Chemo-Enzymatic Fluorescence Labeling Of Genomic DNA For Simultaneous Detection Of Global 5-Methylcytosine And 5-Hydroxymethylcytosine.

5-Methylcytosine and 5-hydroxymethylcytosine are epigenetic modifications involved in gene regulation and cancer. We present a new, simple, and high-throughput platform for multi-color epigenetic analysis. The novelty of our approach is the ability to multiplex methylation and de-methylation signals in the same assay. We utilize an engineered methyltransferase enzyme that recognizes and labels all unmodified CpG sites with a fluorescent cofactor. In combination with the already established labeling of the de-methylation mark 5-hydroxymethylcytosine via enzymatic glycosylation, we obtained a robust platform for simultaneous epigenetic analysis of these marks. We assessed the global epigenetic levels in multiple samples of colorectal cancer and observed a 3.5-fold reduction in 5hmC levels but no change in DNA methylation levels between sick and healthy individuals. We also measured epigenetic modifications in chronic lymphocytic leukemia and observed a decrease in both modification levels (5-hydroxymethylcytosine: whole blood 30 %; peripheral blood mononuclear cells (PBMCs) 40 %. 5-methylcytosine: whole blood 53 %; PBMCs 48 %). Our findings propose using a simple blood test as a viable method for analysis, simplifying sample handling in diagnostics. Importantly, our results highlight the assay's potential for epigenetic evaluation of clinical samples, benefiting research and patient management.

A Chemical Model of a TET Enzyme for Selective Oxidation of Hydroxymethyl Cytosine to Formyl Cytosine.

Methylation/demethylation of cytosines in DNA is central to epigenetics, which plays crucial roles in the regulation of about half of all human genes. Although the methylation mechanism, which downregulates gene expression, has been sufficiently decoded; the demethylation pathway, which upregulates gene expression, still holds questions to be answered. Demethylation of 5-methylcytosine by ten-eleven translocation (TET) enzymes yields understudied but epigenetically relevant intermediates, 5-hydroxymethyl (5-hmC), 5-formyl (5-fC), and 5-carboxyl (5-caC) cytosines. Here we report an iron complex, FeIIITAML (TAML = tetraamido macrocyclic ligand), which can facilitate selective oxidation of 5-hmC to its oxidative derivatives by forming a high-valent Fe-oxo intermediate in the presence of H2O2 under physiologically relevant conditions. Detailed HPLC analyses supported by a wide reaction condition optimization for the 5-hmC → 5-fC oxidation provides us with a chemical model of the TET enzyme. This study shines light on future efforts for a better understanding of the roles of 5-hmC and the TET enzyme mechanism and potentially novel therapeutic methods.

A novel one-pot fluorescence tagging and depyrimidination strategy for quantification of global DNA methylation.

DNA methylation is intensively studied in medical science. Current HPLC methods for quantification of global DNA methylation involve digestion of a DNA sample and HPLC determination of both cytosine (C) and 5-methylcytosine (5mC) so that percentage of 5mC in total cytosine can be calculated as DNA methylation level. Herein we report a novel HPLC method based on a one-pot fluorescence tagging and depyrimidination reaction between DNA and chloroacetaldehyde (CAA) for highly sensitive quantification of global DNA methylation. In the one-pot reaction, C and 5mC residues in a DNA sequence react with CAA, forming fluorescent etheno-adducts that are then released from the sequence through depyrimidination. Interestingly, etheno-5mC (ε-5mC) is ∼20 times more fluorescent than ε-C and other ε-nucleobases resulting from the reaction, which greatly facilitates the quantification. Further, due to the tagging-induced increase in structural aromaticity, ε-nucleobases are far more separable by HPLC than intact nucleobases. The proposed HPLC method with fluorescence detection (HPLC-FD) is quick (i.e., < 1h per assay) and highly sensitive with a detection limit of 0.80 nM (or 250 fg on column) for 5mC. Using the method, DNA samples isolated from yeast, HCT-116 cells, and tissues were analyzed. Global DNA methylation was measured to be in the range from 0.35% to 2.23% in the samples analyzed. This sensitive method allowed accurate analyses of minute DNA samples (∼100 ng) isolated from milligrams of tissues.

