Cytogenetic characteristics of seed progeny of old-aged trees of Pinus pallasiana and Picea abies (Pinaceae)

Information on cytogenetic changes in the seed offspring of old-aged trees is insufficient and inconsistent. In our studies, 150–200-year old trees of Picea abies and Pinus pallasiana were used. We analyzed peculiarities of their karyotype, nucleus-forming region, and nucleolus in the cells of seedlings of P. abies and P. pallasiana emerged from seeds in natural populations and plantations of introduced plants. As a result, age-dependent cytogenetic disorders were observed, such as the chromosome bridges, lag, premature segregation, and agglutination. Peculiarities with regard to number and structure of secondary chromosome constriction are demonstrated. The identified properties of the cell structure of seeds of old-aged trees of P. abies and P. pallasiana indicate that more resources are needed to maintain their protein synthesis at a normal level. The increased number of abnormalities indicates a significant impact of accumulated intracellular metabolites and cytopathological phenomena in mother plants on the quality of seed offspring.

Evaluation of Crossability between Nicotiana benthamiana and Nicotiana excelsior

Wild tobacco species in the Nicotiana section Suaveolentes are promising genetic resources to introduce their disease resistance to cultivated tobacco, Nicotiana tabacum. However, hybrid lethality is observed in hybrid seedlings from crosses between most Suaveolentes species and N. tabacum. In particular, N. benthamiana belonging to the section Suaveolentes produces only viable hybrids after crossing with N. tabacum. In the present study, crossability between N. benthamiana and N. excelsior (section Suaveolentes) was investigated to test the possible usefulness of N. benthamiana as the bridge parent to transfer desirable genes of N. excelsior to N. tabacum via bridge crossing. After reciprocal crosses using three accessions of N. benthamiana and N. excelsior each, several crossing barriers such as cross-incompatibility, seed abortion, and male and female hybrid sterility were observed. Although reciprocal hybrids between N. benthamiana and N. excelsior showed a high degree of chromosome pairing in meiosis, univalents and multivalents, as well as chromosome bridges and lagging chromosomes, were observed. These meiotic abnormalities were thought to cause hybrid sterility. The possible usefulness of reciprocal hybrids between N. benthamiana and N. excelsior is discussed.

Whole chromosome loss and genomic instability in mouse embryos after CRISPR-Cas9 genome editing

Karyotype alterations have emerged as on-target complications from CRISPR-Cas9 genome editing. However, the events that lead to these karyotypic changes in embryos after Cas9-treatment remain unknown. Here, using imaging and single-cell genome sequencing of 8-cell stage embryos, we track both spontaneous and Cas9-induced karyotype aberrations through the first three divisions of embryonic development. We observe the generation of abnormal structures of the nucleus that arise as a consequence of errors in mitosis, including micronuclei and chromosome bridges, and determine their contribution to common karyotype aberrations including whole chromosome loss that has been recently reported after editing in embryos. Together, these data demonstrate that Cas9-mediated germline genome editing can lead to unwanted on-target side effects, including major chromosome structural alterations that can be propagated over several divisions of embryonic development.

Role of the Topoisomerase IIα Chromatin Tether domain in Nucleosome Binding & Chromosome Segregation

Due to the intrinsic nature of DNA replication, replicated genomes retain catenated genomic loci that must be resolved to ensure faithful segregation of sister chromatids in mitosis. Type II DNA Topoisomerase (TopoII) decatenates the catenated genomic DNA through its unique Strand Passage Reaction (SPR). Loss of SPR activity results in anaphase chromosome bridges and formation of Polo-like Kinase Interacting Checkpoint Helicase (PICH)-coated ultra-fine DNA bridges (UFBs) whose timely resolution is required to prevent micronuclei formation. Vertebrates have two TopoII isoforms– TopoIIα and TopoIIβ, that share a conserved catalytic core. However, the essential mitotic function of TopoIIα cannot be compensated by TopoIIβ, due to differences in their catalytically inert C-terminal domains (CTDs). Using genome-edited human cells, we show that specific binding of TopoIIα to methylated histone, tri-methylated H3K27 (H3K27me3), via its Chromatin Tether (ChT) domain within the CTD contributes critically to avoid anaphase UFB formation. Reducing H3K27 methylation prior to mitosis increases UFBs, revealing a requirement for proper establishment of H3K27me3 after DNA replication to facilitate TopoIIα-ChT dependent UFB prevention. We propose that interaction of the TopoIIα-ChT with H3K27me3 is a key factor that ensures the complete resolution of catenated loci to permit faithful chromosome segregation in human cells.

