Unfaithful Maintenance of Methylation Imprints Due to Loss of Maternal Nuclear Dnmt1 during Somatic Cell Nuclear Transfer

The low success rate of somatic cell nuclear transfer (SCNT) in mammalian cloning is largely due to imprinting problems. However, little is known about the mechanisms of reprogramming imprinted genes during SCNT. Parental origin-specific DNA methylation regulates the monoallelic expression of imprinted genes. In natural fertilization, methylation imprints are established in the parental germline and maintained throughout embryonic development. However, it is unclear whether methylation imprints are protected from global changes of DNA methylation in cloned preimplantation embryos. Here, we demonstrate that cloned porcine preimplantation embryos exhibit demethylation at differentially methylated regions (DMRs) of imprinted genes; in particular, demethylation occurs during the first two cell cycles. By RNAi-mediated knockdown, we found that Dnmt1 is required for the maintenance of methylation imprints in porcine preimplantation embryos. However, no clear signals were detected in the nuclei of oocytes and preimplantation embryos by immunofluorescence. Thus, Dnmt1 is present at very low levels in the nuclei of porcine oocytes and preimplantation embryos and maintains methylation imprints. We further showed that methylation imprints were rescued in nonenucleated metaphase II (MII) oocytes. Our results indicate that loss of Dnmt1 in the maternal nucleus during SCNT significantly contributes to the unfaithful maintenance of methylation imprints in cloned embryos

Transgene Expression Is Associated with Copy Number and Cytomegalovirus Promoter Methylation in Transgenic Pigs

Transgenic animals have been used for years to study gene function, produce important proteins, and generate models for the study of human diseases. However, inheritance and expression instability of the transgene in transgenic animals is a major limitation. Copy number and promoter methylation are known to regulate gene expression, but no report has systematically examined their effect on transgene expression. In the study, we generated two transgenic pigs by somatic cell nuclear transfer (SCNT) that express green fluorescent protein (GFP) driven by cytomegalovirus (CMV). Absolute quantitative real-time PCR and bisulfite sequencing were performed to determine transgene copy number and promoter methylation level. The correlation of transgene expression with copy number and promoter methylation was analyzed in individual development, fibroblast cells, various tissues, and offspring of the transgenic pigs. Our results demonstrate that transgene expression is associated with copy number and CMV promoter methylation in transgenic pigs

Determination of Multicomponents in Rubi Fructus by Near-Infrared Spectroscopy Technique

Objective. Rubi Fructus (RF) is a dry aggregate fruit of Rubus (Rosaceae). It has shown significant pharmacological effects such as anti-oxidation, hypoglycemic, and anti-inflammatory. A combination of near-infrared (NIR) spectroscopy and partial least squares regression (PLSR) under seven different spectral data preprocessing techniques was used to determine the performance of quantitative analysis correction models which employed moisure, ellagic acid, and total flavonoids as indicators of RF. Methods. Ninety-seven different RF batches were collected for NIR spectra. By using primary analysis techniques such as drying method, high-performance liquid chromatography (HPLC), and ultraviolet visible spectrophotometry (UV-Vis), the contents of moisure, ellagic acid, and total flavonoids were determined. The NIR spectral data and the primary analysis method data were correlated through PLSR. Seven methods were used for pretreating the spectral data, including no spectral pretreatment, first derivative, standard normalized variate, multiple scattering corrections, elimination of constant offset, and minimum maximum normalization. The quantitative analysis correction models adopted PLSR chemometrics for moisture, ellagic acid and total flavonoids were developed, and their effectiveness was evaluated using the correlation coefficient (R), ratio of prediction to deviation (RPD), and root mean square error (RMSE). Results. The first derivative was combined with variable standardization, elimination of constant offset, and multiple scattering corrections, respectively, to pretreat the PLSR models for moisture, ellagic acid, and total flavonoids. The R-values of the PLSR models for moisture, ellagic acid, and total flavonoids were, respectively, 0.9788, 0.9468, and 0.9748, all of which were higher than 0.90, and the RPD values were 4.9, 3.1, and 4.5, respectively, which were all larger than 3.0. The RMSE ratios of the calibration set and the test set were 0.98, 0.94, and 1.0, respectively. Conclusion. The R-values of the NIR-PLSR models for moisture, ellagic acid, and total flavonoids are all greater than 0.90 after suitable pretreatments, indicating that the models are reliable. The RPD values are more than 3.0, which indicate that the models are good and useable for quality control. The RMSE ratios are closed to 1, indicating that the calibration set and test set had same distribution and the models were not overfitting indicating good predictability

The expression and location of Dnmt1 in oocytes.