Magnetic multi-enzyme cascade combined with liquid chromatography tandem mass spectrometry for fast DNA digestion and quantitative analysis of 5-hydroxymethylcytosine in genome of human bladder cancer T24 cells induced by tetrachlorobenzoquinone.

Long-term exposure to halobenzoquinones (HBQs) can induce genomic damages and abnormal epigenetic modifications. High-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) has shown unique advantages in identification and sensitive analysis of these structurally modified DNA lesions. Prior to MS analysis, genomic DNA needs to be fully digested into mono-nucleosides. Here, we prepared Supernuclease (SN)-, snake venom phosphodiesterase (SVP)- and calf intestinal alkaline phosphatase (CIP)- individually immobilized magnetic nanoparticles (MNPs), and combined them according to certain formula to construct a recyclable SN-SVP-CIP magnetic nanoparticles (SNSC-MNPs) cascade for rapid and efficient DNA digestion. The SNSC-MNPs cascade can fully digest genomic DNA into mono-nucleosides within 30 min. The SNSC-MNPs cascade coupled with HPLC-MS/MS method can accurately and sensitively detect 5-hydroxymethylcytosine (5hmC) changes in genome of human bladder cancer T24 cells induced by tetrachlorobenzoquinone. The immobilization of enzymes on MNPs can enhance the stability and enzymatic activity of the three enzymes, which guarantees the reusability and longtime preservation of the cascades. The relative digestive efficiencies are among 86% -106% up to ten times of reuse. The newly synthesized SNSC-MNPs cascade coupled with HPLC-MS/MS method is promising for fast identification and analysis of structural modifications in genomic DNA.

Single-molecule analysis of genome-wide DNA methylation by fiber FISH coupled with atomic force microscopy.

DNA methylation (mainly at 5-methylcytosine, 5mC) plays an essential role in embryonic development and cellular biology. Alterations in DNA methylation are associated with disease development, especially hematologic malignancies. To investigate the potential of 5mC for diagnosis and treatment, accurate determination of 5mC is essential. Standard bisulfite sequencing-based methodologies or various optical/electrochemical biosensors for identifying 5mC have limitations, such as high cost, severe DNA degradation, over-estimation of the true 5mC level, being able to only display the average 5mC level, etc. Here we propose a single-molecule strategy for the direct identification of whole-genome 5mC by the combination of DNA fiber-based fluorescence in situ hybridization (DNA fiber FISH) and atomic force microscopy (AFM). Using extended DNA fibers and anti-5mC antibody for the detection of 5mC, it is possible to map the physical location of 5mC within the genome DNA. Together with AFM, this method can present the morphology of anti-5mC-DNA complexes and detailed spacing distribution of two neighboring 5mC sites on a single DNA molecule. Furthermore, this approach can be used for reporting other epigenetic modifications, not limited to 5mC or one single epigenetic modification. It can be anticipated to contribute to the development of clinical diagnosis of epigenetic-related diseases.

Nanopore Sequencing and Data Analysis for Base-Resolution Genome-Wide 5-Methylcytosine Profiling.

Whole-genome bisulfite sequencing (WGBS) is currently the gold standard for DNA methylation (5-methylcytosine, 5mC) profiling; however, the destructive nature of sodium bisulfite results in DNA fragmentation and subsequent biases in sequencing data. Such issues have led to the development of bisulfite-free methods for 5mC detection. Nanopore sequencing is a long read nondestructive approach that directly analyzes DNA and RNA fragments in real time. Recently, computational tools have been developed that enable base-resolution detection of 5mC from Oxford Nanopore sequencing data. In this chapter, we provide a detailed protocol for preparation, sequencing, read assembly, and analysis of genome-wide 5mC using Nanopore sequencing technologies.

