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ELife Aug 2022Btg3-associated nuclear protein (Banp) was originally identified as a nuclear matrix-associated region (MAR)-binding protein and it functions as a tumor suppressor. At...
Btg3-associated nuclear protein (Banp) was originally identified as a nuclear matrix-associated region (MAR)-binding protein and it functions as a tumor suppressor. At the molecular level, Banp regulates transcription of metabolic genes via a CGCG-containing motif called the Banp motif. However, its physiological roles in embryonic development are unknown. Here, we report that Banp is indispensable for the DNA damage response and chromosome segregation during mitosis. Zebrafish mutants show mitotic cell accumulation and apoptosis in developing retina. We found that DNA replication stress and tp53-dependent DNA damage responses were activated to induce apoptosis in mutants, suggesting that Banp is required for regulation of DNA replication and DNA damage repair. Furthermore, consistent with mitotic cell accumulation, chromosome segregation was not smoothly processed from prometaphase to anaphase in morphants, leading to a prolonged M-phase. Our RNA- and ATAC-sequencing identified 31 candidates for direct Banp target genes that carry the Banp motif. Interestingly, a DNA replication fork regulator, and two chromosome segregation regulators, and , are included in this list. Thus, Banp directly regulates transcription of for recovery from DNA replication stress, and and for chromosome segregation during mitosis. Our findings provide the first in vivo evidence that Banp is required for cell-cycle progression and cell survival by regulating DNA damage responses and chromosome segregation during mitosis.
Topics: Animals; Cell Cycle; Chromosome Segregation; Chromosomes; DNA Damage; Mitosis; Nuclear Proteins; Retina; Zebrafish
PubMed: 35942692
DOI: 10.7554/eLife.74611 -
Development (Cambridge, England) Sep 2022Mutations that occur in RNA-splicing machinery may contribute to hematopoiesis-related diseases. How splicing factor mutations perturb hematopoiesis, especially in the...
Mutations that occur in RNA-splicing machinery may contribute to hematopoiesis-related diseases. How splicing factor mutations perturb hematopoiesis, especially in the differentiation of erythro-myeloid progenitors (EMPs), remains elusive. Dhx38 is a pre-mRNA splicing-related DEAH box RNA helicase, for which the physiological functions and splicing mechanisms during hematopoiesis currently remain unclear. Here, we report that Dhx38 exerts a broad effect on definitive EMPs as well as the differentiation and maintenance of hematopoietic stem and progenitor cells (HSPCs). In dhx38 knockout zebrafish, EMPs and HSPCs were found to be arrested in mitotic prometaphase, accompanied by a 'grape' karyotype, owing to the defects in chromosome alignment. Abnormal alternatively spliced genes related to chromosome segregation, the microtubule cytoskeleton, cell cycle kinases and DNA damage were present in the dhx38 mutants. Subsequently, EMPs and HSPCs in dhx38 mutants underwent P53-dependent apoptosis. This study provides novel insights into alternative splicing regulated by Dhx38, a process that plays a crucial role in the proliferation and differentiation of fetal EMPs and HSPCs.
Topics: Alternative Splicing; Animals; Hematopoiesis; Hematopoietic Stem Cells; Myeloid Progenitor Cells; Zebrafish
PubMed: 35929537
DOI: 10.1242/dev.200450 -
Life (Basel, Switzerland) May 2022Variants of linker histone H1 are tissue-specific and are responsible for chromatin compaction accompanying cell differentiation, mitotic chromosome condensation, and...
The Highest Density of Phosphorylated Histone H1 Appeared in Prophase and Prometaphase in Parallel with Reduced H3K9me3, and HDAC1 Depletion Increased H1.2/H1.3 and H1.4 Serine 38 Phosphorylation.
BACKGROUND
Variants of linker histone H1 are tissue-specific and are responsible for chromatin compaction accompanying cell differentiation, mitotic chromosome condensation, and apoptosis. Heterochromatinization, as the main feature of these processes, is also associated with pronounced trimethylation of histones H3 at the lysine 9 position (H3K9me3).
METHODS
By confocal microscopy, we analyzed cell cycle-dependent levels and distribution of phosphorylated histone H1 (H1ph) and H3K9me3. By mass spectrometry, we studied post-translational modifications of linker histones.
