ABSTRACT
The spindle assembly checkpoint (SAC) delays anaphase until all chromosomes are bioriented on the mitotic spindle. Under current models, unattached kinetochores transduce the SAC by catalyzing the intramitotic production of a diffusible inhibitor of APC/C(Cdc20) (the anaphase-promoting complex/cyclosome and its coactivator Cdc20, a large ubiquitin ligase). Here we show that nuclear pore complexes (NPCs) in interphase cells also function as scaffolds for anaphase-inhibitory signaling. This role is mediated by Mad1-Mad2 complexes tethered to the nuclear basket, which activate soluble Mad2 as a binding partner and inhibitor of Cdc20 in the cytoplasm. Displacing Mad1-Mad2 from nuclear pores accelerated anaphase onset, prevented effective correction of merotelic errors, and increased the threshold of kinetochore-dependent signaling needed to halt mitosis in response to spindle poisons. A heterologous Mad1-NPC tether restored Cdc20 inhibitor production and normal M phase control. We conclude that nuclear pores and kinetochores both emit "wait anaphase" signals that preserve genome integrity.
Subject(s)
Anaphase , Cell Cycle Proteins/metabolism , M Phase Cell Cycle Checkpoints , Mad2 Proteins/metabolism , Nuclear Pore/metabolism , Nuclear Proteins/metabolism , Active Transport, Cell Nucleus , Cell Cycle Proteins/genetics , Dimerization , HCT116 Cells , HeLa Cells , Humans , Interphase , Kinetochores/metabolism , Mitosis , Nuclear Proteins/geneticsABSTRACT
The human genome functions as a three-dimensional chromatin polymer, driven by a complex collection of chromosome interactions1-3. Although the molecular rules governing these interactions are being quickly elucidated, relatively few proteins regulating this process have been identified. Here, to address this gap, we developed high-throughput DNA or RNA labelling with optimized Oligopaints (HiDRO)-an automated imaging pipeline that enables the quantitative measurement of chromatin interactions in single cells across thousands of samples. By screening the human druggable genome, we identified more than 300 factors that influence genome folding during interphase. Among these, 43 genes were validated as either increasing or decreasing interactions between topologically associating domains. Our findings show that genetic or chemical inhibition of the ubiquitous kinase GSK3A leads to increased long-range chromatin looping interactions in a genome-wide and cohesin-dependent manner. These results demonstrate the importance of GSK3A signalling in nuclear architecture and the use of HiDRO for identifying mechanisms of spatial genome organization.
Subject(s)
Chromatin , Chromosome Positioning , Chromosomes, Human , Genome, Human , Glycogen Synthase Kinases , High-Throughput Screening Assays , Single-Cell Analysis , Humans , Chromatin/drug effects , Chromatin/genetics , Chromatin/metabolism , Chromosome Positioning/drug effects , Chromosomes, Human/drug effects , Chromosomes, Human/genetics , Chromosomes, Human/metabolism , DNA/analysis , DNA/metabolism , Genome, Human/drug effects , Genome, Human/genetics , Glycogen Synthase Kinases/antagonists & inhibitors , Glycogen Synthase Kinases/deficiency , Glycogen Synthase Kinases/genetics , High-Throughput Screening Assays/methods , Interphase , Reproducibility of Results , RNA/analysis , RNA/metabolism , Signal Transduction/drug effects , Single-Cell Analysis/methods , CohesinsABSTRACT
DNA replication occurs through an intricately regulated series of molecular events and is fundamental for genome stability1,2. At present, it is unknown how the locations of replication origins are determined in the human genome. Here we dissect the role of topologically associating domains (TADs)3-6, subTADs7 and loops8 in the positioning of replication initiation zones (IZs). We stratify TADs and subTADs by the presence of corner-dots indicative of loops and the orientation of CTCF motifs. We find that high-efficiency, early replicating IZs localize to boundaries between adjacent corner-dot TADs anchored by high-density arrays of divergently and convergently oriented CTCF motifs. By contrast, low-efficiency IZs localize to weaker dotless boundaries. Following ablation of cohesin-mediated loop extrusion during G1, high-efficiency IZs become diffuse and delocalized at boundaries with complex CTCF motif orientations. Moreover, G1 knockdown of the cohesin unloading factor WAPL results in gained long-range loops and narrowed localization of IZs at the same boundaries. Finally, targeted deletion or insertion of specific boundaries causes local replication timing shifts consistent with IZ loss or gain, respectively. Our data support a model in which cohesin-mediated loop extrusion and stalling at a subset of genetically encoded TAD and subTAD boundaries is an essential determinant of the locations of replication origins in human S phase.
