Presence Of DNA Methyltransferases Research Articles

Chidamide, Decitabine, Cytarabine, Aclarubicin, and Granulocyte Colony-stimulating Factor Therapy for Patients with Relapsed/Refractory Acute Myeloid Leukemia: A Retrospective Study from a Single-Center.

Preclinical evidence and clinical trials have suggested synergistic effects of epigenetic modifiers in combination with cytotoxic agents for the treatment of leukemia. However, their efficacy in patients with relapsed/refractory acute myeloid leukemia (R/R AML) remains unclear. Clinical data of R/R AML patients who received a CDCAG regimen (chidamide, decitabine, cytarabine, aclarubicin, and granulocyte colony-stimulating factor) from July 1, 2018 to October 31, 2021 at our center were retrospectively assessed, and the safety and efficacy of the CDCAG regimen were evaluated. Patients were followed up until November 30, 2021, with a median follow-up of 21.6 months (95% CI: 10.0-33.2 months). A total of 67 patients were enrolled. Two patients died within 3 weeks after the initiation, and therefore only 65 patients underwent the assement for clinical response and survival. It was found that 56.9% patients achieved complete remission with a median overall survival (OS) of 9.6 months. The median OS of responders was 25.9 months, while that of non-responders was 5.0 months (P<0.0001). Patients with gene mutations had a superior overall response rate (ORR) (80.4% vs. 45.5%, P=0.043) compared to those without gene mutations. The presence of DNA methyltransferase 3 A (DNMT3A), ten-eleven translocation-2 (TET2), and isocitrate dehydrogenase 1/2 (IDH1/2) mutations did not affect the response rate (88.2% vs. 68.9%, P=0.220) and reflected a better OS (not attained vs. 9.0 months, P=0.05). The most common non-hematologic adverse events were pulmonary infection (73.1%), followed by febrile neutropenia (23.9%) and sepsis (19.4%). The CDCAG regimen was effective and well-tolerated in R/R AML patients, increasing the potential for allogeneic hematopoietic stem cell transplantation. Moreover, patients with DNMT3A, TET2, and IDH1/2 mutations might benefit from this regimen.

Effect of Demethylation on the Presence of DNA Methyltransferases in Complex with Histone Methylation Markers in Two Myeloma Cell Lines

Background: Enzymes that catalyze CpG methylation in DNA, including the DNA methyltransferases 1 (DNMT1), 3A (DNMT3A) and 3B (DNMT3B) are also implicated in human developmental disorders and cancers, supporting the critical role of DNA methylation. Posttranslational modifications of histones influence the representation of DNMTs in protein complexes including specific histone markers. H3K4me3, H3K36me3 are histone markers of active gene transcription, while for example H3K9me3, H3K27me3 are histone markers of inactive chromatin associated with repressive transcription of the respective genes. Objective: The aim of the study was to find whether demethylation of myeloma cells can affect 1) the formation of a complex of the repressive histone marker - H3K9me3 with DNMT3A and/or DNMT3B; and 2) association of the transcriptionally active histone marker H3K4me3 with DNMT1. Material &amp; Methods: Chromatin lysate was isolated from two myeloma cell lines - KMS12-BM and KMS12-PE, after their 72 hours treatment with 5-Azacytidine (AZA) and/or 5-Aza-2´-deoxycitidine (DAC). Precipitation was performed with antibodies against H3K9me3 and/or H3K4me3. The occurrence of DNMT3A and/or DNMT3B in precipitated complex with H3K9me3 was quantified by qPCR (SYBR Green I) with primers specific for both promoter or coding regions of the respective DNMT genes. The assesment of DNMT1 in complex precipitated with H3K4me3 was quantified by qPCR (SYBR Green I) with primers specific for DNMT1. For result calculation, the 2-delta Ct values of immunoprecipitated samples were normalized to the 2-delta Ct value of IgG respective antibodies, and then the obtained 2(-(delta delta Ct)) value of analyzed samples was normalized to the 2(-(delta delta Ct)) value of control untreated cells (DMSO). Results: In KMS12-PE myeloma cells, we determined the 2(-(delta delta Ct)) values of DNMT3B in precipitated complex with H3K9me3 - 1.52 and 1.47, in cells treated with 0.5 µM AZA and 0.5 µM DAC, respectively. The obtained 2(-(delta delta Ct) values were higher than that of control untreated (DMSO) myeloma cells [with the of 2(-(delta delta Ct)) value of 0.85]. Increased occurrence of DNMT3A was found in H3K9me3 complex of all analyzed samples in comparison to untreated control cells [with the of 2(-(delta delta Ct)) value of 0.89]; the highest 2(-(delta delta Ct) values - 1.07 and 1.21 were determined after DAC treatment. The obtained 2(-(delta delta Ct)) values of DNMT1 from analyzed precipitated samples with H3K4me3 were lower than that of DMSO treated KMS12-PE cells [with the of 2(-(delta delta Ct)) value of 0.85]. In KMS12-BM, we did not detect any changes in the abundance of DNMT3B in precipitated complexes including H3K4me3. Conclusion: In KMS12-PE, but not in KMS12-BM, both demethylation agents used, 5-Aza-2´-deoxycitidine and 5-Azacytidine, affected the appearance of DNMT3A and DNMT3B in a protein complex containing the repressive histone marker H3K9me3 with possible potential to inactivate gene transcription.

