Abnormal Chromosome Condensation Research Articles

Mutations in NCAPG2 Cause a Severe Neurodevelopmental Syndrome that Expands the Phenotypic Spectrum of Condensinopathies

The use of whole-exome and whole-genome sequencing has been a catalyst for a genotype-first approach to diagnostics. Under this paradigm, we have implemented systematic sequencing of neonates and young children with a suspected genetic disorder. Here, we report on two families with recessive mutations in NCAPG2 and overlapping clinical phenotypes that include severe neurodevelopmental defects, failure to thrive, ocular abnormalities, and defects in urogenital and limb morphogenesis. NCAPG2 encodes a member of the condensin II complex, necessary for the condensation of chromosomes prior to cell division. Consistent with a causal role for NCAPG2, we found abnormal chromosome condensation, augmented anaphase chromatin-bridge formation, and micronuclei in daughter cells of proband skin fibroblasts. To test the functional relevance of the discovered variants, we generated an ncapg2 zebrafish model. Morphants displayed clinically relevant phenotypes, such as renal anomalies, microcephaly, and concomitant increases in apoptosis and altered mitotic progression. These could be rescued by wild-type but not mutant human NCAPG2 mRNA and were recapitulated in CRISPR-Cas9 F0 mutants. Finally, we noted that the individual with a complex urogenital defect also harbored a heterozygous NPHP1 deletion, a common contributor to nephronophthisis. To test whether sensitization at the NPHP1 locus might contribute to a more severe renal phenotype, we co-suppressed nphp1 and ncapg2, which resulted in significantly more dysplastic renal tubules in zebrafish larvae. Together, our data suggest that impaired function of NCAPG2 results in a severe condensinopathy, and they highlight the potential utility of examining candidate pathogenic lesions beyond the primary disease locus.

SET Nuclear Oncogene Associates with Microcephalin/MCPH1 and Regulates Chromosome Condensation

Primary microcephaly is an autosomal recessive disorder characterized by marked reduction in human brain size. Microcephalin (MCPH1), one of the genes mutated in primary microcephaly, plays an important role in DNA damage checkpoint control and mitotic entry. Additionally, MCPH1 ensures the proper temporal activation of chromosome condensation during mitosis, by acting as a negative regulator of the condensin II complex. We previously found that deletion of the of the MCPH1 N terminus leads to the premature chromosome condensation (PCC) phenotype. In the present study, we unexpectedly observed that a truncated form of MCPH1 appears to be expressed in MCPH1(S25X/S25X) patient cells. This likely results from utilization of an alternative translational start codon, which would produce a mutant MCPH1 protein with a small deletion of its N-terminal BRCT domain. Furthermore, missense mutations in the MCPH1 cluster at its N terminus, suggesting that intact function of this BRCT protein-interaction domain is required both for coordinating chromosome condensation and human brain development. Subsequently, we identified the SET nuclear oncogene as a direct binding partner of the MCPH1 N-terminal BRCT domain. Cells with SET knockdown exhibited abnormal condensed chromosomes similar to those observed in MCPH1-deficient mouse embryonic fibroblasts. Condensin II knockdown rescued the abnormal chromosome condensation phenotype in SET-depleted cells. In addition, MCPH1 V50G/I51V missense mutations, impair binding to SET and fail to fully rescue the abnormal chromosome condensation phenotype in Mcph1(-/-) mouse embryonic fibroblasts. Collectively, our findings suggest that SET is an important regulator of chromosome condensation/decondensation and that disruption of the MCPH1-SET interaction might be important for the pathogenesis of primary microcephaly.

Oocyte maturation failure: a syndrome of bad eggs

To show that disruption of meiotic competence results in cell cycle arrest, and the production of immature oocytes that are not capable of fertilization. Through an extensive review of animal studies and clinical case reports, we define the syndrome of oocyte maturation failure as a distinct oocyte disorder, present a classification system based on clinical parameters, and discuss the potential molecular origins for the disease.

