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Plants have several mechanisms to adapt or acclimate to environmental stress. Morphological, physiological, or genetic changes are examples of complex plant responses. In recent years, our understanding of the role of epigenetic regulation, which encompasses changes that do not alter the DNA sequence, as an adaptive mechanism in response to stressful conditions has advanced significantly. Some studies elucidated and synthesized epigenetic mechanisms and their relationships with environmental change, while others explored the interplay between epigenetic modifications and environmental shifts, aiming to deepen our understanding of these complex processes. In this study, we performed a systematic review of the literature to analyze the progression of epigenetics studies on plant species' responses to abiotic factors. We also aimed to identify the most studied species, the type of abiotic factor studied, and the epigenetic technique most used in the scientific literature. For this, a search for articles in databases was carried out, and after analyzing them using pre-established inclusion criteria, a total of 401 studies were found. The most studied species were Arabidopsis thaliana and Oryza sativa, highlighting the gap in studies of non-economic and tropical plant species. Methylome DNA sequencing is the main technique used for the detection of epigenetic interactions in published studies. Furthermore, most studies sought to understand the plant responses to abiotic changes in temperature, water, and salinity. It is worth emphasizing further research is necessary to establish a correlation between epigenetic responses and abiotic factors, such as extreme temperatures and light, associated with climate change. [ABSTRACT FROM AUTHOR]

Plants have several mechanisms to adapt or acclimate to environmental stress. Morphological, physiological, or genetic changes are examples of complex plant responses. In recent years, our understanding of the role of epigenetic regulation, which encompasses changes that do not alter the DNA sequence, as an adaptive mechanism in response to stressful conditions has advanced significantly. Some studies elucidated and synthesized epigenetic mechanisms and their relationships with environmental change, while others explored the interplay between epigenetic modifications and environmental shifts, aiming to deepen our understanding of these complex processes. In this study, we performed a systematic review of the literature to analyze the progression of epigenetics studies on plant species’ responses to abiotic factors. We also aimed to identify the most studied species, the type of abiotic factor studied, and the epigenetic technique most used in the scientific literature. For this, a search for articles in databases was carried out, and after analyzing them using pre-established inclusion criteria, a total of 401 studies were found. The most studied species were Arabidopsis thaliana and Oryza sativa, highlighting the gap in studies of non-economic and tropical plant species. Methylome DNA sequencing is the main technique used for the detection of epigenetic interactions in published studies. Furthermore, most studies sought to understand the plant responses to abiotic changes in temperature, water, and salinity. It is worth emphasizing further research is necessary to establish a correlation between epigenetic responses and abiotic factors, such as extreme temperatures and light, associated with climate change.

Schematic diagram of the preparation of CON@Au-Probe nanoprobe and the mechanism for target methylated DNA detection. [Display omitted] Covalent-organic framework nanosheets (CONs) have received increasing attention in various fields. However, large-scale and high-yield preparation and flexible functionalization of CONs remain a challenging task. Herein, we designed and fabricated a novel ultrathin (<5 nm) vinyl-functionalized CON in large scale (>80 mg) and high yield (>60 %) via an imine-exchange synthesis strategy. Through post-modification strategy, the vinyl-functionalized CONs were sequentially modified with thiols and Au nanoparticles with high distribution density (mass ratio of CONs to Au was about 4:1) and narrow size distribution (3.5 ± 0.5 nm). The synthesized hybrid nanosheets were employed as material platform to design a novel nanoprobe for ultrasensitive detection towards cancer-associated methylated DNA. Integrating site-specific base oxidation damage strategy and target cyclic amplification effect, the well-designed CONs-based nanoprobe achieved high sensitivity for detecting methylated DNA as low as 10 fM and ultrahigh specificity for distinguishing 0.0001% methylation level. Noteworthy, the nanoprobe was successfully applied to the analysis of DNA methylation in human colon cancer cells, expanding the application scope of CONs and opening a new horizon to develop novel CONs-based nanoprobes. [ABSTRACT FROM AUTHOR]

