Mobility of kinetochore proteins measured by FRAP analysis in living cells

The kinetochore is essential for faithful chromosome segregation during mitosis and is assembled through dynamic processes involving numerous kinetochore proteins. Various experimental strategies have been used to understand kinetochore assembly processes. Fluorescence recovery after photobleaching (FRAP) analysis is also a useful strategy for revealing the dynamics of kinetochore assembly. In this study, we introduced fluorescence protein-tagged kinetochore protein cDNAs into each endogenous locus and performed FRAP analyses in chicken DT40 cells. Centromeric protein (CENP)-C was highly mobile in interphase, but immobile during mitosis. CENP-C mutants lacking the CENP-A-binding domain became mobile during mitosis. In contrast to CENP-C, CENP-T and CENP-H were immobile during both interphase and mitosis. The mobility of Dsn1, which is a component of the Mis12 complex and directly binds to CENP-C, depended on CENP-C mobility during mitosis. Thus, our FRAP assays provide dynamic aspects of how the kinetochore is assembled.

Targeting chromosome trisomy for chromosome editing

A trisomy is a type of aneuploidy characterised by an additional chromosome. The additional chromosome theoretically accepts any kind of changes since it is not necessary for cellular proliferation. This advantage led us to apply two chromosome manipulation methods to autosomal trisomy in chicken DT40 cells. We first corrected chromosome 2 trisomy to disomy by employing counter-selection markers. Upon construction of cells carrying markers targeted in one of the trisomic chromosome 2s, cells that have lost markers integrated in chromosome 2 were subsequently selected. The loss of one of the chromosome 2s had little impacts on the proliferative capacity, indicating unsubstantial role of the additional chromosome 2 in DT40 cells. We next tested large-scale truncations of chromosome 2 to make a mini-chromosome for the assessment of chromosome stability by introducing telomere repeat sequences to delete most of p-arm or q-arm of chromosome 2. The obtained cell lines had 0.7 Mb mini-chromosome, and approximately 0.2% of mini-chromosome was lost per cell division in wild-type background while the rate of chromosome loss was significantly increased by the depletion of DDX11, a cohesin regulatory protein. Collectively, our findings propose that trisomic chromosomes are good targets to make unique artificial chromosomes.

Vertebrate CTF18 and DDX11 essential function in cohesion is bypassed by preventing WAPL-mediated cohesin release

The alternative PCNA loader containing CTF18-DCC1-CTF8 facilitates sister chromatid cohesion (SCC) by poorly defined mechanisms. Here we found that in DT40 cells, CTF18 acts complementarily with the Warsaw breakage syndrome DDX11 helicase in mediating SCC and proliferation. We uncover that the lethality and cohesion defects of ctf18 ddx11 mutants are associated with reduced levels of chromatin-bound cohesin and rescued by depletion of WAPL, a cohesin-removal factor. On the contrary, high levels of ESCO1/2 acetyltransferases that acetylate cohesin to establish SCC do not rescue ctf18 ddx11 phenotypes. Notably, the tight proximity of sister centromeres and increased anaphase bridges characteristic of WAPL-depleted cells are abrogated by loss of both CTF18 and DDX11. The results reveal that vertebrate CTF18 and DDX11 collaborate to provide sufficient amounts of chromatin-loaded cohesin available for SCC generation in the presence of WAPL-mediated cohesin-unloading activity. This process modulates chromosome structure and is essential for cellular proliferation in vertebrates.

PRDX1 is essential for the viability and maintenance of reactive oxygen species in chicken DT40

Background Peroxiredoxin 1 (PRDX1) is a member of a ubiquitous family of thiol peroxidases that catalyze the reduction of peroxides, including hydrogen peroxide. It functions as an antioxidant enzyme, similar to catalase and glutathione peroxidase. PRDX1 was recently shown act as a sensor of reactive oxygen species (ROS) and play a role in ROS-dependent intracellular signaling pathways. To investigate its physiological functions, PRDX1 was conditionally disrupted in chicken DT40 cells in the present study. Results The depletion of PRDX1 resulted in cell death with increased levels of intracellular ROS. PRDX1-depleted cells did not show the accumulation of chromosomal breaks or sister chromatid exchange (SCE). These results suggest that cell death in PRDX1-depleted cells was not due to DNA damage. 2-Mercaptoethanol protected against cell death in PRDX1-depleted cells and also suppressed elevations in ROS. Conclusions PRDX1 is essential in chicken DT40 cells and plays an important role in maintaining intracellular ROS homeostasis (or in the fine-tuning of cellular ROS levels). Cells deficient in PRDX1 may be used as an endogenously deregulated ROS model to elucidate the physiological roles of ROS in maintaining proper cell growth.

Targeting Chromosome Trisomy for Chromosome Editing

A trisomy is a type of aneuploidy characterised by an additional chromosome. The additional chromosome theoretically accepts any kind of changes since it is not necessary for cellular proliferation. This advantage led us to apply two chromosome manipulation methods to autosomal trisomy in chicken DT40 cells. We first corrected chromosome 2 trisomy to disomy by employing counter-selection makers. Upon construction of cells carrying makers targeted in one of the trisomic chromosome 2s, cells that have lost makers integrated in chromosome 2 were subsequently selected. The loss of one of the chromosome 2s had little impacts on the proliferative capacity, indicating unsubstantial role of the additional chromosome 2 in DT40 cells. We next tested large-scale truncations of chromosome 2 to make a mini-chromosome for the assessment of chromosome stability by introducing telomere repeat sequences to delete most of p-arm or q-arm of chromosome 2. The obtained cell lines had 0.7 Mb mini-chromosome, and approximately 0.2% of mini-chromosome was lost per cell division in wild-type background while the rate of chromosome loss was significantly increased by the depletion of DDX11, a cohesin regulatory protein. Collectively, our findings propose that trisomic chromosomes are good targets to make unique artificial chromosomes. (197 words)

