Mechanism Of Demethylation Research Articles

A New Isoform of the Histone Demethylase JMJD2A/KDM4A Is Required for Skeletal Muscle Differentiation

In proliferating myoblasts, muscle specific genes are silenced by epigenetic modifications at their promoters, including histone H3K9 methylation. Derepression of the promoter of the gene encoding the myogenic factor myogenin (Myog) is key for initiation of muscle differentiation. The mechanism of H3K9 demethylation at the Myog promoter is unclear, however. Here, we identify an isoform of the histone demethylase JMJD2A/KDM4A that lacks the N-terminal demethylase domain (ΔN-JMJD2A). The amount of ΔN-JMJD2A increases during differentiation of C2C12 myoblasts into myotubes. Genome-wide expression profiling and exon-specific siRNA knockdown indicate that, in contrast to the full-length protein, ΔN-JMJD2A is necessary for myotube formation and muscle-specific gene expression. Moreover, ΔN-JMJD2A promotes MyoD-induced conversion of NIH3T3 cells into muscle cells. ChIP-on-chip analysis indicates that ΔN-JMJD2A binds to genes mainly involved in transcriptional control and that this binding is linked to gene activation. ΔN-JMJD2A is recruited to the Myog promoter at the onset of differentiation. This binding is essential to promote the demethylation of H3K9me2 and H3K9me3. We conclude that induction of the ΔN-JMJD2A isoform is crucial for muscle differentiation: by directing the removal of repressive chromatin marks at the Myog promoter, it promotes transcriptional activation of the Myog gene and thus contributes to initiation of muscle-specific gene expression.

X-ray absorption spectroscopy structural investigation of early intermediates in the mechanism of DNA repair by human ABH2.

Human ABH2 repairs DNA lesions by using an Fe(II)- and αKG-dependent oxidative demethylation mechanism. The structure of the active site features the facial triad of protein ligands consisting of the side chains of two histidine residues and one aspartate residue that is common to many non-heme Fe(II) oxygenases. X-ray absorption spectroscopy (XAS) of metallated (Fe and Ni) samples of ABH2 was used to investigate the mechanism of ABH2 and its inhibition by Ni(II) ions. The data are consistent with a sequential mechanism that features a five-coordinate metal center in the presence and absence of the α-ketoglutarate cofactor. This aspect is not altered in the Ni(II)-substituted enzyme, and both metals are shown to bind the cofactor. When the substrate is bound to the native Fe(II) complex with α-ketoglutarate bound, a five-coordinate Fe(II) center is retained that features an open coordination position for O(2) binding. However, in the case of the Ni(II)-substituted enzyme, the complex that forms in the presence of the cofactor and substrate is six-coordinate and, therefore, features no open coordination site for oxygen activation at the metal.

5-Aza-2'-deoxycytidine inhibiting metastasis to lungs of triple-negative breast cancer cells

Objective To investigate the effect of 5-Aza-2'-deoxycytidine (5-Aza-CdR) on metastasis to lungs of triple-negative breast cancer (TNBC) MDA-MB-231 cells and the mechanisms. Methods MDA-MB-231 cells were divided into two groups: control group and 5-Aza-CdR-treated group. The mRNA expression and promoter methylation status of breast cancer metastasis suppressor-1 (BRMS1 ) and CXC chemokine receptor-4 (CXCR4) in the MDA-MB-231 cells were evaluated by semi-quantitative reverse transcription-polymerase chain reaction ( SqRT-PCR) and methylation specific polymerase chain reaction (MSP) respectively. Then 2 x 105 cells were injected into each BALB/C nu/nu mouse (5 mice in each group) through lateral tail veins. Five weeks later, the metastasis of lungs was evaluated through the mRNA abundance of the target gene HPRT in the lungs tissue from the mice detected by fluorescent quantitative RT-PCR (FqRT-PCR). Results 5-Aza-CdR up-regulated the BRMS1 mRNA expression [0 versus 0. 390 ±0. 001 (relative integrated density value, IDV against GAPDH) ,P ＜0. 05] and demethylated the methylated CpG island B in promoter, while the CXCR4 mRNA expression (0. 580 ±0. 003 versus 0. 580 ±0. 010,P＞0. 05) and the status of unmethylated CXCR4 CpG island 1 in promoter had no significant change. The Ct values of HPRT and GAPDH in control and 5-Aza-CdR-treated groups were 24. 75 ± 1. 55,16. 19 ±0. 69 versus 27. 61 ± 1. 67, 17. 48 ±0. 96 respectively, 2-△△Ct =0. 34, and the inhibitory rate of metastasis was 66% . The mRNA abundance of the HPRT was lower and there were fewer metastases in the lungs of 5-Aza-CdR-treated group than in control group. Conclusion 5-Aza-CdR reactivated tumor metastasis suppressor gene BRMS1 and decreased the metastasis to lungs of TNBC MDA-MB-231 cells by demethylation mechanism. Key words: 5-Aza-CdR; Methylation; Breast carcinoma; Metastasis