Simple Summary: The high mobility group box1 (HMGB1) protein operates as an alarm for danger. We explore the correlation between HMGB1 expression and the clinical survival status of patients receiving antitumor immunotherapy toward assessing the prognostic value in a multicancer context. We further conducted screening and identified four interacting genes that are targeted for binding by HMGB1-proteins and validate and contextualize the results in vitro. Additionally, HMGB1 function is also remarkably related to the tumor immune microenvironment and immune infiltration, especially regarding the high purity of stromal and immune cells in the tumor microenvironment (TME). In multicancer analysis, HMGB1 is also found linked with frequently genetic mutations and deletions, including in TMB and MSI which lead to resistance against antitumor immunotherapy and worse clinical prognosis among patients. The combination of HMGB1 expression with immune checkpoint molecules (ICPs), such as PD-L1, might present a novel immunology-based antitumor immunotherapy strategy. Objective: To assess the correlation between HMGB1 expression and the patient prognosis in a multicancer context. Methods: The potential oncogenic role of HMGB1 was explored in forty tumors through the TCGA, GEO, and Oncomine datasets. We analyzed the clinical prognostic value and antitumor immunotherapy of HMGB1 in a multicancer context using GEO (GSE111636). Results: High expression of HMGB1 is present in multicancer cases, and its low expression is closely associated with the prognostic survival of patients, in terms of both overall and disease-free survival in ACC and LUAD. Further investigation revealed that the high expression of gastric and lung cancer is closely associated with low risk and better prognosis of patients based on COX and Kaplan–Meier analysis of OS, FP and PPS. HMGB1 expression was found to be significantly correlated with cancer-associated fibroblast and CD8+ T cell infiltration in the TME. The analysis of GO functional annotation/KEGG pathways indicates that HMGB1 may regulate tumor immunity-related pathways, such as the tumor immunotherapy response in colorectal cancer. The function of four genes as hubs are confirmed by in vitro HMGB1 knockdown which led to inhibition of cell proliferation and metastasis in SW620 and SW480 cells. Conclusion: HMGB1 is a potential novel biomarker for improving clinical prognosis and antitumor immunotherapy efficacy. CDK1, HMGB2, SSRP1, and H2AFV may serve as key nodes for HMGB1 in colorectal cancer. [ABSTRACT FROM AUTHOR]

Transcription factors play key roles in cell-fate decisions by regulating 3D genome conformation and gene expression. The traditional view is that methylation of DNA hinders transcription factors binding to them, but recent research has shown that many transcription factors prefer to bind to methylated DNA. Therefore, identifying such transcription factors and understanding their functions is a stepping-stone for studying methylation-mediated biological processes. In this paper, a two-step discriminated method was proposed to recognize transcription factors and their preference for methylated DNA based only on sequences information. In the first step, the proposed model was used to discriminate transcription factors from non-transcription factors. The areas under the curve (AUCs) are 0.9183 and 0.9116, respectively, for the 5-fold cross-validation test and independent dataset test. Subsequently, for the classification of transcription factors that prefer methylated DNA and transcription factors that prefer non-methylated DNA, our model could produce the AUCs of 0.7744 and 0.7356, respectively, for the 5-fold cross-validation test and independent dataset test. Based on the proposed model, a user-friendly web server called TFPred was built, which can be freely accessed at http://lin-group.cn/server/TFPred/.

Transcription factors (TFs) are proteins specifically involved in gene expression regulation. It is generally accepted in epigenetics that methylated nucleotides could prevent the TFs from binding to DNA fragments. However, recent studies have confirmed that some TFs have capability to interact with methylated DNA fragments to further regulate gene expression. Although biochemical experiments could recognize TFs binding to methylated DNA sequences, these wet experimental methods are time-consuming and expensive. Machine learning methods provide a good choice for quickly identifying these TFs without experimental materials. Thus, this study aims to design a robust predictor to detect methylated DNA-bound TFs. We firstly proposed using tripeptide word vector feature to formulate protein samples. Subsequently, based on recurrent neural network with long short-term memory, a two-step computational model was designed. The first step predictor was utilized to discriminate transcription factors from non-transcription factors. Once proteins were predicted as TFs, the second step predictor was employed to judge whether the TFs can bind to methylated DNA. Through the independent dataset test, the accuracies of the first step and the second step are 86.63% and 73.59%, respectively. In addition, the statistical analysis of the distribution of tripeptides in training samples showed that the position and number of some tripeptides in the sequence could affect the binding of TFs to methylated DNA. Finally, on the basis of our model, a free web server was established based on the proposed model, which can be available at https://bioinfor.nefu.edu.cn/TFPM/. [ABSTRACT FROM AUTHOR]

