Chemoenzymatic labeling of DNA methylation patterns for single-molecule epigenetic mapping.

DNA methylation, specifically, methylation of cytosine (C) nucleotides at the 5-carbon position (5-mC), is the most studied and significant epigenetic modification. Here we developed a chemoenzymatic procedure to fluorescently label non-methylated cytosines in CpG context, allowing epigenetic profiling of single DNA molecules spanning hundreds of thousands of base pairs. We used a CpG methyltransferase with a synthetic S-adenosyl-l-methionine cofactor analog to transfer an azide to cytosines instead of the natural methyl group. 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 up to 2-fold by the addition of a nucleosidase, presumably by degrading the inactive by-product of the cofactor after labeling, preventing its inhibitory effect. We used the method to determine the decline in global DNA methylation in a chronic lymphocytic leukemia patient and then performed whole-genome methylation mapping of the model plant Arabidopsis thaliana. Our genome maps show high concordance with published bisulfite sequencing methylation maps. Although mapping resolution is limited by optical detection to 500-1000 bp, the labeled DNA molecules produced by this approach are hundreds of thousands of base pairs long, allowing access to long repetitive and structurally variable genomic regions.

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 demethylation 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.

Epigenetic Optical Mapping of 5-Hydroxymethylcytosine in Nanochannel Arrays.

The epigenetic mark 5-hydroxymethylcytosine (5-hmC) is a distinct product of active DNA demethylation that is linked to gene regulation, development, and disease. In particular, 5-hmC levels dramatically decline in many cancers, potentially serving as an epigenetic biomarker. The noise associated with next-generation 5-hmC sequencing hinders reliable analysis of low 5-hmC containing tissues such as blood and malignant tumors. Additionally, genome-wide 5-hmC profiles generated by short-read 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, highly sensitive 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 single-molecule read-length of 100 kbp to 1 Mbp produces 5-hmC profiles across variable genomic regions that failed to show up in the sequencing data. In addition, optical 5-hmC mapping shows a 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.

Selective nanopore sequencing of human BRCA1 by Cas9-assisted targeting of chromosome segments (CATCH).

Next generation sequencing (NGS) is challenged by structural and copy number variations larger than the typical read length of several hundred bases. Third-generation sequencing platforms such as single-molecule real-time (SMRT) and nanopore sequencing provide longer reads and are able to characterize variations that are undetected in NGS data. Nevertheless, these technologies suffer from inherent low throughput which prohibits deep sequencing at reasonable cost without target enrichment. Here, we optimized Cas9-Assisted Targeting of CHromosome segments (CATCH) for nanopore sequencing of the breast cancer gene BRCA1. A 200 kb target containing the 80 kb BRCA1 gene body and its flanking regions was isolated intact from primary human peripheral blood cells, allowing long-range amplification and long-read nanopore sequencing. The target was enriched 237-fold and sequenced at up to 70× coverage on a single flow-cell. Overall performance and single-nucleotide polymorphism (SNP) calling were directly compared to Illumina sequencing of the same enriched sample, highlighting the benefits of CATCH for targeted sequencing. The CATCH enrichment scheme only requires knowledge of the target flanking sequence for Cas9 cleavage while providing contiguous data across both coding and non-coding sequence and holds promise for characterization of complex disease-related or highly variable genomic regions.

One-Pot Chemoenzymatic Cascade for Labeling of the Epigenetic Marker 5-Hydroxymethylcytosine.

The epigenetic DNA modification 5-hydroxymethylcytosine (5-hmC) is important for the regulation of gene expression during development and in tumorigenesis. 5-hmC can be selectively glycosylated by T4 ß-glucosyltransferase (ß-GT); introduction of an azide on the attached sugar provides a chemical handle for isolation or fluorescent tagging of 5-hmC residues by click chemistry. This approach has not been broadly adopted because of the challenging synthesis and limited commercial availability of the glycosylation substrate, 6-deoxy-6-azido-α-D-glucopyranoside. We report the enzyme-assisted synthesis of this precursor by the uridylyltransferase from Pasteurella multocida (PmGlmU). We were able to directly label 5-hmC in genomic DNA by an enzymatic cascade involving successive action of PmGlmU and ß-GT. This is a facile and cost-effective one-pot chemoenzymatic methodology for 5-hmC analysis.