High histone crotonylation modification in bovine fibroblasts promotes cell proliferation and the developmental efficiency of preimplantation nuclear transfer embryos.

Lysine crotonylation (Kcr) is a recently discovered histone acylation modification that is closely associated with gene expression, cell proliferation, and the maintenance of stem cell pluripotency and indicates the transcriptional activity of genes and the regulation of various biological processes. During cell culture, the introduction of exogenous croconic acid disodium salt (Nacr) has been shown to modulate intracellular Kcr levels. Although research on Kcr has increased, its role in cell growth and proliferation and its potential regulatory mechanisms remain unclear compared to those of histone methylation and acetylation. Our investigation demonstrated that the addition of 5 mM Nacr to cultured bovine fibroblasts increased the expression of genes associated with Kcr modification, ultimately promoting cell growth and stimulating cell proliferation. Somatic cell nuclear transfer of donor cells cultured in 5 mM Nacr resulted in 38.1% blastocyst development, which was significantly greater than that in the control group (25.2%). This research is important for elucidating the crotonylation modification mechanism in fibroblast proliferation to promote the efficacy of somatic cell nuclear transfer.

The signature of cancer methylation markers in maternal plasma: Factors influencing the development and application of cancer liquid biopsy assay.

BACKGROUND: DNA methylation is highly correlated with cancer and embryo development, and plasma-based methylation markers have been widely used for cancer early detection. However, whether the commonly used cancer methylation markers cause "false positives" in the plasma of pregnant women has not been comprehensively evaluated. METHODS: We conducted a case-control study from February 2021 to March 2023, which included 138 pregnant women and 44 control women. Plasma cell-free DNA (cfDNA) was isolated and bisulfite-converted, and then the methylation levels of eight methylated markers related to gastrointestinal cancer (SEPT9, SDC2, C9orf50, KCNQ5, CLIP4, TFPI2, ELMO1 and ZNF582) and three markers related to lung cancer (SHOX2, RASSF1A and PTGER4) were analyzed. RESULTS: When comparing the plasma of pregnant women to that of control women, SEPT9, CLIP4, ZNF582, SHOX2, RASSF1A and PTGER showed significantly higher levels of methylation (p < 0.05). These positive signals originate from the placenta/fetus rather than the mother. We found no discernible difference in DNA methylation levels between fetal cfDNA fractions of < 10 % and ≥ 10 % in pregnant women (p > 0.05), while CLIP4 and PTGER4 showed high methylation levels in the assisted fertilization group compared to the natural fertilization group (p < 0.05). CONCLUSION: Our study shows that cancer and fetus/placenta exhibit similar DNA methylation patterns, and some gastrointestinal cancer and lung cancer-related methylation markers also show positives in maternal plasma. This is an important consideration in the design and application of plasma-based cancer liquid biopsy assays.

A sensitive and robust plasma-based DNA methylation panel for early detection of target gastrointestinal cancers.

BACKGROUND: Target gastrointestinal cancers (GICs), encompassing esophageal cancer (EC), gastric cancer (GC), and colorectal cancer (CRC), originate within a single readily accessible luminal organ system and are diagnosable using endoscopy. However, endoscopy is an invasive procedure with low compliance and no plasma-based DNA methylation assay for the early detection of GICs. METHODS: Nine potential DNA methylation markers were identified and evaluated in tissue (n=60) and plasma (n=155) cohorts to select the most suitable markers. A training cohort (n=244) and a validation cohort (n=199), including GICs patients, benign tumors, gastrointestinal polyps, and controls, were enrolled to develop and validate a DNA methylation panel. An independent prospective cohort (n=158) was used to validate the panel's performance and compare it with blood protein tumor markers. RESULTS: Six out of nine candidate methylation markers with excellent discrimination abilities in both tissue and plasma cohorts were selected for the DNA methylation panel. The panel demonstrated high AUC values of 0.937 (EC), 0.968 (GC), and 0.987 (CRC) in training cohort, and achieved AUC values of 0.921 (EC), 0.921 (GC), and 0.959 (CRC) in validation cohort. Notably, it achieved impressive AUC values of 0.971 and 0.843 for identifying stage I GICs in the training and validation cohorts, respectively. In the prospective cohort, the six-marker panel showed comparable AUC values to CEA, AFP, and CA19-9 (0.935, 0.769, 0.663, and 0.668, respectively). CONCLUSION: This study successfully developed and validated a novel, robust, sensitive, and specific plasma-based DNA methylation panel, offering a promising strategy for the early detection of GICs.

A Novel Plasma-Based Methylation Panel for Upper Gastrointestinal Cancer Early Detection.

