Cytosine Methyltransferase Research Articles

DNA cytosine methylation suppresses meiotic recombination at the sex-determining region.

Meiotic recombination between homologous chromosomes is vital for maximizing genetic variation among offspring. However, sex-determining regions are often rearranged and blocked from recombination. It remains unclear whether rearrangements or other mechanisms might be responsible for recombination suppression. Here, we uncover that the deficiency of the DNA cytosine methyltransferase DNMT1 in the green alga Chlamydomonas reinhardtii causes anomalous meiotic recombination at the mating-type locus (MT), generating haploid progeny containing both plus and minus mating-type markers due to crossovers within MT. The deficiency of a histone methyltransferase for H3K9 methylation does not lead to anomalous recombination. These findings suggest that DNA methylation, rather than rearrangements or histone methylation, suppresses meiotic recombination, revealing an unappreciated biological function for DNA methylation in eukaryotes.

Virus-encoded glycosyltransferases hypermodify DNA with diverse glycans

Enzymatic modification of DNA nucleobases can coordinate gene expression, nuclease protection, or mutagenesis. We recently discovered a clade of phage-specific cytosine methyltransferase (MT) and 5-methylpyrimidine dioxygenase (5mYOX) enzymes that produce 5-hydroxymethylcytosine (5hmC) as a precursor for enzymatic hypermodifications on viral genomes. Here, we identify phage MT- and 5mYOX-associated glycosyltransferases (GTs) that catalyze linkage of diverse sugars to 5hmC nucleobase substrates. Metavirome mining revealed thousands of biosynthetic gene clusters containing enzymes with predicted roles in cytosine sugar hypermodification. We developed a platform for high-throughput screening of GT-containing pathways, relying on the Escherichiacoli metabolome as a substrate pool. We successfully reconstituted several pathways and isolated diverse sugar modifications appended to cytosine, including mono-, di-, or tri-saccharides comprised of hexoses, N-acetylhexosamines, or heptose. These findings expand our knowledge of hypermodifications on nucleic acids and the origins of corresponding sugar-installing enzymes.

Translocating bacteria in SIV infection are not stochastic and preferentially express cytosine methyltransferases

Microbial translocation is a significant contributor to chronic inflammation in people living with HIV (PLWH) and is associated with increased mortality and morbidity in individuals treated for long periods with antiretrovirals. The use of therapeutics to treat microbial translocation has yielded mixed effects, in part, because the species and mechanisms contributing to translocation in HIV remain incompletely characterized. To characterize translocating bacteria, we cultured translocators from chronically SIV-infected rhesus macaques. Proteomic profiling of these bacteria identified cytosine-specific methyltransferases as a common feature and therefore, a potential driver of translocation. Treatment of translocating bacteria with the cytosine methyltransferase inhibitor decitabine significantly impaired growth for several species in vitro. In rhesus macaques, oral treatment with decitabine led to some transient decreases in translocator taxa in the gut microbiome. These data provide mechanistic insight into bacterial translocation in lentiviral infection and explore a novel therapeutic intervention that may improve the prognosis of PLWH.

DNA cytosine methyltransferases differentially regulate genome-wide hypermutation and interhomolog recombination in Trichoderma reesei meiosis.

Trichoderma reesei is an economically important enzyme producer with several unique meiotic features. spo11, the initiator of meiotic double-strand breaks (DSBs) in most sexual eukaryotes, is dispensable for T. reesei meiosis. T. reesei lacks the meiosis-specific recombinase Dmc1. Rad51 and Sae2, the activator of the Mre11 endonuclease complex, promote DSB repair and chromosome synapsis in wild-type and spo11Δ meiosis. DNA methyltransferases (DNMTs) perform multiple tasks in meiosis. Three DNMT genes (rid1, dim2and dimX) differentially regulate genome-wide cytosine methylation and C:G-to-T:A hypermutations in different chromosomal regions. We have identified two types of DSBs: type I DSBs require spo11 or rid1 for initiation, whereas type II DSBs do not rely on spo11 and rid1 for initiation. rid1 (but not dim2) is essential for Rad51-mediated DSB repair and normal meiosis. rid1 and rad51 exhibit a locus heterogeneity (LH) relationship, in which LH-associated proteins often regulate interconnectivity in protein interaction networks. This LH relationship can be suppressed by deleting dim2 in a haploid rid1Δ (but not rad51Δ) parental strain, indicating that dim2 and rid1 share a redundant function that acts earlier than rad51 during early meiosis. In conclusion, our studies provide the first evidence of the involvement of DNMTs during meiotic initiation and recombination.