In non-metastatic colorectal cancer (CRC), we evaluated prospectively the pertinence of longitudinal detection and quantification of circulating tumor DNA (ctDNA) as a prognostic marker of recurrence. The presence of ctDNA was assessed from plasma collected before and after surgery for 184 patients classified as stage II or III and at each visit during 3–4 years of follow-up. The ctDNA analysis was performed by droplet-based digital polymerase chain reaction, targeting mutation and methylation markers, blindly from the clinical outcomes. Multivariate analyses were adjusted on age, gender, stage, and adjuvant chemotherapy. Before surgery, 27.5% of patients were positive for ctDNA detection. The rate of recurrence was 32.7% and 11.6% in patients with or without detectable ctDNA respectively (P = 0.001). Time to recurrence (TTR) was significantly shorter in patients with detectable ctDNA before (adjusted hazard ratio [HR] = 3.58, 95% confidence interval [CI] 1.71–7.47) or immediately after surgery (adjusted HR = 3.22, 95% CI 1.32–7.89). The TTR was significantly shorter in patients with detectable ctDNA during the early postoperative follow-up (1–6 months) (adjusted HR = 5, 95% CI 1.9–12.9). Beyond this period, ctDNA remained a prognostic marker with a median anticipated diagnosis of recurrence of 13.1 weeks (interquartile range 28 weeks) when compared to imaging follow-up. The rate of ctDNA+ might be underestimated knowing that consensus pre-analytical conditions were not described at initiation of the study. This prospective study confirms the relevance of ctDNA as a recurrence risk factor in stage II and III CRC before surgery and as a marker of minimal residual disease after surgery that may predict recurrence several months before imaging techniques. • Longitudinal ctDNA evaluation is a prognostic marker of recurrence in a prospective series. • The rate of ctDNA detection was 27.5% before surgery and 10.5% after surgery. • Patients with preoperative ctDNA detection recurred in 32.7% of the cases. • Patients with postoperative ctDNA detection recurred in 44.4% of the cases. • Patients with preoperative and postoperative negative ctDNA recurred in 10.4% of the cases. [ABSTRACT FROM AUTHOR]

The way in which living organisms mobilize a combination of long-term adaptive mechanisms and short-term phenotypic plasticity to face environmental variations is still largely unknown. In the context of climate change, understanding the genetic and epigenetic bases for adaptation and plasticity is a major stake for preserving genomic resources and the resilience capacity of livestock populations. We characterized both epigenetic and genetic variations by contrasting 22 sheep and 21 goats from both sides of a climate gradient, focusing on free-ranging populations from Morocco. We produced for each individual Whole-Genome Sequence at 12X coverage and MeDIP-Seq data, to identify regions under selection and those differentially methylated. For both species, the analysis of genetic differences (FST) along the genome between animals from localities with high vs. low temperature annual variations detected candidate genes under selection in relation to environmental perception (5 genes), immunity (4 genes), reproduction (8 genes) and production (11 genes). Moreover, we found for each species one differentially methylated gene, namely AGPTA4 in goat and SLIT3 in sheep, which were both related, among other functions, to milk production and muscle development. In both sheep and goats, the comparison between genomic regions impacted by genetic and epigenetic variations suggests that climatic variations impacted similar biological pathways but different genes.

The way in which living organisms mobilize a combination of long-term adaptive mechanisms and short-term phenotypic plasticity to face environmental variations is still largely unknown. In the context of climate change, understanding the genetic and epigenetic bases for adaptation and plasticity is a major stake for preserving genomic resources and the resilience capacity of livestock populations. We characterized both epigenetic and genetic variations by contrasting 22 sheep and 21 goats from both sides of a climate gradient, focusing on free-ranging populations from Morocco. We produced for each individual Whole-Genome Sequence at 12X coverage and MeDIP-Seq data, to identify regions under selection and those differentially methylated. For both species, the analysis of genetic differences (FST) along the genome between animals from localities with high vs. low temperature annual variations detected candidate genes under selection in relation to environmental perception (5 genes), immunity (4 genes), reproduction (8 genes) and production (11 genes). Moreover, we found for each species one differentially methylated gene, namely AGPTA4 in goat and SLIT3 in sheep, which were both related, among other functions, to milk production and muscle development. In both sheep and goats, the comparison between genomic regions impacted by genetic and epigenetic variations suggests that climatic variations impacted similar biological pathways but different genes. [ABSTRACT FROM AUTHOR]

Abstract Fluorescence imaging of nucleic acids is a very important technique necessary to understand gene expression and the resulting changes in cell function. This mini-review focuses on sequence-specific fluorescence imaging of intracellular RNA and methylated DNA using fluorescent nucleic acid probes. A couple of functional fluorescent nucleic acid probes developed by our laboratory are introduced and the examples of their application to fluorescence imaging of intracellular nucleic acids are described.

