DPPA3 facilitates genome-wide DNA demethylation in mouse primordial germ cells.

BACKGROUND: Genome-wide DNA demethylation occurs in mammalian primordial germ cells (PGCs) as part of the epigenetic reprogramming important for gametogenesis and resetting the epigenetic information for totipotency. Dppa3 (also known as Stella or Pgc7) is highly expressed in mouse PGCs and oocytes and encodes a factor essential for female fertility. It prevents excessive DNA methylation in oocytes and ensures proper gene expression in preimplantation embryos: however, its role in PGCs is largely unexplored. In the present study, we investigated whether or not DPPA3 has an impact on CG methylation/demethylation in mouse PGCs. RESULTS: We show that DPPA3 plays a role in genome-wide demethylation in PGCs even before sex differentiation. Dppa3 knockout female PGCs show aberrant hypermethylation, most predominantly at H3K9me3-marked retrotransposons, which persists up to the fully-grown oocyte stage. DPPA3 works downstream of PRDM14, a master regulator of epigenetic reprogramming in embryonic stem cells and PGCs, and independently of TET1, an enzyme that hydroxylates 5-methylcytosine. CONCLUSIONS: The results suggest that DPPA3 facilitates DNA demethylation through a replication-coupled passive mechanism in PGCs. Our study identifies DPPA3 as a novel epigenetic reprogramming factor in mouse PGCs.

Epigenetic plasticity safeguards heterochromatin configuration in mammals.

Heterochromatin is a key architectural feature of eukaryotic chromosomes critical for cell type-specific gene expression and genome stability. In the mammalian nucleus, heterochromatin segregates from transcriptionally active genomic regions and exists in large, condensed, and inactive nuclear compartments. However, the mechanisms underlying the spatial organization of heterochromatin need to be better understood. Histone H3 lysine 9 trimethylation (H3K9me3) and lysine 27 trimethylation (H3K27me3) are two major epigenetic modifications that enrich constitutive and facultative heterochromatin, respectively. Mammals have at least five H3K9 methyltransferases (SUV39H1, SUV39H2, SETDB1, G9a and GLP) and two H3K27 methyltransferases (EZH1 and EZH2). In this study, we addressed the role of H3K9 and H3K27 methylation in heterochromatin organization using a combination of mutant cells for five H3K9 methyltransferases and an EZH1/2 dual inhibitor, DS3201. We showed that H3K27me3, which is normally segregated from H3K9me3, was redistributed to regions targeted by H3K9me3 after the loss of H3K9 methylation and that the loss of both H3K9 and H3K27 methylation resulted in impaired condensation and spatial organization of heterochromatin. Our data demonstrate that the H3K27me3 pathway safeguards heterochromatin organization after the loss of H3K9 methylation in mammalian cells.

Conservation and divergence of canonical and non-canonical imprinting in murids.

BACKGROUND: Genomic imprinting affects gene expression in a parent-of-origin manner and has a profound impact on complex traits including growth and behavior. While the rat is widely used to model human pathophysiology, few imprinted genes have been identified in this murid. To systematically identify imprinted genes and genomic imprints in the rat, we use low input methods for genome-wide analyses of gene expression and DNA methylation to profile embryonic and extraembryonic tissues at allele-specific resolution. RESULTS: We identify 14 and 26 imprinted genes in these tissues, respectively, with 10 of these genes imprinted in both tissues. Comparative analyses with mouse reveal that orthologous imprinted gene expression and associated canonical DNA methylation imprints are conserved in the embryo proper of the Muridae family. However, only 3 paternally expressed imprinted genes are conserved in the extraembryonic tissue of murids, all of which are associated with non-canonical H3K27me3 imprints. The discovery of 8 novel non-canonical imprinted genes unique to the rat is consistent with more rapid evolution of extraembryonic imprinting. Meta-analysis of novel imprinted genes reveals multiple mechanisms by which species-specific imprinted expression may be established, including H3K27me3 deposition in the oocyte, the appearance of ZFP57 binding motifs, and the insertion of endogenous retroviral promoters. CONCLUSIONS: In summary, we provide an expanded list of imprinted loci in the rat, reveal the extent of conservation of imprinted gene expression, and identify potential mechanisms responsible for the evolution of species-specific imprinting.

Noncanonical imprinting: intergenerational epigenetic inheritance mediated by Polycomb complexes.

