Yuval Ebenstein | Tel Aviv University (original) (raw)

Papers by Yuval Ebenstein

bioRxiv (Cold Spring Harbor Laboratory), Oct 31, 2022

5-methylcytosine and 5-hydroxymethylcytosine are epigenetic modifications involved in gene regula... more 5-methylcytosine and 5-hydroxymethylcytosine are epigenetic modifications involved in gene regulation and cancer. Here, we describe a new, simple, and high-throughput platform for multi-colour 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 labelling 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 reduction in 5hydroxymethylcytosine levels, but no change in DNA methylation levels between sick and healthy individuals. We also measured epigenetic modifications in chronic lymphocytic leukaemia and observed a decrease in both modification levels. Our results indicate that this assay may be used for the epigenetic characterization of clinical samples for research and patient management.

bioRxiv (Cold Spring Harbor Laboratory), Oct 12, 2022

Cancer cells display complex genomic aberrations that include large-scale genetic rearrangements ... more Cancer cells display complex genomic aberrations that include large-scale genetic rearrangements and epigenetic modulation that are not easily characterized by short-read sequencing. We present a method for simultaneous profiling of long-range genetic/epigenetic changes in matched cancer samples. Clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Most ccRCC cases demonstrate somatic genomic alterations involving the short arm of chromosome 3 (3p), most often targeting the von Hippel-Lindau (VHL) gene. Aiming to identify somatic alterations that characterize early stage ccRCC, we performed comprehensive genetic, cytogenetic and epigenetic analyses comparing ccRCC tumor to adjacent non-tumorous tissue. Optical genome mapping identified genomic aberrations such as structural and copy number variations, complementing exome-sequencing results. Single-molecule methylome and hydroxymethylome mapping revealed multiple differential regions, some of them known to be associated with ccRCC pathogenesis. Among them, metabolic pathways were significantly enriched. Moreover, significant global epigenetic differences were detected between the tumor and the adjacent nontumorous tissue, and a correlation between epigenetic signals and gene expression was found. This is the first reported comparison of a human tumor and a matched tissue by optical genome/epigenome mapping, revealing well-established and novel somatic aberrations.

Essays in Biochemistry, Apr 1, 2021

The human genome contains multiple layers of information that extend beyond the genetic sequence.... more The human genome contains multiple layers of information that extend beyond the genetic sequence. In fact, identical genetics do not necessarily yield identical phenotypes as evident for the case of two different cell types in the human body. The great variation in structure and function displayed by cells with identical genetic background is attributed to additional genomic information content. This includes large-scale genetic aberrations, as well as diverse epigenetic patterns that are crucial for regulating specific cell functions. These genetic and epigenetic patterns operate in concert in order to maintain specific cellular functions in health and disease. Single-molecule optical genome mapping is a high-throughput genome analysis method that is based on imaging long chromosomal fragments stretched in nanochannel arrays. The access to long DNA molecules coupled with fluorescent tagging of various genomic information presents a unique opportunity to study genetic and epigenetic patterns in the genome at a single-molecule level over large genomic distances. Optical mapping entwines synergistically chemical, physical, and computational advancements, to uncover invaluable biological insights, inaccessible by sequencing technologies. Here we describe the method's basic principles of operation, and review the various available mechanisms to fluorescently tag genomic information. We present some of the recent biological and clinical impact enabled by optical mapping and present recent approaches for increasing the method's resolution and accuracy. Finally, we discuss how multiple layers of genomic information may be mapped simultaneously on the same DNA molecule, thus paving the way for characterizing multiple genomic observables on individual DNA molecules.