Recent studies have revealed an unexpected role of DNA methylation at promoter regions in transcription activation. However, whether DNA methylation at enhancer regions activates gene expression and influences cellular functions remains to be determined. In this study, by employing the transcription factor krÜppel-like factor 4 (KLF4) that binds to methylated CpGs (mCpGs), we investigated the molecular outcomes of the recruitment of KLF4 to mCpGs at enhancer regions in human glioblastoma cells. First, by integrating KLF4 ChIP-seq, whole-genome bisulfite sequence, and H3K27ac ChIP-seq datasets, we found 1,299 highly methylated (β >0.5) KLF4 binding sites, three-quarters of which were located at putative enhancer regions, including gene bodies and intergenic regions. In the meantime, by proteomics, we identified 16 proteins as putative targets upregulated by KLF4-mCpG binding at enhancer regions. By chromosome conformation capture (3C) analysis, we demonstrated that KLF4 bound to methylated CpGs at the enhancer regions of the B-cell lymphocyte kinase (BLK) and Lim domain only protein 7 (LMO7) genes, and activated their expression via 3D chromatin loop formation with their promoter regions. Expression of mutant KLF4, which lacks KLF4 ability to bind methylated DNA, or removal of DNA methylation in enhancer regions by a DNA methyltransferase inhibitor abolished chromatin loop formation and gene expression, suggesting the essential role of DNA methylation in enhancer-promoter interactions. Finally, we performed functional assays and showed that BLK was involved in glioblastoma cell migration. Together, our study established the concept that DNA methylation at enhancer regions interacts with transcription factors to activate gene expression and influence cellular functions.

MeCP2 is an essential transcriptional repressor that mediates transcriptional inhibition by binding to methylated DNA. The binding specificity of MeCP2 protein to methylated DNA was considered to depend on its methyl-CpG binding domain (MBD). In this study, we used atomic force microscope based single-molecular force spectroscopy to investigate the interaction of MeCP2 MBD and methylated DNA. The specific interaction forces of the MeCP2 MBD-methylated DNA complexes were measured for the first time. The dynamics was also investigated by measuring the unbinding force of the complex at different loading rates. In addition, the distribution of unbinding forces and binding probabilities of MeCP2 MBD and different DNA were studied at the same loading rate. It was found that MeCP2 MBD had weak interaction with hemi-methylated and unmethylated DNA compared to methylated DNA. This work revealed the binding characteristics of MeCP2 MBD and methylated DNA at the single-molecule level. It provides a new idea for exploring the molecular mechanism of MeCP2 in regulating methylation signals. Image 1 • The interaction forces between MeCP2 MBD and methylated DNA were obtained at single-molecule level. • The dynamic parameters between MeCP2 MBD and methylated DNA were obtained at different loading rates. • MeCP2 MBD interacted specifically with DNA containing symmetrical methyl-CpG pairs. • It provides a new idea for exploring the binding mechanism between MeCP2 protein and methylated DNA. [ABSTRACT FROM AUTHOR]

Purpose: Methylation in IKZF1 and BCAT1 are common events in colorectal cancer (CRC). They are often detected in blood as circulating tumor DNA (ctDNA) at diagnosis and disappear after surgery in most CRC patients. A prospective study was conducted to determine the relationship between detection of these markers following surgery and risk for residual disease and for recurrence.Methods: ctDNA status with methylated BCAT1 and IKZF1 was determined within 12 months of surgical resection of CRC, and was related to presence of or risk for residual disease (margins involved, metastases present or nature of node involvement), and to recurrence-free survival.Results: Blood was collected from 172 CRC patients after surgery and 28 (16%) were ctDNA positive. Recurrence was diagnosed in 23 of the 138 with clinical follow-up after surgery (median follow-up 23.3 months, IQR 14.3-29.5). Multivariate modeling indicated that features suggestive of residual disease were an independent predictor of post-surgery ctDNA status: cases with any of three features (close resection margins, apical node involved, or distant metastases) were 5.3 times (95% CI 1.5-18.4, p = 0.008) more likely to be ctDNA positive. Multivariate analysis showed that post-surgery ctDNA positivity was independently associated with an increased risk of recurrence (HR 3.8, 1.5-9.5, p = 0.004).Conclusions: CRC cases positive for methylated ctDNA after surgery are at increased risk of residual disease and subsequently recurrence. This could have implications for guiding recommendations for adjuvant therapy and surveillance strategies. Randomized studies are now indicated to determine if monitoring cases with these biomarkers leads to survival benefit. [ABSTRACT FROM AUTHOR]

Abstract: We report a rapid and sensitive electrochemical strategy for the detection of gene‐specific 5‐methylcytosine DNA methylation. Magnetic beads (MBs) modified with an antibody for 5‐methylcytosines (5‐mC) are used for the capture of any 5‐mC methylated single‐stranded (ss)DNA sequence. A flanking region next to the 5‐mCs of the captured methylated ssDNA is recognized by hybridization with a synthetic biotinylated DNA sequence. Amperometric transduction at disposable screen‐printed carbon electrodes (SPCEs) is employed. The developed biosensor has a dynamic range from 3.9 to 500 p m and a limit of detection of 1.2 p m for the methylated synthetic sequence of the tumor suppressor gene O‐6‐methylguanine‐DNA methyltransferase (MGMT) promoter region. The method is applied in the 45‐min analysis of specific methylation in the MGMT promoter region directly in raw spiked human serum samples and in genomic DNA extracted from U‐87 glioblastoma cells and paraffin‐embedded brain tumor tissues without any amplification and pretreatment step. [ABSTRACT FROM AUTHOR]