A Pocket on the Surface of the N-Terminal BRCT Domain of Mcph1 Is Required to Prevent Abnormal Chromosome Condensation

Mcph1 is mutated in autosomal recessive primary microcephaly and premature chromosome condensation (PCC) syndrome. Increased chromosome condensation is a common feature of cells isolated from patients afflicted with either disease. Normal cells depleted of Mcph1 also exhibit PCC phenotype. Human Mcph1 contains three BRCA1-carboxyl terminal (BRCT) domains, the first of which (Mcph1N) is necessary for the prevention of PCC. The only known disease-associated missense mutation in Mcph1 resides in this domain (T27R). We have determined the X-ray crystal structure of human Mcph1N to 1.6 Å resolution. Compared with other BRCT domain structures, the most striking differences are an elongated, ordered β1–α1 loop and an adjacent hydrophobic pocket. This pocket is in the equivalent structural position to the phosphate binding site of BRCT domains that recognize phospho-proteins, although the phosphate-binding residues are absent in Mcph1N. Mutations in the pocket abrogate the ability of full-length Mcph1 to rescue the PCC phenotype of Mcph1 −/− mouse embryonic fibroblast cells, suggesting that it forms an essential part of a protein–protein interaction site necessary to prevent PCC.

Functional analysis of an Orc6 mutant in Drosophila

The origin recognition complex (ORC) is a 6-subunit complex required for the initiation of DNA replication in eukaryotic organisms. ORC is also involved in other cell functions. The smallest Drosophila ORC subunit, Orc6, is important for both DNA replication and cytokinesis. To study the role of Orc6 in vivo, the orc6 gene was deleted by imprecise excision of P element. Lethal alleles of orc6 are defective in DNA replication and also show abnormal chromosome condensation and segregation. The analysis of cells containing the orc6 deletion revealed that they arrest in both the G(1) and mitotic stages of the cell cycle. Orc6 deletion can be rescued to viability by a full-length Orc6 transgene. The expression of mutant transgenes of Orc6 with deleted or mutated C-terminal domain results in a release of mutant cells from G(1) arrest and restoration of DNA replication, indicating that the DNA replication function of Orc6 is associated with its N-terminal domain. However, these mutant cells accumulate at mitosis, suggesting that the C-terminal domain of Orc6 is important for the passage through the M phase. In a cross-species complementation experiment, the expression of human Orc6 in Drosophila Orc6 mutant cells rescued DNA replication, suggesting that this function of the protein is conserved among metazoans.

Multiple functions of the Drosophila Orc6 protein

The Origin recognition complex (ORC) is important for the DNA replication in eukaryotic cells. ORC is also involved in other cell functions. The smallest Drosophila ORC subunit, Orc6, consists of two functional domains. Larger N‐terminal domain directly involved in DNA binding and is important for ORC‐dependent DNA replication. Smaller C‐terminal domain is important for cytokinesis.Deletion of Orc6 gene was created in Drosophila. Mutant lethal alleles of Orc6 are defective in both DNA replication and cell proliferation during larval stages of development. Homozygous Orc6 mutants display disc‐less phenotype and show abnormal chromosome condensation and segregation. Mutant cells arrest in both G1 and mitosis stages of the cell cycle. Orc6 mutation can be rescued to viability by full‐length Orc6 transgene. The expression of a transgene carrying the core replicative N‐terminal domain of the Orc6 protein results a restoration of DNA replication in mutant larval tissues. The mutant cells carrying this transgene do not exhibit G1 arrest but nevertheless accumulate at mitosis.The cytokinetic function of DmOrc6 is associated with the C‐terminal domain of the protein. Orc6 interacts with the septin protein Pnut, which together with the septins Sep1 and Sep2 forms a septin complex important for cytokinesis. Septin complexes exhibit GTPase activity and can assemble into large filaments. The interaction of Orc6 with the septin complex is dependent on the coiled‐coil domain of Pnut. Furthermore, the binding of Orc6 to Pnut increases the intrinsic GTPase activity of the Drosophila septin complex, while in the absence of GTP it enhances septin complex filament formation.Together our results suggest an active role for Orc6 in DNA replication and septin complex function. Orc6 might be a part of a control mechanism directing the cytokinesis machinery during the final steps of mitosis.

