The impact of selective HDAC inhibitors on the transcriptome of early mouse embryos.

BACKGROUND: Histone acetylation, which is regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs), plays a crucial role in the control of gene expression. HDAC inhibitors (HDACi) have shown potential in cancer therapy; however, the specific roles of HDACs in early embryos remain unclear. Moreover, although some pan-HDACi have been used to maintain cellular undifferentiated states in early embryos, the specific mechanisms underlying their effects remain unknown. Thus, there remains a significant knowledge gap regarding the application of selective HDACi in early embryos. RESULTS: To address this gap, we treated early embryos with two selective HDACi (MGCD0103 and T247). Subsequently, we collected and analyzed their transcriptome data at different developmental stages. Our findings unveiled a significant effect of HDACi treatment during the crucial 2-cell stage of zygotes, leading to a delay in embryonic development after T247 and an arrest at 2-cell stage after MGCD0103 administration. Furthermore, we elucidated the regulatory targets underlying this arrested embryonic development, which pinpointed the G2/M phase as the potential period of embryonic development arrest caused by MGCD0103. Moreover, our investigation provided a comprehensive profile of the biological processes that are affected by HDACi, with their main effects being predominantly localized in four aspects of zygotic gene activation (ZGA): RNA splicing, cell cycle regulation, autophagy, and transcription factor regulation. By exploring the transcriptional regulation and epigenetic features of the genes affected by HDACi, we made inferences regarding the potential main pathways via which HDACs affect gene expression in early embryos. Notably, Hdac7 exhibited a distinct response, highlighting its potential as a key player in early embryonic development. CONCLUSIONS: Our study conducted a comprehensive analysis of the effects of HDACi on early embryonic development at the transcriptional level. The results demonstrated that HDACi significantly affected ZGA in embryos, elucidated the distinct actions of various selective HDACi, and identified specific biological pathways and mechanisms via which these inhibitors modulated early embryonic development.

Role of DNA dioxygenase Ten-Eleven translocation 3 (TET3) in rheumatoid arthritis progression.

BACKGROUND: Rheumatoid arthritis (RA) patients present with abnormal methylation patterns in their fibroblast-like synoviocytes (FLS). Given that DNA demethylation is critical for producing DNA methylation patterns, we hypothesized that DNA demethylation may facilitate RA progression. Therefore, we designed this study to examine the role of DNA dioxygenase family, Ten-Eleven translocation (TET1/2/3), in the pathological process of RA. METHODS: Synovial tissues and FLS were obtained from patients with RA and Osteoarthritis. K/BxN serum-induced arthritis was induced in Wild-type (WT) and TET3 heterozygous-deficient (TET3+/-) C57BL/6 mice. RESULTS: We found that both TET3 and 5-hydroxymethylcytosine (5hmC) were upregulated in synovitis tissues from RA patients and confirmed this upregulation in the cultured FLS derived from synovitis tissues. Tumor necrosis factor α (TNFα) upregulated TET3 and 5hmC levels in cultured FLS, and the stimulated FLS exhibited high cell mobility with increased transcription of cellular migration-related factors such as C-X-C motif chemokine ligand 8 (CXCL8) and C-C motif chemokine ligand 2 (CCL2) in a TET3-dependent manner. In addition, TET3 haploinsufficiency lowered RA progression in a mouse model of serum-induced arthritis. CONCLUSIONS: Based on these findings, we can assume that TET3-mediated DNA demethylation acts as an epigenetic regulator of RA progression.

Detection of NO3- introduced in plasma-irradiated dry lettuce seeds using liquid chromatography-electrospray ionization quantum mass spectrometry (LC-ESI QMS).

Discharge plasma irradiates seeds with reactive oxygen and nitrogen species (RONS). However, RONS introduced in seeds by plasma irradiation have not been successfully detected thus far. This study provides experimental evidence that nitrate ion NO3- is introduced in lettuce seeds as RONS upon irradiation with atmospheric-pressure air dielectric barrier discharge plasma. Plasma irradiation for 5 min promotes seed germination. The components of the plasma-irradiated seeds were examined using electrospray ionization quantum mass spectrometry (ESI QMS), which revealed that the plasma irradiation introduced an ion with a mass of 62 m/z in detectable amounts. This ion was identified as NO3- by liquid chromatography (LC), multiple wavelength detector (MWD), and LC-ESI QMS. A one-dimensional simulation at electron temperature Te = 1 eV, electron density Ne = 1013/m3, and gas temperature Tg = 300 K indicated the introduction of NO3-, involving nitric oxide NO. NO3- is one of the most important ions that trigger signal transduction for germination when introduced in seeds. The scanning electron microscopy (SEM) images revealed that there was no change on the surface of the seeds after plasma irradiation. Plasma irradiation is an effective method of introducing NO3- in seeds in a dry process without causing damage.