A novel variant in NSUN2 causes intellectual disability in a Chinese family

NSUN2-intellectual disability syndrome, also known as intellectual disability type 5 (MRT5), is an autosomal recessive disorder that is characterized by intellectual disability (ID), postnatal growth retardation, dysmorphic facies, microcephaly, short stature, developmental delay, language impairment and other congenital abnormalities. The disease is caused by mutations in the NSUN2 gene, which encodes a tRNA cytosine methyltransferase that has an important role in spindle assembly during mitosis and chromosome segregation. In this study, we recruited a family that had two individuals with ID. Whole exome sequencing was performed to identify a homozygous frameshift variant (c.1171_1175delACCAT(p.Thr391fs*18*)) in NSUN2 (NM_017755.5) in the proband. The varint was confirmed as segregating in his affected brother and his parents by Sanger sequencing. The individuals that we described showed a similar dysmorphology profile to that associated with MRT5. To analyze the correlations between genotypes of NSUN2 and phenotypes of individuals with ID, we examined 17 variants and the associated phenotypes from 32 ID individuals in current and previous studies. We concluded that mutations in NSUN2 cause a wide range of phenotypic defects. Although some clinical manifestations were highly variable, the core phenotypes associated with NSUN2 mutations were dysmorphic facies, microcephaly, short stature, ID, growth restriction, language impairment, hypotonia and delayed puberty. Our study expands the genetic spectrum of NSUN2 mutations and helps to further define the genotype-phenotype correlations in MRT5.

Dynamic deformylation of 5-formylcytosine and decarboxylation of 5-carboxylcytosine during differentiation of mouse embryonic stem cells into mouse neurons

Regulation of cell fate requires the establishment and erasure of 5-methylcytosine (5mC) in genomic DNA. The formation of 5mC is achieved by DNA cytosine methyltransferases (DNMTs), whereas the removal of 5mC can be accomplished by various pathways. Aside from ten-eleven translocation (TET)-mediated oxidation of 5mC followed by thymine DNA glycosylase (TDG)-initiated base excision repair (BER), the direct deformylation of 5-formylcytosine (5fC) and decarboxylation of 5-carboxylcytosine (5caC) have also been discovered as the novel DNA demethylation pathways. Although these novel demethylation pathways have been identified in stem cells and somatic cells, their precise roles in regulating cell fate remain unclear. Here, we differentiate mouse embryonic stem cells (mESCs) into mouse embryoid bodies (mEBs), followed by further differentiation into mouse neural stem cells (mNSCs) and finally into mouse neurons (mNeurons). During this sequential differentiation process, we employ probe molecules, namely 2′-fluorinated 5-formylcytidine (F-5fC) and 2′-fluorinated 5-carboxyldeoxycytidine (F-5caC), for metabolic labeling. The results of mass spectrometry (MS) analysis demonstrate the deformylation and decarboxylation activities are progressively decreased and increased respectively during differentiation process, and this opposite demethylation tendency is not associated with DNMTs and TETs.

Conversion of the CG specific M.MpeI DNA methyltransferase into an enzyme predominantly methylating CCA and CCC sites.

We used structure guided mutagenesis and directed enzyme evolution to alter the specificity of the CG specific bacterial DNA (cytosine-5) methyltransferase M.MpeI. Methylation specificity of the M.MpeI variants was characterized by digestions with methylation sensitive restriction enzymes and by measuring incorporation of tritiated methyl groups into double-stranded oligonucleotides containing single CC, CG, CA or CT sites. Site specific mutagenesis steps designed to disrupt the specific contacts between the enzyme and the non-substrate base pair of the target sequence (5'-CG/5'-CG) yielded M.MpeI variants with varying levels of CG specific and increasing levels of CA and CC specific MTase activity. Subsequent random mutagenesis of the target recognizing domain coupled with selection for non-CG specific methylation yielded a variant, which predominantly methylates CC dinucleotides, has very low activity on CG and CA sites, and no activity on CT sites. This M.MpeI variant contains a one amino acid deletion (ΔA323) and three substitutions (N324G, R326G and E305N) in the target recognition domain. The mutant enzyme has very strong preference for A and C in the 3' flanking position making it a CCA and CCC specific DNA methyltransferase.

