Establishing Chromatin Regulatory Landscape during Mouse Preimplantation Development.

How the chromatin regulatory landscape in the inner cell mass cells is established from differentially packaged sperm and egg genomes during preimplantation development is unknown. Here, we develop a low-input DNase I sequencing (liDNase-seq) method that allows us to generate maps of DNase I-hypersensitive site (DHS) of mouse preimplantation embryos from 1-cell to morula stage. The DHS landscape is progressively established with a drastic increase at the 8-cell stage. Paternal chromatin accessibility is quickly reprogrammed after fertilization to the level similar to maternal chromatin, while imprinted genes exhibit allelic accessibility bias. We demonstrate that transcription factor Nfya contributes to zygotic genome activation and DHS formation at the 2-cell stage and that Oct4 contributes to the DHSs gained at the 8-cell stage. Our study reveals the dynamic chromatin regulatory landscape during early development and identifies key transcription factors important for DHS establishment in mammalian embryos.

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.

Embryonic development following somatic cell nuclear transfer impeded by persisting histone methylation.

Mammalian oocytes can reprogram somatic cells into a totipotent state enabling animal cloning through somatic cell nuclear transfer (SCNT). However, the majority of SCNT embryos fail to develop to term due to undefined reprogramming defects. Here, we identify histone H3 lysine 9 trimethylation (H3K9me3) of donor cell genome as a major barrier for efficient reprogramming by SCNT. Comparative transcriptome analysis identified reprogramming resistant regions (RRRs) that are expressed normally at 2-cell mouse embryos generated by in vitro fertilization (IVF) but not SCNT. RRRs are enriched for H3K9me3 in donor somatic cells and its removal by ectopically expressed H3K9me3 demethylase Kdm4d not only reactivates the majority of RRRs, but also greatly improves SCNT efficiency. Furthermore, use of donor somatic nuclei depleted of H3K9 methyltransferases markedly improves SCNT efficiency. Our study thus identifies H3K9me3 as a critical epigenetic barrier in SCNT-mediated reprogramming and provides a promising approach for improving mammalian cloning efficiency.

Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity.

Targeted genome editing technologies have enabled a broad range of research and medical applications. The Cas9 nuclease from the microbial CRISPR-Cas system is targeted to specific genomic loci by a 20 nt guide sequence, which can tolerate certain mismatches to the DNA target and thereby promote undesired off-target mutagenesis. Here, we describe an approach that combines a Cas9 nickase mutant with paired guide RNAs to introduce targeted double-strand breaks. Because individual nicks in the genome are repaired with high fidelity, simultaneous nicking via appropriately offset guide RNAs is required for double-stranded breaks and extends the number of specifically recognized bases for target cleavage. We demonstrate that using paired nicking can reduce off-target activity by 50- to 1,500-fold in cell lines and to facilitate gene knockout in mouse zygotes without sacrificing on-target cleavage efficiency. This versatile strategy enables a wide variety of genome editing applications that require high specificity.

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.

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.

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.

Simultaneous mapping of active DNA demethylation and sister chromatid exchange in single cells.

To understand mammalian active DNA demethylation, various methods have been developed to map the genomic distribution of the demethylation intermediates 5-formylcysotine (5fC) and 5-carboxylcytosine (5caC). However, the majority of these methods requires a large number of cells to begin with. In this study, we describe low-input methylase-assisted bisulfite sequencing (liMAB-seq ) and single-cell MAB-seq (scMAB-seq), capable of profiling 5fC and 5caC at genome scale using ∼100 cells and single cells, respectively. liMAB-seq analysis of preimplantation embryos reveals the oxidation of 5mC to 5fC/5caC and the positive correlation between chromatin accessibility and processivity of ten-eleven translocation (TET) enzymes. scMAB-seq captures the cell-to-cell heterogeneity of 5fC and 5caC and reveals the strand-biased distribution of 5fC and 5caC. scMAB-seq also allows the simultaneous high-resolution mapping of sister chromatid exchange (SCE), facilitating the study of this type of genomic rearrangement. Therefore, our study not only establishes new methods for the genomic mapping of active DNA demethylation using limited numbers of cells or single cells but also demonstrates the utilities of the methods in different biological contexts.

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.

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.

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.

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.

Tet1 controls meiosis by regulating meiotic gene expression.

Meiosis is a germ-cell-specific cell division process through which haploid gametes are produced for sexual reproduction. Before the initiation of meiosis, mouse primordial germ cells undergo a series of epigenetic reprogramming steps, including the global erasure of DNA methylation at the 5-position of cytosine (5mC) in CpG-rich DNA. Although several epigenetic regulators, such as Dnmt3l and the histone methyltransferases G9a and Prdm9, have been reported to be crucial for meiosis, little is known about how the expression of meiotic genes is regulated and how their expression contributes to normal meiosis. Using a loss-of-function approach in mice, here we show that the 5mC-specific dioxygenase Tet1 has an important role in regulating meiosis in mouse oocytes. Tet1 deficiency significantly reduces female germ-cell numbers and fertility. Univalent chromosomes and unresolved DNA double-strand breaks are also observed in Tet1-deficient oocytes. Tet1 deficiency does not greatly affect the genome-wide demethylation that takes place in primordial germ cells, but leads to defective DNA demethylation and decreased expression of a subset of meiotic genes. Our study thus establishes a function for Tet1 in meiosis and meiotic gene activation in female germ cells.

Evaluation of modal noise in graded-index silica and plastic optical fiber links for radio over multimode fiber systems.

We have evaluated and compared modal noise induced in a graded-index silica multimode fiber (GI-MMF) link and a graded-index plastic optical fiber (GI-POF) link with the misaligned fiber connections. In radio over fiber (RoF) systems using these optical fibers, modal noise appears as unwanted amplitude modulation in the received signal, and results in degradation of the RoF transmission performance. In this work, we have evaluated the modal noise induced in GI-MMFs and GI-POFs with its same core diameter of 50 µm. Our results show that GI-POFs have an inherently higher tolerance to misaligned connection and less modal noise than GI-MMFs in terms of both the error-vector magnitude and the speckle pattern of the transmitted signals.

Reflection noise reduction effect of graded-index plastic optical fiber in multimode fiber link.

We experimentally demonstrate that a graded-index plastic optical fiber (GI POF) can significantly reduce reflection noise in a multimode fiber link with a vertical-cavity surface-emitting laser (VCSEL). By directly observing beams backreflected to the VCSEL, we show that the noise reduction effect is closely related to random mode coupling because of light scattering by microscopic heterogeneities in the GI POF core material. This suggests that intrinsic mode coupling can lower the self-coupling efficiency of the light backreflected to the VCSEL cavity through beam quality degradation. Using GI POFs, low-cost radio-over-fiber systems for indoor networks can be realized without optical isolators or fiber end-face polishing.

Efficient group delay averaging in graded-index plastic optical fiber with microscopic heterogeneous core.

Intrinsic mode coupling in a graded-index plastic optical fiber (GI POF) is investigated using the developed coupled power theory for a GI POF with a microscopic heterogeneous core. The results showed that the intrinsic material properties can induce random power transitions between all the guided modes, whereas the structural deformation of microbending results in nearest-neighbor coupling. It was numerically demonstrated that efficient group-delay averaging due to intrinsic mode coupling brings the pronounced bandwidth enhancement in fibers with much shorter length than the case of glass multimode fibers.

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.

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.