2C Gene Research Articles

Lactylation of Hdac1 regulated by Ldh prevents the pluripotent-to-2C state conversion.

Cellular metabolism regulates the pluripotency of embryonic stem cells (ESCs). Yet, how metabolism regulates the transition among different pluripotent states remains elusive. It has been shown that protein lactylation, which uses lactate, a metabolic product of glycolysis, as a substrate, plays a critical role in various biological events. Here we focused on that glycolysis regulates the conversion between ESCs and 2-cell-like cells (2CLCs) through protein lactylation. RNA-seq revealed the activation of 2-cell (2C) genes by suppression of Ldh. Stable isotope labeling by amino acids in cell culture (SILAC) coupled with lactylated peptide enrichment and quantitative mass spectrometric analysis was carried out to investigate the mechanism how protein lactylation regulates the pluripotent-to-2C transition. And we focused on Hdac1. Lactylation of Hdac1 required for silencing 2C genes was proved by quantitative reverse-transcription PCR (qRT-PCR), immunofluorescence (IF), Western blot and chimeric embryos. Chromatin immunoprecipitation coupled with sequencing (ChIP-seq) and in vitro deacetylation assay confirmed lactylation of Hdac1 promoting its binding at 2C genes and enhancing its deacetylase activity, thereby facilitating the removal of H3K27ac and the silencing of 2C genes. We found that inhibition or depletion of Ldha, the enzyme converting pyruvate to lactate, leads to the activation of 2C genes, as well as reduced global lactylation in ESCs. To investigate the mechanism how protein lactylation regulates the pluripotent-to-2C transition, quantitative lactylome analysis was performed, and 1716 lactylated proteins were identified. We then focused on Hdac1, a histone deacetylase involved in the silencing of 2C genes. Lactylation of Hdac1 promotes its binding at 2C genes and enhances its deacetylase activity, thus facilitating the removal of H3K27ac and the silencing of 2C genes. In summary, our study reveals a mechanistic link between cellular metabolism and pluripotency regulation through protein lactylation. Our research is the first time to reveal that quantitative lactylome analysis in mouse ESCs. We found that lactylated Hdac1 promotes its binding at 2C genes and enhances its deacetylase activity, thus facilitating the removal of H3K27ac and the silencing of 2C genes.

Isolated Lateralized Overgrowth - Phenotypic Spectrum and Molecular Alterations.

To evaluate the molecular aberrations at 11p15.5 locus in thirty-two patients with isolated lateralized overgrowth (ILO). Among selected 32 cases of ILO, methylation-sensitive multiplex ligation-dependent probe amplification (MS-MLPA) was performed initially followed by short tandem repeats (STR) marker analysis to confirm uniparental disomy (UPD). In those patients with normal MLPA reports, cyclin dependent kinase inhibitor 1C (CDKN1C) gene and whole exome sequencing was performed. Molecular analysis by MS-MLPA showed methylation aberrations in 28% (9/32) of patients. Gain of methylation at IC1 imprinting center (H4, H7) and loss of methylation at IC2 (H6, H9) was observed in 2 patients each. Uniparental disomy was observed in 9% cases. Except one, all patients with methylation aberration had more than one limb hypertrophy. Two patients (H22/H29) also had loss of methylation at IC1. Though this molecular alteration is specifically associated with Silver Russel syndrome (SRS), but the affected children did not completely fulfill the diagnostic criteria for SRS. In a recent study, a discrepancy was reported between the diagnosis of Beckwith-Wiedemann syndrome (BWS)/SRS and the molecular findings in the patients. Many times, it is very difficult to differentiate between hemi hypertrophy/hemi hypotrophy. Patients, in whom no aberrations were detected on MS-MLPA, whole exome sequencing (WES) was performed and no pathogenic variant was identified. Thus, ILO may be considered as a mild presentation on the extreme edge of BWS spectrum with methylation aberration and UPDin one thirdof cases which has implications in follow up.

Evaluating the potential impact of sodium-glucose cotransporter-2 inhibitor "canagliflozin" on the hepatic damage triggered by hypertension in rats.

