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Molecular Cell Feb 2022We have used a combination of chemical genetics, chromatin proteomics, and imaging to map the earliest chromatin transactions during vertebrate cell entry into mitosis....
We have used a combination of chemical genetics, chromatin proteomics, and imaging to map the earliest chromatin transactions during vertebrate cell entry into mitosis. Chicken DT40 CDK1 cells undergo synchronous mitotic entry within 15 min following release from a 1NM-PP1-induced arrest in late G. In addition to changes in chromatin association with nuclear pores and the nuclear envelope, earliest prophase is dominated by changes in the association of ribonucleoproteins with chromatin, particularly in the nucleolus, where pre-rRNA processing factors leave chromatin significantly before RNA polymerase I. Nuclear envelope barrier function is lost early in prophase, and cytoplasmic proteins begin to accumulate on the chromatin. As a result, outer kinetochore assembly appears complete by nuclear envelope breakdown (NEBD). Most interphase chromatin proteins remain associated with chromatin until NEBD, after which their levels drop sharply. An interactive proteomic map of chromatin transactions during mitotic entry is available as a resource at https://mitoChEP.bio.ed.ac.uk.
Topics: Animals; CDC2 Protein Kinase; Cell Line, Tumor; Chickens; Chromatin; Chromatin Assembly and Disassembly; Chromosomes; DNA; Lamin Type B; Lymphoma, B-Cell; Nuclear Proteins; Prophase; Protein Binding; Proteome; Proteomics; RNA, Ribosomal; Time Factors
PubMed: 35090599
DOI: 10.1016/j.molcel.2021.12.039 -
Genes & Development Feb 2022Mechanisms regulating meiotic progression in mammals are poorly understood. The -methyladenosine (mA) reader and 3' → 5' RNA helicase YTHDC2 switches cells from...
Mechanisms regulating meiotic progression in mammals are poorly understood. The -methyladenosine (mA) reader and 3' → 5' RNA helicase YTHDC2 switches cells from mitotic to meiotic gene expression programs and is essential for meiotic entry, but how this critical cell fate change is accomplished is unknown. Here, we provide insight into its mechanism and implicate YTHDC2 in having a broad role in gene regulation during multiple meiotic stages. Unexpectedly, mutation of the mA-binding pocket of YTHDC2 had no detectable effect on gametogenesis and mouse fertility, suggesting that YTHDC2 function is mA-independent. Supporting this conclusion, CLIP data defined YTHDC2-binding sites on mRNA as U-rich and UG-rich motif-containing regions within 3' UTRs and coding sequences, distinct from the sites that contain mA during spermatogenesis. Complete loss of YTHDC2 during meiotic entry did not substantially alter translation of its mRNA binding targets in whole-testis ribosome profiling assays but did modestly affect their steady-state levels. Mutation of the ATPase motif in the helicase domain of YTHDC2 did not affect meiotic entry, but it blocked meiotic prophase I progression, causing sterility. Our findings inform a model in which YTHDC2 binds transcripts independent of mA status and regulates gene expression during multiple stages of meiosis by distinct mechanisms.
Topics: Animals; Gene Expression Regulation; Male; Mammals; Meiosis; Mice; RNA Helicases; RNA, Messenger; Spermatogenesis
PubMed: 35058317
DOI: 10.1101/gad.349190.121 -
Cell Reports Dec 2021Mechanisms driving the prolonged meiotic prophase I in mammals are poorly understood. RNA helicase YTHDC2 is critical for mitosis to meiosis transition. However, YTHDC2...
Mechanisms driving the prolonged meiotic prophase I in mammals are poorly understood. RNA helicase YTHDC2 is critical for mitosis to meiosis transition. However, YTHDC2 is highly expressed in pachytene cells. Here we identify an essential role for YTHDC2 in meiotic progression. Specifically, YTHDC2 deficiency causes microtubule-dependent telomere clustering and apoptosis at the pachytene stage of prophase I. Depletion of YTHDC2 results in a massively dysregulated transcriptome in pachytene cells, with a tendency toward upregulation of genes normally expressed in mitotic germ cells and downregulation of meiotic transcripts. Dysregulation does not correlate with mA status, and YTHDC2-bound mRNAs are enriched in genes upregulated in mutant germ cells, revealing that YTHDC2 primarily targets mRNAs for degradation. Furthermore, altered transcripts in mutant pachytene cells encode microtubule network proteins. Our results demonstrate that YTHDC2 regulates the pachytene stage by perpetuating a meiotic transcriptome and preventing microtubule network changes that could lead to telomere clustering.