Survivability and subsequent development of vitrified early-stage mouse embryos after warming at different temperatures.

Cryopreservation of embryos is a useful method for stably preserving various strains for a long time, and the cryopreserved embryos can be used at any time by simple warming. However, the viability of cryopreserved embryos, particularly vitrification at an early stage, is low compared to that of fresh embryos. As the warming process during vitrification is known to affect the survivability and subsequent development of embryos, the present study aimed to examine the viability and subsequent development of vitrified early-stage mouse embryos after warming at different temperatures. The survival rate of pronuclear and 2-cell stage embryos warmed at 60 °C (97% and 88%, respectively) was significantly higher than that of the embryos warmed at 37 °C (46% and 48%, respectively). The pronuclear and 2-cell stage embryos warmed at 60 °C (86% and 100%) showed better development to the blastocyst stage than the embryos warmed at 37 °C (72% and 84%, respectively). The development of offspring of the surviving embryos was similar at both the warming temperatures. These results showed that the survivability and subsequent development of vitrified early-stage mouse embryos were obviously increased upon rapid warming. This improved warming process could be helpful for the maintenance and reproduction of genetic resources.

Totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage.

In multicellular organisms, oocytes and sperm undergo fusion during fertilization and the resulting zygote gives rise to a new individual. The ability of zygotes to produce a fully formed individual from a single cell when placed in a supportive environment is known as totipotency. Given that totipotent cells are the source of all multicellular organisms, a better understanding of totipotency may have a wide-ranging impact on biology. The precise delineation of totipotent cells in mammals has remained elusive, however, although zygotes and single blastomeres of embryos at the two-cell stage have been thought to be the only totipotent cells in mice. We now show that a single blastomere of two- or four-cell mouse embryos can give rise to a fertile adult when placed in a uterus, even though blastomere isolation disturbs the transcriptome of derived embryos. Single blastomeres isolated from embryos at the eight-cell or morula stages and cultured in vitro manifested pronounced defects in the formation of epiblast and primitive endoderm by the inner cell mass and in the development of blastocysts, respectively. Our results thus indicate that totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage.

Loss of TET proteins in regulatory T cells promotes abnormal proliferation, Foxp3 destabilization and IL-17 expression.

Ten-eleven translocation (TET) proteins regulate DNA methylation and gene expression by converting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Although Tet2/Tet3 deficiency has been reported to lead to myeloid cell, B-cell and invariant natural killer T (iNKT) cell malignancy, the effect of TET on regulatory T cells (Tregs) has not been elucidated. We found that Tet2/Tet3 deficiency in Tregs led to lethal hyperproliferation of CD4+Foxp3+ T cells in the spleen and mesenteric lymph nodes after 5 months of age. Additionally, in aged Treg-specific Tet2/Tet3-deficient mice, serum IgG1, IgG3, IgM and IgE levels were markedly elevated. High IL-17 expression was observed in both Foxp3+ and Fopx3- CD4+ T cells, and adoptive transfer of Tet2/Tet3-deficient Tregs into lymphopenic mice inhibited Foxp3 expression and caused conversion into IL-17-producing cells. However, the conserved non-coding DNA sequence-2 (CNS2) region of the Foxp3 gene locus, which has been shown to be particularly important for stable Foxp3 expression, was only partly methylated. We identified novel TET-dependent demethylation sites in the Foxp3 upstream enhancer, which may contribute to stable Foxp3 expression. Together, these data indicate that Tet2 and Tet3 are involved in Treg stability and immune homeostasis in mice.

Improvement of Foxp3 stability through CNS2 demethylation by TET enzyme induction and activation.

