Vitamin E (Tocopherols) is lipid-soluble antioxidants and essential for human health. Gamma-tocopherol methyltransferase (delta-TMT), one of the key enzymes in tocopherol biosynthetic pathway in plants, converts delta,sigma-tocopherols into alpha,beta-tocopherols. In this study, we isolated the 1552 bp promoter of Arabidopsis TMT gene. The promoter was fused with GUS reporter gene and this expression cassette was introduced into wild Arabidopsis thaliana by Agrobacterium-mediated transformation. GUS staining shows that GUS gene is expressed in leaves, stems and flowers, with the highest expression in young leaves, stamens and stem apices, while not observable in roots, seeds and siliques. The data indicate that gamma-TMT gene promoter is likely to be expressed preferentially in some of the tissues of Arabidopsis.
Arabidopsis ; genetics ; Base Sequence ; Genes, Reporter ; Immunohistochemistry ; Methyltransferases ; genetics ; Molecular Sequence Data ; Promoter Regions, Genetic ; genetics

OBJECTIVETo evaluate the effects of DNMT3A gene mutation on prognosis of patients with acute myeloid leukemia (AML) by a meta-analysis.

METHODSMethods of Cochrane systematic review was followed by 7 databases,including PubMed, Embase, Ovid, CNKI, CBM, WanFang Data and VIP, were searched for peer-reviewed articles related to DNMT3A gene mutations and prognosis of patients with AML.Then manual retrieval was applied into literature references. After the evaluation of quality and extract of clinical trialliterature data, Stata 11.0 was employed to perform meta-analysis.

RESULTSSeven randomized controlled trials involving 1493 cases were included in the meta-analysis. The prognosis of patients with DNMT3A mutations and without DNMT3A mutations was compared. There was no statistically significant difference in complete remission(CR) rate (OR=1.034, 95%CI: 0.596~1.796, P=0.905 between two groups, but the overall survival (OS（HR=1.990, 95%CI: 1.463~2.510, P=0.000 and disease free survival (DFS) (HR= 2.840, 95%CI: 1.063~4.613, P=0.002) of patients without DNMT3A mutations were longer than those with DNMT3A mutation.

CONCLUSIONDNMT3A gene mutation is an independent risk factor of poor prognosis of patients with acute myeloid leukemia.

DNA (Cytosine-5-)-Methyltransferases ; genetics ; Humans ; Leukemia, Myeloid, Acute ; diagnosis ; genetics ; Mutation ; Prognosis ; Risk Factors

Epigenetic changes, including abnormal DNA methylation, have been identified to play significant roles in tumor initiation and progression. Recently, mutations of DNMT3A were identified in acute myeloid leukemia (AML), which possibly caused changes in DNA methylation, and indicated a poor prognosis. Sequencing analysis showed that most of the mutations were single nucleotide variations, including a hotspot Arg882. DNMT3A mutations were detected in about 20% AML patients, and closely associated with the age over 60, the M(4), M(5) subtypes and intermediate-risk cytogenetics. Others showed that these alterations also present in myelodysplastic syndrome (MDS) and primary myelofibrosis (PMF) prior to development of the obvious leukemia, indicating that these mutations might contribute to leukemogenesis. However, its prognostic value of minimal residual disease and role of therapeutic targets are still unclear, focusing on a large cohort of AML patients will solve these issues. In this review, the achievement in studying DNMT3A gene mutation are summarized, and the latest research progress is briefly discussed.
DNA (Cytosine-5-)-Methyltransferases ; genetics ; DNA Methylation ; Humans ; Leukemia, Myeloid, Acute ; genetics ; Mutation

OBJECTIVETo study the effects of suppressor of variegation 3-9 homolog 1 (SUV39H1) gene silencing by small interfering RNA (siRNA) on the proliferation, tumor suppressor gene p15 expression and histone modification in acute myeloid leukemia cell line KG-1 cells, and to explore novel therapeutic target of leukemia.

METHODSThe SUV39H1 gene specific siRNA was synthesized in vitro and transfected into KG-1 cells by Lipofectamine(TM) 2000. The SUV39H1 mRNA and protein were detected by RT-PCR and Western blot. Cell growth affected by SUV39H1 siRNA was determined by MTS. The expressions of tumor suppressor gene p15, histone methylation of H3K9 and histone acetylation of H3, H3K9, H3K14, H3K27 and H4 were detected by Western blot.