Topics: Animals; Female; Male; Meiosis; Mice; Mice, Inbred C57BL; Mice, Knockout; Microtubules; Pachytene Stage; RNA Helicases; Spermatocytes; Telomere; Transcriptome
PubMed: 34910909
DOI: 10.1016/j.celrep.2021.110110 -
Journal of Enzyme Inhibition and... Dec 2022Microtubules play an important role in the process of cell mitosis and can form a spindle in the mitotic prophase of the cell, which can pull chromosomes to the ends of... (Review)
Review
Microtubules play an important role in the process of cell mitosis and can form a spindle in the mitotic prophase of the cell, which can pull chromosomes to the ends of the cell and then divide into two daughter cells to complete the process of mitosis. Tubulin inhibitors suppress cell proliferation by inhibiting microtubule dynamics and disrupting microtubule homeostasis. Thereby inducing a cell cycle arrest at the G2/M phase and interfering with the mitotic process. It has been found that a variety of chalcone derivatives can bind to microtubule proteins and disrupt the dynamic balance of microtubules, inhibit the proliferation of tumour cells, and exert anti-tumour effects. Consequently, a great number of studies have been conducted on chalcone derivatives targeting microtubule proteins. In this review, synthetic or natural chalcone microtubule inhibitors in recent years are described, along with their structure-activity relationship (SAR) for anticancer activity.
Topics: Antineoplastic Agents; Cell Proliferation; Chalcones; Dose-Response Relationship, Drug; Humans; Molecular Structure; Neoplasms; Polymerization; Structure-Activity Relationship; Tubulin; Tubulin Modulators
PubMed: 34894980
DOI: 10.1080/14756366.2021.1976772 -
G3 (Bethesda, Md.) Oct 2021Meiosis-specific chromatin structures, guided by histone modifications, are critical mediators of a meiotic transient transcription program and progression through...
Meiosis-specific chromatin structures, guided by histone modifications, are critical mediators of a meiotic transient transcription program and progression through prophase I. Histone H3K4 can be methylated up to three times by the Set1-containing COMPASS complex and each methylation mark corresponds to a different chromatin conformation. The level of H3K4 modification is directed by the activity of additional COMPASS components. In this study, we characterized the role of the COMPASS subunits during meiosis in Saccharomyces cerevisiae. In vegetative cells, previous studies revealed a role for subunits Swd2, Sdc1, and Bre2 for H3K4me2 while Spp1 supported trimethylation. However, we found that Bre2 and Sdc1 are required for H3K4me3 as yeast prepare to enter meiosis while Spp1 is not. Interestingly, we identified distinct meiotic functions for the core COMPASS complex members that required for all H3K4me, Set1, Swd1, and Swd3. While Set1 and Swd1 are required for progression through early meiosis, Swd3 is critical for late meiosis and spore morphogenesis. Furthermore, the meiotic requirement for Set1 is independent of H3K4 methylation, suggesting the presence of nonhistone substrates. Finally, checkpoint suppression analyses indicate that Set1 and Swd1 are required for both homologous recombination and chromosome segregation. These data suggest that COMPASS has important new roles for meiosis that are independent of its well-characterized functions during mitotic divisions.
Topics: DNA-Binding Proteins; Histone-Lysine N-Methyltransferase; Meiosis; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 34849786
DOI: 10.1093/g3journal/jkab283 -
The Journal of Cell Biology Dec 2021The dynein motor performs multiple functions in mitosis by engaging with a wide cargo spectrum. One way to regulate dynein's cargo-binding selectivity is through the...