Hydroxylation of 5-Methylcytosine by TET1 Promotes Active DNA Demethylation in the Adult Brain

Cytosine methylation is the major covalent modification of mammalian genomic DNA and plays important roles in transcriptional regulation. The molecular mechanism underlying the enzymatic removal of this epigenetic mark, however, remains elusive. Here, we show that 5-methylcytosine (5mC) hydroxylase TET1, by converting 5mCs to 5-hydroxymethylcytosines (5hmCs), promotes DNA demethylation in mammalian cells through a process that requires the base excision repair pathway. Though expression of the 12 known human DNA glycosylases individually did not enhance removal of 5hmCs in mammalian cells, demethylation of both exogenously introduced and endogenous 5hmCs is promoted by the AID (activation-induced deaminase)/APOBEC (apolipoprotein B mRNA-editing enzyme complex) family of cytidine deaminases. Furthermore, Tet1 and Apobec1 are involved in neuronal activity-induced, region-specific, active DNA demethylation and subsequent gene expression in the dentate gyrus of the adult mouse brain in vivo. Our study suggests a TET1-induced oxidation-deamination mechanism for active DNA demethylation in mammals.

The in vivo induction of sister chromatid exchange by the demethylating agent 5-aza-2'-deoxycytidine

Previously, we observed that the demethylating agent 5-azacytidine (azaC) induces a constant and limited low frequency of sister chromatid exchange (SCE), seemingly due to a limited number of SCE-prone sites whose expression is related to DNA demethylation. An alternative explanation for the low frequency of SCE induction may be its inefficient incorporation into DNA, as it has a higher incorporation into RNA. The aim of the present work is to determine if the frequency of SCE induction is increased after exposure to 5-aza-2'-deoxycytidine (azadC), a compound with the same mechanism of demethylation as azaC but more efficiently incorporated into DNA. Groups of mice were treated with 2.2, 4.4, 6.6 and 8.8 μmol azadC per kilogram body weight, and the SCE frequency, the mitotic index and the average generation time were determined after two cell division cycles. The dose-response data of SCE induction showed two components: (i) a dose-dependent increase between 0 and 4.4 μmol and (ii) almost a same level of two SCEs per cell at 4.4 and 8.8 μmol. Although azadC is incorporated more efficiently into DNA, as shown by a lower dose required for a maximal effect, the highest frequency of SCE induction is similar to that observed with azaC. These results indicate that the low incorporation of azaC into DNA seems not to be the factor that limits SCE induction, but the limited number of specific SCE-prone sites in demethylated DNA. Perhaps, there are a restricted number of sites prone to homologous recombination due to DNA demethylation.