Construction of a Dye-Sensitized and Gold Plasmon-Enhanced Cathodic Photoelectrochemical Biosensor for Methyltransferase Activity Assay.

DNA methyltransferases may function as important biomarkers of cancers and genetic diseases. Herein, we develop a dye-sensitized and gold plasmon-enhanced cathodic photoelectrochemical (PEC) biosensor on the basis of p-type covalent organic polymers (COPs) for the signal-on measurement of M.SssI methyltransferase (M.SssI MTase). The cathodic PEC biosensor is constructed by the in situ growth of p-type COP films onto a glass coated with indium tin oxide and the subsequent assembly of biotin- and HS-labeled double-stranded DNA (dsDNA) probes onto the COP film via biotin-streptavidin interaction. The dsDNA probe contains the recognition sequence of M.SssI MTase. The COP thin films possess a porous ultrathin nanosheet structure with abundant active sites, facilitating the generation of a high photocurrent compared with the hydrothermally synthesized ones. The presence of DNA methyltransferases can prevent the digestion of restriction endonuclease HpaII, consequently inducing the introduction of gold nanoparticles (AuNPs) to the dsDNA probes via the S-Au bond and the intercalation of rhodamine B (RhB) into the DNA grooves to produce a high photocurrent due to the dye-photosensitized enhancement and AuNP-mediated surface plasmon resonance. However, in the absence of M.SssI MTase, HpaII digests the dsDNA probes, and neither AuNPs nor RhB can be introduced onto the electrode surface, leading to a low photocurrent. This cathodic PEC biosensor possesses high sensitivity and good selectivity, and it can screen the inhibitors and detect M.SssI MTase in serum as well.

Antimicrobial activity and acetylcholinesterase inhibition by extracts from chromatin modulated fungi.

Major health challenges as the increasing number of cases of infections by antibiotic multiresistant microorganisms and cases of Alzheimer's disease have led to searching new control drugs. The present study aims to verify a new way of obtaining bioactive extracts from filamentous fungi with potential antimicrobial and acetylcholinesterase inhibitory activities, using epigenetic modulation to promote the expression of genes commonly silenced. For such finality, five filamentous fungal species (Talaromyces funiculosus, Talaromyces islandicus, Talaromyces minioluteus, Talaromyces pinophilus, Penicillium janthinellum) were grown or not with DNA methyltransferases inhibitors (procainamide or hydralazine) and/or a histone deacetylase inhibitor (suberohydroxamic acid). Extracts from T. islandicus cultured or not with hydralazine inhibited Listeria monocytogenes growth in 57.66 ± 5.98% and 15.38 ± 1.99%, respectively. Increment in inhibition of acetylcholinesterase activity was observed for the extract from P. janthinellum grown with procainamide (100%), when compared to the control extract (39.62 ± 3.76%). Similarly, inhibition of acetylcholinesterase activity increased from 20.91 ± 3.90% (control) to 92.20 ± 3.72% when the tested extract was obtained from T. pinophilus under a combination of suberohydroxamic acid and procainamide. Concluding, increases in antimicrobial activity and acetylcholinesterase inhibition were observed when fungal extracts in the presence of DNA methyltransferases and/or histone deacetylase modulators were tested.