The dynein motor performs multiple functions in mitosis by engaging with a wide cargo spectrum. One way to regulate dynein's cargo-binding selectivity is through the C-terminal domain (CTD) of its light intermediate chain 1 subunit (LIC1), which binds directly with cargo adaptors. Here we show that mitotic phosphorylation of LIC1-CTD at its three cdk1 sites is required for proper mitotic progression, for dynein loading onto prometaphase kinetochores, and for spindle assembly checkpoint inactivation in human cells. Mitotic LIC1-CTD phosphorylation also engages the prolyl isomerase Pin1 predominantly to Hook2-dynein-Nde1-Lis1 complexes, but not to dynein-spindly-dynactin complexes. LIC1-CTD dephosphorylation abrogates dynein-Pin1 binding, promotes prophase centrosome-nuclear envelope detachment, and impairs metaphase chromosome congression and mitotic Golgi fragmentation, without affecting interphase membrane transport. Phosphomutation of a conserved LIC1-CTD SP site in zebrafish leads to early developmental defects. Our work reveals that LIC1-CTD phosphorylation differentially regulates distinct mitotic dynein pools and suggests the evolutionary conservation of this phosphoregulation.
Topics: Animals; Cell Line, Tumor; Centrosome; Cytoplasmic Dyneins; Dynactin Complex; Evolution, Molecular; Golgi Apparatus; Humans; Interphase; Kinetochores; Metaphase; Microtubule-Associated Proteins; Mitosis; Mutant Proteins; NIMA-Interacting Peptidylprolyl Isomerase; Nuclear Envelope; Phosphorylation; Protein Binding; Protein Subunits; Rats; Zebrafish
PubMed: 34709360
DOI: 10.1083/jcb.202005184 -
Cell Cycle (Georgetown, Tex.) Oct 2021To ensure genome stability, chromosomes need to undergo proper condensation into two linked sister chromatids from prophase to prometaphase, followed by equal...
To ensure genome stability, chromosomes need to undergo proper condensation into two linked sister chromatids from prophase to prometaphase, followed by equal segregation at anaphase. Emerging evidence has shown that persistent DNA entanglements connecting the sister chromatids lead to the formation of ultrafine anaphase bridges (UFBs). If UFBs are not resolved soon after anaphase, they can induce chromosome missegregation. PICH (PLK1-interacting checkpoint helicase) is a DNA translocase that localizes on chromosome arms, centromeres and UFBs. It plays multiple essential roles in mitotic chromosome organization and segregation. PICH also recruits other associated proteins to UFBs, and together they mediate UFB resolution. Here, the proposed mechanism behind PICH's functions in chromosome organization and UFB resolution will be discussed. We summarize the regulation of PICH action at chromosome arms and centromeres, how PICH recognizes UFBs and recruits other UFB-associated factors, and finally how PICH promotes UFB resolution together with other DNA processing enzymes.
Topics: Anaphase; Centromere; Chromatids; Chromosome Segregation; Genomic Instability; Humans; Mitosis
PubMed: 34530686
DOI: 10.1080/15384101.2021.1970877 -
PLoS Genetics Aug 2021Meiosis is essential for the generation of gametes and sexual reproduction, yet the factors and underlying mechanisms regulating meiotic progression remain largely...
Meiosis is essential for the generation of gametes and sexual reproduction, yet the factors and underlying mechanisms regulating meiotic progression remain largely unknown. Here, we showed that MTL5 translocates into nuclei of spermatocytes during zygotene-pachytene transition and ensures meiosis advances beyond pachytene stage. MTL5 shows strong interactions with MuvB core complex components, a well-known transcriptional complex regulating mitotic progression, and the zygotene-pachytene transition of MTL5 is mediated by its direct interaction with the component LIN9, through MTL5 C-terminal 443-475 residues. Male Mtl5c-mu/c-mu mice expressing the truncated MTL5 (p.Ser445Arg fs*3) that lacks the interaction with LIN9 and is detained in cytoplasm showed male infertility and spermatogenic arrest at pachytene stage, same as that of Mtl5 knockout mice, indicating that the interaction with LIN9 is essential for the nuclear translocation and function of MTL5 during meiosis. Our data demonstrated MTL5 translocates into nuclei during the zygotene-pachytene transition to initiate its function along with the MuvB core complex in pachytene spermatocytes, highlighting a new mechanism regulating the progression of male meiosis.
