Polycomb directs timely activation of germline genes in spermatogenesis.

During spermatogenesis, a large number of germline genes essential for male fertility are coordinately activated. However, it remains unknown how timely activation of this group of germline genes is accomplished. Here we show that Polycomb-repressive complex 1 (PRC1) directs timely activation of germline genes during spermatogenesis. Inactivation of PRC1 in male germ cells results in the gradual loss of a stem cell population and severe differentiation defects, leading to male infertility. In the stem cell population, RNF2, the dominant catalytic subunit of PRC1, activates transcription of Sall4, which codes for a transcription factor essential for subsequent spermatogenic differentiation. Furthermore, RNF2 and SALL4 together occupy transcription start sites of germline genes in the stem cell population. Once differentiation commences, these germline genes are activated to enable the progression of spermatogenesis. Our study identifies a novel mechanism by which Polycomb directs the developmental process by activating a group of lineage-specific genes.

The joy of the 11th International Fission Yeast Meeting in Hiroshima (POMBE2023 Hiroshima) after a long wait due to the COVID-19 pandemic.

The 11th International Fission Yeast Meeting took place at Astel Plaza in Hiroshima, Japan, from May 28th to June 2nd, 2023. This highly anticipated gathering, originally scheduled for May 2021, had been postponed for 2 years due to the COVID-19 pandemic. Researchers from 21 countries, including 211 overseas and 157 domestic participants (overall gender ratio is roughly 60% male vs. 40% female), eagerly awaited the opportunity to meet in person, as virtual interactions had been the only means of communication during this challenging period. The meeting featured four kick-off special lectures, 101 regular talks, and 152 poster presentations. Additionally, a discussion session on upfront frontier research in fission yeast provided an interactive platform for both speakers and attendees. Throughout the event, participants shared cutting-edge knowledge, celebrated significant research findings, and relished the invaluable experience of an in-person meeting. The vibrant and friendly atmosphere, characteristic of this esteemed international conference, fostered collaboration and reinforced the significance of studying this exceptional model organism. Undoubtedly, the outcomes of this meeting will greatly contribute to our understanding of complex biological systems, not only in fission yeast but also in general eukaryotes.

Complementation of fission yeast kinesin-5/Cut7 with human Eg5 provides a versatile platform for screening of anticancer compounds.

Kinesin-5 family proteins are essential for bipolar spindle assembly to ensure mitotic fidelity. Here, we demonstrate evolutionary functional conservation of kinesin-5 between human and fission yeast. Human Eg5 expressed in the nucleus replaces fission yeast counterpart Cut7. Intriguingly, Eg5 overproduction results in cytotoxicity. This phenotype provides a useful platform for the development of novel kinesin-5 inhibitors as anticancer drugs.

KIFC1 regulates ZWINT to promote tumor progression and spheroid formation in colorectal cancer.

Colorectal cancer (CRC) is the second leading cause of cancer-related mortality worldwide. Kinesin Family Member C1 (KIFC1) has been proposed as a promising therapeutic target due to its pivotal role in centrosome clustering to mediate cancer cell progression. This study aimed to analyze the expression and biological function of KIFC1 in CRC. Immunohistochemically, 67 (52%) of 129 CRC cases were positive for KIFC1 and statistically associated with poorer overall survival. KIFC1 small interfering RNA (siRNA)-transfected cells demonstrated lower cell proliferation as compared to the negative control cells. A specific KIFC1 inhibitor, kolavenic acid analog (KAA) drastically inhibited CRC cell proliferation. Microarray analysis revealed that KAA-treated CRC cells presented reduced ZW10 interacting kinetochore protein (ZWINT) expression as compared to control cells. Immunohistochemical analysis demonstrated that 61 (47%) of 129 CRC cases were positive for ZWINT and ZWINT expression was significantly correlated with KIFC1 expression. ZWINT-positive cases exhibited significantly worse overall survival. KIFC1 siRNA-transfected cells showed reduced ZWINT expression while ZWINT siRNA-transfected cells decreased cell proliferation. Both KIFC1 and ZWINT knockdown cells attenuated spheroid formation ability. This study provides new insights into KIFC1 regulating ZWINT in CRC progression and its potential as a therapeutic target.