RESULTS
Phosphorylated histone H1, similarly to H3K9me3, has a comparable level in the G1, S, and G2 phases of the cell cycle. A high density of phosphorylated H1 was inside nucleoli of mouse embryonic stem cells (ESCs). H1ph was also abundant in prophase and prometaphase, while H1ph was absent in anaphase and telophase. H3K9me3 surrounded chromosomal DNA in telophase. This histone modification was barely detectable in the early phases of mitosis. Mass spectrometry revealed several ESC-specific phosphorylation sites of H1. HDAC1 depletion did not change H1 acetylation but potentiated phosphorylation of H1.2/H1.3 and H1.4 at serine 38 positions.
CONCLUSIONS
Differences in the level and distribution of H1ph and H3K9me3 were revealed during mitotic phases. ESC-specific phosphorylation sites were identified in a linker histone.
PubMed: 35743829
DOI: 10.3390/life12060798 -
Cell Death & Disease Jun 2022CCAR2 (cell cycle and apoptosis regulator 2) is a multifaceted protein involved in cell survival and death following cytotoxic stress. However, little is known about the...
CCAR2 (cell cycle and apoptosis regulator 2) is a multifaceted protein involved in cell survival and death following cytotoxic stress. However, little is known about the physiological functions of CCAR2 in regulating cell proliferation in the absence of external stimuli. The present study shows that CCAR2-deficient cells possess multilobulated nuclei, suggesting a defect in cell division. In particular, the duration of mitotic phase was perturbed. This disturbance of mitotic progression resulted from premature loss of cohesion with the centromere, and inactivation of the spindle assembly checkpoint during prometaphase and metaphase. It resulted in the formation of lagging chromosomes during anaphase, leading ultimately to the activation of the abscission checkpoint to halt cytokinesis. The CCAR2-dependent mitotic progression was related to spatiotemporal regulation of active Aurora B. In conclusion, the results suggest that CCAR2 governs mitotic events, including proper chromosome segregation and cytokinetic division, to maintain chromosomal stability.
Topics: Aurora Kinase B; Cell Cycle Proteins; Centromere; Chromosome Segregation; Mitosis; Protein Serine-Threonine Kinases; Spindle Apparatus
PubMed: 35672287
DOI: 10.1038/s41419-022-04990-8 -
European Review For Medical and... May 2022We have previously reported the novel off-target microtubules destabilizing activity of SB225002, a compound that was originally designed as a selective and potent IL-8...
OBJECTIVE
We have previously reported the novel off-target microtubules destabilizing activity of SB225002, a compound that was originally designed as a selective and potent IL-8 receptor B antagonist. In the present study we investigated the reversibility of SB225002 antimitotic effect and provided additional mechanistic insights underlying cell death induction in SW480 human colorectal adenocarcinoma cells.
MATERIALS AND METHODS
Mitotically arrested cells by SB225002 treatment were isolated by shake-off, and their identity was verified by both flow cytometry and immunoblotting. The reversibility of SB225002 antimitotic effects was investigated by flow cytometry and immunoblotting. Prometaphase arrested cells were imaged via indirect immunofluorescence and confocal microscopy. Activation of CHK1 in mitotically arrested cells was assessed by immunoblotting, and the relationship between CHK1 and mitotic arrest was examined via siRNA-mediated knockdown of CHK1. JNK signaling was evaluated via immunoblotting as well as pharmacological inhibition, followed by flow cytometry. The role of reactive oxygen species (ROS) in cytotoxicity was evaluated by ROS scavenging and flow cytometry.
RESULTS
Following SB225002 washout, the mitotic checkpoint was abrogated, and cell cycle perturbations were gradually restored with induction of cell death. Mechanistically, CHK1 checkpoint was activated by SB225002 and occurred downstream of the mitotic checkpoint. In addition, SB225002 activated JNK signaling which contributed to cell death and restrained polyploidy. Furthermore, SB225002 increased intracellular ROS which played a role in mediating SB225002 cytotoxicity.