<p>A, the cytoplasmic location of Dnmt1 in GV and MII stage oocytes (×400), and B, relative Dnmt1 transcription levels during oocyte maturation. No obvious changes of Dnmt1 expression were observed in GV, GVBD, MI and MII stage oocytes. GV, GVBD, MI and MII stage oocytes were collected at 0 h, 19 h, 24 h and 42 h, respectively.</p

A Novel Role for <i>DNA Methyltransferase 1</i> in Regulating Oocyte Cytoplasmic Maturation in Pigs

<div><p>Maternal factors are required for oocyte maturation and embryo development. To better understand the role of <i>DNA methyltransferase 1</i> (<i>Dnmt1</i>) in oocyte maturation and embryo development, small interfering RNA (siRNA) was conducted in porcine oocytes. In this study, our results showed that <i>Dnmt1</i> localized in oocyte cytoplasm and its expression displayed no obvious change during oocyte maturation. When siRNAs targeting <i>Dnmt1</i> were injected into germinal vesicle (GV) stage oocytes, <i>Dnmt1</i> transcripts significantly decreased in matured oocytes (P<0.05). After <i>Dnmt1</i> knockdown in GV stage oocytes, the significant reduction of glutathione content, mitochondrial DNA copy number, glucose-6-phosphate dehydrogenase activity and expression profiles of maternal factors and the severely disrupted distribution of cortical granules were observed in MII stage oocytes (P<0.05), leading to the impaired oocyte cytoplasm. Further study displayed that <i>Dnmt1</i> knockdown in GV stage oocytes significantly reduced the development of early embryos generated through parthenogenetic activation, <i>in vitro</i> fertilization and somatic cell nuclear transfer (P<0.05). In conclusion, <i>Dnmt1</i> was indispensable for oocyte cytoplasmic maturation, providing a novel role for <i>Dnmt1</i> in the regulation of oocyte maturation.</p></div

Blastocysts derived from PA, IVF and SCNT after Dnmt1 knockdown.

<p>After siRNA injection into GV stage oocytes, the matured oocytes were subjected to PA, IVF and SCNT, and the morphologies of responding blastocysts (×40) were observed at 156 h after embryo culture.</p

The interference efficiencies among different siRNAs.

<p>siRNA-RFD, siRNA targeting the replication foci domain (RFD) of Dnmt1, siRNA-BAH, siRNA targeting the bromo adjacent homology domain (BAH) of Dnmt1, and siRNA-DCM, siRNA targeting the cytosine-C5 specific DNA methylase domain (DCM) of Dnmt1. After siRNAs were injected into GV stage oocytes, the interference efficiencies were mensurated in matured MII stage oocytes. siRNA-RFD, siRNA-BAH and siRNA-DCM significantly reduced the expression of Dnmt1, and the interference level of siRNA-DCM was the highest. ^a-eValues with different superscripts differed significantly (P<0.05).</p

Partial erosion on under-methylated regions and chromatin reprogramming contribute to oncogene activation in IDH mutant gliomas

Abstract Background IDH1/2 hotspot mutations are well known to drive oncogenic mutations in gliomas and are well-defined in the WHO 2021 classification of central nervous system tumors. Specifically, IDH mutations lead to aberrant hypermethylation of under-methylated regions (UMRs) in normal tissues through the disruption of TET enzymes. However, the chromatin reprogramming and transcriptional changes induced by IDH-related hypermethylation in gliomas remain unclear. Results Here, we have developed a precise computational framework based on Hidden Markov Model to identify altered methylation states of UMRs at single-base resolution. By applying this framework to whole-genome bisulfite sequencing data from 75 normal brain tissues and 15 IDH mutant glioma tissues, we identified two distinct types of hypermethylated UMRs in IDH mutant gliomas. We named them partially hypermethylated UMRs (phUMRs) and fully hypermethylated UMRs (fhUMRs), respectively. We found that the phUMRs and fhUMRs exhibit distinct genomic features and chromatin states. Genes related to fhUMRs were more likely to be repressed in IDH mutant gliomas. In contrast, genes related to phUMRs were prone to be up-regulated in IDH mutant gliomas. Such activation of phUMR genes is associated with the accumulation of active H3K4me3 and the loss of H3K27me3, as well as H3K36me3 accumulation in gene bodies to maintain gene expression stability. In summary, partial erosion on UMRs was accompanied by locus-specific changes in key chromatin marks, which may contribute to oncogene activation. Conclusions Our study provides a computational strategy for precise decoding of methylation encroachment patterns in IDH mutant gliomas, revealing potential mechanistic insights into chromatin reprogramming that contribute to oncogenesis

The GSH content and mtDNA copy number per oocyte.

<p>A, the GSH content, and B, the mtDNA copy number per matured oocyte after Dnmt1 knockdown. After siRNA injection into GV stage oocytes, the GSH content and mtDNA copy number per oocyte were significantly reduced in MII stage oocytes. The number of oocytes detected was on the top of the column chart, and ^※Values with a star marker in the same column chart differed significantly (P<0.05).</p