Subject(s)
Cell Cycle Proteins , Chromatin , Chromosomal Proteins, Non-Histone , Replication Origin , Cell Cycle Proteins/metabolism , Chromatin/genetics , Chromosomal Proteins, Non-Histone/metabolism , DNA Replication , Humans , Replication Origin/genetics , S Phase , CohesinsABSTRACT
DNA repair by homologous recombination (HR) is essential for genomic integrity, tumor suppression, and the formation of gametes. HR uses DNA synthesis to repair lesions such as DNA double-strand breaks and stalled DNA replication forks, but despite having a good understanding of the steps leading to homology search and strand invasion, we know much less of the mechanisms that establish recombination-associated DNA polymerization. Here, we report that C17orf53/HROB is an OB-fold-containing factor involved in HR that acts by recruiting the MCM8-MCM9 helicase to sites of DNA damage to promote DNA synthesis. Mice with targeted mutations in Hrob are infertile due to depletion of germ cells and display phenotypes consistent with a prophase I meiotic arrest. The HROB-MCM8-MCM9 pathway acts redundantly with the HELQ helicase, and cells lacking both HROB and HELQ have severely impaired HR, suggesting that they underpin two major routes for the completion of HR downstream from RAD51. The function of HROB in HR is reminiscent of that of gp59, which acts as the replicative helicase loader during bacteriophage T4 recombination-dependent DNA replication. We therefore propose that the loading of MCM8-MCM9 by HROB may similarly be a key step in the establishment of mammalian recombination-associated DNA synthesis.
Subject(s)
DNA Repair/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Homologous Recombination/genetics , Minichromosome Maintenance Proteins/metabolism , Animals , Cell Line , DNA Helicases/metabolism , Female , HCT116 Cells , HEK293 Cells , HeLa Cells , Humans , Infertility/genetics , Male , Mice, Inbred C57BL , Sequence Deletion , Sf9 CellsABSTRACT
Termination is a ubiquitous phase in every transcription cycle but is incompletely understood and a subject of debate. We used gene editing as a new approach to address its mechanism through engineered conditional depletion of the 5' â 3' exonuclease Xrn2 or the polyadenylation signal (PAS) endonuclease CPSF73 (cleavage and polyadenylation specificity factor 73). The ability to rapidly control Xrn2 reveals a clear and general role for it in cotranscriptional degradation of 3' flanking region RNA and transcriptional termination. This defect is characterized genome-wide at high resolution using mammalian native elongating transcript sequencing (mNET-seq). An Xrn2 effect on termination requires prior RNA cleavage, and we provide evidence for this by showing that catalytically inactive CPSF73 cannot restore termination to cells lacking functional CPSF73. Notably, Xrn2 plays no significant role in either Histone or small nuclear RNA (snRNA) gene termination even though both RNA classes undergo 3' end cleavage. In sum, efficient termination on most protein-coding genes involves CPSF73-mediated RNA cleavage and cotranscriptional degradation of polymerase-associated RNA by Xrn2. However, as CPSF73 loss caused more extensive readthrough transcription than Xrn2 elimination, it likely plays a more underpinning role in termination.
Subject(s)
Cleavage And Polyadenylation Specificity Factor/metabolism , Exoribonucleases/physiology , RNA Polymerase II/metabolism , Transcription Termination, Genetic , 3' Untranslated Regions , Cell Line , Cleavage And Polyadenylation Specificity Factor/antagonists & inhibitors , Cleavage And Polyadenylation Specificity Factor/genetics , Exoribonucleases/antagonists & inhibitors , Exoribonucleases/metabolism , High-Throughput Nucleotide Sequencing , Histones , Humans , Indoleacetic Acids/pharmacology , Mutation , RNA, Small Nuclear/genetics , Sequence Analysis, RNAABSTRACT
The conditional depletion of a protein of interest (POI) is useful not only for loss-of-function studies, but also for the modulation of biological pathways. Technologies that work at the level of DNA, mRNA, and protein are available for temporal protein depletion. Compared with technologies targeting the pretranslation steps, direct protein depletion (or protein knockdown approaches) is advantageous in terms of specificity, reversibility, and time required for depletion, which can be achieved by fusing a POI with a protein domain called a degron that induces rapid proteolysis of the fusion protein. Conditional degrons can be activated or inhibited by temperature, small molecules, light, or the expression of another protein. The conditional degron-based technologies currently available are described and discussed.