BACKGROUND: Upper gastrointestinal cancer (UGC) is an important cause of cancer death in China, with low five-year survival rates due to the majority of UGC patients being diagnosed at an advanced stage. Therefore, there is an urgent need to develop cost-effective, reliable and non-invasive methods for the early detection of UGC. METHODS: A novel plasma-based methylation panel combining simultaneous detection of three methylated biomarkers (ELMO1, ZNF582 and TFPI2) and an internal control gene were developed and used to examine plasma samples from 186 UGC patients and 190 control subjects. RESULTS: The results indicated excellent PCR amplification efficiency and reproducibility of ELMO1, ZNF582 and TFPI2 in the range of 10-100,000 copies per PCR reaction of fully methylated genomic DNA. The methylation levels of ELMO1, ZNF582 and TFPI2 were significantly higher in UGC samples than those in control subjects. The sensitivities of ELMO1, ZNF582 and TFPI2 alone for UGC detection were 32.3%, 61.3% and 30.6%, respectively; when three markers were combined, the sensitivity was improved to 71.0%, with a specificity of 90.0%, and the area under the curve (AUC) was 0.870 (95% CI: 0.832-0.902). CONCLUSION: Methylated ELMO1, ZNF582 and TFPI2 were specific for UGC and the three-methylated gene panel provided an alternative non-invasive choice for UGC early detection.

Growth Traits and Sperm Proteomics Analyses of Myostatin Gene-Edited Chinese Yellow Cattle.

Chinese Yellow Cattle, an ancient and domesticated breed for draft service, provide unique animal genetic resources with excellent genetic features, including crude feed tolerance, good stress resistance, strong adaptability, and tender meat quality; however, their production performance and meat yield are significantly inferior. Herein, the myostatin gene (MSTN), a negative regulator of skeletal muscle development, was knocked out by CRISPR/Cas9 technology. Eight MSTN gene-edited bull calves (MT) were born, and six of them are well-developed. Compared with the control cattle (WT), the growth trait indexes of MT cattle were generally increased, and the hindquarters especially were significantly improved. The biochemical indexes and the semen characteristics demonstrated that MT bulls were healthy and fertile. Consistent with our conjecture, the wobble and beating of MT bull spermatozoa were significantly higher than that of WT. Nine sperm motility-related proteins and nineteen mitochondrial-related proteins were identified by up-regulation in MT bull spermatozoa using FLQ proteomic technique and act to govern sperm flagellum assembly, organization, and beating and provide sufficient energy for sperm motility. The current study confirmed that the MSTN gene-edited Chinese Yellow cattle have improved growth traits and normal fertility, which can be used for beef cattle production and breeding.

Comparative analysis morphology, anatomical structure and transcriptional regulatory network of chlorophyll biosynthesis in Oryza longistaminata, O. sativa and their F1 generation.

Oryza longistaminata, a perennial wild species, is widely distributed in the African continent. It has strong tolerance to biotic and abiotic stresses, and high biomass production on poor soils. Chlorophyll biosynthesis is important for photosynthesis in rice. However, the chlorophyll biosynthesis and related gene profiles of O. longistaminata and its descendants remained unclear. Here, the F1 generation of O. sativa and O. longistaminata were obtained. Then, the comparative analysis morphology, anatomical structure, and transcriptional regulatory networks of chlorophyll biosynthesis were detected and analyzed. Results showed that the F1 generation has obvious long awn, similar with that of the male parent. The purple color of the long awn is different from that of the male parent. Microstructural results showed that the flag leaves of F1 have large mesophyll cell gaps in the upper- and lower-positions, small mesophyll cell gaps in the middle position, and more chloroplasts. Increased chlorophyll content was also observed in the F1 generation. In the lower-position flag leaves, the total chlorophyll contents of F1 were 1.55 and 1.5 times those of O. sativa and O. longistaminata, respectively. POR, MgCH and HEMA1 showed higher expression levels than the other related genes selected in the chlorophyll biosynthesis pathway. The HEMA1 expression level in the middle-position flag leaves of O. longistaminata was the highest, and it was 2.83 and 2.51 times that of O. sativa and F1, respectively. The expression level of DVR gene in lower-position flag leaves of F1 were 93.16% and 95.06% lower than those of O. sativa and O. longistaminata, respectively. This study provided a potential reference for studying the photosynthesis and heterosis utilization of O. longistaminata.

Transient Dux expression facilitates nuclear transfer and induced pluripotent stem cell reprogramming.