Polycomb protein SCML2 facilitates H3K27me3 to establish bivalent domains in the male germline.

Repressive H3K27me3 and active H3K4me2/3 together form bivalent chromatin domains, molecular hallmarks of developmental potential. In the male germline, these domains are thought to persist into sperm to establish totipotency in the next generation. However, it remains unknown how H3K27me3 is established on specific targets in the male germline. Here, we demonstrate that a germline-specific Polycomb protein, SCML2, binds to H3K4me2/3-rich hypomethylated promoters in undifferentiated spermatogonia to facilitate H3K27me3. Thus, SCML2 establishes bivalent domains in the male germline of mice. SCML2 regulates two major classes of bivalent domains: Class I domains are established on developmental regulator genes that are silent throughout spermatogenesis, while class II domains are established on somatic genes silenced during late spermatogenesis. We propose that SCML2-dependent H3K27me3 in the male germline prepares the expression of developmental regulator and somatic genes in embryonic development.

The Putative RNA-Binding Protein Dri1 Promotes the Loading of Kinesin-14/Klp2 to the Mitotic Spindle and Is Sequestered into Heat-Induced Protein Aggregates in Fission Yeast.

Cells form a bipolar spindle during mitosis to ensure accurate chromosome segregation. Proper spindle architecture is established by a set of kinesin motors and microtubule-associated proteins. In most eukaryotes, kinesin-5 motors are essential for this process, and genetic or chemical inhibition of their activity leads to the emergence of monopolar spindles and cell death. However, these deficiencies can be rescued by simultaneous inactivation of kinesin-14 motors, as they counteract kinesin-5. We conducted detailed genetic analyses in fission yeast to understand the mechanisms driving spindle assembly in the absence of kinesin-5. Here, we show that deletion of the dri1 gene, which encodes a putative RNA-binding protein, can rescue temperature sensitivity caused by cut7-22, a fission yeast kinesin-5 mutant. Interestingly, kinesin-14/Klp2 levels on the spindles in the cut7 mutants were significantly reduced by the dri1 deletion, although the total levels of Klp2 and the stability of spindle microtubules remained unaffected. Moreover, RNA-binding motifs of Dri1 are essential for its cytoplasmic localization and function. We have also found that a portion of Dri1 is spatially and functionally sequestered by chaperone-based protein aggregates upon mild heat stress and limits cell division at high temperatures. We propose that Dri1 might be involved in post-transcriptional regulation through its RNA-binding ability to promote the loading of Klp2 on the spindle microtubules.

Two spatially distinct kinesin-14 proteins, Pkl1 and Klp2, generate collaborative inward forces against kinesin-5 Cut7 in S. pombe.

Kinesin motors play central roles in bipolar spindle assembly. In many eukaryotes, spindle pole separation is driven by kinesin-5, which generates outward force. This outward force is balanced by antagonistic inward force elicited by kinesin-14 and/or dynein. In fission yeast, two kinesin-14 proteins, Pkl1 and Klp2, play an opposing role against the kinesin-5 motor protein Cut7. However, how the two kinesin-14 proteins coordinate individual activities remains elusive. Here, we show that although deletion of either pkl1 or klp2 rescues temperature-sensitive cut7 mutants, deletion of only pkl1 can bypass the lethality caused by cut7 deletion. Pkl1 is tethered to the spindle pole body, whereas Klp2 is localized along the spindle microtubule. Forced targeting of Klp2 to the spindle pole body, however, compensates for Pkl1 functions, indicating that cellular localizations, rather than individual motor specificities, differentiate between the two kinesin-14 proteins. Interestingly, human kinesin-14 (KIFC1 or HSET) can replace either Pkl1 or Klp2. Moreover, overproduction of HSET induces monopolar spindles, reminiscent of the phenotype of Cut7 inactivation. Taken together, this study has uncovered the biological mechanism whereby two different Kinesin-14 motor proteins exert their antagonistic roles against kinesin-5 in a spatially distinct manner.

Genomic integration of Wnt/ß-catenin and BMP/Smad1 signaling coordinates foregut and hindgut transcriptional programs.