Loss of 5-hydroxymethylcytosine as a Poor Prognostic Factor for Primary Testicular Diffuse Large B-cell Lymphoma.

Background: 5-Hydroxymethylcytosine (5-hmC), a stable epigenetic marker, is closely related to tumor staging, recurrence and survival, but the prognostic value of 5-hmC in primary testicular diffuse large B-cell lymphoma (PT-DLBCL) remains unclear. This study aimed to investigate the 5-hmC expression in PT-DLBCL and evaluate its prognostic value. Methods: A total of 34 patients with PT-DLBCL treated in the Department of Hematology from August 2000 to August 2020 were included in this study. The expression of 5-hmC in PT-DLBCL tissues and normal testicular tissues were assessed by immunohistochemistry. 5-hmC staining is estimated as a percentage under every nuclear staining intensity score (0-3), 0 or 1 of which were regarded as 5-hmC reduction. The quantification of 5-hmC reduction is defined as the percentage of cells with 5-hmC staining scores of 0 and 1. According 5-hmC reduction of 80%, a 5-hmC reduction of <80% is regarded as "5-hmC high-level group", and a 5-hmC reduction of ≥80% is regarded as "5-hmC low-level group". Furthermore, Cox regression model was used to evaluate the prognostic value of all covariates. Results: The median percentage of 5-hmC reduction in the PT-DLBCL group was 77.50% (60%-90%), the median 5-hmC reduction in the normal testicular tissues was 30% (20%-50%). Compared with normal testicular tissue, 5-hmC levels in PT-DLBCL tissue were significantly decreased (p<0.05). Of the 34 PT-DLBCL patients, 17 had tumors with relatively low 5-hmC expression (5-hmC reduction of ≥80%) and 17 had tumors with relatively high 5-hmC expression (5-hmC reduction of < 80%). 5-hmC expression was negatively correlated with international prognostic index (p = 0.037), while there was no significant difference in 5-hmC decrease among different groups of age at diagnosis, lactate dehydrogenase, testicular lymphoma involvement (unilateral or bilateral), Ki-67 and tumor diameter. Relatively low 5-hmC expression indicated shorter overall survival (OS) (5-year OS 50.2% vs 81.3%, p=0.022) and progression-free survival (PFS) (5-year PFS 38.5% vs 70.7%, p=0.001). Cox multivariate analysis of IPI (2-3 vs. 0-1), intrathecal prophylaxis (No vs. Yes), and 5-hmC reduction (≥80% vs. <80%) showed that 5-hmC reduction ≥80% (hazard ratio: 7.252, p = 0.005) and not receiving intrathecal prophylaxis (hazard ratio: 7.207, p =0.001) are independent risk factors for poor prognosis of PT-DLBCL. Conclusion: Our results suggested that 5-hmC decline can be identified as a poor prognostic predictor for PT-DLBCL. It is necessary to further explore the underlying mechanism of this epigenetic marker to identify methods to re-establish 5-hmC levels and provide new targets for cancer therapy.

Integrated 5-hydroxymethylcytosine and fragmentation signatures as enhanced biomarkers in lung cancer.

