Genes to Cells

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.

Nuclear structure and nucleocytoplasmic interaction are closely linked to the regulation of cellular and higher-order biological processes. An alternative pathway for nucleocytoplasmic transport involving perinuclear vesicle formation and release, termed nuclear envelope budding (NEB), has been observed in diverse species and cell types. NEB-like events have also been reported in mammalian zygotes, including mice. However, their molecular basis remains unclear. Recent results suggest that protein kinase C (PKC) signaling regulates perinuclear vesicle biogenesis during NEB. While multiple PKC isoforms are expressed in mouse oocytes, their functions in zygotes are not fully understood. We investigated the effects of pharmacological PKC inhibition on zygotic development and NEB-like perinuclear vesicle formation. Our results suggest that NEB-like events in mouse zygotes may involve PKC-dependent mechanisms and that PKC activity might be critical for the 1-to 2-cell transition.

Heterochromatin is a highly condensed chromatin structure that represses gene expression. In eukaryotes, maintenance of the heterochromatin structure during cell proliferation is essential for guaranteeing cell identity. However, how heterochromatin is maintained and transmitted to the daughter cells remains largely unknown. In this study, we constructed a reporter system to study the maintenance of heterochromatin in the subtelomeric region of the fission yeast, Schizosaccharomyces pombe. We demonstrated that once subtelomeric heterochromatin was established, it tended to be maintained as a metastable structure through cell proliferation. Using this system, we screened an S. pombe genome-wide gene deletion library and identified 57 factors required for the maintenance of subtelomeric heterochromatin. We focused on Mrc1Claspin, a mediator of DNA replication checkpoint. We found that Mrc1 maintains heterochromatin structure not only at the subtelomeres but also at other heterochromatic loci, such as the pericentromeres and mating-type regions. Furthermore, we showed that Mrc1 facilitates the hypoacetylation state of histone H3K14 by recruiting the Snf2/Hdac-containing Repressor Complex (SHREC), via physical interaction. In addition, depletion of Mst2, an H3K14 acetyltransferase, restored heterochromatin integrity in mrc1 mutants. This is the first report to show a link between DNA replication factors and H3K14 deacetylation in heterochromatin.

The bacterial condensin MukB facilitates proper chromosome segregation in Escherichia coli. A portion of the MukB proteins localize at a specific chromosome region, binding to DNA in a non-sequence-specific manner. However, it is unclear how MukB localizes at a particular site without sequence specificity. Like other structural maintenance of chromosome (SMC) proteins, MukB topologically loads onto DNA, and It has an intrinsic property of preferential topological loading onto the single-stranded DNA (ssDNA). We consider it crucial for the localization of a specific region. To investigate the property of MukB, we attempted to identify positively charged amino acid residues responsible for ssDNA binding. We created a series of mutated MukB proteins in which a single positively charged amino acid was replaced with a negatively charged one. The results showed that some substitutions located on the inner surface of the MukB head domain impacted ssDNA-binding activity, leading to deficiencies in cell growth and nucleoid segregation. The efficiency of topological loading onto ssDNA was also decreased when the positive charges were replaced with negative ones. These amino acid residues align with and bind to ssDNA when the MukB dimer secures ssDNA within its ring, thereby likely strengthening the ssDNA-binding ability of MukB.

Heterochromatin marks such as H3K9me3 undergoes global erasure and re-establishment after fertilization, and the proper reprogramming of H3K9me3 is essential for early development. Despite the widely conserved dynamics of heterochromatin reprogramming in invertebrates and non-mammalian vertebrates, previous studies have shown that the underlying mechanisms may differ between species. In this study, we investigated the molecular mechanism of H3K9me3 dynamics in medaka (Japanese killifish, Oryzias latipes) as a non-mammalian vertebrate model, and found that rapid cell cycle during the cleavage stages causes DNA replication-dependent passive erasure of H3K9me3. We also found that cell cycle slowing, toward the mid-blastula transition, permits increasing nuclear accumulation of H3K9me3 histone methyltransferase Setdb1, leading to the onset of H3K9me3 re-accumulation. We further demonstrated that cell cycle length in early development regulates H3K9me3 reprogramming in zebrafish and Xenopus laevis as well. Together with the previous studies in invertebrates, we propose that the cell cycle length-dependent mechanism for both global erasure and re-accumulation of H3K9me3 is widely conserved among rapid-cleavage species of non-mammalian vertebrates and invertebrates such as Drosophila, C. elegans and teleost fish.

