Genes to Cells

CENP-A, a variant of histone H3, determines the identity of the centromere, a chromosome locus at which a microtubule attachment site termed kinetochore is assembled. Because the position and size of the centromere and its number per chromosome must be maintained for the faithful segregation of chromosomes, the distribution of CENP-A is strictly regulated. In this study, we have aimed to understand mechanisms to regulate the distribution of CENP-A by a genetic approach in the fission yeast Schizosaccharomyces pombe. A mutant of the ufd1+ gene (ufd1-73) encoding a cofactor of Cdc48 ATPase is sensitive to CENP-A expressed at a high level and allows mislocalization of CENP-A. ChIP analysis has revealed that the level of CENP-A in centromeric chromatin is increased in the ufd1-73 mutant even when CENP-A is expressed at a normal level. A preexisting mutant of the cdc48+ gene (cdc48-353) phenocopies the ufd1-73 mutant. We have also shown that Cdc48 and Ufd1 proteins physically interact with centromeric chromatin. Finally, Cdc48 ATPase with Ufd1 artificially recruited to the centromere of a mini-chromosome (Ch16) induce a loss of CENP-A from Ch16, resulting in an increased rate of chromosome loss. It appears that Cdc48 ATPase, together with its cofactor Ufd1 segregates excess CENP-A from chromatin, likely in a direct manner, to maintain proper distribution of CENP-A. This mechanism may play an important role in centromere disassembly, a process to eliminate CENP-A massively to inactivate the kinetochore function during development, differentiation, and stress response in other organisms. Significance statementMaintaining the proper distribution of CENP-A is crucial for centromere identity. This process involves accurate positioning of CENP-A and removal of excess CENP-A from chromatin. The Cdc48-Ufd1 complex is essential for this regulation as it acts as a segregase that directly eliminates surplus CENP-A from chromatin. These findings have therapeutic significance as targeting the Cdc48 complex can potentially correct abnormal karyotypes in disomic embryos and prevent trisomic disorders like Down syndrome. This research advances our understanding of centromeric chromatin regulation and its potential therapeutic applications.

EWSR1 (Ewing sarcoma breakpoint region 1) was originally identified as a part of an aberrant EWSR1/FLI1 fusion gene in Ewing sarcoma, the second most common pediatric bone cancer. Due to formation of the EWSR1/FLI1 fusion gene in the tumor genome, the cell loses one wild type EWSR1 allele. Our previous study demonstrated that the loss of ewsr1a (homologue of human EWSR1) in zebrafish leads to the high incidence of mitotic dysfunction, of aneuploidy, and of tumorigenesis in the tp53 mutant background. To dissect the molecular function of EWSR1, we successfully established a stable DLD-1 cell line that enables a conditional knockdown of EWSR1 using Auxin Inducible Degron (AID) system. When both EWSR1 genes of DLD-1 cell were tagged with mini-AID at its 5-end using CRISPR/Cas9 system, treatment of the (AID-EWSR1/AID-EWSR1) DLD-1 cells with a plant-based Auxin (AUX) led to the significant levels of degradation of AID-EWSR1 proteins. During anaphase, the EWSR1 knockdown (AUX+) cells displayed higher incidence of lagging chromosomes compared to the control (AUX-) cells. This defect was proceeded by a lower incidence of the localization of Aurora B at inner centromeres, and by a higher incidence of the protein at kinetochores compared to the control cells during pro/metaphase. Despite these defects, the EWSR1 knockdown cells did not undergo mitotic arrest, suggesting that the cell lacks the error correction mechanism. Significantly, the EWSR1 knockdown (AUX+) cells induced higher incidence of aneuploidy compared to the control (AUX-) cells. Since our previous study demonstrated that EWSR1 interacts with the key mitotic kinase, Aurora B, we generated replacement lines of EWSR1-mCherry and EWSR1:R565A-mCherry (a mutant that has low affinity for Aurora B) in the (AID-EWSR1/AID-EWSR1) DLD-1 cells. The EWSR1-mCherry rescued the high incidence of aneuploidy of EWSR1 knockdown cells, whereas EWSR1-mCherry:R565A failed to rescue the phenotype. Together, we demonstrate that EWSR1 is essential to prevent aneuploidy through interaction with Aurora B, most likely by regulating the localization of Aurora B at centromere.

