Biology subjects

Ishiguro, K.-i.

Publications and source records attributed to Ishiguro, K.-i..

18 recordsLinked to original sources

bioRxiv2026

Firrm functions as a multiprotein interaction platform that coordinates Mcm8-9-Hrob recruitment and Fignl1-mediated Rad51 unloading

Rad51 forms nucleoprotein filaments to catalyze homology search and strand exchange. The transition to recombination-associated DNA synthesis is accompanied by Rad51 unloading and recombination helicase recruitment. However, how these reactions are coordinated remains unclear. Using Xenopus egg extracts, we identify a mechanistic coupling between Rad51 unloading and recombination helicase recruitment. Proteomic analysis revealed that Rad51 filaments assembled on double-stranded DNA recruit a limited set of recombination regulators, including C1orf112/Firrm, Fignl1, Mcm8, Mcm9, and Hrob. Firrm forms a stoichiometric complex with Fignl1 and directly binds short acidic motifs in the intrinsically disordered regions of both Mcm9 and Hrob. The Firrm-Mcm9 interaction promotes recruitment of Mcm8-9 and Hrob to Rad51 filaments. Purified Mcm8-9 stimulates the ATPase activity of Fignl1 through Firrm in a DNA-dependent manner. Rad51 unloading from double-stranded DNA depends primarily on Fignl1 ATPase activity and is stimulated by Firrm-Mcm9 interaction. In human cells, FIRRM-FIGNL1 and MCM8-9-HROB act in the same genetic pathway to confer resistance to DNA interstrand crosslinks. These results support a model in which Rad51 filaments and Firrm-Fignl1 promote recruitment of Mcm8-9-Hrob, which, in turn, stimulates Fignl1 ATPase activity, thereby coupling recombination helicase recruitment with Rad51 unloading.

molecular biology↗

bioRxiv2026

Replication protein A prevents unregulated priming and Rad51 loading on single-stranded DNA in nuclear extracts of Xenopus eggs

In eukaryotes, single-stranded DNA (ssDNA) generated during DNA replication, recombination, and repair is rapidly bound and protected by the major single-stranded DNA-binding protein replication protein A (RPA). RPA not only stabilizes ssDNA but also acts as a central platform that coordinates diverse DNA transactions. Exhaustion of RPA due to unregulated ssDNA production leads to replication fork breakage and replication catastrophe, underscoring its critical role in genome stability. However, the direct consequences of RPA limitation remain incompletely understood. Using Xenopus egg extracts, we show that excess ssDNA induces spontaneous priming, a reaction that is otherwise prevented in a physiological nuclear environment. We provide evidence that priming suppression is mediated by stoichiometric binding of RPA to ssDNA. Analysis of the ssDNA-binding proteome reveals that RPA promotes the association of ATR checkpoint factors, Pol-primase, and the RFWD3 ubiquitin ligase with ssDNA. In contrast, RPA depletion induces the recruitment of Rad51, Rad51 paralogs, and Fbh1, a DNA helicase that interacts with both RPA and Rad51 and promotes fork breakage under replication stress. Collectively, our findings suggest that RPA contributes to genome stability by protecting ssDNA from inappropriate DNA synthesis and unscheduled recruitment of recombination and fork-processing factors.

biochemistry↗

bioRxiv2026

MEIOSIN retains an intrinsic STRA8-independent activity that drives meiotic entry across vertebrate evolution

Meiotic entry in vertebrates has been viewed as a transcriptional switch driven by the MEIOSIN-STRA8 axis, but whether this represents the ancestral regulatory logic of meiosis has remained unclear. Here we combine comparative genomics with genetic and single-cell analyses in zebrafish and mice to show that MEIOSIN retains an intrinsic STRA8-independent activity. We identify Meiosin orthologs in zebrafish and hagfish, vertebrate lineages that lack Stra8, and show that these proteins retain the HMG domain but have lost the bHLH domain required for the canonical MEIOSIN-STRA8 interaction. In zebrafish, meiosin is transiently induced at meiotic entry in both sexes, yet its loss selectively disrupts the female germ line, causing failure of oogenesis and female-to-male sex reversal. In mice, MEIOSIN lacking the bHLH domain still initiates key features of meiotic entry and partially activates meiotic target genes, although it fails to support full meiotic progression. Together, these findings identify HMG-containing MEIOSIN as a conserved core regulator of meiotic entry and position STRA8 as a later-acting module that enhances the efficiency and robustness of meiotic gene activation.