Genomic imprinting is illustrative of intergenerational epigenetic inheritance. The passage of parental genomes into the embryo is accompanied by epigenetic modifications, resulting in imprinted monoallelic gene expression in mammals. Some imprinted genes are regulated by maternal inheritance of H3K27me3, which is termed noncanonical imprinting. Noncanonical imprinting is established by Polycomb repressive complexes during oogenesis and maintained in preimplantation embryos and extraembryonic tissues, including the placenta. Recent studies of noncanonical imprinting have contributed to our understanding of chromatin regulation in oocytes and early embryos, imprinted X-chromosome inactivation, secondary differentially DNA-methylated regions, and the anomalies of cloned mice. Here, I summarize the current knowledge of noncanonical imprinting and remark on analogous mechanisms in invertebrates and plants.

Low-Input CUT&RUN for Mouse Oocytes and Preimplantation Embryos.

Cleavage Under Target & Release Using Nuclease (CUT&RUN) enables the detection of DNA regions that are bound by a protein of interest. This method is suitable for low-input materials because of the absence of an immunoprecipitation step. However, it sometimes fails when applying it to fragile cells, such as mouse oocytes. Here we describe our low-input CUT&RUN protocol optimized for mouse oocyte and preimplantation embryo samples in which the primary antibody and protein A-MNase binding steps are completed before the cells are bound to Concanavalin A-coated magnetic beads. This modification prevents crush of oocytes and early embryos and unwanted loss of chromatin during CUT&RUN procedures.

Transcriptomic signatures in trophectoderm and inner cell mass of human blastocysts classified according to developmental potential, maternal age and morphology.

Selection of high-quality embryos is important to achieve successful pregnancy in assisted reproductive technology (ART). Recently, it has been debated whether RNA-sequencing (RNA-Seq) should be applied to ART to predict embryo quality. However, information on genes that can serve as markers for pregnant expectancy is limited. Furthermore, there is no information on which transcriptome of trophectoderm (TE) or inner cell mass (ICM) is more highly correlated with pregnant expectancy. Here, we performed RNA-Seq analysis of TE and ICM of human blastocysts, the pregnancy expectation of which was retrospectively determined using the clinical outcomes of 1,890 cases of frozen-thawed blastocyst transfer. We identified genes that were correlated with the expected pregnancy rate in ICM and TE, respectively, with a larger number of genes identified in TE than in ICM. Downregulated genes in the TE of blastocysts that were estimated to have lower expectation of pregnancy included tight junction-related genes such as CXADR and ATP1B1, which have been implicated in peri-implantation development. Moreover, we identified dozens of differentially expressed genes by regrouping the blastocysts based on the maternal age and the Gardner score. Additionally, we showed that aneuploidy estimation using RNA-Seq datasets does not correlate with pregnancy expectation. Thus, our study provides an expanded list of candidate genes for the prediction of pregnancy in human blastocyst embryos.

Integrative analysis of scRNA-seq and scATAC-seq revealed transit-amplifying thymic epithelial cells expressing autoimmune regulator.

Medullary thymic epithelial cells (mTECs) are critical for self-tolerance induction in T cells via promiscuous expression of tissue-specific antigens (TSAs), which are controlled by the transcriptional regulator, AIRE. Whereas AIRE-expressing (Aire+) mTECs undergo constant turnover in the adult thymus, mechanisms underlying differentiation of postnatal mTECs remain to be discovered. Integrative analysis of single-cell assays for transposase-accessible chromatin (scATAC-seq) and single-cell RNA sequencing (scRNA-seq) suggested the presence of proliferating mTECs with a specific chromatin structure, which express high levels of Aire and co-stimulatory molecules, CD80 (Aire+CD80hi). Proliferating Aire+CD80hi mTECs detected using Fucci technology express a minimal number of Aire-dependent TSAs and are converted into quiescent Aire+CD80hi mTECs expressing high levels of TSAs after a transit amplification. These data provide evidence for the existence of transit-amplifying Aire+mTEC precursors during the Aire+mTEC differentiation process of the postnatal thymus.

Noncanonical imprinting sustains embryonic development and restrains placental overgrowth.