ACS Photonics, Jun 7, 2019

Fluorescent peptide nanodots (PNDs) are bioorganic nanoparticles self-assembled from peptide biom... more Fluorescent peptide nanodots (PNDs) are bioorganic nanoparticles self-assembled from peptide biomolecules of different origin and complexity. These recently discovered nanodots of biological origin are highly promising for biomedical imaging applications due to their biocompatibility, bright and tunable fluorescence over the entire visible range and photostability. Here we apply single-particle microscopy methods to study the photophysical properties of individual PNDs. We show that the fluorescence spectrum tunability, studied previously only for PND ensembles in solutions, origins at the single-particle level. Temporal dynamics measurements of the single particles reveal fluorescence lifetime in the range of nanoseconds and pronounced fluorescence blinking with continuous bright states of seconds. The latter provides a first evidence of quantum emitter transitions between two states (ON and OFF) in fluorescent PNDs. All these findings advance the understanding of the fluorescence mechanism of PNDs and provide strong motivation for using PNDs as fluorescent agents for various bioimaging and super-resolution techniques.

Cambridge University Press eBooks, May 26, 2022

ChemBioChem, Jul 30, 2023

5‐Methylcytosine and 5‐hydroxymethylcytosine are epigenetic modifications involved in gene regula... more 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.

Optical mapping relies on the preparation of fluorescent DNA. DNA must be imaged with good signal... more Optical mapping relies on the preparation of fluorescent DNA. DNA must be imaged with good signal to noise and therefore the background of unwanted DNA fragments, fluorescent dyes and other reagents need to be removed. We use deterministic lateral displacement to separate 48.5 kbp DNA from 50 kbp molecules from a background of shorter digested fragments. In both cases improving signal to noise during imaging.

BackgroundAging is linked to hypermethylation of CpG sites on promoters and enhancers, along with... more BackgroundAging is linked to hypermethylation of CpG sites on promoters and enhancers, along with loss of methylation in intergenic zones. That such changes are not necessarily a continuous process is exemplified by the extensive changes in DNA methylation during development with another significant time of change during adolescence. However, the relation between age and DNA methylation during adult life has not been systematically evaluated. In particular, potential changes in methylation trends in the same CpGs over the years that may occur with aging remain largely unexplored.MethodsHere we set out to determine the average trends by age of the CpG sites represented in the Illumina 450 platform, based on data from 2143 subjects of the age range of 20 to 80 years, compiled from 24 different cohorts. Using several mathematical procedures, we initially separated stationary probes from probes whose methylation changes with age. Among the latter, representing ∼20% of the probes, we the...

Nanopore sequencing platforms combined with supervised machine learning (ML) have been effective ... more Nanopore sequencing platforms combined with supervised machine learning (ML) have been effective at detecting base modifications in DNA such as 5mC and 6mA. These ML-based nanopore callers have typically been trained on data that span all modifications on all possible DNA k-mer backgrounds—a complete training dataset. However, as nanopore technology is pushed to more and more epigenetic modifications, such complete training data will not be feasible to obtain. Nanopore calling has historically been performed with Hidden Markov Models (HMMs) that cannot make successful calls for k-mer contexts not seen during training because of their independent emission distributions. However, deep neural networks (DNNs), which share parameters across contexts, are increasingly being used as callers, often outperforming their HMM cousins. It stands to reason that a DNN approach should be able to better generalize to unseen k-mer contexts. Indeed, herein we demonstrate that a common DNN approach (De...

Mapping DNA damage and its repair has immense potential in understanding environmental exposures,... more Mapping DNA damage and its repair has immense potential in understanding environmental exposures, their genotoxicity, and their impact on human health. Monitoring changes in genomic stability also aids in the diagnosis of numerous DNA-related diseases, like cancer, and assists in monitoring their progression and prognosis. However, genome-wide maps of DNA damage distribution are challenging to produce. Here we describe the localization of DNA damage and repair loci by Repair Assisted Damage Detection sequencing – RADD-Seq. Based on the enrichment of damage lesions coupled with a pull-down assay and followed by next generation sequencing, this method is easy to perform and can produce compelling results with minimal coverage. RADD-seq enables the localization of both DNA damage and repair sites for a wide range of single-strand damage types. Using this technique, we created a genome-wide map of oxidative DNA damage loci before and after repair. Oxidative lesions were heterogeneously ...