Protective effect of quercetin, EGCG, catechin and betaine against oxidative stress induced by ethanol in vitro

There is a need for a nontoxic antioxidant agent to be identified which will prevent alcoholic liver disease (ALD) in alcoholic patients. We tested 4 candidate agents: quercetin, EGCG, catechin and betaine, all of which occur naturally in food. HepG2 cells overexpressing CYP2E1 were subjected to arachidonic acid, iron and 100 mM ethanol with or without the antioxidant agent. All the agents prevented oxidative stress and MDA/4HNE formation induced by ethanol, except for EGCG. Catechin prevented CYP2E1 induction by ethanol. All the agents tended to down-regulate the ethanol-induced increased expression of glutathionine peroxidase 4 (GPX4). All the agents, except catechin, tended to reduce the expression of SOD2 induced by ethanol. Heat shock protein 70 was up-regulated by ethanol alone and betaine tended to prevent this. All 4 agents down-regulated the expression of Gadd45b in the presence of ethanol, which could explain the mechanism of DNA demethylation associated with the up-regulation of the gene expression observed in experimental ALD. In conclusion, the in vitro model of oxidative stress induced by ethanol provided evidence that all 4 agents tested prevented some aspect of liver cell injury caused by ethanol.

Regulation and function of DNA methylation in plants and animals

DNA methylation is an important epigenetic mark involved in diverse biological processes. In plants, DNA methylation can be established through the RNA-directed DNA methylation pathway, an RNA interference pathway for transcriptional gene silencing (TGS), which requires 24-nt small interfering RNAs. In mammals, de novo DNA methylation occurs primarily at two developmental stages: during early embryogenesis and during gametogenesis. While it is not clear whether establishment of DNA methylation patterns in mammals involves RNA interference in general, de novo DNA methylation and suppression of transposons in germ cells require 24-32-nt piwi-interacting small RNAs. DNA methylation status is dynamically regulated by DNA methylation and demethylation reactions. In plants, active DNA demethylation relies on the repressor of silencing 1 family of bifunctional DNA glycosylases, which remove the 5-methylcytosine base and then cleave the DNA backbone at the abasic site, initiating a base excision repair (BER) pathway. In animals, multiple mechanisms of active DNA demethylation have been proposed, including a deaminase- and DNA glycosylase-initiated BER pathway. New information concerning the effects of various histone modifications on the establishment and maintenance of DNA methylation has broadened our understanding of the regulation of DNA methylation. The function of DNA methylation in plants and animals is also discussed in this review.

MicroRNA in Osteoarthritis

Osteoarthritis (OA) is the most prevalent degenerative joint disease and is accompanied by pain and joint dysfunction. Its clinical treatment tends to be unsatisfactory. Novel targets in OA include genes that are involved in OA pathophysiology and have been discovered using gene network, epigenetic and microRNA (miRNA) approaches. miRNA has been implicated in important cellular processes such as lipid metabolism, apoptosis, differentiation and organ development. The importance of miRNA regulation in cellular function is becoming increasingly clear as new miRNA targets are revealed. The present review summarizes the current evidence of the important role played by miRNA in determining the complex gene expression patterns of OA chondrocytes and their role in the regulation of transcription, and possible demethylation mechanisms that might be applicable in OA. In summary, miRNA may have important diagnostic and therapeutic potential, and might provide a novel means of treating OA.

Reactivating aberrantly hypermethylated p15 gene in leukemic T cells by a phenylhexyl isothiocyanate mediated inter-active mechanism on DNA and chromatin