developmental biology↗

bioRxiv2026

The Nkx2.3-Nr5a1 gene cascade plays a crucial role in spleen-specific vascular architecture and marginal zone formation

NR5A1 is a nuclear receptor and master regulator of steroidogenic tissue development and steroid hormone biosynthesis in the adrenal glands and gonads. Although NR5A1 is expressed in splenic vascular endothelial cells, its function in the adult spleen remains unclear. Using single-cell transcriptomics in mice, we show that NR5A1-positive endothelial cells are heterogeneous and exhibit distinct phenotypes according to their localization in the splenic sinus or marginal sinus. To assess NR5A1 function, we generated a spleen-specific Nr5a1 enhancer deletion model. Enhancer loss resulted in reduced spleen size and ablation of the marginal zone and marginal sinus. In addition, 21-hydroxylase activity in splenic sinusoidal endothelial cells was reduced, particularly in females, leading to decreased levels of adrenocortical steroids in the spleen. Enhancer deletion also induced ectopic lymphatic-like and high endothelial venule-like vasculature similar to those observed in Nkx2.3-deficient mice. Consistently, mutation of the NKX2.3-binding motif within the enhancer abolished NR5A1 expression. Together, these findings identify an Nkx2.3[->]Nr5a1 regulatory axis essential for spleen-specific endothelial cell differentiation and splenic architecture required for blood filtration and marginal zone formation.

developmental biology↗

bioRxiv2025

MAP7-driven cytoskeletal cross-talk establishes Sertoli cell polarity crucial for germ cell meiotic progression

How specialized apical membrane domains are assembled in vivo remains poorly understood. In the mouse testis, Sertoli cells build a luminal apical domain after birth that organizes the seminiferous epithelium and supports germ cell differentiation, distinct from fetal polarity established during cord formation. Here, we identify the microtubule-associated protein MAP7 as a key regulator of cytoskeletal organization during apical domain formation. MAP7 preferentially localizes to apical microtubules in Sertoli cells, showing limited overlap with stable microtubule markers. Native-tissue imaging indicates that MAP7-decorated microtubules, initially lacking obvious directionality, become increasingly aligned along the tubule axis as the apical domain matures. In Map7-deficient testes, microtubule abundance is maintained, but higher-order organization is disrupted and accompanied by persistent luminal F-actin accumulation. These defects compromise apical domain maturation and lumen formation during the first wave of spermatogenesis and are associated with altered tight-junction patterning. Proteomic analysis identifies the non-muscle myosin II heavy chains MYH9 and MYH10 among MAP7-associated proteins. MYH9 becomes enriched at luminal regions where microtubules and F-actin converge, but remains diffuse in Map7-deficient Sertoli cells despite ectopic luminal cytoskeletal assemblies, suggesting that MAP7 helps establish a cytoskeletal context that supports spatially restricted NMII enrichment. Single-cell RNA sequencing shows that Sertoli transcriptional differentiation largely proceeds without MAP7, whereas germ cells exhibit delayed meiotic progression with expansion of pachytene-stage subpopulations. Together, these findings establish MAP7-dependent microtubule remodeling as an organizing mechanism for postnatal apical domain maturation and suggest how epithelial cytoskeletal architecture shapes a niche that helps pace developmental progression of neighboring germ cells.

cell biology↗

bioRxiv2025

Functional redundancy between UTY and UTX in regulating the localization of transcription factors involved in pluripotency