Sensitive detection of DNA methyltransferase using the dendritic rolling circle amplification-induced fluorescence

The analysis of DNA methylation and MTase activities is very important in the early clinical diagnosis of cancer, on purposes of providing insights into the mechanism of gene repression and developing novel drugs of treating methylation-related diseases. Combining the dendritic rolling circle amplification and Mg2+-dependent DNAzyme with a function of catalyzing the generation of a fluorophore-labeled nucleic acid acting as readout signal for the analyses, a new fluorescent method for DNA methyltransferase detection was reported. In the presence of DNA methyltransferases (MTase), the methylation-responsive sequence of double-stranded DNA probe was methylated and then cleaved by the methylation-sensitive restriction endonuclease DpnI. The cleaved hybrid DNA probe then functioned as a signal primer to initiate the dendritic rolling circle amplification reaction, containing a circular DNA and a structurally tailored hairpin structure. Subsequently, the circular nucleic acid template produced a complementary sequence to the Mg2+-dependent DNAzyme and a sequence identical to the loop region of the co-added hairpin structure. At last, a fluorescence readout signal was afforded by the DNAzyme-catalyzed cleavage of a fluorophore/quencher-modified substrate. This method enabled the analysis of the target MTase with a detection limit up to 0.36 U mL−1, and a dynamic range was obtained from 1.0 to 10 U mL−1. Moreover, the proposed strategy was successfully applied in real sample assay. With this assay, the inhibitors of MTase were evaluated and screened which might be helpful for the discovery of anticancer drugs.

DNA methylation in an engineered heart tissue model of cardiac hypertrophy: common signatures and effects of DNA methylation inhibitors.

DNA methylation affects transcriptional regulation and constitutes a drug target in cancer biology. In cardiac hypertrophy, DNA methylation may control the fetal gene program. We therefore investigated DNA methylation signatures and their dynamics in an in vitro model of cardiac hypertrophy based on engineered heart tissue (EHT). We exposed EHTs from neonatal rat cardiomyocytes to a 12-fold increased afterload (AE) or to phenylephrine (PE 20 µM) and compared DNA methylation signatures to control EHT by pull-down assay and DNA methylation microarray. A 7-day intervention sufficed to induce contractile dysfunction and significantly decrease promoter methylation of hypertrophy-associated upregulated genes such as Nppa (encoding ANP) and Acta1 (α-skeletal actin) in both intervention groups. To evaluate whether pathological consequences of AE are affected by inhibiting de novo DNA methylation we applied AE in the absence and presence of DNA methyltransferase (DNMT) inhibitors: 5-aza-2'-deoxycytidine (aza, 100 µM, nucleosidic inhibitor), RG108 (60 µM, non-nucleosidic) or methylene disalicylic acid (MDSA, 25 µM, non-nucleosidic). Aza had no effect on EHT function, but RG108 and MDSA partially prevented the detrimental consequences of AE on force, contraction and relaxation velocity. RG108 reduced AE-induced Atp2a2 (SERCA2a) promoter methylation. The results provide evidence for dynamic DNA methylation in cardiac hypertrophy and warrant further investigation of the potential of DNA methylation in the treatment of cardiac hypertrophy.

Progresses of DNA methylation in common ocular tumor

DNA methylation is a major event of epigenetic modifications. In the presence of DNA methyltransferases (DNMT), the addition of methyl groups from cytosines can impact gene expression and alter cell and organism function. Methyl groups of CpG sites in the promoter can directly interfere with binding of transcription factors to their recognition cis elements or, on the other side, can facilitate interaction with a family of methyl-binding proteins and result in gene silencing. Analysis of DNA methylation includes genome-wide DNA methylation assessment, analysis of gene-specific DNA methylation and finding of new DNA methylation sites. The implication of DNA methylation has been suggested with the pathogenesis of retinoblastoma, uveal tract melanoma and ocular surface tumor. We focus on the progresses of DNA methylation on ocular tumors in this article.

