Mapping epigenetic modifications by sequencing technologies.

The "epigenetics" concept was first described in 1942. Thus far, chemical modifications on histones, DNA, and RNA have emerged as three important building blocks of epigenetic modifications. Many epigenetic modifications have been intensively studied and found to be involved in most essential biological processes as well as human diseases, including cancer. Precisely and quantitatively mapping over 100 [1], 17 [2], and 160 [3] different known types of epigenetic modifications in histone, DNA, and RNA is the key to understanding the role of epigenetic modifications in gene regulation in diverse biological processes. With the rapid development of sequencing technologies, scientists are able to detect specific epigenetic modifications with various quantitative, high-resolution, whole-genome/transcriptome approaches. Here, we summarize recent advances in epigenetic modification sequencing technologies, focusing on major histone, DNA, and RNA modifications in mammalian cells.

DMRT1 regulates human germline commitment.

Germline commitment following primordial germ cell (PGC) specification during early human development establishes an epigenetic programme and competence for gametogenesis. Here we follow the progression of nascent PGC-like cells derived from human embryonic stem cells in vitro. We show that switching from BMP signalling for PGC specification to Activin A and retinoic acid resulted in DMRT1 and CDH5 expression, the indicators of migratory PGCs in vivo. Moreover, the induction of DMRT1 and SOX17 in PGC-like cells promoted epigenetic resetting with striking global enrichment of 5-hydroxymethylcytosine and locus-specific loss of 5-methylcytosine at DMRT1 binding sites and the expression of DAZL representing DNA methylation-sensitive genes, a hallmark of the germline commitment programme. We provide insight into the unique role of DMRT1 in germline development for advances in human germ cell biology and in vitro gametogenesis.

Modular Oxidation of Cytosine Modifications and Their Application in Direct and Quantitative Sequencing of 5-Hydroxymethylcytosine.

Selective, efficient, and controllable oxidation of cytosine modifications is valuable for epigenetic analyses, yet only limited progress has been made. Here, we present two modular chemical oxidation reactions: conversion of 5-hydroxymethylcytosine (5hmC) into 5-formylcytosine (5fC) using 4-acetamido-2,2,6,6-tetramethylpiperidine-1-oxoammonium tetrafluoroborate (ACT+BF4-) and further transformation of 5fC into 5-carboxycytosine (5caC) through Pinnick oxidation. Both reactions are mild and efficient on double-stranded DNA. We integrated these two oxidations with borane reduction to develop chemical-assisted pyridine borane sequencing plus (CAPS+), for direct and quantitative mapping of 5hmC. Compared with CAPS, CAPS+ improved the conversion rate and false-positive rate. We applied CAPS+ to mouse embryonic stem cells, human normal brain, and glioblastoma DNA samples and demonstrated its superior sensitivity in analyzing the hydroxymethylome.

Sarcoidosis with the livedo reticularis-like appearance.

Sarcoidosis is a multisystem inflammatory disease manifesting as noncaseating epithelioid cell granulomas. 25 to 30% of individuals with systemic sarcoidosis show variable cutaneous manifestations. A 59-year-old female was seen with reticular purplish-red nodules and plaques on the legs for three months. A skin biopsy of the livedo area revealed non-caseating epithelioid cell granulomas surrounding blood vessels in the dermis. She was diagnosed with sarcoidosis livedo, and cutaneous lesions subsided with oral prednisone. Sarcoidosis livedo (SL) assumes a uncommon livedo reticularis-like presentation. This is the first Chinese patient with SL, and more patients are needed to unveil the unique characters of SL.

Whole-genome long-read TAPS deciphers DNA methylation patterns at base resolution using PacBio SMRT sequencing technology.

Long-read sequencing provides valuable information on difficult-to-map genomic regions, which can complement short-read sequencing to improve genome assembly, yet limited methods are available to accurately detect DNA methylation over long distances at a whole-genome scale. By combining our recently developed TET-assisted pyridine borane sequencing (TAPS) method, which enables direct detection of 5-methylcytosine and 5-hydroxymethylcytosine, with PacBio single-molecule real-time sequencing, we present here whole-genome long-read TAPS (wglrTAPS). To evaluate the performance of wglrTAPS, we applied it to mouse embryonic stem cells as a proof of concept, and an N50 read length of 3.5 kb is achieved. By sequencing wglrTAPS to 8.2× depth, we discovered a significant proportion of CpG sites that were not covered in previous 27.5× short-read TAPS. Our results demonstrate that wglrTAPS facilitates methylation profiling on problematic genomic regions with repetitive elements or structural variations, and also in an allelic manner, all of which are extremely difficult for short-read sequencing methods to resolve. This method therefore enhances applications of third-generation sequencing technologies for DNA epigenetics.