BackgroundWe have previously demonstrated that phenylhexyl isothiocyanate (PHI), a synthetic isothiocyanate, inhibits histone deacetylases and remodels chromatins to induce growth arrest in HL-60 myeloid leukemia cells in a concentration-dependent manner.MethodsTo investigate the effect of PHI, a novel histone deacetylases inhibitor (HDACi), on demethylation and activation of transcription of p15 in acute lymphoid leukemia cell line Molt-4, and to further decipher the potential mechanism of demethylation, DNA sequencing and modified methylation specific PCR (MSP) were used to screen p15-M and p15-U mRNA after Molt-4 cells were treated with PHI, 5-Aza and TSA. DNA methyltransferase 1 (DNMT1), 3A (DNMT3A), 3B (DNMT3B) and p15 mRNA were measured by RT-PCR. P15 protein, acetylated histone H3 and histone H4 were detected by Western Blot.ResultsThe gene p15 in Molt-4 cells was hypermethylated and inactive. Hypermethylation of gene p15 was attenuated and p15 gene was activated de novo after 5 days exposure to PHI in a concentration-dependent manner. DNMT1 and DNMT3B were inhibited by PHI (P < 0.05). Alteration of DNMT3A was not significant at those concentrations. Acetylated histone H3 and histone H4 were accumulated markedly after exposure to PHI.ConclusionPHI could induce both DNA demethylation and acetylated H3 and H4 accumulation in Molt-4 cells. Hypermethylation of gene p15 was reversed and p15 transcription could be reactivated de novo by PHI.

Global DNA Demethylation During Physiological Erythropoiesis In Vivo

AbstractAbstract 2083In the mammalian genome cytosine residues that are followed by guanine (5‘-CpG-3‘ dinucleotides) are frequently methylated, a modification that is associated with transcriptional silencing. Two genome-wide waves of demethylation, in primordial germ cells and in the early pre-implantation embryo, erase methylation marks and are each followed by de novo methylation, setting up a pattern subsequently inherited throughout development [1]. While no global methylation changes are thought to occur during further somatic development, methylation does alter at gene-specific loci, contributing to tissue-specific patterns of gene expression.We set out to study dynamic changes in DNA methylation during erythropoiesis. We used flow cytometry and the cell surface markers CD71 and Ter119 to subdivide freshly isolated fetal liver cells into a developmental sequence of six subsets, from the least mature Subset 0 (S0), to the most mature Subset 5 (S5) [2]. We measured DNA methylation in genomic DNA prepared from freshly sorted S0 to S5 cells. Surprisingly, we found that demethylation at the erythroid-specific β-globin locus control region (LCR) was coincident with progressive genome-wide methylation loss. Both global demethylation as well as demethylation at the β-globin LCR began with the upregulation of CD71 at the onset of erythroid terminal differentiation, and continued with erythroid maturation, with global hypomethylation persisting during enucleation.We employed several distinct methodologies to measure global DNA methylation level. Using Enzyme-Linked Immunosorbent Assay (ELISA), we found that genomic DNA isolated from increasingly mature erythroblasts had progressively reduced binding to a 5-methylcytosine-specific antibody. We also used the LUminometric Methylation Assay (LUMA) to compare the genome-wide cleavage of CCGG sites by each of the isoschizomers HpaII and MspI, which are methylation sensitive and insensitive, respectively. Both the ELISA and LUMA assays showed a global, progressive and significant loss of DNA methylation with erythroid differentiation: 70% of CpG dinucleotides genome-wide were methylated in S0, decreasing to 40–50% by S4/5 (p<0.01). Further, using pyrosequencing of bisulfite-converted DNA, we found a similar decrease in CpG methylation in the promoters of genes whose transcription is silenced with erythroid maturation, notably PU.1 and Fas.To characterize the global loss in methylation further, we examined the status of imprinted genes and of repetitive transposable elements, since both represent genetic loci that are usually stably and highly methylated in somatic cells. We found loss of methylation in imprinted loci, including PEG3 and the H19 Differentially Methylated Region (DMR). We also found a significant loss of methylation at the Long Interspersed Nuclear Element (LINE-1), a repetitive retrotransposon, whose methylation level decreased from over 90% in S0 cells, to 70% in S4/5.Mechanistically, global demethylation was associated with a rapid decline in the DNA methyltransferases DNMT3a and DNMT3b. However, exogenous re-expression of these enzymes in vitro was not sufficient to reverse the process. Both global and erythroid-specific demethylation required rapid DNA replication, triggered with the onset of erythroid terminal differentiation. We were able to slow down demethylation quantitatively by slowing down the rate of DNA replication with aphidicolin, an inhibitor of DNA polymerase α.Global loss of DNA methylation was not associated with a global increase in transcription, as determined by GeneChip analysis, nor was it associated with increased transcription of the LINE-1 retrotransposon. We propose that global demethylation is a consequence of global cellular mechanisms required for the rapid demethylation and induction of β-globin and other erythroid genes. Our findings suggest mechanisms of global demethylation in development and disease, and show that contrary to previously held dogma, DNA demethylation occurs globally during physiological somatic cell differentiation.