Chromosomes structure changes dramatically upon entering meiosis to ensure the successful progression of meiosis-specific events. During this process, a multilayer proteinaceous structure called synaptonemal complex (SC) is formed in many eukaryotes. Instead, in the fission yeast Schizosaccharomyces pombe, linear elements (LinEs), which are structures related to an axial element of SC, form on the meiotic cohesin-based chromosome axis and are required for the formation of DNA double-strand breaks. In contrast to the well-organized SC structure, LinE structure had been observed only by silver-stained electron micrographs or in immuno-fluorescence stained spread nuclei. Thus, their fine structure and dynamics in intact living cells remain to be elucidated. In this study, we performed live cell imaging with wide-field fluorescence microscopy as well as 3D structured illumination microscopy (3D-SIM) for the four components of LinE, the Rec10, Rec25, Rec27 and Mug20. We found that LinEs consist of threads formed along the chromosome axes during the meiotic prophase. Rec10 binds to the chromosome itself and shapes into LinEs only in the presence of all the other LinE components. Rec25, Rec27, and Mug20 attach to the chromosome in the presence of Rec10. LinEs are stable in a short-time treatment with 1,6-hexanediol; and fluorescence recovery after photobleaching (FRAP) experiment reveals slow recovery from photobleaching, indicating a stable property of LinEs.Competing Interest StatementThe authors have declared no competing interest.View Full Text

Body color plays key roles in fitness, communication with others, and hiding. In poikilothermal vertebrates, the body color is mainly determined by types and distributions of chromatophores. Among them, carotenoid color of xanthophores/erythrophores is important for interspecific diversity and colorful males as sexual dimorphism. Most vertebrates cannot synthesize carotenoids in their bodies and must ingest them from food. However, the genes involved in the uptake process are not fully understood. Therefore, we tried to identify the causal gene of the carotenoid color mutant of medaka. The HdrR-II1 strain used in the genome project has orange body color in males and white body color in females. The orange and white body color was known to be controlled by the sex-linked R locus, but the causal gene of this was unknown. In this study, we identified that the causal gene of the R locus is tetratricopeptide repeat domain 39b like (ttc39bl). In the HdrR-II1, the ttc39bl on the Y chromosome is normal, but the ttc39bl on the X chromosome has an 821 bases insertion in exon 3 and is broken. This insertion is also present on both the X and Y chromosomes of commercially available white medaka.

Break-induced DNA replication (BIR) have been detected not only in the genome of rare disease patients but also in cancer cells, however, the mechanisms of BIR formation havent been explained in details. In the late G2 phase-like mouse oocytes, we found DNA double-strand breaks (DSBs) could induce Rad51 dependent small-scale DNA replication. In addition, we also found the DSBs could be amplified in mouse oocytes, and the amplification could be inhibited by Rad51 inhibitor IBR2 and DNA replication inhibitor ddATP. Lastly, we found the DSB repair was relatively inefficiency in hybrid mouse oocytes compared with that of the purebred mouse oocytes. We found DSBs could induce BIR more easier in hybrid mouse oocytes, indicating the DNA repair in oocytes could be affected by the sequence differences between homologous chromatids. In summary, our results indicated that the condensed chromatin configuration in late G2 phase and the sequence similarity between broken DNA and template DNA are causing factors of BIR in mammalian genome, and the DNA damage could be amplified in late G2 phase cells.