BACKGROUND: Lung cancer is one of most common cancers worldwide, with a 5-year survival rate of less than 20%, which is mainly due to late-stage diagnosis. Noninvasive methods using 5-hydroxymethylation of cytosine (5hmC) modifications and fragmentation profiles from 5hmC cell-free DNA (cfDNA) sequencing provide an opportunity for lung cancer detection and management. RESULTS: A total of 157 lung cancer patients were recruited to generate the largest lung cancer cfDNA 5hmC dataset, which mainly consisted of 62 lung adenocarcinoma (LUAD), 48 lung squamous cell carcinoma (LUSC) and 25 small cell lung cancer (SCLC) patients, with most patients (131, 83.44%) at advanced tumor stages. A 37-feature 5hmC model was constructed and validated to distinguish lung cancer patients from healthy controls, with areas under the curve (AUCs) of 0.8938 and 0.8476 (sensitivity = 87.50% and 72.73%, specificity = 83.87% and 80.60%) in two distinct validation sets. Furthermore, fragment profiles of cfDNA 5hmC datasets were first explored to develop a 48-feature fragmentation model with good performance (AUC = 0.9257 and 0.822, sensitivity = 87.50% and 78.79%, specificity = 80.65% and 76.12%) in the two validation sets. Another diagnostic model integrating 5hmC signals and fragment profiles improved AUC to 0.9432 and 0.8639 (sensitivity = 87.50% and 83.33%, specificity = 90.30% and 77.61%) in the two validation sets, better than models based on either of them alone and performing well in different stages and lung cancer subtypes. Several 5hmC markers were found to be associated with overall survival (OS) and disease-free survival (DFS) based on gene expression data from The Cancer Genome Atlas (TCGA). CONCLUSIONS: Both the 5hmC signal and fragmentation profiles in 5hmC cfDNA data are sensitive and effective in lung cancer detection and could be incorporated into the diagnostic model to achieve good performance, promoting research focused on clinical diagnostic models based on cfDNA 5hmC data.

BiLSTM-5mC: A Bidirectional Long Short-Term Memory-Based Approach for Predicting 5-Methylcytosine Sites in Genome-Wide DNA Promoters.

An important reason of cancer proliferation is the change in DNA methylation patterns, characterized by the localized hypermethylation of the promoters of tumor-suppressor genes together with an overall decrease in the level of 5-methylcytosine (5mC). Therefore, identifying the 5mC sites in the promoters is a critical step towards further understanding the diverse functions of DNA methylation in genetic diseases such as cancers and aging. However, most wet-lab experimental techniques are often time consuming and laborious for detecting 5mC sites. In this study, we proposed a deep learning-based approach, called BiLSTM-5mC, for accurately identifying 5mC sites in genome-wide DNA promoters. First, we randomly divided the negative samples into 11 subsets of equal size, one of which can form the balance subset by combining with the positive samples in the same amount. Then, two types of feature vectors encoded by the one-hot method, and the nucleotide property and frequency (NPF) methods were fed into a bidirectional long short-term memory (BiLSTM) network and a full connection layer to train the 22 submodels. Finally, the outputs of these models were integrated to predict 5mC sites by using the majority vote strategy. Our experimental results demonstrated that BiLSTM-5mC outperformed existing methods based on the same independent dataset.

Epigenetic Quantification of DNA 5-Hydroxymethylcytosine Using DNA Hybridization-Based Single-Molecule Immunofluorescent Imaging.

5-Hydroxymethylcytosine (5hmC) is a deoxyribonucleic acid (DNA) epigenetic modification that has an important function in embryonic development and human diseases. However, the numerous methods that have been developed to detect and quantify 5hmC, require large amounts of DNA sample to be modified via chemical reactions, which considerably limits their application with cell-free DNA (cfDNA). Meanwhile, other antibody-based methods of detecting 5hmC do not offer information about the DNA sequence. Here, in this article DNA hybridization-based single-molecule immunofluorescent imaging is presented, an ultrasensitive method of detecting 5hmC modification in DNA. Via using the probe DNA to capture the DNA fragment of interest and the 5hmC antibody to detect the 5hmC modification in DNA, the fluorescent response signal of the 5hmC modification from the secondary antibody at the single-molecule level is successfully detected. Using the method, one could determine the quantity of 5hmC in the gene of interest within 6 h. In addition, it requires only 3 pg of the DNA sample and minimal experience and training for operation and analysis.

Cell-free DNA 5-hydroxymethylcytosine profiles of long non-coding RNA genes enable early detection and progression monitoring of human cancers.