Hypertension (HTN) is a prevalent chronic disease. HTN and liver disease association is extensively noted. Thus, finding a medication that can alleviate HTN and its accompanying liver insult would be promising. This study investigated the potential impacts of canagliflozin "sodium-glucose cotransporter-2 inhibitor" on the liver of the Nω-nitro-L-arginine methyl ester (L-NAME)-induced HTN rat model. Twenty-four adult male rats were divided into four groups; negative control group, canagliflozin group, L-NAME group: 50 mg/kg of L-NAME was injected daily for 5 weeks and L-NAME + canagliflozin group: 1 week after L-NAME injection both L-NAME + canagliflozin (40 mg/kg) were given concomitantly daily for further 4 weeks. Liver functions, serum lipid profile, hepatic oxidative/nitrative stress biomarkers, gene expression of lipogenic enzymes, B-cell lymphoma 2 (Bcl2), and DNA fragmentation, were measured. Besides, hepatic histology and immunohistochemistry of nuclear factor kappa B (NF-κB) and endothelial nitric oxide synthase (eNOS) were assessed. Canagliflozin improved hepatic lipogenesis via the downregulation of fatty acid synthase (FAS) and transcriptional regulatory element binding protein 1c (SREBP1c) genes leading to an improved serum lipid profile. Further, canagliflozin modified the eNOS/inducible nitric oxide synthase (iNOS) pathway and decreased the NF-κB immunoreactivity besides restoring the oxidants-antioxidants balance; increased reduced glutathione concomitant with declined malondialdehyde. This improvement of the liver was mirrored by the significant restoration of liver architecture and confirmed by the preserved liver DNA content and upregulation of the antiapoptotic Bcl2 mRNA level and attenuation of the alanine transaminase, aspartate aminotransferase. In conclusion, canagliflozin is a promising anti-hypertensive and hepatic-supportive medication. RESEARCH HIGHLIGHTS: Canagliflozin's antioxidant, anti-inflammatory, anti-lipogenic, and antiapoptotic characteristics mitigate remote liver compromise caused by hypertension. Canagliflozin can be exploited as a hepatoprotective and antihypertensive medication.

FOXO3 Inhibits the Cisplatin Resistance and Progression of Melanoma Cells by Promoting CDKN1C Transcription.

Forkhead box O3 (FOXO3) and cyclin dependent kinase inhibitor 1C Gene (CDKN1C) have been shown to be involved in the melanoma process, but their roles in the cisplatin (DDP) resistance of melanoma remain unclear. The mRNA levels of CDKN1C and FOXO3 were measured using quantitative real-time PCR. The protein levels of CDKN1C, FOXO3 and mitochondrial oxidative phosphorylation (mtOXPHOS)-related markers were determinant by western blot analysis. The DDP resistance, proliferation, and apoptosis of melanoma cells were assessed by cell counting kit 8 assay, colony formation assay and flow cytometry. Glucose consumption, lactate production and ATP level were detected to assess glycolysis. The regulation of FOXO3 on CDKN1C was confirmed by ChIP assay and dual-luciferase reporter assay. In vivo experiments were performed to evaluate the effect of FOXO3 on DDP sensitivity in melanoma tumor tissues. CDKN1C and FOXO3 were downregulated in chemoresistant melanoma tissues, and their low expression levels were related to the poor prognosis of melanoma patients. Overexpression of CDKN1C and FOXO3 repressed DDP resistance, proliferation, and glycolysis, while promoted apoptosis and mtOXPHOS in DDP-resistant melanoma cells. Further analysis suggested that FOXO3 could bind to CDKN1C promoter region to enhance its transcription. Besides, CDKN1C knockdown reversed the regulation of FOXO3 on melanoma cell DDP resistance and progression. Moreover, FOXO3 overexpression enhanced the DDP sensitivity of melanoma tumor tissues in vivo. FOXO3 promoted the transcription of CDKN1C, thereby inhibiting the DDP resistance and progression of melanoma cells.

Abstract 5606: The role of KMT2C in the homologous recombination repair in epithelial ovarian cancer