Cloned animals generated by somatic cell nuclear transfer (SCNT) have been reported for many years; however, SCNT is extremely inefficient, and zygotic genome activation (ZGA) is required for SCNT-mediated somatic cell reprogramming. To identify candidate factors that facilitate ZGA in SCNT-mediated reprogramming, we performed siRNA-repressor and mRNA-inducer screenings, which reveal Dux, Dppa2, and Dppa4 as key factors enhancing ZGA in SCNT. We show that direct injection of ZGA inducers has no significant effect on SCNT blastocyst formation; however, following the establishment of an inducible Dux transgenic mouse model, we demonstrate that transient overexpression of Dux not only improves SCNT efficiency but also increases that of chemically induced pluripotent stem cell reprogramming. Moreover, transcriptome profiling reveals that Dux-treated SCNT embryos are similar to fertilized embryos. Furthermore, transient overexpression of Dux combined with inactivation of DNA methyltransferases (Dnmts) further promotes the full embryonic development of SCNT-derived animals. These findings enhance our understanding of ZGA-regulator function in somatic reprogramming.

Melatonin restores the pluripotency of long-term-cultured embryonic stem cells through melatonin receptor-dependent m6A RNA regulation.

N6-methyladenosine (m6A) methylation is the most common and abundant modification on mammalian messenger RNA (mRNA) and regulates the pluripotency of embryonic stem cells (ESCs). Research has shown that melatonin plays a fundamental role in DNA and histone modifications. However, the effect of melatonin on RNA modification is unknown. Here, for the first time, we investigated the effect of melatonin on m6A modifications in long-term-cultured ESCs. Pluripotency studies indicated that 10 µmol/L melatonin sufficiently maintained ESCs with stemness features over 45 passages (more than 90 days). Notably, treatment of ESCs with melatonin led to a significant decrease in the nuclear presence of m6A methyltransferase complex and decreased global m6A modification. Depletion of melatonin receptor 1 (MT1) by CRISPR/Cas9 significantly reduced the effects of melatonin on ESC pluripotency and m6A modification. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) revealed that melatonin promotes stabilization of core pluripotency factors, such as Nanog, Sox2, Klf4, and c-Myc, by preventing m6A-dependent mRNA decay. Using cell signaling pathway profiling systems, melatonin was shown to regulate m6A modification predominantly through the MT1-JAK2/STAT3-Zfp217 signal axis. This study reveals a new dimension regarding melatonin regulation of gene expression at the RNA level.

Histone Demethylase UTX is an Essential Factor for Zygotic Genome Activation and Regulates Zscan4 Expression in Mouse Embryos.

Following fertilization, the zygotic genome is activated through a process termed zygotic genome activation (ZGA), which enables zygotic gene products to replace the maternal products and initiates early embryonic development. During the ZGA period, the embryonic epigenome experiences extensive recodifications. The H3K27me3 demethylase UTX is essential for post-implantation embryonic development. However, it remains unclear whether UTX participates in preimplantation development, especially during the ZGA process. In the present study, we showed that either knockdown or overexpression of UTX led to embryonic development retardation, whereas simultaneous depletion of UTX and overexpression of ZSCAN4D rescued the embryonic development, indicating that UTX positively regulated Zscan4d expression. Using a transgenic mice model, we also found that UTX was required for preimplantation embryonic development. In conclusion, these results indicate that UTX functions as a novel regulator and plays critical roles during ZGA in addition to early embryonic development.

Generation of Fad2 and Fad3 transgenic mice that produce n-6 and n-3 polyunsaturated fatty acids.

Linoleic acid (18 : 2, n-6) and α-linolenic acid (18 : 3, n-3) are polyunsaturated fatty acids (PUFAs), which are essential for mammalian health, development and growth. However, the majority of mammals, including humans, are incapable of synthesizing n-6 and n-3 PUFAs. Mammals must obtain n-6 and n-3 PUFAs from their diet. Fatty acid desaturase (Fad) plays a critical role in plant PUFA biosynthesis. Therefore, we generated plant-derived Fad3 single and Fad2-Fad3 double transgenic mice. Compared with wild-type mice, we found that PUFA levels were greatly increased in the single and double transgenic mice by measuring PUFA levels. Moreover, the concentration of n-6 and n-3 PUFAs in the Fad2-Fad3 double transgenic mice were greater than in the Fad3 single transgenic mice. These results demonstrate that the plant-derived Fad2 and Fad3 genes can be expressed in mammals. To clarify the mechanism for Fad2 and Fad3 genes in transgenic mice, we measured the PUFAs synthesis-related genes. Compared with wild-type mice, these Fad transgenic mice have their own n-3 and n-6 PUFAs biosynthetic pathways. Thus, we have established a simple and efficient method for in vivo synthesis of PUFAs.

Inhibiting repressive epigenetic modification promotes telomere rejuvenation in somatic cell reprogramming.

