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Stem Cell Reports Jun 2024Removal of somatic histone H3 lysine 9 trimethylation (H3K9me3) from the embryonic genome can improve the efficiency of mammalian cloning using somatic cell nuclear...
Removal of somatic histone H3 lysine 9 trimethylation (H3K9me3) from the embryonic genome can improve the efficiency of mammalian cloning using somatic cell nuclear transfer (SCNT). However, this strategy involves the injection of histone demethylase mRNA into embryos, which is limiting because of its invasive and labor-consuming nature. Here, we report that treatment with an inhibitor of G9a (G9ai), the major histone methyltransferase that introduces H3K9me1/2 in mammals, greatly improved the development of mouse SCNT embryos. Intriguingly, G9ai caused an immediate reduction of H3K9me1/2, a secondary loss of H3K9me3 in SCNT embryos, and increased the birth rate of cloned pups about 5-fold (up to 3.9%). G9ai combined with the histone deacetylase inhibitor trichostatin A further improved this rate to 14.5%. Mechanistically, G9ai and TSA synergistically enhanced H3K9me3 demethylation and boosted zygotic genome activation. Thus, we established an easy, highly effective SCNT protocol that would enhance future cloning research and applications.
Topics: Animals; Histones; Nuclear Transfer Techniques; Mice; Histone-Lysine N-Methyltransferase; Methylation; Cloning, Organism; Embryo, Mammalian; Embryonic Development; Hydroxamic Acids; Female; Histone Deacetylase Inhibitors
PubMed: 38729154
DOI: 10.1016/j.stemcr.2024.04.003 -
Neuroscience Research May 2024Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are characterized by nuclear depletion and cytoplasmic aggregation of TAR DNA-binding protein-43... (Review)
Review
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are characterized by nuclear depletion and cytoplasmic aggregation of TAR DNA-binding protein-43 (TDP-43). TDP-43 plays a key role in regulating the splicing of numerous genes, including TARDBP. This review aims to delineate two aspects of ALS/FTD pathogenesis associated with TDP-43 function. First, we provide novel mechanistic insights into the splicing of UNC13A, a TDP-43 target gene. Single nucleotide polymorphisms (SNPs) in UNC13A are the most common risk factors for ALS/FTD. We found that TDP-43 represses "cryptic exon" inclusion during UNC13A RNA splicing. A risk-associated SNP in this exon results in increased RNA levels of UNC13A retaining the cryptic exon. Second, we described the perturbation of the TDP-43 autoregulatory mechanism caused by age-related DNA demethylation. Aging is a major risk factor for sporadic ALS/FTD. Typically, TDP-43 levels are regulated via alternative splicing of TARDBP mRNA. We hypothesized that TARDBP methylation is altered by aging, thereby disrupting TDP-43 autoregulation. We found that demethylation reduces the efficiency of alternative splicing and increases TARDBP mRNA levels. Moreover, we demonstrated that, with aging, this region is demethylated in the human motor cortex and is associated with the early onset of ALS.
PubMed: 38723906
DOI: 10.1016/j.neures.2024.05.001 -
Bioorganic Chemistry Jun 2024Diabetes mellitus is a metabolic disease characterized by hyperglycemia, which can be counteracted by the inhibition of α-glucosidase (α-Glu) and α-amylase (α-Amy),...
Diabetes mellitus is a metabolic disease characterized by hyperglycemia, which can be counteracted by the inhibition of α-glucosidase (α-Glu) and α-amylase (α-Amy), enzymes responsible for the hydrolysis of carbohydrates. In recent decades, many natural compounds and their bioinspired analogues have been studied as α-Glu and α-Amy inhibitors. However, no studies have been devoted to the evaluation of α-Glu and α-Amy inhibition by the neolignan obovatol (1). In this work, we report the synthesis of 1 and a library of new analogues. The synthesis of these compounds was achieved by implementing methodologies based on: phenol allylation, Claisen/Cope rearrangements, methylation, Ullmann coupling, demethylation, phenol oxidation and Michael-type addition. Obovatol (1) and ten analogues were evaluated for their in vitro inhibitory activity towards α-Glu and α-Amy. Our investigation highlighted that the naturally occurring 1 and four neolignan analogues (11, 22, 26 and 27) were more effective inhibitors than the hypoglycemic drug acarbose (α-Amy: 34.6 µM; α-Glu: 248.3 µM) with IC value of 6.2-23.6 µM toward α-Amy and 39.8-124.6 µM toward α-Glu. Docking investigations validated the inhibition outcomes, highlighting optimal compatibility between synthesized neolignans and both the enzymes. Concurrently circular dichroism spectroscopy detected the conformational changes in α-Glu induced by its interaction with the studied neolignans. Detailed studies through fluorescence measurements and kinetics of α-Glu and α-Amy inhibition also indicated that 1, 11, 22, 26 and 27 have the greatest affinity for α-Glu and 1, 11 and 27 for α-Amy. Surface plasmon resonance imaging (SPRI) measurements confirmed that among the compounds studied, the neolignan 27 has the greater affinity for both enzymes, thus corroborating the results obtained by kinetics and fluorescence quenching. Finally, in vitro cytotoxicity of the investigated compounds was tested on human colon cancer cell line (HCT-116). All these results demonstrate that these obovatol-based neolignan analogues constitute promising candidates in the pursuit of developing novel hypoglycemic drugs.
