The Experts below are selected from a list of 23724 Experts worldwide ranked by ideXlab platform
Norbert O Reich - One of the best experts on this subject based on the ideXlab platform.
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a novel class of selective non nucleoside inhibitors of human DNA Methyltransferase 3A
2021Co-Authors: Sunzeyu Huang, Nathaniel J Stillson, Jonathan E Sandoval, Chitoh Yung, Norbert O ReichAbstract:Screening of a small chemical library (Medicines for Malaria Venture Pathogen Box) identified two structurally related pyrazolone (inhibitor 1) and pyridazine (inhibitor 2) DNMT3A inhibitors with low micromolar inhibition constants. The uncompetitive and mixed type inhibition patterns with DNA and AdoMet suggest these molecules act through an allosteric mechanism, and thus are unlikely to bind to the enzyme's active site. Unlike the clinically used mechanism based DNMT inhibitors such as decitabine or azacitidine that act via the enzyme active site, the inhibitors described here could lead to the development of more selective drugs. Both inhibitors show promising selectivity for DNMT3A in comparison to DNMT1 and bacterial DNA cytosine Methyltransferases. With further study, this could form the basis of preferential targeting of de novo DNA methylation over maintenance DNA methylation.
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rna modulation of the human DNA Methyltransferase 3A
2012Co-Authors: Celeste Holzschietinger, Norbert O ReichAbstract:DNA Methyltransferase 3A (DNMT3A) is one of two human de novo DNA Methyltransferases essential for transcription regulation during cellular development and differentiation. There is increasing evidence that RNA plays a role in directing DNA methylation to specific genomic locations within mammalian cells. Here, we describe two modes of RNA regulation of DNMT3A in vitro. We show a single-stranded RNA molecule that is antisense to the E-cadherin promoter binds tightly to the catalytic domain in a structurally dependent fashion causing potent inhibition of DNMT3A activity. Two other RNA molecules bind DNMT3A at an allosteric site outside the catalytic domain, causing no change in catalysis. Our observation of the potent and specific in vitro modulation of DNMT3A activity by RNA supports in vivo data that RNA interacts with DNMT3A to regulate transcription.
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rna modulation of the human DNA Methyltransferase 3A
2012Co-Authors: Celeste Holzschietinger, Norbert O ReichAbstract:DNA Methyltransferase 3A (DNMT3A) is one of two human de novo DNA Methyltransferases essential for transcription regulation during cellular development and differentiation. There is increasing evidence that RNA plays a role in directing DNA methylation to specific genomic locations within mammalian cells. Here, we describe two modes of RNA regulation of DNMT3A in vitro. We show a single-stranded RNA molecule that is antisense to the E-cadherin promoter binds tightly to the catalytic domain in a structurally dependent fashion causing potent inhibition of DNMT3A activity. Two other RNA molecules bind DNMT3A at an allosteric site outside the catalytic domain, causing no change in catalysis. Our observation of the potent and specific in vitro modulation of DNMT3A activity by RNA supports in vivo data that RNA interacts with DNMT3A to regulate transcription.
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identification of a second DNA binding site in human DNA Methyltransferase 3A by substrate inhibition and domain deletion
2010Co-Authors: Matthew M Purdy, Celeste Holzschietinger, Norbert O ReichAbstract:The human DNA Methyltransferase 3A (DNMT3A) is essential for establishing DNA methylation patterns. Knowing the key factors involved in the regulation of mammalian DNA methylation is critical to furthering understanding of embryonic development and designing therapeutic approaches targeting epigenetic mechanisms. We observe substrate inhibition for the full length DNMT3A but not for its isolated catalytic domain, demonstrating that DNMT3A has a second binding site for DNA. Deletion of recognized domains of DNMT3A reveals that the conserved PWWP domain is necessary for substrate inhibition and forms at least part of the allosteric DNA binding site. The PWWP domain is demonstrated here to bind DNA in a cooperative manner with muM affinity. No clear sequence preference was observed, similar to previous observations with the isolated PWWP domain of Dnmt3b but with one order of magnitude weaker affinity. Potential roles for a low affinity, low specificity second DNA binding site are discussed.