There has been a great deal of research on cell division and its mechanisms; however, its processes have not yet been entirely elucidated. To find novel proteins that regulate cell division, we performed the screening using siRNAs and/or the expression plasmid of the target genes and identified leucine zipper protein 1 (LUZP1). Recent studies have shown that LUZP1 interacts with various proteins and stabilizes the actin cytoskeleton; however, the function of LUZP1 in mitosis is not known. In this study, we found that LUZP1 colocalized with the chromosomal passenger complex (CPC) at the centromere in metaphase and at the central spindle in anaphase and that these LUZP1 localizations were regulated by CPC activity and kinesin family member 20A (KIF20A). Mass spectrometry analysis identified that LUZP1 interacted with death-associated protein kinase 3 (DAPK3), one regulator of the cleavage furrow ingression in cytokinesis. In addition, we found that LUZP1 also interacted with myosin light chain 9 (MYL9), a substrate of DAPK3, and comprehensively inhibited MYL9 phosphorylation by DAPK3. In line with a known role for MYL9 in the actin-myosin contraction, LUZP1 suppression accelerated the constriction velocity at the division plane in our timelapse analysis. Our study indicates that LUZP1 is a novel regulator for cytokinesis that regulates the constriction velocity of the contractile ring.

Cell cycle division 25B (CDC25B) belongs to the family of cell cycle regulatory proteins. It drives G2/M transition by activating cyclin-dependent protein kinases (CDK1), also known as CDC2, whose activity is directly related to its subcellular localization and phosphorylation state.14-3-3 (YHWA) regulates cell division cycle by binding to Cdc25B as a chaperone protein in mammals. Previously, we found that Cdc25B-Ser149 plays an important role in G2/M transition of mouse fertilized eggs, but the molecular mechanism of this transition remains unclear. In this study, we assessed the role of 14-3-3{varepsilon} (YHWAE) interaction with phosphorylated Cdc25B-Ser149 in G2/M transition of mouse fertilized eggs. Co-expression of Cdc25B-Ser149A and 14-3-3{varepsilon} could effectively activate maturation promoting factor (MPF) through direct dephosphorylation of Cdc2-Tyr15, and induce G2 fertilized eggs to enter mitosis rapidly. However, co-expression of the phosphomimic Cdc25B-Ser149D or Cdc25B-WT and 14-3-3{varepsilon} showed no significant difference in comparison with control groups. 14-3-3{varepsilon} binds to Cdc25B-WT, which is abolished when Ser149 is mutated to Ala. In addition, we found that 14-3-3{varepsilon} and Cdc25B were co-localized in the cytoplasm at the G1, S and early G2 phases. Cdc25B was translocated from the cytoplasm to the nucleus at the late G2 phase. However, when Ser149 is mutated to Ala, the cytoplasmic localization of Cdc25B is completely abolished. Our findings suggest that Cdc25B-Ser149 is another specific binding site for 14-3-3{varepsilon} in G2/M transition of one-cell fertilized mouse eggs, which plays essential roles in the regulation of early development of fertilized mouse eggs.

As a versatile genome editing tool, the CRISPR-Cas9 system induces DNA double-strand breaks at targeted sites to activate mainly two DNA repair pathways: HDR which allows precise editing via recombination with a homologous template DNA, and NHEJ which connects two ends of the broken DNA, which is often accompanied by random insertions and deletions. Therefore, how to enhance HDR while suppressing NHEJ is a key to successful applications that require precise genome editing. Histones are small proteins with a lot of basic amino acids that generate electrostatic affinity to DNA. Since H2A.X is involved in DNA repair processes, we fused H2A.X to Cas9 and found that this fusion protein could improve the HDR/NHEJ ratio. As various post-translational modifications of H2A.X play roles in the regulation of DNA repair, we also fused H2A.X mimicry variants to replicate these post-translational modifications including phosphorylation, methylation, and acetylation. However, none of them were effective to improve the HDR/NHEJ ratio. We further fused other histone variants to Cas9 and found that H2A.1 exhibited the improved HDR/NHEJ ratio better than H2A.X. Thus, the fusion of histone variants to Cas9 is a promising option to enhance precise genome editing.