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.

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.

Primordial germ cells (PGCs) are the precursors of sperms and oocytes. Proper development of PGCs is crucial for the survival of the species. In many organisms, factors responsible for PGC development are synthesized during early oogenesis and assembled into the germ plasm. During early embryonic development, germ plasm is inherited by a few cells, leading to the formation of PGCs. While germline development has been extensively studied, how components of the germ plasm regulate PGC development is not fully understood. Here, we report that Dzip1 is dynamically expressed in vertebrate germline and is a novel component of the germ plasm in Xenopus and zebrafish. Knockdown of Dzip1 impairs PGC development in Xenopus embryos. At the molecular level, Dzip1 physically interacts with Dazl, an evolutionarily conserved RNA-binding protein that plays a multifaced role during germline development. We further showed that the sequence between amino acid residues 282 and 550 of Dzip1 is responsible for binding to Dazl. Disruption of the binding between Dzip1 and Dazl leads to defective PGC development. Taken together, our results presented here demonstrate that Dzip1 is dynamically expressed in the vertebrate germline and plays a novel function during Xenopus PGC development.

Organisms use a specialized cell division called meiosis for the creation of haploid gametes. Multiple carefully orchestrated steps must occur at specific times and places for meiosis to be successful, including chromosome pairing, meiotic entry, recombination, synapsis, and two rounds of chromosome segregation. The regulation and molecular mechanisms for many of the steps of meiosis have not been fully elucidated. During synapsis, the synaptonemal complex (SC) builds along the entire lengths of the homologs to maintain the pairing of the homologs and promote the formation of the crossovers that help ensure proper segregation of homologs at the meiosis I division in many organisms. The SC is a large tripartite structure that is believed to function as a biomolecular condensate. To attempt to identify proteins that interact with SC components during female meiosis in Drosophila melanogaster, a protein of the lateral element, C(2)M, and a protein of the central element, Cona, were tagged with the APEX2 enzyme, which can biotinylate nearby proteins under the appropriate conditions. Under biotinylating promoting conditions, biotin labeled proteins were observed to be associated with the SC by immunofluorescence. Biotinylated proteins were isolated for mass spectrometry analysis, and multiple proteins were found to be enriched compared to control samples. RNAi knockdown lines targeting a subset of enriched proteins were examined for phenotypes in early Drosophila female meiosis. RNAi knockdown of Cpsf5, an mRNA cleavage factor, caused delayed or defective SC formation, as well as additional meiotic defects, indicating a role for maturation of mRNA in regulating processes of female meiosis. These results support proximity labeling as a strategy for identifying additional meiotic proteins.

The mechanism by which transcriptional machinery is recruited to enhancers and promoters to regulate gene expression is one of the most challenging and extensively studied questions in modern biology. Here, we ask if inter-allelic interactions between two homologous alleles can affect gene regulation. Using MS2- and PP7-based, allele-specific live imaging assay, we visualized de novo transcription of a reporter gene in hemizygous and homozygous Drosophila embryos. Surprisingly, each homozygous allele produced fewer RNAs than the hemizygous allele, suggesting the possibility of allelic competition in homozygotes. Moreover, the MS2-yellow reporter gene showed reduced transcriptional activity when a partial transcription unit (enhancer or promoter only) was in the homologous position. We propose that the transcriptional machinery that binds to both the enhancer and promoter region, such as RNA Pol II or preinitiation complexes, may be responsible for the allelic competition. To support this idea, we showed that the homologous alleles did not interfere with each other in earlier nuclear cycles when Pol II is in excess, while the degree of interference gradually increased in nuclear cycle 14. Such allelic competition was observed for endogenous snail as well. Our study provides new insights into the role of 3D inter-allelic interactions in gene regulation.

Spermatogenesis requires high regulation of germ cell morphology. The spermatogonia regulates its differentiation state by its own migration. The male germ cells differentiate and mature with the formation of syncytia, failure of forming the appropriate syncytia results in the arrest of spermatogenesis at the spermatocyte stage. However, the detailed molecular mechanisms of male germ cell morphological regulation are unknown. Here, we found that EXOC1 is important for the pseudopod formation of spermatogonia and spermatocyte syncytia in mice. We found that while EXOC1 contributes to the inactivation of Rac1 in the pseudopod formation of spermatogonia, in spermatocyte syncytium formation, EXOC1 and SNAP23 cooperate with STX2. Our results showed that EXOC1 functions in concert with various cell morphology regulators in spermatogenesis. Since EXOC1 is known to bind to several cell morphogenesis factors, this study is expected to be the starting point for the discovery of many morphological regulators of male germ cells.

