Biology subjects

Ebenstein, Y.

Publications and source records attributed to Ebenstein, Y..

23 records · Page 2Linked to original sources

bioRxiv2019

Global Modulation in DNA Epigenetics during Pro-Inflammatory Macrophage Activation

DNA methylation patterns create distinct gene expression profiles. These patterns are maintained after cell division, thus enabling the differentiation and maintenance of multiple cell types from the same genome sequence. The advantage of this mechanism for transcriptional control is that chemical-encoding allows to rapidly establish new epigenetic patterns \"on-demand\" through enzymatic methylation and de-methylation of DNA. Here we show that this feature is associated with the fast response of macrophages during their pro-inflammatory activation. By using a combination of mass spectroscopy and single-molecule imaging to quantify global epigenetic changes in the genomes of primary macrophages, we followed three distinct DNA marks (methylated, hydroxymethylated and unmethylated), involved in establishing new DNA methylation patterns during pro-inflammatory activation. The observed epigenetic modulation together with gene expression data generated for the involved enzymatic machinery, may suggest that de-methylation upon LPS-activation starts with oxidation of methylated CpGs, followed by excision-repair of these oxidized bases and their replacement with unmodified cytosine.

immunology↗

bioRxiv2018

Genome-wide epigenetic profiling of 5-hydroxymethylcytosine by long-read optical mapping

The epigenetic mark 5-hydroxymethylcytosine (5-hmC) is a distinct product of active enzymatic demethylation that is linked to gene regulation, development and disease. Genome-wide 5-hmC profiles generated by short-read next-generation sequencing are limited in providing long-range epigenetic information relevant to highly variable genomic regions, such as the 3.7 Mbp disease-related Human Leukocyte Antigen (HLA) region. We present a long-read, single-molecule mapping technology that generates hybrid genetic/epigenetic profiles of native chromosomal DNA. The genome-wide distribution of 5- hmC in human peripheral blood cells correlates well with 5-hmC DNA immunoprecipitation (hMeDIP) sequencing. However, the long read length of 100 kbp-1Mbp produces 5-hmC profiles across variable genomic regions that failed to showup in the sequencing data. In addition, optical 5-hmC mapping shows strong correlation between the 5-hmC density in gene bodies and the corresponding level of gene expression. The single molecule concept provides information on the distribution and coexistence of 5-hmC signals at multiple genomic loci on the same genomic DNA molecule, revealing long-range correlations and cell-to-cell epigenetic variation.

genomics↗

bioRxiv2017

Reduced representation optical methylation mapping (R2OM2).

Reduced representation methylation profiling is a method of analysis in which a subset of CpGs is used to report the overall methylation status of the probed genomic regions. This approach has been widely adopted for genome-scale bisulfite sequencing since it requires fewer sequencing reads and uses significantly less starting material than whole-genome analysis. Consequently, this method is suitable for profiling medical samples and single cells at high throughput and reduced costs. Here, we use this concept in order to create a pattern of fluorescent optical methylation profiles along individual DNA molecules. Reduced representation optical methylation mapping (R2OM2) in combination with Bionano Genomics next generation genome mapping (NGM) technology provides a hybrid genetic/epigenetic genome map of individual chromosome segments spanning hundreds of kilobase pairs (kbp). These long reads, along with the single-molecule resolution, allow for epigenetic variation calling and methylation analysis of large structural aberrations such as pathogenic macrosatellite arrays not accessible to single-cell next generation sequencing (NGS). We apply this method to facioscapulohumeral dystrophy (FSHD) showing both structural variation and hypomethylation status of a disease-associated, highly repetitive locus on chromosome 4q.

genetics↗

bioRxiv2017

Reduced representation optical methylation mapping (R2OM2).

Reduced representation methylation analysis utilizes a subset of CpGs in order to report the overall methylation status of the probed genomic regions. Here, we use this concept in order to create fluorescent optical methylation profiles along chromosomal DNA molecules for epigenetic profiling. Reduced representation optical methylation mapping (R2OM2) in combination with Bionano Genomics next generation genome mapping (NGM) technology provides a hybrid genetic/epigenetic genome map of individual chromosome segments spanning hundreds of kilobase pairs (kbp). These long reads, along with the single-molecule resolution, allow for epigenetic variation calling and methylation analysis of large structural aberrations such as pathogenic macrosatellite arrays not accessible to single-cell next generation sequencing (NGS). We show that in addition to the inherent long-read benefits of R2OM2, it provides genomic methylation patterns comparable to whole genome bisulfite sequencing (WGBS) while retaining single-molecule information. The method is applied here to detect methylation along genes, around regulatory histone marks and to study facioscapulohumeral muscular dystrophy (FSHD), simultaneously recording the haplotype, copy number and methylation status of the disease-associated, highly repetitive locus onchromosome 4q.

genetics↗

bioRxiv2017

Cas9-Assisted Targeting of CHromosome segments (CATCH) for targeted nanopore sequencing and optical genome mapping

Variations in the genetic code, from single point mutations to large structural or copy number alterations, influence susceptibility, onset, and progression of genetic diseases and tumor transformation. Next-generation sequencing analyses are unable to reliably capture aberrations larger than the typical sequencing read length of several hundred bases. Long-read, single-molecule sequencing methods such as SMRT and nanopore sequencing can address larger variations, but require costly whole genome analysis. Here we describe a method for isolation and enrichment of a large genomic region of interest for targeted analysis based on Cas9 excision of two sites flanking the target region and isolation of the excised DNA segment by pulsed field gel electrophoresis. The isolated target remains intact and is ideally suited for optical genome mapping and long-read sequencing at high coverage. In addition, analysis is performed directly on native genomic DNA that retains genetic and epigenetic composition without amplification bias. This method enables detection of mutations and structural variants as well as detailed analysis by generation of hybrid scaffolds composed of optical maps and sequencing data at a fraction of the cost of whole genome sequencing.

genomics↗