Sirtuin 3 (SIRT3) is a potential therapeutic target for cardiovascular, metabolic, and other aging-related diseases. In this study, we investigated the role of SIRT3 in diabetic cardiomyopathy (DCM). ...Mice were injected with streptozotocin (STZ, 60 mg/kg, ip) to induce diabetes mellitus. Our proteomics analysis revealed that SIRT3 expression in the myocardium of diabetic mice was lower than that of control mice, as subsequently confirmed by real-time PCR and Western blotting. To explore the role of SIRT3 in DCM, SIRT3-knockout mice and 129S1/SvImJ wild-type mice were injected with STZ. We found that diabetic mice with SIRT3 deficiency exhibited aggravated cardiac dysfunction, increased lactate dehydrogenase (LDH) level in the serum, decreased adenosine triphosphate (ATP) level in the myocardium, exacerbated myocardial injury, and promoted myocardial reactive oxygen species (ROS) accumulation. Neonatal rat cardiomyocytes were transfected with SIRT3 siRNA, then exposed to high glucose (HG, 25.5 mM). We found that downregulation of SIRT3 further increased LDH release, decreased ATP level, suppressed the mitochondrial membrane potential, and elevated oxidative stress in HG-treated cardiomyocytes. SIRT3 deficiency further raised expression of necroptosis-related proteins including receptor-interacting protein kinase 1 (RIPK1), RIPK3, and cleaved caspase 3, and upregulated the expression of inflammation-related proteins including NLR family pyrin domain-containing protein 3 (NLRP3), caspase 1 p20, and interleukin-1β both in vitro and in vivo. Collectively, SIRT3 deficiency aggravated hyperglycemia-induced mitochondrial damage, increased ROS accumulation, promoted necroptosis, possibly activated the NLRP3 inflammasome, and ultimately exacerbated DCM in the mice. These results suggest that SIRT3 can be a molecular intervention target for the prevention and treatment of DCM.
DNA demethylation can occur passively by “dilution” of methylation marks by DNA replication, or actively and independently of DNA replication. Direct conversion of 5‐methylcytosine (5mC) to cytosine ...(C), as originally proposed, does not occur. Instead, active DNA methylation involves oxidation of the methylated base by ten‐eleven translocations (TETs), or deamination of the methylated or a nearby base by activation induced deaminase (AID). The modified nucleotide, possibly together with surrounding nucleotides, is then replaced by the BER pathway. Recent data clarify the roles and the regulation of well‐known enzymes in this process. They identify base excision repair (BER) glycosylases that may cooperate with or replace thymine DNA glycosylase (TDG) in the base excision step, and suggest possible involvement of DNA damage repair pathways other than BER in active DNA demethylation. Here, we review these new developments.
DNA methylation is not forever. It can be lost passively during DNA replication or removed actively. Active erase of methylation begins with modification of the methylated or a nearby nucleotide by AID‐mediated deamination or TET‐mediated oxidation, and ends with replacement of the modified nucleotide by DNA damage repair pathways.
The repertoire of nucleobase methylation in DNA and RNA, introduced by chemical agents or enzymes, is large. Most methylation can be reversed either directly by restoration of the original nucleobase ...or indirectly by replacement of the methylated nucleobase with an unmodified nucleobase. In many direct and indirect demethylation reactions, ALKBH (AlkB homolog) and TET (ten eleven translocation) hydroxylases play a role. Here, we suggest a chemical classification of methylation types. We then discuss pathways for removal, emphasizing oxidation reactions. We highlight the recently expanded repertoire of ALKBH- and TET-catalyzed reactions and describe the discovery of a TET-like protein that resembles the hydroxylases but uses an alternative co-factor and catalyzes glyceryl transfer rather than hydroxylation.
The prevalent DNA modification in higher organisms is the methylation of cytosine to 5-methylcytosine (5mC), which is partially converted to 5-hydroxymethylcytosine (5hmC) by the Tet (ten eleven ...translocation) family of dioxygenases. Despite their importance in epigenetic regulation, it is unclear how these cytosine modifications are reversed. Here, we demonstrate that 5mC and 5hmC in DNA are oxidized to 5-carboxylcytosine (5caC) by Tet dioxygenases in vitro and in cultured cells. 5caC is specifically recognized and excised by thymine-DNA glycosylase (TDG). Depletion of TDG in mouse embyronic stem cells leads to accumulation of 5caC to a readily detectable level. These data suggest that oxidation of 5mC by Tet proteins followed by TDG-mediated base excision of 5caC constitutes a pathway for active DNA demethylation.