RESULTSSUV39H1 mRNA was markedly suppressed by the SUV39H1 specific siRNA in a concentration-dependent manner. SUV39H1 siRNA inhibited the proliferation of KG-1 cells. Proliferation inhibition rate was (23.57 ± 1.98)%, (48.69 ± 1.84)%, (62.69 ± 1.61)% and (81.06 ± 3.22)% after transfected with SUV39H1 siRNA at 30, 60, 120 and 240 nmol/L for 48 hours, respectively. SUV39H1 siRNA down-regulated histone tri-methylated-H3K9 by 25%, 33% and 49% compared to control group when treated with SUV39H1 siRNA at 30, 60 and 120 nmol/L for 48 hours, while up-regulated histone acetylated H3K9 by 1.83, 2.16 and 3.07 folds, and global histone H3 in 1.35, 1.87 and 2.37 folds, but no changes were observed in histone acetylation of H3K14, H3K27 and H4. Expression of p15 increased 1.52, 2.89 and 3.08 folds after SUV39H1 siRNA treatment.

CONCLUSIONSSUV39H1 gene silencing could induce the re-expression of p15 and inhibit cell proliferation by down-regulation of histone methylation of H3K9, up-regulation of histone acetylation of H3K9 and global H3. SUV39H1 might be a new target for cancer therapy.

Cell Line, Tumor ; Gene Silencing ; Histones ; genetics ; Humans ; Leukemia ; genetics ; Methyltransferases ; genetics ; RNA, Small Interfering ; Repressor Proteins ; genetics

The aim of the present study was to gain an insight into the thiopurine methytransferase (TPMT) genotyping assay, which was based on polymerase chain reaction (PCR), allele-specific PCR, restriction digestion of PCR products, denaturing high-performance liquid chromatography (DHPLC) and SNaPshot sequencing and in combination with direct DNA sequencing. Among the f our methods to test TPMT genetic SNPs based on PCR, allele specific PCR was not able to differentiate wild type from varied type. BsiYI, MwoI and AccI to digest PCR products were used so that SNP in TPMT exon 5, 7 and 10 tested. It showed that there were no differences between the results of digestion of PCR products and those of DNA sequence analysis. Therefore, this method was reliable. But some other methods were still needed to look for a compensation, because no restriction map changing resulted from the 2 SNPs in TPMT promotor was found. As to the results of DHPLC, those for the screening of TPMT exon-5 and -10 for SNPs were the same as restriction analysis of PCR products and direct DNA sequencing. But the variation of the heterozygotes in exon-7 was high, which was different from the results of direct DNA sequencing. After changing the Tm of DNA step by step, It was found that all the samples showed single peak when the temperature was 54 degrees C. But this result was unbelievable because a heterozygote in exon 7 as positive control could not be found. Therefore, it was necessary to test the sensitivity and accuracy of DHPLC, though DHPLC could be used as an effective method of SNPs screening. The results of the SNaPshot sequencing were also same as those of restriction analysis of PCR products and direct DNA sequencing. And the results showed that the bases of TPMT promoter -91 and -168 were G, instead of A and T. The results of the four methods to detect TPMT genetic SNPs based on PCR showed that SNPs analysis technique should be a combination of the techniques above-mentioned. One technique alone could not satisfy the need in clinics and research. The compensation of each other was very important.
Chromatography, High Pressure Liquid ; Exons ; Humans ; Methyltransferases ; genetics ; Polymerase Chain Reaction ; Polymorphism, Single Nucleotide

As an important drug during maintenance treatment of acute lymphoblastic leukemia (ALL), 6-mercaptopurine (6-MP) has several side effects, including hepatotoxicity and bone marrow suppression. Since its tolerability varies from person to person, 6-MP treatment should be individualized. The deficiency of thiopurine methyltransferase (TPMT) enzyme activity is associated with 6-MP intolerance. There is a lower frequency of mutation in TPMT alleles among Asian patients. Recent studies have shown that in ALL patients with NUDT15 gene mutation, the maximum tolerated dose of 6-MP is lower than the conventional dose. The article reviews the significance of NUDT15 gene in individualized treatment with 6-MP in children with ALL.
Antimetabolites, Antineoplastic ; Child ; Humans ; Mercaptopurine ; Methyltransferases ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; Pyrophosphatases ; genetics

