Project description:Senescence, a state of permanent cell cycle arrest, can be induced by DNA damage. This process, which was initially described in fibroblasts, is now recognized to occur in stem cells. It has been well characterized in cell lines, but there is currently very limited data available on human senescence in vivo. We recently reported that the expression of transposable elements (TE), including endogenous retroviruses, was up-regulated along with inflammatory genes in human senescent hematopoietic stem and progenitor cells (HSPCs) in vivo. The mechanism of regulation of TE expression is not completely understood, but changes in DNA methylation and chromatin modifications are known to alter their expression. In order to elucidate the molecular mechanisms for TE up-regulation after senescence of HSPCs, we employed whole-genome bisulfite sequencing in paired senescent and active human HSPCs in vivo from healthy subjects. We found that the senescent HSPCs exhibited hypomethylated regions in the genome, which were enriched for TEs. This is the first report characterizing the methylome of senescent human HSPCs.
Project description:Hematopoietic stem cells (HSC) produce all blood cell lineages by virtue of their capacity to self-renew and differentiate into progenitors with decreasing cellular potential. Recent studies suggest that epigenetic mechanisms play an important role in controlling stem cell potency and cell fate decisions. To investigate this hypothesis in HSC, we have modified the conventional chromatin immunoprecipitation assay allowing for the analysis of 50,000 prospectively purified stem and progenitor cells. Together with bisulfite sequencing analysis, we found that methylated H3K4 and AcH3 and unmethylated CpG dinucleotides colocalize across defined regulatory regions of lineage-affiliated genes in HSC. These active epigenetic histone modifications either accumulated or were replaced by increased DNA methylation and H3K27 trimethylation in committed progenitors consistent with gene expression. We also observed bivalent histone modifications at a lymphoid-affiliated gene in HSC and downstream transit-amplifying progenitors. Together, these data support a model in which epigenetic modifications serve as an important mechanism to control HSC multipotency.
Project description:DNA methylation is an important epigenetic mark relevant to normal development and disease genesis. A common approach to characterizing genome-wide DNA methylation is using Next Generation Sequencing technology to sequence bisulfite treated DNA. The short sequence reads are mapped to the reference genome to determine the methylation statuses of Cs. However, despite intense effort, a much smaller proportion of the reads derived from bisulfite treated DNA (usually about 40-80%) can be mapped than regular short reads mapping (> 90%), and it is unclear what factors lead to this low mapping efficiency.To address this issue, we used the hairpin bisulfite sequencing technology to determine sequences of both DNA double strands simultaneously. This enabled the recovery of the original non-bisulfite-converted sequences. We used Bismark for bisulfite read mapping and Bowtie2 for recovered read mapping. We found that recovering the reads improved unique mapping efficiency by 9-10% compared to the bisulfite reads. Such improvement in mapping efficiency is related to sequence entropy.The hairpin recovery technique improves mapping efficiency, and sequence entropy relates to mapping efficiency.
Project description:The existence of cytosine methylation in mammalian mitochondrial DNA (mtDNA) is a controversial subject. Because detection of DNA methylation depends on resistance of 5'-modified cytosines to bisulfite-catalyzed conversion to uracil, examined parameters that affect technical adequacy of mtDNA methylation analysis. Negative control amplicons (NCAs) devoid of cytosine methylation were amplified to cover the entire human or mouse mtDNA by long-range PCR. When the pyrosequencing template amplicons were gel-purified after bisulfite conversion, bisulfite pyrosequencing of NCAs did not detect significant levels of bisulfite-resistant cytosines (brCs) at ND1 (7 CpG sites) or CYTB (8 CpG sites) genes (CI95 = 0%-0.94%); without gel-purification, significant false-positive brCs were detected from NCAs (CI95 = 4.2%-6.8%). Bisulfite pyrosequencing of highly purified, linearized mtDNA isolated from human iPS cells or mouse liver detected significant brCs (~30%) in human ND1 gene when the sequencing primer was not selective in bisulfite-converted and unconverted templates. However, repeated experiments using a sequencing primer selective in bisulfite-converted templates almost completely (< 0.8%) suppressed brC detection, supporting the false-positive nature of brCs detected using the non-selective primer. Bisulfite-seq deep sequencing of linearized, gel-purified human mtDNA detected 9.4%-14.8% brCs for 9 CpG sites in ND1 gene. However, because all these brCs were associated with adjacent non-CpG brCs showing the same degrees of bisulfite resistance, DNA methylation in this mtDNA-encoded gene was not confirmed. Without linearization, data generated by bisulfite pyrosequencing or deep sequencing of purified mtDNA templates did not pass the quality control criteria. Shotgun bisulfite sequencing of human mtDNA detected extremely low levels of CpG methylation (<0.65%) over non-CpG methylation (<0.55%). Taken together, our study demonstrates that adequacy of mtDNA methylation analysis using methods dependent on bisulfite conversion needs to be established for each experiment, taking effects of incomplete bisulfite conversion and template impurity or topology into consideration.