Neonatal Nutrition and Consequences on Adult Health

After completing this article, readers should be able to: 1. Describe aberrations in adult health that can result from nutrition in the fetal period and in neonatal life. 2. Review the results of studies from human neonates regarding early nutritional interventions designed for “catch-up” growth. 3. Review results from animal studies regarding early nutrition and “programming” for adult metabolic syndrome. 4. Describe some of the potential epigenetic mechanisms that may be responsible for “programming.” 5. Examine methods for preventing growth delays, the consequences of undernutrition, and the metabolic syndrome related to adverse “programming.” One of the traditional goals of neonatal nutrition is to maintain growth within standardized limits. In the preterm infant, growth between the 10th and 90th percentiles of intrauterine rate has been an ideal goal. More recently, maintenance of lean body mass and bone density; prevention of complications such as chronic lung disease, necrotizing enterocolitis, and infection; and optimization of neurodevelopment and adult health through early nutritional programming have become recognized as more meaningful goals than mere somatic growth. To meet these goals, current nutritional practices require thorough scrutiny in terms of quantity and quality (composition) and how they are customized, if not for the individual patient (which would be ideal), at least for subgroups such as those who are small or appropriate for gestational age, sick or healthy, preterm or term, or male or female. Fetal nutrition and development frequently are used as a template for the low-birthweight infant. A large body of literature on the consequences of fetal undernutrition shows that infants born small for gestational age (SGA) not only have short-term morbidity, but long-term morbidity manifested in what has been termed the “metabolic syndrome.” This syndrome includes abdominal obesity, arterial hypertension, and insulin resistance. Other clinical manifestations can be observed, including thyroid dysfunction, hirsutism, ovarian hyperandrogenism and infertility, dyslipidemia (increased …

DNA mismatch-specific base flipping by a bisacridine macrocycle.

Most, if not all, enzymes that chemically modify nucleobases in DNA flip their target base from the inside of the double helix into an extrahelical position. This energetically unfavorable conformation is partly stabilized by specific binding of the apparent abasic site being formed. Thus, DNA base-flipping enzymes, like DNA methyltransferases and DNA glycosylases, generally bind very strongly to DNA containing abasic sites or abasic-site analogues. The macrocyclic bisacridine BisA has previously been shown to bind abasic sites. Herein we demonstrate that it is able to specifically recognize DNA base mismatches and most likely induces base flipping. Specific binding of BisA to DNA mismatches was studied by thermal denaturation experiments by using short duplex oligodeoxynucleotides containing central TT, TC, or TG mismatches or a TA match. In the presence of the macrocycle a strong increase in the melting temperature of up to 7.1 degrees C was observed for the mismatch-containing duplexes, whereas the melting temperature of the fully matched duplex was unaffected. Furthermore, BisA binding induced an enhanced reactivity of the mispaired thymine residue in the DNA toward potassium permanganate oxidation. A comparable reactivity has previously been observed for a TT target base mismatch in the presence of DNA methyltransferase M.TaqI. This similarity to a known base-flipping enzyme suggests that insertion of BisA into the DNA helix displaces the mispaired thymine residue into an extrahelical position, where it should be more prone to chemical oxidation. Thus, DNA base flipping does not appear to be limited to DNA-modifying enzymes but it is likely to also be induced by a small synthetic molecule binding to a thermodynamically weakened site in DNA.

Regulated synthesis and localization of DNA methyltransferase during spermatogenesis.

Differences in the methylation patterns of male and female gamete DNA are likely to be involved in genomic imprinting. However, little is known of the mechanisms that regulate de novo methylation and demethylation during gametogenesis. We report here that the well-characterized M(r) 190,000 form of DNA methyltransferase (the only known form) is present in isolated mitotic, meiotic, and postmeiotic male germ cells, with the exception of meiotic pachytene spermatocytes, where the protein is undetectable by immunoblot analysis and a novel 6.2-kb DNA methyltransferase transcript is present. Whereas replication and methylation are coupled in somatic cells, the presence of DNA methyltransferase in postreplicative male germ cells is consistent with previously observed de novo methylation events in these cells. Immunofluorescence experiments revealed that DNA methyltransferase is localized to the nuclei of male germ cells, with a subset of spermatogonia and postreplicative leptotene/zygotene spermatocytes displaying prominent nuclear foci that are strongly enriched in DNA methyltransferase. The data suggest that down-regulation of DNA methyltransferase expression during the pachytene stage of meiosis utilizes an mechanism that is associated with the production of a larger mRNA, and that de novo methylation in leptotene/zygotene spermatocytes may take place in spatially restricted nuclear domains that are enriched in DNA methyltransferase.