BACKGROUND: 5-Hydroxymethylcytosine (5hmC) is a significant DNA epigenetic modification. However, the 5hmC modification alterations in genomic regions encoding long non-coding RNA (lncRNA) and their clinical significance remain poorly characterized. RESULTS: A three-phase discovery-modeling-validation study was conducted to explore the potential of the plasma-derived 5hmC modification level in genomic regions encoding lncRNAs as a superior alternative biomarker for cancer diagnosis and surveillance. Genome-wide 5hmC profiles in the plasma circulating cell-free DNA of 1632 cancer and 1379 non-cancerous control samples from different cancer types and multiple centers were repurposed and characterized. A large number of altered 5hmC modifications were distributed at genomic regions encoding lncRNAs in cancerous compared with healthy subjects. Furthermore, most 5hmC-modified lncRNA genes were cancer-specific, with only a relatively small number of 5hmC-modified lncRNA genes shared by various cancer types. A 5hmC-LncRNA diagnostic score (5hLD-score) comprising 39 tissue-shared 5hmC-modified lncRNA gene markers was developed using elastic net regularization. The 5hLD-score was able to accurately distinguish tumors from healthy controls with an area under the curve (AUC) of 0.963 [95% confidence interval (CI) 0.940-0.985] and 0.912 (95% CI 0.837-0.987) in the training and internal validation cohorts, respectively. Results from three independent validations confirmed the robustness and stability of the 5hLD-score with an AUC of 0.851 (95% CI 0.786-0.916) in Zhang's non-small cell lung cancer cohort, AUC of 0.887 (95% CI 0.852-0.922) in Tian's esophageal cancer cohort, and AUC of 0.768 (95% CI 0.746-0.790) in Cai's hepatocellular carcinoma cohort. In addition, a significant association was identified between the 5hLD-score and the progression from hepatitis to liver cancer. Finally, lncRNA genes modified by tissue-specific 5hmC alteration were again found to be capable of identifying the origin and location of tumors. CONCLUSION: The present study will contribute to the ongoing effort to understand the transcriptional programs of lncRNA genes, as well as facilitate the development of novel invasive genomic tools for early cancer detection and surveillance.

DNA methylation-calling tools for Oxford Nanopore sequencing: a survey and human epigenome-wide evaluation.

BACKGROUND: Nanopore long-read sequencing technology greatly expands the capacity of long-range, single-molecule DNA-modification detection. A growing number of analytical tools have been developed to detect DNA methylation from nanopore sequencing reads. Here, we assess the performance of different methylation-calling tools to provide a systematic evaluation to guide researchers performing human epigenome-wide studies. RESULTS: We compare seven analytic tools for detecting DNA methylation from nanopore long-read sequencing data generated from human natural DNA at a whole-genome scale. We evaluate the per-read and per-site performance of CpG methylation prediction across different genomic contexts, CpG site coverage, and computational resources consumed by each tool. The seven tools exhibit different performances across the evaluation criteria. We show that the methylation prediction at regions with discordant DNA methylation patterns, intergenic regions, low CG density regions, and repetitive regions show room for improvement across all tools. Furthermore, we demonstrate that 5hmC levels at least partly contribute to the discrepancy between bisulfite and nanopore sequencing. Lastly, we provide an online DNA methylation database ( https://nanome.jax.org ) to display the DNA methylation levels detected by nanopore sequencing and bisulfite sequencing data across different genomic contexts. CONCLUSIONS: Our study is the first systematic benchmark of computational methods for detection of mammalian whole-genome DNA modifications in nanopore sequencing. We provide a broad foundation for cross-platform standardization and an evaluation of analytical tools designed for genome-scale modified base detection using nanopore sequencing.

iR5hmcSC: Identifying RNA 5-hydroxymethylcytosine with multiple features based on stacking learning.

