scholarly journals Identification of DNMT3B2 as the Predominant Isoform of DNMT3B in Porcine Alveolar Macrophages and Its Involvement in LPS-Stimulated TNF-α Expression

Genes ◽  
2020 ◽  
Vol 11 (9) ◽  
pp. 1065
Author(s):  
Yanbing Zhang ◽  
Hui Li ◽  
Xiao Xiang ◽  
Yan Lu ◽  
Mona Sharma ◽  
...  
Keyword(s):  
Alveolar Macrophages ◽  
Dna Methyltransferase ◽  
Biological Function ◽  
De Novo ◽  
Splice Variants ◽  
Expression Patterns ◽  
Gene Promoter ◽  
Dna Methyltransferase 3B ◽  
Tnf Α ◽  
Dnmt Family

DNA methyltransferase 3B (DNMT3B) as one member of the DNMT family functions as a de novo methyltransferase, characterized as more than 30 splice variants in humans and mice. However, the expression patterns of DNMT3B in pig as well as the biological function of porcine DNMT3B remain to be determined. In this study, we first examined the expression patterns of DNMT3B in porcine alveolar macrophages (PAM). We demonstrated that only DNMT3B2 and DNMT3B3 were the detectable isoforms in PAM. Furthermore, we revealed that DNTM3B2 was the predominant isoform in PAM. Next, in the model of LPS (lipopolysaccharide)-activated PAM, we showed that in comparison to the unstimulated PAM, (1) expression of DNTM3B is reduced; (2) the methylation level of TNF-α gene promoter is decreased. We further establish that DNMT3B2-mediated methylation of TNF-α gene promoter restricts induction of TNF-α in the LPS-stimulated PAM. In summary, these findings reveal that DNMT3B2 is the predominant isoform in PAM and its downregulation contributes to expression of TNF-α via hypomethylation of TNF-α gene promoter in the LPS-stimulated PAM.

99 EXTRACELLULAR VESICLES OF BOVINE OVIDUCTAL FLUID MODIFY THE GENE EXPRESSION ON BOVINE IN VITRO-DERIVED EMBRYOS

2016 ◽  
Vol 28 (2) ◽  
pp. 179 ◽  
Author(s):  
R. Lopera-Vasquez ◽  
M. Hamdi ◽  
V. Maillo ◽  
C. Nunez ◽  
M. Yanez-Mo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Embryo Development ◽  
Extracellular Vesicles ◽  
Membrane Trafficking ◽  
Dna Methyltransferase ◽  
De Novo ◽  
Expression Patterns ◽  
Water Channel ◽  
Developmental Competence