Subject(s)
Gene Expression Regulation , Proteasome Endopeptidase Complex/metabolism , Protein Biosynthesis , Proteomics/methods , Animals , CRISPR-Cas Systems , Gene Expression Regulation/drug effects , Gene Expression Regulation/radiation effects , Humans , Integrases/genetics , Integrases/metabolism , Light , Morpholinos/genetics , Morpholinos/metabolism , Plants/genetics , Plants/metabolism , Protein Biosynthesis/drug effects , Protein Biosynthesis/radiation effects , Protein Domains , Protein Engineering , Proteolysis/drug effects , Proteolysis/radiation effects , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Small Molecule Libraries/pharmacologyABSTRACT
The c-Jun N-terminal kinase-associated leucine zipper protein (JLP), a scaffold protein of mitogen-activated protein kinase signaling pathways, is a multifunctional protein involved in a variety of cellular processes. It has been reported that JLP is overexpressed in various types of cancer and is expected to be a potential therapeutic target. However, whether and how JLP overexpression affects non-transformed cells remain unknown. Here, we aimed to investigate the effect of JLP overexpression on chromosomal stability in human non-transformed cells and the mechanisms involved. We found that aneuploidy was induced in JLP-overexpressed cells. Moreover, we established JLP-inducible cell lines and observed an increased frequency of chromosome missegregation, reduced time from nuclear envelope breakdown to anaphase onset, and decreased levels of the spindle assembly checkpoint (SAC) components at the prometaphase kinetochore in cells overexpressing the wild-type JLP. In contrast, we observed that a point mutant JLP lacking the ability to interact with dynein light intermediate chain 1 (DLIC1) failed to induce chromosomal instability. Our results suggest that overexpression of the wild-type JLP facilitates premature SAC silencing through interaction with DLIC1, leading to aneuploidy. This study provides a novel insight into the mechanism through which JLP overexpression is associated with cancer development and progression.
Subject(s)
Adaptor Proteins, Signal Transducing , Neoplasms , Humans , Adaptor Proteins, Signal Transducing/metabolism , Leucine Zippers , Dyneins/genetics , Dyneins/metabolism , Neoplasms/metabolism , Chromosomal Instability , Aneuploidy , MitosisABSTRACT
The eukaryotic genome is organized within cells as chromatin. For proper information output, higher-order chromatin structures can be regulated dynamically. How such structures form and behave in various cellular processes remains unclear. Here, by combining super-resolution imaging (photoactivated localization microscopy [PALM]) and single-nucleosome tracking, we developed a nuclear imaging system to visualize the higher-order structures along with their dynamics in live mammalian cells. We demonstrated that nucleosomes form compact domains with a peak diameter of â¼160 nm and move coherently in live cells. The heterochromatin-rich regions showed more domains and less movement. With cell differentiation, the domains became more apparent, with reduced dynamics. Furthermore, various perturbation experiments indicated that they are organized by a combination of factors, including cohesin and nucleosome-nucleosome interactions. Notably, we observed the domains during mitosis, suggesting that they act as building blocks of chromosomes and may serve as information units throughout the cell cycle.
Subject(s)
Chromatin Assembly and Disassembly , Heterochromatin/metabolism , Microscopy, Video/methods , Mitosis , Nucleosomes/metabolism , Animals , Cell Cycle Proteins/metabolism , Cell Differentiation , Chromosomal Proteins, Non-Histone/metabolism , Chromosomes, Human , HCT116 Cells , HeLa Cells , Heterochromatin/chemistry , Humans , Mice , Motion , Nucleic Acid Conformation , Nucleosomes/chemistry , Protein Conformation , RNA Interference , Structure-Activity Relationship , Time Factors , Transcription, Genetic , Transfection , CohesinsABSTRACT
To conduct comprehensive characterization of molecular properties in organisms, we established an efficient method to produce knockout (KO)-rescue mice within a single generation. We applied this method to produce 20 strains of almost completely embryonic stem cell (ESC)-derived mice ("ES mice") rescued with wild-type and mutant Cry1 gene under a Cry1-/-:Cry2-/- background. A series of both phosphorylation-mimetic and non-phosphorylation-mimetic CRY1 mutants revealed that multisite phosphorylation of CRY1 can serve as a cumulative timer in the mammalian circadian clock. KO-rescue ES mice also revealed that CRY1-PER2 interaction confers a robust circadian rhythmicity in mice. Surprisingly, in contrast to theoretical predictions from canonical transcription/translation feedback loops, the residues surrounding the flexible P loop and C-lid domains of CRY1 determine circadian period without changing the degradation rate of CRY1. These results suggest that CRY1 determines circadian period through both its degradation-dependent and -independent pathways.