Germ cell fate is believed to be determined by the signaling from sexually differentiated somatic cell. However, the molecular mechanism remains elusive. In this study, ectopic initiation of meiosis in male germ cells was observed during embryonic stage by over-activating CTNNB1 in Sertoli cells. Somatic cell transcriptome and single germ cell RNA-seq analysis indicated that TGF-{beta} signaling was activated after CTNNB1 over-activation. In vitro and in vivo experiments confirmed somatic cell-derived BMPs played crucial roles in germ cell meiosis initiation. Further studies revealed that Dazl was significantly increased in germ cells of CTNNB1 over-activated testes and induced by BMP signaling. DNMT3a and DNA methylation was also reduced in germ cells of CTNNB1 over-activated testes and increased by BMP signaling inhibitor treatment. Taken together, this study demonstrates that germ cell fate could be reprogrammed after sex determination. BMP signaling pathway is involved in germ cell meiosis initiation via up-regulating Dazl expression.

CTP synthase (CTPS) forms cytoophidia in all three domains of life. Here we focus on the function of cytoophidia in cell proliferation using Schizosaccharomyces pombe as a model system. We find that converting His359 of CTPS into Ala359 leads to cytoophidium disassembly. By reducing the level of CTPS protein or specific point mutations, the loss of cytoophidia prolongs the G2 phase and expands cell size. In addition, the loss-filament mutant of CTPS leads to a decrease in the expression of genes related to G2/M transition and cell growth, including slm9. The overexpression of slm9 alleviates the G2 phase elongation and cell size enlargement induced by CTPS loss-filament mutant. Overall, our results connect cytoophidia with cell cycle and cell size control in Schizosaccharomyces pombe.

The mouse xenograft model is one of the most widely used animal model for biomedicine research. It is vital to distinguish the cells from different species, especially for the spatial distribution information. However, the available strategies of species-specific detection are either inapplicable in situ or of low specificity. Here, we reported a method based on DAPI staining, which offers an effective, convenient way that accurately identifies human and mouse nuclei at single-cell level in situ. This method was proven to be effective in cell co-culture and tumor xenograft tissue section. Microscopic imaging results shows obvious DAPI plaques-like structures in mouse nuclei, but absent in human nuclei. Moreover, we found these structures are co-localized with mouse major satellite DNA, which is located pericentromere in mouse, but absent in human. Our study provides a high-performance method that can be widely used for distinguish human and mouse cell in situ.

A retron is a distinct system encoding reverse transcriptase and a unique single-stranded DNA/RNA hybrid called multicopy single-stranded DNA (msDNA). The ability of msDNA to serve as a homologous recombination donor for gene editing has attracted great interest. However, the mechanism by which msDNA expression affects editing efficiency remains unclear. In this study, we show that an increase in msd number increased msDNA yield but was not necessarily accompanied by an increase in editing efficiency. Mechanistic studies indicate that msd and genomic regions competed for msDNA during recombination. As the number of msd increased, the amount of msDNA allocated to the genomic targets decreased, resulting in a decrease in editing efficiency. Finally, we reduced msd editing by expressing msDNA corresponding to the plasmid replication leading strand sequence, thus constructing a retron-based gene editing system that achieved 100% editing efficiency in the shortest time reported to date. The above results reveal a completely different features between retron-based gene editing system and oligonucleotide-mediated gene editing system and will provide theoretical guidance for the design and application of the retron system.

Mitochondrial biogenesis is a cell response to external stimuli which is generally believed to suppress apoptosis. However, during the process of apoptosis, whether mitochondrial biogenesis occurs in the early stage of the apoptotic cells remains unclear. To address this question, we constructed the COX8-EGFP-ACTIN-mCherry HeLa cells with recombinant fluorescent proteins respectively tagged on the nucleus and mitochondria, and monitored the mitochondrial changes in living cells exposed to gamma-ray radiation. Besides in situ detection of mitochondrial fluorescence changes, we also examined the cell viability, nuclear DNA damage, reactive oxygen species (ROS), Mitochondrial superoxide, citrate synthase activity, ATP, cytoplasmic and mitochondrial calcium, mitochondrial DNA copy number and expression of transcription genes related to mitochondrial biogenesis as well as the apoptosis biomarkers. As a result, we confirmed that significant mitochondrial biogenesis took place preceding the radiation-induced apoptosis, and the change of mitochondrial biogenesis at early time was closely correlated with the apoptotic cells at late stage. The involved mechanism was also discussed. HighlightsO_LIA dual fluorescence reporter system was successfully constructed for in-situ observation of mitochondrial biogenesis in living cells. C_LIO_LIThe whole process of radiation-induced mitochondrial biogenesis and apoptosis was scrutinized. C_LIO_LIThe conception of the relationship between mitochondrial biogenesis and apoptosis was revised. C_LIO_LIAssessment of the early event of mitochondrial biogenesis is critical for prediction of the late fate of cells. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/263152v3_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@122afb8org.highwire.dtl.DTLVardef@3e87c6org.highwire.dtl.DTLVardef@143fa24org.highwire.dtl.DTLVardef@1db5d6a_HPS_FORMAT_FIGEXP M_FIG C_FIG