Since induced regulatory T cells (iTregs) can be produced in a large quantity in vitro, these cells are expected to be clinically useful to induce immunological tolerance in various immunological diseases. Foxp3 (Forkhead box P3) expression in iTregs is, however, unstable due to the lack of demethylation of the CpG island in the conserved non-coding sequence 2 (CNS2) of the Foxp3 locus. To facilitate the demethylation of CNS2, we over-expressed the catalytic domain (CD) of the ten-eleven translocation (TET) protein, which catalyzes the steps of the iterative demethylation of 5-methylcytosine. TET-CD over-expression in iTregs resulted in partial demethylation of CNS2 and stable Foxp3 expression. We also discovered that TET expression was enhanced under low oxygen (5%) culture conditions, which facilitated CNS2 DNA demethylation and stabilization of Foxp3 expression in a TET2- and TET3-dependent manner. In combination with vitamin C treatment, which has been reported to enhance TET catalytic activity, iTregs generated under low oxygen conditions retained more stable Foxp3 expression in vitro and in vivo and exhibited stronger suppression activity in a colitis model compared with untreated iTregs. Our data indicate that the induction and activation of TET enzymes in iTregs would be an effective method for Treg-mediated adoptive immunotherapy.

Mild Amide-Cleavage Reaction Mediated by Electrophilic Benzylation.

An extremely mild method for amide-cleavage by using the triazine-based benzylating reagent 4-(4,6-diphenoxy-1,3,5-triazin-2-yl)-4-benzylmorpholinium trifluoromethanesulfonate (DPT-BM), which spontaneously releases benzyl cation species when being dissolved at room temperature, has been developed. O-Benzylation of the amide with DPT-BM and the subsequent hydrolysis of the resulting intermediate benzyl imidate salt afford the corresponding amine and benzyl ester, which can be converted by hydrogenolysis into a carboxylic acid under neutral conditions. O-Benzylation proceeds depending on both steric and electronic factors around the amide group. Thus, some amides have been selectively cleaved over other amides. Furthermore, intramolecular chemoselective cleavage of an amide group in the presence of an ester group was achieved. Such selective hydrolytic reactions cannot be performed with Meerwein reagents as well as under acidic or basic hydrolytic conditions.

Histone chaperone CAF-1 mediates repressive histone modifications to protect preimplantation mouse embryos from endogenous retrotransposons.

Substantial proportions of mammalian genomes comprise repetitive elements including endogenous retrotransposons. Although these play diverse roles during development, their appropriate silencing is critically important in maintaining genomic integrity in the host cells. The major mechanism for retrotransposon silencing is DNA methylation, but the wave of global DNA demethylation that occurs after fertilization renders preimplantation embryos exceptionally hypomethylated. Here, we show that hypomethylated preimplantation mouse embryos are protected from retrotransposons by repressive histone modifications mediated by the histone chaperone chromatin assembly factor 1 (CAF-1). We found that knockdown of CAF-1 with specific siRNA injections resulted in significant up-regulation of the retrotransposons long interspersed nuclear element 1, short interspersed nuclear element B2, and intracisternal A particle at the morula stage. Concomitantly, increased histone H2AX phosphorylation and developmental arrest of the majority (>95%) of embryos were observed. The latter was caused at least in part by derepression of retrotransposons, as treatment with reverse transcriptase inhibitors rescued some embryos. Importantly, ChIP analysis revealed that CAF-1 mediated the replacement of H3.3 with H3.1/3.2 at the retrotransposon regions. This replacement was associated with deposition of repressive histone marks, including trimethylation of histone H3 on lysine 9 (H3K9me3), H3K9me2, H3K27me3, and H4K20me3. Among them, H4K20me3 and H3K9me3 seemed to play predominant roles in retrotransposon silencing, as assessed by knockdown of specific histone methyltransferases and forced expression of unmethylatable mutants of H3.1K9 and H4K20. Our data thus indicate that CAF-1 is an essential guardian of the genome in preimplantation mouse embryos by deposition of repressive histone modifications via histone variant replacement.

Maternal TET3 is dispensable for embryonic development but is required for neonatal growth.

The development of multicellular organisms is accompanied by reprogramming of the epigenome in specific cells, with the epigenome of most cell types becoming fixed after differentiation. Genome-wide reprogramming of DNA methylation occurs in primordial germ cells and in fertilized eggs during mammalian embryogenesis. The 5-methylcytosine (5mC) content of DNA thus undergoes a marked decrease in the paternal pronucleus of mammalian zygotes. This loss of DNA methylation has been thought to be mediated by an active demethylation mechanism independent of replication and to be required for development. TET3-mediated sequential oxidation of 5mC has recently been shown to contribute to the genome-wide loss of 5mC in the paternal pronucleus of mouse zygotes. We now show that TET3 localizes not only to the paternal pronucleus but also to the maternal pronucleus and oxidizes both paternal and maternal DNA in mouse zygotes, although these phenomena are less pronounced in the female pronucleus. Genetic ablation of TET3 in oocytes had no significant effect on oocyte development, maturation, or fertilization or on pregnancy, but it resulted in neonatal sublethality. Our results thus indicate that zygotic 5mC oxidation mediated by maternal TET3 is required for neonatal growth but is not essential for development.