Cell-free DNA TAPS provides multimodal information for early cancer detection.

Multimodal, genome-wide characterization of epigenetic and genetic information in circulating cell-free DNA (cfDNA) could enable more sensitive early cancer detection, but it is technologically challenging. Recently, we developed TET-assisted pyridine borane sequencing (TAPS), which is a mild, bisulfite-free method for base-resolution direct DNA methylation sequencing. Here, we optimized TAPS for cfDNA (cfTAPS) to provide high-quality and high-depth whole-genome cell-free methylomes. We applied cfTAPS to 85 cfDNA samples from patients with hepatocellular carcinoma (HCC) or pancreatic ductal adenocarcinoma (PDAC) and noncancer controls. From only 10 ng of cfDNA (1 to 3 ml of plasma), we generated the most comprehensive cfDNA methylome to date. We demonstrated that cfTAPS provides multimodal information about cfDNA characteristics, including DNA methylation, tissue of origin, and DNA fragmentation. Integrated analysis of these epigenetic and genetic features enables accurate identification of early HCC and PDAC.

Endonuclease enrichment TAPS for cost-effective genome-wide base-resolution DNA methylation detection.

Whole genome base-resolution methylome sequencing allows for the most comprehensive analysis of DNA methylation, however, the considerable sequencing cost often limits its applications. While reduced representation sequencing can be an affordable alternative, over 80% of CpGs in the genome are not covered. Building on our recently developed TET-assisted pyridine borane sequencing (TAPS) method, we here described endonuclease enrichment TAPS (eeTAPS), which utilizes dihydrouracil (DHU)-cleaving endonuclease digestion of TAPS-converted DNA to enrich methylated CpG sites (mCpGs). eeTAPS can accurately detect 87% of mCpGs in the mouse genome with a sequencing depth equivalent to 4× whole genome sequencing. In comparison, reduced representation TAPS (rrTAPS) detected less than 4% of mCpGs with 2.5× sequencing depth. Our results demonstrate eeTAPS to be a new strategy for cost-effective genome-wide methylation analysis at single-CpG resolution that can fill the gap between whole-genome and reduced representation sequencing.

Subtraction-free and bisulfite-free specific sequencing of 5-methylcytosine and its oxidized derivatives at base resolution.

Although various methods have been developed for sequencing cytosine modifications, it is still challenging for specific and quantitative sequencing of individual modification at base-resolution. For example, to obtain both true 5-methylcytosine (5mC) and true 5-hydroxymethylcytosine (5hmC) information, the two major epigenetic modifications, it usually requires subtraction of two methods, which increases noise and requires high sequencing depth. Recently, we developed TET-assisted pyridine borane sequencing (TAPS) for bisulfite-free direct sequencing of 5mC and 5hmC. Here we demonstrate that two sister methods, TAPSß and chemical-assisted pyridine borane sequencing (CAPS), can be effectively used for subtraction-free and specific whole-genome sequencing of 5mC and 5hmC, respectively. We also demonstrate pyridine borane sequencing (PS) for whole-genome profiling of 5-formylcytosine and 5-carboxylcytosine, the further oxidized derivatives of 5mC and 5hmC. This work completes the set of versatile borane reduction chemistry-based methods as a comprehensive toolkit for direct and quantitative sequencing of all four cytosine epigenetic modifications.

Mapping the epigenetic modifications of DNA and RNA.

Over 17 and 160 types of chemical modifications have been identified in DNA and RNA, respectively. The interest in understanding the various biological functions of DNA and RNA modifications has lead to the cutting-edged fields of epigenomics and epitranscriptomics. Developing chemical and biological tools to detect specific modifications in the genome or transcriptome has greatly facilitated their study. Here, we review the recent technological advances in this rapidly evolving field. We focus on high-throughput detection methods and biological findings for these modifications, and discuss questions to be addressed as well. We also summarize third-generation sequencing methods, which enable long-read and single-molecule sequencing of DNA and RNA modification.

Accurate targeted long-read DNA methylation and hydroxymethylation sequencing with TAPS.

We present long-read Tet-assisted pyridine borane sequencing (lrTAPS) for targeted base-resolution sequencing of DNA methylation and hydroxymethylation in regions up to 10 kb from nanogram-level input. Compatible with both Oxford Nanopore and PacBio Single-Molecule Real-Time (SMRT) sequencing, lrTAPS detects methylation with accuracy comparable to short-read Illumina sequencing but with long-range epigenetic phasing. We applied lrTAPS to sequence difficult-to-map regions in mouse embryonic stem cells and to identify distinct methylation events in the integrated hepatitis B virus genome.