ELUSIVE ENZYME SPECIES TRAPPED

WORKING INDEPENDENTLY and after years of unsuccessful efforts, two groups have captured and characterized critical intermediates in oxidation reactions catalyzed by two key enzyme families: cytochrome P450 monooxygenases and DNA-repair dioxygenases. The P450 monooxygenase study helps confirm researchers’ understanding of the workings of a ubiquitous group of enzymes that play a critical role in nutrient and drug metabolism. And the DNA-repair dioxygenase report helps nail down the detailed molecular mechanism of oxidative demethylation, a process that plays a major role in epigenetic gene programming and in conditions such as obesity and cancer. Cytochrome P450 enzymes incorporate a heme iron center and use O 2 to hydroxylate aliphatic carbon-hydrogen bonds, with the remaining oxygen reduced to water. Reearchers have tried for decades to pin down a key intermediate in P450 catalysis, a postulated iron(IV)-oxoporphyrin cation radical known as compound I. Jonathan Rittle and Michael T. Green of Pennsylvania...

Iron-catalysed oxidation intermediates captured in a DNA repair dioxygenase

Mononuclear iron-containing oxygenases conduct a diverse variety of oxidation functions in biology1,2, including the oxidative demethylation of methylated nucleic acids and histones3,4. E. coli AlkB is the first such enzyme that was discovered to repair methylated nucleic acids (Fig. 1)5,6, which are otherwise cytotoxic and/or mutagenic. AlkB human homologues are known to play pivotal roles in various processes7–11. Presented here is the first structural characterization of oxidation intermediates for these demethylases. Employing a chemical cross-linking strategy12,13, complexes of AlkB-dsDNA containing 1,N6-etheno adenine (εA), N3-methyl thymine (3-meT), and N3-methyl cytosine (3-meC) were stabilized and crystallized, respectively. Exposing these crystals, grown under anaerobic conditions containing iron(II) and α-ketoglutarate (αKG), to dioxygen initiates oxidation in crystallo (Supplementary Fig. 1). A glycol (from εA) and a hemiaminal (from 3-meT) intermediates are captured; a zwitterionic intermediate (from 3-2 meC) is also proposed, based on crystallographic observations and computational analysis. The observation of these unprecedented intermediates provides direct support for the oxidative demethylation mechanism for these demethylases. This study also depicts a general mechanistic view of how a methyl group is oxidatively removed from different biological substrates.

Domain structure of the DEMETER 5-methylcytosine DNA glycosylase

DNA glycosylases initiate the base excision repair (BER) pathway by excising damaged, mismatched, or otherwise modified bases. Animals and plants independently evolved active BER-dependent DNA demethylation mechanisms important for epigenetic reprogramming. One such DNA demethylation mechanism is uniquely initiated in plants by DEMETER (DME)-class DNA glycosylases. Arabidopsis DME family glycosylases contain a conserved helix-hairpin-helix domain present in both prokaryotic and eukaryotic DNA glycosylases as well as two domains A and B of unknown function that are unique to this family. Here, we employed a mutagenesis approach to screen for DME residues critical for DNA glycosylase activity. This analysis revealed that amino acids clustered in all three domains, but not in the intervening variable regions, are required for in vitro 5-methylcytosine excision activity. Amino acids in domain A were found to be required for nonspecific DNA binding, a prerequisite for 5-methylcytosine excision. In addition, mutational analysis confirmed the importance of the iron-sulfur cluster motif to base excision activity. Thus, the DME DNA glycosylase has a unique structure composed of three essential domains that all function in 5-methylcytosine excision.