Functional and non-functional spindle formation affecting mitosis and cytokinesis in Fucus distichus zygotes: the role of the centrosome

Abstract Functional (with centrioles) and non-functional (without centrioles) mitotic spindle formation was studied in Fucus distichus zygotes. In normally fertilized zygotes, a pair of centrioles derived from sperm existed at both mitotic poles. Both centrin and γ-tubulin were associated with centrioles. In multipolar spindles of fertilized multinucleate eggs, mitotic poles with centrioles were stained with an anti-γ-tubulin antibody, but the other poles were not, and cytokinesis proceeded abnormally. When karyogamy was blocked with colchicine, centrioles remained around the sperm nucleus. A monastral spindle or a bipolar, barrel-shaped spindle was formed on the egg nucleus; however, there were no centrioles at these mitotic poles. When cytoplasmic fragments containing only an egg or a sperm nucleus were separately prepared and cultured, the egg nucleus appeared to enter, but did not complete, mitosis. In cytoplasm containing only a sperm nucleus, mitosis and cytokinesis did not occur. We concluded that the egg nucleus has the potential to form a non-functional spindle without the sperm centrioles. However, this acentrosomal spindle was non-functional; mitosis remained at the stage of chromosome condensation and did not proceed to cytokinesis. Even though some cells proceeded to cytokinesis, they showed abnormal chromosome condensation.

Regulation of spindle and chromatin dynamics during early and late stages of oocyte maturation by aurora kinases

Examination of factors regulating oocyte chromatin remodeling is crucial to circumvent embryonic aneuploidy and resulting defects. Aurora kinases (AURK) are involved in regulation of chromatin remodeling, however, little attention has been paid to AURKs in regard to oocyte maturation. Meiotically incompetent mouse oocytes contain transcripts for all three Aurk isoforms: A, B and C. Upon achieving meiotic competence, oocytes showed significant increases in transcript levels of all three Aurk isoforms and transcript levels remained unchanged as oocytes progressed through meiosis, with AurkA being the predominant isoform. Inhibition of oocyte AURKs during the prophase–metaphase I (MI) transition via inhibitor ZM447439 (ZM) had no effect on germinal vesicle breakdown. However, meiotic spindles were malformed, and microtubule organizing centers and chromatin were scattered. Chromosomal spreads of MI oocytes indicated AURK inhibition resulted in abnormal chromosome condensation. Furthermore, inhibition of AURK during prophase I–MII prevented completion of MII and extrusion of the polar body. Inhibition of AURKs during the MI–MII transition resulted in significantly fewer cells progressing to MII and induced aberrant chromatin remodeling. Further analysis indicated that inhibition of AURKs resulted in absence of histone-H3 phosphorylation at serine 10 and 28. These data suggest a ZM-sensitive AURK may be an oocyte histone-H3 kinase capable of regulating chromatin remodeling throughout oocyte meiosis, both pre- and post-MI.

Zinc Induces Mixed Types of Cell Death, Necrosis, and Apoptosis, in Molt-4 Cells

To investigate the mode of zinc-induced cell death, the associated morphological changes, and biological events were examined in zinc-treated Molt-4 cells. Fluorescence microscope observations with double staining of zinc-treated cells with Hoechst 33342 and propidium iodide (PI) indicated that the metal induced both necrosis and apoptosis. To confirm this, cells were stained with both PI and FITC-labeled annexin V, which binds phosphatidylserine, and then analyzed by flow cytometry. The results also confirmed that zinc induces mixed types of cell death, necrosis and apoptosis, and that the former induction occurs earlier and at a greater frequency. Hallmarks of apoptosis such as abnormal chromosome condensation and release of cytochrome c, as well as the appearance of annexin-positive cells, appeared along with the expression of mitochondrial membrane protein 7A6. However, zinc did not induce increases in caspase-3 like protease and caspase-8 activities, and caused slightly hypodiploid cells. Furthermore, the induction of cell death and annexin-positive cells was not blocked by the caspase inhibitors Ac-YVAD-CHO and Ac-DEVD-CHO. These results indicate that zinc induces both necrosis and apoptosis, without caspase-3 activation.