Kevin V Morris - One of the best experts on this subject based on the ideXlab platform.
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the molecular dynamics of long noncoding rna control of transcription in pten and its pseudogene
2017Co-Authors: Nicholas Lister, Per Johnsson, Linda Vidarsdottir, Dan Grander, Galina Shevchenko, James L Walshe, Jessica N Groen, Sandro F Ataide, Kevin V MorrisAbstract:Abstract RNA has been found to interact with chromatin and modulate gene transcription. In human cells, little is known about how long noncoding RNAs (lncRNAs) interact with target loci in the context of chromatin. We find here, using the phosphatase and tensin homolog (PTEN) pseudogene as a model system, that antisense lncRNAs interact first with a 5′ UTR-containing promoter-spanning transcript, which is then followed by the recruitment of DNA Methyltransferase 3A (DNMT3A), ultimately resulting in the transcriptional and epigenetic control of gene expression. Moreover, we find that the lncRNA and promoter-spanning transcript interaction are based on a combination of structural and sequence components of the antisense lncRNA. These observations suggest, on the basis of this one example, that evolutionary pressures may be placed on RNA structure more so than sequence conservation. Collectively, the observations presented here suggest a much more complex and vibrant RNA regulatory world may be operative in the regulation of gene expression.
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a pseudogene long noncoding rna network regulates pten transcription and translation in human cells
2013Co-Authors: Per Johnsson, Amanda Ackley, Linda Vidarsdottir, Wengonn Lui, Martin Corcoran, Dan Grander, Kevin V MorrisAbstract:PTEN is a tumor-suppressor gene that has been shown to be under the regulatory control of a PTEN pseudogene expressed noncoding RNA, PTENpg1. Here, we characterize a previously unidentified PTENpg1-encoded antisense RNA (asRNA), which regulates PTEN transcription and PTEN mRNA stability. We find two PTENpg1 asRNA isoforms, α and β. The α isoform functions in trans, localizes to the PTEN promoter and epigenetically modulates PTEN transcription by the recruitment of DNA Methyltransferase 3A and Enhancer of Zeste. In contrast, the β isoform interacts with PTENpg1 through an RNA-RNA pairing interaction, which affects PTEN protein output through changes of PTENpg1 stability and microRNA sponge activity. Disruption of this asRNA-regulated network induces cell-cycle arrest and sensitizes cells to doxorubicin, which suggests a biological function for the respective PTENpg1 expressed asRNAs.
Aline Renneville - One of the best experts on this subject based on the ideXlab platform.
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prognostic significance of DNA Methyltransferase 3A mutations in cytogenetically normal acute myeloid leukemia a study by the acute leukemia french association
2012Co-Authors: Aline Renneville, Nicolas Boissel, Olivier Nibourel, Celine Berthon, Nathalie Helevaut, Claude Gardin, J M CayuelaAbstract:Recently, DNA Methyltransferase 3A (DNMT3A) mutations have been identified in acute myeloid leukemia (AML), the highest frequency being found within cytogenetically normal (CN) AML. In this study, diagnostic samples from 123 adults younger than 60 years with primary CN-AML homogeneously treated in the Acute Leukemia French Association-9801 and -9802 trials were screened for mutations in DNMT3A-conserved domains by direct sequencing. Patients were also assessed for the presence of FLT3 (fms-like tyrosine kinase receptor-3), NPM1 (nucleophosmin), CEBPA, WT1 (Wilms tumor 1), IDH1 (isocitrate dehydrogenase 1) and IDH2 mutations. Thirty-eight mutations were detected in 36 patients (29%): 36 nucleotide substitutions, mostly affecting amino-acid residue R882 and two frameshift deletions. DNMT3A mutations were significantly associated with the French–American–British subtypes M4/M5 and the presence of NPM1 mutations. In the whole cohort, DNMT3A mutated patients had a shorter event-free survival (5-year EFS: 13% vs 32%, P=0.02) and overall survival (5-year OS: 23% vs 45%, P=0.02) compared with DNMT3A wild-type patients. In multivariate analysis including age, white blood cell count, NPM1/FLT3-internal tandem duplication/CEBPA risk group and DNMT3A mutational status, the presence of a DNMT3A mutation remained an independent adverse prognostic factor for EFS and OS, suggesting that testing for DNMT3A mutations could help further improve risk stratification in CN-AML.