Identification of RING finger protein 4 (RNF4) as a modulator of DNA demethylation through a functional genomics screen

DNA methylation is an important epigenetic modification involved in transcriptional regulation, nuclear organization, development, aging, and disease. Although DNA methyltransferases have been characterized, the mechanisms for DNA demethylation remain poorly understood. Using a cell-based reporter assay, we performed a functional genomics screen to identify genes involved in DNA demethylation. Here we show that RNF4 (RING finger protein 4), a SUMO-dependent ubiquitin E3-ligase previously implicated in maintaining genome stability, plays a key role in active DNA demethylation. RNF4 reactivates methylation-silenced reporters and promotes global DNA demethylation. Rnf4 deficiency is embryonic lethal with higher levels of methylation in genomic DNA. Mechanistic studies show that RNF4 interacts with and requires the base excision repair enzymes TDG and APE1 for active demethylation. This activity appears to occur by enhancing the enzymatic activities that repair DNA G:T mismatches generated from methylcytosine deamination. Collectively, our study reveals a unique function for RNF4, which may serve as a direct link between epigenetic DNA demethylation and DNA repair in mammalian cells.

UTX mediates demethylation of H3K27me3 at muscle-specific genes during myogenesis

Polycomb (PcG) and Trithorax (TrxG) group proteins act antagonistically to establish tissue-specific patterns of gene expression. The PcG protein Ezh2 facilitates repression by catalysing histone H3-Lys27 trimethylation (H3K27me3). For expression, H3K27me3 marks are removed and replaced by TrxG protein catalysed histone H3-Lys4 trimethylation (H3K4me3). Although H3K27 demethylases have been identified, the mechanism by which these enzymes are targeted to specific genomic regions to remove H3K27me3 marks has not been established. Here, we demonstrate a two-step mechanism for UTX-mediated demethylation at muscle-specific genes during myogenesis. Although the transactivator Six4 initially recruits UTX to the regulatory region of muscle genes, the resulting loss of H3K27me3 marks is limited to the region upstream of the transcriptional start site. Removal of the repressive H3K27me3 mark within the coding region then requires RNA Polymerase II (Pol II) elongation. Interestingly, blocking Pol II elongation on transcribed genes leads to increased H3K27me3 within the coding region, and formation of bivalent (H3K27me3/H3K4me3) chromatin domains. Thus, removal of repressive H3K27me3 marks by UTX occurs through targeted recruitment followed by spreading across the gene.

DNA methylation reprogramming and DNA repair in the mouse zygote

Here, we summarize current knowledge about epigenetic reprogramming during mammalian preimplantation development, as well as the potential mechanisms driving these processes. We will particularly focus on changes taking place in the zygote, where the paternally derived DNA and chromatin undergo the most striking alterations, such as replacement of protamines by histones, histone modifications and active DNA demethylation. The putative mechanisms of active paternal DNA demethylation have been studied for over a decade, accumulating a lot of circumstantial evidence for enzymatic activities provided by the oocyte, protection of the maternal genome against such activities and possible involvement of DNA repair. We will discuss the various facets of dynamic epigenetic changes related to DNA methylation with an emphasis on the putative involvement of DNA repair in DNA demethylation.

Biochemistry and occurrence of O-demethylation in plant metabolism

Demethylases play a pivitol role in numerous biological processes from covalent histone modification and DNA repair to specialized metabolism in plants and microorganisms. Enzymes that catalyze O- and N-demethylation include 2-oxoglutarate (2OG)/Fe(II)-dependent dioxygenases, cytochromes P450, Rieske-domain proteins and flavin adenine dinucleotide (FAD)-dependent oxidases. Proposed mechanisms for demethylation by 2OG/Fe(II)-dependent enzymes involve hydroxylation at the O- or N-linked methyl group followed by formaldehyde elimination. Members of this enzyme family catalyze a wide variety of reactions in diverse plant metabolic pathways. Recently, we showed that 2OG/Fe(II)-dependent dioxygenases catalyze the unique O-demethylation steps of morphine biosynthesis in opium poppy, which provides a rational basis for the widespread occurrence of demethylases in benzylisoquinoline alkaloid metabolism.