Subject(s)
Circadian Clocks , Circadian Rhythm , Cryptochromes/metabolism , Embryonic Stem Cells/metabolism , Animals , Behavior, Animal , Cryptochromes/chemistry , Cryptochromes/deficiency , Cryptochromes/genetics , Genotype , HEK293 Cells , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Molecular , Motor Activity , Mutation , NIH 3T3 Cells , Period Circadian Proteins/genetics , Period Circadian Proteins/metabolism , Phenotype , Phosphorylation , Protein Conformation , Signal Transduction , Structure-Activity Relationship , Time Factors , TransfectionABSTRACT
DNA replication fork progression can be disrupted at difficult to replicate loci in the human genome, which has the potential to challenge chromosome integrity. This replication fork disruption can lead to the dissociation of the replisome and the formation of DNA damage. To model the events stemming from replisome dissociation during DNA replication perturbation, we used a degron-based system for inducible proteolysis of a subunit of the replicative helicase. We show that MCM2-depleted cells activate a DNA damage response pathway and generate replication-associated DNA double-strand breaks (DSBs). Remarkably, these cells maintain some DNA synthesis in the absence of MCM2, and this requires the MCM8-9 complex, a paralog of the MCM2-7 replicative helicase. We show that MCM8-9 functions in a homologous recombination-based pathway downstream from RAD51, which is promoted by DSB induction. This RAD51/MCM8-9 axis is distinct from the recently described RAD52-dependent DNA synthesis pathway that operates in early mitosis at common fragile sites. We propose that stalled replication forks can be restarted in S phase via homologous recombination using MCM8-9 as an alternative replicative helicase.
Subject(s)
DNA Replication/genetics , DNA/biosynthesis , Minichromosome Maintenance Proteins/metabolism , Cell Line, Tumor , DNA Breaks, Double-Stranded , Enzyme Activation/genetics , HCT116 Cells , Homologous Recombination/genetics , Humans , Minichromosome Maintenance Complex Component 2/genetics , Minichromosome Maintenance Complex Component 2/metabolism , Minichromosome Maintenance Proteins/genetics , Mutation , Rad52 DNA Repair and Recombination Protein/metabolism , S Phase/geneticsABSTRACT
Two mitotic cyclin types, cyclin A and B, exist in higher eukaryotes, but their specialised functions in mitosis are incompletely understood. Using degron tags for rapid inducible protein removal, we analyse how acute depletion of these proteins affects mitosis. Loss of cyclin A in G2-phase prevents mitotic entry. Cells lacking cyclin B can enter mitosis and phosphorylate most mitotic proteins, because of parallel PP2A:B55 phosphatase inactivation by Greatwall kinase. The final barrier to mitotic establishment corresponds to nuclear envelope breakdown, which requires a decisive shift in the balance of cyclin-dependent kinase Cdk1 and PP2A:B55 activity. Beyond this point, cyclin B/Cdk1 is essential for phosphorylation of a distinct subset of mitotic Cdk1 substrates that are essential to complete cell division. Our results identify how cyclin A, cyclin B and Greatwall kinase coordinate mitotic progression by increasing levels of Cdk1-dependent substrate phosphorylation.