Abstract LB_A20: Overcoming cancer drug resistance with AI-designed RNA epigenetic inhibitors

Abstract Background: Drug resistance is a major obstacle to successful treatment of cancer and leukemia. The role of RNA epigenetics in cancer drug resistance is largely unknown. Our previous studies demonstrated that specific RNA cytosine methyltransferases (RCMTs), namely NSUN1 (aka NOL1, NPO2) and NSUN2, mediate the formation of drug-resistant active chromatin structures (ACS) at nascent RNAs to regulate drug resistance in leukemia cells (Cheng, J.X. et al. Nat Commun, 2018). The Cancer Genome Atlas Program (TCGA) data demonstrate that elevated expression levels of NSUN1 and NSUN2 are correlated with significantly shorter patients’ survival in almost all cancer subtypes (https://www.proteinatlas.org/ENSG00000111641-NOP2/pathology). However, due to the lack of NSUN1 and NSUN2 proteins’ crystal structures, no NSUN1/2-targeting therapeutics have been developed yet. Methods: 1. Argonne artificial intelligence (AI)/supercomputer design of NSUN1/2-targeting small-molecule compound libraries, simulation of compound-protein interactions and analysis of structure activity relationship (SAR). 2. Novel RNA epigenetic technologies, including NSUN1/2-targeting fluorescence releasing assay (FRA), 5-ethynyl uridine click chemistry (EC) and proximity ligation rolling cycle amplification (PL-RCA)-coupled confocal microscopy (CM), flow cytometry (FCM) and RNA sequencing (RNA-seq). 3. Functional drug screening, H&E histology and immunohistochemistry (IHC). 4. Enforced NSUN1/2 expression and knockout (KO) cell lines and syngeneic AML mouse models. Results:1. Identification of the ligand-binding modules/surfaces in NSUN1 and NSUN2 proteins and generation of NSUN1/2-targeting small-molecule libraries by AI/supercomputing. 2. Identification of several NSUN1/2 inhibitors that effectively inhibit growth of the venetoclax-resistant human monocytic leukemia (SC), erythroid leukemia (K562) and lung cancer (H82) cells. 3. Demonstration of the lead inhibitors execute their effects through disruption of NSUN1/2-mediated (ACS) and translational complexes (TLC). 4. Characterization of the lead inhibitors’ binding modules with AI/supercomputing SAR analysis. 5. In vivo validation of the efficacies of the identified lead inhibitors using syngeneic AML mouse models. Conclusion:By leveraging the power of the Argonne AI/supercomputer and our functional assays, we identified several small-molecule inhibitors that can disrupt the NSUN1/2-mediated ACS and TLC to overcome venetoclax resistance in AML cell lines and syngeneic mice. This study has not only illustrated the importance of NSUN1 and NSUN2 in oncogenic bioprocesses and drug (venetoclax) resistance, but also paved the way to the development of novel RNA epigenetics-driven therapeutics for leukemia and other cancer subtypes. Citation Format: Jason X Cheng, Shaun Wood, Derrick Tang, Linda Degenstein, Andrzej Joachimiak, Rick Stevens. Overcoming cancer drug resistance with AI-designed RNA epigenetic inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_A20.

Transposon-aided capture (TRACA) of plasmids from the human gut.

The gut microbiota consists of a vast and diverse assemblage of microorganisms that play a pivotal role in maintaining host health. Nevertheless, a significant portion of the human gut microbiota remains uncultivated. Plasmids, a type of MGE, assume a critical function in the biological evolution and adaptation of bacteria to varying environments. To investigate the plasmids present within the gut microbiota community, we used the transposon-aided capture method (TRACA) to explore plasmids derived from the gut microbiota. In this study, fecal samples were collected from two healthy human volunteers and subsequently subjected to the TRACA method for plasmid isolation. Then, the complete sequence of the plasmids was obtained using the genome walking method, and sequence identity was also analyzed. A total of 15 plasmids were isolated. At last, 13 plasmids were successfully sequenced, of which 12 plasmids were highly identical to the plasmids in the National Center for Biotechnology Information (NCBI) database and were all small plasmids. Furthermore, a putative novel plasmid, named pMRPHD, was isolated, which had mobilized elements (oriT and oriV) and a potential type II restriction-modification (R-M) system encoded by DNA cytosine methyltransferase and type II restriction enzyme (Ban I), whose specific functions and applications warrant further exploration.

