5-methylcytidine Research Articles

WITHDRAWN: Detection of N3-methylcytidine in RNA at single base resolution using Protoscript II to read through and generate high mutation rates

N3-methylcytidine (m3C), an RNA modification involving methylation of cytidine at the Watson-Crick face, has been identified in human tRNA, mitochondrial tRNA (mt-tRNA), and mRNA. In tRNA and mt-tRNA, it has been shown to play important roles in tRNA structure and translation. m3C remains understudied in mRNA and other low-abundance RNAs due to its markedly lower levels, the lack of high-resolution techniques to reproducibly map its location, and under-characterization of its effector proteins on these RNA types. Here we describe a new approach which utilizes the high readthrough and mutation rates of Protoscript II (PSII) at m3C sites in combination with AlkB demethylation to detect m3C at single-base resolution. We first validate the method on synthetic probes. Then, using this method, we successfully identify the known m3C sites on tRNA and mt-tRNA and show that none of the m3C sites identified in cytosolic tRNA are likely METTL8 substrates. In contrast, the two m3C sites detected in mt-tRNA are installed by METTL8 since they are sensitive to METTL8 knockdown (KD) and over expression (OE). These results demonstrate that the method can be used to investigate changes in the m3C level in many sites using an unbiased approach. Overall, this study introduces a convenient high-resolution method for m3C mapping with high readthrough rates, paving the way for future mechanistic studies on the biological function of m3C and its effector proteins.

Epitranscriptomic cytidine methylation of the hepatitis B viral RNA is essential for viral reverse transcription and particle production

Epitranscriptomic RNA modifications have emerged as important regulators of the fate and function of viral RNAs. One prominent modification, the cytidine methylation 5-methylcytidine (m5C), is found on the RNA of HIV-1, where m5C enhances the translation of HIV-1 RNA. However, whether m5C functionally enhances the RNA of other pathogenic viruses remains elusive. Here, we surveyed a panel of commonly found RNA modifications on the RNA of hepatitis B virus (HBV) and found that HBV RNA is enriched with m5C as well as ten other modifications, at stoichiometries much higher than host messenger RNA (mRNA). Intriguingly, m5C is mostly found on the epsilon hairpin, an RNA element required for viral RNA encapsidation and reverse transcription, with these m5C mainly deposited by the cellular methyltransferase NSUN2. Loss of m5C from HBV RNA due to NSUN2 depletion resulted in a partial decrease in viral core protein (HBc) production, accompanied by a near-complete loss of the reverse transcribed viral DNA. Similarly, mutations introduced to remove the methylated cytidines resulted in a loss of HBc production and reverse transcription. Furthermore, pharmacological disruption of m5C deposition led to a significant decrease in HBV replication. Thus, our data indicate m5C methylations as a critical mediator of the epsilon elements' function in HBV virion production and reverse transcription, suggesting the therapeutic potential of targeting the m5C methyltransfer process on HBV epsilon as an antiviral strategy.

An on-line SPE-LC-MS/MS method for quantification of nucleobases and nucleosides present in biological fluids.

Solid phase extraction (SPE) and liquid chromatographic (LC) separation of nucleobases and nucleosides are challenging due to the high hydrophilicity of these compounds. Herein we report a novel on-line SPE-LC-MS/MS method for their quantification after pre-column derivatization with chloroacetaldehyde (CAA). The method proposed is selective and sensitive with limits of detection at the nano-molar level. Analysis of urine and saliva samples by using this method is demonstrated. Adenine, guanine, cytosine, adenosine, guanosine, and cytidine were found in the range from 0.19 (guanosine) to 1.83 μM (cytidine) in urine and from 0.015 (guanosine) to 0.79 μM (adenine) in saliva. Interestingly, methylation of cytidine was found to be significantly different in urine from that in saliva. While 5-hydroxymethylcytidine was detected at a very low level (<0.05 μM) in saliva, it was found to be the most prominent methylated cytidine in urine at a high level of 3.33 μM. Since on-line SPE is deployed, the proposed LC-MS/MS quantitative assay is convenient to carry out and offers good assay accuracy and repeatability.