Topics: alpha-Amylases; alpha-Glucosidases; Glycoside Hydrolase Inhibitors; Lignans; Structure-Activity Relationship; Humans; Molecular Structure; Dose-Response Relationship, Drug; Molecular Docking Simulation; Hypoglycemic Agents; Enzyme Inhibitors
PubMed: 38723423
DOI: 10.1016/j.bioorg.2024.107392 -
Microbial Biotechnology May 2024Pectin structures have received increasing attention as emergent prebiotics due to their capacity to promote beneficial intestinal bacteria. Yet the collective activity...
A methyl esterase from Bifidobacterium longum subsp. longum reshapes the prebiotic properties of apple pectin by triggering differential modulatory capacity in faecal cultures.
Pectin structures have received increasing attention as emergent prebiotics due to their capacity to promote beneficial intestinal bacteria. Yet the collective activity of gut bacterial communities to cooperatively metabolize structural variants of this substrate remains largely unknown. Herein, the characterization of a pectin methylesterase, BpeM, from Bifidobacterium longum subsp. longum, is reported. The purified enzyme was able to remove methyl groups from highly methoxylated apple pectin, and the mathematical modelling of its activity enabled to tightly control the reaction conditions to achieve predefined final degrees of methyl-esterification in the resultant pectin. Demethylated pectin, generated by BpeM, exhibited differential fermentation patterns by gut microbial communities in in vitro mixed faecal cultures, promoting a stronger increase of bacterial genera associated with beneficial effects including Lactobacillus, Bifidobacterium and Collinsella. Our findings demonstrate that controlled pectin demethylation by the action of a B. longum esterase selectively modifies its prebiotic fermentation pattern, producing substrates that promote targeted bacterial groups more efficiently. This opens new possibilities to exploit biotechnological applications of enzymes from gut commensals to programme prebiotic properties.
Topics: Malus; Pectins; Prebiotics; Feces; Carboxylic Ester Hydrolases; Fermentation; Humans; Bifidobacterium longum; Gastrointestinal Microbiome; Bifidobacterium
PubMed: 38722820
DOI: 10.1111/1751-7915.14443 -
The Journal of Clinical Investigation May 2024Newborn mammalian cardiomyocytes quickly transition from a fetal to an adult phenotype that utilizes mitochondrial oxidative phosphorylation but loses mitotic capacity....
Newborn mammalian cardiomyocytes quickly transition from a fetal to an adult phenotype that utilizes mitochondrial oxidative phosphorylation but loses mitotic capacity. We tested whether forced reversal of adult cardiomyocytes back to a fetal glycolytic phenotype would restore proliferative capacity. We deleted Uqcrfs1 (mitochondrial Rieske iron-sulfur protein, RISP) in hearts of adult mice. As RISP protein decreased, heart mitochondrial function declined, and glucose utilization increased. Simultaneously, the hearts underwent hyperplastic remodeling during which cardiomyocyte number doubled without cellular hypertrophy. Cellular energy supply was preserved, AMPK activation was absent, and mTOR activation was evident. In ischemic hearts with RISP deletion, new cardiomyocytes migrated into the infarcted region, suggesting the potential for therapeutic cardiac regeneration. RNA sequencing revealed upregulation of genes associated with cardiac development and proliferation. Metabolomic analysis revealed a decrease in α-ketoglutarate (required for TET-mediated demethylation) and an increase in S-adenosylmethionine (required for methyltransferase activity). Analysis revealed an increase in methylated CpGs near gene transcriptional start sites. Genes that were both differentially expressed and differentially methylated were linked to upregulated cardiac developmental pathways. We conclude that decreased mitochondrial function and increased glucose utilization can restore mitotic capacity in adult cardiomyocytes, resulting in the generation of new heart cells, potentially through the modification of substrates that regulate epigenetic modification of genes required for proliferation.