Recent studies have revealed an unexpected role of DNA methylation at promoter regions in transcription activation. However, whether DNA methylation at enhancer regions activates gene expression and influences cellular functions remains to be determined. In this study, by employing the transcription factor krÜppel-like factor 4 (KLF4) that binds to methylated CpGs (mCpGs), we investigated the molecular outcomes of the recruitment of KLF4 to mCpGs at enhancer regions in human glioblastoma cells. First, by integrating KLF4 ChIP-seq, whole-genome bisulfite sequence, and H3K27ac ChIP-seq datasets, we found 1,299 highly methylated (β >0.5) KLF4 binding sites, three-quarters of which were located at putative enhancer regions, including gene bodies and intergenic regions. In the meantime, by proteomics, we identified 16 proteins as putative targets upregulated by KLF4-mCpG binding at enhancer regions. By chromosome conformation capture (3C) analysis, we demonstrated that KLF4 bound to methylated CpGs at the enhancer regions of the B-cell lymphocyte kinase (BLK) and Lim domain only protein 7 (LMO7) genes, and activated their expression via 3D chromatin loop formation with their promoter regions. Expression of mutant KLF4, which lacks KLF4 ability to bind methylated DNA, or removal of DNA methylation in enhancer regions by a DNA methyltransferase inhibitor abolished chromatin loop formation and gene expression, suggesting the essential role of DNA methylation in enhancer-promoter interactions. Finally, we performed functional assays and showed that BLK was involved in glioblastoma cell migration. Together, our study established the concept that DNA methylation at enhancer regions interacts with transcription factors to activate gene expression and influence cellular functions. [ABSTRACT FROM AUTHOR]

Methylated DNA is a covalent post-translational modification, which plays a crucial role in pathological and physiological processes including several diseases, such as cardiovascular disease, diabetes or cancer. Despite that, methylated DNA presents a new generation of biomarkers, which brings a promising alternative for using in point-care diagnostic. Regarding this fact, DNA based electrochemical sensors enable fast, reliable, low-cost, time-consuming and efficient detection. The application of these biosensors as possible alternatives for the determination of methylated DNA is recently growing. Therefore, a biosensor for the determination of methylated DNA was fabricated. This study was aimed to develop an efficient biosensor, with an amplified electrochemical signal which is suited for the detection of the low-level concentration of methylated DNA. The bare gold electrode was first covered with the graphene oxide modified with gold, silver and copper nanoparticles. These composites have a strong affinity to DNA probe and their effect on the sensitivity and selectivity of the biosensor was investigated. The developed biosensor shows promising analytical characteristics with a wide detection linear range. The electrochemical impedance spectroscopy (EIS) was used to detect the hybridization of the DNA probe with methylated DNA target.

We determine, by using a tight-binding model Hamiltonian, the characteristic current–voltage (IxV) curves of a 5-methylated cytosine single strand poly-GC DNA-like finite segment, considering the methyl groups attached laterally to a random fraction of the cytosine basis. Striking, we found that the methylation significantly impacts the ohmic resistance ( R ) of the DNA-like segments, indicating that measurements of R can be used as a biosensor tool to probe the presence of anomalous methylation. [ABSTRACT FROM AUTHOR]

Recognition of the methylated regions of the DNA plays an important role in the epigenetic processes. We analyze the interactions between the methylated DNA and the methyl-CpG-binding proteins using two models. The first model was built from a methylated or non-methylated cytosine, a guanine and an arginine residue in the experimental arrangement. We applied the M06L density functional method with a small, polarized double-ζ basis set for the geometry optimizations, and the MP2 method with polarized triple-ζ basis set for the energy calculations. The second model was built from two methylcytosines, guanines, guanidinium groups plus an additional carboxyl group in the experimental arrangement. We applied the B3LYP method with a small, polarized double-ζ basis set for the geometry optimizations and thermal corrections. The single-point energies were obtained from dual-hybrid dRPA75 and dRPA@PBE0 calculations supplemented by a moderately large polarized triple-ζ basis set. The hydration effects were modeled by adding explicit water molecules. These calculations revealed that the hydrophobic interaction has the largest contribution to the Gibbs interaction energy and turns the arginine side chains into hydrogen bonding position. Our results show that the translation of the protein along the DNA double helix is sterically hindered by the contact of its arginine side chains with the methyl groups of the methyl cytosines. This supports a hopping mechanism for the searching movement of the protein along the DNA. [ABSTRACT FROM AUTHOR]