Phosphorylation of Histone H3 Is Required for Proper Chromosome Condensation and Segregation

Phosphorylation of histone H3 at serine 10 occurs during mitosis in diverse eukaryotes and correlates closely with mitotic and meiotic chromosome condensation. To better understand the function of H3 phosphorylation in vivo, we created strains of Tetrahymena in which a mutant H3 gene (S10A) was the only gene encoding the major H3 protein. Although both micronuclei and macronuclei contain H3 in typical nucleosomal structures, defects in nuclear divisions were restricted to mitotically dividing micronuclei; macronuclei, which are amitotic, showed no defects. Strains lacking phosphorylated H3 showed abnormal chromosome segregation, resulting in extensive chromosome loss during mitosis. During meiosis, micronuclei underwent abnormal chromosome condensation and failed to faithfully transmit chromosomes. These results demonstrate that H3 serine 10 phosphorylation is causally linked to chromosome condensation and segregation in vivo and is required for proper chromosome dynamics.

Topoisomerase II expression and VM-26 induction of DNA breaks during spermatogenesis in Xenopus laevis.

The relative content of topoisomerase II (topo II) and the induction of topo-II-mediated DNA damage and cellular abnormalities have been characterized in developing spermatogenic cells of Xenopus laevis to gain an insight into the role of topo II during spermatogenesis. Decatenation assays identified topo II activity in nuclear extracts from spermatocytes and pre-elongate spermatids, but not in extracts from elongate spermatids or sperm. Extracts from early-mid spermatids contained 14% (per cell) of the decatenation activity found in spermatocyte extracts. Immunoblots of SDS extracts from whole cells and nuclei from both spermatocytes and pre-elongate spermatids, but not elongate spermatids or sperm, resolved a 180 kDa polypeptide that reacts with polyclonal antisera to Xenopus oocyte topo II, an antipeptide antibody (FHD29) to human topo II alpha and beta, and an antipeptide antibody to human topo II alpha, suggesting homology between Xenopus spermatogenic cell topo II and mammalian topo II alpha. Immunofluorescence microscopy of topo II in testis cryosections revealed the presence of topo II in nuclei of all spermatogenic stages, but not in sperm. The relative levels of topo II estimated from fluorescence intensity were highest in spermatogonia and spermatocytes, then early-mid spermatids, followed by elongate spermatids and somatic cells. Incubation of isolated spermatogenic cells with teniposide (VM-26), a topo II-targetted drug, resulted in a dose-dependent induction of DNA breaks in all spermatocytes and spermatid stages to nuclear elongation stages, as analyzed by alkaline single cell gel electrophoresis. Addition of 0.5-50 microM VM-26 to spermatogenic cell cultures for 27 hours resulted in stage-dependent abnormalities. Mid-late spermatid stages were relatively resistant to VM-26-induced damage. In contrast, meiotic division stages were arrested and spermatogonia B were killed by VM-26, and VM-26 induced abnormal chromosome condensation in pachytene spermatocytes. The results of these studies show that cellular levels of topo II are stage-dependent during spermatogenesis, that most spermatogenic stages are sensitive to topo II-mediated DNA damage, and that spermatogonia B, meiotic divisions and pachytene spermatocytes are particularly sensitive to induction of morphological abnormalities and cell death during acute exposure to topo II-targetted drugs.

Benomyl-induced mitotic disturbances in Hordeum vulgare

Benomyl [methyl-1-(butylcarbamoyl)-2-benzimidazole carbamate], a benzimidazole derivative systemic fungicide was tested with regard to its ability to induce mitotic disturbances such as the inhibition of spindle formation, chromosome disorientation, abnormal chromosome condensation and chromosome malsegregation and also chromatid aberrations in root-meristem cells at the reconstructed karyotype MK 14/2034 of Hordeum vulgare. Benomyl at the concentrations of 10 −5 M and 3 × 10 −5 M induced mitotic abnormalities which ranged from partial disturbance of centromeric activity of one or several chromatids followed by missegregation, as well as chromosome lagging and micronuclei formation, to complete inhibition of spindle formation. No statistically significant increase of chromatid aberrations was, however, observed.