Extracellular vesicles (EVs) act as intercellular communicators through their protein, lipid, and mRNA content. The interaction of EVs from oviducal environment and the first stages of embryo development is currently an enigma. The aim of the present study was to evaluate the developmental competence and the expression profile of bovine blastocysts cultured with previously purified EVs recovered from ampullary and isthmic oviducal fluid (OF) under different centrifugal forces. OF-EVs recovered from oviducts of slaughtered heifers in early luteal phase were quantified with a nanoparticle tracking analysis system, and their integrity and size were assessed by electron microscopy. In vitro-produced zygotes were cultured in SOF+3 mg mL–1 BSA (C–), C– with 3 × 105 OF-EVs/mL from the ampulla (A) and isthmus (I) isolated at 1 × 103 (A10k and I10k, respectively) and 1 × 105 (A100k and I100k, respectively) × g. A control culture group of SOF+5% FCS (C+) was included. Blastocyst development was recorded on Day 7, 8, and 9 (D0: day of fertilization). Blastocysts on Days 7/8 cultured in C–, C+, I10k, and I100k were used to measure the relative mRNA expression of genes related with membrane trafficking (AQP3, AQP11, and ATP1A1), metabolism (LDLR and LDHA), and epigenetics (DNMT3A, IGF2R, GRB10, and SNRPN) by RT-qPCR. One-way ANOVA was used for statistical analysis. The size of ampullary and isthmic OF-EVs was similar with a mean of 220 nm. The concentration of I10k was significantly lower compared with A100k (3.6 × 108 v. 10.5 × 108 EVs/mL, respectively; P < 0.05); however, no differences were found in the rest of the groups with a mean concentration of 7.6 × 108 EVs/mL. EVs and C– groups showed a delayed embryo development at Day 7 compared with C+ (range: 12.0–13.8 v. 20.6%, respectively, P < 0.05); however, it was compensated at Days 8 and 9 (Day 9 range: 28.5–30.8%). The water channel related protein AQP3, associated with blastocoel formation, water, and cryoprotectant movement during cryopreservation, was up-regulated in I10k and I100k blastocysts compared with C+. The lipid receptor LDLR, proposed as a regulator of lipid uptake in blastocysts, was significantly down-regulated in C+ compared with the other groups, a possible consequence of a higher concentration of lipids in the C+ group. The de novo DNA methyltransferase DNMT3A and the imprinting gene SNRPN were down-regulated in the C+ compared with I100k, suggesting alterations in imprinting. In conclusion, bovine isthmic OF-EVs supplementation in in vitro embryo culture has a positive effect on gene expression patterns of developmental related genes compared with serum supplementation, suggesting an association between the oviducal environment and the developing embryo. Funded by the Spanish Ministry of Science and Innovation (AGL2012–37510 and AGL2012–39652-C02–01).

scholarly journals DNA Methyltransferase 3B Gene Promoter and Interleukin-1 Receptor Antagonist Polymorphisms in Childhood Immune Thrombocytopenia

2012 ◽  
Vol 2012 ◽  
pp. 1-6 ◽  
Author(s):  
Margarita Pesmatzoglou ◽  
Marilena Lourou ◽  
George N. Goulielmos ◽  
Eftichia Stiakaki
Keyword(s):  
Receptor Antagonist ◽  
Immune Thrombocytopenia ◽  
Dna Methyltransferase ◽  
Interleukin 1 ◽  
Gene Promoter ◽  
Control Group ◽  
Genotype Distribution ◽  
Genotype I ◽  
Vntr Polymorphism ◽  
Dna Methyltransferase 3B

Primary immune thrombocytopenia (ITP) is one of the most common blood diseases as well as the commonest acquired bleeding disorder in childhood. Although the etiology of ITP is unclear, in the pathogenesis of the disease, both environmental and genetic factors including polymorphisms of TNF-a, IL-10, and IL-4 genes have been suggested to be involved. In this study, we investigated the rs2424913 single-nucleotide polymorphism (SNP) (C46359T) in DNA methyltransferase 3B (DNMT3B) gene promoter and the VNTR polymorphism of IL-1 receptor antagonist (IL-1 Ra) intron-2 in 32 children (17 boys) with the diagnosis of ITP and 64 healthy individuals. No significant differences were found in the genotype distribution ofDNMT3Bpolymorphism between the children with ITP and the control group, whereas the frequency of allele T appeared significantly increased in children with ITP (P = 0.03, OR = 2, 95% CI: 1.06–3.94). In case ofIL-1 Rapolymorphism, children with ITP had a significantly higher frequency of genotype I/II, compared to control group (P = 0.043, OR = 2.60, 95% CI: 1.02–6.50). Moreover, genotype I/I as well as allele I was overrepresented in the control group, suggesting that allele I may have a decreased risk for development of ITP. Our findings suggest that rs2424913DNMT3BSNP as well asIL-1 RaVNTR polymorphism may contribute to the susceptibility to ITP.