Topics: Active Transport, Cell Nucleus; Animals; Cell Cycle Proteins; Chromosome Pairing; Cytoplasm; DNA-Binding Proteins; Fertility; Infertility, Male; Male; Meiosis; Meiotic Prophase I; Metallothionein; Mice; Mice, Inbred C57BL; Pachytene Stage; Spermatocytes; Spermatogenesis; Testis; Tumor Suppressor Proteins
PubMed: 34388164
DOI: 10.1371/journal.pgen.1009753 -
PLoS Genetics Jul 2021Homologous recombination is a high-fidelity repair pathway for DNA double-strand breaks employed during both mitotic and meiotic cell divisions. Such repair can lead to...
Homologous recombination is a high-fidelity repair pathway for DNA double-strand breaks employed during both mitotic and meiotic cell divisions. Such repair can lead to genetic exchange, originating from crossover (CO) generation. In mitosis, COs are suppressed to prevent sister chromatid exchange. Here, the BTR complex, consisting of the Bloom helicase (HIM-6 in worms), topoisomerase 3 (TOP-3), and the RMI1 (RMH-1 and RMH-2) and RMI2 scaffolding proteins, is essential for dismantling joint DNA molecules to form non-crossovers (NCOs) via decatenation. In contrast, in meiosis COs are essential for accurate chromosome segregation and the BTR complex plays distinct roles in CO and NCO generation at different steps in meiotic recombination. RMI2 stabilizes the RMI1 scaffolding protein, and lack of RMI2 in mitosis leads to elevated sister chromatid exchange, as observed upon RMI1 knockdown. However, much less is known about the involvement of RMI2 in meiotic recombination. So far, RMI2 homologs have been found in vertebrates and plants, but not in lower organisms such as Drosophila, yeast, or worms. We report the identification of the Caenorhabditis elegans functional homolog of RMI2, which we named RMIF-2. The protein shows a dynamic localization pattern to recombination foci during meiotic prophase I and concentration into recombination foci is mutually dependent on other BTR complex proteins. Comparative analysis of the rmif-2 and rmh-1 phenotypes revealed numerous commonalities, including in regulating CO formation and directing COs toward chromosome arms. Surprisingly, the prevalence of heterologous recombination was several fold lower in the rmif-2 mutant, suggesting that RMIF-2 may be dispensable or less strictly required for some BTR complex-mediated activities during meiosis.
Topics: Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Cell Cycle Proteins; Chromosomal Proteins, Non-Histone; Chromosome Segregation; Chromosomes; Crossing Over, Genetic; DNA Breaks, Double-Stranded; DNA Repair; DNA Topoisomerases, Type I; DNA-Binding Proteins; Homologous Recombination; Meiosis; Sister Chromatid Exchange
PubMed: 34252074
DOI: 10.1371/journal.pgen.1009663 -
Developmental Cell Jun 2021Nuclear envelope assembly during late mitosis includes rapid formation of several thousand complete nuclear pore complexes (NPCs). This efficient use of NPC components...
Nuclear envelope assembly during late mitosis includes rapid formation of several thousand complete nuclear pore complexes (NPCs). This efficient use of NPC components (nucleoporins or "NUPs") is essential for ensuring immediate nucleocytoplasmic communication in each daughter cell. We show that octameric subassemblies of outer and inner nuclear pore rings remain intact in the mitotic endoplasmic reticulum (ER) after NPC disassembly during prophase. These "inherited" subassemblies then incorporate into NPCs during post-mitotic pore formation. We further show that the stable subassemblies persist through multiple rounds of cell division and the accompanying rounds of NPC mitotic disassembly and post-mitotic assembly. De novo formation of NPCs from newly synthesized NUPs during interphase will then have a distinct initiation mechanism. We postulate that a yet-to-be-identified modification marks and "immortalizes" one or more components of the specific octameric outer and inner ring subcomplexes that then template post-mitotic NPC assembly during subsequent cell cycles.
Topics: Cell Cycle; Cell Nucleus; Endoplasmic Reticulum; Humans; Interphase; Mitosis; Nuclear Envelope; Nuclear Pore; Nuclear Pore Complex Proteins
PubMed: 34129835
DOI: 10.1016/j.devcel.2021.05.015