Computational mutation prediction to enhance or recover enzymatic activity.

Atomistic computational simulations have become a useful tool for biomolecular research. These tools are now becoming sufficiently accurate to predict single point mutations that can enhance or recover enzymatic activity. In this talk I will present the use of computational approaches to perform these predictions, and examples coupled with experimental validation. Two examples of applications of these methods to DNA transaction enzymes will be discussed. The first example involves an enzyme from the APOBEC3 family, which are host restriction factors that inhibit lentiviruses, such as HIV-1. For APOBEC3C (A3C), the chimpanzee and gorilla orthologues are more active than human A3C, and the Old World Monkey A3C from rhesus macaque (rh) is not active against HIV-1. Using a combination of molecular dynamics (MD), direct coupling analysis (DCA) and experimental techniques we have investigated the key residues for antivirala ctivities. Overall, our results determine the basis for why rhA3C is less active than human A3C and establish the amino acid network for dimerization and increased activity. The second example involves the methyltransferase (MTase) from Mycoplasma penetrans, which has recently been shown to synthesize the modified nucleotide 5-carboxymethylcytosine (5cxmC), formed as a trace byproduct of cytidine methylation by M.MpeI with the secondary metabolite carboxy-S-adenosyl-L-methionine (CxSAM) in place of the normal reaction cofactor, SAM. The use of computational simulations to investigate this new selectivity and predict additional mutations to enhance this new function, and experimental confirmation of these predictions will be presented and discussed.

Discovery of Inhibitors of DNA Methyltransferase 2, an Epitranscriptomic Modulator and Potential Target for Cancer Treatment.

Selective manipulation of the epitranscriptome could be beneficial for the treatment of cancer and also broaden the understanding of epigenetic inheritance. Inhibitors of the tRNA methyltransferase DNMT2, the enzyme catalyzing the S-adenosylmethionine-dependent methylation of cytidine 38 to 5-methylcytidine, were designed, synthesized, and analyzed for their enzyme-binding and -inhibiting properties. For rapid screening of potential DNMT2 binders, a microscale thermophoresis assay was established. Besides the natural inhibitors S-adenosyl-l-homocysteine (SAH) and sinefungin (SFG), we identified new synthetic inhibitors based on the structure of N-adenosyl-2,4-diaminobutyric acid (Dab). Structure-activity relationship studies revealed the amino acid side chain and a Y-shaped substitution pattern at the 4-position of Dab as crucial for DNMT2 inhibition. The most potent inhibitors are alkyne-substituted derivatives, exhibiting similar binding and inhibitory potencies as the natural compounds SAH and SFG. CaCo-2 assays revealed that poor membrane permeabilities of the acids and rapid hydrolysis of an ethylester prodrug might be the reasons for the insufficient activity in cellulo.

DNA Methylation of Patatin-Like Phospholipase Domain-Containing Protein 6 Gene Contributes to the Risk of Intracranial Aneurysm in Males.

Objective: This study is aimed to investigate the contribution of patatin-like phospholipase domain-containing protein 6 (PNPLA6) DNA methylation to the risk of intracranial aneurysm (IA) in the Han Chinese population.Methods: A total of 96 age- and sex-matched participants were recruited to evaluate PNPLA6 methylation via bisulfite pyrosequencing. The PNPLA6 mRNA expression in the plasma was determined using real-time quantitative reverse transcription-polymerase chain reaction. Human primary artery smooth muscle cells (HPCASMC) were used for the in vitro function study.Results: PNPLA6 methylation was significantly higher in patients with IA than in healthy controls (p < 0.01). Sex group analysis showed that this correlation appeared in the male group (p < 0.01) but not in the female group (p > 0.05). PNPLA6 methylation was significantly associated with age in all participants (r = 0.306, p = 0.003) and in the control group (r = 0.377, p = 0.008) but not in the IA group (r = 0.127, p = 0.402). Furthermore, the PNPLA6 mRNA expression significantly decreased in patients with IA than that in the controls (p = 0.016). PNPLA6 expression was significantly inversely correlated with elevated DNA methylation in participants (r = −0.825, p < 0.0001). In addition, PNPLA6 transcription was significantly enhanced following treatment with 5-aza-2’-deoxycytidine methylation inhibitor in HPCASMC.The receiver operating characteristic analyses of curves showed that the PNPLA6 mean methylation [area under the curve (AUC) = 0.74, p < 0.001] and mRNA expression (AUC = 0.86, p < 0.001) could have a diagnostic value for patients with IA.Conclusion: Although future functional experiments are required to test our hypothesis, our study demonstrated that PNPLA6 methylation and mRNA expression were significantly associated with the risk of IA; thus, they show potential for use in the early diagnosis of IA.