Recognition of methylated DNA sites would be useful strategy due to the important roles of methylation in disease occurrence and developmental processes. The interaction of CpG rich methylated and unmethylated DNA hybrid with methylene blue (MB) as an optical probe has been investigated by absorption, emission, circular dichorism and fluorescence anisotropy analysis. Titration of MB with both sequences caused a hypsochromism and decreased the absorption of MB that indicating an intercalative mode of interaction. The experimental results revealed that MB as the optical indicator could distinguish between the methylated and unmethylated DNA sequences. Under optimum conditions, upon the addition of methylated dsDNA, the fluorescence intensity increased in linear range from 1.0 × 10 to 1.0 × 10 M with detection limit of 7.2 × 10 M and on the other hand, the intensity of MB showed no change with addition of unmethylated dsDNA. [ABSTRACT FROM AUTHOR]

Molecular biology has evolved far beyond that which could have been predicted at the time DNA identity testing was established. Indeed we should now perhaps be referring to forensic molecular biology.Aside from DNA’s established role in identifying the who in crime investigations, other developments in medical and developmental molecular biology are now ripe for application to forensic challenges. The impact of DNA methylation and other post-fertilization DNA modifications, plus the emerging role of small RNAs in the control of gene expression, is re-writing our understanding of human biology. It is apparent that these emerging technologies will expand forensic molecular biology to allow for inferences about when a crime took place and what took place.However, just as the introduction of DNA identity testing engendered many challenges, so the expansion of molecular biology into these domains will raise again the issues of scientific validity, interpretation, probative value, and infringement of personal liberties. This Commentary ponders some of these emerging issues, and presents some ideas on how they will affect the conduct of forensic molecular biology in the foreseeable future.

Hypermethylation at promoter regions of tumour suppressor genes is diagnostic for many cancers. Many genomic regions that may be the targets for clinical diagnostic assays have been identified through use of measuring systems reliant on bisulphite conversion, but few of these promising markers are in clinical use. The comparability of a widely used DNA methylation measuring system involving bisulphite conversion was evaluated by supplying three experienced centres with methylated DNA reference material mixtures that were independently prepared and characterised by mass spectrometry and high-pressure liquid chromatography. A replication scheme was designed to evaluate reproducibility of key analytical steps within and between laboratories by regression analysis. In general, methylation was underestimated and methylation ratio values were highly variable. The difference in methylation ratio between CpG sites was the key contributor to variable results. The CpG site effect followed a similar pattern at all centres and at all methylation levels examined indicating that sequence context had a major effect on methylation ratio measurement using the bisulphite conversion process. The magnitude of underestimation combined with the variability of measurements between CpG sites compromises the concept of measuring genomic regional methylation by averaging the methylation ratios of many CpG sites. There were no significant differences in replicate bisulphite conversions or sample work-up and instrument analysis at each centre thus making this technique suitable for comparative intralaboratory investigations. However, it may not be suitable for a routine diagnostic assay without extensive standardisation efforts. [ABSTRACT FROM AUTHOR]

Aberrantly methylated genes are increasingly being established as biomarkers for the detection of colorectal cancer (CRC). In the present study, the methylation levels of the secreted frizzled-related protein gene 2 (SFRP2), GATA binding protein 4/5 (GATA4/5), N-Myc downstream-regulated gene 4 (NDRG4) and vimentin (VIM) promoters were evaluated for their use as markers in the noninvasive detection of CRC. Methylation-specific polymerase chain reaction was performed to analyze promoter CpG methylation of SFRP2, GATA4/5, NDRG4 and VIM in the fecal DNA of 56 patients with CRC and 40 individuals exhibiting normal colonoscopy results. Promoter methylation levels of SFRP2, GATA4/5, NDRG4 and VIM in CRC patients were 57.1% (32/56), 42.9% (24/56), 83.9% (47/56), 28.6% (16/56) and 41.1% (23/56), respectively. Furthermore, the specificity of the genes were 90.0% (4/40), 95.0% (2/40), 82.5% (7/40), 97.5% (4/40) and 85.0% (6/40), respectively. The overall sensitivity of detection for fecal DNA with at least one methylated gene was 96.4% (54/56) in CRC patients. By contrast, only 14 of the 40 normal individuals exhibited methylated DNA in the aforementioned promoter regions. Methylation of the SFRP2, GATA4/5, NDRG4 and VIM promoters in fecal DNA is associated with the presence of colorectal tumors. Therefore, the detection of aberrantly methylated DNA in fecal samples may present a promising, noninvasive screening method for CRC. [ABSTRACT FROM AUTHOR]

