Research

Our research focuses on epigenetic dynamics during mammalian germ cell development and fertilization. Epigenetic inheritance from parents to progeny has been catching a huge attention, thus our research can contribute to elucidate the mechanism.

Research 2024 version

Understanding the global architecture of sperm-retained histones

In mammals, sperm is the only cell possessing chromatin without histones. However, a small amount of histones are still retained in sperm, and their function has been a big question in the field (Okada & Yamaguchi, Cell Mol Life Sci, 2017). We previously generated transgenic mice, in which histone eviction and retention during spermiogenesis can be visualized in live mice (Makino, Front Cell Dev Biol, 2014; Kojima, Biotechnol Bioeng, 2018). We also identified novel phosphorylation in a germ cell-specific H2A variant (known as TH2A) and Haspin as its kinase (Hada, Sci Rep., 2017; Hada, Chromosoma, 2017). The pTH2A was specifically accumulated in condensed sperm chromatin and (peri) centromere of the meiotic chromosome (Figure 1). Interestingly, centromeric pTH2A was significantly decreased in aged oocytes, suggesting that pTH2A is a new indicator of chromatin condensation and oocyte aging (Figure 2).
We recently developed a method for efficient solubilization of highly condensed sperm chromatin by using Nucleoplasmin. This method enabled us to determine the genome-wide localization of sperm-retained histones by ChIP-seq and found that sperm-retained histones are mainly localized in gene desert, while modified histones are enriched in specific gene elements depending on the modification (Yamaguchi, Cell Rep, 2018) (Figure 3). More recently, we also demonstrated the critical role of histone ubiquitylation in histone-protamine replacement during spermiogenesis (Kim et al., 2020).

Figure 1. Immunostaining of the seminiferous tubules. pTH2A was detected in condensed spermatids. pTH2A (green), PNA (red), and DNA (blue) are displayed. The white dashed line indicates the periphery of tubules, and the roman numerals indicate differentiation stages of seminiferous tubules.

Figure 2. Immunostaining of Metaphase I chromosomes from aged mouse oocytes. Centromeric pTH2A was decreased in aged oocytes. pTH2A (green) and DNA (blue) are displayed. The white arrowhead indicates a magnified area.

Figure 3. A model of modification-specific localization of retained histones in sperm genome

Functional analyses of factors involved in meiotic axis formation

Meiosis is a specialized cell division that generates genetic diversity in the next generation by exchanging paternal and maternal genetic information. During meiotic prophase I, homologous chromosomes undergo pairing/synapsis, recombination, crossing over, and division on the chromosome axis. The chromosome axis consists of meiotic cohesins (RAD21L and REC8) and synaptonemal complex (SYCP2 and SYCP3). It is also known that HORMAD proteins (HORMAD1 and HORMAD2) localize along the unsynapsed chromosome axes and that they promote the recruitment of DNA double-strand break (DSB) factors such as SPO11 to chromosomal axes in early axis formation and monitor homolog synapsis. However, the molecular mechanism by which HORMAD proteins, which do not contain a definitive DNA-binding domain, localize to the chromosomal axes is still unclear. This study aims to clarify how HORMAD1 is recruited on chromosomal axes and reveal its molecular mechanism. The image acquisition using N-STORM super-resolution microscope clarified that HORMAD1 also shows localization patterns that are close to RAD21L and REC8 in the subsequent axis formation process (Figure 4). Moreover, from the analysis of mice in which each factor was knocked out, it became clear that the localization of HORMAD1 on chromatin was mediated by meiotic cohesin prior to axis formation, and the interaction between HORMAD1 and cohesin was partially supported by SYCP2 (Fujiwara, PLoS Genetics, 2020).

Figure 4. A chromosome sample of a preleptotene spermatocyte in which early synapsis occurs. The specimen was fluorescently labeled with HORMAD1 and RAD21L by immunostaining and observed using a super-resolution microscope.