Digestive system development is orchestrated by combinatorial signaling interactions between endoderm and mesoderm, but how these signals are interpreted in the genome is poorly understood. Here we identified the transcriptomes of Xenopus foregut and hindgut progenitors, which are conserved with mammals. Using RNA-seq and ChIP-seq we show that BMP/Smad1 regulates dorsal-ventral gene expression in both the endoderm and mesoderm, whereas Wnt/ß-catenin acts as a genome-wide toggle between foregut and hindgut programs. Unexpectedly, ß-catenin and Smad1 binding were associated with both transcriptional activation and repression, with Wnt-repressed genes often lacking canonical Tcf DNA binding motifs, suggesting a novel mode of direct repression. Combinatorial Wnt and BMP signaling was mediated by Smad1 and ß-catenin co-occupying hundreds of cis-regulatory DNA elements, and by a crosstalk whereby Wnt negatively regulates BMP ligand expression in the foregut. These results extend our understanding of gastrointestinal organogenesis and of how Wnt and BMP might coordinate genomic responses in other contexts.

Kolavenic acid analog restores growth in HSET-overproducing fission yeast cells and multipolar mitosis in MDA-MB-231 human cells.

Although cancer cells often harbor supernumerary centrosomes, they form pseudo-bipolar spindles via centrosome clustering, instead of lethal multipolar spindles, and thus avoid cell death. Kinesin-14 HSET/KIFC1 is a crucial protein involved in centrosome clustering. Accordingly, a compound that targets HSET could potentially inhibit cancer cell proliferation in a targeted manner. Here, we report three natural compounds derived from Solidago altissima that restored the growth of fission yeast cells exhibiting lethal HSET overproduction (positive screening), namely solidagonic acid (SA) (1), kolavenic acid analog (KAA: a stereo isomer at C-9 and C-10 of 6ß-tigloyloxykolavenic acid) (2), and kolavenic acid (KA) (3). All three compounds suppressed fission yeast cell death and enabled reversion of the mitotic spindles from a monopolar to bipolar morphology. Compound 2, which exerted the strongest activity against HSET-overproducing yeast cells, also inhibited centrosome clustering in MDA-MB-231 human breast adenocarcinoma cells, which contained large numbers of supernumerary centrosomes. These natural compounds may be useful as bioprobes in studies of HSET function. Moreover, compound 2 is a prime contender in the development of novel agents for cancer treatment.

Dynamic reorganization of open chromatin underlies diverse transcriptomes during spermatogenesis.

During spermatogenesis, germ cells undergo massive cellular reconstruction and dynamic chromatin remodeling to facilitate highly diverse transcriptomes, which are required for the production of functional sperm. However, it remains unknown how germline chromatin is organized to promote the dynamic, complex transcriptomes of spermatogenesis. Here, using ATAC-seq, we establish the varied landscape of open chromatin during spermatogenesis. We identify the reorganization of accessible chromatin in intergenic and intronic regions during the mitosis-to-meiosis transition. During the transition, mitotic-type open chromatin is closed while the de novo formation of meiotic-type open chromatin takes place. Contrastingly, differentiation processes such as spermatogonial differentiation and the meiosis-to-postmeiosis transition involve chromatin closure without the de novo formation of accessible chromatin. In spermiogenesis, the germline-specific Polycomb protein SCML2 promotes the closure of open chromatin at autosomes for gene suppression. Paradoxically, we identify the massive de novo formation of accessible chromatin when the sex chromosomes undergo meiotic sex chromosome inactivation, and this is also mediated by SCML2. These results reveal meiotic sex chromosome inactivation as an active process for chromatin organization. Together, our results unravel the genome-wide, dynamic reorganization of open chromatin and reveal mechanisms that underlie diverse transcriptomes during spermatogenesis.

Generation of temperature sensitive mutations with error-prone PCR in a gene encoding a component of the spindle pole body in fission yeast.

Temperature-sensitive (ts) mutants provide powerful tools for investigation of cellular functions of essential genes. We report here asimple procedure to generate ts mutations using error-prone PCR within pcp1 that encodes aspindle pole body (SPB) component in Schizosaccharomyces pombe. This manipulation is not restricted to pcp1, and can be suited to any essential genes involved in other processes.