RNA-Cleaving Deoxyribozymes Differentiate Methylated Cytidine Isomers in RNA.

Deoxyribozymes are emerging as modification‐specific endonucleases for the analysis of epigenetic RNA modifications. Here, we report RNA‐cleaving deoxyribozymes that differentially respond to the presence of natural methylated cytidines, 3‐methylcytidine (m3C), N 4‐methylcytidine (m4C), and 5‐methylcytidine (m5C), respectively. Using in vitro selection, we found several DNA catalysts, which are selectively activated by only one of the three cytidine isomers, and display 10‐ to 30‐fold accelerated cleavage of their target m3C‐, m4C‐ or m5C‐modified RNA. An additional deoxyribozyme is strongly inhibited by any of the three methylcytidines, but effectively cleaves unmodified RNA. The mXC‐detecting deoxyribozymes are programmable for the interrogation of natural RNAs of interest, as demonstrated for human mitochondrial tRNAs containing known m3C and m5C sites. The results underline the potential of synthetic functional DNA to shape highly selective active sites.

RNA‐Cleaving Deoxyribozymes Differentiate Methylated Cytidine Isomers in RNA

AbstractDeoxyribozymes are emerging as modification‐specific endonucleases for the analysis of epigenetic RNA modifications. Here, we report RNA‐cleaving deoxyribozymes that differentially respond to the presence of natural methylated cytidines, 3‐methylcytidine (m3C), N4‐methylcytidine (m4C), and 5‐methylcytidine (m5C), respectively. Using in vitro selection, we found several DNA catalysts, which are selectively activated by only one of the three cytidine isomers, and display 10‐ to 30‐fold accelerated cleavage of their target m3C‐, m4C‐ or m5C‐modified RNA. An additional deoxyribozyme is strongly inhibited by any of the three methylcytidines, but effectively cleaves unmodified RNA. The mXC‐detecting deoxyribozymes are programmable for the interrogation of natural RNAs of interest, as demonstrated for human mitochondrial tRNAs containing known m3C and m5C sites. The results underline the potential of synthetic functional DNA to shape highly selective active sites.

High throughput and low bias DNA methylation and hydroxymethylation analysis by direct injection mass spectrometry

We present a direct injection mass spectrometry (DI-MS) platform that accurately, precisely, and quickly quantitates global levels of DNA cytidine methylation (5 mC) and hydroxymethylation (5hmC). Our platform combines an Advion TriVersa NanoMate coupled online to a Thermo Scientific Orbitrap Fusion Lumos. Following digestion to nucleosides, the DNA samples are analyzed at the rate of <1 min per injection with comparable detection limits of 0.63 ng/μL and 0.31 ng/μL, respectively. In contrast, the detection limits for 5 mC and 5hmC in state-of-art nano liquid chromatography (LC) coupled to online mass spectrometry (nLC-MS) are notably different (0.04 ng/μL and 2.5 ng/μL, respectively). The high sensitivity of DI-MS is achieved by optimizing sample buffer composition, the source fragmentation energy, and the radio frequency of the instrument ion funnel. DI-MS accurately reports the relative abundance of 5 mC and 5hmC over a range of 1%–7% (R2 > 0.98) and 0.13%–1.75% (R2 > 0.99), respectively. Accurate measurement of C, 5 mC and 5hmC is achieved by optimizing in-source fragmentation to obtain a population of up to 93% of just the nucleoside base. This protocol minimizes base dimer formation and partial base-deoxyribose dissociation in gas phase and greatly improves modified base quantitation. We also demonstrate that DI-MS overcomes biases in differential chromatographic retention and issues of sample degradation in the autosampler due to its high throughput. Finally, we present an application of our workflow to quantify DNA modifications on a batch of 81 samples in about 1.5 h.