Subject(s)
CDC2 Protein Kinase/metabolism , Cyclin A/metabolism , Cyclin B/metabolism , Mitosis , Protein Phosphatase 2/metabolism , CDC2 Protein Kinase/genetics , Cell Line , Cyclin A/genetics , Cyclin B/genetics , Humans , Protein Phosphatase 2/geneticsABSTRACT
The mini-chromosome maintenance proteins 2-7 (MCM2-7) hexamer is a protein complex that is key for eukaryotic DNA replication, which occurs only once per cell cycle. To achieve DNA replication, eukaryotic cells developed multiple mechanisms that control the timing of the loading of the hexamer onto chromatin and its activation as the replicative helicase. MCM2-7 is highly abundant in proliferating cells, which confers resistance to replication stress. Thus, the presence of an excess of MCM2-7 is important for maintaining genome integrity. However, the mechanism via which high MCM2-7 levels are achieved, other than the transcriptional upregulation of the MCM genes in the G1 phase, remained unknown. Recently, we and others reported that the MCM-binding protein (MCMBP) plays a role in the maintenance of high MCM2-7 levels and hypothesized that MCMBP functions as a chaperone in the assembly of the MCM2-7 hexamer. In this review, we discuss the roles of MCMBP in the control of MCM proteins and propose a model of the assembly of the MCM2-7 hexamer. Furthermore, we discuss a potential mechanism of the licensing checkpoint, which arrests the cells in the G1 phase when the levels of chromatin-bound MCM2-7 are reduced, and the possibility of targeting MCMBP as a chemotherapy for cancer.
Subject(s)
Cell Cycle Proteins , Minichromosome Maintenance Proteins , Minichromosome Maintenance Proteins/metabolism , Cell Cycle , Cell Cycle Proteins/metabolism , Chromatin , DNA ReplicationABSTRACT
Fanconi anemia (FA) is a hereditary disorder caused by mutations in any 1 of 22 FA genes. The disease is characterized by hypersensitivity to interstrand crosslink (ICL) inducers such as mitomycin C (MMC). In addition to promoting ICL repair, FA proteins such as RAD51, BRCA2, or FANCD2 protect stalled replication forks from nucleolytic degradation during replication stress, which may have a profound impact on FA pathophysiology. Recent studies showed that expression of the putative DNA/RNA helicase SLFN11 in cancer cells correlates with cell death on chemotherapeutic treatment. However, the underlying mechanisms of SLFN11-mediated DNA damage sensitivity remain unclear. Because SLFN11 expression is high in hematopoietic stem cells, we hypothesized that SLFN11 depletion might ameliorate the phenotypes of FA cells. Here we report that SLFN11 knockdown in the FA patient-derived FANCD2-deficient PD20 cell line improved cell survival on treatment with ICL inducers. FANCD2-/-SLFN11-/- HAP1 cells also displayed phenotypic rescue, including reduced levels of MMC-induced chromosome breakage compared with FANCD2-/- cells. Importantly, we found that SLFN11 promotes extensive fork degradation in FANCD2-/- cells. The degradation process is mediated by the nucleases MRE11 or DNA2 and depends on the SLFN11 ATPase activity. This observation was accompanied by an increased RAD51 binding at stalled forks, consistent with the role of RAD51 antagonizing nuclease recruitment and subsequent fork degradation. Suppression of SLFN11 protects nascent DNA tracts even in wild-type cells. We conclude that SLFN11 destabilizes stalled replication forks, and this function may contribute to the attrition of hematopoietic stem cells in FA.
Subject(s)
DNA Replication , Fanconi Anemia/pathology , Nuclear Proteins/metabolism , Cell Cycle Checkpoints , Cell Line , Chromosome Breakage , Cross-Linking Reagents/pharmacology , DNA Helicases/metabolism , Fanconi Anemia Complementation Group D2 Protein/genetics , Gene Knockdown Techniques , Humans , MRE11 Homologue Protein/metabolism , Models, Biological , Mutation/genetics , Phenotype , RNA, Small Interfering/metabolism , Rad51 Recombinase/metabolismABSTRACT
We have recently discovered Japanese children with a novel Fanconi anemia-like inherited bone marrow failure syndrome (IBMFS). This disorder is likely caused by the loss of a catabolic system directed toward endogenous formaldehyde due to biallelic variants in ADH5 combined with a heterozygous ALDH2*2 dominant-negative allele (rs671), which is associated with alcohol-induced Asian flushing. Phytohemagglutinin-stimulated lymphocytes from these patients displayed highly increased numbers of spontaneous sister chromatid exchanges (SCEs), reflecting homologous recombination repair of formaldehyde damage. Here, we report that, in contrast, patient-derived fibroblasts showed normal levels of SCEs, suggesting that different cell types or conditions generate various amounts of formaldehyde. To obtain insights about endogenous formaldehyde production and how defects in ADH5/ALDH2 affect human hematopoiesis, we constructed disease model cell lines, including induced pluripotent stem cells (iPSCs). We found that ADH5 is the primary defense against formaldehyde, and ALDH2 provides a backup. DNA repair capacity in the ADH5/ALDH2-deficient cell lines can be overwhelmed by exogenous low-dose formaldehyde, as indicated by higher levels of DNA damage than in FANCD2-deficient cells. Although ADH5/ALDH2-deficient cell lines were healthy and showed stable growth, disease model iPSCs displayed drastically defective cell expansion when stimulated into hematopoietic differentiation in vitro, displaying increased levels of DNA damage. The expansion defect was partially reversed by treatment with a new small molecule termed C1, which is an agonist of ALDH2, thus identifying a potential therapeutic strategy for the patients. We propose that hematopoiesis or lymphocyte blastogenesis may entail formaldehyde generation that necessitates elimination by ADH5/ALDH2 enzymes.