Breakage of CRISPR/Cas9-Induced Chromosome Bridges in Mitotic Cells

Chromosomal instability, the most frequent form of plasticity in cancer cells, often proceeds through the formation of chromosome bridges. Despite the importance of these bridges in tumor initiation and progression, debate remains over how and when they are resolved. In this study, we investigated the behavior and properties of chromosome bridges to gain insight into the potential mechanisms underlying bridge-induced genome instability. We report that bridges may break during mitosis or may remain unbroken until the next interphase. During mitosis, we frequently observed discontinuities in the bridging chromatin, and our results strongly suggest that a substantial fraction of chromosome bridges are broken during this stage of the cell cycle. This notion is supported by the observation that the chromatin flanking mitotic bridge discontinuities is often decorated with the phosphorylated form of the histone H2AX, a marker of DNA breaks, and by MDC1, an early mediator of the cell response to DNA breaks. Also, free 3′OH DNA ends were detected in more than half of the bridges during the final stages of cell division. However, even if detected, the DNA ends of broken bridges are not repaired in mitosis. To investigate whether mitotic bridge breakage depends on mechanical stress, we used experimental models in which chromosome bridges with defined geometry are formed. Although there was no association between spindle pole separation or the distance among non-bridge kinetochores and bridge breakage, we found a direct correlation between the distance between bridge kinetochores and bridge breakage. Altogether, we conclude that the discontinuities observed in bridges during mitosis frequently reflect a real breakage of the chromatin and that the mechanisms responsible for chromosome bridge breakage during mitosis may depend on the separation between the bridge kinetochores. Considering that previous studies identified mechanical stress or biochemical digestion as possible causes of bridge breakage in interphase cells, a multifactorial model emerges for the breakage of chromosome bridges that, according to our results, can occur at different stages of the cell cycle and can obey different mechanisms.

The chromatin-binding domain of Ki-67 together with p53 protects human chromosomes from mitotic damage

Vertebrate mammals express a protein called Ki-67 which is most widely known as a clinically useful marker of highly proliferative cells. Previous studies of human cells indicated that acute depletion of Ki-67 can elicit a delay at the G1/S boundary of the cell cycle, dependent on induction of the checkpoint protein p21. Consistent with those observations, we show here that acute Ki-67 depletion causes hallmarks of DNA damage, and the damage occurs even in the absence of checkpoint signaling. This damage is not observed in cells traversing S phase but is instead robustly detected in mitotic cells. The C-terminal chromatin-binding domain of Ki-67 is necessary and sufficient to protect cells from this damage. We also observe synergistic effects when Ki-67 and p53 are simultaneously depleted, resulting in increased levels of chromosome bridges at anaphase, followed by the appearance of micronuclei. Therefore, these studies identify the C terminus of Ki-67 as an important module for genome stability.

Traip Mitotic Function Controls Brain Size

Microcephaly is a developmental failure to achieve proper brain size and neuron number. Mutations in diverse genes are linked to microcephaly, including several with DNA damage repair (DDR) functions; however, it is not well understood how these DDR gene mutations limit brain size. One such gene is TRAIP, which has multiple known functions in DDR. We characterized the Drosophila ortholog Traip, finding that loss of Traip causes a brain-specific defect in the Mushroom Body (MB). Traip mutant (traip-) MBs had reduced size and fewer neurons, but no neurodegeneration, consistent with human primary microcephaly disorders. Reduced neuron numbers in traip- were explained by premature caspase-dependent cell death of MB neuroblasts (MB-NBs). Many traip- MB-NBs had prominent chromosome bridges in anaphase, along with polyploidy, aneuploidy, or micronuclei. We found no evidence for an interphase DNA repair role for Traip in MB-NBs; instead, proper MB development requires Traip function during mitosis, where Traip localizes to centrosomes and mitotic spindles. Our results suggest that proper brain size is ensured by the recently described role for TRAIP in unloading stalled replication forks in mitosis, which suppresses DNA bridges and neural stem cell death to promote proper neuron number. Further, the mitotic nature of traip- MB-NB defects and Traip localization suggest a closer etiological link between DDR microcephaly genes like Traip and the centrosome/spindle-related genes more commonly associated with microcephaly.

Meiotic behaviour and pollen fertility of F1, F2 and BC1 progenies of Iris dichotoma and I. domestica

Pollen characteristics are very important for Iris interspecific hybridisation. In this study, the pollen viability and male meiosis were studied in yellow-flowered Iris dichotoma (Y2), I. domestica (S3) and their hybrids F1, F2 and BC1 (BC1-Y and BC1-S). The BC1-Y hybrids showed higher pollen viability than that of F1, F2 and BC1-S hybrids, which were between I. dichotoma (26.1%) and I. domestica (35.1%). Two sterile hybrids, F2-1 and BC1-S-1, exhibited more meiotic abnormalities (57.3% and 58.7%) than other individuals. During the first meiotic division, a diffuse diplotene stage was observed for the first time in the genus Iris. The meiotic abnormalities included non-congressed chromosomes, chromosome bridges, lagging chromosomes, unequal division, abnormally oriented spindle fibres, nonsynchronous division and polyad, and resulted in reduced pollen fertility. The relatively high frequency of 2n pollen grains was found in hybrids of BC1-Y-2, BC1-Y-1, BC1-S-2, BC1-S-3 and BC1-S-4. Our research provides a new resource for meiotic behaviour and pollen fertility of the genus Iris.

The chromatin-binding domain of Ki-67 together with p53 protects human chromosomes from mitotic damage

Vertebrate mammals express a protein called Ki-67 which is most widely known as a clinically useful marker of highly proliferative cells. Previous studies of human cells indicated that acute depletion of Ki-67 can elicit a delay at the G1/S boundary of the cell cycle, dependent on induction of the checkpoint protein p21. Consistent with those observations, we show here that acute Ki-67 depletion causes hallmarks of DNA damage, and the damage occurs even in the absence of checkpoint signaling. This damage is not observed in cells traversing S phase but is instead robustly detected in mitotic cells. The C-terminal chromatin binding domain of Ki-67 is necessary and sufficient to protect cells from this damage. We also observe synergistic effects when Ki-67 and p53 are simultaneously depleted, resulting in increased levels of chromosome bridges at anaphase, followed by the appearance of micronuclei. Therefore, these studies identify the C-terminus of Ki-67 as an important module for genome stability.