Abstract Background: Epithelial ovarian cancer (EOC) with homologous recombination (HR) deficiency is susceptible to PARP inhibitor therapy due to synthetic lethality. Identification of the patient population for PARP inhibitor therapy is imperative to reduce recurrence of EOC and improve the survival outcomes of patients. Current biomarkers for PARP inhibitor therapy consist of an array of genes involved in the HR repair pathway in EOC, including BRCA1, BRCA2 and PALB2. Through TCGA analysis, we found that the lysine (K)-specific methyltransferase 2C (KMT2C) gene is mutated in 10.3% of all EOC patients and considerably enriched in BRCA1 mutated EOC patients. However, the role of KMT2C in HR repair and PARP inhibitor sensitivity of EOC remains largely undefined. Methods: BRCA2-mutated PEO1 and BRCA2-wild type PEO1-NR cells were transfected with KMT2C siRNA and assayed for KMT2C protein expression by western blotting. DR-GFP (HR) and EJ5-GFP (NHEJ)-SKOV3 cell lines were co-transfected with the ISceI-expressing plasmid pCBA-ISceI and KMT2C siRNA. GFP-positive cells were analyzed by flow cytometry. SKOV3 cells were transfected with KMT2C siRNA and the expression of BRCA2 and Rad51 mRNA was determined using quantitative RT-PCR analysis. Results: siRNA-mediated knockdown of KMT2C resulted in marked down-regulation of KMT2C protein in PEO1 and PEO1-NR cells. KMT2C knockdown caused a significant decrease in ISceI-induced HR activity in DR-GFP-SKOV3 cells (p < 0.05). In contrast, KMT2C knockdown had no effects on ISceI-induced NHEJ activity in EJ5-GFP-SKOV3 cells. As a control, Rad51 knockdown caused complete suppression of HR activity and had no effects on NHEJ activity. Furthermore, quantitative RT-PCR analysis of key HR genes showed that KMT2C knockdown resulted in a moderate but significant down-regulation of BRCA2 (p < 0.05) and Rad51 (p < 0.01) expression in SKOV3 cells. Conclusion: Our results suggest that KMT2C partakes in the HR repair pathway in EOC. However, the contribution of KMT2C to HR activity is not rate-limiting. Based on the known function of KMT2C in histone modification, we speculate that KMT2C serves to positively regulate the transcription of HR genes and therefore promote HR activity. Future investigation into the exact roles of KMT2C in HR repair, etiology of EOC, and diagnostic biomarkers for the PARP inhibitor sensitivity of EOC is warranted. Citation Format: Mirielle C. Ma, Z. Ping Lin, Elena S. Ratner. The role of KMT2C in the homologous recombination repair in epithelial ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5606.

Genome-wide identification and characterization of protein phosphatase 2C (PP2C) gene family in sunflower (Helianthus annuus L.) and their expression profiles in response to multiple abiotic stresses.

Plant protein phosphatase 2C (PP2C) plays vital roles in responding to various stresses, stimulating growth factors, phytohormones, and metabolic activities in many important plant species. However, the PP2C gene family has not been investigated in the economically valuable plant species sunflower (Helianthus annuus L.). This study used comprehensive bioinformatics tools to identify and characterize the PP2C gene family members in the sunflower genome (H. annuus r1.2). Additionally, we analyzed the expression profiles of these genes using RNA-seq data under four different stress conditions in both leaf and root tissues. A total of 121 PP2C genes were identified in the sunflower genome distributed unevenly across the 17 chromosomes, all containing the Type-2C phosphatase domain. HanPP2C genes are divided into 15 subgroups (A-L) based on phylogenetic tree analysis. Analyses of conserved domains, gene structures, and motifs revealed higher structural and functional similarities within various subgroups. Gene duplication and collinearity analysis showed that among the 53 HanPP2C gene pairs, 48 demonstrated segmental duplications under strong purifying selection pressure, with only five gene pairs showing tandem duplications. The abundant segmental duplication was observed compared to tandem duplication, which was the major factor underlying the dispersion of the PP2C gene family in sunflowers. Most HanPP2C proteins were localized in the nucleus, cytoplasm, and chloroplast. Among the 121 HanPP2C genes, we identified 71 miRNAs targeting 86 HanPP2C genes involved in plant developmental processes and response to abiotic stresses. By analyzing cis-elements, we identified 63 cis-regulatory elements in the promoter regions of HanPP2C genes associated with light responsiveness, tissue-specificity, phytohormone, and stress responses. Based on RNA-seq data from two sunflower tissues (leaf and root), 47 HanPP2C genes exhibited varying expression levels in leaf tissue, while 49 HanPP2C genes showed differential expression patterns in root tissue across all stress conditions. Transcriptome profiling revealed that nine HanPP2C genes (HanPP2C12, HanPP2C36, HanPP2C38, HanPP2C47, HanPP2C48, HanPP2C53, HanPP2C54, HanPP2C59, and HanPP2C73) exhibited higher expression in leaf tissue, and five HanPP2C genes (HanPP2C13, HanPP2C47, HanPP2C48, HanPP2C54, and HanPP2C95) showed enhanced expression in root tissue in response to the four stress treatments, compared to the control conditions. These results suggest that these HanPP2C genes may be potential candidates for conferring tolerance to multiple stresses and further detailed characterization to elucidate their functions. From these candidates, 3D structures were predicted for six HanPP2C proteins (HanPP2C47, HanPP2C48, HanPP2C53, HanPP2C54, HanPP2C59, and HanPP2C73), which provided satisfactory models. Our findings provide valuable insights into the PP2C gene family in the sunflower genome, which could play a crucial role in responding to various stresses. This information can be exploited in sunflower breeding programs to develop improved cultivars with increased abiotic stress tolerance.