Active DNA Demethylation Mediated by DNA Glycosylases

Active DNA demethylation is involved in many vital developmental and physiological processes of plants and animals. Recent genetic and biochemical studies in Arabidopsis have demonstrated that a subfamily of DNA glycosylases function to promote DNA demethylation through a base excision-repair pathway. These specialized bifunctional DNA glycosylases remove the 5-methylcytosine base and then cleave the DNA backbone at the abasic site, resulting in a gap that is then filled with an unmethylated cytosine nucleotide by as yet unknown DNA polymerase and ligase enzymes. Evidence suggests that active DNA demethylation in mammalian cells is also mediated at least in part by a base excision repair pathway where the AID/Apobec family of deaminases convert 5-methylcytosine to thymine followed by G/T mismatch repair by the DNA glycosylase MBD4 or TDG. This review also discusses other possible mechanisms of active DNA demethylation, how genome DNA methylation status might be sensed to regulate the expression of demethylase genes, and the targeting of demethylases by small RNAs.

O6-Methylguanine Repair by O6-Alkylguanine-DNA Alkyltransferase

O6-alkylguanine-DNA alkyltransferase (AGT) repairs O6-methylguanine (O6mG) in DNA that is known to cause mutation and cancer. On the basis of calculations performed using density functional theory involving the active site of AGT, a mechanism for catalytic demethylation of O6mG to guanine has been proposed. In this mechanism, roles of six amino acids, i.e., Cys145, His146, Glu172, Tyr114, Lys165, and Ser159 in catalytic demethylation of O6mG are involved. This mechanism has three steps as follows. At the first step, Cys145 in the Cys145-water-His146-Glu172 tetrad is converted to cysteine thiolate anion while at the second step, abstraction of the Tyr114 proton by the N3 site of O6mG occurs in a barrierless manner. In the third step, abstraction of Lys165 proton by deprotonated Tyr114 and transfer of the methyl group of O6mG to the thiolate group of Cys145 anion occur simultaneously. As AGT is a major target in cancer therapy, identification of the roles of the different amino acids in demethylation of O6mG is expected to be useful in designing efficient AGT inhibitors.

Effects of 5-aza-2'-deoxycytidine on the amount of exosomes and secreted immuno-associated proteins by hepatoma cells

To investigate the effects of 5-Aza-deoxycytidine (5-Aza-CdR) on the amount of exosomes and immuno-associated proteins produced in hepatoma HepG2 and Hep3B cells. Exosomes derived from HepG2 and Hep3B cells with or without treatment by 5-Aza-CdR were isolated and purified by combination of ultrafiltration centrifugation and sucrose gradient ultracentrifugation. The number of exosomes was counted under the electron microscope. The concentration of proteins in exosomes was detected by BCA. The expression of HSP70, HLA-I and NY-ESO-1 proteins in exosomes was assayed by Western blot and immuno-electron microscopy. The mRNA expression of p53 gene was observed by reverse transcription-polymerase chain reaction (RT-PCR). The mRNA expression of p53 gene was increased in both hepatoma cell lines after treatment with 5-Aza-CdR. The number of exosomes and the concentration of total proteins in exosomes were significantly increased after treatment by 5-Aza-CdR (P < 0.05). The immuno-electron microscopy and Western blotting showed that after treatment with 5-Aza-CdR, the contents of HSP70, HLA-I and NY-ESO-1 proteins were increased in exosomes in both HepG2 and Hep3B hepatoma cells. 5-Aza-CdR, an inhibitor of DNA methyltransferase, can increase the amount of exosomes and exosome-containning immuno-associated proteins secreted by hepatoma cells. It may be contributed by up-regulation of p53 gene and demethylation mechanism of 5-Aza-CdR.