RNA 5-hydroxymethylcytosine (5hmC) modification is the basis of the translation of genetic information and the biological evolution. The study of its distribution in transcriptome is fundamentally crucial to reveal the biological significance of 5hmC. Biochemical experiments can use a variety of sequencing-based technologies to achieve high-throughput identification of 5hmC; however, they are labor-intensive, time-consuming, as well as expensive. Therefore, it is urgent to develop more effective and feasible computational methods. In this paper, a novel and powerful model called iR5hmcSC is designed for identifying 5hmC. Firstly, we extract the different features by K-mer, Pseudo Structure Status Composition and One-Hot encoding. Subsequently, the combination of chi-square test and logistic regression is utilized as the feature selection method to select the optimal feature sets. And then stacking learning, an ensemble learning method including random forest (RF), extra trees (EX), AdaBoost (Ada), gradient boosting decision tree (GBDT), and support vector machine (SVM), is used to recognize 5hmC and non-5hmC. Finally, 10-fold cross-validation test is performed to evaluate the model. The accuracy reaches 85.27% and 79.92% on benchmark dataset and independent dataset, respectively. The result is better than the state-of-the-art methods, which indicates that our model is a feasible tool to identify 5hmC. The datasets and source code are freely available at https://github.com/HongyanShi026/iR5hmcSC.

Short history of 5-methylcytosine: from discovery to clinical applications.

Covalent modifications of nucleotides in genetic material have been known from the beginning of the last century. Currently, one of those modifications referred to as DNA methylation, is impacting personalised medicine both as a treatment target and a biomarker source for clinical disease management. In this short review, we describe landmark discoveries that led to the elucidation of the DNA methylation importance in the cell's physiology and clarification of its role as one of the major processes in disease pathology. We also describe turning points in the development of methodologies to study this modification, which ultimately resulted in the development of in-vitro diagnostic kits targeting disease related DNA methylation changes as biomarkers.

Analysis of 5-Hydroxymethyluracil Levels Using Flow Cytometry.

5-hydroxymethyluracil was originally identified as an oxidatively modified DNA base derivative. Recent evidence suggests that its formation may result from the oxidation of thymine in a reaction that is catalyzed by TET proteins. Alternatively, it could be generated through the deamination of 5-hydroxymethylcytosine by activation-induced cytidine deaminase. The standard method for evaluating 5-hydroxymethyluracil content is the highly sensitive and highly specific isotope-dilution automated online two-dimensional ultraperformance liquid chromatography with tandem mass spectrometry (2D-UPLC-MS/MS). Despite many advantages, this method has one great limitation. It is not able to measure compounds at a single-cell level. Our goal was to develop and optimize a method based on flow cytometry that allows the evaluation of 5-hydroxymethyluracil levels at a single cell level in peripheral leukocytes.

High-Resolution Analysis of 5-Hydroxymethylcytosine by TET-Assisted Bisulfite Sequencing.

DNA cytosine modification is an important epigenetic mechanism that serves critical functions in a variety of biological processes in development and disease. 5-Methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are the two most common epigenetic marks found in the mammalian genome. 5hmC is generated from 5mC by the ten-eleven translocation (TET) family of dioxygenase enzymes. This modification can reach substantial levels in certain cell types such as embryonic stem cells and neurons. Standard bisulfite sequencing techniques cannot distinguish between 5mC and 5hmC. Therefore, the method of TET-assisted bisulfite sequencing has been developed for detecting 5hmC specifically. The method is based on protection of 5hmC by glycosylation followed by complete oxidation of both 5mC and 5fC to 5caC, which converts to uracil after bisulfite treatment leaving only 5hmC remaining as a cytosine signal after PCR and sequencing. The method requires a highly active TET protein for the conversion steps. Here, we present an efficient TET protein purification method and a streamlined TAB-sequencing protocol for 5hmC analysis at single base resolution.

Oxidative Bisulfite Sequencing: An Experimental and Computational Protocol.