The Y chromosome harbors few protein-coding genes, and their roles in early human development remain largely unclear. Here, we demonstrate that UTY, a Y-linked homolog of the histone demethylase UTX, plays a critical, demethylase-independent role in maintaining pluripotency in human embryonic stem cells (hESCs). Despite its low expression and weak enzymatic activity, UTY co-occupies active cis-regulatory elements with UTX and promotes the transcription of key pluripotency genes, including NODAL and LEFTY. Integrated genomic analyses of single and double knockout hESCs reveal that UTY and UTX function redundantly to maintain chromatin accessibility and ensure proper recruitment of core transcription factors such as OCT4 and SOX2. The combined loss of UTY and UTX disrupts transcription factor localization, induces widespread gene expression changes, and leads to a collapse of the pluripotent state, without detectable changes in H3K27 methylation. Instead, these defects are associated with impaired recruitment of ATP-dependent chromatin remodelers and reduced histone acetylation, suggesting a demethylase-independent mechanism. Our findings uncover an essential role for the evolutionarily conserved UTY in safeguarding enhancer function and transcription factor occupancy, highlighting Y-linked regulatory mechanisms that are critical for maintaining human pluripotency. Summary statementThis study reveals how UTX and UTY cooperate to maintain stable gene regulation in human stem cells and guide proper developmental programs.

developmental biology↗

bioRxiv2024

Meioc-Piwil1 complexes regulate rRNA transcription for differentiation of spermatogonial stem cells

Ribosome biogenesis is vital for sustaining stem cell properties, yet its regulatory mechanisms are obscure. Herein, we show unique properties of zebrafish meioc mutants in which spermatogonial stem cells (SSCs) do not differentiate or upregulate rRNAs. Meioc colocalized with Piwil1 in perinuclear germ granules, but Meioc depletion resulted in Piwil1 accumulation in nucleoli. Nucleolar Piwil1 interacted with 45S pre-rRNA. piwil1+/-spermatogonia with reduced Piwil1 upregulated rRNAs, and piwil1+/-;meioc-/- spermatogonia recovered differentiation later than those in meioc-/-. Further, Piwil1 interacted with Setdb1 and HP1, and meioc-/- spermatogonia exhibited high levels of H3K9me3 and methylated CpG in the 45S-rDNA region. These results indicate that zebrafish SSCs maintain low levels of rRNA transcription with repressive marks similar to Drosophila piRNA targets of RNA polymerase II, and that Meioc has a unique function on preventing localization of Piwil1 in nucleoli to upregulate rRNA transcripts and to promote SSC differentiation.

developmental biology↗

bioRxiv2023

ATF7IP2/MCAF2 directs H3K9 methylation and meiotic gene regulation in the male germline

H3K9 tri-methylation (H3K9me3) plays emerging roles in gene regulation, beyond its accumulation on pericentric constitutive heterochromatin. It remains a mystery why and how H3K9me3 undergoes dynamic regulation in male meiosis. Here, we identify a novel, critical regulator of H3K9 methylation and spermatogenic heterochromatin organization: the germline-specific protein ATF7IP2 (MCAF2). We show that, in male meiosis, ATF7IP2 amasses on autosomal and X pericentric heterochromatin, spreads through the entirety of the sex chromosomes, and accumulates on thousands of autosomal promoters and retrotransposon loci. On the sex chromosomes, which undergo meiotic sex chromosome inactivation (MSCI), the DNA damage response pathway recruits ATF7IP2 to X pericentric heterochromatin, where it facilitates the recruitment of SETDB1, a histone methyltransferase that catalyzes H3K9me3. In the absence of ATF7IP2, male germ cells are arrested in meiotic prophase I. Analyses of ATF7IP2-deficient meiosis reveal the proteins essential roles in the maintenance of MSCI, suppression of retrotransposons, and global upregulation of autosomal genes. We propose that ATF7IP2 is a downstream effector of the DDR pathway in meiosis that coordinates the organization of heterochromatin and gene regulation through the spatial regulation of SETDB1-mediated H3K9me3 deposition.

developmental biology↗

bioRxiv2023

Atypical heat shock transcription factor HSF5 is critical for male meiotic prophase under non-stress conditions

Meiotic prophase progression is differently regulated in males and females. In males, pachytene transition during meiotic prophase is accompanied by robust alteration in gene expression. However, how gene expression is regulated differently to ensure meiotic prophase completion in males remains elusive. Herein, we identified HSF5 as a male germ cell-specific heat shock transcription factor (HSF) for meiotic prophase progression. Genetic analyses and single-cell RNA-sequencing demonstrated that HSF5 is essential for progression beyond the pachytene stage under non-stress conditions rather than heat stress. Chromatin binding analysis in vivo and DNA-binding assays in vitro suggested that HSF5 binds to promoters in a subset of genes associated with chromatin organization. HSF5 recognizes a DNA motif different from typical heat shock elements recognized by other canonical HSFs. This study suggests that HSF5 is an atypical HSF that enforces the gene expression program for pachytene transition during meiotic prophase in males.