There are two meiotic cohesin pathways that regulate synaptonemal complex (SC) assembly in Drosophila. We previously proposed that C(2)M, which is required for SC assembly, is the only meiosis-specific component of a complex that includes Stromalin (SA), Nipped-B, SMC1 and SMC3. This model also predicts that specific residues within the C-terminus and N-terminus of C(2)M should interact with SMC1 and SMC3 to form a ring structure that may regulate the ability of C(2)M to facilitate SC assembly. Through mutant analysis, our results show several residues known to interact with SMC1 or SMC3 are critical for SC formation, suggesting that C(2)M may require a ring structure to perform meiosis-specific functions such as the formation of SC. We also show that SA colocalizes with and depends on C(2)M. However, the dynamics of C(2)M differ from SA and the SMCs in a way that suggests C(2)M regulates the dynamics and chromosome loading of the other cohesin proteins SMC1 and SA. Consistent with this conclusion, our results suggest that C(2)M can promote chromosome localization of the other cohesin components, and can induce SC assembly when ectopically expressed in germline mitotic cells.

In lepidopteran insects, sperm polymorphism is a remarkable feature, in which males exhibit two different types of sperms. Both sperm morphs are essential for fertilization as eupyrene (nucleate) sperm carries DNA and fertilizes the egg, while apyrene (anucleate) sperm is necessary for transporting eupyrene sperm into females. To date, the functional genetic study on dichotomous spermatogenesis has been limited. It is known that, in the model species including mice, worms, and flies, the components in piRNA biogenesis pathway play an important role in gonad development. In this study, we characterize BmHen1 as a new critical component involved in the regulation of eupyrene sperm development in B. mori. We generated the loss-of-function mutant of BmHen1 ({Delta}BmHen1) through CRISPR/Cas9-based gene editing, and found that it is both female- and male-sterile.{Delta} BmHen1 females lay significantly fewer eggs than wild-type, which display morphological defects. Fluorescence staining assays show that the{Delta} BmHen1 eupyrene sperms exhibit severe defects in nuclei formation, while its apyrene sperms are normal. We then constructed the loss-of-function mutants of Siwi and BmAgo3 ({Delta}Siwi and{Delta} BmAgo3) through CRISPR/Cas9-based gene editing, which encode PIWI proteins acting as the core elements in piRNA biogenesis, and explored whether they might be involved in spermatogenesis. To our surprise,{Delta} Siwi and{Delta} BmAgo3 mutants develop normal male reproduction system, indicating that they dont participate in sperm development. As the activity of BmHen1 depends on BmPnldc1 during piRNA biogenesis, and{Delta} BmHen1 and{Delta} BmPnldc1 mutants display similar defects in sperm development, we performed RNA sequencing analysis to look for the genes that might be co-regulated by BmHen1 and BmPnldc1. Our results indicate that the defects in{Delta} BmHen1 and{Delta} BmPnldc1 eupyrene sperms could be attributed to dysregulated genes involved in energy metabolism and cell differentiation. Furthermore, we found that the piRNA biogenesis is inhibited in{Delta} BmHen1 and{Delta} BmPnldc1 sperm bundles, whereas the transposon activity was induced. Taken together, our findings suggest that BmHen1 is a new crucial component regulating eupyrene sperm development in B. mori, whereas the PIWI proteins Siwi and BmAgo3 are not involved in this process. Our results may provide a potential gene target for genetic modification of sterility in B. mori.

The histone variant H2AX phosphorylated on serine 139, named {gamma}-H2AX, is a canonical DNA double-strand breaks marker. During mammalian meiotic prophase I, {gamma}-H2AX participates in meiotic recombination, meiotic sex chromosome inactivation and meiotic silencing of unsynapsed chromatin. In this study, we have analyzed the distribution of {gamma}-H2AX during male mouse meiosis by immunofluorescence on spread and squashed spermatocytes. We have found that {gamma}-H2AX locates at the inner kinetochore plate of meiotic kinetochores in both meiotic divisions. Therefore our results, for the first time, uncover a novel role for {gamma}-H2AX at mammalian meiotic kinetochores.