Two XMAP215/TOG Microtubule Polymerases, Alp14 and Dis1, Play Non-Exchangeable, Distinct Roles in Microtubule Organisation in Fission Yeast.

Proper bipolar spindle assembly underlies accurate chromosome segregation. A cohort of microtubule-associated proteins orchestrates spindle microtubule formation in a spatiotemporally coordinated manner. Among them, the conserved XMAP215/TOG family of microtubule polymerase plays a central role in spindle assembly. In fission yeast, two XMAP215/TOG members, Alp14 and Dis1, share essential roles in cell viability; however how these two proteins functionally collaborate remains undetermined. Here we show the functional interplay and specification of Alp14 and Dis1. Creation of new mutant alleles of alp14, which display temperature sensitivity in the absence of Dis1, enabled us to conduct detailed analyses of a double mutant. We have found that simultaneous inactivation of Alp14 and Dis1 results in early mitotic arrest with very short, fragile spindles. Intriguingly, these cells often undergo spindle collapse, leading to a lethal "cut" phenotype. By implementing an artificial targeting system, we have shown that Alp14 and Dis1 are not functionally exchangeable and as such are not merely redundant paralogues. Interestingly, while Alp14 promotes microtubule nucleation, Dis1 does not. Our results uncover that the intrinsic specification, not the spatial regulation, between Alp14 and Dis1 underlies the collaborative actions of these two XMAP215/TOG members in mitotic progression, spindle integrity and genome stability.

An unconventional interaction between Dis1/TOG and Mal3/EB1 in fission yeast promotes the fidelity of chromosome segregation.

Dynamic microtubule plus-ends interact with various intracellular target regions such as the cell cortex and the kinetochore. Two conserved families of microtubule plus-end-tracking proteins, the XMAP215, ch-TOG or CKAP5 family and the end-binding 1 (EB1, also known as MAPRE1) family, play pivotal roles in regulating microtubule dynamics. Here, we study the functional interplay between fission yeast Dis1, a member of the XMAP215/TOG family, and Mal3, an EB1 protein. Using an in vitro microscopy assay, we find that purified Dis1 autonomously tracks growing microtubule ends and is a bona fide microtubule polymerase. Mal3 recruits additional Dis1 to microtubule ends, explaining the synergistic enhancement of microtubule dynamicity by these proteins. A non-canonical binding motif in Dis1 mediates the interaction with Mal3. X-ray crystallography shows that this new motif interacts in an unconventional configuration with the conserved hydrophobic cavity formed within the Mal3 C-terminal region that typically interacts with the canonical SXIP motif. Selectively perturbing the Mal3-Dis1 interaction in living cells demonstrates that it is important for accurate chromosome segregation. Whereas, in some metazoans, the interaction between EB1 and the XMAP215/TOG family members requires an additional binding partner, fission yeast relies on a direct interaction, indicating evolutionary plasticity of this critical interaction module.

Fission yeast cells overproducing HSET/KIFC1 provides a useful tool for identification and evaluation of human kinesin-14 inhibitors.

Many human cancer cells contain more than two centrosomes, yet these cancer cells can form pseudo-bipolar spindles through the mechanism, called centrosome clustering, and survive, instead of committing lethal multipolar mitoses. Kinesin-14/HSET, a minus end-directed motor, plays a crucial role in centrosome clustering. Accordingly, HSET is deemed to be a promising chemotherapeutic target to selectively kill cancer cells. Recently, three HSET inhibitors (AZ82, CW069 and SR31527) have been reported, but their specificity and efficacy have not been evaluated rigorously. This downside partly stems from the lack of robust systems for the assessment of these drugs. Yeasts and filamentous fungi provide not only powerful models for basic and applied biology but also versatile tools for drug discovery and evaluation. Here we show that these three inhibitors on their own are cytotoxic to fission yeast, suggesting that they have off-targets in vivo except for kinesin-14. Nonetheless, intriguingly, AZ82 can neutralize otherwise toxic overproduced HSET; this includes a substantial reduction in the percentage of HSET-driven abnormal mitotic cells and partial suppression of its lethality. SR31527 also displays modest neutralizing activity, while we do not detect such activity in CW069. As an experimental proof-of-principle study, we have treated HSET-overproducing fission yeast cells with extracts prepared from various plant species and found activities that rescue HSET-driven lethality in those from Chamaecyparis pisifera and Toxicodendron trichocarpum. This methodology of protein overproduction in fission yeast, therefore, provides a convenient, functional assay system by which to screen for not only selective human kinesin-14 inhibitors but also those against other molecules of interest.