developmental biology↗

bioRxiv2022

MCMBP promotes the assembly of the MCM2-7 hetero-hexamer to ensure robust DNA replication in human cells

The MCM2-7 hetero-hexamer is the replicative DNA helicase that plays a central role in eukaryotic DNA replication. In proliferating cells, the expression level of the MCM2-7 hexamer is kept high, which safeguards the integrity of the genome. However, how the MCM2-7 hexamer is assembled in living cells remains unknown. Here, we revealed that the MCM-binding protein (MCMBP) plays a critical role in the assembly of this hexamer in human cells. MCMBP associates with MCM3 which is essential for maintaining the level of the MCM2-7 hexamer. Acute depletion of MCMBP demonstrated that it contributes to MCM2-7 assembly using nascent MCM3. Cells depleted of MCMBP gradually ceased to proliferate because of reduced replication licensing. Under this condition, p53-positive cells exhibited arrest in the G1 phase, whereas p53-null cells entered the S phase and lost their viability because of the accumulation of DNA damage, suggesting that MCMBP is a potential target for killing p53-deficient cancers.

molecular biology↗

bioRxiv2021

FBXO47 is essential for preventing the synaptonemal complex from premature disassembly in mouse male meiosis

Meiotic prophase is a prolonged G2 phase that ensures the completion of numerous meiosis-specific chromosome events. During meiotic prophase, homologous chromosomes undergo synapsis to facilitate meiotic recombination yielding crossovers. It remains largely elusive how homolog synapsis is temporally maintained and destabilized during meiotic prophase. Here we show that FBXO47 is the stabilizer of synaptonemal complex during male meiotic prophase. Disruption of FBXO47 shows severe impact on homologous chromosome synapsis and DSB repair processes, leading to male infertility. Notably, in the absence of FBXO47, although once homologous chromosomes are synapsed, the synaptonemal complex is precociously disassembled before progressing beyond pachytene. Remarkably, Fbxo47 KO spermatocytes remain in earlier stage of meiotic prophase and lack crossovers, despite apparently exhibiting diplotene-like chromosome morphology. We propose that FBXO47 functions independently of SCF E3 ligase, and plays a crucial role in preventing synaptonemal complex from premature disassembly during cell cycle progression of meiotic prophase.

cell biology↗

bioRxiv2021

Trim41 is essential for preventing X chromosome chaotic synapsis in male mice

Meiosis is a hallmark event in germ cell development that accompanies sequential chromosome events executed by numerous molecules. Therefore, characterization of these factors is one of the best strategies to clarify the mechanism of meiosis. Here, we report tripartite motif-containing 41 (TRIM41), a ubiquitin ligase E3, as an essential factor for proper meiotic progression and fertility in male mice. Trim41 KO spermatocytes exhibited synaptonemal complex protein 3 (SYCP3) overloading, especially on the X chromosome, showing extensive self-synapsis of X chromosome and non-homologous synapsis between the X chromosome and autosomes. Furthermore, the mutant mice lacking the RING domain of TRIM41, required for the ubiquitin ligase E3 activity, phenocopied Trim41 KO mice. We then examined the behavior of mutant TRIM41 ({Delta}RING-TRIM41) and found that {Delta}RING-TRIM41 accumulated on the chromosome axes with overloaded SYCP3. This result showed that TRIM41 exerts the function on the chromosome axes. In summary, our study revealed that Trim41 is essential for preventing SYCP3 overloading and chaotic synapsis of the X chromosome, suggesting a TRIM41-mediated mechanism for regulating unsyapsed axes during male meiotic progression. Summary statementTrim41-disruption caused abnormal synapsis configuration of the X chromosome and complete infertility in male mice. Thus, TRIM41 prevents the sex chromosome from chaotic synapsis.

developmental biology↗

bioRxiv2021

Map7D2 and Map7D1 facilitate MT stabilization through distinct mechanisms to control cell motility and neurite outgrowth