The genome of Haberlea rhodopensis provides insights into the mechanisms for tolerance to multiple extreme environments

Haberlea rhodopensis, a resurrection species, is the only plant known to be able to survive multiple extreme environments, including desiccation, freezing temperatures, and long-term darkness. However, the molecular mechanisms underlying tolerance to these stresses are poorly studied. Here, we present a high-quality genome of Haberlea and found that ~ 23.55% of the 44,306 genes are orphan. Comparative genomics analysis identified 89 significantly expanded gene families, of which 25 were specific to Haberlea. Moreover, we demonstrated that Haberlea preserves its resurrection potential even in prolonged complete darkness. Transcriptome profiling of plants subjected to desiccation, darkness, and low temperatures revealed both common and specific footprints of these stresses, and their combinations. For example, PROTEIN PHOSPHATASE 2C (PP2C) genes were substantially induced in all stress combinations, while PHYTOCHROME INTERACTING FACTOR 1 (PIF1) and GROWTH RESPONSE FACTOR 4 (GRF4) were induced only in darkness. Additionally, 733 genes with unknown functions and three genes encoding transcription factors specific to Haberlea were specifically induced/repressed upon combination of stresses, rendering them attractive targets for future functional studies. The study provides a comprehensive understanding of the genomic architecture and reports details of the mechanisms of multi-stress tolerance of this resurrection species that will aid in developing strategies that allow crops to survive extreme and multiple abiotic stresses.

Strigolactone and abscisic acid synthesis and signaling pathways are enhanced in the wheat oligo-tillering mutant ot1

Tiller number greatly contributes to grain yield in wheat. Using ethylmethanesulfonate mutagenesis, we previously discovered the oligo-tillering mutant ot1. The tiller number was significantly lower in ot1 than in the corresponding wild type from the early tillering stage until the heading stage. Compared to the wild type, the thousand-grain weight and grain length were increased by 15.41% and 31.44%, respectively, whereas the plant height and spike length were decreased by 26.13% and 37.25%, respectively. Transcriptomic analysis was conducted at the regreening and jointing stages to identify differential expressed genes (DEGs). Functional enrichment analysis with the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) databases showed differential expression of genes associated with ADP binding, transmembrane transport, and transcriptional regulation during tiller development. Differences in tiller number in ot1 led to the upregulation of genes in the strigolactone (SL) and abscisic acid (ABA) pathways. Specifically, the SL biosynthesis genes DWARF (D27), D17, D10, and MORE AXILLARY GROWTH 1 (MAX1) were upregulated by 3.37- to 8.23-fold; the SL signal transduction genes D14 and D53 were upregulated by 1.81- and 1.32-fold, respectively; the ABA biosynthesis genes 9-CIS-EPOXICAROTENOID DIOXIGENASE 3 (NCED3) and NCED5 were upregulated by 1.66- and 3.4-fold, respectively; and SNF1-REGULATED PROTEIN KINASE2 (SnRK2) and PROTEIN PHOSPHATASE 2C (PP2C) genes were upregulated by 1.30- to 4.79-fold. This suggested that the tiller number reduction in ot1 was due to alterations in plant hormone pathways. Genes known to promote tillering growth were upregulated, whereas those known to inhibit tillering growth were downregulated. For example, PIN-FORMED 9 (PIN9), which promotes tiller development, was upregulated by 8.23-fold in ot1; Ideal Plant Architecture 1 (IPA1), which inhibits tiller development, was downregulated by 1.74-fold. There were no significant differences in the expression levels of TILLER NUMBER 1 (TN1) or TEOSINTE BRANCHED 1 (TB1), indicating that the tiller reduction in ot1 was not controlled by known genes. Our findings provide valuable data for subsequent research into the genetic bases and regulatory mechanisms of wheat tillering.

A novel 11 base pair deletion in KMT2C resulting in Kleefstra syndrome 2.

