Biology subjects

Sakashita, A.

Publications and source records attributed to Sakashita, A..

11 recordsLinked to original sources

bioRxiv2026

Paternal folate deficiency reveals meiosis as a metabolic sensing window in the male germline

Folate-dependent one-carbon metabolism supplies methyl donors required for chromatin modification, yet how metabolic conditions shape epigenome establishment in the male germline remains poorly understood. Here, using a folate-deficient mouse model, we identify meiotic prophase I as a metabolically sensitive window in the male germline. By integrating in vivo germline analysis with bulk and single-cell transcriptomic and epigenomic profiling, we show that folate deficiency disturbs transcriptional programs in pachytene spermatocytes and preferentially perturbs CpG island (CGI)-associated promoters, which are characterized by bivalent H3K4me3 and H3K27me3 during meiosis. Consistent with this selective vulnerability, active chromatin marks, including H3K4me3 and H3K27ac, are markedly reduced at CGI-associated promoters under folate-deficient conditions. Notably, loci that later exhibit altered H3K4me3 enrichment in mature sperm show earlier chromatin perturbations during meiosis, suggesting that these sperm epigenomic alterations may originate during meiotic development. Together, these findings establish a mechanistic link between paternal folate deficiency and dynamic epigenomic remodeling of CGI-associated chromatin in the male germline.

developmental biology↗

bioRxiv2025

REFEE Shapes ZGA Through Post-Transcriptional Regulation of Repetitive RNAs in Mouse Embryos

During early mammalian embryonic development, repetitive elements such as transposons and duplicated genes are transcriptionally active and play key roles in embryogenesis. MERVL, an endogenous retrovirus, is essential for shaping the early transcriptome, particularly during the transition from totipotency to pluripotency. However, the function of many repetitive elements remains poorly understood. Here, we add a new repetitive player, REFEE, that regulates repetitive RNAs and contributes to totipotency regulation through post-transcriptional mechanisms. REFEE was initially identified as a paralogue of Alyref, a nucleocytoplasmic RNA exporter, transiently expressed during the 2-cell stage of mouse embryogenesis. REFEE preferentially binds and stabilizes MERVL RNA, promoting its export. REFEE knockdown embryos fail to complete zygotic genome activation (ZGA), accompanied by a significant reduction in MERVL RNA levels. REFEE primarily influences transcript levels of ZGA genes, upregulating or downregulating them in a manner correlated with their genomic proximity to MERVL. Proteomic analysis further revealed that REFEE knockdown also decreases protein levels of its RNA targets, indicating an additional layer of post-transcriptional regulation beyond RNA stability. Our findings identify REFEE as an essential post-transcriptional regulator of early embryonic development and highlight the functional interplay between repetitive elements in shaping the zygotic transcriptome.

developmental biology↗

bioRxiv2025

Site-specific DNA demethylation during spermatogenesis presets the sites of nucleosome retention in mouse sperm

DNA methylation patterns are inherited from the parental germline to the embryo. In mature sperm, the sites of unmethylated DNA are tightly coupled to sites of histone retention at gene regulatory elements that are implicated in paternal epigenetic inheritance. The timing and mechanism of site-specific DNA demethylation in the male germline remains unknown. Here, we perform genome-wide profiling of DNA methylation during spermatogenesis by capturing methylated DNA through interaction with a methyl-DNA binding protein domain (MBD). Our data demonstrate a site-specific change in DNA methylation during the mitosis-to-meiosis transition. Importantly, demethylation at these genomic sites during this transition precedes nucleosome retention in spermatozoa. These results suggest that site-specific DNA demethylation during the mitosis-to-meiosis transition of spermatogenesis contributes to the establishment of sperm epigenetic states associated with embryonic gene expression. We therefore propose DNA demethylation during spermatogenesis as a novel phase of epigenetic reprogramming that contributes to embryonic gene regulation.

developmental biology↗

bioRxiv2023

CTCF-mediated 3D chromatin predetermines the gene expression program in the male germline