The compound eyes of Drosophila are widely used to gain valuable insights into genetics, developmental biology, cell biology, disease biology, and gene regulation. Various parameters, such as eye size, pigmentation loss, formation of necrotic patches, and disorientation, fusion or disruption of ommatidial arrays are commonly assessed to evaluate eye development and degeneration. In this study, we developed an improved optical alignment imaging technique named "Low Angle Ring Illumination Stereomicroscopy" (LARIS), which provides high-contrast images of the Drosophila compound eye. By capturing images of the same eye using different optical alignments of the stereomicroscope, we achieved the highest resolution with minimal reflection through the LARIS method. The images captured using LARIS clearly show ommatidial fusion, disorientation, and pigmentation loss in the Drosophila eye compared to those obtained with conventional imaging method. We believe that LARIS will open new avenues for improved imaging of the compound eyes of Drosophila and other insects.

Ovarian somatic tissues around oocytes are essential for oogenesis in many metazoans. The functional ovarian somatic tissues sometimes have structural properties specific to their roles. In contrast, there is no evidence of such structural modularity of the follicle epithelium in Myriapoda, suggesting that myriapod ovarian soma may not participate in oogenesis. The ultrastructural nature of follicle cells also supports solitary oogenesis in most myriapods investigated. In contrast, here, we report two structurally and developmentally distinct domains of the follicle epithelium in the Japanese pill millipede, Hyleoglomeris japonica. The follicle epithelium of H. japonica has a thick cell mass on the apex of the follicle. These thick cells contain the rich rough endoplasmic reticulum, mitochondria, and Goldi bodies and possess many microvilli, indicating synthetic/secretory activities, and become thicker along with the oogenetic progress. Another region of epithelium does not exhibit these features. These results show the structural and functional modularity specific to some functions of the follicle epithelium of H. japonica. Therefore, the follicle epithelium of Myriapoda is divided into 3 types: physiologically functional uniformly, nonfunctional only, and those with both. We suggest the need to reconsider the nature and roles of ovarian somatic tissues of Myriapoda and Arthropoda.

Activation-induced cytidine deaminase (AID) somatically hypermutates the immunoglobulin heavy chain variable region (IGHV) gene to create the antibody diversity required to resist infections. This hypermutational process involves many pathways including transcription, DNA structural change and repair. While many of the proteins involved have been identified, their relative abundance, organization and regulation have not been resolved and additional factors and pathways need to be identified. To identify the proteome occupying IGHV, we have utilized dCas9-APEX targeted by guide RNAs to biotinylate and enrich the proteins associated with the mutating V region chromatin in the Ramos human B cell line and compared them to the non-mutating downstream constant region (C) chromatin. We identified hundreds of proteins specifically enriched on the V or C region. We confirmed the functionality of selected factors by examining the changes in the V region-specific proteome after inhibiting transcriptional elongation and somatic mutation with the Dot1L inhibitor EPZ004777. SummaryLocus-specific proteomics using dCas9-APEX identifies new aspects of the chromatin context involved in V region somatic hypermutation (SHM) in the human Ramos B cell line. An inhibitor of Dot1L which participates in SHM is used to identify functional SHM-related factors.