Haploinsufficiency of the Lysine Methyltransferase 2C (KMT2C) gene results in the autosomal dominant disorder, Kleefstra syndrome 2. It is an extremely rare neurodevelopmental condition, with 14 previous reports describing varied clinical manifestations including dysmorphic features, delayed psychomotor development and delayed growth. Here, we describe a female with global developmental delay, attention deficit disorder, dyspraxia, short stature and subtle non-specific dysmorphic features. To identify causative mutations, whole exome sequencing was performed on the proband and her younger brother with discrete clinical presentation. Whole exome sequencing identified a novel de novo heterozygous 11 bp deletion in KMT2C (c.1759_1769del), resulting in a frameshift mutation and early termination of the protein (p.Gln587SerfsTer7). This variant is the second-most N-terminal reported mutation, located 4171 amino acids upstream of the critical enzymatically active SET domain (required for chromatin modification and histone methylation). The majority of the other reported mutations are frameshift mutations upstream of the SET domain and are predicted to result in protein truncation. It is thought that truncation of the SET domain, results functionally in an inability to modify chromatin through histone methylation. This report expands the clinical and genetic characterisation of Kleefstra syndrome 2.

Frequency of Vancomycin and Aminoglycoside Resistance Genes in Enterococcus spp. Isolated from Chicken Raw Meat Samples

Background: Enterococci are main normal microbial flora of both humans and animals and can survive in a diverse range of environments. These bacteria carry out aminoglycoside and vancomycin resistance genes and spread them in environment by many routs such as chicken meat products. The present study was aimed to determine the frequency of aminoglycoside and vancomycin resistant genes in Enterococcus species isolated from chicken meat specimens.
 Methods: A total of 250 chicken raw meat specimens was prepared from slaughterhouses at Zanjan province, cultured at BHI broth and incubated at 37°C for 24h. The positive cultures were sub-cultured in blood agar plates and grown colonies identified using phenotypical and biochemical tests. Antibiotic susceptibility was determined according to the Clinical and Laboratory Standards Institute (CLSI) standards and PCR assays were performed to detect vanA, vanB, aph (2")1c, aph (2")1b, aph (2")1d, ant(3'), aph(3')IIIa, ant(4')1a, ant(6') and aac(6') genes.
 Results: In total, 100 Enterococcus species isolated from 250 specimens and 35% of them belonged to E. faecalis and the others were E. faecium (65%). The prevalence of the vanA, vanB, aph (2")1c, aph (2")1b, aph (2")1d, ant(3'), aph(3')IIIa, ant(4')1a, ant(6') and aac(6') genes among the 100 Enterococcus species was 14%, 12%, 10%, 1%, 2%, 50%, 26%, 9%, 18% and 22%, respectively.
 Conclusion: The current study revealed that the rate of antimicrobial resistance genes to aminoglycosides and vancomycin was worrying and health measurements in meat products industries must be performed to prevent spread of antimicrobial resistance elements among bacteria.

Prognostic analysis of mutated genes and insight into effects of DNA damage and repair on mutational strand asymmetries in gastric cancer

Gastric cancer (GACA) is a complex and multifaceted disease influenced by a variety of environmental and genetic factors. Somatic mutations play a major role in its development, and their characteristics, including the asymmetry between two DNA strands, are of great interest and appear as a signal of information and guidance, revealing mechanisms of DNA damage and repair. Here, we analyzed the impact of High-frequency mutated genes on patient prognosis and found that the effect of expression levels of tumor protein p53 (TP53) and lysine methyltransferase 2C (KMT2C) genes remained high throughout the development of GACA, with similar expression patterns. After investigating mutation asymmetry across mutagenic processes, we found that transcriptional asymmetry was dominated by T > G mutations under the influence of transcription couples repair and damage. The apolipoprotein B mRNA editing enzyme catalytic polypeptide like (APOBEC) enzyme that induces mutations during DNA replication has been identified here and we identified a replicative asymmetry, which was dominated by C > A mutations in left-replicating. Strand bias in different mutation classes at transcription factor binding sites and enhancer regions were also confirmed, which implies the important role of non-coding regulatory elements in the occurrence of mutations. This work systematically describes mutational strand asymmetries in specific genomic regions, shedding light on the DNA damage and repair mechanisms underlying somatic mutations in cohorts of GACA patients with gastric cancer.