Methyl-CpG-binding domain (MBD) proteins can specifically recognize and bind methylated CpG sites of DNA, thus repress gene transcription. In this study, we designed and expressed two recombinant proteins, MBD2b and SNAP-MBD2b, in E. coli. An optimized protocol was developed to purify the proteins using Ni-NTA affinity cartridge and cation exchange resin. The engineered proteins purified by this method exhibited more than 93% purity and high binding avidity. We found that both SNAP-MBD2b and MBD2b were prone to aggregate during dialysis. However, this could be prevented by the use of 0.3 mol/L NaCl. The fusion of SNAP-tag with MBD2b significantly enhanced the expression of MBD2b protein in E. coli and reduced the adsorption of MBD2b on solid interfaces involved in protein purification and immobilization. The engineered proteins can be used for the study of interaction with methylated DNA and the assays for DNA methylation. [ABSTRACT FROM AUTHOR]

Abstract: DNA methylation is an important epigenetic modification of genomes and is associated with various human diseases. Here, we develop a sensitive approach for DNA methylation assay using the short linear quencher–fluorophore DNA probe (QF probe) and two-stage isothermal amplification. With bisulfite treatment, the methylated DNA target is able to hybridize with the template to initiate the strand displacement amplification (SDA), generating abundant triggers which can further hybridize with the QF probes to form the DNA duplexes. The subsequent recognition of DNA duplexes and the cleavage of QF probes by the nicking enzyme can initiate the nicking enzyme signal amplification (NESA), inducing a significant fluorescence enhancement. While in the absence of methylated DNA, neither SDA nor NESA reaction can be initiated and no fluorescence enhancement is observed. This method exhibits high sensitivity with a detection limit of 0.78pM, and can distinguish as low as 0.1% methylation level from the mixture of methylated and unmethylated DNA. Notably, the introduction of SDA into NESA can improve the detection sensitivity by up to 2 orders of magnitude as compared with the NESA assay, and it can even discriminate single-base mismatched methylated DNA. [Copyright &y& Elsevier]

Methyl-directed site-specific DNA endonucleases (MD endonucleases) are a small group of enzymes that specifically cleave only methylated DNA. The group includes N6-methyladenine- and 5-methylcytosine-directed enzymes. Although poorly understood, MD endonucleases are of interest for both basic research and application in biotechnology and epigenomics. The review for the first time summarizes the properties of MD endonucleases and considers their role in the bacterial cell and their possible uses in biotechnology and epigenomics. [ABSTRACT FROM AUTHOR]

Abstract: Kaiso is a Cys2His2 zinc finger (ZF) protein that mediates methyl-CpG-dependent and sequence-specific transcriptional repression. As a first step towards elucidating the structural and molecular basis for recognition of these disparate DNA sequences, the minimal binding region of Kaiso was identified and optimal DNA sequences for high-affinity interactions were characterized. Contrary to previous findings, Kaiso requires all three zinc fingers plus adjacent protein regions for DNA recognition. An N-terminal extension contributes to structural stability, while an extended C-terminal region augments DNA binding. Complexes formed between the optimized Kaiso construct and both DNA sequences are suitable for future structural evaluation. [Copyright &y& Elsevier]

Since methylation at the N-7 and O positions of guanine and the N-3 position of adenine in DNA are the predominant reaction sites, N -methylguanine ( N -MeG), O -methylguanine ( O -MeG), and N -methyladenine ( N -MeA) have been suggested as good biomarkers for assessing exposure to methylating agents. Here, we report the development of a sensitive and selective assay based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) to simultaneously measure N -MeG, O -MeG, and N -MeA in DNA hydrolysates. With the use of isotope internal standards (N- N -MeG, d - O -MeG, and d - N -MeA) and online solid-phase extraction, DNA hydrolysates can be directly analyzed within 12 min without prior sample purification. The limits of detection were 0.02, 0.002, and 0.01 ng/mL on-column (6.1, 0.6, and 3.4 fmol) for N -MeG, O -MeG, and N -MeA, respectively. Inter- and intraday imprecision (CV) were 3.6-9.6% and 2.7-13.6%, respectively. Mean recoveries were 96-109%. This method was then applied to quantitate the amounts of methylated purines in calf thymus DNA treated with methyl methanesulfonate (MMS). The levels of N -MeG, O -MeG, and N -MeA in calf thymus DNA increase with MMS concentration and incubation time. The ratio of relative yields of N -MeG, O -MeG, and N -MeA in MMS-treated DNA was found to be 1.00:0.0032:0.119, respectively. This LC-MS/MS assay provides the sensitivity and high throughput required to evaluate the extent of methylated lesions in DNA induced by methylating agents. [ABSTRACT FROM AUTHOR]