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prognostic significance of DNA Methyltransferase 3A mutations in cytogenetically normal acute myeloid leukemia a study by the acute leukemia french association
2011Co-Authors: Aline Renneville, Nicolas Boissel, Olivier Nibourel, Celine Berthon, Nathalie Helevaut, Claude Gardin, Jeanmichel Cayuela, Sandrine Hayette, Oumedaly Reman, Nathalie ContentinAbstract:Abstract 2506 Introduction: The development of massively parallel sequencing technologies has led to the identification of somatic DNA Methyltransferase 3A (DNMT3A) gene mutations in acute myeloid leukemia (AML), with the highest frequency being found in cytogenetically normal (CN) AML. DNMT3A mutations have been suggested to predict poor clinical outcome in AML, but only few data are available on their prognostic significance within CN-AML. The aim of this study was to determine the frequency, the main associated features, and the prognostic significance of DNMT3A mutations in CN-AML. Patients and methods: This retrospective study was performed in 123 young adult patients (16–60 years) with previously untreated primary CN-AML and enrolled on two concomitant protocols of the Acute French Leukemia Association (ALFA), the ALFA-9801 and ALFA-9802 trials. DNMT3A mutations were screened on genomic DNA by PCR and direct Sanger sequencing. We focused our screening on the 3 conserved domains of DNMT3A (the proline-tryptophane-tryptophane-proline (PWWP) domain, the ADD-type zinc finger domain, and the C5-Methyltransferase domain), corresponding to exons 8–9 and 11–23. The patients were also assessed for the presence of FLT3 internal tandem duplication (FLT3-ITD), FLT3 tyrosine kinase domain (FLT3-TKD), NPM1, CEBPA, WT1, IDH1, and IDH2 mutations. Results: Thirty-eight DNMT3A mutations were identified in 36 of the 123 (29%) patients. These alterations consisted of 36 nucleotide substitutions and 2 frameshift deletions. Thirty out of 36 (83%) nucleotide substitutions affected the amino acid residue R882 (R882H, n = 21; R882C, n = 7; R882P, n = 2), 5 represented other missense alterations, and 1 was a nonsense mutation. Two patients exhibited 2 heterozygous missense mutations in different exons, and one patient had a homozygous missense mutation. DNMT3A mutated and wild-type cases did not differ in terms of age, gender, and white blood cell (WBC) count at presentation. DNMT3A mutations were strongly associated with the French-American-British (FAB) subtypes M4/M5 (P =.0002) and the presence of NPM1 mutations (P =.0006), and tended to often co-occur with IDH1R132 mutations (P = .09). In the entire cohort, complete remission rate was found lower in DNMT3A mutated patients than in DNMT3A wild-type patients, but without reaching statistical significance (80% vs 90%, P =.24). DNMT3A mutated patients had a shorter event-free survival (5-year EFS: 13% vs 32%, P =.02) and overall survival (5-year OS: 23% vs 45%, P =.02) compared to DNMT3A wild-type patients. We next performed subgroup analysis according to the NPM1/FLT3-ITD genotypes. In patients with the non-favorable genotypes, that is NPM1 mutated/FLT3-ITD positive, NPM1 wild-type/FLT3-ITD positive, NPM1 wild-type/FLT3-ITD negative (n = 86), 18 (21%) had a concomitant DNMT3A mutation. In