Obox4 promotes 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 two-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 preimplantation development.

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.

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.

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.

Polycomb protein SCML2 mediates paternal epigenetic inheritance through sperm chromatin.

Sperm chromatin retains small amounts of histones, and chromatin states of sperm mirror gene expression programs of the next generation. However, it remains largely unknown how paternal epigenetic information is transmitted through sperm chromatin. Here, we present a novel mouse model of paternal epigenetic inheritance, in which deposition of Polycomb repressive complex 2 (PRC2) mediated-repressive H3K27me3 is attenuated in the paternal germline. By applying modified methods of assisted reproductive technology using testicular sperm, we rescued infertility of mice missing Polycomb protein SCML2, which regulates germline gene expression by establishing H3K27me3 on bivalent promoters with other active marks H3K4me2/3. We profiled epigenomic states (H3K27me3 and H3K4me3) of testicular sperm and epididymal sperm, demonstrating that the epididymal pattern of the sperm epigenome is already established in testicular sperm and that SCML2 is required for this process. In F1 males of X-linked Scml2-knockout mice, which have a wild-type genotype, gene expression is dysregulated in the male germline during spermiogenesis. These dysregulated genes are targets of SCML2-mediated H3K27me3 in F0 sperm. Further, dysregulation of gene expression was observed in the mutant-derived wild-type F1 preimplantation embryos. Together, we present functional evidence that the classic epigenetic regulator Polycomb mediates paternal epigenetic inheritance through sperm chromatin.

The EZH2-PRC2-H3K27me3 axis governs the endometrial cell cycle and differentiation for blastocyst invasion.

Infertility occurs in 15% of couples worldwide. Recurrent implantation failure (RIF) is one of the major problems in in vitro fertilization and embryo transfer (IVF-ET) programs, and how to manage patients with RIF to achieve successful pregnancy outcomes remains unresolved. Here, a uterine polycomb repressive complex 2 (PRC2)-regulated gene network was found to control embryo implantation. Our RNA-seq analyses of the human peri-implantation endometrium obtained from patients with RIF and fertile controls revealed that PRC2 components, including its core enzyme enhancer of zeste homolog 2 (EZH2)-catalyzing H3K27 trimethylation (H3K27me3) and their target genes are dysregulated in the RIF group. Although fertility of uterine epithelium-specific knockout mice of Ezh2 (eKO mice) was normal, Ezh2-deleted mice in the uterine epithelium and stroma (uKO mice) exhibited severe subfertility, suggesting that stromal Ezh2 plays a key role in female fertility. The RNA-seq and ChIP-seq analyses revealed that H3K27me3-related dynamic gene silencing is canceled, and the gene expression of cell-cycle regulators is dysregulated in Ezh2-deleted uteri, causing severe epithelial and stromal differentiation defects and failed embryo invasion. Thus, our findings indicate that the EZH2-PRC2-H3K27me3 axis is critical to preparing the endometrium for the blastocyst invasion into the stroma in mice and humans.

Transcription of MERVL retrotransposons is required for preimplantation embryo development.

Zygotic genome activation (ZGA) is a critical postfertilization 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 two-cell stage during ZGA. Although MERVL expression is widely used as a marker of totipotency, the role of this retrotransposon in mouse embryogenesis remains elusive. Here, we show that full-length MERVL transcripts, but not encoded retroviral proteins, are essential for accurate regulation of the host transcriptome and chromatin state during preimplantation development. Both knockdown and CRISPRi-based repression of MERVL result in embryonic lethality due to defects in differentiation and genomic stability. Furthermore, transcriptome and epigenome analysis revealed that loss of MERVL transcripts led to retention of an accessible chromatin state at, and aberrant expression of, a subset of two-cell-specific genes. Taken together, our results suggest a model in which an endogenous retrovirus plays a key role in regulating host cell fate potential.

Bioinformatics Pipelines for Identification of Super-Enhancers and 3D Chromatin Contacts.