Overcoming Venetoclax Resistance in Leukemia with AI-Designed RNA Epigenetic Inhibitors

Background : Venetoclax resistance is a major obstacle to successful treatment of acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). Its underlying mechanisms are multifaceted and have been linked to monocytic differentiation, reprograming mitochondrial metabolism, and increased expression of BRD4 and other BCL2-like proteins, etc. Our previous studies demonstrated that specific RNA cytosine methyltransferase, namely NSUN1 (aka NOL1, NPO2) and NSUN2, mediate the formation of drug-resistant active chromatin structures (ACS) at nascent RNAs to regulate drug resistance in leukemia cells (Cheng, J.X. et al. Nat Commun, 2018) ( Figure 1A). The Cancer Genome Atlas Program (TCGA) data demonstrated that elevated expression of NSUN1 and NSUN2 is correlated with significantly shorter patients' survival in almost all cancer subtypes. However, due to the lack of the crystal structures of NSUN1 and NSUN2 proteins, no NSUN1/2-targeting therapeutics have been developed yet. Methods: AI/supercomputing design of NSUN1/2-targeting drug libraries and analysis of structure activity relationship (SAR). Newly developed functional technologies, including NSUN1/2-targeting fluorescence releasing assay (FRA), 5-ethynyl uridine click chemistry (EC) and proximity ligation rolling cycle amplification (PL-RCA)-coupled confocal microscopy (CM), flow cytometry (FCM) and RNA sequencing (RNA-seq). NSUN1/2-targeting functional drug screening (NSUN1/2-FDS) assay and drug growth inhibition (MTT) assay. H&E histology and immunohistochemistry (IHC). Enforced NSUN1/2 expression and knockout (KO) cell lines and syngeneic AML mouse models. Results: Identification of several ligan-binding modules/surfaces in NSUN1 and NSUN2 proteins and generation of small-molecule libraries to targe these ligand-binding surfaces/modules with AI/supercomputing structural prediction and ligand-receptor interaction simulation. Identification of several effective small-molecule inhibitors of NSUN1 and NSUN2 by functional screening of the AI-designed drug libraries in venetoclax-resistant human monocytic leukemia (SC), erythroid leukemia (K562) and lung cancer (H82) cells. Defining the binding modules of the identified NSUN1/2 inhibitors and selecting lead inhibitors through supercomputer/AI-directed SAR analysis. Elucidation of the action mechanisms of NSUN1/2 lead inhibitors thorough disruption of NSUN1/2-mediated ACS in the nucleus and translational complexes (TLC) in the ribosomes and mitochondria ( Figure 1A). In vivo validation of the efficacies of the identified lead inhibitors using a syngeneic AML mouse model. As shown in Figure 1B, NSUN1-17, one of the lead inhibitors, efficiently eliminated the AML cells/blasts from the blood vessels and parenchyma of the lungs of the syngeneic AML mice. In addition, our siRNA knock-down experiments demonstrated that downregulation of NSUN1/2 expression re-sensitizes venetoclax-resistant monocytic leukemia cells to a low dose of venetoclax. Our NIH/3T3 soft-agarose assay confirmed the oncogenic transformation capability of NSUN1 and NSUN2. We have also successfully established the homozygous ( Nsun2-/-) mouse model with markedly reduced body weight, but a normal lifespan, compared to the heterozygous mice ( Nsun2+/-). Conclusion: By leveraging the Argonne AI/supercomputer and our functional assays, we identified several small-molecule inhibitors that can disrupt the NSUN1/2-mediated ACS and TLC to overcome venetoclax resistance in AML cell lines and syngeneic mice. This study has not only illustrated the importance of NSUN1 and NSUN2 in maintaining oncogenic transcription and translation as well as venetoclax resistance in AML, but also paved the way to the development of novel RNA epigenetics-driven therapeutics for leukemia and other cancers.

Identification of a DNA-cytosine methyltransferase that impacts global transcription to promote group B streptococcal vaginal colonization.

