only altered the chromatin structure of the URE region, but also interefered with the physical interaction between the URE and PU.1 promoter, as assessed by chromosome capture configuration (3C) assays. Interestingly, the AML1/ETO9a fusion oncogene has a unique role on the epigenetic status of the PU.1 URE in addition to its dominant effect on the 3 AML1 sites. AML1-ETO9 blocks the autoregulation of PU.1 through the PU.1 site in the URE. In summary, our data suggests that AML1 regulates PU.1 expression through 3 AML1 binding sites in the PU.1 URE by modifying chromatin structure in the URE region. In addition, PU.1 can autoregulate itself by facilitating similar epigenetic changes. Dysregulation of the epigenetic status by chromosome translocation products such as AML1-ETO might play an important role in leukemogenesis.First two authors contribute equally to this work.

Grasping at Origins

Chromosome segregation in the bacterium Caulobacter crescentus involves propulsion of the replication origin and its capture at one pole of the cell. Bowman et al. (2008) and Ebersbach et al. (2008) now report the discovery of a protein called PopZ that mediates this chromosome capture.

Cis-Regulatory Elements within the Odorant Receptor Coding Region

Complex regulatory mechanisms lead to the expression in each olfactory neuron of one allele of only one of the 1000 odorant receptor (OR) genes. In this issue, Nguyen et al. (2007) provide evidence that regulatory elements residing within the coding region of OR genes are involved in the singularity of OR gene expression.

Farnesyl transferase inhibitors impair chromosomal maintenance in cell lines and human tumors by compromising CENP-E and CENP-F function

Farnesyl transferase inhibitors (FTI) exhibit anticancer activity as a single agent in preclinical studies and show promise in combination with other therapeutics in clinical trials. Previous studies show that FTIs arrest cancer cells in mitosis; however, the mechanism by which this occurs is unclear. Here, we observed that treatment of various cancer cell lines with the FTI lonafarnib caused mitotic chromosomal alignment defects, leaving cells in a pseudometaphase state, whereby both aligned chromosomes and chromosomes juxtaposed to the spindle poles (termed "lagging chromosomes") were observed in the same cell. To determine how this occurs, we investigated the functionality of two farnesylated mitotic proteins, CENP-E and CENP-F, which mediate chromosomal capture and alignment. The data show that lonafarnib in proliferating cancer cells depletes CENP-E and CENP-F from metaphase but not prometaphase kinetochores. Loss of CENP-E and CENP-F metaphase localization triggered aberrant chromosomal maintenance, causing aligned chromosomes to be prematurely released from the spindle equator and become lagging chromosomes, resulting in a mitotic delay. Furthermore, lonafarnib treatment reduces sister kinetochore tension and activates the BubR1 spindle checkpoint, suggesting that farnesylation of CENP-E and CENP-F is critical for their functionality in maintaining kinetochore-microtubule interactions. Importantly, apparently similar chromosomal alignment defects were observed in head and neck tumors samples from a phase I trial with lonafarnib, providing support that lonafarnib disrupts chromosomal maintenance in human cancers. Lastly, to examine how farnesylation could regulate CENP-E in mediating kinetochore-microtubule attachments, we examined possible docking motifs of a farnesyl group on the outer surface of the microtubule. This analysis revealed three hydrophobic patches on the tubulin dimer for insertion of a farnesyl group, alluding to the possibility of an association between a farnesyl group and the microtubule.

Pharmacologic inhibition of CDC25 phosphatases impairs interphase microtubule dynamics and mitotic spindle assembly

The CDC25 cell cycle regulators are promising targets for new pharmacologic approaches in cancer therapy. Inhibitory compounds such as BN82685 have proven to be effective in specifically targeting CDC25 in cultured cells and in inhibiting tumor cell growth. Here, we report that BN82685 impairs microtubule dynamic instability and alters microtubule organization and assembly at the centrosome in interphase cells. Treatment of mitotic cells with BN82685 delays mitotic spindle assembly, chromosome capture, and metaphase plate formation. Furthermore, we show that combining low concentrations of both BN82685 and paclitaxel inhibits the proliferation of HT29 human colon cancer cells. Our results show a role for CDC25 phosphatases in regulating microtubule dynamics throughout the cell cycle and suggest that combinations of CDC25 inhibitors with microtubule-targeting agents may be of therapeutic value.

Dynamic microtubules are essential for efficient chromosome capture and biorientation in S. cerevisiae

Attachment of chromosomes to the mitotic spindle has been proposed to require dynamic microtubules that randomly search three-dimensional space and become stabilized upon capture by kinetochores. In this study, we test this model by examining chromosome capture in Saccharomyces cerevisiae mutants with attenuated microtubule dynamics. Although viable, these cells are slow to progress through mitosis. Preanaphase cells contain a high proportion of chromosomes that are attached to only one spindle pole and missegregate in the absence of the spindle assembly checkpoint. Measurement of the rates of chromosome capture and biorientation demonstrate that both are severely decreased in the mutants. These results provide direct evidence that dynamic microtubules are critical for efficient chromosome capture and biorientation and support the hypothesis that microtubule search and capture plays a central role in assembly of the mitotic spindle.

Molecular Requirements for Kinetochore-Associated Microtubule Formation in Mammalian Cells

In centrosome-containing cells, microtubules nucleated at centrosomes are thought to play a major role in spindle assembly. In addition, microtubule formation at kinetochores has also been observed, most recently under physiological conditions in live cells. The relative contributions of microtubule formation at kinetochores and centrosomes to spindle assembly, and their molecular requirements, remain incompletely understood. Using mammalian cells released from nocodazole-induced disassembly, we observed microtubule formation at centrosomes and at Bub1-positive sites on chromosomes. Kinetochore-associated microtubules rapidly coalesced into pole-like structures in a dynein-dependent manner. Microinjection of excess importin-beta or depletion of the Ran-dependent spindle assembly factor, TPX2, blocked kinetochore-associated microtubule formation, enhanced centrosome-associated microtubule formation, but did not prevent chromosome capture by centrosomal microtubules. Depletion of the chromosome passenger protein, survivin, reduced microtubule formation at kinetochores in an MCAK-dependent manner. Microtubule formation in cells depleted of Bub1 or Nuf2 was indistinguishable from that in controls. Our data demonstrate that microtubule assembly at centrosomes and kinetochores is kinetically distinct and differentially regulated. The presence of microtubules at kinetochores provides a mechanism to reconcile the time required for spindle assembly in vivo with that observed in computer simulations of search and capture.

Chromosome Congression: Another Fine Mesh We’ve Gotten into

Collecting chromosomes prior to their accurate distribution by the mitotic spindle is widely believed to be a microtubule-driven process. However, a recent study in Nature by Lénart et al. (2005) has revealed that a contractile actin network makes an essential contribution to chromosome capture in animal oocytes.

A contractile nuclear actin network drives chromosome congression in oocytes

Chromosome capture by microtubules is widely accepted as the universal mechanism of spindle assembly in dividing cells. However, the observed length of spindle microtubules and computer simulations of spindle assembly predict that chromosome capture is efficient in small cells, but may fail in cells with large nuclear volumes such as animal oocytes. Here we investigate chromosome congression during the first meiotic division in starfish oocytes. We show that microtubules are not sufficient for capturing chromosomes. Instead, chromosome congression requires actin polymerization. After nuclear envelope breakdown, we observe the formation of a filamentous actin mesh in the nuclear region, and find that contraction of this network delivers chromosomes to the microtubule spindle. We show that this mechanism is essential for preventing chromosome loss and aneuploidy of the egg--a leading cause of pregnancy loss and birth defects in humans.