Mammalian sperm and oocytes have unique epigenetic landscapes that are extremely different from each other. These epigenomes are genome-widely reprogrammed after fertilization and become largely symmetric between the paternal and maternal alleles by the blastocyst stage. Nevertheless, certain genomic regions resist the reprogramming and carry the gametic epigenetic information into offspring. These regions can serve as a platform of intergenerational epigenetic inheritance. We recently found that a post-translational modification of histone proteins, H3K27me3, in oocytes can be intergenerationally inherited and regulate mono-allelic gene expression in preimplantation embryos and extra-embryonic cells (e.g. placentae) in a germline DNA methylation-independent manner. We are now trying to understand how maternal histone modifications resist reprogramming at post-fertilization, what the functions of intergenerational epigenomes are, and whether/how maternal environment could alter the oocyte epigenome and affect offspring’s health, including metabolic traits.
Keywords: Epigenetic inheritance, Maternal histone-mediated imprinting, Preimplantation embryo, Placenta , DOHaD
A new life begins when a sperm enters an oocyte. The two originally different cells are now fused to form a single cell, called zygote, in which two nuclei co-exist (paternal pronucleus and maternal pronucleus). The zygote divides to form a 2-cell embryo and the subsequent cleavages give rise to 4-cell, morula (8-16 cells), and blastocyst (32-128 cells) embryos. This developmental process takes place in the oviduct and the uterus before implantation and therefore is called ‘pre-implantation development’. Preimplantation embryos can be cultured in vitro up to the blastocyst stage, and they can give rise to live animals after transplanted into surrogate mothers. Such 'ex vivo' permissive characteristics of preimplantation embryos allow researchers to easily manipulate gene function in a physiological context. Importantly, the epigenetic landscape is genome-widely reprogrammed in preimplantation embryos, providing an excellent model to study epigenetics.
The epigenetics field is getting bigger and bigger. Epigenetics is nowadays an infrastructure to study life sciences. There are various definitions of epigenetics, but my understanding of epigenetics is a gene regulatory mechanism beyond the layers of genomic sequence and transcription factors. The nature of epigenetics include chemical modifications on DNA and histones. Epigenetic modifications compartmentalizes chromatin into transcriptionally-permissive and -repressive states. Each cell type has its own epigenetic landscape to enable a cell-type specific gene expression program. See more information about Epigenetics elsewhere if needed.
The epigenetic landscapes of sperm and oocytes are very different from each other. Shortly after fertilization, the sperm-derived paternal chromatin and the oocyte-derived maternal chromatin show epigenetic modifications totally different from each other. During preimplantation development, the epigenetic asymmetry between the parental chromatins is gradually resolved. This resetting process, called epigenetic reprogramming, is believed to be important to prepare a white canvas to establish new epigenetic information in a cell type-specific manner during cellular differentiation at post-implantation.
Reprogramming resistance & Genomic imprinting
While the epigenetic landscapes of gametes are genome-widely reprogrammed during preimplantation development, certain genomic loci escape the reprogramming wave. Such reprogramming resistance results in transmission of parental epigenetic information into the next generation. When the epigenetic modifications at reprogramming resistant loci are parent-of-origin specific, it causes imprinted expression. The representatives of these loci are imprinting control regions that are marked by oocyte- or sperm-specific DNA methylation.