Subject(s)
Aldehyde Dehydrogenase, Mitochondrial/genetics , Congenital Bone Marrow Failure Syndromes/genetics , Fanconi Anemia/genetics , Induced Pluripotent Stem Cells/pathology , CRISPR-Cas Systems , Cell Line , Cells, Cultured , Congenital Bone Marrow Failure Syndromes/diagnosis , Congenital Bone Marrow Failure Syndromes/pathology , DNA Damage , Fanconi Anemia/diagnosis , Fanconi Anemia/pathology , Gene Deletion , Humans , Induced Pluripotent Stem Cells/metabolism , MutationABSTRACT
RIF1 controls both DNA replication timing and the DNA double-strand break (DSB) repair pathway to maintain genome integrity. However, it remains unclear how RIF1 links these two processes following exposure to ionizing radiation (IR). Here, we show that inhibition of homologous recombination repair (HRR) by RIF1 occurs in a dose-dependent manner and is controlled via DNA replication. RIF1 inhibits both DNA end resection and RAD51 accumulation after exposure to high doses of IR. Contrastingly, HRR inhibition by RIF1 is antagonized by BRCA1 after a low-dose IR exposure. At high IR doses, RIF1 suppresses replication initiation by dephosphorylating MCM helicase. Notably, the dephosphorylation of MCM helicase inhibits both DNA end resection and HRR, even without RIF1. Thus, our data show the importance of active DNA replication for HRR and suggest a common suppression mechanism for DNA replication and HRR at high IR doses, both of which are controlled by RIF1.This article has an associated First Person interview with the first author of the paper.
Subject(s)
Recombinational DNA Repair , Telomere-Binding Proteins , DNA Repair/genetics , DNA Replication , Homologous Recombination/genetics , Humans , Radiation Dosage , Telomere-Binding Proteins/genetics , Telomere-Binding Proteins/metabolism , Tumor Suppressor p53-Binding Protein 1/metabolismABSTRACT
In eukaryotic nuclei, chromatin loops mediated through cohesin are critical structures that regulate gene expression and DNA replication. Here, we demonstrate a new method to see endogenous genomic loci using synthetic zinc-finger proteins harboring repeat epitope tags (ZF probes) for signal amplification via binding of tag-specific intracellular antibodies, or frankenbodies, fused with fluorescent proteins. We achieve this in two steps: First, we develop an anti-FLAG frankenbody that can bind FLAG-tagged proteins in diverse live-cell environments. The anti-FLAG frankenbody complements the anti-HA frankenbody, enabling two-color signal amplification from FLAG- and HA-tagged proteins. Second, we develop a pair of cell-permeable ZF probes that specifically bind two endogenous chromatin loci predicted to be involved in chromatin looping. By coupling our anti-FLAG and anti-HA frankenbodies with FLAG- and HA-tagged ZF probes, we simultaneously see the dynamics of the two loci in single living cells. This shows a close association between the two loci in the majority of cells, but the loci markedly separate from the triggered degradation of the cohesin subunit RAD21. Our ability to image two endogenous genomic loci simultaneously in single living cells provides a proof of principle that ZF probes coupled with frankenbodies are useful new tools for exploring genome dynamics in multiple colors.