Topics: Animals; Myocytes, Cardiac; Mice; Cell Proliferation; Mitochondria, Heart; Mice, Knockout; Electron Transport Complex III; Glucose
PubMed: 38722697
DOI: 10.1172/JCI165482 -
Free Neuropathology Jan 2024L-2-hydroxyglutaric aciduria (L-2-HGA) is a rare neurometabolic disorder characterized by accumulation of L2-hydroxyglutarate (L-2-HG) due to mutations in the gene....
L-2-hydroxyglutaric aciduria (L-2-HGA) is a rare neurometabolic disorder characterized by accumulation of L2-hydroxyglutarate (L-2-HG) due to mutations in the gene. L-2-HGA patients have a significantly increased lifetime risk of central nervous system (CNS) tumors. Here, we present a 16-year-old girl with L-2-HGA who developed a tumor in the right cerebral hemisphere, which was discovered after left-sided neurological deficits of the patient. Histologically, the tumor had a high-grade diffuse glioma phenotype. DNA sequencing revealed the inactivating homozygous germline mutation as well as inactivating mutations in , and . Genome-wide DNA-methylation analysis was unable to classify the tumor with high confidence. More detailed analysis revealed that this tumor clustered amongst IDH-wildtype gliomas by methylation profiling and did not show the glioma CpG island methylator phenotype (G-CIMP) in contrast to IDH-mutant diffuse gliomas with accumulated levels of D-2-HG, the stereoisomer of L-2-HD. These findings were against all our expectations given the inhibitory potential of 2-HG on DNA-demethylation enzymes. Our final integrated histomolecular diagnosis of the tumor was diffuse pediatric-type high-grade glioma, H3-wildtype and IDH-wildtype. Due to rapid tumor progression the patient died nine months after initial diagnosis. In this manuscript, we provide extensive molecular characterization of the tumor as well as a literature review focusing on oncogenetic considerations of L-2-HGA-associated CNS tumors.
PubMed: 38716347
DOI: 10.17879/freeneuropathology-2024-5377 -
IScience May 2024Ten-eleven translocation (TET) proteins are DNA dioxygenases that mediate active DNA demethylation. TET3 is the most highly expressed TET protein in thymic developing...
Ten-eleven translocation (TET) proteins are DNA dioxygenases that mediate active DNA demethylation. TET3 is the most highly expressed TET protein in thymic developing T cells. TET3, either independently or in cooperation with TET1 or TET2, has been implicated in T cell lineage specification by regulating DNA demethylation. However, TET-deficient mice exhibit complex phenotypes, suggesting that TET3 exerts multifaceted roles, potentially by interacting with other proteins. We performed liquid chromatography with tandem mass spectrometry in primary developing T cells to identify TET3 interacting partners in endogenous, conditions. We discover TET3 interacting partners. Our data establish that TET3 participates in a plethora of fundamental biological processes, such as transcriptional regulation, RNA polymerase elongation, splicing, DNA repair, and DNA replication. This resource brings in the spotlight emerging functions of TET3 and sets the stage for systematic studies to dissect the precise mechanistic contributions of TET3 in shaping T cell biology.
PubMed: 38711449
DOI: 10.1016/j.isci.2024.109782 -
British Journal of Cancer Jun 2024Tregs trafficking is controlled by CXCR4. In Renal Cell Carcinoma (RCC), the effect of the new CXCR4 antagonist, R54, was explored in peripheral blood (PB)-Tregs...
BACKGROUND
Tregs trafficking is controlled by CXCR4. In Renal Cell Carcinoma (RCC), the effect of the new CXCR4 antagonist, R54, was explored in peripheral blood (PB)-Tregs isolated from primary RCC patients.
METHODS
PB-Tregs were isolated from 77 RCC patients and 38 healthy donors (HDs). CFSE-T effector-Tregs suppression assay, IL-35, IFN-γ, IL-10, TGF-β1 secretion, and Tregs frequency were evaluated. Tregs were characterised for CTLA-4, PD-1, CD40L, PTEN, CD25, TGF-β1, FOXP3, DNMT1 transcriptional profile. PTEN-pAKT signalling was evaluated in the presence of R54 and/or triciribine (TCB), an AKT inhibitor. Methylation of TSDR (Treg-Specific-Demethylated-Region) was conducted.