The mutagenic activity of razoxane (ICRF 159): an anticancer agent.

The mutagenic activity of razoxane (ICRF 159) was studied using the Salmonella/microsome assay and rodent bone-marrow micronucleus and metaphase assays. Razoxane (up to 5000 micrograms/plate) did not cause an increase in the mutation frequency in the Salmonella/microsome assay. In the mouse micronucleus assay razoxane (200 and 400 mg kg-1 i.p.) was cytotoxic to the bone marrow cells (which limited the analysis) but an increase in micronucleated polychromatic erythrocytes was observed in razoxane dosed animals (5-fold compared to control value). In the Chinese hamster metaphase assay razoxane (up to 500 mg kg-1 orally) induced abnormal chromosome condensation and an increase in structural chromosome aberrations (7 fold compared to control value) as well as an increase in the number of polypoid cells (8-fold compared to control value). The mutagenic effect of razoxane was restricted to eukaryotic organisms and was associated with specific chromosomal changes.

A temperature-sensitive mutant of cultured mouse cells defective in chromosome condensation

A temperature-sensitive mutant, designated ts85, was isolated from a mouse mammary carcinoma cell line, FM3A. The ts85 cells grew at 33 °C (permissive temperature) with a doubling time of 18 h, which was almost the same as with wild-type cells, whereas the cell number scarcely increased at all at 39 °C (non-permissive temperature). When the ts85 cells were shifted from 33 to 39 °C, their DNA synthesis fell to below 1% of the initial value in 14 h. RNA or protein synthesis, however, was maintained at the initial levels for at least 14 h at 39 °C. Cytofluorometric analysis of asynchronous cultures and studies with synchronous cultures suggested that the bulk of the cells cultured at 39 °C for 12–18 h were arrested in late S and G2 phases. Electron microscopic observations revealed that chromatin was abnormally condensed into fragmented and compact forms, particularly around nucleoli, in about 80% of cells of an asynchronous culture incubated at 39 °C for 16 h. Cells in mitosis were not detected in such cultures and nuclear membrane and nucleoli were still intact. Such abnormal chromosome condensation was not observed in the ts85 cells at 33 °C or in wild-type cells at either temperature. Since these findings suggest that a ts gene product of ts85 cells is necessary for chromosome condensation, ts85 cells may represent a useful tool for establishing the mechanisms of chromosome condensation. The interrelationship between abnormal chromosome condensation and reduction in DNA synthesis of the ts85 cells is discussed.

Evidence for the involvement of H1 histone phosphorylation in chromosome condensation.

Changes in the structural organisation of chromatin are necessary for the progression of the cell cycle. These changes are thought to be regulated mainly by the modification of chromosomal proteins by reactions such as phosphorylation1–8, acetylation9–12, methylation13,14 and poly(ADP-ribosyl)-ation15,16. Among these modifications, phosphorylation of histones, especially that of the H1 histone, is the most likely candidate for a factor which regulates chromosome condensation1–8. It has been proposed that the phosphorylated form of H1 histone may be involved in the initiation of chromosome condensation1–4 or in the maintenance of the condensed state of chromatin8. The latter possibility is unlikely because zinc chloride, which inhibits phosphatase in vivo and preserves the highly phosphorylated form of H1 histone at metaphase, does not prevent the dispersion of chromosomes at the end of mitosis17. As for the former possibility, Bradbury et al. reported2 that the activity of H1 histone phosphorylation is closely correlated with mitotic triggering in the cell cycle and suggest3,4 that H1 histone phosphokinase is involved in the initiation of mitosis. However, no causal relationship has been established. To analyse the mechanism of the progression of the cell cycle, we have isolated several temperature-sensitive (ts) growth mutants from FM3A, a cell line derived from C3H mouse mammary carcinoma18. We report here on one of these ts mutants, designated the ts85 strain, which shows an abnormal chromosome condensation as well as deficiency in H1 histone phosphorylation at the non-permissive temperature. Our results support the hypothesis that H1 histone phosphorylation controls the initiation step of mitosis through chromosome condensation.