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 epigenetic 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 IVF but not SCNT. RRRs are enriched for H3K9me3 in donor somatic cells, and its removal by ectopic expression of the 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

Crystal Structure of the Plant Epigenetic Protein Arginine Methyltransferase 10

Protein arginine methyltransferase 10 (PRMT10) is a type-I arginine methyltransferase essential for regulating flowering time in Arabidopsis thaliana (At). We present a 2.6 Å resolution crystal structure of AtPRMT10 in complex with a reaction product, S-adenosylhomocysteine. The structure reveals a dimerization arm 12–20 residues longer than PRMT structures elucidated previously; as a result, the essential AtPRMT10 dimer exhibits a large central cavity and a distinctly accessible active site. We employ molecular dynamics to examine how dimerization facilitates AtPRMT10 motions necessary for activity, and show that these motions are conserved in other PRMT enzymes. Finally, functional data reveal that the N-terminal ten residues of AtPRMT10 influence substrate specificity, and that enzyme activity is dependent on substrate protein sequences distal from the methylation site. Taken together, these data provide insights into the molecular mechanism of Arabidopsis thaliana PRMT10 as well as other members of the PRMT family of enzymes. They highlight differences between AtPRMT10 and other PRMTs, but also indicate that motions are a conserved element of PRMT function

Loss of HDAC-Mediated Repression and Gain of NF-κB Activation Underlie Cytokine Induction in ARID1A- and PIK3CA-Mutation-Driven Ovarian Cancer

ARID1A is frequently mutated in ovarian clear cell carcinoma (OCCC) and often co-exists with activating mutations of PIK3CA. Although induction of pro-inflammatory cytokines has been observed in this cancer, the mechanism by which the two mutations synergistically activate cytokine genes remains elusive. Here, we established an in vitro model of OCCC by introducing ARID1A knockdown and mutant PIK3CA into a normal human ovarian epithelial cell line, resulting in cell transformation and cytokine gene induction. We demonstrate that loss of ARID1A impairs the recruitment of the Sin3A-HDAC complex, while the PIK3CA mutation releases RelA from IκB, leading to cytokine gene activation. We show that an NF-κB inhibitor partly attenuates the proliferation of OCCC and improves the efficacy of carboplatin both in cell culture and in a mouse model. Our study thus reveals the mechanistic link between ARID1A/PIK3CA mutations and cytokine gene induction in OCCC and suggests that NF-κB inhibition could be a potential therapeutic option

Role of Tet proteins in enhancer activity and telomere elongation

DNA methylation at the C-5 position of cytosine (5mC) is one of the best-studied epigenetic modifications and plays important roles in diverse biological processes. Iterative oxidation of 5mC by the ten-eleven translocation (Tet) family of proteins generates 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5fC and 5caC are selectively recognized and excised by thymine DNA glycosylase (TDG), leading to DNA demethylation. Functional characterization of Tet proteins has been complicated by the redundancy between the three family members. Using CRISPR/Cas9 technology, we generated mouse embryonic stem cells (ESCs) deficient for all three Tet proteins (Tet triple knockout [TKO]). Whole-genome bisulfite sequencing (WGBS) analysis revealed that Tet-mediated DNA demethylation mainly occurs at distally located enhancers and fine-tunes the transcription of genes associated with these regions. Functional characterization of Tet TKO ESCs revealed a role for Tet proteins in regulating the two-cell embryo (2C)-like state under ESC culture conditions. In addition, Tet TKO ESCs exhibited increased telomere–sister chromatid exchange and elongated telomeres. Collectively, our study reveals a role for Tet proteins in not only DNA demethylation at enhancers but also regulating the 2C-like state and telomere homeostasis

Tet3 and DNA Replication Mediate Demethylation of Both the Maternal and Paternal Genomes in Mouse Zygotes