Centromere chromatin has been implicated in silencing the mating type (MAT) locus, which resides within a chromosomal region occupied by OpCse4, the CENP-A homolog, in the Flip-flop type of Mating Type Switching (MTS) in the methylotrophic yeast Ogataea polymorpha. In this study, we investigated the role of centromere chromatin in the recombination event between MAT-adjacent inverted repeats (IRs) that drives MTS. Our results demonstrate that the position of the centromere-proximal IR is critical for efficient recombination between the IRs. Mutants lacking functional OpCse4 or its chaperone OpScm3 exhibit MTS-deficient phenotypes, supporting an active role for centromere chromatin in facilitating MTS. Additionally, we identified OpRad6 and OpBre1 as essential for MTS, and found that deletion of the C-terminal acidic tail of OpRad6 alone is sufficient to disrupt switching. This suggests that monoubiquitylated histone H2B may contribute to MTS either directly or indirectly. Collectively, our findings highlight a direct and functional involvement of centromere chromatin in promoting Flip-flop type MTS in O. polymorpha.

Micronuclei (MN) are membrane-enclosed chromatin bodies and hallmarks of genome instability. Here, we report that importin , a key nuclear transport factor, is highly concentrated in a distinct subset of MN in cultured human cancer cells. This selective localization is not governed by classical nuclear transport pathways. Live-cell photobleaching revealed remarkably reduced mobility of importin between MN and cytoplasm. In addition, the subset of importin -positive MN exhibited collapsed nuclear envelopes and compromised barrier functions. Importin was also enriched in euchromatin regions, where it colocalized with chromatin-regulating molecules. Importantly, importin and DNA repair/sensing molecules such as RAD51, RPA2, and cGAS showed mutually exclusive localization in MN, indicating that MN comprise distinct internal environments. These findings identify importin as a molecular marker of the restricted MN state, representing a previously unrecognized microenvironment distinct from subsets characterized by conventional molecular markers of disrupted MN. This framework provides new insights into how MN heterogeneity underlies genome instability and immune evasion during cancer progression. Summary statementAccumulation of importin in micronuclei, followed by modulation of the microenvironment of the micronuclei, suggests a non-canonical function of importin in genomic instability and cancer development.

Histone acetyltransferase Gcn5 plays an important role in transcription activation, DNA replication-coupled nucleosome assembly and nucleotide excision repair (NER). However, its functions on the heterochromatin are unexplored. Here, we find that removal of Gcn5 leads to more condensed heterochromatin structure, as revealed by topology analysis of HML circles. Importantly, the altered heterochromatin structure is restored by re-expression of Gcn5 in the gcn5{Delta} cells. As a result of the more compact heterochromatin, gene silencing at the HML locus is increased and NER efficiency at HML is impaired in the absence of Gcn5. Interestingly, while the association of SIR complex with HML is enhanced in cells lacking Gcn5, the altered compaction of HML heterochromatin is also observed due to the deletion of Gcn5 from Sir- cells. These findings reveal a role of Gcn5 in the regulation of heterochromatin structure, gene silencing and NER efficiency at the heterochromatic HML locus in yeast.

During mammalian oocyte meiosis, spindle migration and asymmetric cytokinesis are unique steps for the successful polar body extrusion. The asymmetry defects of oocytes will lead to the failure of fertilization and embryo implantation. In present study we reported that an actin nucleating factor formin-like 2 (FMNL2) played critical roles in the regulation of spindle migration and organelle distribution. Our results showed that FMNL2 mainly localized at the oocyte cortex and periphery of spindle. Depletion of FMNL2 led to the failure of polar body extrusion and large polar bodies in oocytes. Live-cell imaging revealed that the spindle failed to migrate to the oocyte cortex, which caused polar body formation defects, and this might be due to the decreased polymerization of cytoplasmic actin by FMNL2 depletion. Furthermore, mass spectrometry analysis indicated that FMNL2 was associated with mitochondria and endoplasmic reticulum-related proteins, and FMNL2 depletion disrupted the function and distribution of mitochondria and endoplasmic reticulum, showing with decreased mitochondrial membrane potential and the occurrence of endoplasmic reticulum stress. Microinjecting Fmnl2-EGFP mRNA into FMNL2-depleted oocytes significantly rescued these defects. Thus, our results indicate that FMNL2 is essential for the actin assembly, which further involves into meiotic spindle migration and ER/mitochondria functions in mouse oocytes.