DNA methylation and hydroxymethylation analysis using a high throughput and low bias direct injection mass spectrometry platform

DNA modifications are small covalent chemical groups that modify nucleotides to regulate DNA readout. Anomalous abundance and genome-wide localization of these modifications can negatively tune gene expression and propagate into unbalanced epigenetics regulation, which is known to be associated with multiple conditions such as cancer, diabetes and aging. We present a direct injection mass spectrometry (DI-MS) platform that offers fast, accurate and precise quantitation of global levels of DNA cytidine methylation (mC) and hydroxymethylation (hmC) in less than one minute per sample. On the contrary to most methods adopting mass spectrometry for the analysis of nucleotide modifications, in this DI-MS approach we eliminate the use of liquid chromatography, increasing throughput, eliminating issues of carryover and batch effects caused by column contamination across samples. In addition, potential biases in detection efficiency of modified nucleotides with different binding efficiency to stationary phases is eliminated, as no chromatographic separation is adopted. This method can analyze >1000 samples per day, overcoming the throughput of next-generation sequencing.•Direct injection mass spectrometry improves throughput and precision compared to liquid chromatography.•Direct injection can be used to quantify in less than one minute global levels of DNA methylation and hydroxymethylation.•The unbiased acquisition can be potentially utilized to analyze other nucleotide modifications.

Quantitative changes in DNA methylation induced by monochromatic light in barley regenerants obtained by androgenesis

Changes in DNA methylation are one of the best known mechanisms of epigenetic regulation of gene expression, which in the process of induced androgenesis is associated with reprogramming of haploid microspores development towards the formation of embryos, as a result of exposure of anthers in ears and then anthers culture in vitro to stress factors. The aim of the study was to test the hypothesis of whether the use of monochromatic light during induced androgenesis might be associated with epigenetic phenomena. The experiments were carried out on DH plants of spring barley (Hordeum vulgare L.) obtained by androgenesis modified by monochromatic light: blue, green and red. A quantitative evaluation of the effect of light on the degree of DNA methylation was performed using RP-HPLC for the comparison of regenerants obtained under standard, control conditions (darkness) with those obtained with light usage. The differences in the amount of methylated cytidine in comparison to the control were: 0.40%, 0.16% and -0.55%, for blue, red and green light, respectively. The level of global genomic DNA methylation from control plants was in the range 21.32-21.52%. Methylation changes in response to monochromatic light used during callus formation in anthers culture, determined by RP-HPLC, are significant although small.

N 1-Methylpseudouridine substitution enhances the performance of synthetic mRNA switches in cells.

Synthetic messenger RNA (mRNA) tools often use pseudouridine and 5-methyl cytidine as substitutions for uridine and cytidine to avoid the immune response and cytotoxicity induced by introducing mRNA into cells. However, the influence of base modifications on the functionality of the RNA tools is poorly understood. Here we show that synthetic mRNA switches containing N1-methylpseudouridine (m1Ψ) as a substitution of uridine substantially out-performed all other modified bases studied, exhibiting enhanced microRNA and protein sensitivity, better cell-type separation ability, and comparably low immune stimulation. We found that the observed phenomena stem from the high protein expression from m1Ψ containing mRNA and efficient translational repression in the presence of target microRNAs or proteins. In addition, synthetic gene circuits with m1Ψ significantly improve performance in cells. These findings indicate that synthetic mRNAs with m1Ψ modification have enormous potentials in the research and application of biofunctional RNA tools.

Elevated methylation of cyclin dependent kinase inhibitor 2B contributes to the risk of coronary heart disease in women.