Genomic imprinting regulates parental origin-dependent monoallelic gene expression. It is mediated by either germline differential methylation of DNA (canonical imprinting) or oocyte-derived H3K27me3 (noncanonical imprinting) in mice. Depletion of Eed, an essential component of Polycomb repressive complex 2, results in genome-wide loss of H3K27me3 in oocytes, which causes loss of noncanonical imprinting (LOI) in embryos. Although Eed maternal KO (matKO) embryos show partial lethality after implantation, it is unknown whether LOI itself contributes to the developmental phenotypes of these embryos, which makes it unclear whether noncanonical imprinting is developmentally relevant. Here, by combinatorial matKO of Xist, a noncanonical imprinted gene whose LOI causes aberrant transient maternal X-chromosome inactivation (XCI) at preimplantation, we show that prevention of the transient maternal XCI greatly restores the development of Eed matKO embryos. Moreover, we found that the placentae of Eed matKO embryos are remarkably enlarged in a manner independent of Xist LOI. Heterozygous deletion screening of individual autosomal noncanonical imprinted genes suggests that LOI of the Sfmbt2 miRNA cluster chromosome 2 miRNA cluster (C2MC), solute carrier family 38 member 4 (Slc38a4), and Gm32885 contributes to the placental enlargement. Taken together, our study provides evidence that Xist imprinting sustains embryonic development and that autosomal noncanonical imprinting restrains placental overgrowth.

H2AK119ub1 guides maternal inheritance and zygotic deposition of H3K27me3 in mouse embryos.

Parental epigenomes are established during gametogenesis. While they are largely reset after fertilization, broad domains of Polycomb repressive complex 2 (PRC2)-mediated formation of lysine 27-trimethylated histone H3 (H3K27me3) are inherited from oocytes in mice. How maternal H3K27me3 is established and inherited by embryos remains elusive. Here, we show that PRC1-mediated formation of lysine 119-monoubiquititinated histone H2A (H2AK119ub1) confers maternally heritable H3K27me3. Temporal profiling of H2AK119ub1 dynamics revealed that atypically broad H2AK119ub1 domains are established, along with H3K27me3, during oocyte growth. From the two-cell stage, H2AK119ub1 is progressively deposited at typical Polycomb targets and precedes H3K27me3. Reduction of H2AK119ub1 by depletion of Polycomb group ring finger 1 (PCGF1) and PCGF6-essential components of variant PRC1 (vPRC1)-leads to H3K27me3 loss at a subset of genes in oocytes. The gene-selective H3K27me3 deficiency is irreversibly inherited by embryos, causing loss of maternal H3K27me3-dependent imprinting, embryonic sublethality and placental enlargement at term. Collectively, our study unveils preceding dynamics of H2AK119ub1 over H3K27me3 at the maternal-to-zygotic transition, and identifies PCGF1/6-vPRC1 as an essential player in maternal epigenetic inheritance.

Formation of α clusters in dilute neutron-rich matter.

The surface of neutron-rich heavy nuclei, with a neutron skin created by excess neutrons, provides an important terrestrial model system to study dilute neutron-rich matter. By using quasi-free α cluster-knockout reactions, we obtained direct experimental evidence for the formation of α clusters at the surface of neutron-rich tin isotopes. The observed monotonous decrease of the reaction cross sections with increasing mass number, in excellent agreement with the theoretical prediction, implies a tight interplay between α-cluster formation and the neutron skin. This result, in turn, calls for a revision of the correlation between the neutron-skin thickness and the density dependence of the symmetry energy, which is essential for understanding neutron stars. Our result also provides a natural explanation for the origin of α particles in α decay.

Low-noise radio over graded-index plastic optical fiber.

We developed a graded-index plastic optical fiber (GI POF) that enables lower-noise radio frequency (RF) transmission than conventional multimode fibers for short-distance household applications (<100m). It is shown that reflection noise degrades RF transmission, regardless of the carrier frequency, through the spurious generation that accompanies the RF modulation of a vertical-cavity surface-emitting laser. The GI POF with distinctive mode coupling, which is closely related to its microscopic polymer structure, suppresses noise and spurious generation to improve transmission quality. Our low-noise radio-over-GI-POF technology will offer significant advantages for optical wiring systems for broadcast and communication in small- and medium-scale buildings.

Loss of Ikbkap/Elp1 in mouse oocytes causes spindle disorganization, developmental defects in preimplantation embryos and impaired female fertility.