Long G2 accumulates recombination intermediates and disturbs chromosome segregation at dysfunction telomere in Schizosaccharomyces pombe.

Protection of telomere (Pot1) is a single-stranded telomere binding protein which is essential for chromosome ends protection. Fission yeast Rqh1 is a member of RecQ helicases family which has essential roles in the maintenance of genomic stability and regulation of homologous recombination. Double mutant between fission yeast pot1Δ and rqh1 helicase dead (rqh1-hd) maintains telomere by homologous recombination. In pot1Δ rqh1-hd double mutant, recombination intermediates accumulate near telomere which disturb chromosome segregation and make cells sensitive to microtubule inhibitors thiabendazole (TBZ). Deletion of chk1(+) or mutation of its kinase domain shortens the G2 of pot1Δ rqh1-hd double mutant and suppresses both the accumulation of recombination intermediates and the TBZ sensitivity of that double mutant. In this study, we asked whether the long G2 is the reason for the TBZ sensitivity of pot1Δ rqh1-hd double mutant. We found that shortening the G2 of pot1Δ rqh1-hd double mutant by additional mutations of wee1 and mik1 or gain of function mutation of Cdc2 suppresses both the accumulation of recombination intermediates and the TBZ sensitivity of pot1Δ rqh1-hd double mutant. Our results suggest that long G2 of pot1Δ rqh1-hd double mutant may allow time for the accumulation of recombination intermediates which disturb chromosome segregation and make cells sensitive to TBZ.

The yeast chromatin remodeler Rsc1-RSC complex is required for transcriptional activation of autophagy-related genes and inhibition of the TORC1 pathway in response to nitrogen starvation.

The yeast RSC, an ATP-dependent chromatin-remodeling complex, is essential for mitotic and meiotic growth. There are two distinct isoforms of this complex defined by the presence of either Rsc1 or Rsc2; however, the functional differences between these complexes are unclear. Here we show that the RSC complex containing Rsc1, but not Rsc2, functions in autophagy induction. Rsc1 was required not only for full expression of ATG8 mRNA but also for maintenance of Atg8 protein stability. Interestingly, decreased autophagic activity and Atg8 protein stability in rsc1Δ cells, but not the defect in ATG8 mRNA expression, were partially suppressed by deletion of TOR1. In addition, we found that rsc1Δ impaired the binding between the Rho GTPase Rho1 and the TORC1-specific component Kog1, which is required for down-regulation of TORC1 activity. These results suggest that the Rsc1-containing RSC complex plays dual roles in the proper induction of autophagy: 1) the transcriptional activation of autophagy-related genes independent of the TORC1 pathway and 2) the inactivation of TORC1, possibly through enhancement of Rho1-Kog1 binding.

ZSCAN4-binding motif-TGCACAC is conserved and enriched in CA/TG microsatellites in both mouse and human genomes.

The Zinc finger and SCAN domain containing 4 (ZSCAN4) protein, expressed transiently in pluripotent stem cells, gametes, and early embryos, extends telomeres, enhances genome stability, and improves karyotypes in mouse embryonic stem (mES) cells. To gain insights into the mechanism of ZSCAN4 function, we identified genome-wide binding sites of endogenous ZSCAN4 protein using ChIP-seq technology in mouse and human ES cells, where the expression of endogenous ZSCAN4 was induced by treating cells with retinoic acids or by overexpressing DUX4. We revealed that both mouse and human ZSCAN4 bind to the TGCACAC motif located in CA/TG microsatellite repeats, which are known to form unstable left-handed duplexes called Z-DNA that can induce double-strand DNA breaks and mutations. These ZSCAN4 binding sites are mostly located in intergenic and intronic regions of the genomes. By generating ZSCAN4 knockout in human ES cells, we showed that ZSCAN4 does not seem to be involved in transcriptional regulation. We also found that ectopic expression of mouse ZSCAN4 enhances the suppression of chromatin at ZSCAN4-binding sites. These results together suggest that some of the ZSCAN4 functions are mediated by binding to the error-prone regions in mouse and human genomes.