Maternal inheritance of a histone modification
Since the first identification of imprinted genes in mammals in 1991, DNA methylation had been the only known mechanism governing genomic imprinting. Nevertheless, a few studies have demonstrated that several paternally-expressed imprinted genes (PEGs) maintain their imprinted expression even in the lack of oocyte DNA methylation, suggesting the existence of a DNA methylation-independent imprinting mechanism. In 2017, we discovered that oocyte H3K27me3 is transmitted into embryos and the oocyte-derived H3K27me3 controls all of the PEGs reported to be independent of maternal DNA methylation (Inoue et al., 2017 Nature, Inoue et al., 2017 Genes Dev). These PEGs include Xist, an X-linked long non-coding RNA critical for X chromosome inactivation (XCI). Loss of maternal H3K27me3 leads to bi-allelic expression of Xist and bi-allelic XCI in preimplantation embryos. Interestingly, while DNA methylation-mediated imprinting is generally maintained in both embryonic and extra-embryonic cell lineages, maternal H3K27me3-mediated imprinting is maintained only in extra-embryonic cells such as placentae. We created a mouse model in which H3K27me3-, but not DNA methylation-, mediated imprinting is disrupted, and found that loss of H3K27me3 imprinting causes developmental retardation at post-implantation and embryonic sublethality (Inoue et al., 2018 Genes Dev), suggesting the importance of this new imprinting mechanism in mouse development. A follow-up study revealed that DNA methylation is newly established in the maternal allele at peri-implantation to maintain imprinted expression in placentae (Chen et al., 2019 Sci Adv). It is fascinating to study how maternal H3K27me3-mediated imprinting is regulated during development, what its functions are, and whether it is conserved in the other organisms.
Current research directions
1. Mechanisms and functions of intergenerationally-heritable maternal histone modifications.
2. Functions and regulatory mechanisms of maternal H3K27me3-mediated imprinting
3. Understand the mechanisms of intergenerational inheritance of maternal metabolic disorders.
Unique research tools
In addition to general molecular biology tools, we have unique research tools as listed below. The ability to integrate these allows us to conduct high-originality researches.
1. Epigenome analysis technologies applicable to a small number of cells
Low-input ChIP-seq (Inoue et al., 2017 Genes Dev)
2. Micromanipulation & Developmental engineering
mRNA/siRNA injection, pronuclear isolation, pronuclear transfer, chromosome transfer, somatic cell nuclear transfer, in vitro fertilization (IVF), in vitro maturation (IVM), in vitro growth (IVG), intracytoplasmic sperm injection (ICSI), embryo transplantation, dissection of E6.5 epiblast/extra-embryonic ectoderm/visceral endoderm, etc.
3. Maternal protein depletion system
siRNA injection into GV oocytes followed by IVM-IVF
Since most of biological events occurring in zygotes and early preimplantation embryos are regulated by maternally-stored proteins, it is essential to deplete them to understand their mechanisms and functions. One way is to generate an oocyte-specific KO model, which is time-consuming and thereby extremely low throughput. An alternative is RNA interference-mediated knockdown of interested genes. Although siRNA injection into MII-stage oocytes is commonly used due to its relative easiness, it often fails to deplete maternal proteins because these proteins have already been accumulated in MII oocytes. To achieve efficient knockdown of maternal proteins, we inject siRNA into GV-stage oocytes and then let the oocytes reach the MII-stage using in vitro maturation (IVM). This allows a 20-42 hrs time window for injected siRNA to act before fertilization. The usefulness of this approach has been proved by our past works in which maternally-stored proteins, Tet3 and Nyfa, were efficiently depleted (Inoue et al., 2012 Cell Res; Lu et al., 2016 Cell). The detailed protocol will be distributed upon request.
siRNA injection into growing oocytes followed by IVG-IVM-IVF
Nevertheless, it sometimes happens that the above approach fails to knockdown targeted maternal proteins when the proteins are highly stable. To overcome this issue, we inject siRNA into growing oocytes followed by in vitro growth (Inoue et al, 2014 Protocol Ex). In this system, secondary follicles are collected from ovaries of 12 day-old mice, oocytes within the follicles are injected with siRNA, and then the follicles are cultured in vitro for 12 days until the oocytes reach the fully-grown GV-stage. Then, the oocytes are matured in vitro (IVM) and fertilized in vitro (IVF) to obtain zygotes. This system provides a long time window (~2 weeks) for injected siRNA to act. The injected siRNA can act for such a long period because the oocyte is a non-dividing cell and siRNA is not diluted by cell division. By using this method, we have succeeded to knockdown NPM2 and HIRA in mouse oocytes (Inoue and Aoki, 2010 FASEB J; Inoue and Zhang, 2014 NSMB).