Springer Protocols Handbooks, 2021

T4 β-glucosyltransferase is used to tag 5hmC sites with a fluorescent reporter molecule. The enzy... more T4 β-glucosyltransferase is used to tag 5hmC sites with a fluorescent reporter molecule. The enzyme is fed with a synthetic cofactor UDP-6-N3-Glu, resulting in covalent attachment of a functional azide at the 5hmC site. This azide is further reacted with a DBCO functionalized fluorophore via a copper-free "click" chemistry reaction to generate the fluorescently labeled 5hmC. enzymatic fluorescent Chemo hmC 5 labeling of nm wide 45 DNA is stretched in silicon nanochannels

ABSTRACTDNA methylation, specifically, methylation of cytosine (C) nucleotides at the 5-carbon po... more ABSTRACTDNA methylation, specifically, methylation of cytosine (C) nucleotides at the 5-carbon position (5-mC), is the most studied and among the most significant epigenetic modifications. Here we developed a chemoenzymatic procedure to fluorescently label non-methylated cytosines in the CpG context allowing epigenetic profiling of single DNA molecules spanning hundreds of thousands of base pairs. For this method, a CpG methyltransferase was used to transfer an azide to cytosines from a synthetic S-adenosyl-l-methionine cofactor analog. A fluorophore was then clicked onto the DNA, reporting on the amount and position of non-methylated CpGs. We found that labeling efficiency was increased two-fold by the addition of a nucleosidase that degrades the inactive by-product of the azide-cofactor after labeling, and prevents its inhibitory effect. We first used the method to determine the decline in global DNA methylation in chronic lymphocytic leukemia patients and then performed whole gen...

MotivationWhile promoter methylation is associated with reinforcing fundamental tissue identities... more MotivationWhile promoter methylation is associated with reinforcing fundamental tissue identities, the methylation status of distant enhancers was shown by genome-wide association studies to be a powerful determinant of cell-state and cancer. With recent availability of long-reads that report on the methylation status of enhancer-promoter pairs on the same molecule, we hypothesized that probing these pairs on the single-molecule level may serve the basis for detection of rare cancerous transformations in a given cell population. We explore various analysis approaches for deconvolving cell-type mixtures based on their genome-wide enhancer-promoter methylation profiles.ResultsTo evaluate our hypothesis we examine long-read optical methylome data for the GM12787 cell line and myoblast cell lines from two donors. We identified over 100,000 enhancer-promoter pairs that co-exist on at least 30 individual DNA molecules per pair. We developed a detailed methodology for mixture deconvolution...

Essays in Biochemistry, 2021

Nanoscale, 2020

The DNA methyltransferase M.TaqI is presented as a new tool to label DNA nanostructures covalentl... more The DNA methyltransferase M.TaqI is presented as a new tool to label DNA nanostructures covalently with fluorophores and azide groups.

Analytical Chemistry, 2020

Knowing the amount and type of DNA damage is of great significance for a broad range of clinical ... more Knowing the amount and type of DNA damage is of great significance for a broad range of clinical and research applications. However, existing methods either lack in their ability to distinguish between types of DNA damage, or are limited in their sensitivity and reproducibility. The method described herein enables rapid and robust quantification of type-specific single-strand DNA damage. The method is based on Repair-Assisted Damage Detection (RADD) by which fluorescent nucleotides are incorporated into DNA damage sites using type-specific repair enzymes. Up to 90 DNA samples are then deposited on a multi-well glass slide, and analyzed by Page 2 of 24 ACS Paragon Plus Environment Analytical Chemistry 3 a conventional slide scanner for quantification of DNA damage levels. Accurate and sensitive measurements of oxidative or UV-induced DNA damage levels and repair kinetics are presented for both in-vitro and in-vivo models.

Chemical Communications, 2019

Specific and simultaneous quantitation of DNA adducts is a major obstacle. Using repair enzymes, ... more Specific and simultaneous quantitation of DNA adducts is a major obstacle. Using repair enzymes, we present a protocol to quantify two types of DNA lesions simultaneously on the same DNA molecule and examine repair dynamics by single-molecule imaging.