this high-risk subgroup of CN-AML, DNMT3A mutations conferred a worse clinical outcome (5-year EFS: 0% vs 23%, P =.02; 5-year OS: 14% vs 37%, P =.06). In patients with the favorable genotype NPM1 mutated/FLT3-ITD negative (n = 37), 18 (49%) were found to display a concomitant DNMT3A mutation. Within this favorable subgroup, patients carrying a DNMT3A mutation had a significantly inferior EFS and OS compared to DNMT3A wild-type patients (5-year EFS: 25% vs 65%, P =.01; 5-year OS: 29% vs 76%, P =.02). Furthermore, in multivariate analysis including age, WBC count, NPM1/FLT3-ITD genotypes, and DNMT3A mutational status, the presence of a DNMT3A mutation remained an independent adverse prognostic factor for EFS (hazard ratio = 2.29; 95% CI, 1.42 to 3.70; P =.0007) and OS (hazard ratio = 2.34; 95% CI, 1.37 to 4.00; P =.002). Conclusion: DNMT3A mutations are one of the most common gene mutations in CN-AML and independently predict poor clinical outcome. Testing for DNMT3A mutations could help further improve risk stratification in CN-AML. Disclosures: No relevant conflicts of interest to declare.
Celeste Holzschietinger - One of the best experts on this subject based on the ideXlab platform.
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rna modulation of the human DNA Methyltransferase 3A
2012Co-Authors: Celeste Holzschietinger, Norbert O ReichAbstract:DNA Methyltransferase 3A (DNMT3A) is one of two human de novo DNA Methyltransferases essential for transcription regulation during cellular development and differentiation. There is increasing evidence that RNA plays a role in directing DNA methylation to specific genomic locations within mammalian cells. Here, we describe two modes of RNA regulation of DNMT3A in vitro. We show a single-stranded RNA molecule that is antisense to the E-cadherin promoter binds tightly to the catalytic domain in a structurally dependent fashion causing potent inhibition of DNMT3A activity. Two other RNA molecules bind DNMT3A at an allosteric site outside the catalytic domain, causing no change in catalysis. Our observation of the potent and specific in vitro modulation of DNMT3A activity by RNA supports in vivo data that RNA interacts with DNMT3A to regulate transcription.
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rna modulation of the human DNA Methyltransferase 3A
2012Co-Authors: Celeste Holzschietinger, Norbert O ReichAbstract:DNA Methyltransferase 3A (DNMT3A) is one of two human de novo DNA Methyltransferases essential for transcription regulation during cellular development and differentiation. There is increasing evidence that RNA plays a role in directing DNA methylation to specific genomic locations within mammalian cells. Here, we describe two modes of RNA regulation of DNMT3A in vitro. We show a single-stranded RNA molecule that is antisense to the E-cadherin promoter binds tightly to the catalytic domain in a structurally dependent fashion causing potent inhibition of DNMT3A activity. Two other RNA molecules bind DNMT3A at an allosteric site outside the catalytic domain, causing no change in catalysis. Our observation of the potent and specific in vitro modulation of DNMT3A activity by RNA supports in vivo data that RNA interacts with DNMT3A to regulate transcription.
