The question of whether retained histones in the sperm genome localize to gene-coding regions or gene deserts has been debated for years. Previous contradictory observations are likely caused by the non-uniform sensitivity of sperm chromatin to micrococcal nuclease (MNase) digestion. Sperm chromatin has a highly condensed but heterogeneous structure and is composed of 90%∼99% protamines and 1%∼10% histones. In this study, we utilized nucleoplasmin (NPM) to improve the solubility of sperm chromatin by removing protamines in vitro. NPM treatment efficiently solubilized histones while maintaining quality and quantity. Chromatin immunoprecipitation sequencing (ChIP-seq) analyses using NPM-treated sperm demonstrated the predominant localization of H4 to distal intergenic regions, whereas modified histones exhibited a modification-dependent preferential enrichment in specific genomic elements, such as H3K4me3 at CpG-rich promoters and H3K9me3 in satellite repeats, respectively, implying the existence of machinery protecting modified histones from eviction.

Gene expression is controlled by alterations in the epigenome, including DNA methylation and histone modification. Recently, it was reported that 5-methylcytosine (5mC) is converted to 5-hydroxymethylcytosine (5hmC) by proteins in the ten-eleven translocation (TET) family. This conversion is believed to be part of the mechanism by which methylated DNA is demethylated. Moreover, histones undergo modifications such as phosphorylation and acetylation. In addition, modification with O-linked-N-acetylglucosamine (O-GlcNAc) by O-GlcNAc transferase (OGT) was recently identified as a novel histone modification. Herein, we focused on TET3, the regulation of which is still unclear. We attempted to elucidate the mechanism of its regulation by biochemical approaches. First, we conducted mass spectrometric analysis in combination with affinity purification of FLAG-TET3, which identified OGT as an important partner of TET3. Co-immunoprecipitation assays using a series of deletion mutants showed that the C-terminal H domain of TET3 was required for its interaction with OGT. Furthermore, we showed that TET3 is GlcNAcylated by OGT, although the GlcNAcylation did not affect the global hydroxylation of methylcytosine by TET3. Moreover, we showed that TET3 enhanced its localization to chromatin through the stabilization of OGT protein. Taken together, we showed a novel function of TET3 that likely supports the function of OGT.

Cellular differentiation is associated with dynamic chromatin remodeling in establishing a cell-type-specific epigenomic landscape. Here, we find that mouse testis-specific and replication-dependent histone H3 variant H3t is essential for very early stages of spermatogenesis. H3t gene deficiency leads to azoospermia because of the loss of haploid germ cells. When differentiating spermatogonia emerge in normal spermatogenesis, H3t appears and replaces the canonical H3 proteins. Structural and biochemical analyses reveal that H3t-containing nucleosomes are more flexible than the canonical nucleosomes. Thus, by incorporating H3t into the genome during spermatogonial differentiation, male germ cells are able to enter meiosis and beyond.

Somatic cell nuclear transfer (SCNT) in mammals is an inefficient process that is frequently associated with abnormal phenotypes, especially in placentas. Recent studies demonstrated that mouse SCNT placentas completely lack histone methylation (H3K27me3)-dependent imprinting, but how it affects placental development remains unclear. Here, we provide evidence that the loss of H3K27me3 imprinting is responsible for abnormal placental enlargement and low birth rates following SCNT, through upregulation of imprinted miRNAs. When we restore the normal paternal expression of H3K27me3-dependent imprinted genes (Sfmbt2, Gab1, and Slc38a4) in SCNT placentas by maternal knockout, the placentas remain enlarged. Intriguingly, correcting the expression of clustered miRNAs within the Sfmbt2 gene ameliorates the placental phenotype. Importantly, their target genes, which are confirmed to cause SCNT-like placental histology, recover their expression level. The birth rates increase about twofold. Thus, we identify loss of H3K27me3 imprinting as an epigenetic error that compromises embryo development following SCNT.

In vivo fluorescent imaging technique is a strong tool to visualize the various cellular events such as the proliferation, differentiation, migration, and a lineage tracing in living cells requiring no further experimental procedure such as immunostaining. During spermatogenesis, unique and dynamic histone exchanges occur. Since the timing and types of histone exchanges defines the particular stages of spermatogenesis, visualizing certain types of histones in testes is useful not only for researching specific histone dynamics, but also for monitoring the stages of spermatogenesis in vivo. In this study, we report the establishment of two transgenic (Tg) mouse lines expressing histone H4-Venus (H4V) and histone H3.3-mCherry (H33C) fusion proteins in testicular germ cells, and demonstrated their utility for monitoring germ cell development in vivo. Because of the choice of promoter as well as the nature of these histones, H4V and H33C were exclusively expressed in the germ cells of the distinct stages, which allowed the determination of spermatogenic stages in real time. In addition, disappearance of H4V and H33C at particular stages of differentiation/fertilization also represented dynamic histone removal. Collectively, these Tg mice are a valuable resource not only for monitoring differentiation stages, but also for studying the chromatin dynamics of post-natal testicular germ cell development in vivo.