The epigenomes of mammalian sperm and oocytes, characterized by gamete-specific 5-methylcytosine (5mC) patterns, are reprogrammed during early embryogenesis to establish full developmental potential. ...Previous studies have suggested that the paternal genome is actively demethylated in the zygote while the maternal genome undergoes subsequent passive demethylation via DNA replication during cleavage. Active demethylation is known to depend on 5mC oxidation by Tet dioxygenases and excision of oxidized bases by thymine DNA glycosylase (TDG). Here we show that both maternal and paternal genomes undergo widespread active and passive demethylation in zygotes before the first mitotic division. Passive demethylation was blocked by the replication inhibitor aphidicolin, and active demethylation was abrogated by deletion of Tet3 in both pronuclei. At actively demethylated loci, 5mCs were processed to unmodified cytosines. Surprisingly, the demethylation process was unaffected by the deletion of TDG from the zygote, suggesting the existence of other demethylation mechanisms downstream of Tet3-mediated oxidation.
Display omitted
•Maternal and paternal genomes both undergo active demethylation in mouse zygotes•Both zygotic genomes also undergo replication-dependent passive demethylation•At actively demethylated loci, 5mCs are processed to unmodified cytosines•Active demethylation depends on Tet3 dioxygenase, but not on TDG glycosylase
In one-cell mouse embryos, the maternal and paternal genomes both undergo global Tet3-dependent active demethylation and replication-mediated passive demethylation. Surprisingly, the active demethylation pathway does not require TDG.
Postnatal growth of mammalian oocytes is accompanied by a progressive gain of DNA methylation, which is predominantly mediated by DNMT3A, a de novo DNA methyltransferase
. Unlike the genome of sperm ...and most somatic cells, the oocyte genome is hypomethylated in transcriptionally inert regions
. However, how such a unique feature of the oocyte methylome is determined and its contribution to the developmental competence of the early embryo remains largely unknown. Here we demonstrate the importance of Stella, a factor essential for female fertility
, in shaping the oocyte methylome in mice. Oocytes that lack Stella acquire excessive DNA methylation at the genome-wide level, including in the promoters of inactive genes. Such aberrant hypermethylation is partially inherited by two-cell-stage embryos and impairs zygotic genome activation. Mechanistically, the loss of Stella leads to ectopic nuclear accumulation of the DNA methylation regulator UHRF1
, which results in the mislocalization of maintenance DNA methyltransferase DNMT1 in the nucleus. Genetic analysis confirmed the primary role of UHRF1 and DNMT1 in generating the aberrant DNA methylome in Stella-deficient oocytes. Stella therefore safeguards the unique oocyte epigenome by preventing aberrant de novo DNA methylation mediated by DNMT1 and UHRF1.
Tet-mediated DNA oxidation is a recently identified mammalian epigenetic modification, and its functional role in cell-fate transitions remains poorly understood. Here, we derive mouse embryonic ...fibroblasts (MEFs) deleted in all three Tet genes and examine their capacity for reprogramming into induced pluripotent stem cells (iPSCs). We show that Tet-deficient MEFs cannot be reprogrammed because of a block in the mesenchymal-to-epithelial transition (MET) step. Reprogramming of MEFs deficient in TDG is similarly impaired. The block in reprogramming is caused at least in part by defective activation of key miRNAs, which depends on oxidative demethylation promoted by Tet and TDG. Reintroduction of either the affected miRNAs or catalytically active Tet and TDG restores reprogramming in the knockout MEFs. Thus, oxidative demethylation to promote gene activation appears to be functionally required for reprogramming of fibroblasts to pluripotency. These findings provide mechanistic insight into the role of epigenetic barriers in cell-lineage conversion.
Display omitted
•Tet dioxygenases and TDG glycosylase are essential for fibroblast reprogramming•Tet and TDG mediate demethylation and reactivation of miRNAs critical for MET•Tet enzymes are not required for the reactivation of pluripotency loci
Using triple Tet gene knockout cells, Hu et al. show that Tet activity is required for reprogramming of fibroblasts to iPSCs for activation of key miRNA expression during the MET step.