Group B Streptococcus (GBS) colonizes the female reproductive tract (FRT) in one-third of women, and carriage leads to numerous adverse pregnancy outcomes including the preterm premature rupture of membranes, chorioamnionitis, and stillbirth. The presence of GBS in the FRT during pregnancy is also the largest predisposing factor for the transmission of GBS and invasive neonatal diseases, including pneumonia, sepsis, and meningitis. The factors contributing to GBS colonization are still being elucidated. Here, we show for the first time that GBS transcription is regulated by an orphan DNA cytosine methyltransferase (Dcm). Many GBS factors are regulated by Dcm, especially those involved in carbohydrate transport and metabolism. We show that GBS persistence in the FRT is dependent on the catabolism of sugars found on the vaginal mucin MUC5B. Collectively, this work highlights the regulatory importance of a DNA methyltransferase and identifies both host and bacterial factors required for GBS colonization.

Base Editor Scanning Reveals Activating Mutations of DNMT3A.

DNA methyltransferase 3A (DNMT3A) is a de novo cytosine methyltransferase responsible for establishing proper DNA methylation during mammalian development. Loss-of-function (LOF) mutations to DNMT3A, including the hotspot mutation R882H, frequently occur in developmental growth disorders and hematological diseases, including clonal hematopoiesis and acute myeloid leukemia. Accordingly, identifying mechanisms that activate DNMT3A is of both fundamental and therapeutic interest. Here, we applied a base editor mutational scanning strategy with an improved DNA methylation reporter to systematically identify DNMT3A activating mutations in cells. By integrating an optimized cellular recruitment strategy with paired isogenic cell lines with or without the LOF hotspot R882H mutation, we identify and validate three distinct hyperactivating mutations within or interacting with the regulatory ADD domain of DNMT3A, nominating these regions as potential functional target sites for pharmacological intervention. Notably, these mutations are still activating in the context of a heterozygous R882H mutation. Altogether, we showcase the utility of base editor scanning for discovering functional regions of target proteins.

Relative expression of key genes involved in nucleic acids methylation in Anopheles gambiae sensu stricto.

In vertebrates, enzymes responsible for DNA methylation, one of the epigenetic mechanisms, are encoded by genes falling into the cytosine methyltransferases genes family (Dnmt1, Dnmt3a,b and Dnmt3L). However, in Diptera, only the methyltransferase Dnmt2 was found, suggesting that DNA methylation might act differently for species in this order. Moreover, genes involved in epigenetic dynamics, such as Ten-eleven Translocation dioxygenases (TET) and Methyl-CpG-binding domain (MBDs), present in vertebrates, might play a role in insects. This work aimed at investigating nucleic acids methylation in the malaria vector Anopheles gambiae (Diptera: Culicidae) by analysing the expression of Dnmt2, TET2 and MBDs genes using quantitative real-time polymerase chain reaction (qRT-PCR) at pre-immature stages and in reproductive tissues of adult mosquitoes. In addition, the effect of two DNA methylation inhibitors on larval survival was evaluated. The qPCR results showed an overall low expression of Dnmt2 at all developmental stages and in adult reproductive tissues. In contrast, MBD and TET2 showed an overall higher expression. In adult mosquito reproductive tissues, the expression level of the three genes in males' testes was significantly higher than that in females' ovaries. The chemical treatments did not affect larval survival. The findings suggest that mechanisms other than DNA methylation underlie epigenetic regulation in An. gambiae.

Novel RNA epigenetics-driven technologies for rapid prediction of cancer drug resistance.