Cyclin dependent kinase inhibitor 2B (CDKN2B) encodes a cyclin-dependent kinase inhibitor that may enhance the formation of atherosclerotic plaques. The aim of the present study was to investigate the contribution of CDKN2B promoter methylation on the risk of coronary heart disease (CHD). The present results indicated a significant association between increased CDKN2B methylation and the risk of CHD (adjusted P=0.043). A breakdown analysis according to sex demonstrated that CDKN2B methylation was significantly associated with the risk of CHD in women (adjusted P=0.010), but not in men. A further breakdown analysis by age indicated a significant association of CHD in the women >60 years (P=0.024). Luciferase reporter gene assay results indicated that the CDKN2B promoter fragment significantly enhanced luciferase activity (P<0.001). In addition, CDKN2B transcription was significantly enhanced following treatment with 5-aza-2′-deoxycytidine methylation inhibitor in human aortic endothelial cells (HAEC) and human primary coronary artery smooth muscle cells (HPCASMC; P<0.05 and P<0.01), but not in 293 cells. Notably, estrogen treatment reduced CDKN2B methylation of several CpGs and significantly increased CDKN2B gene expression levels in HAEC, HPCASMC and 293 cells (P<0.05 and P<0.01). Additionally, treatment of HAEC and HPCASMC with simvastatin and γ-carboxy-L-glutamic acid reduced CDKN2B promoter methylation and increased CDKN2B transcription concomitantly. The present study suggests that CDKN2B promoter methylation may be associated with sex dimorphism in the pathogenesis of CHD.

Modeling p K Shift in DNA Triplexes Containing Locked Nucleic Acids.

The protonation states for nucleic acid bases are difficult to assess experimentally. In the context of DNA triplex, the protonation state of cytidine in the third strand is particularly important, because it needs to be protonated in order to form Hoogsteen hydrogen bonds. A sugar modification, locked nucleic acid (LNA), is widely used in triplex forming oligonucleotides to target sites in the human genome. In this study, the parameters for LNA are developed in line with the CHARMM nucleic acid force field and validated toward the available structural experimental data. In conjunction, two computational methods were used to calculate the protonation state of the third strand cytidine in various DNA triplex environments: λ-dynamics and multiple pH regime. Both approaches predict p K of this cytidine shifted above physiological pH when cytidine is in the third strand in a triplex environment. Both methods show an upshift due to cytidine methylation, and a small downshift when the sugar configuration is locked. The predicted p K values for cytidine in DNA triplex environment can inform the design of better-binding oligonucleotides.

Abstract 4351: Effects of epigenetic agents on methylation of DNA in vitro and in vivo, as measured with stable isotopically labeled methionine

Abstract Background: Modulation of cytidine methylation in DNA continues to be actively investigated as a target for chemotherapy. The primary source of methyl groups is methionine, via S-adenosyl-methionine (SAM) as the direct methyl donor for DNA methyl transferases, e.g., DNMT1. Methods were developed using stable isotopes and mass spec detection to examine the flux of the DNMT reaction and modification to this flux by cytidine analogs such as decitabine, an established hypomethylation agent, and 5-aza-4’-thio-2’-deoxy-cytidine (AzaTdC), currently under development at NCI. Methods: For in vitro studies; A549, H23, HCT116, H522, and OVCAR3 cell lines grown in RPMI1640 media supplemented with 10% FBS were exposed to D3-methionine(20uM) and either decitabine or AzaTdC (NSC77586) at 0 to 5uM in the media for 0 to 48hr. For in vivo studies; mice were maintained under standard ACUC protocols and practices. Tumors were grown SC in the flank area. Stable labeled methionine, decitabine and AzaTdC were administered as an oral bolus. At set times, mice were sacrificed and tumors, tissues and plasma samples collected. DNA was extracted and purified from cells and tissues using PureLink Genomic DNA kits (Invitrogen) according to manufacturer’s instruction. The purified DNA was digested to nucleosides for analysis. The isotopic enrichment in 5-methyl-deoxy-cytidine (mdC) was determined by analysis using HPLC coupled to a QE mass spectrometer (Thermo Scientific). Results: D3-methionine was found to be an efficient precursor via SAM for the methylation of cytidine in DNA. Fortification of basal media (100uM methionine) with 20uM D3-methionine yielded D3-mdC enrichment of 5-15% in 24 hr, depending on cell line. All cell lines were found to have some enrichment. Treatment of cells with either decitabine or AzaTdC caused a concentration dependent reduction of the enrichment for mdC in DNA. In mice, after an oral dose of D3-methionine, enrichment of methionine and SAM in plasma peaked within 1hr and fell to 5% of peak levels within 5hr. Methionine and SAM enrichment in tissues followed similar time curves. At 50mg/kg of D3-methionine, no perturbation was observed for endogenous levels of either methionine or SAM. Enrichment of mdC in DNA was detectable in 1hr, maximal in 5hr, and remained constant for up to 48hr. When mice were treated with 1-2 mg/kg of either decitabine or AzaTdC along with D3-methionine, the level of enrichment of mdC in DNA was reduced by 20-70% in tumors, bone marrow and intestine. Conclusions: Stable labeled methionine can be efficiently used to monitor the flux of methyl groups into mdC in DNA, both in vitro and in vivo. Changes in the flux of the methylation reaction can be used to assess the extent and time course for inhibition caused by cytidine analogs. These techniques may have relevance for improvement of doses and schedules to produce epigenetic modulation. Funded by NCI Contract No. HHSN261200800001E Citation Format: Lawrence Anderson, Eva Majerova, Kimberly D. Hill, John Carter, Jesse Stottlemyer, Howard Stotler, Melinda G. Hollingshead, Jerry M. Collins. Effects of epigenetic agents on methylation of DNA in vitro and in vivo, as measured with stable isotopically labeled methionine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4351. doi:10.1158/1538-7445.AM2017-4351