RESULTS
R54 impaired PB-RCC-Tregs function, reduced Tregs frequency, the release of IL-35, IL-10, and TGF-β1, while increased IFN-γ Teff-secretion. The CXCR4 ligand, CXCL12, recruited Tregs in RCC while R54 significantly reduced it. IL-2/PMA activates Tregs reducing Tregs while R54 increases it. The AKT inhibitor, TCB, prevented the increase in Tregs R54-mediated. Moreover, R54 significantly reduced FOXP3-TSDR demethylation with DNMT1 and FOXP3 downregulation.
CONCLUSION
R54 impairs Tregs function in primary RCC patients targeting PTEN/PI3K/AKT pathway, reducing TSDR demethylation and FOXP3 and DNMT1 expression. Thus, CXCR4 targeting is a strategy to inhibit Tregs activity in the RCC tumour microenvironment.
Topics: Humans; T-Lymphocytes, Regulatory; PTEN Phosphohydrolase; Receptors, CXCR4; Kidney Neoplasms; Carcinoma, Renal Cell; Female; Male; Middle Aged; Aged; Adult; Signal Transduction; Forkhead Transcription Factors
PubMed: 38704478
DOI: 10.1038/s41416-024-02702-x -
BMC Biology May 2024Gonadotropin precisely controls mammalian reproductive activities. Systematic analysis of the mechanisms by which epigenetic modifications regulate the synthesis and...
BACKGROUND
Gonadotropin precisely controls mammalian reproductive activities. Systematic analysis of the mechanisms by which epigenetic modifications regulate the synthesis and secretion of gonadotropin can be useful for more precise regulation of the animal reproductive process. Previous studies have identified many differential mA modifications in the GnRH-treated adenohypophysis. However, the molecular mechanism by which mA modification regulates gonadotropin synthesis and secretion remains unclear.
RESULTS
Herein, it was found that GnRH can promote gonadotropin synthesis and secretion by promoting the expression of FTO. Highly expressed FTO binds to Foxp2 mRNA in the nucleus, exerting a demethylation function and reducing mA modification. After Foxp2 mRNA exits the nucleus, the lack of mA modification prevents YTHDF3 from binding to it, resulting in increased stability and upregulation of Foxp2 mRNA expression, which activates the cAMP/PKA signaling pathway to promote gonadotropin synthesis and secretion.
CONCLUSIONS
Overall, the study reveals the molecular mechanism of GnRH regulating the gonadotropin synthesis and secretion through FTO-mediated mA modification. The results of this study allow systematic interpretation of the regulatory mechanism of gonadotropin synthesis and secretion in the pituitary at the epigenetic level and provide a theoretical basis for the application of reproductive hormones in the regulation of animal artificial reproduction.
Topics: Alpha-Ketoglutarate-Dependent Dioxygenase FTO; Gonadotropin-Releasing Hormone; Animals; Gonadotropins; Mice; RNA, Messenger; RNA Methylation
PubMed: 38702712
DOI: 10.1186/s12915-024-01905-1 -
Nature Communications May 2024Mutations in DNA damage response (DDR) factors are associated with human infertility, which affects up to 15% of the population. The DDR is required during germ cell...
Mutations in DNA damage response (DDR) factors are associated with human infertility, which affects up to 15% of the population. The DDR is required during germ cell development and meiosis. One pathway implicated in human fertility is DNA translesion synthesis (TLS), which allows replication impediments to be bypassed. We find that TLS is essential for pre-meiotic germ cell development in the embryo. Loss of the central TLS component, REV1, significantly inhibits the induction of human PGC-like cells (hPGCLCs). This is recapitulated in mice, where deficiencies in TLS initiation (Rev1 or Pcna) or extension (Rev7 ) result in a > 150-fold reduction in the number of primordial germ cells (PGCs) and complete sterility. In contrast, the absence of TLS does not impact the growth, function, or homeostasis of somatic tissues. Surprisingly, we find a complete failure in both activation of the germ cell transcriptional program and in DNA demethylation, a critical step in germline epigenetic reprogramming. Our findings show that for normal fertility, DNA repair is required not only for meiotic recombination but for progression through the earliest stages of germ cell development in mammals.
Topics: Animals; Humans; DNA Demethylation; Mice; DNA Repair; Germ Cells; DNA-Directed DNA Polymerase; Male; Nucleotidyltransferases; Female; DNA Damage; Mice, Knockout; Meiosis; DNA Replication; Proliferating Cell Nuclear Antigen; Epigenesis, Genetic; Translesion DNA Synthesis
PubMed: 38702312
DOI: 10.1038/s41467-024-47219-2