e19001 Background: The human genome comprises ~99% non-coding DNA sequences (ncDNAs), most of which are actively transcribed into various forms of non-coding RNAs (ncRNAs) that undergo extensive chemical modifications to regulate various functions of the subcellular organelles in human cells ( Statello, et al. Nat Rev Mol Cell Biol 2020). However, the clinical potential of RNA modifications and RNA modifying proteins, i.e., RNA epigenetics, is largely unexplored. In a recent study ( Cheng et al. Nat Commun 2018), we first demonstrated that specific RNA cytosine methyltransferases (RCMTs), namely NSUN1 and NSUN2, directly interact with elongating RNA polymerase II (eRNAPII) and the bromodomain-containing protein 4 (BRD4) to form distinct active chromatin structures (ACS) that regulate drug resistance in leukemia cells. This proof-of-concept study enabled us to develop the first-generation of RNA epigenetics-driven drug-predictive system. The primary goal of this study is to validate and optimize this novel drug-predictive system with clinical myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Methods: We developed new technologies, including 5-ethynyl uridine click chemistry (EC) and proximity ligation rolling cycle amplification (PL-RCA)-coupled confocal microscopy (CM), flow cytometry (FCM) and RNA sequencing (RNA-seq). Immunohistochemistry (IHC), western blot (WB), co-immunoprecipitation (co-IP), and dot blot were also used in this study. Results: Our data demonstrated significantly increased expression of the NSUN1, NSUN2 and other key components of the drug-resistant ACS in MDS/AML, which is correlated with disease progression and drug resistance in MDS and AML. We have developed novel technologies, i.e., EC- and PL-RCA-coupled CM and FCM (EC-/PL-RCA/CM & FCM) to visualize and quantify RCMTs-mediated ACS and nascent RNA synthesis in MDS/AML. We discovered nascent RNA synthesis as a highly sensitive and super-fast drug-predictive marker that is independent of cell death and apoptosis. Our data demonstrated that PL-RCA/FCM and EC-/CM have a very high sensitivity to detect the NSUN1/2-mediated ACS in leukemia cell lines and clinical MDS/AML specimens. Our preliminary data demonstrated that increased levels of NSUN1/2-mediated ACS detected by PL-RCA/FCM in pre-treatment MDS/AML can rapidly predict the responses to venetoclax and/or azacitidine. We are actively pursuing RNA-seq targeting NSUN1/2-associated nascent RNAs and ACS, which provides a novel strategy to dissect the functional genomics and RNA epigenetics underlying cancer drug resistance. Conclusions: We have developed a novel RCMTs/NSUN1/2-targeting system for rapid prediction and in-depth analysis of drug resistance in MDS/AML. A large sized clinical study is needed to further validate its usefulness in clinical settings.

RNA cytosine methyltransferase NSUN5 promotes protein synthesis and tumorigenic phenotypes in glioblastoma.

Glioblastoma (GBM) is the most common and aggressive malignant primary brain tumor in adults. The standard treatment achieves a median overall survival for GBM patients of only 15 months. Hence, novel therapies based on an increased understanding of the mechanistic underpinnings of GBM are desperately needed. In this study, we show that elevated expression of 28S rRNA (cytosine-C(5))-methyltransferase NSUN5, which methylates cytosine 3782 of 28S rRNA in GBM cells, is strongly associated with the poor survival of GBM patients. Moreover, we demonstrate that overexpression of NSUN5 increases protein synthesis in GBM cells. NSUN5 knockdown decreased protein synthesis, cell proliferation, sphere formation, migration, and resistance to temozolomide in GBM cell lines. NSUN5 knockdown also decreased the number and size of GBM neurospheres in vitro. As a corollary, mice harboring U251 tumors wherein NSUN5 was knocked down survived longer than mice harboring control tumors. Taken together, our results suggest that NSUN5 plays a protumorigenic role in GBM by enabling the enhanced protein synthesis requisite for tumor progression. Accordingly, NSUN5 may be a hitherto unappreciated target for the treatment of GBM.

Epigenetic changes of Arabidopsis MET1 cytosine methyltransferase mutants under salt stress

In this study, we investigated the morphological and molecular responses of Arabidopsis met1-7 and met1-3 mutants under salinity stress. Global DNA methylation changes of mutants were compared and genes known to be involved in DNA methylation in plants were analyzed. Also, the expression of the telomerase reverse transcriptase (TERT) gene as a stress response indicator was analyzed in order to examine the effects of stress on the telomerase enzyme in the mutants. We found that mutants have a higher rate of hypomethylation than Col-0, under all conditions. According to the qPCR analysis, expression levels of Domains Rearranged Methylase 2 (DRM2), Polymerase IV (Pol IV) and Polymerase V (Pol V) genes were higher in mutants than Col-0 plants. Contrary to expectations for the TERT gene, there was an increase in expression in the stress-applied mutant plants. Taken together, results suggest that the RNA-directed DNA methylation (RdDM) pathway is enhanced in mutants in order to deal with the lack of DNA methylation in CG sites. In conclusion, we believe that the morphological and molecular data obtained regarding the effects of NaCl application to met1 mutants will help us to understand the epigenetic basis of stress mechanisms.