With the exception of imprinted genes and certain repeats, DNA methylation is globally erased during preimplantation development. Recent studies have suggested that Tet3-mediated oxidation of 5-methylcytosine (5mC) and DNA replication-dependent dilution both contribute to global paternal DNA demethylation, but demethylation of the maternal genome occurs via replication. Here we present genome-scale DNA methylation maps for both the paternal and maternal genomes of Tet3-depleted and/or DNA replication-inhibited zygotes. In both genomes, we found that inhibition of DNA replication blocks DNA demethylation independently from Tet3 function and that Tet3 facilitates DNA demethylation largely by coupling with DNA replication. For both genomes, our data indicate that replication-dependent dilution is the major contributor to demethylation, but Tet3 plays an important role, particularly at certain loci. Our study thus defines the respective functions of Tet3 and DNA replication in paternal DNA demethylation and reveals an unexpected contribution of Tet3 to demethylation of the maternal genome. •Tet3 only partially mediates paternal DNA demethylation•DNA replication is the major contributor to paternal DNA demethylation•Tet3-dependent DNA demethylation also occurs on the maternal genome•Zygotic gene activation is independent of Tet3 activity Using genome-scale DNA methylation analyses of manually isolated paternal and maternal pronuclei, Zhang and colleagues show that zygotic demethylation of both genomes is mediated by Tet3 and DNA replication, with the latter as the major contributor

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

Regulation of flowering time by the protein arginine methyltransferase AtPRMT10

In plants, histone H3 lysine methyltransferases are important in gene silencing and developmental regulation; however, the roles of histone H4 methylation in plant development remain unclear. Recent studies found a type II histone arginine methyltransferase, AtPRTM5, which is involved in promoting growth and flowering. Here, we purified a dimerized plant-specific histone H4 methyltransferase, plant histone arginine methyltransferase 10 (PHRMT10), from cauliflower. Arabidopsis thaliana protein arginine methyltransferase 10 (AtPRMT10)—the Arabidopsis homologue of PHRMT10—was shown to be a type I PRMT, which preferentially asymmetrically methylated histone H4R3 in vitro. Genetic disruption of AtPRMT10 resulted in late flowering by upregulating FLOWERING LOCUS C (FLC) transcript levels. In addition, we show that AtPRMT10 functions genetically separate from AtPRMT5, but that each acts to fine-tune expression of FLC. This work adds an extra layer of complexity to flowering-time regulation and also sheds light on the importance of asymmetric arginine methylation in plant development

Microbial Community Shifts with Soil Properties and Enzyme Activities in Inter-/Mono-Cropping Systems in Response to Tillage

No-till and cereal–legume intercropping have been recognized as favorable cropping practices to increase crop yields while maintaining soil quality in arid and semiarid environments, but the biological mechanisms are poorly understood. The present study aimed to determine the response of yields, soil properties, enzyme activities, and microbial community diversity and composition in mono- and inter-cropping under conventional and no-tillage conditions. We initiated a field experiment in Wuwei, a typical arid area of China, in 2014. Soil was sampled in August 2022 and, yields, soil properties, enzyme activities, and the microbial community diversity and composition were determined in the maize and pea strips in inter- and mono-cropping systems. Results revealed that the maize and pea strips in the no-till intercropping significantly increased yields, total and organic carbon stocks, decreased NO3−-N, and obtained the highest total and organic P in the soil. No-tillage significantly enhanced the Shannon index and Pielou evenness of the bacterial community and total microbial community over conventional tillage, with the α-diversity of the bacterial community and total microbial community distinctly higher in the NTIM treatment than in the CTIM treatment. The α-diversity of the total microbial community was significantly related to yield, soil IC and OC, and the α-diversity of the archaea community was significantly related to soil TC, TC/TP, TN/TP, and BX. Meanwhile, the α-diversity of the eukaryote community was significantly related to soil yield, soil TC/TP. Both no-tillage and intercropped maize significantly increased the abundance of archaea phylum Thaumarchaeota and bacterial phylum Nitrospirae, and were significantly positively associated with soil OC and NH4+-N, benefiting nitrogen fixation of intercropped pea from the atmosphere under the no-tillage cereal/legume intercropping. No-till intercropping was conducive to the accumulation of organic carbon, while decreasing the abundance of Proteobacteria, Acidobacteria, and Verrucomicrobia. Limited soil enzyme activities (ACP, ALP, DP, NAG, BG, AG, CB) led to decreases in organic carbon turnover and utilization. Intercropping altered soil microbial community diversity and composition due to changes in soil properties and enzyme activities. These findings suggest that no-tilled cereal–legume intercropping is a sustainable cropping practice for improving soil properties and enhancing microbial (archaea, bacterial, eukaryota) diversity, but the persistence is not conducive to rapid turnover of soil nutrients due to limited enzyme activities