TRPM7 is an essential regulator for volume-sensitive outwardly rectifying anion channel

Animal cells can regulate their volume after swelling by the regulatory volume decrease (RVD) mechanism. In epithelial cells, RVD is attained through KCl release mediated via volume-sensitive outwardly rectifying Cl− channels (VSOR) and Ca2+-activated K+ channels. Swelling-induced activation of TRPM7 cation channels leads to Ca2+ influx, thereby stimulating the K+ channels. Here, we examined whether TRPM7 plays any role in VSOR activation. When TRPM7 was knocked down in human HeLa cells or knocked out in chicken DT40 cells, not only TRPM7 activity and RVD efficacy but also VSOR activity were suppressed. Heterologous expression of TRPM7 in TRPM7-deficient DT40 cells rescued both VSOR activity and RVD, accompanied by an increase in the expression of LRRC8A, a core molecule of VSOR. TRPM7 exerts the facilitating action on VSOR activity first by enhancing molecular expression of LRRC8A mRNA through the mediation of steady-state Ca2+ influx and second by stabilizing the plasmalemmal expression of LRRC8A protein through the interaction between LRRC8A and the C-terminal domain of TRPM7. Therefore, TRPM7 functions as an essential regulator of VSOR activity and LRRC8A expression.

Evidence for divergence of DNA methylation maintenance and a conserved inhibitory mechanism from DNA demethylation in chickens and mammals

Background DNA methylation is a significant epigenetic modification that is evolutionarily conserved in various species and often serves as a repressive mark for transcription. DNA methylation levels and patterns are regulated by a balance of opposing enzyme functions, DNA methyltransferases, DNMT1/3A/3B and methylcytosine dioxygenases, TET1/2/3. In mice, the TET enzyme converts DNA cytosine methylation (5mC) to 5-hydroxymethylcytosine (5hmC) at the beginning of fertilisation and gastrulation and initiates a global loss of 5mC, while the 5mC level is increased on the onset of cell differentiation during early embryonic development. Objective Global loss and gain of DNA methylation may be differently regulated in diverged species. Methods Chicken B-cell lymphoma DT40 cells were used as an avian model to compare differences in the overall regulation of DNA modification with mammals. Results We found that DNA methylation is maintained at high levels in DT40 cells through compact chromatin formation, which inhibits TET-mediated demethylation. Human and mouse chromosomes introduced into DT40 cells by cell fusion lost the majority of 5mC, except for human subtelomeric repeats. Conclusion Our attempt to elucidate the differences in the epigenetic regulatory mechanisms between birds and mammals explored the evidence that they share a common chromatin-based regulation of TET–DNA access, while chicken DNMT1 is involved in different target sequence recognition systems, suggesting that factors inducing DNMT–DNA association have already diverged.

A super-sensitive auxin-inducible degron system with an engineered auxin-TIR1 pair

The auxin-inducible degron (AID) system enables rapid depletion of target proteins within the cell by applying the natural auxin IAA. The AID system is useful for investigating the physiological functions of essential proteins; however, this system generally requires high dose of auxin to achieve effective depletion in vertebrate cells. Here, we describe a super-sensitive AID system that incorporates the synthetic auxin derivative 5-Ad-IAA and its high-affinity-binding partner OsTIR1F74A. The super-sensitive AID system enabled more than a 1000-fold reduction of the AID inducer concentrations in chicken DT40 cells. To apply this system to various mammalian cell lines including cancer cells containing multiple sets of chromosomes, we utilized a single-step method where CRISPR/Cas9-based gene knockout is combined with insertion of a pAID plasmid. The single-step method coupled with the super-sensitive AID system enables us to easily and rapidly generate AID-based conditional knockout cells in a wide range of vertebrate cell lines. Our improved method that incorporates the super-sensitive AID system and the single-step method provides a powerful tool for elucidating the roles of essential genes.

Cisplatin is more mutagenic than carboplatin or oxaliplatin at equitoxic concentrations

ABSTRACTPlatinum-based drugs are a mainstay of cancer chemotherapy. However, their mutagenic effect can increase tumour heterogeneity, contribute to the evolution of treatment resistance, and also induce secondary malignancies. We coupled whole genome sequencing with phenotypic investigations on two cell line models to compare the magnitude and understand the mechanism of mutagenicity of cisplatin, carboplatin and oxaliplatin. Cisplatin induced significantly more base substitution mutations than carboplatin or oxaliplatin when used at equitoxic concentrations on human TK6 or chicken DT40 cells, and also induced the highest number of short insertions and deletions. Assessment through histone H2AX phosphorylation and single cell agarose gel electrophoresis suggested that cisplatin caused more DNA damage than carboplatin or oxaliplatin. The analysis of base substitution spectra revealed that all three tested platinum drugs elicit both a direct mutagenic effect at purine dinucleotides, and an indirect effect of accelerating endogenous mutagenic processes. Whereas the direct mutagenic effect correlated with the level of DNA damage caused, the indirect mutagenic effects were equal. The different mutagenicity and DNA damaging effect of equitoxic platinum drug treatments suggests that DNA damage independent mechanisms significantly contribute to their cytotoxicity. Thus, the comparatively high mutagenicity of cisplatin should be taken into account in the design of chemotherapeutic regimens.