Precise regulation of gene expression is integral in development. Emerging studies have highlighted that super-enhancers (SEs), which are clusters of multiple enhancers, play critical roles in regulating cell type-specific gene expression via 3D chromatin, thereby defining the cellular identities of given cells. Here we provide optimized bioinformatics pipelines to identify SEs and 3D chromatin contacts. Our pipelines encompass the processing of chromatin immunoprecipitation sequencing (ChIP-seq) data to identify SEs and the processing of genome-wide chromosome conformation capture (Hi-C) data. We can then infer long-range chromatin contacts between SEs and other genomic regions. This integrative computational approach, which can be applied to CUT&RUN and CUT&Tag, alternative technologies to ChIP-seq, allows us to identify genomic locations of SEs and their 3D genome configuration, whereby multiple SEs act in concert. We show an analysis of mouse spermatogenesis as an example of this application.

Suppression of endogenous retroviral enhancers in mouse embryos derived from somatic cell nuclear transfer.

Endogenous retroviruses (ERVs) in the mammalian genome play diverse roles in embryonic development. These developmentally related ERVs are generally repressed in somatic cells and therefore are likely repressed in embryos derived from somatic cell nuclear transfer (SCNT). In this study, we sought to identify ERVs that are repressed in SCNT-derived morulae, which might cause previously unexplained embryonic deaths shortly after implantation. Our transcriptome analysis revealed that, amongst ERV families, ERVK was specifically, and strongly downregulated in SCNT-derived embryos while other transposable elements including LINE and ERVL were unchanged. Among the subfamilies of ERVK, RLTR45-int was most repressed in SCNT-derived embryos despite its highest expression in control fertilized embryos. Interestingly, the nearby genes (within 5-50 kb, n = 18; 50-200 kb, n = 63) of the repressed RLTR45-int loci were also repressed in SCNT-derived embryos, with a significant correlation between them. Furthermore, lysine H3K27 acetylation was enriched around the RLTR45-int loci. These findings indicate that RLTR45-int elements function as enhancers of nearby genes. Indeed, deletion of two sequential RLTR45-int loci on chromosome 4 or 18 resulted in downregulations of nearby genes at the morula stage. We also found that RLTR45-int loci, especially SCNT-low, enhancer-like loci, were strongly enriched with H3K9me3, a repressive histone mark. Importantly, these H3K9me3-enriched regions were not activated by overexpression of H3K9me3 demethylase Kdm4d in SCNT-derived embryos, suggesting the presence of another epigenetic barrier repressing their expressions and enhancer activities in SCNT embryos. Thus, we identified ERVK subfamily RLTR45-int, putative enhancer elements, as a strong reprogramming barrier for SCNT (253 words).

PRC1-mediated epigenetic programming is required to generate the ovarian reserve.

The ovarian reserve defines the female reproductive lifespan, which in humans spans decades due to robust maintenance of meiotic arrest in oocytes residing in primordial follicles. Epigenetic reprogramming, including DNA demethylation, accompanies meiotic entry, but the chromatin changes that underpin the generation and preservation of ovarian reserves are poorly defined. We report that the Polycomb Repressive Complex 1 (PRC1) establishes repressive chromatin states in perinatal mouse oocytes that directly suppress the gene expression program of meiotic prophase-I and thereby enable the transition to dictyate arrest. PRC1 dysfuction causes depletion of the ovarian reserve and leads to premature ovarian failure. Our study demonstrates a fundamental role for PRC1-mediated gene silencing in female reproductive lifespan, and reveals a critical window of epigenetic programming required to establish ovarian reserve.

CRISPR-Mediated Activation of Transposable Elements in Embryonic Stem Cells.

Mounting evidence has established that subsets of transposable elements (TEs) function as gene regulatory elements in a cell type- and species-specific manner. Here we describe an in vitro system to ectopically activate TEs using CRISPR-mediated activation (CRISPRa) for functional studies in mouse embryonic stem cells (ESCs). We established a stable mouse CRISPRa ESC line, in which expression of guide RNA enables the activation of TE-derived enhancers and the expression of their adjacent genes. We show an example of ectopic activation of TE-derived enhancers that function in male meiosis, as well as the expression of adjacent germline genes in ESCs. This system can also be applied to functional studies of TEs that are not active in ESCs.