In silico study of selective inhibition mechanism of S-adenosyl-L-methionine analogs for human DNA methyltransferase 3A

Epigenetic mechanisms leading to transcriptional regulation, including DNA methylation, are frequently dysregulated in diverse cancers. Interfering with aberrant DNA methylation performed by DNA cytosine methyltransferases (DNMTs) is a clinically validated approach. In particular, the selective inhibition of the de novo DNMT3A and DNMT3B enzymes, whose expression is limited to early embryogenesis, adult stem cells, and in cancers, is particularly attractive; such selectivity is likely to attenuate the dose limiting toxicity shown by current, non-selective DNMT inhibitors. We use molecular dynamics (MD) based computational analysis to study known small molecule binders of DNMT3A, then propose reversible, tight binding, and selective inhibitors that exploit the Asn1192/Arg688 difference between the maintenance DNMT1 and DNMT3A near the active site. A similar strategy exploiting the presence of a unique active site cysteine Cys666 is used to propose DNMT3A-selective irreversible inhibitors. We report our results of relative binding energies of the known and proposed compounds estimated using MM/GBSA and umbrella sampling (US) techniques, and our evaluation of other end-point binding free energy calculation methods for these receptors. These calculations offer insight into the potential for small molecules to selectively target the active site of DNMT3A.

Identification, expression, and purification of DNA cytosine 5-methyltransferases with short recognition sequences

BackgroundDNA methyltransferases (MTases) are enzymes that induce methylation, one of the representative epigenetic modifications of DNA, and are also useful tools for analyzing epigenomes. However, regarding DNA cytosine 5-methylation, MTases identified so far have drawbacks in that their recognition sequences overlap with those for intrinsic DNA methylation in mammalian cells and/or that the recognition sequence is too long for fine epigenetic mapping. To identify MTases with short recognition sequences that never overlap with the CG dinucleotide, we systematically investigated the 25 candidate enzymes identified using a database search, which showed high similarity to known cytosine 5-MTases recognizing short sequences.ResultsWe identified MTases with six new recognition sequences, including TCTG, CC, CNG, TCG, GCY, and GGCA. Because the recognition sequence never overlapped with the CG dinucleotide, MTases recognizing the CC dinucleotide were promising.ConclusionsIn the current study, we established a procedure for producing active CC-methylating MTases and applied it to nucleosome occupancy and methylome sequencing to prove the usefulness of the enzyme for fine epigenetic mapping. MTases that never overlap with CG dinucleotides would allow us to profile multiple epigenomes simultaneously.

Single-Molecule Mapping of Chromatin Accessibility Using NOMe-seq/dSMF.

The bulk of gene expression regulation in most organisms is accomplished through the action of transcription factors (TFs) on cis-regulatory elements (CREs). In eukaryotes, these CREs are generally characterized by nucleosomal depletion and thus higher physical accessibility of DNA. Many methods exploit this property to map regions of high average accessibility, and thus putative active CREs, in bulk. However, these techniques do not provide information about coordinated patterns of accessibility along the same DNA molecule, nor do they map the absolute levels of occupancy/accessibility. SMF (Single-Molecule Footprinting) fills these gaps by leveraging recombinant DNA cytosine methyltransferases (MTase) to mark accessible locations on individual DNA molecules. In this chapter, we discuss current methods and important considerations for performing SMF experiments.

Abstract 3265: Discovery of a first-in-class reversible DNMT1-selective inhibitor

Abstract DNA methylation, a key epigenetic driver of transcriptional silencing, is universally dysregulated in cancer. Reversal of DNA methylation by hypomethylating agents, such as the cytidine analogs decitabine or azacytidine, has demonstrated clinical benefit in hematologic malignancies. These nucleoside analogs are incorporated into replicating DNA where they inhibit DNA cytosine methyltransferases DNMT1, DNMT3A, and DNMT3B through irreversible covalent interactions. These agents induce significant toxicity to normal blood cells thus limiting their clinical doses. We report the discovery of a series of potent first-in-class DNMT1-selective inhibitors that compete with the active-site loop of DNMT1 for penetration into hemi-methylated DNA. These compounds induce robust loss of DNA methylation, transcriptional activation, and cancer cell growth inhibition in vitro leading to superior tumor regression and survival in mouse models of acute myeloid leukemia due to improved in vivo tolerability compared with decitabine. Citation Format: Melissa B. Pappalardi. Discovery of a first-in-class reversible DNMT1-selective inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3265.