Spermatogenesis is a unidirectional differentiation process that generates haploid sperm, but how the gene expression program that directs this process is established is largely unknown. Here we determine the high-resolution 3D chromatin architecture of male germ cells during spermatogenesis and show that CTCF-mediated 3D chromatin predetermines the gene expression program required for spermatogenesis. In undifferentiated spermatogonia, CTCF-mediated chromatin contacts on autosomes pre-establish meiosis-specific super-enhancers (SE). These meiotic SE recruit the master transcription factor A-MYB in meiotic spermatocytes, which strengthens their 3D contacts and instructs a burst of meiotic gene expression. We also find that at the mitosis-to-meiosis transition, the germline-specific Polycomb protein SCML2 resolves chromatin loops that are specific to mitotic spermatogonia. Moreover, SCML2 and A-MYB establish the unique 3D chromatin organization of sex chromosomes during meiotic sex chromosome inactivation. We propose that CTCF-mediated 3D chromatin organization enforces epigenetic priming that directs unidirectional differentiation, thereby determining the cellular identity of the male germline.

developmental biology↗

bioRxiv2023

KRAB-zinc-finger proteins regulate endogenous retroviruses to sculpt germline transcriptomes and genome evolution

As transposable elements (TEs) coevolved with the host genome, the host genome exploited TEs as functional regulatory elements. What remains largely unknown are how the activity of TEs, namely, endogenous retroviruses (ERVs), are regulated and how TEs evolved in the germline. Here we show that KRAB domain-containing zinc-finger proteins (KZFPs), which are highly expressed in mitotically dividing spermatogonia, bind to suppressed ERVs that function following entry into meiosis as active enhancers. These features are observed for independently evolved KZFPs and ERVs in mice and humans, i.e., are evolutionarily conserved in mammals. Further, we show that meiotic sex chromosome inactivation (MSCI) antagonizes the coevolution of KZFPs and ERVs in mammals. Our study uncovers a mechanism by which KZFPs regulate ERVs to sculpt germline transcriptomes. We propose that epigenetic programming in the mammalian germline during the mitosis-to-meiosis transition facilitates coevolution of KZFPs and TEs on autosomes and is antagonized by MSCI.

genomics↗

bioRxiv2022

Transcription of Murine Endogenous Retrovirus MERVL Is Required for Progression of Development in Early Preimplantation Embryos

Zygotic genome activation (ZGA) is a critical post-fertilization step that promotes totipotency and allows different cell fates to emerge in the developing embryo. MERVL, murine endogenous retrovirus-L, is transiently upregulated at the 2-cell stage around the time of ZGA. Although MERVL expression is widely used as a marker of totipotency, the role of this retrotransposon in mouse embryogenesis remains elusive. Here, we develop a method for consistent knockdown of interspersed copies of MERVL and show that MERVL RNA, but not encoded retroviral proteins and their long terminal repeat (LTR) promoter, is essential for accurate regulation of the host transcriptome and chromatin state during preimplantation development. MERVL knockdown (KD) results in embryonic lethality due to defects in differentiation and genomic stability. Furthermore, transcriptome and epigenome analysis revealed that MERVL-KD embryos retained an accessible chromatin state at, and aberrant expression of, a subset of 2-cell specific genes at mid-preimplantation stages. Taken together, our results suggest a model in which an endogenous retrovirus plays a critical role in regulating host cell fate potential.

developmental biology↗

bioRxiv2022

Obox4 secures zygotic genome activation upon loss of Dux

Once fertilized, mouse zygotes rapidly proceed to zygotic genome activation (ZGA), during which long terminal repeats (LTRs) of murine endogenous retroviruses with leucine tRNA primer (MERVL) are activated by a conserved homeodomain-containing transcription factor, DUX. However, Dux-knockout embryos produce fertile mice, suggesting that ZGA is redundantly driven by an unknown factor(s). Here we present multiple lines of evidence that the multicopy homeobox gene, Obox4, encodes a transcription factor that is highly expressed in mouse 2-cell embryos and redundantly drives ZGA. Genome-wide profiling revealed that OBOX4 specifically binds and activates MERVL LTRs as well as a subset of murine endogenous retroviruses with lysine tRNA primer (MERVK) LTRs. Depletion of Obox4 is tolerated by embryogenesis, whereas concomitant Obox4/Dux depletion markedly compromises embryonic development. Our study identified OBOX4 as a transcription factor that provides genetic redundancy to pre-implantation development.