Systematic allelic analysis defines the interplay of key pathways in X chromosome inactivation.

Xist RNA, the master regulator of X chromosome inactivation, acts in cis to induce chromosome-wide silencing. Whilst recent studies have defined candidate silencing factors, their relative contribution to repressing different genes, and their relationship with one another is poorly understood. Here we describe a systematic analysis of Xist-mediated allelic silencing in mouse embryonic stem cell-based models. Using a machine learning approach we identify distance to the Xist locus and prior gene expression levels as key determinants of silencing efficiency. We go on to show that Spen, recruited through the Xist A-repeat, plays a central role, being critical for silencing of all except a subset of weakly expressed genes. Polycomb, recruited through the Xist B/C-repeat, also plays a key role, favouring silencing of genes with pre-existing H3K27me3 chromatin. LBR and the Rbm15/m6A-methyltransferase complex make only minor contributions to gene silencing. Together our results provide a comprehensive model for Xist-mediated chromosome silencing.

Bisulfite-free direct detection of 5-methylcytosine and 5-hydroxymethylcytosine at base resolution.

Bisulfite sequencing has been the gold standard for mapping DNA modifications including 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) for decades1-4. However, this harsh chemical treatment degrades the majority of the DNA and generates sequencing libraries with low complexity2,5,6. Here, we present a bisulfite-free and base-level-resolution sequencing method, TET-assisted pyridine borane sequencing (TAPS), for detection of 5mC and 5hmC. TAPS combines ten-eleven translocation (TET) oxidation of 5mC and 5hmC to 5-carboxylcytosine (5caC) with pyridine borane reduction of 5caC to dihydrouracil (DHU). Subsequent PCR converts DHU to thymine, enabling a C-to-T transition of 5mC and 5hmC. TAPS detects modifications directly with high sensitivity and specificity, without affecting unmodified cytosines. This method is nondestructive, preserving DNA fragments over 10 kilobases long. We applied TAPS to the whole-genome mapping of 5mC and 5hmC in mouse embryonic stem cells and show that, compared with bisulfite sequencing, TAPS results in higher mapping rates, more even coverage and lower sequencing costs, thus enabling higher quality, more comprehensive and cheaper methylome analyses.

Bisulfite-free and base-resolution analysis of 5-methylcytidine and 5-hydroxymethylcytidine in RNA with peroxotungstate.

5-Methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), two of the best-studied DNA modifications, play crucial roles in normal development and disease in mammals. Although 5-methylcytidine (m5C) and 5-hydroxymethylcytidine (hm5C) have also been identified in RNA, their distribution and biological function in RNA remain largely unexplored, due to the lack of suitable sequencing methods. Here, we report a base-resolution sequencing method for hm5C in RNA. We applied the selective oxidation of hm5C to trihydroxylated-thymine (thT) mediated by peroxotungstate. thT was subsequently converted to T during cDNA synthesis using a thermostable group II intron reverse transcriptase (TGIRT). Base-resolution analysis of the hm5C sites in RNA was performed using Sanger sequencing. Furthermore, in combination with the TET enzyme oxidation of m5C to hm5C in RNA, we expand the use of peroxotungstate oxidation to detect m5C in RNA at base-resolution. By using this method, we confirmed three known m5C sites in human tRNA, demonstrating the applicability of our method in analyzing real RNA samples.

5-Carboxylcytosine is resistant towards phosphodiesterase I digestion: implications for epigenetic modification quantification by mass spectrometry.

DNA cytosine modifications are important epigenetic modifications in gene regulation and pathogenesis. DNA hydrolysis followed by HPLC-MS/MS is the gold standard in DNA modification quantification. In particular, it is the only sensitive and accurate method for low abundance modifications, such as 5-carboxylcytosine (5caC). Here, we report the discovery of the nuclease resistance property of 5caC to snake venom phosphodiesterase I (PDE1), a 3' to 5' exonuclease commonly used in several DNA hydrolysis protocols. We conducted a systematic evaluation of six commonly used hydrolysis protocols and found that all protocols that use PDE1 underestimate the level of 5caC. Finally, we identified the best method for cytosine modification quantification of biological samples, which leads to an over 10-fold higher amount of 5caC being detected compared with other methods. Our results highlight that caution should be taken when choosing a DNA hydrolysis protocol to quantify certain DNA modifications.

Glucose and glutamine availability regulate HepG2 transcriptional responses to low oxygen.