The efficiency of somatic cell nuclear transfer (SCNT) reprogramming is extremely low in terms of production of cloned animals. Here, we found that telomere rejuvenation is a critical event for SCNT reprogramming. Through small-molecule screening, we identified that melatonin significantly improved the in vitro and in vivo developmental competence of SCNT-derived embryos. Through use of embryonic biopsy, single-cell RNA sequencing, and quantitative FISH experiments, we revealed that melatonin not only attenuated the zygotic genome activation defect but also facilitated telomere elongation in the SCNT embryos. Further investigation indicated that melatonin inhibited heterochromatic epigenetic modification related to gene silencing including DNA methylation and histone H3 lysine 9 trimethylation. In addition, melatonin could increase the level of activation markers such as acetylated histone H3. This is the first study to characterize melatonin-treatment and telomere rejuvenation in SCNT-mediated reprogramming. Moreover, combinational use of melatonin-treated donor embryos and pseudopregnant recipients achieved synergistic enhancement of the production of cloned animals.-Yang, L., Liu, X., Song, L., Su, G., Di, A., Bai, C., Wei, Z., Li, G. Inhibiting repressive epigenetic modification promotes telomere rejuvenation in somatic cell reprogramming.

KDM6A and KDM6B play contrasting roles in nuclear transfer embryos revealed by MERVL reporter system.

Despite the success of animal cloning by somatic cell nuclear transfer (SCNT) in many species, the method is limited by its low efficiency. After zygotic genome activation (ZGA) during mouse development, a large number of endogenous retroviruses (ERVs) are expressed, including the murine endogenous retrovirus-L (MuERVL/MERVL). In this study, we generate a series of MERVL reporter mouse strains to detect the ZGA event in embryos. We show that the majority of SCNT embryos do not undergo ZGA, and H3K27me3 prevents SCNT reprogramming. Overexpression of the H3K27me3-specific demethylase KDM6A, but not of KDM6B, improves the efficiency of SCNT Conversely, knockdown of KDM6B not only facilitates ZGA, but also impedes ectopic Xist expression in SCNT reprogramming. Furthermore, knockdown of KDM6B increases the rate of SCNT-derived embryonic stem cells from Duchenne muscular dystrophy embryos. These results not only provide insight into the mechanisms underlying failures of SCNT, but also may extend the applications of SCNT.

The Maternal Effect Genes UTX and JMJD3 Play Contrasting Roles in Mus musculus Preimplantation Embryo Development.

During the process of embryonic development in mammals, epigenetic modifications must be erased and reconstructed. In particular, the trimethylation of histone 3 lysine 27 (H3K27me3) is associated with gene-specific transcriptional repression and contributes to the maintenance of the pluripotent embryos. In this study, we determined that the global levels of the H3K27me3 marker were elevated in MII oocyte chromatin and decrease to minimal levels at the 8-cell and morula stages. When the blastocyst hatched, H3K27me3 was re-established in the inner cell mass. We also determined that H3K27me3-specific demethylases, UTX and JMJD3, were observed at high transcript and protein levels in mouse preimplantation embryos. In the activated oocytes, when the H3K27me3 disappeared at the 8-cell stage, the UTX (but not JMJD3) protein levels were undetectable. Using RNA interference, we suppressed UTX and JMJD3 gene expression in the embryos and determined that the functions of UTX and JMJD3 were complementary. When JMJD3 levels were decreased by RNA interference, the embryo development rate and quality were improved, but the knockdown of UTX produced the opposite results. Understanding the epigenetic mechanisms controlling preimplantation development is critical to comprehending the basis of embryonic development and to devise methods and approaches to treat infertility.

Expression levels of GSTA2 and APOD genes might be associated with carotenoid coloration in golden pheasant (Chrysolophus pictus) plumage.

Carotenoids, which generate yellow, orange, and red colors, are crucial pigments in avian plumage. Investigations into genes associated with carotenoidbased coloration in avian species are important; however, such research is difficult because carotenoids cannot be synthetized in vertebrates as they are only derived from dietary sources. Here, the golden pheasant (Chrysolophus pictus) was used as a model in analysis of candidate gene expression profiles implicated in carotenoid binding and deposition. Using mass and Raman spectrometry to confirm the presence of carotenoids in golden pheasant feathers, we found C40H54O and C40H56O2 in feathers with yellow to red colors, and in the rachis of iridescent feathers. The global gene expression profiles in golden pheasant skins were analyzed by RNA-seq and all six carotenoid binding candidate genes sequenced were studied by realtime PCR. StAR4, GSTA2, Scarb1, and APOD in feather follicles showed different expressions in red breast and orange nape feathers compared with that of iridescent mantle feathers. Further comparison of golden pheasant yellow rump and Lady Amherst's pheasant (Chrysolophus amherstiae) white nape feathers suggested that GSTA2 and APOD played a potential role in carotenoid-based coloration in golden pheasant.