Expression of DNMT Isoforms Is Differentially Associated with Aberrant Promotor Methylation in MDS Hematopoietic Progenitor Cells during Lineage Specific Differentiation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2628-2628 ◽  
Author(s):  
Olaf J. Hopfer ◽  
Martina Komor ◽  
Ina S. Koehler ◽  
Matthias Schulze ◽  
Claudia Freitag ◽  
...  
Keyword(s):  
High Risk ◽  
Dna Methyltransferase ◽  
De Novo ◽  
Disease Risk ◽  
Expression Patterns ◽  
Survivin Expression ◽  
Low Risk ◽  
Dnmt1 Expression ◽  
Time Point

Abstract Recent findings suggest that in myelodysplastic syndrome (MDS) several key regulatory genes are affected by aberrant promotor methylation. To explore the molecular basis of this impairment we have generated an in vitro model of MDS lineage-specific hematopoietic differentiation by culturing CD34+ cells from healthy donors (n=7) and MDS patients (low risk: RA/n=6, RARS/n=3; high risk: RAEB/n=4, RAEB-T/n=2) with EPO, TPO and GCSF. Cell harvest was at days 0, 4, 7 and 11. Promotor methylation analysis of key genes involved in the control of apoptosis (p73, survivin, DAPK), DNA-repair (hMLH1), differentiation (RARb, WT1) and cell cycle control (p14, p15, p16, CHK2) was performed by methylation specific PCR of bisulfite treated genomic DNA for each lineage at each time point. In addition, expression of DNMT1 (maintenance DNA methyltransferase), DNMT3a and DNMT3b (both de novo DNA methyltransferase) was analyzed by real time RT-PCR and correlated with gene promotor methylation at any time point. DNMT1 expression was increased during erythropoiesis in both, normal controls and MDS patients. On the other hand, expression of de novo DNMTs was elevated during thrombopoiesis at all time points. During erythropoiesis hypermethylation of p73, hMLH1 and RARb was associated with elevated DNMT1, hypermethylation of p15, p16, p73 and survivin was positively associated with increasing DNMT3 expression. Interestingly, DNMT1 was only elevated in low risk MDS, but not further increased in high risk MDS patients. Surprisingly, MDS specific survivin promotor methylation was inverse correlated with DNTM1 and DNMT3a expression. However, a negative correlation of DNMT3a with survivin expression was found in low risk MDS but not in high risk MDS. In summary our data indicate that all mammalian DNMT isoforms may be involved in the aberrant DNA-methylation phenotype in MDS. Elevated DNMT1 expression may in particular contribute to ineffective erythropoiesis in low risk MDS. DNMT3a and 3b were elevated during megakaryopoiesis and their expression was inversely correlated with MDS disease risk (IPSS). We conclude that the knowledge about distinct expression patterns of DNMT isoforms in hematopoiesis may be of help for further strategies to implicate DNMT-inhibitors in the treatment of patients with MDS.

54 EXPRESSION PATTERN OF DNMT1 AND DNMT3a GENES DURING INTERGENERIC SOMATIC CELL NUCLEAR TRANSFER EMBRYO DEVELOPMENT

2013 ◽  
Vol 25 (1) ◽  
pp. 174
Author(s):  
M. Morovic ◽  
F. Strejcek ◽  
O. Ostrup ◽  
A. Lucas-Hahn ◽  
B. Petersen ◽  
...  
Keyword(s):  
Expression Pattern ◽  
Nuclear Transfer ◽  
Dna Methyltransferase ◽  
De Novo ◽  
Expression Patterns ◽  
Preimplantation Embryos ◽  
Specific Primers ◽  
Cell Stage ◽  
Nuclear Transfer Embryo ◽  
Species Specific