The methyl-specific restriction system of Streptomyces coelicolor A3(2) was analyzed by carrying out transformations with unmethylated and methylated pSET152 DNA. Streptomyces coelicolor was found to strongly restrict DNA methylated in vivo by the Dam, Dcm and Hsd modification systems of Escherichia coli. Hsd-modified DNA was restricted as strongly as Dam-modified DNA, even though there are significantly fewer sites on the plasmid; Dcm-modified plasmid was restricted more strongly then either Dam- or Hsd-modified DNA. Restriction of plasmid DNA modified in vitro by different methylases also showed a greater dependence on the methylated sequence than on the number of methylated sites. Streptomyces coelicolor mutants were constructed that lacked genes identified as the likely candidates for encoding methyl-specific restriction nucleases (the products of the SCO4213, SCO4631 and SCO2863 genes, as well as the SCO3261–SCO3262 operon) that are located in the laterally acquired genomic islands of the S. coelicolor chromosome; these mutants showed partial alleviation of methylated DNA restriction. Cloning of these genes in the close relative Streptomyces lividans increased the restriction of methylated DNA by this species, confirming their role as part of the methyl-specific restriction system of S. coelicolor. [ABSTRACT FROM AUTHOR]

It is now widely accepted that there is a need for the development of molecular markers of cancer that can be used for clinical prognostication and monitoring. Approximately a decade ago tumor-derived circulating nucleic acids in the plasma or serum (CNAPS) of cancer patients were introduced as a noninvasive tool for cancer detection. This review focuses on the various types of CNAPS of patients with solid neoplasias (genetic alterations in circulating DNA, microsatellites, methylated DNA, viral DNA, nucleosomes, mitochondrial DNA and cell-free mRNA) and their putative potential as prognostic or predictive parameter or even as a tool for therapy monitoring during follow-up. Additionally, this review aims to point out the difference between a prognostic and a predictive factor in patient bloodstream. However, with rapid technical improvement and well-designed studies we conclude that the next years will see CNAPS analysis integrated in the prognostication and monitoring of cancer patients, thus producing more specific treatment regimens for patients with various stages of neoplastic disease and ultimately longer survival and better quality of life. [ABSTRACT FROM AUTHOR]

The polymerase chain reaction (PCR) is used to copy DNA in vitro for a variety of applications including amplifying a target DNA, mutating a base, adding tags, and sequencing by synthesis applications. Next-generation sequencing (NGS) is a DNA sequencing technology that has been applied to screening cancer and tissue variants, deep sequencing, and gene expression analysis, and more recently, it has been applied to DNA typing for human identification, estimating age, and detecting and differentiating body fluids. Body fluids are normally identified using color tests, microscopy, and immunochromatographic assays. Pyrosequencing is an NGS approach that has been applied to body fluid analysis. The pyrosequencing assays can detect one or several mixed body fluids by analysis of their tissue-specific differentially methylated regions (tDMRs). Here, the process of designing pyrosequencing primers for forensic biology applications is described., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

DNA methylation serves to mark DNA as either a directed epigenetic signaling modification or in response to DNA lesions. Methods for detecting DNA methylation have become increasingly more specific and sensitive over time. Conventional methods for detecting DNA methylation, ranging from paper chromatography to differential restriction enzyme digestion preference to dot blots, have more recently been supplemented by ultrahigh performance liquid chromatography coupled with mass spectrometry (UHPLC-MS/MS) to accurately quantify specific DNA methylation. Methylated DNA can also be sequenced by either methylated DNA immunoprecipitation followed by sequencing (MeDIP-seq) or single-molecule real-time sequencing (SMRTseq) for identifying genomic locations of DNA methylation. Here we describe a protocol for the detection and quantification of epigenetic signaling DNA methylation modifications including, N6-methyladenine (6mA), N4-methylcytosine (4mC) and C5-methylcytosine (5mC) in genomic DNA by triple quadrupole liquid chromatography coupled with tandem mass spectrometry (QQQ-LC-MS/MS). The high sensitivity of the UHPLC-MS/MS methodology and the use of calibration standards of pure nucleosides allow for the accurate quantification of DNA methylation.

Transcription factors play key roles in cell-fate decisions by regulating 3D genome conformation and gene expression. The traditional view is that methylation of DNA hinders transcription factors binding to them, but recent research has shown that many transcription factors prefer to bind to methylated DNA. Therefore, identifying such transcription factors and understanding their functions is a stepping-stone for studying methylation-mediated biological processes. In this paper, a two-step discriminated method was proposed to recognize transcription factors and their preference for methylated DNA based only on sequences information. In the first step, the proposed model was used to discriminate transcription factors from non-transcription factors. The areas under the curve (AUCs) are 0.9183 and 0.9116, respectively, for the 5-fold cross-validation test and independent dataset test. Subsequently, for the classification of transcription factors that prefer methylated DNA and transcription factors that prefer non-methylated DNA, our model could produce the AUCs of 0.7744 and 0.7356, respectively, for the 5-fold cross-validation test and independent dataset test. Based on the proposed model, a user-friendly web server called TFPred was built, which can be freely accessed at http://lin-group.cn/server/TFPred/., (© 2020 The Author(s).)