Subject(s)
Cell Nucleus , Chromatin , Chromatin/genetics , Epitopes , Genomics , ZincABSTRACT
Cancer stem-like cells (CSCs) induce drug resistance and recurrence of tumors when they experience DNA replication stress. However, the mechanisms underlying DNA replication stress in CSCs and its compensation remain unclear. Here, we demonstrate that upregulated c-Myc expression induces stronger DNA replication stress in patient-derived breast CSCs than in differentiated cancer cells. Our results suggest critical roles for mini-chromosome maintenance protein 10 (MCM10), a firing (activating) factor of DNA replication origins, to compensate for DNA replication stress in CSCs. MCM10 expression is upregulated in CSCs and is maintained by c-Myc. c-Myc-dependent collisions between RNA transcription and DNA replication machinery may occur in nuclei, thereby causing DNA replication stress. MCM10 may activate dormant replication origins close to these collisions to ensure the progression of replication. Moreover, patient-derived breast CSCs were found to be dependent on MCM10 for their maintenance, even after enrichment for CSCs that were resistant to paclitaxel, the standard chemotherapeutic agent. Further, MCM10 depletion decreased the growth of cancer cells, but not of normal cells. Therefore, MCM10 may robustly compensate for DNA replication stress and facilitate genome duplication in cancer cells in the S-phase, which is more pronounced in CSCs. Overall, we provide a preclinical rationale to target the c-Myc-MCM10 axis for preventing drug resistance and recurrence of tumors.
Subject(s)
Breast Neoplasms/genetics , Minichromosome Maintenance Proteins/metabolism , Neoplasm Recurrence, Local/genetics , Neoplastic Stem Cells/pathology , Proto-Oncogene Proteins c-myc/metabolism , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Breast/pathology , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , DNA Damage/drug effects , DNA Replication/drug effects , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Female , Humans , Minichromosome Maintenance Proteins/genetics , Neoplasm Recurrence, Local/pathology , Neoplasm Recurrence, Local/prevention & control , Neoplastic Stem Cells/drug effects , Primary Cell Culture , Proto-Oncogene Proteins c-myc/antagonists & inhibitors , Spheroids, Cellular , Tumor Cells, Cultured , Up-RegulationABSTRACT
Chk1 (encoded by CHEK1 in mammals) is an evolutionarily conserved protein kinase that transduces checkpoint signals from ATR to Cdc25A during the DNA damage response (DDR). In mammals, Chk1 also controls cellular proliferation even in the absence of exogenous DNA damage. However, little is known about how Chk1 regulates unperturbed cell cycle progression, and how this effect under physiological conditions differs from its regulatory role in DDR. Here, we have established near-diploid HCT116 cell lines containing endogenous Chk1 protein tagged with a minimum auxin-inducible degron (mAID) through CRISPR/Cas9-based gene editing. Establishment of these cells enabled us to induce specific and rapid depletion of the endogenous Chk1 protein, which resulted in aberrant accumulation of DNA damage factors that induced cell cycle arrest at S or G2 phase. Cdc25A was stabilized upon Chk1 depletion before the accumulation of DNA damage factors. Simultaneous depletion of Chk1 and Cdc25A partially suppressed the defects caused by Chk1 single depletion. These results indicate that, similar to its function in DDR, Chk1 controls normal cell cycle progression mainly by inducing Cdc25A degradation.
Subject(s)
Checkpoint Kinase 1/metabolism , DNA Damage , G2 Phase Cell Cycle Checkpoints , Proteolysis , S Phase Cell Cycle Checkpoints , cdc25 Phosphatases/metabolism , CRISPR-Cas Systems , Checkpoint Kinase 1/genetics , Gene Editing , HCT116 Cells , Humans , cdc25 Phosphatases/geneticsABSTRACT
At the onset of procentriole formation, a structure called the cartwheel is formed adjacent to the pre-existing centriole. SAS-6 proteins are thought to constitute the hub of the cartwheel structure. However, the exact function of the cartwheel in the process of centriole formation has not been well characterized. In this study, we focused on the functions of human SAS-6 (HsSAS-6, also known as SASS6). By using an in vitro reconstitution system with recombinant HsSAS-6, we first observed its conserved molecular property of forming the central part of the cartwheel structure. Furthermore, we uncovered critical functions of HsSAS-6 by using a combination of an auxin-inducible HsSAS-6-degron (AID) system and super-resolution microscopy in human cells. Our results demonstrate that the HsSAS-6 is required not only for the initiation of centriole formation, but also for the stabilization of centriole intermediates. Moreover, after procentriole formation, HsSAS-6 is necessary for limiting Plk4 accumulation at the centrioles and thereby suppressing the formation of initiation sites that would otherwise promote the development of extra procentrioles. Overall, these findings illustrate the conserved and fundamental functions of the cartwheel in centriole duplication.