One of the most-discussed reasons for developmental incompetence of embryos constructed by the cloning procedure is inadequate reprogramming of the transferred nucleus to a state equivalent to that of an early embryonic nucleus. Previous studies have shown species-dependent expression patterns of DNA methyltransferase (DNMT) genes in mammalian oocytes and preimplantation embryos, and also a correlation between incomplete DNA methylation and the lack of NT success in mammals. In the present study, the expression pattern of DNMT1 and DNTM3a genes at the 2-cell and 4-cell stages of bovine versus porcine intergeneric nuclear transfer (iSCNT) embryos was observed by reverse-transcriptase (RT) PCR. All pools were done in triplicate and contained 10 iSCNT embryos. The species-specific primers for DNMT1 and DNMT3a genes were designed for determination of de novo synthesis of epigenetic enzymes. As positive controls, porcine and bovine parthenogenetic embryos were used. Gene transcription for bovine DNMT1 (bDNMT1) and DNMT3a (bDNMT3a) was not observed in 2- and 4-cell stage embryos generated by bovine fibroblast transfer into the porcine ooplasm; however, using primers for pig DNMT1 (pDNMT1) and DNMT3a (pDNMT3a), positive results were obtained. In the 2- and 4-cell-stage embryos constructed using porcine fibroblast and bovine ooplasm, only the bovine-specific primers showed positive signals. Based on the different timing of major genome activation during embryonic development in bovine and porcine embryos, the strong influence of ooplasm on introduced fibroblast was expected. Despite the mRNA presence of DNMT1 and DNMT3a enzymes of oocyte origin, de novo transcription of somatic DNMT1 and DNMT3a genes was not detected and iSCNT embryos did not develop beyond the 4-cell stage. These results strongly suggest species-specific and maternally driven regulation of epigenetic reprogramming during early embryogenesis. This work was supported by VEGA 1/0077/11.

scholarly journals Characterization of CiWRI1 from Carya illinoinensis in Seed Oil Biosynthesis

Forests ◽  
2020 ◽  
Vol 11 (8) ◽  
pp. 818
Author(s):  
Xiaofeng Zhou ◽  
Yuqiu Dai ◽  
Haijun Wu ◽  
Peiqiao Zhong ◽  
Linjie Luo ◽  
...  
Keyword(s):  
Seed Oil ◽  
Unsaturated Fatty Acids ◽  
De Novo ◽  
Germination Rate ◽  
Expression Patterns ◽  
Lipid Synthesis ◽  
Gene Promoter ◽  
Carya Illinoinensis ◽  
Oil Biosynthesis ◽  
Commercially Important

Pecan (Carya illinoinensis) is a widely consumed edible woody oil species that is rich in unsaturated fatty acids (FAs) that are beneficial to human health. However, the genes and mechanisms regulating seed oil biosynthesis in pecan are not well understood. Here, we analyzed the expression patterns of genes involved in seed oil biosynthesis in two different varieties of pecan with distinct fruit maturation schedules and oil contents. We cloned the C. illinoinensis WRINKLED 1 (CiWRI1) gene, a homolog of ArabidopsisWRINKLED1 (AtWRI1), which plays a key role in FA synthesis. Overexpressing CiWRI1 restored lipid synthesis in the Arabidopsiswri1-1 mutant and rescued other phenotypic defects such as plant height, root length, and germination rate, suggesting that CiWRI1 is an ortholog of the AtWRI1 and is involved in the regulation of FA synthesis. To investigate the mechanism of CiWRI1 regulation, we cloned C. illinoinensis BIOTIN CARBOXYL CARRIER PROTEIN ISOFORM2 (CiBCCP2) and determined that the CiWRI1 protein directly binds to an ASML1/WRI1 (AW)-box motif in the CiBCCP2 gene promoter and thereby activates its transcription. CiBCCP2 overexpression partly rescued the phenotypic defects of the wri1-1 mutant, indicating that it is directly regulated by CiWRI1. Thus, de novo FA biosynthesis in seed is conserved across plant species; moreover, CiWRI1 regulates oil synthesis by directly controlling CiBCCP2 expression. These findings present novel potential targets for molecular-marker-assisted breeding of this commercially important plant.

scholarly journals Expression and Potential Role of microRNA-29b in Mouse Early Embryo Development

2015 ◽  
Vol 35 (3) ◽  
pp. 1178-1187 ◽  
Author(s):  
Junqiang Zhang ◽  
Ying Wang ◽  
Xiaoguang Liu ◽  
Shenglin Jiang ◽  
Chun Zhao ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Embryonic Development ◽  
Dna Methyltransferase ◽  
Biological Function ◽  
Expression Patterns ◽  
Early Embryo ◽  
Early Embryonic Development ◽  
Preimplantation Embryos ◽  
Early Embryos