CONCLUSIONS
Findings of the present study warrants further development of SB225002 as a lead compound that uniquely targets microtubules dynamics and IL-8 signaling.
Topics: Antimitotic Agents; Humans; Microtubules; Phenylurea Compounds; Reactive Oxygen Species; Receptors, Interleukin-8
PubMed: 35647855
DOI: 10.26355/eurrev_202205_28869 -
Journal of Cell Science Jun 2022Mitotic kinesin-like protein 2 (MKLP2; also known as KIF20A) is a motor protein with a well-established function in promoting cytokinesis. However, our results with...
Mitotic kinesin-like protein 2 (MKLP2; also known as KIF20A) is a motor protein with a well-established function in promoting cytokinesis. However, our results with siRNAs targeting MKLP2 and small-molecule inhibitors of MKLP2 (MKLP2i) suggest that it also has a function earlier in mitosis, prior to anaphase. In this study, we provide direct evidence that MKLP2 facilitates chromosome congression in prometaphase. We employed live imaging to observe HeLa cells with fluorescently tagged histones treated with MKLP2i and discovered a pronounced chromosome congression defect. We show that MKLP2 facilitates error correction, as inhibited cells have a significant increase in unstable, syntelic kinetochore-microtubule attachments. We find that the aberrant attachments are accompanied by elevated Aurora kinase (A and B) activity and phosphorylation of the downstream target HEC1 (also known as NDC80) at Ser55. Finally, we show that MKLP2 inhibition results in aneuploidy, confirming that MKLP2 safeguards cells against chromosomal instability. This article has an associated First Person interview with the first author of the paper.
Topics: Aurora Kinase B; Chromosome Segregation; Chromosomes; HeLa Cells; Humans; Kinesins; Kinetochores; Microtubules; Mitosis; Spindle Apparatus
PubMed: 35638575
DOI: 10.1242/jcs.259560 -
Cell Discovery May 2022Noncoding RNAs are known to associate with mitotic chromosomes, but the identities and functions of chromosome-associated RNAs in mitosis remain elusive. Here, we show...
Noncoding RNAs are known to associate with mitotic chromosomes, but the identities and functions of chromosome-associated RNAs in mitosis remain elusive. Here, we show that rRNA species associate with condensed chromosomes during mitosis. In particular, pre-rRNAs such as 45S, 32S, and 30S are highly enriched on mitotic chromosomes. Immediately following nucleolus disassembly in mitotic prophase, rRNAs are released and associate with and coat each condensed chromosome at prometaphase. Using unbiased mass spectrometry analysis, we further demonstrate that chromosome-bound rRNAs are associated with Ki-67. Moreover, the FHA domain and the repeat region of Ki-67 recognize and anchor rRNAs to chromosomes. Finally, suppression of chromosome-bound rRNAs by RNA polymerase I inhibition or by using rRNA-binding-deficient Ki-67 mutants impair mitotic chromosome dispersion during prometaphase. Our study thus reveals an important role of rRNAs in preventing chromosome clustering during mitosis.
PubMed: 35637200
DOI: 10.1038/s41421-022-00400-7 -
Bioinformatics (Oxford, England) Jun 2022Lattice light-sheet microscopy (LLSM) is revolutionizing cell biology since it enables fast, high-resolution extended imaging in three dimensions combined with a drastic...
MOTIVATION
Lattice light-sheet microscopy (LLSM) is revolutionizing cell biology since it enables fast, high-resolution extended imaging in three dimensions combined with a drastic reduction in photo-toxicity and bleaching. However, analysis of such datasets still remains a major challenge.
RESULTS
Automated tracking of kinetochores, the protein complex facilitating and controlling microtubule attachment of the chromosomes within the mitotic spindle, provides quantitative assessment of chromosome dynamics in mitosis. Here, we extend existing open-source kinetochore tracking software (KiT) to track (and pair) kinetochores throughout prometaphase to anaphase in LLSM data. One of the key improvements is a regularization term in the objective function to enforce biological information about the number of kinetochores in a human mitotic cell, as well as improved diagnostic tools. This software provides quantitative insights into how kinetochores robustly ensure congression and segregation of chromosomes during mitosis.