Changes in the nuclear deposition of histone H2A variants during pre-implantation development in mice.

Histone H2A has several variants, and changes in chromatin composition associated with their replacement might involve chromatin structure remodeling. We examined the dynamics of the canonical histone H2A and its three variants, H2A.X, H2A.Z and macroH2A, in the mouse during oogenesis and pre-implantation development when genome remodeling occurs. Immunocytochemistry with specific antibodies revealed that, although H2A and all variants were deposited in the nuclei of full-grown oocytes, only histone H2A.X was abundant in the pronuclei of one-cell embryos after fertilization, in contrast with the low abundance of histone H2A and the absence of H2A.Z. The decline in H2A and the depletion of H2A.Z and macroH2A after fertilization were confirmed using Flag epitope-tagged H2A, H2A.Z and macroH2A transgenic mouse lines. Microinjection experiments with mRNA encoding the Flag-tagged proteins revealed a similar pattern of nuclear incorporation of the H2A variants. Fusion protein experiments using H2A, H2A.Z and macroH2A fused with the C-terminal 23 amino acids of H2A.X showed that the C-terminal amino acids of H2A.X function specifically to target this variant histone into chromatin in embryos after fertilization and that the absence of H2A.Z and macroH2A from the chromatin is required for normal development. These results suggest that global changes in the composition of histone H2A variants in chromatin play a role in genome remodeling after fertilization.

An atlas of gene regulatory networks for memory CD4 + T cells in youth and old age.

Aging profoundly affects immune-system function, promoting susceptibility to pathogens, cancers and chronic inflammation. We previously identified a population of IL-10-producing, T follicular helper-like cells (" Tfh10 "), linked to suppressed vaccine responses in aged mice. Here, we integrate single-cell ( sc )RNA-seq, scATAC-seq and genome-scale modeling to characterize Tfh10 - and the full CD4 + memory T cell ( CD4 + TM ) compartment - in young and old mice. We identified 13 CD4 + TM populations, which we validated through cross-comparison to prior scRNA-seq studies. We built gene regulatory networks ( GRNs ) that predict transcription-factor control of gene expression in each T-cell population and how these circuits change with age. Through integration with pan-cell aging atlases, we identified intercellular-signaling networks driving age-dependent changes in CD4 + TM. Our atlas of finely resolved CD4 + TM subsets, GRNs and cell-cell communication networks is a comprehensive resource of predicted regulatory mechanisms operative in memory T cells, presenting new opportunities to improve immune responses in the elderly.

PRC1 suppresses a female gene regulatory network to ensure testicular differentiation.

Gonadal sex determination and differentiation are controlled by somatic support cells of testes (Sertoli cells) and ovaries (granulosa cells). In testes, the epigenetic mechanism that maintains chromatin states responsible for suppressing female sexual differentiation remains unclear. Here, we show that Polycomb repressive complex 1 (PRC1) suppresses a female gene regulatory network in postnatal Sertoli cells. We genetically disrupted PRC1 function in embryonic Sertoli cells after sex determination, and we found that PRC1-depleted postnatal Sertoli cells exhibited defective proliferation and cell death, leading to the degeneration of adult testes. In adult Sertoli cells, PRC1 suppressed specific genes required for granulosa cells, thereby inactivating the female gene regulatory network. Chromatin regions associated with female-specific genes were marked by Polycomb-mediated repressive modifications: PRC1-mediated H2AK119ub and PRC2-mediated H3K27me3. Taken together, this study identifies a critical Polycomb-based mechanism that suppresses ovarian differentiation and maintains Sertoli cell fate in adult testes.