Innate immunity is the first line of defense against infections, which functions as a significant role in resisting pathogen invasion. Rapid immune response is initiated by pattern recognition ...receptors (PRRs) quickly distinguishing "self" and "non-self." Upon evolutionarily conserved pathogen-associated molecular pattern (PAMP) is recognized by PRRs, innate immune response against infection is triggered via an orchestration of molecular interaction, cytokines cascades, and immune cells. RIG-I plays a critical role in type I interferon (IFN-I) production by direct recognition of cytoplasmic double-stranded viral RNA. However, the activation mechanism of RIG-I is incompletely understood. In this study, we reported RNA-binding protein ZFP36 as a positive regulator of RIG-I-mediated IFN-I production. ZFP36 is a member of Zinc finger proteins (ZFPs) characterized by the zinc finger (ZnF) motif, which broadly involved gene transcription and signal transduction. However, its role in regulating antiviral innate immune signaling is still unclear. We found that ZFP36 associates with RIG-I and potentiates the FN-β production induced by SeV. Mechanistically, ZFP36 promotes K63-linked polyubiquitination of RIG-I, mostly at K154/K164/K172, thereby facilitating the activation of RIG-I during infection. While the mutant ZFP36 (C118S/C162S) failed to increase polyubiquitination of RIG-I and SeV induced FN-β. Our findings collectively demonstrated that ZFP36 acts as a positive regulator in antiviral innate immunity by targeting RIG-I for K63-linked ubiquitination, thus improving our understanding of the activation mechanism of RIG-I.
Accurate and reliable monthly runoff forecasting plays an important role in making full use of water resources. In recent years, long short-term memory neural networks (LSTM), as a deep learning ...technology, has been successfully applied in forecasting monthly runoff. However, the hyperparameters of LSTM are predetermined, which has a significant influence on model performance. In this study, given that the decomposition of monthly runoff series may provide a more accurate prediction, as revealed by many previous studies, a hybrid model, namely, VMD-GWO-LSTM, is proposed for monthly runoff forecasting. The proposed hybrid model comprises two main components, namely, variational mode decomposition (VMD) coupled with the gray wolf optimizer (GWO)-based LSTM. First, VMD is utilized to decompose raw monthly runoff series into several subsequences. Second, GWO is implemented to optimize the hyperparameters of the LSTM for each subsequence on the condition that the inputs are determined. Finally, the total output of all subsequences is aggregated as the final forecast result. Four quantitative indices are employed to evaluate the model performance. The proposed model is demonstrated using 73 and 62 years of monthly runoff series data derived from the Xinfengjiang and Guangzhao Reservoirs in China's Pearl River system, respectively. To identify the feasibility and superiority of the proposed model, backpropagation neural networks (BPNN), support vector machine (SVM), LSTM, EMD-LSTM, VMD-LSTM and GWO-LSTM are also utilized for comparison. The results indicate that the proposed hybrid model can yield best forecast accuracy among these models, making it a promising new method for monthly runoff forecasting.
SUMMARY
Precise gene‐editing using CRISPR/Cas9 technology remains a long‐standing challenge, especially for genes with low expression and no selectable phenotypes in Chlamydomonas reinhardtii, a ...classic model for photosynthesis and cilia research. Here, we developed a multi‐type and precise genetic manipulation method in which a DNA break was generated by Cas9 nuclease and the repair was mediated using a homologous DNA template. The efficacy of this method was demonstrated for several types of gene editing, including inactivation of two low‐expression genes (CrTET1 and CrKU80), the introduction of a FLAG‐HA epitope tag into VIPP1, IFT46, CrTET1 and CrKU80 genes, and placing a YFP tag into VIPP1 and IFT46 for live‐cell imaging. We also successfully performed a single amino acid substitution for the FLA3, FLA10 and FTSY genes, and documented the attainment of the anticipated phenotypes. Lastly, we demonstrated that precise fragment deletion from the 3′‐UTR of MAA7 and VIPP1 resulted in a stable knock‐down effect. Overall, our study has established efficient methods for multiple types of precise gene editing in Chlamydomonas, enabling substitution, insertion and deletion at the base resolution, thus improving the potential of this alga in both basic research and industrial applications.
Significance Statement
Efficient precision gene‐editing system was established in Chlamydomonas, including target gene inactivation, single amino acid substitution, knock‐in of an epitope or a YFP tag, and the precise deletion of a DNA fragment in the genome. The methods established in this study will facilitate generating multiple types of genetically modified strains, re‐constructing the metabolism pathway for producing the bio‐fuel and high‐value algal compounds, and modifying the structure of the cell wall for bio‐refinery.
PDF