Role of Pseudoisocytidine Tautomerization in Triplex-Forming Oligonucleotides: In Silico and in Vitro Studies.

Pseudoisocytidine (ΨC) is a synthetic cytidine analogue that can target DNA duplex to form parallel triplex at neutral pH. Pseudoisocytidine has mainly two tautomers, of which only one is favorable for triplex formation. In this study, we investigated the effect of sequence on ΨC tautomerization using λ-dynamics simulation, which takes into account transitions between states. We also performed in vitro binding experiments with sequences containing ΨC and furthermore characterized the structure of the formed triplex using molecular dynamics simulation. We found that the neighboring methylated or protonated cytidine promotes the formation of the favorable tautomer, whereas the neighboring thymine or locked nucleic acid has a poor effect, and consecutive ΨC has a negative influence. The deleterious effect of consecutive ΨC in a triplex formation was confirmed using in vitro binding experiments. Our findings contribute to improving the design of ΨC-containing triplex-forming oligonucleotides directed to target G-rich DNA sequences.

The RNA methyltransferase Dnmt2 methylates DNA in the structural context of a tRNA

ABSTRACTThe amino acid sequence of Dnmt2 is very similar to the catalytic domains of bacterial and eukaryotic DNA-(cytosine 5)-methyltransferases, but it efficiently catalyzes tRNA methylation, while its DNA methyltransferase activity is the subject of controversial reports with rates varying between zero and very weak. By using composite nucleic acid molecules as substrates, we surprisingly found that DNA fragments, when presented as covalent DNA-RNA hybrids in the structural context of a tRNA, can be more efficiently methylated than the corresponding natural tRNA substrate. Furthermore, by stepwise development of tRNAAsp, we showed that this natural Dnmt2 substrate could be engineered to employ RNAs that act like guide RNAs in vitro. The 5’-half of tRNAAsp was able to efficiently guide methylation toward a single stranded tRNA fragment as would result from tRNA cleavage by tRNA specific nucleases. In a more artificial setting, a composite system of guide RNAs could ultimately be engineered to enable the enzyme to perform cytidine methylation on single stranded DNA in vitro.