Patulin alleviates hepatic lipid accumulation by regulating lipogenesis and mitochondrial respiration

AimsThe aim of this study is to evaluate the effects of patulin on hepatic lipid metabolism and mitochondrial oxidative function and elucidate the underlying molecular mechanisms. Main methodsThe effects of patulin on hepatic lipid accumulation were evaluated in free fatty acid-treated AML12 or HepG2 cells through oil red O staining, triglyceride assay, real-time polymerase chain reaction, and western blotting. Alteration of mitochondrial oxidative capacity by patulin treatment was determined using Seahorse analysis to measure the oxygen consumption rate. Key findingsThe increased amounts of lipid droplets induced by free fatty acids were significantly reduced by patulin treatment. Patulin markedly activated the CaMKII/AMP-activated protein kinase (AMPK)/proliferator-activated receptor-γ coactivator (PGC)-1α signaling pathway in hepatocytes, reduced the expression of sterol regulatory element binding protein 1c (SREBP-1c) and lipogenic genes, and increased the expression of genes related to mitochondrial fatty acid oxidation. In addition, patulin treatment enhanced the mitochondrial consumption rate and increased the expression of mitochondrial oxidative phosphorylation proteins in HepG2 hepatocytes. The effects of patulin on anti-lipid accumulation; SREBP-1c, PGC-1α, and carnitine palmitoyltransferase 1 expression; and mitochondrial oxidative capacity were significantly prevented by compound C, an AMPK inhibitor. SignificancePatulin is a potent inducer of the AMPK pathway, and AMPK-mediated mitochondrial activation is required for the efficacy of patulin to inhibit hepatic lipid accumulation. This study is the first to report that patulin is a promising bioactive compound that prevents the development and worsening of fatty liver diseases, including non-alcoholic fatty liver disease, by improving mitochondrial quality and lipid metabolism.

Nucleolus assembly impairment leads to two-cell transcriptional repression via NPM1-mediated PRC2 recruitment.

The nucleolus is a compartmentalized organelle in eukaryotic cells known to form during embryogenesis, yet how its layered architecture is transformed from homogenous precursor bodies is unclear, and any impacts of this formation on embryonic cell fate determination remain unknown. Here, we demonstrate that lncRNA LoNA tethers granular-component-enriched NPM1 to dense-fibrillar-component-enriched FBL and drives the formation of compartmentalized nucleolus via facilitating liquid-liquid phase separation of those two nucleolar proteins. Phenotypically, LoNA-deficient embryos show developmental arrest at the two-cell (2C) stage. Mechanistically, we demonstrate that LoNA deficiency leads to nucleolar formation failure, resulting in mislocalization and acetylation of NPM1 in the nucleoplasm. Acetylated NPM1 recruits and guides PRC2 complex to 2C genes, where PRC2 complex trimethylates H3K27, leading to transcriptional repression of these genes. Collectively, our findings reveal that lncRNA is required for the establishment of nucleolar structure, and this process has an impact on two-cell embryonic development via 2C transcriptional activation.

Dppa3 Improves the Germline Competence of Pluripotent Stem Cells.

Chimera formation and germline competence are critical features of mouse pluripotent stem cells (PSCs). However, the factors that contribute to germline competence in the chimeras remain to be understood. To determine the role of Dppa3 in PSCs, we first constructed Dppa3 knockout (Dppa3 KO) and Dppa3 overexpression (Dppa3 OE) PSCs, respectively. Using Dppa3 KO and Dppa3 OE PSCs, we then investigated the role of Dppa3 in PSCs by evaluating the chimera generation, DNA methylation, and pluripotent state conversion. We show that Dppa3 plays an important role in chimera formation and germline competence of mouse PSCs. PSC lines with high expression of Dppa3 show high germline competence. In contrast, Dppa3 deficiency reduces chimera formation and abrogates the germline transmission capacity of PSCs. Molecularly, Dppa3 facilitates establishing global DNA hypomethylation in PSCs. High levels of Dppa3 in PSCs reduce the expression of Dnmt3a/b and impede Uhrf1-Dnmt1 complex binding to DNA replication forks, maintaining DNA hypomethylation. Additionally, Dppa3 facilitates two-cell-stage (2C) genes expression and promotes conversion to a 2C-like state. These data show that Dppa3 is involved in maintaining DNA hypomethylation homeostasis and is required for high chimera formation and germline competence of PSCs.

RBBP4 is an epigenetic barrier for the induced transition of pluripotent stem cells into totipotent 2C-like cells.