Development of a new benzylating reagent spontaneously releasing benzyl cation equivalents at room temperature.

A new O-benzylating reagent, that is, 4-(4,6-diphenoxy-1,3,5-triazin-2-yl)-4-benzylmorpholinium trifluoromethanesulfonate (DPT-BM), has been developed. Benzyl cation equivalents are generated from DPT-BM by dissolving the compound in a solvent at room temperature under non-acidic conditions. The benzylation of various alcohols by using a combination of DPT-BM and magnesium oxide provided the benzyl ethers in good yields.

The discovery of histone demethylases.

Histone methylation is a key element of the eukaryotic epigenome. Since the discovery of the first histone demethylase (HDM) in 2004, more than 20 demethylases have been identified and characterized. They belong to either the LSD family or the JmjC family, demonstrating the reversibility of all methylation states at almost all major histone lysine methylation sites. These findings ended decades of debate about the reversibility of histone methylation, representing a major breakthrough that shifts our understanding of epigenetic inheritance and regulation of genome function. Here, we summarize the discovery of HDMs and more recent advances, challenges, and future prospects of HDM research.

Identification of the KDM2/7 histone lysine demethylase subfamily inhibitor and its antiproliferative activity.

Histone N(ε)-methyl lysine demethylases KDM2/7 have been identified as potential targets for cancer therapies. On the basis of the crystal structure of KDM7B, we designed and prepared a series of hydroxamate analogues bearing an alkyl chain. Enzyme assays revealed that compound 9 potently inhibits KDM2A, KDM7A, and KDM7B, with IC50s of 6.8, 0.2, and 1.2 µM, respectively. While inhibitors of KDM4s did not show any effect on cancer cells tested, the KDM2/7-subfamily inhibitor 9 exerted antiproliferative activity, indicating the potential for KDM2/7 inhibitors as anticancer agents.

Hydroxylation mediates chromatin demethylation.

Methylation of DNA and histones in chromatin has been implicated in numerous biological processes. For many years, methylation has been recognized as static and stable modification, as compared with other covalent modifications of chromatin. Recently, however, several mechanisms have been demonstrated to be involved in demethylation of chromatin, suggesting that chromatin methylation is more dynamically regulated. One chemical reaction that mediates demethylation of both DNA and histones is hydroxylation, catalysed by Fe(II) and α-ketoglutarate (KG)-dependent hydroxylase/dioxygenase. Given that methylation of chromatin is an important epigenetic mark involved in fundamental biological processes such as cell fate determination, understanding how chromatin methylation is dynamically regulated has implications for human diseases and regenerative medicine.

In vitro histone demethylase assay.

Post-translational modifications of histones play an important role in regulating chromatin dynamics and function. One of the modifications, methylation, occurs on both lysine and arginine residues, and methylation status defines the epigenetic program of a cell by determining chromatin structure and thereby regulating DNA-dependent processes such as transcription. Until recently, histone methylation was considered to be irreversible. However, the discovery of histone demethylases revealed that histone methylation is more dynamic than previously recognized. This protocol describes two different in vitro histone demethylase enzyme reactions and three different methods for measuring histone demethylase activity. The first reaction (type I) uses the Fe(II)- and α-ketoglutarate-dependent dioxygenase family of histone demethylase (represented by JmjC domain-containing histone demethylase [JHDM]); the second (type II) is for the flavin adenine dinucleotide (FAD)-dependent amine oxidase family (represented by lysine-specific demethylase 1 [LSD1]). Histone demethylase activity can then be detected by measuring the release of radiolabeled formaldehyde from (3)H-labeled methylated histone substrates, by monitoring the change in methylation levels of histone substrates by immunoblotting with site-specific methylhistone antibodies, or by using mass spectrometry to detect reductions in histone peptide masses that correspond to methyl groups. These assays can be applied to a wide range of histone demethylase studies, including the measurement of histone demethylase activity in tissue and cell lysates, identification of novel histone demethylases, and screening for inhibitors of histone demethylases.

KDM7 is a dual demethylase for histone H3 Lys 9 and Lys 27 and functions in brain development.