Bisulfite sequencing (BS-seq) remains the gold standard technique to quantitively map DNA methylation at a single-base resolution. However, BS-seq cannot discriminate between 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Oxidative bisulfite sequencing (oxBS-seq) was one of the first techniques that enabled absolute quantification of 5mC and 5hmC at single-base resolution. OxBS-seq uses chemical oxidation of 5hmC prior to bisulfite treatment to provide a direct readout of 5mC; comparison with BS-seq data can then be used to infer 5hmC levels. Here we describe in detail an updated version of our laboratory's oxBS-seq protocol, which uses potassium perruthenate (KRuO4) as an oxidant. We also describe a bioinformatics pipeline designed to handle Illumina short read sequencing data from whole-genome oxBS-seq.

Bisulfite-Free Sequencing of 5-Hydroxymethylcytosine with APOBEC-Coupled Epigenetic Sequencing (ACE-Seq).

Here, we provide a detailed protocol for our previously published technique, APOBEC-Coupled Epigenetic Sequencing (ACE-Seq), which localizes 5-hydroxymethylcytosine at single nucleotide resolution using nanogram quantities of input genomic DNA. In addition to describing suggested troubleshooting workflows, these methods include four important updates which should facilitate widespread implementation of the technique: (1) additionally optimized reaction conditions; (2) redesigned quality controls which can be performed prior to resource-consumptive deep sequencing; (3) confirmation that the less active, uncleaved APOBEC3A (A3A) fusion protein, which is easier to purify, can be used to perform ACE-Seq ; and (4) an example bioinformatic pipeline with suggested filtering strategies. Finally, we have provided a supplementary video which gives a narrated overview of the entire method and focuses on how best to perform the snap cool and A3A deamination steps central to successful execution of the method.

Engineered TALE Repeats for Enhanced Imaging-Based Analysis of Cellular 5-Methylcytosine.

Transcription-activator-like effectors (TALEs) are repeat-based, programmable DNA-binding proteins that can be engineered to recognize sequences of canonical and epigenetically modified nucleobases. Fluorescent TALEs can be used for the imaging-based analysis of cellular 5-methylcytosine (5 mC) in repetitive DNA sequences. This is based on recording fluorescence ratios from cell co-stains with two TALEs: an analytical TALE targeting the cytosine (C) position of interest through a C-selective repeat that is blocked by 5 mC, and a control TALE targeting the position with a universal repeat that binds both C and 5 mC. To enhance this approach, we report herein the development of novel 5 mC-selective repeats and their integration into TALEs that can replace universal TALEs in imaging-based 5 mC analysis, resulting in a methylation-dependent response of both TALEs. We screened a library of size-reduced repeats and identified several 5 mC binders. Compared to the 5 mC-binding repeat of natural TALEs and to the universal repeat, two repeats containing aromatic residues showed enhancement of 5 mC binding and selectivity in cellular transcription activation and electromobility shift assays, respectively. In co-stains of cellular SATIII DNA with a corresponding C-selective TALE, this selectivity results in a positive methylation response of the new TALE, offering perspectives for studying 5 mC functions in chromatin regulation by in situ imaging with increased dynamic range.

Targeted bisulfite sequencing for biomarker discovery.

Cytosine methylation is one of the best studied epigenetic modifications. In mammals, DNA methylation patterns vary among cells and is mainly found in the CpG context. DNA methylation is involved in important processes during development and differentiation and its dysregulation can lead to or is associated with diseases, such as cancer, loss-of-imprinting syndromes and neurological disorders. It has been also shown that DNA methylation at the cellular, tissue and organism level varies with age. To overcome the costs of Whole-Genome Bisulfite Sequencing, the gold standard method to detect 5-methylcytosines at a single base resolution, DNA methylation arrays have been developed and extensively used. This method allows one to assess the status of a fraction of the CpG sites present in the genome of an organism. In order to combine the relatively low cost of Methylation Arrays and digital signals of bisulfite sequencing, we developed a Targeted Bisulfite Sequencing method that can be applied to biomarker discovery for virtually any phenotype. Here we describe a comprehensive step-by-step protocol to build a DNA methylation-based epigenetic clock.