Cellular totipotency is critical for whole-organism generation, yet how totipotency is established remains poorly illustrated. Abundant transposable elements (TEs) are activated in totipotent cells, which is critical for embryonic totipotency. Here, we show that the histone chaperone RBBP4, but not its homolog RBBP7, is indispensable for maintaining the identity of mouse embryonic stem cells (mESCs). Auxin-induced degradation of RBBP4, but not RBBP7, reprograms mESCs to the totipotent 2C-like cells. Also, loss of RBBP4 enhances transition from mESCs to trophoblast cells. Mechanistically, RBBP4 binds to the endogenous retroviruses (ERVs) and functions as an upstream regulator by recruiting G9a to deposit H3K9me2 on ERVL elements, and recruiting KAP1 to deposit H3K9me3 on ERV1/ERVK elements, respectively. Moreover, RBBP4 facilitates the maintenance of nucleosome occupancy at the ERVK and ERVL sites within heterochromatin regions through the chromatin remodeler CHD4. RBBP4 depletion leads to the loss of the heterochromatin marks and activation of TEs and 2C genes. Together, our findings illustrate that RBBP4 is required for heterochromatin assembly and is a critical barrier for inducing cell fate transition from pluripotency to totipotency.

Behavioral and Transcriptomic Changes Following Brain-Specific Loss of Noradrenergic Transmission.

Noradrenaline (NE) plays an integral role in shaping behavioral outcomes including anxiety/depression, fear, learning and memory, attention and shifting behavior, sleep-wake state, pain, and addiction. However, it is unclear whether dysregulation of NE release is a cause or a consequence of maladaptive orientations of these behaviors, many of which associated with psychiatric disorders. To address this question, we used a unique genetic model in which the brain-specific vesicular monoamine transporter-2 (VMAT2) gene expression was removed in NE-positive neurons disabling NE release in the entire brain. We engineered VMAT2 gene splicing and NE depletion by crossing floxed VMAT2 mice with mice expressing the Cre-recombinase under the dopamine β-hydroxylase (DBH) gene promotor. In this study, we performed a comprehensive behavioral and transcriptomic characterization of the VMAT2DBHcre KO mice to evaluate the role of central NE in behavioral modulations. We demonstrated that NE depletion induces anxiolytic and antidepressant-like effects, improves contextual fear memory, alters shifting behavior, decreases the locomotor response to amphetamine, and induces deeper sleep during the non-rapid eye movement (NREM) phase. In contrast, NE depletion did not affect spatial learning and memory, working memory, response to cocaine, and the architecture of the sleep-wake cycle. Finally, we used this model to identify genes that could be up- or down-regulated in the absence of NE release. We found an up-regulation of the synaptic vesicle glycoprotein 2c (SV2c) gene expression in several brain regions, including the locus coeruleus (LC), and were able to validate this up-regulation as a marker of vulnerability to chronic social defeat. The NE system is a complex and challenging system involved in many behavioral orientations given it brain wide distribution. In our study, we unraveled specific role of NE neurotransmission in multiple behavior and link it to molecular underpinning, opening future direction to understand NE role in health and disease.

Design and Construction of Carboxylesterase 2c Gene Knockout Rats by CRISPR/Cas9.

Carboxylesterase 2 (CES2) is mainly distributed in the human liver and gut, and plays an active role in the metabolic activation of many prodrugs and lipid metabolism. Although CES2 is of great significance, there are still few animal models related to CES2. This research aims to construct Ces2c gene knockout (KO) rats and further study the function of CES2. CRISPR/Cas9 gene editing technology was used to target and cleave the rat Ces2c gene. Compensatory effects of major CES subtypes both in the liver and small intestine of KO rats were detected at mRNA levels. Meanwhile, diltiazem and aspirin were used as substrates to test the metabolic capacity of Ces2c in KO rats. This Ces2c KO rat model showed normal growth and breeding without off-target effects. The metabolic function of Ces2c KO rats was verified by the metabolic study of CES2 substrates in vitro. The results showed that the metabolic capacity of diltiazem in KO rats was weakened, while the metabolic ability of aspirin did not change significantly. In addition, the serum physiological indexes showed that the Ces2c deletion did not affect the liver function of rats.. The Ces2c KO rat model was successfully constructed by CRISPR/Cas9 system. This rat model can not only be used as an important tool to study the drug metabolism mediated by CES2, but also as an important animal model to study the physiological function of CES2.