Elongator complexes are well known to be involved in a wide variety of cellular processes; however, their functions in mammalian oocytes have not been characterized. Here, we demonstrated in mice that specific deletion of one of the core subunits, Ikbkap/Elp1, in oocytes resulted in spindle defects and chromosome disorganization without affecting folliculogenesis. In accordance with these findings, we observed that Ikbkap mutant female mice are subfertile. Further analyses uncovered that kinetochore-microtubule attachments are severely compromised in Ikbkap-deficient oocytes. Moreover, we revealed that Ikbkap modulates the acetylation status of α-tubulin in oocytes, which may at least in part mediate the meiotic phenotypes described above by affecting microtubule dynamics and kinetochore function. Finally, we showed that embryos derived from Ikbkap-deficient oocytes exhibit an increased frequency of aneuploidy, digyny, progressive delays in preimplantation development, and severe degeneration before reaching the blastocyst stage. In summary, we identify Ikbkap as an important player in regulating oocyte meiosis by modulating tubulin acetylation for chromosome/spindle organization.

Acyl-CoA synthetase 6 regulates long-chain polyunsaturated fatty acid composition of membrane phospholipids in spermatids and supports normal spermatogenic processes in mice.

Long-chain polyunsaturated fatty acids (LCPUFAs), such as docosahexaenoic acid (DHA, 22:6) and docosapentaenoic acid (DPA, 22:5), have versatile physiologic functions. Studies have suggested that DHA and DPA are beneficial for maintaining sperm quality. However, their mechanisms of action are still unclear because of the poor understanding of DHA/DPA metabolism in the testis. DHA and DPA are mainly stored as LCPUFA-containing phospholipids and support normal spermatogenesis. Long-chain acyl-conenzyme A (CoA) synthetase (ACSL) 6 is an enzyme that preferentially converts LCPUFA into LCPUFA-CoA. Here, we report that ACSL6 knockout (KO) mice display severe male infertility due to attenuated sperm numbers and function. ACSL6 is highly expressed in differentiating spermatids, and ACSL6 KO mice have reduced LCPUFA-containing phospholipids in their spermatids. Delayed sperm release and apoptosis of differentiated spermatids were observed in these mice. The results of this study indicate that ACSL6 contributes to the local accumulation of DHA- and DPA-containing phospholipids in spermatids to support normal spermatogenesis.-Shishikura, K., Kuroha, S., Matsueda, S., Iseki, H., Matsui, T., Inoue, A., Arita, M. Acyl-CoA synthetase 6 regulates long-chain polyunsaturated fatty acid composition of membrane phospholipids in spermatids and supports normal spermatogenic processes in mice.

Unconventional plastic optical fiber design for very short multimode fiber link.

We introduce a graded-index plastic optical fiber (GI POF) design for very short-distance household applications, in which the transmission quality is predominantly determined by system noise rather than the loss and bandwidth. The developed GI POF has strong mode coupling with low accompanying scattering loss, which is closely related to the specific microscopic heterogeneities in the core material. Such characteristic mode coupling significantly decreases reflection noise, improving the transmission quality compared with silica GI multimode fiber (MMF) for lengths below 30 m. Moreover, in the GI POF link, the transmission quality tends to improve with increasing fiber length, despite the increased loss and decreased bandwidth. This feature suggests that the system noise can be controlled by the microscopic heterogeneous properties of the GI POF for a very short MMF link, where the fiber loss and bandwidth are sufficiently low and high, respectively. This unconventional concept for optical-fiber design can advance fiber-optic communication in emerging applications in households located near optical network terminals.

Allelic H3K27me3 to allelic DNA methylation switch maintains noncanonical imprinting in extraembryonic cells.

Faithful maintenance of genomic imprinting is essential for mammalian development. While germline DNA methylation-dependent (canonical) imprinting is relatively stable during development, the recently found oocyte-derived H3K27me3-mediated noncanonical imprinting is mostly transient in early embryos, with some genes important for placental development maintaining imprinted expression in the extraembryonic lineage. How these noncanonical imprinted genes maintain their extraembryonic-specific imprinting is unknown. Here, we report that maintenance of noncanonical imprinting requires maternal allele-specific de novo DNA methylation [i.e., somatic differentially methylated regions (DMRs)] at implantation. The somatic DMRs are located at the gene promoters, with paternal allele-specific H3K4me3 established during preimplantation development. Genetic manipulation revealed that both maternal EED and zygotic DNMT3A/3B are required for establishing somatic DMRs and maintaining noncanonical imprinting. Thus, our study not only reveals the mechanism underlying noncanonical imprinting maintenance but also sheds light on how histone modifications in oocytes may shape somatic DMRs in postimplantation embryos.