Microtubule (MT) dynamics are modulated through the coordinated action of various MT-associated proteins (MAPs). However, the regulatory mechanisms underlying MT dynamics remain unclear. We show that the MAP7 family protein Map7D2 stabilizes MTs to control cell motility and neurite outgrowth. Map7D2 directly bound to MTs through its N-terminal half and stabilized MTs in vitro. Map7D2 localized prominently to the centrosome and partially on MTs in mouse N1-E115 neuronal cells, which expresses two of the four MAP7 family members, Map7D2 and Map7D1. Map7D2 loss decreased the resistance to the MT-destabilizing agent, nocodazole without affecting acetylated/detyrosinated stable MTs, suggesting that Map7D2 stabilizes MTs via direct binding. In addition, Map7D2 loss increased the rate of random cell migration and neurite outgrowth, presumably by disturbing the balance between MT stabilization and destabilization. Map7D1 exhibited similar subcellular localization and gene knock-down phenotypes to Map7D2. However, in contrast to Map7D2, Map7D1 was required for the maintenance of acetylated stable MTs. Taken together, our data suggest that Map7D2 and Map7D1 facilitate MT stabilization through distinct mechanisms in cell motility and neurite outgrowth.

cell biology↗

bioRxiv2021

The polyol pathway is an evolutionarily conserved system for sensing glucose uptake

Cells must adjust the expression levels of metabolic enzymes in response to fluctuating nutrient supply. For glucose, such metabolic remodeling is highly dependent on a master transcription factor ChREBP/MondoA. However, it remains elusive how glucose fluctuations are sensed by ChREBP/MondoA despite the stability of major glycolytic pathways. Here we show that in both flies and mice, ChREBP/MondoA activation in response to glucose ingestion depends on an evolutionarily conserved glucose-metabolizing pathway: the polyol pathway. The polyol pathway converts glucose to fructose via sorbitol. It has been believed that this pathway is almost silent, and its activation in hyperglycemic conditions has deleterious effects on human health. We show that the polyol pathway is required for the glucose-induced nuclear translocation of Mondo, a Drosophila homologue of ChREBP/MondoA, which directs gene expression for organismal growth and metabolism. Likewise, inhibition of the polyol pathway in mice impairs ChREBPs nuclear localization and reduces glucose tolerance. We propose that the polyol pathway is an evolutionarily conserved sensing system for the glucose uptake that allows metabolic remodeling.

physiology↗

bioRxiv2021

KCTD19 associates with ZFP541 and HDAC1 and is required for meiotic exit in male mice

Meiosis is a cell division process with complex chromosome events where various molecules must work in tandem. To find meiosis-related genes, we screened evolutionarily conserved and reproductive tract-enriched genes using the CRISPR/Cas9 system and identified potassium channel tetramerization domain containing 19 (Kctd19) as an essential factor for meiosis. In prophase I, Kctd19 deficiency did not affect synapsis or the DNA damage response, and chiasma structures were also observed in metaphase I spermatocytes of Kctd19 KO mice. However, spermatocytes underwent apoptotic elimination during the metaphase-anaphase transition. We were able to rescue the Kctd19 KO phenotype with an epitope-tagged Kctd19 transgene. Immunoprecipitation-mass spectrometry identified zinc finger protein 541 (ZFP541) and histone deacetylase 1 (HDAC1) as binding partners of KCTD19, indicating that KCTD19 is involved in chromatin modification. Phenotyping of Zfp541 KO spermatocytes demonstrated XY chromosome asynapsis and recurrent DNA damage in the late pachytene stage, leading to apoptosis. In summary, our study reveals that KCTD19 associates with ZFP541 and HDAC1, and that both KCTD19 and ZFP541 were essential for meiotic exit in male mice. Author summaryMeiosis is a fundamental process that consisting of one round of genomic DNA replication and two rounds of chromosome segregation producing four haploid cells. To properly distribute their genetic material, cells need to undergo complex chromosome events such as a physical linkage of homologous chromosomes (termed synapsis) and meiotic recombination. The molecules involved in these events have not been fully characterized yet, especially in mammals. Using a CRISPR/Cas9-screening system, we identified the potassium channel tetramerization domain containing 19 (Kctd19) as an essential factor for meiosis in male mice. Further, we identified zinc finger protein 541 (ZFP541) and histone deacetylase 1 (HDAC1) as binding partners of KCTD19. By observing meiosis of Zfp541 knockout germ cells, we found that Zfp541 was also essential for meiotic completion. These results show that the KCTD19/ZFP541 complex plays a critical role and is indispensable for male meiosis and fertility.