METTL3 and METTL14 are two components that form the core heterodimer of the main RNA m6A methyltransferase complex (MTC) that installs m6A. Surprisingly, depletion of METTL3 or METTL14 displayed distinct effects on stemness maintenance of mouse embryonic stem cell (mESC). While comparable global hypo-methylation in RNA m6A was observed in Mettl3 or Mettl14 knockout mESCs, respectively. Mettl14 knockout led to a globally decreased nascent RNA synthesis, whereas Mettl3 depletion resulted in transcription upregulation, suggesting that METTL14 might possess an m6A-independent role in gene regulation. We found that METTL14 colocalizes with the repressive H3K27me3 modification. Mechanistically, METTL14, but not METTL3, binds H3K27me3 and recruits KDM6B to induce H3K27me3 demethylation independent of METTL3. Depletion of METTL14 thus led to a global increase in H3K27me3 level along with a global gene suppression. The effects of METTL14 on regulation of H3K27me3 is essential for the transition from self-renewal to differentiation of mESCs. This work reveals a regulatory mechanism on heterochromatin by METTL14 in a manner distinct from METTL3 and independently of m6A, and critically impacts transcriptional regulation, stemness maintenance, and differentiation of mESCs.
Animals ; Mice ; Methylation ; Chromatin ; Histones/metabolism* ; RNA, Messenger/genetics* ; Methyltransferases/metabolism* ; RNA/metabolism*

This study was aimed to investigate the influence of short hairpin RNA (shRNA) on proliferation of human leukemia cell line THP-1. The shRNA targeting the site 732-752 of DOT1L mRNA was designed and chemically synthesized, then a single-vector lentiviral, tet-inducible shRNA-DOT1L system (Plko-Tet-On) was generated. Thereafter, the THP-1 cells with lentivirus were infected to create stable cell line with regulatable shRNA expression. The expression of DOT1L in the THP-1 cell line was assayed by RT-PCR. Effect of shRNA-DOT1L on the proliferation of THP-1 cells was detected with MTT method,and the change of colony forming potential of THP-1 cells was analyzed by colony forming unit test. Cell cycle distribution was tested by flow cytometry. The results indicated that the expression of DOT1L was statistically lower than that in the control groups. The proliferation and colony forming capacity of THP-1 cells were significantly inhibited. The percentage of cells at G0/G1 phase increased in THP-1/shRNA cells treated with Dox while the percentage of cells at S phase significantly decreased as compared with that in the control group. It is concluded that the shRNA targeting DOT1L can effectively inhibit the proliferation of acute monocytic leukemia cell line THP-1.
Cell Line, Tumor ; Cell Proliferation ; Genetic Vectors ; Humans ; Lentivirus ; genetics ; Leukemia, Monocytic, Acute ; genetics ; Methyltransferases ; genetics ; RNA, Small Interfering

Asian Continental Ancestry Group ; genetics ; China ; DNA (Cytosine-5-)-Methyltransferases ; genetics ; Humans ; Parkinson Disease ; genetics ; Polymorphism, Single Nucleotide

Thiopurine S-methyltransferase (TPMT) methylates purine analogues, showing TPMT activity in inverse relation to concentrations of active metabolites such as 6-thioguanine nucleotide (6-TGN). With conventional dosing of thiopurines, patients with homozygous variant TPMT alleles consistently suffer from severe myelosuppression. Here, we report a patient with TPMT*3C/*3C who managed successfully with monitoring of thiopurine metabolites. The patient was an 18-year-old male diagnosed with Crohn's disease. The standard dose of azathioprine (AZA) (1.8 mg/kg/day) with mesalazine (55.6 mg/kg/day) was prescribed. Two weeks after starting AZA treatment, the patient developed leukopenia. The DNA sequence analysis of TPMT identified a homozygous missense variation (NM_000367.2: c.719A>G; p.Tyr240Cys), TPMT*3C/*3C. He was treated with adjusted doses of azathioprine (0.1-0.2 mg/kg/day) and his metabolites were closely monitored. Leukopenia did not reoccur during the follow-up period of 24 months. To our knowledge, this is the first case of a patient homozygous for TPMT*3C successfully treated with azathioprine in Korea. While a TPMT genotyping test may be helpful to determine a safe starting dose, it may not completely prevent myelosuppression. Monitoring metabolites as well as routine laboratory tests can contribute to assessing drug metabolism and optimizing drug dosing with minimized drug-induced toxicity.
Adolescent ; Azathioprine/adverse effects/*therapeutic use ; Homozygote ; Humans ; Inflammatory Bowel Diseases/*drug therapy/*enzymology/*genetics/metabolism ; Male ; Methyltransferases/*genetics