Methylation of histone H3 Lys 9 and Lys 27 (H3K9 and H3K27) is associated with transcriptional silencing. Here we show that KDM7, a JmjC domain-containing protein, catalyzes demethylation of both mono- or dimethylated H3K9 and H3K27. Inhibition of KDM7 orthologs in zebrafish resulted in developmental brain defects. KDM7 interacts with the follistatin gene locus, and KDM7 depletion in mammalian neuronal cells suppressed follistatin gene transcription in association with increased levels of dimethylated H3K9 and H3K27. Our findings identify KDM7 as a dual demethylase for H3K9 and H3K27 that functions as an eraser of silencing marks on chromatin during brain development.

CHD8 suppresses p53-mediated apoptosis through histone H1 recruitment during early embryogenesis.

The chromodomain helicase DNA-binding (CHD) family of enzymes is thought to regulate gene expression, but their role in the regulation of specific genes has been unclear. Here we show that CHD8 is expressed at a high level during early embryogenesis and prevents apoptosis mediated by the tumour suppressor protein p53. CHD8 was found to bind to p53 and to suppress its transactivation activity. CHD8 promoted the association of p53 and histone H1, forming a trimeric complex on chromatin that was required for inhibition of p53-dependent transactivation and apoptosis. Depletion of CHD8 or histone H1 resulted in p53 activation and apoptosis. Furthermore, Chd8(-/-) mice died early during embryogenesis, manifesting widespread apoptosis, whereas deletion of p53 ameliorated this developmental arrest. These observations reveal a mode of p53 regulation mediated by CHD8, which may set a threshold for induction of apoptosis during early embryogenesis by counteracting p53 function through recruitment of histone H1.

The H3K36 demethylase Jhdm1b/Kdm2b regulates cell proliferation and senescence through p15(Ink4b).

The Ink4a-Arf-Ink4b locus has a crucial role in both cellular senescence and tumorigenesis. JmjC domain-containing histone demethylase 1b (Jhdm1b, also known as Kdm2b and Fbxl10), the mammalian paralog of the histone demethylase Jhdm1a (also known as Kdm2a and Fbxl11), has been implicated in cell-cycle regulation and tumorigenesis. In this report, we show that Jhdm1b is a histone H3 lysine 36 (H3K36) demethylase. Knockdown of Jhdm1b in primary mouse embryonic fibroblasts inhibits cell proliferation and induces cellular senescence in a pRb- and p53 pathway-dependent manner. Notably, the effect of Jhdm1b on cell proliferation and cellular senescence is mediated through derepression of p15(Ink4b), as loss of p15(Ink4b) function rescues cell-proliferation defects in Jhdm1b-knockdown cells. Chromatin immunoprecipitation on ectopically expressed Jhdm1b demonstrates that Jhdm1b targets the p15(Ink4b) locus and regulates its expression in an enzymatic activity-dependent manner. Alteration of Jhdm1b level affects Ras-induced neoplastic transformation. Collectively, our results indicate that Jhdm1b is an H3K36 demethylase that regulates cell proliferation and senescence through p15(Ink4b).

Up-regulation of p21CIP1 expression mediated by ERK-dependent and -independent pathways contributes to hepatocyte growth factor-induced inhibition of HepG2 hepatoma cell proliferation.

Strong activation of the ERK signal is required for hepatocyte growth factor (HGF) to inhibit proliferation of the human hepatocellular carcinoma cell line HepG2. However, it is still to be elucidated whether the activation alone is sufficient to induce the inhibitory effect. In this study, we constructed HepG2 cell clones expressing a high level of epidermal growth factor receptor (EGFR), and examined the effect of the strong activation of ERK on the proliferation of the cell clones. EGF treatment of the cell clones induced strong activation of ERK similar to HGF treatment, but did not inhibit cell proliferation. HGF treatment of the cell clones up-regulated the expression of a Cdk inhibitor p16(INK4a), which has previously been shown to be required to inhibit the proliferation of HepG2 cells, but EGF treatment did not. Furthermore, EGF treatment of the cell clones did not induce the up-regulation of another Cdk inhibitor p21(CIP1), whereas HGF treatment did. Knockdown of p21 by siRNA restored the proliferation of HepG2 cells inhibited by HGF, and restored Cdk2 activity suppressed in HGF-treated HepG2 cells. These results suggest that strong activation of ERK alone is not sufficient, and some other pathway(s), which is activated through the HGF receptor but not through EGFR, is also required to induce the up-regulation of p16 and p21 expression, and also suggest that in addition to the up-regulated expression of p16, that of p21 contributes to the suppression of Cdk2 activity leading to the inhibition of proliferation of HGF-treated HepG2 cells.