Background/Aims: MicroRNA-29b (miR29b) has been previously identified in early mouse embryos through miRNA microarray analysis. Recent research has indicated that miR29b participates in DNA methylation by regulating DNA methyltransferase 3a/3b (Dnmt3a/3b) expression. However, the expression pattern and biological function of miR29b in mouse preimplantation embryonic development remain unknown. Methods: In this study, we examined the expression patterns of miR29b and Dnmt3a/3b in mouse early embryos at different developmental stages. Subsequently, expression and localization of DNMT3A/3B protein was analyzed in mouse early embryos by immunofluorescence staining. The biological function of miR29b in mouse early embryos was analyzed by microinjection of commercially available miRNA-specific inhibitors and mimics. Results: Our data showed that Dnmt3a/3b mRNA expression is negatively regulated by miR29b in mouse early embryos. Immunofluorescence analysis revealed that DNMT3A/3B protein expression is predominantly localized within the nucleoplasm of embryos. Alterations to the activity of miR29b could change the DNA methylation levels in mouse preimplantation embryos and lead to a developmental blockade, from the morula to the blastocyst stage. Conclusion: These results indicated a role for the miR29b-Dnmt3a/3b-DNA methylation axis in mouse early embryonic development, and we provide evidence that miR29b is indispensable for mouse early embryonic development. This study contributes to a preliminary understanding of the role of miR29b during mouse embryonic development.

scholarly journals PSIII-38 Butyric acid and derivatives: In vitro anti-inflammatory effects tested in porcine alveolar macrophages

2020 ◽  
Vol 98 (Supplement_4) ◽  
pp. 370-370
Author(s):  
Lauren L Kovanda ◽  
Monika Hejna ◽  
Yanhong Liu
Keyword(s):  
Organic Acid ◽  
Butyric Acid ◽  
Sodium Butyrate ◽  
Alveolar Macrophages ◽  
Block Design ◽  
Lps Challenge ◽  
Tnf Α ◽  
Anti Inflammatory ◽  
Challenge Group

Abstract The aim of this experiment was to examine the anti-inflammatory effects of butyric acid, sodium butyrate, monobutyrin and tributyrin using porcine alveolar macrophages (PAMs). PAMs were isolated from the bronchial lavage of 6 piglets at 6 weeks of age, and then seeded at 106 cells/mL in 24-well plates. After 24 h incubation, cells were treated with different treatments in a randomized complete block design with 10 replicates. The treatments were in a factorial arrangement with 2 doses of lipopolysaccharide (LPS, 0 or 1 μg/mL) and 5 levels of organic acid (0, 0.5, 1, 2, 4 mM for butyric acid and tributyrin and 0, 1, 2, 4, 8 mM for sodium butyrate and monobutyrin). Supernatants were collected after another 24 h incubation and analyzed for tumor necrosis factor alpha (TNF-α). Cell viability was also tested by the MTT assay. Data were analyzed using the MIXED procedure of SAS. No cytotoxic effect was observed in LPS challenge and each organic acid with the percentage of live cells was more than 76% in comparison to the sham control. Sodium butyrate at 2 and 4 mM dose exhibited (P &lt; 0.01) a stimulatory effect on cell proliferation. LPS challenge remarkably stimulated (P &lt; 0.0001) TNF-α secretion from PAMs. In the non-challenge group, butyric acid, monobutyrin, and tributyrin linearly reduced TNF-α production from PAMs, whereas 2 mM sodium butyrate tended to increase (P = 0.056) TNF-α secretion from PAMs. In the LPS challenge group, all tested organic acid dose-dependently reduced (P &lt; 0.001) TNF-α production from LPS-challenged PAMs, with the strongest inhibiting effect observed at the highest dose. Results indicated that butyric acid and its derivatives that were tested in the current experiment all had strong anti-inflammatory activities in vitro.

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