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identification of a second DNA binding site in human DNA Methyltransferase 3A by substrate inhibition and domain deletion
2010Co-Authors: Matthew M Purdy, Celeste Holzschietinger, Norbert O ReichAbstract:The human DNA Methyltransferase 3A (DNMT3A) is essential for establishing DNA methylation patterns. Knowing the key factors involved in the regulation of mammalian DNA methylation is critical to furthering understanding of embryonic development and designing therapeutic approaches targeting epigenetic mechanisms. We observe substrate inhibition for the full length DNMT3A but not for its isolated catalytic domain, demonstrating that DNMT3A has a second binding site for DNA. Deletion of recognized domains of DNMT3A reveals that the conserved PWWP domain is necessary for substrate inhibition and forms at least part of the allosteric DNA binding site. The PWWP domain is demonstrated here to bind DNA in a cooperative manner with muM affinity. No clear sequence preference was observed, similar to previous observations with the isolated PWWP domain of Dnmt3b but with one order of magnitude weaker affinity. Potential roles for a low affinity, low specificity second DNA binding site are discussed.
Arumugam Rajavelu - One of the best experts on this subject based on the ideXlab platform.
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identification of novel inhibitors of DNA methylation by screening of a chemical library
2013Co-Authors: Alexandre Ceccaldi, Arumugam Rajavelu, Sergey Ragozin, Catherine Senamaudbeaufort, Noe Testa, Hana Daliali, Christine Maulaybailly, Severine Amand, Pavel Bashtrykov, Dominique GuianvarchAbstract:In order to discover new inhibitors of the DNA Methyltransferase 3A/3L complex, we used a medium-throughput nonradioactive screen on a random collection of 1120 small organic compounds. After a primary hit detection against DNA methylation activity of the murine Dnmt3A/3L catalytic complex, we further evaluated the EC50 of the 12 most potent hits as well as their cytotoxicity on DU145 prostate cancer cultured cells. Interestingly, most of the inhibitors showed low micromolar activities and little cytotoxicity. Dichlone, a small halogenated naphthoquinone, classically used as pesticide and fungicide, showed the lowest EC50 at 460 nM. We briefly assessed the selectivity of a subset of our new inhibitors against hDNMT1 and bacterial Dnmts, including M. SssI and EcoDam, and the protein lysine Methyltransferase PKMT G9a and the mode of inhibition. Globally, the tested molecules showed a clear preference for the DNA Methyltransferases, but poor selectivity among them. Two molecules including Dichlone efficientl...
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function and disruption of DNA Methyltransferase 3A cooperative DNA binding and nucleoprotein filament formation
2012Co-Authors: Arumugam Rajavelu, Renata Z Jurkowska, Jurgen Fritz, Albert JeltschAbstract:The catalytic domain of Dnmt3A cooperatively multimerizes on DNA forming nucleoprotein filaments. Based on modeling, we identified the interface of Dnmt3A complexes binding next to each other on the DNA and disrupted it by charge reversal of critical residues. This prevented cooperative DNA binding and multimerization of Dnmt3A on the DNA, as shown by the loss of cooperative complex formation in electrophoretic mobility shift assay, the loss of cooperativity in DNA binding in solution, the loss of a characteristic 8- to 10-bp periodicity in DNA methylation and direct imaging of protein–DNA complexes by scanning force microscopy. Non-cooperative Dnmt3A-C variants bound DNA well and retained methylation activity, indicating that cooperative DNA binding and multimerization of Dnmt3A on the DNA are not required for activity. However, one non-cooperative variant showed reduced heterochromatic localization in mammalian cells. We propose two roles of Dnmt3A cooperative DNA binding in the cell: (i) either nucleofilament formation could be required for periodic DNA methylation or (ii) favorable interactions between Dnmt3A complexes may be needed for the tight packing of Dnmt3A at heterochromatic regions. The complex interface optimized for tight packing would then promote the cooperative binding of Dnmt3A to naked DNA in vitro .