genetics↗

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

Super-enhancer switching drives a burst in germline gene expression at the mitosis-to-meiosis transition

The testis has the most diverse and complex transcriptome of all organs due to bursts in expression of thousands of germline-specific genes. Much of this unique gene expression takes place when mitotic germ cells differentiate and enter into meiotic prophase. Here, we demonstrate that the genome-wide reorganization of super-enhancers (SEs) drives bursts of germline genes after the mitosis-to-meiosis transition. At the mitosis-to-meiosis transition, mitotic SEs dissolve while meiotic SEs are established. Meiotic SEs are associated with the activation of key germline genes, defining the cellular identity of germ cells. This SE switching is regulated by the establishment of meiotic SEs via A-MYB (MYBL1), a key transcription factor for germline genes, and by the resolution of mitotic SEs via SCML2, a germline-specific Polycomb protein required for spermatogenesis-specific gene expression. Prior to the entry into meiosis, meiotic SEs are preprogrammed in mitotic spermatogonia, serving to direct the unidirectional differentiation of spermatogenesis. We identify key regulatory factors for both mitotic and meiotic enhancers, revealing a molecular logic for the concurrent activation of mitotic enhancers and suppression of meiotic enhancers in the somatic and/or mitotically proliferating phase.

developmental biology↗

bioRxiv2020

Endogenous retroviruses drive species-specific germline transcriptomes in mammals

Gene regulation in the germline ensures the production of high-quality gametes, long-term maintenance of the species, and speciation. Germline transcriptomes undergo dynamic changes after the mitosis-to-meiosis transition in males and have been subject to evolutionary divergence among mammals. However, the mechanism that underlies germline regulatory divergence remains undetermined. Here, we show that endogenous retroviruses influence species-specific germline transcriptomes in mammals. We show that the expression of endogenous retroviruses, particularly the evolutionarily young K family (ERVK), is associated with gene activation after the mitosis-to-meiosis transition in male mice. We demonstrate that accessible chromatin and H3K27ac, a marker of active enhancers, are tightly associated with ERVK loci as well as with the activation of neighboring evolutionarily young germline genes. Thus, ERVKs serve as evolutionarily novel enhancers in mouse spermatogenesis. These ERVK loci bear binding motifs for critical regulators of spermatogenesis such as A-MYB. The genome-wide transposition of ERVKs might have rewired germline gene expression in a species-specific manner. Notably, these features are present in human spermatogenesis, but independently evolved ERVs are associated with expression of germline genes, demonstrating the prevalence of ERV-driven mechanisms in mammals. Together, we propose a model whereby species-specific transcriptomes are fine-tuned by endogenous retroviruses in the mammalian germline.

developmental biology↗

bioRxiv2017

Stepwise approach to oocyte depletion in Sry mutated XY female mice

Little is known regarding the mechanisms underlying infertility in male-to-female sex-reversed females. To gain a better understanding of germ cell dysfunction in this condition, we produced XYsry--females via the CRISPR/Cas9 system in C57BL/6 inbred strain mice. Mutant mice showed severe attrition of germ cells during foetal development, resulting in depletion of ovarian germ cells before sexual maturation. Comprehensive transcriptome analysis of embryonic day 13.5 primordial germ cells (PGCs) and postnatal day 1 oocytes demonstrated that XYsry--PGCs had already deviated from the developmental process at the mitotic stage. In addition, XYsry--oocytes resulted in meiotic disruption and developmental failure, and the genes related to these processes were significantly changed. This grievous disruption, which caused germ cell deterioration in XYsry--females, was shown to proceed from the germ cells themselves. These results provide novel insight into the germ cell depletion of sex-reversed mice as well as into disorders of sex differentiation in human, such as Swyer syndrome, wherein patients present as typical females albeit with an XY karyotype and infertile.

developmental biology↗