Knockout analysis is a common tool to reveal transcription factor (TF) functions. However, such a reverse genetic analysis based on observed phenotype changes in mutant cell may lead to a misunderstanding of TF wild-type functions. Here, a model was proposed, in which the knockout-observed TF-target regulatory relationships might only occur in mutant cell, and they do not reflect TF normal functions in wild-type cell. Actually, the knockout of one TF might release another TF which is the protein-protein interaction partner of the deleted TF. The free TF could bind its new target genes and cause their significant expression changes. These seemingly TF knockout affected genes are thus not directly regulated by the deleted TF, but are gain-of-regulated genes of the latter TF in mutant cell. Based on this model, multiple sources of genome-wide data were used to identify 20 such TF pairs, and one pair was validated using other independent data. TF wild-type regulatory genes are not associated with their gain-of-regulated genes. My findings revealed TF-target relationships complicated by TF knockout analysis.

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.

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.

DNA topoisomerase II (Top2) is a nuclear protein that resolves DNA topological problems and plays critical roles in multiple nuclear processes. Human cells have two Top2 proteins, Top2A and Top2B, that are localized in both the nucleoplasm and nucleolus. Previously, ATP depletion was shown to augment the nucleolar localization of Top2B, but the molecular details of subnuclear distributions, particularly of Top2A, remained to be fully elucidated in relation to the status of cellular ATP. Here, we analyzed the nuclear dynamics of human Top2A and Top2B in ATP-depleted cells. Both proteins rapidly translocated from the nucleoplasm to the nucleolus in response to ATP depletion. FRAP analysis demonstrated that they were highly mobile in the nucleoplasm and nucleolus. The nucleolar retention of both proteins was sensitive to the RNA polymerase I inhibitor BMH-21, and the Top2 proteins in the nucleolus were immediately dispersed into the nucleoplasm by BMH-21. Under ATP-depleted conditions, the Top2 poison etoposide was less effective, indicating the therapeutic relevance of Top2 subnuclear distributions. These results give novel insights into the subnuclear dynamics of Top2 in relation to cellular ATP levels and also provide discussions about its possible mechanisms and biological significance.

Aneuploidy is caused by chromosomal missegregation and is frequently observed in cancers and hematological diseases. Pericentromeric heterochromatin formation is essential for proper chromosome segregation; however, it is unclear how heterochromatin is targeted to pericentromeres. In this study, we investigated the involvement of two homologous zinc-finger proteins (ZNF518A and ZNF518B) in heterochromatin formation at pericentromeres by determining the cellular localization of ZNF518, centromeric proteins such as CENP-A and CENP-B, and heterochromatin factors, including HP1 and histone H3K9 trimethylation. The results showed that various segments in ZNF518 interact with CENP-B, HP1, and G9a histone H3K9 methyltransferase. In conclusion, we identified a novel mechanism underlying pericentromeric heterochromatin formation mediated by ZNF518.

Formin1 (FMN1), a member of the non-diaphanous formin family, is essential for development and neuronal function. Unlike diaphanous-related formins, FMN1 is not subject to canonical autoinhibition through the DID and DAD domains, nor is it activated by Rho GTPase binding. Recent studies suggest that formins also play roles in the nucleus, influencing DNA damage response and transcriptional regulation. However, the mechanisms regulating nuclear formins particularly non-diaphanous ones like FMN1 remain poorly understood. Our previous research identified the interaction between FMN1 and FNBP4, prompting further investigation into its functional role in regulating actin dynamics. Results reveal that FNBP4 inhibits FMN1-mediated actin assembly in vitro. It is shown that FNBP4 prevents FMN1 from displacing the capping protein CapZ at the growing barbed end of actin filaments. Additionally, FNBP4 inhibits FMN1s bundling activity in a concentration-dependent manner. Further analysis indicates that FNBP4 interacts with the FH1 domain and the interdomain connector between the FH1 and FH2 domains, creating spatial constraints on the FH2 domain. We propose that FNBP4 acts as a stationary inhibitor of FMN1. In addition, we identify a monopartite nuclear localization signal (NLS) in FNBP4, and subcellular localization studies show that FNBP4 colocalizes with FMN1. This study provides new insights into the regulatory role of FNBP4 in modulating FMN1-mediated actin dynamics, suggesting that FNBP4 may function as a nuclear inhibitor of actin polymerization and shedding light on regulatory mechanisms specific to non-diaphanous formins.