AVAILABILITY AND IMPLEMENTATION
KiT is free, open-source software implemented in MATLAB and can be downloaded as a package from https://github.com/cmcb-warwick/KiT. The source repository is available at https://bitbucket.org/jarmond/kit (tag v2.4.0) and under continuing development.
SUPPLEMENTARY INFORMATION
Supplementary data are available at Bioinformatics online.
Topics: Humans; Kinetochores; Spindle Apparatus; Anaphase; Microtubules; Software; Chromosome Segregation
PubMed: 35579370
DOI: 10.1093/bioinformatics/btac330 -
Cells May 2022The process of chromosome congression and alignment is at the core of mitotic fidelity. In this review, we discuss distinct spatial routes that the chromosomes take to... (Review)
Review
The process of chromosome congression and alignment is at the core of mitotic fidelity. In this review, we discuss distinct spatial routes that the chromosomes take to align during prometaphase, which are characterized by distinct biomolecular requirements. Peripheral polar chromosomes are an intriguing case as their alignment depends on the activity of kinetochore motors, polar ejection forces, and a transition from lateral to end-on attachments to microtubules, all of which can result in the delayed alignment of these chromosomes. Due to their undesirable position close to and often behind the spindle pole, these chromosomes may be particularly prone to the formation of erroneous kinetochore-microtubule interactions, such as merotelic attachments. To prevent such errors, the cell employs intricate mechanisms to preposition the spindle poles with respect to chromosomes, ensure the formation of end-on attachments in restricted spindle regions, repair faulty attachments by error correction mechanisms, and delay segregation by the spindle assembly checkpoint. Despite this protective machinery, there are several ways in which polar chromosomes can fail in alignment, mis-segregate, and lead to aneuploidy. In agreement with this, polar chromosomes are present in certain tumors and may even be involved in the process of tumorigenesis.
Topics: Chromosome Segregation; Kinetochores; Microtubules; Mitosis; Spindle Apparatus
PubMed: 35563837
DOI: 10.3390/cells11091531 -
Molecular Biology Reports Jun 2022This study served as the pioneer in studying the anti-cancer role of chicken cathelicidin peptides.
BACKGROUND
This study served as the pioneer in studying the anti-cancer role of chicken cathelicidin peptides.
METHODS AND RESULTS
Chicken cathelicidins were used as anticancer agent against the breast cancer cell line (MCF-7) and human colon cancer cell line (HCT116). In addition, the mechanism of action of the interaction of cationic peptides with breast cancer cell line MCF-7 was also investigated. An in vivo investigation was also achieved to evaluate the role of chicken cathelicidin in Ehrlich ascites cell (EAC) suppression as a tumor model after subcutaneous implantation in mice. It was found during the study that exposure of cell lines to 40 µg/ml of chicken cathelicidin for 72 h reduced cell lines growth rate by 90-95%. These peptides demonstrated down-regulation of (cyclin A1 and cyclin D genes) of MCF-7 cells. The study showed that two- and three-fold expression of both of caspase-3 and - 7 genes in untreated MCF-7 cells compared to treated MCF-7 cells with chicken cathelicidin peptides. Our data showed that chicken (CATH-1) enhance releasing of TNFα, INF-γ and upregulation of granzyme K in treated mice groups, in parallel, the tumor size and volume was reduced in the treated EAC-bearing groups. Tumor of mice groups treated with chicken cathelicidin displayed high area of necrosis compared to untreated EAC-bearing mice. Based on histological analysis and immunohistochemical staining revealed that the tumor section in Ehrlich solid tumor exhibited a strong Bcl2 expression in untreated control compared to mice treated with 10 & 20 µg of cathelicidin. Interestingly, low expression of Bcl2 were observed in mice taken 40 µg/mL of CATH-1.
CONCLUSIONS
This study drive intention in treatment of cancer through the efficacy of anticancer efficacy of chicken cathelicidin peptides.
Topics: Animals; Antineoplastic Agents; Cathelicidins; Cell Line, Tumor; Chickens; Humans; MCF-7 Cells; Mice; Neoplasms; Proto-Oncogene Proteins c-bcl-2
PubMed: 35449320
DOI: 10.1007/s11033-022-07267-7