Background: Little is known about the impact of nutrients on cellular transcriptional responses, especially in face of environmental stressors such as oxygen deprivation. Hypoxia-inducible factors (HIF) coordinate the expression of genes essential for adaptation to oxygen-deprived environments. A second family of oxygen-sensing genes known as the alpha-ketoglutarate-dependent dioxygenases are also implicated in oxygen homeostasis and epigenetic regulation. The relationship between nutritional status and cellular response to hypoxia is understudied. In vitro cell culture systems frequently propagate cells in media that contains excess nutrients, and this may directly influence transcriptional response in hypoxia. Methods: We studied the effect of glucose and glutamine concentration on HepG2 hepatoma transcriptional response to low oxygen and expression of hypoxia inducible factor-1α (HIF-1α). Mass spectrometry confirmed low oxygen perturbation of dioxygenase transcripts resulted in changes in DNA methylation. Results: Under normoxic conditions, we observed a significant upregulation of both HIF-target genes and oxygen-dependent dioxygenases in HepG2 cells cultured with physiological levels of glucose or glutamine relative to regular DMEM media, demonstrating that excess glutamine/glucose can mask changes in gene expression. Under hypoxic conditions, CA9 was the most upregulated gene in physiological glutamine media while TETs and FTO dioxygenases were downregulated in physiological glucose. Hypoxic regulation of these transcripts did not associate with changes in HIF-1α protein expression. Downregulation of TETs suggests a potential for epigenetic modulation. Mass-spectrometry quantification of modified DNA bases confirmed our transcript data. Hypoxia resulted in decreased DNA hydroxymethylation, which correlated with TETs downregulation. Additionally, we observed that TET2 expression was significantly downregulated in patients with hepatocellular carcinoma, suggesting that tumour hypoxia may deregulate TET2 expression resulting in global changes in DNA hydroxymethylation.   Conclusion: Given the dramatic effects of nutrient availability on gene expression, future in vitro experiments should be aware of how excess levels of glutamine and glucose may perturb transcriptional responses.

Simultaneously Monitoring Immune Response and Microbial Infections during Pregnancy through Plasma cfRNA Sequencing.

BACKGROUND: Plasma cell-free RNA (cfRNA) encompasses a broad spectrum of RNA species that can be derived from both human cells and microbes. Because cfRNA is fragmented and of low concentration, it has been challenging to profile its transcriptome using standard RNA-seq methods. METHODS: We assessed several recently developed RNA-seq methods on cfRNA samples. We then analyzed the dynamic changes of both the human transcriptome and the microbiome of plasma during pregnancy from 60 women. RESULTS: cfRNA reflects a well-orchestrated immune modulation during pregnancy: an up-regulation of antiinflammatory genes and an increased abundance of antimicrobial genes. We observed that the plasma microbiome remained relatively stable during pregnancy. The bacteria Ureaplasma shows an increased prevalence and increased abundance at postpartum, which is likely to be associated with postpartum infection. We demonstrated that cfRNA-seq can be used to monitor viral infections. We detected a number of human pathogens in our patients, including an undiagnosed patient with a high load of human parvovirus B19 virus (B19V), which is known to be a potential cause of complications in pregnancy. CONCLUSIONS: Plasma cfRNA-seq demonstrates the potential to simultaneously monitor immune response and microbial infections during pregnancy.

5-Hydroxymethylcytosine signatures in cell-free DNA provide information about tumor types and stages.

5-Hydroxymethylcytosine (5hmC) is an important mammalian DNA epigenetic modification that has been linked to gene regulation and cancer pathogenesis. Here we explored the diagnostic potential of 5hmC in circulating cell-free DNA (cfDNA) using a sensitive chemical labeling-based low-input shotgun sequencing approach. We sequenced cell-free 5hmC from 49 patients of seven different cancer types and found distinct features that could be used to predict cancer types and stages with high accuracy. Specifically, we discovered that lung cancer leads to a progressive global loss of 5hmC in cfDNA, whereas hepatocellular carcinoma and pancreatic cancer lead to disease-specific changes in the cell-free hydroxymethylome. Our proof-of-principle results suggest that cell-free 5hmC signatures may potentially be used not only to identify cancer types but also to track tumor stage in some cancers.

Quantitation and mapping of the epigenetic marker 5-hydroxymethylcytosine.

We here review primary methods used in quantifying and mapping 5-hydroxymethylcytosine (5hmC), including global quantification, restriction enzyme-based detection, and methods involving DNA-enrichment strategies and the genome-wide sequencing of 5hmC. As discovered in the mammalian genome in 2009, 5hmC, oxidized from 5-methylcytosine (5mC) by ten-eleven translocation (TET) dioxygenases, is increasingly being recognized as a biomarker in biological processes from development to pathogenesis, as its various detection methods have shown. We focus in particular on an ultrasensitive single-molecule imaging technique that can detect and quantify 5hmC from trace samples and thus offer information regarding the distance-based relationship between 5hmC and 5mC when used in combination with fluorescence resonance energy transfer.