Retrotransposons in the Mammalian Male Germline.

Retrotransposons are a subset of DNA sequences that constitute a large part of the mammalian genome. They can translocate autonomously or non-autonomously, potentially jeopardizing the heritable germline genome. Retrotransposons coevolved with the host genome, and the germline is the prominent battlefield between retrotransposons and the host genome to maximize their mutual fitness. Host genomes have developed various mechanisms to suppress and control retrotransposons, including DNA methylation, histone modifications, and Piwi-interacting RNA (piRNA), for their own benefit. Thus, rapidly evolved retrotransposons often acquire positive functions, including gene regulation within the germline, conferring reproductive fitness in a species over the course of evolution. The male germline serves as an ideal model to examine the regulation and evolution of retrotransposons, resulting in genomic co-evolution with the host genome. In this review, we summarize and discuss the regulatory mechanisms of retrotransposons, stage-by-stage, during male germ cell development, with a particular focus on mice as an extensively studied mammalian model, highlighting suppression mechanisms and emerging functions of retrotransposons in the male germline.

Highly rigid H3.1/H3.2-H3K9me3 domains set a barrier for cell fate reprogramming in trophoblast stem cells.

The placenta is a highly evolved, specialized organ in mammals. It differs from other organs in that it functions only for fetal maintenance during gestation. Therefore, there must be intrinsic mechanisms that guarantee its unique functions. To address this question, we comprehensively analyzed epigenomic features of mouse trophoblast stem cells (TSCs). Our genome-wide, high-throughput analyses revealed that the TSC genome contains large-scale (>1-Mb) rigid heterochromatin architectures with a high degree of histone H3.1/3.2-H3K9me3 accumulation, which we termed TSC-defined highly heterochromatinized domains (THDs). Importantly, depletion of THDs by knockdown of CAF1, an H3.1/3.2 chaperone, resulted in down-regulation of TSC markers, such as Cdx2 and Elf5, and up-regulation of the pluripotent marker Oct3/4, indicating that THDs maintain the trophoblastic nature of TSCs. Furthermore, our nuclear transfer technique revealed that THDs are highly resistant to genomic reprogramming. However, when H3K9me3 was removed, the TSC genome was fully reprogrammed, giving rise to the first TSC cloned offspring. Interestingly, THD-like domains are also present in mouse and human placental cells in vivo, but not in other cell types. Thus, THDs are genomic architectures uniquely developed in placental lineage cells, which serve to protect them from fate reprogramming to stably maintain placental function.

Meiosis-specific ZFP541 repressor complex promotes developmental progression of meiotic prophase towards completion during mouse spermatogenesis.

During spermatogenesis, meiosis is accompanied by a robust alteration in gene expression and chromatin status. However, it remains elusive how the meiotic transcriptional program is established to ensure completion of meiotic prophase. Here, we identify a protein complex that consists of germ-cell-specific zinc-finger protein ZFP541 and its interactor KCTD19 as the key transcriptional regulators in mouse meiotic prophase progression. Our genetic study shows that ZFP541 and KCTD19 are co-expressed from pachytene onward and play an essential role in the completion of the meiotic prophase program in the testis. Furthermore, our ChIP-seq and transcriptome analyses identify 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 demonstrates that a germ-cell specific complex that contains ZFP541 and KCTD19 promotes the progression of meiotic prophase towards completion in male mice, and triggers the reconstruction of the transcriptional network and chromatin organization leading to post-meiotic development.

Isolation of Murine Spermatogenic Cells using a Violet-Excited Cell-Permeable DNA Binding Dye.