Variable presence of 5-methylcytosine in commercial RNA and DNA

Nucleoside methylations and other nucleic acid modifications have recently encountered a surge in interest, prompted, among other things, by the detection of methylation and active demethylation of DNA and mRNA by similar mechanisms. In DNA, deoxycytidine methylation by Dnmt enzymes generates 5-methyldeoxycytidine,1 an important epigenetic mark that typically causes inactivation of transcription of the methylated promoter region. Recent exciting developments have shown that these marks are not concrete-cast, but can be actively removed by the oxidative action of TET enzymes,2 which generate, through a series of 2-electron oxidations, first hydroxymethylcytidine (hm5C), then formyldeoxycytidine (f5C),3 and finally carboxydeoxycytidine (ca5C), which may eventually regenerate deoxycytidine by decarboxylation. The apparent functional homolog in mRNA is m6A, which appears to reduce translation efficiency. Here, too, the methylation can be removed by TET-related enzymes generating first hydroxymethyladenosine (hm6A), then formyladenosine (f6A).4,5,6 Also, the presence of 5-methylcytidine in mRNA has been reported early on, and has recently raised renewed interest, although its function is as yet unclear and putative conversion into hydroxymethylcytidine is yet to be demonstrated.32 These developments are enhanced by the development of highly sensitive detection methods7,8 including the adaptation of the so-called bisulfite sequencing from DNA, where it is well established,9 to RNA, where its application has significantly contributed to the present high level of interest.10,11,12 However, bisulfite sequencing alone does not yield unassailable results13,14 and we have thus looked to expand the limits of detection of m5C in both DNA and RNA by LC-MS/MS. Here we report a straightforward regimen that provides values for the limit of quantification (LOQ) in the triple digit attomol range. Its application to presumed negative controls, namely synthetic oligonucleotides, surprisingly detected significant amounts of m5C in both types of synthetic nucleic acids.

Structures of the Ets Protein DNA-binding Domains of Transcription Factors Etv1, Etv4, Etv5, and Fev: DETERMINANTS OF DNA BINDING AND REDOX REGULATION BY DISULFIDE BOND FORMATION

Ets transcription factors, which share the conserved Ets DNA-binding domain, number nearly 30 members in humans and are particularly involved in developmental processes. Their deregulation following changes in expression, transcriptional activity, or by chromosomal translocation plays a critical role in carcinogenesis. Ets DNA binding, selectivity, and regulation have been extensively studied; however, questions still arise regarding binding specificity outside the core GGA recognition sequence and the mode of action of Ets post-translational modifications. Here, we report the crystal structures of Etv1, Etv4, Etv5, and Fev, alone and in complex with DNA. We identify previously unrecognized features of the protein-DNA interface. Interactions with the DNA backbone account for most of the binding affinity. We describe a highly coordinated network of water molecules acting in base selection upstream of the GGAA core and the structural features that may account for discrimination against methylated cytidine residues. Unexpectedly, all proteins crystallized as disulfide-linked dimers, exhibiting a novel interface (distant to the DNA recognition helix). Homodimers of Etv1, Etv4, and Etv5 could be reduced to monomers, leading to a 40-200-fold increase in DNA binding affinity. Hence, we present the first indication of a redox-dependent regulatory mechanism that may control the activity of this subset of oncogenic Ets transcription factors.

DNA methylation reduces binding and cleavage by bleomycin.

In a recent study, we described the enhanced double-strand cleavage of hairpin DNAs by Fe·bleomycin (Fe·BLM) that accompanies increasingly strong binding of this antitumor agent and suggested that this effect may be relevant to the mechanism by which BLM mediates its antitumor effects. Because the DNA in tumor cells is known to be hypomethylated on cytidine relative to that in normal cells, it seemed of interest to study the possible effects of methylation status on BLM-induced double-strand DNA cleavage. Three hairpin DNAs found to bind strongly to bleomycin, and their methylated counterparts, were used to study the effect of methylation on bleomycin-induced DNA degradation. Under conditions of limited DNA cleavage, there was a significant overall decrease in the cleavage of methylated hairpin DNAs. Cytidine methylation was found to result in decreased BLM-induced cleavage at the site of methylation and to result in enhanced cleavage at adjacent nonmethylated sites. For two of the three hairpin DNAs studied, methylation was accompanied by a dramatic decrease in the binding affinity for Fe·BLM, suggesting the likelihood of diminished double-strand cleavage. The source of the persistent binding of BLM by the third hairpin DNA was identified. Also identified was the probable molecular mechanism for diminished binding and cleavage of the methylated DNAs by BLM. The possible implications of these findings for the antitumor selectivity of bleomycin are discussed.