Genetic variation within the melatonin receptor 1C (MTNR1C) gene in the native chicken ‘Zo-ar’ of Mizoram, India

The study has been aimed to uncover the presence of polymorphism within the MTNR1C gene in native chicken population, ‘Zo-ar’ of the Mizoram state of India and to calculate the allelic and genotypic frequency to screen the variation present within the population with respect to the MTNR1C gene which is associated with various egg production traits in poultry. DNA extraction has been done with the help of blood samples collected from a total of 50 randomly chosen ‘Zo-ar’ chicken irrespective of age or sex from different regions of Mizoram. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay has been used for the detection of SNPs within the MTNR1C gene. Two genotypes AG (0.34) and GG (0.66) have been detected for the MTNR1C gene in the ‘Zo-ar’ chicken population while the AA (0.00) genotype was found to be completely absent in the present study. The G allele (0.83) was found to be predominantly present in the populations. Further, the population was found to be conforming to the Hardy-Weinberg Equilibrium with respect to the MTNR1C locus. From the present study the presence of variation within the MTNR1C locus was seen in the ‘Zo-ar’ chicken of Mizoram. This indicates the possibility of genetic improvement in egg production traits by using proper selection methods and formulating appropriate mating systems.

RNA analysis of diet-induced sarcopenic obesity in rats

Obesity has been suggested as a risk factor for sarcopenia. Sarcopenic obesity (SO), as a new category of obesity, is a high-risk geriatric syndrome in elderly individuals. However, knowledge about the molecular pathomechanisms of SO is still sparse. In the present study, starting at 13 months, male Sprague-Dawley (SD) rats were fed a high-fat diet (HFD) and normal diet (ND) for 28 weeks to establish a rodent animal model of SO with an identical protocol, which was further assessed and verified as a successful SO model. Through RNA-seq analysis of gastrocnemius muscle in SO rats, we found that differentially expressed genes (DEGs) and alternative splicing events (ASEs) focused mainly on inflammatory, immune-response, skeletal muscle cell differentiation, fat cell differentiation and antigen processing and presentation. Furthermore, as the core regulation factor of skeletal muscle, the mef2c (myocyte enhancer Factor 2C) gene also has a significant alternative 3′ splice site (A3SS) and down-regulated expression in HFD-induced SO. The alternative genes targeted by mef2c identified by GO analysis were enriched in transcript regulation of RNA polymerase II promoter. In conclusion, these explorative findings in aging high-fat-fed rats might serve as a firm starting point for understanding the pathway and mechanism of sarcopenic obesity.

First detection and molecular characteristics of bopivirus from goats in China.

A metavirome analysis was performed and detected bopivirus in the diarrhoeal fecal samples of goats in China. A total of 136 fecal samples were collected from yeanlings between the dates of June 2021 and January 2022 in Sichuan province, China. Moreover, "Bopivirus B" strains were detected by a specific RT-PCR targeting the 3D gene of the virus. The results showed that the overall detection rate of "Bopivirus B" was 19.12% (26/136). Additionally, there was a higher detection rate (24.05%, 19/79) in the fecal samples collected from yeanlings with diarrhea compared to those from asymptomatic animals (12.28%, 7/57). In these samples, no other common diarrhea-causing pathogens were detected except for three enteric viruses, namely caprine enterovirus, caprine kobuvirus and caprine hunnivirus (with detection rates of 13.97, 13.97, and 8.82%, respectively). Subsequently, full-length VP4, VP2, VP3, and VP1 genes from "Bopivirus B"-positive samples were amplified, cloned, sequenced, and analyzed. The phylogenetic analysis performed on the VP1 genes revealed that the identified bopivirus belonged to genotype B1 (seven strains) and B2 (three strains) and presented a high genetic diversity. Furthermore, a complete genome sequence of a "Bopivirus B" strain (SWUN/B1/2022) was obtained using PCR from fecal sample of a diarrhoeal yeanling. The complete genome was 7,309 nucleotides in length with a standard picornavirus genome organization, and shares 93.10% and 91.10% nucleotide similarity with bopivirus B1 genotype strain ovine/TB14/2010-HUN and bopivirus B2 genotype strain goat/AGK16/2020-HUN, respectively. According to the species classification criteria put forward by the International Committee on Taxonomy of Viruses and VP1 genotype, the strain SWUN/B1/2022 belongs to the bopivirus B1. This strain has unique amino acid substitutions in the VP4, VP2, VP3, and VP1 genes. Moreover, genomic recombination analysis revealed that this strain may be a minor parental strain of bopivirus B1 ovine/TB14/2010-HUN. Evolutionary analysis based on the 2C and 3CD genes revealed that the new bopivirus B1 strain SWUN/B1/2022 presents a unique evolutionary pattern. This study provided evidence to suggest that "Bopivirus B" is circulating with substantial genetic diversity in goats in China at present, and the mixed infection of "Bopivirus B" with other enteric viruses should be considered to be a composite factor in the occurrence of viral diarrhea in goats.