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established cell isolation protocols using Hoechst 33342 staining in combination with fluorescence-activated cell sorting, it requires cell sorters equipped with an ultraviolet laser. Here we describe a cell isolation protocol using the DyeCycle Violet (DCV) stain, a low cytotoxicity DNA binding dye structurally similar to Hoechst 33342. DCV can be excited by both ultraviolet and violet lasers, which improves the flexibility of equipment choice, including a cell sorter not equipped with an ultraviolet laser. Using this protocol, one can isolate three live-cell subpopulations in meiotic prophase I, including leptotene/zygotene, pachytene, and diplotene spermatocytes, as well as post-meiotic round spermatids. We also describe a protocol to prepare single-cell suspension from mouse testes. Overall, the procedure requires a short time to complete (4-5 hours depending on the number of needed cells), which facilitates many downstream applications.

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. Male germline transcriptomes undergo dynamic changes after the mitosis-to-meiosis transition and have been subject to evolutionary divergence among mammals. However, the mechanisms underlying germline regulatory divergence remain undetermined. Here, we show that endogenous retroviruses (ERVs) influence species-specific germline transcriptomes. After the mitosis-to-meiosis transition in male mice, specific ERVs function as active enhancers to drive germline genes, including a mouse-specific gene set, and bear binding motifs for critical regulators of spermatogenesis, such as A-MYB. This raises the possibility that a genome-wide transposition of ERVs rewired germline gene expression in a species-specific manner. Of note, independently evolved ERVs are associated with the expression of human-specific germline genes, demonstrating the prevalence of ERV-driven mechanisms in mammals. Together, we propose that ERVs fine-tune species-specific transcriptomes in the mammalian germline.

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

Owing to bursts in the expression of thousands of germline-specific genes, the testis has the most diverse and complex transcriptome of all organs. By analyzing the male germline of mice, we demonstrate that the genome-wide reorganization of super-enhancers (SEs) drives bursts in germline gene expression after the mitosis-to-meiosis transition. SE reorganization is regulated by two molecular events: the establishment of meiosis-specific SEs via A-MYB (MYBL1), a key transcription factor for germline genes, and the resolution of SEs in mitotically proliferating cells via SCML2, a germline-specific Polycomb protein required for spermatogenesis-specific gene expression. Before entry into meiosis, meiotic SEs are preprogrammed in mitotic spermatogonia to ensure 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 mitotic proliferation phases.

Comparative analysis of enteroendocrine cells and their hormones between mouse intestinal organoids and native tissues.

Endocrine cells in the gastrointestinal tract secrete multiple hormones to maintain homeostasis in the body. In the present study, we generated intestinal organoids from the duodenum, jejunum, and ileum of Neurogenin 3 (Ngn3)-EGFP mice and examined how enteroendocrine cells (EECs) within organoid cultures resemble native epithelial cells in the gut. Transcriptome analysis of EGFP-positive cells from Ngn3-EGFP organoids showed gene expression pattern comparable to EECs in vivo. We also compared mRNAs of five major hormones, namely, ghrelin (Ghrl), cholecystokinin (Cck), Gip, secretin (Sct), and glucagon (Gcg) in organoids and small intestine along the longitudinal axis and found that expression patterns of these hormones in organoids were similar to those in native tissues. These findings suggest that an intestinal organoid culture system can be utilized as a suitable model to study enteroendocrine cell functions in vitro.

UHRF1 suppresses retrotransposons and cooperates with PRMT5 and PIWI proteins in male germ cells.

DNA methylation, repressive histone marks, and PIWI-interacting RNA (piRNA) are essential for the control of retrotransposon silencing in the mammalian germline. However, it remains unknown how these repressive epigenetic pathways crosstalk to ensure retrotransposon silencing in the male germline. Here, we show that UHRF1 is responsible for retrotransposon silencing and cooperates with repressive epigenetic pathways in male germ cells. Conditional loss of UHRF1 in postnatal germ cells causes DNA hypomethylation, upregulation of retrotransposons, the activation of a DNA damage response, and switches in the global chromatin status, leading to complete male sterility. Furthermore, we show that UHRF1 interacts with PRMT5, an arginine methyltransferase, to regulate the repressive histone arginine modifications (H4R3me2s and H3R2me2s), and cooperates with the PIWI pathway during spermatogenesis. Collectively, UHRF1 regulates retrotransposon silencing in male germ cells and provides a molecular link between DNA methylation, histone modification, and the PIWI pathway in the germline.