developmental biology↗

bioRxiv2021

Meiosis-specific ZFP541 repressor complex promotes meiotic prophase exit during spermatogenesis

During spermatogenesis, meiosis is accompanied by robust alteration in gene expression and chromatin status. However, it remained elusive how meiotic transcriptional program is established to ensure completion of meiotic prophase. Here, we identified a novel protein complex consisting of germ-cell-specific zinc-finger protein ZFP541 and its interactor KCTD19 as the key transcriptional regulator for meiotic prophase exit. Our genetic study showed that ZFP541 and KCTD19 are co-expressed from pachytene onward and play an essential role in the completion of meiotic prophase program in the testis. Furthermore, our ChIP-seq and transcriptome analyses revealed that ZFP541 binds to and suppresses a broad range of genes whose function is associated with biological processes of transcriptional regulation and covalent chromatin modification. The present study demonstrated that germ-cell specific ZFP541-KCTD19 containing complex promotes meiotic prophase exit in males, and triggers reconstruction of the transcription network and chromatin organization leading to post-meiotic development.

developmental biology↗

bioRxiv2020

Phosphorylation of the Anaphase Promoting Complex activator CDH1/FZR regulates the transition from Meiosis I to Meiosis II in mouse male germ cell

CDH1/FZR is an activator of Anaphase promoting complex/Cyclosome (APC/C), best known for its role as E3 ubiquitin ligase that drives the cell cycle. APC/C activity is regulated by CDK-mediated phosphorylation of CDH1 during mitotic cell cycle. Although the critical role of CDH1 phosphorylation has been shown mainly in yeast and in vitro cell culture studies, its biological significance in mammalian tissues in vivo remained elusive. Here, we examined the in vivo role of CDH1 phosphorylation using a mouse model, in which non-phosphorylatable substitutions were introduced in the putative CDK-phosphorylation sites of CDH1. Although ablation of CDH1 phosphorylation did not show substantial consequences in mouse somatic tissues, it led to severe testicular defects resulting in male infertility. In the absence of CDH1 phosphorylation, male juvenile germ cells entered meiosis normally but skipped meiosis II producing diploid spermatid-like cells. In aged testis, male germ cells were overall abolished, showing Sertoli cell-only phenotype. The present study demonstrated that phosphorylation of CDH1 is required for temporal regulation of APC/C activity at the transition from meiosis I to meiosis II, and for spermatoginial stem cell maintenance, which raised an insight into the sexual dimorphism of CDH1-regulation in germ cells.

cell biology↗

bioRxiv2020

A meiosis-specific factor MRM/C19orf57 modulates localization of RAD51 and DMC1 recombinases to DSBs in mouse meiotic recombination

Meiotic recombination is critical for genetic exchange and generation of chiasmata that ensures faithful chromosome segregation during meiosis I. Meiotic recombination is initiated by DNA double-strand break (DSB) followed by multiple processes of DNA repair. The exact mechanisms how recombinases localize to DSB remained elusive. Here we show that C19orf57/4930432K21Rik/BRME1 is a new player for meiotic recombination in mice. C19orf57/4930432K21Rik/BRME1 associates with ssDNA binding proteins, BRCA2 and MEILB2/HSF2BP, critical recruiters of recombinases onto DSB sites. Disruption of C19orf57/4930432K21Rik/BRME1 shows severe impact on DSB repair and male fertility. Remarkably, removal of single stranded DNA (ssDNA) binding proteins from DSB sites is delayed, and reciprocally the loading of RAD51 and DMC1 onto resected ssDNA is impaired in Brme1 KO spermatocytes. We propose that C19orf57/4930432K21Rik/BRME1 modulates localization of recombinases to meiotic DSB sites through the interaction with the BRCA2-MEILB2/HSF2BP complex during meiotic recombination.

molecular biology↗