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Quaternary structure and regulation of mammalian DNA Methyltransferase 3A (Dnmt3A)
2011Co-Authors: Arumugam RajaveluAbstract:The Dnmt3 family includes two catalytically active de novo Methyltransferases (Dnmt3A and Dnmt3b) and one catalytically inactive regulatory factor called Dnmt3L. The N-terminal part of Dnmt3A consists of an ADD and a PWWP domain, we have shown that the ADD domain interacts with H3K4 unmethylated tails and the PWWP domain interacts with H3K36me3 marks. These interactions are responsible for the regulation of Dnmt3A's activity as well as for targeting to heterochromatin. We have identified that Dnmt3A and Dnmt3L form a heterotetramer that methylates two CpG sites on opposite DNA strands in a distance of 8-10 bps. We have shown that Dnmt3A forms a linear multimer and it binds to many DNA molecules oriented in parallel. This process is required for the heterochromatic localization of Dnmt3A in cells. The regulator protein Dnmt3L reorganizes the quaternary structures of Dnmt3A, thereby prevents multimerization. In addition to formation of protein multimers, Dnmt3A complexes bind cooperatively to DNA forming protein-DNA filaments. This might be required for generation of 8-10 bp periodicity patterns at imprinted genes and tight packing of Dnmt3A at the heterochromatic region. In addition, we have identified that the enzymatic activity Dnmt3A is regulated by phosphorylation. Casein kinase 2 mediated phosphorylation of the Dnmt3A negatively regulates its enzymatic activity in cells. Adding to existing evidence, our data uncovered novel mechanism of regulation of Dnmt3A activity, which are required for the proper DNA methylation patterns in cells. Also, using a newly developed high throughput assay, we have identified novel inhibitors to Dnmt3A from chemical libraries and identified new compounds purified from the dietary black tea and coffee polyphenols, which inhibit the Dnmt3A activity moderately.
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The inhibition of the mammalian DNA Methyltransferase 3A (Dnmt3A) by dietary black tea and coffee polyphenols
2011Co-Authors: Arumugam Rajavelu, Zumrad Tulyasheva, Rakesh Jaiswal, Albert Jeltsch, Nikolai KuhnertAbstract:Background Black tea is, second only to water, the most consumed beverage globally. Previously, the inhibition of DNA Methyltransferase 1 was shown by dietary polyphenols and epi-gallocatechin gallate (EGCG), the main polyphenolic constituent of green tea, and 5-caffeoyl quinic acid, the main phenolic constituent of the green coffee bean. Results We studied the inhibition of DNA Methyltransferase 3A by a series of dietary polyphenols from black tea such as theaflavins and thearubigins and chlorogenic acid derivatives from coffee. For theaflavin 3,3 digallate and thearubigins IC_50 values in the lower micro molar range were observed, which when compared to pharmacokinetic data available, suggest an effect of physiological relevance. Conclusions Since Dnnmt3A has been associated with development, cancer and brain function, these data suggest a biochemical mechanism for the beneficial health effect of black tea and coffee and a possible molecular mechanism for the improvement of brain performance and mental health by dietary polyphenols.
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the inhibition of the mammalian DNA Methyltransferase 3A dnmt3A by dietary black tea and coffee polyphenols
2011Co-Authors: Arumugam Rajavelu, Zumrad Tulyasheva, Rakesh Jaiswal, Albert Jeltsch, Nikolai KuhnertAbstract:Black tea is, second only to water, the most consumed beverage globally. Previously, the inhibition of DNA Methyltransferase 1 was shown by dietary polyphenols and epi-gallocatechin gallate (EGCG), the main polyphenolic constituent of green tea, and 5-caffeoyl quinic acid, the main phenolic constituent of the green coffee bean. We studied the inhibition of DNA Methyltransferase 3A by a series of dietary polyphenols from black tea such as theaflavins and thearubigins and chlorogenic acid derivatives from coffee. For theaflavin 3,3 digallate and thearubigins IC50 values in the lower micro molar range were observed, which when compared to pharmacokinetic data available, suggest an effect of physiological relevance. Since Dnnmt3A has been associated with development, cancer and brain function, these data suggest a biochemical mechanism for the beneficial health effect of black tea and coffee and a possible molecular mechanism for the improvement of brain performance and mental health by dietary polyphenols.