Maternal Eed knockout causes loss of H3K27me3 imprinting and random X inactivation in the extraembryonic cells.

Genomic imprinting is essential for mammalian development. Recent studies have revealed that maternal histone H3 Lys27 trimethylation (H3K27me3) can mediate DNA methylation-independent genomic imprinting. However, the regulatory mechanisms and functions of this new imprinting mechanism are largely unknown. Here we demonstrate that maternal Eed, an essential component of the Polycomb group complex 2 (PRC2), is required for establishing H3K27me3 imprinting. We found that all H3K27me3-imprinted genes, including Xist, lose their imprinted expression in Eed maternal knockout (matKO) embryos, resulting in male-biased lethality. Surprisingly, although maternal X-chromosome inactivation (XmCI) occurs in Eed matKO embryos at preimplantation due to loss of Xist imprinting, it is resolved at peri-implantation. Ultimately, both X chromosomes are reactivated in the embryonic cell lineage prior to random XCI, and only a single X chromosome undergoes random XCI in the extraembryonic cell lineage. Thus, our study not only demonstrates an essential role of Eed in H3K27me3 imprinting establishment but also reveals a unique XCI dynamic in the absence of Xist imprinting.

Reprogramming of Chromatin Accessibility in Somatic Cell Nuclear Transfer Is DNA Replication Independent.

Mammalian oocytes have the ability to reset the transcriptional program of differentiated somatic cells into that of totipotent embryos through somatic cell nuclear transfer (SCNT). However, the mechanisms underlying SCNT-mediated reprogramming are largely unknown. To understand the mechanisms governing chromatin reprogramming during SCNT, we profiled DNase I hypersensitive sites (DHSs) in donor cumulus cells and one-cell stage SCNT embryos. To our surprise, the chromatin accessibility landscape of the donor cells is drastically changed to recapitulate that of the in vitro fertilization (IVF)-derived zygotes within 12 hr. Interestingly, this DHS reprogramming takes place even in the presence of a DNA replication inhibitor, suggesting that SCNT-mediated DHS reprogramming is independent of DNA replication. Thus, this study not only reveals the rapid and drastic nature of the changes in chromatin accessibility through SCNT but also establishes a DNA replication-independent model for studying cellular reprogramming.

Transcriptional activation of transposable elements in mouse zygotes is independent of Tet3-mediated 5-methylcytosine oxidation.

This corrects the article DOI: 10.1038/cr.2012.160.

Genomic imprinting of Xist by maternal H3K27me3.

Maternal imprinting at the Xist gene is essential to achieve paternal allele-specific imprinted X-chromosome inactivation (XCI) in female mammals. However, the mechanism underlying Xist imprinting is unclear. Here we show that the Xist locus is coated with a broad H3K27me3 domain that is established during oocyte growth and persists through preimplantation development in mice. Loss of maternal H3K27me3 induces maternal Xist expression and maternal XCI in preimplantation embryos. Our study thus identifies maternal H3K27me3 as the imprinting mark of Xist.

Maternal H3K27me3 controls DNA methylation-independent imprinting.

Mammalian sperm and oocytes have different epigenetic landscapes and are organized in different fashions. After fertilization, the initially distinct parental epigenomes become largely equalized with the exception of certain loci, including imprinting control regions. How parental chromatin becomes equalized and how imprinting control regions escape from this reprogramming is largely unknown. Here we profile parental allele-specific DNase I hypersensitive sites in mouse zygotes and morula embryos, and investigate the epigenetic mechanisms underlying these allelic sites. Integrated analyses of DNA methylome and tri-methylation at lysine 27 of histone H3 (H3K27me3) chromatin immunoprecipitation followed by sequencing identify 76 genes with paternal allele-specific DNase I hypersensitive sites that are devoid of DNA methylation but harbour maternal allele-specific H3K27me3. Interestingly, these genes are paternally expressed in preimplantation embryos, and ectopic removal of H3K27me3 induces maternal allele expression. H3K27me3-dependent imprinting is largely lost in the embryonic cell lineage, but at least five genes maintain their imprinted expression in the extra-embryonic cell lineage. The five genes include all paternally expressed autosomal imprinted genes previously demonstrated to be independent of oocyte DNA methylation. Thus, our study identifies maternal H3K27me3 as a DNA methylation-independent imprinting mechanism.