Project description:DNA methylation plays a key role in epigenetic regulation of eukaryotic genomes. Hence the genome-wide distribution of 5-methylcytosine, or the methylome, has been attracting intense attention. In recent years, whole-genome bisulfite sequencing (WGBS) has enabled methylome analysis at single-base resolution. However, WGBS typically requires microgram quantities of DNA as well as global PCR amplification, thereby precluding its application to samples of limited amounts. This is presumably because bisulfite treatment of adaptor-tagged templates, which is inherent to current WGBS methods, leads to substantial DNA fragmentation. To circumvent the bisulfite-induced loss of intact sequencing templates, we conceived an alternative method termed Post-Bisulfite Adaptor Tagging (PBAT) wherein bisulfite treatment precedes adaptor tagging by two rounds of random primer extension. The PBAT method can generate a substantial number of unamplified reads from as little as subnanogram quantities of DNA. It requires only 100 ng of DNA for amplification-free WGBS of mammalian genomes. Thus, the PBAT method will enable various novel applications that would not otherwise be possible, thereby contributing to the rapidly growing field of epigenomics.
Project description:DNA methylation is an epigenetic modification important for cell fate determination and cell type-specific gene expression. Transcriptional regulatory regions of the mammalian genome contain a large number of tissue/cell type-dependent differentially methylated regions (T-DMRs) with DNA methylation patterns crucial for transcription of the corresponding genes. In general, tissues consist of multiple cell types in various proportions, making it difficult to detect T-DMRs of minor cell types in tissues. The present study attempts to detect T-DMRs of minor cell types in tissues by ultra-deep bisulfite sequencing of cell type-restricted genes and to assume proportions of minor cell types based on DNA methylation patterns of sequenced reads. For this purpose, we focused on transcriptionally active hypomethylated alleles (Hypo-alleles), which can be recognized by the high ratio of unmethylated CpGs in each sequenced read (allele). The pituitary gland contains multiple cell types including five hormone-expressing cell types and stem/progenitor cells, each of which is a minor cell type in the pituitary tissue. By ultra-deep sequencing of more than 100 reads for detection of Hypo-alleles in pituitary cell type-specific genes, we identified T-DMRs specific to hormone-expressing cells and stem/progenitor cells and used them to estimate the proportions of each cell type based on the Hypo-allele ratio in pituitary tissue. Therefore, introduction of the novel Hypo-allele concept enabled us to detect T-DMRs of minor cell types with estimation of their proportions in the tissue by ultra-deep bisulfite sequencing.
Project description:Bisulfite sequencing is a key methodology in epigenetics. However, the standard workflow of bisulfite sequencing involves heat and strongly basic conditions to convert the intermediary product 5,6-dihydrouridine-6-sulfonate (dhU6S) (generated by reaction of bisulfite with deoxycytidine (dC)) to uracil (dU). These harsh conditions generally lead to sample loss and DNA damage while milder conditions may result in incomplete conversion of intermediates to uracil. Both can lead to poor recovery of bisulfite-treated DNA by the polymerase chain reaction (PCR) as either damaged DNA and/or intermediates of bisulfite treatment are poor substrate for standard DNA polymerases. Here we describe an engineered DNA polymerase (5D4) with an enhanced ability to replicate and PCR amplify bisulfite-treated DNA due to an ability to bypass both DNA lesions and bisulfite intermediates, allowing significantly milder conversion conditions and increased sensitivity in the PCR amplification of bisulfite-treated DNA. Incorporation of the 5D4 DNA polymerase into the bisulfite sequencing workflow thus promises significant sensitivity and efficiency gains.
Project description:5-Methylcytosine (5mC) in DNA can be oxidized stepwise to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) by the TET family proteins. Thymine DNA glycosylase can further remove 5fC and 5caC, connecting 5mC oxidation with active DNA demethylation. Here, we present a chemical modification-assisted bisulfite sequencing (CAB-Seq) that can detect 5caC with single-base resolution in DNA. We optimized 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC)-catalyzed amide bond formation between the carboxyl group of 5caC and a primary amine group. We found that the modified 5caC can survive the bisulfite treatment without deamination. Therefore, this chemical labeling coupled with bisulfite treatment provides a base-resolution detection and sequencing method for 5caC.
Project description:We have developed an NGS-based deep bisulfite sequencing protocol for the DNA methylation analysis of genomes. This approach allows the rapid and efficient construction of NGS-ready libraries with a large number of PCR products that have been individually amplified from bisulfite-converted DNA. This approach also employs a bioinformatics strategy to sort the raw sequence reads generated from NGS platforms and subsequently to derive DNA methylation levels for individual loci. The results demonstrated that this NGS-based deep bisulfite sequencing approach provide not only DNA methylation levels but also informative DNA methylation patterns that have not been seen through other existing methods.•This protocol provides an efficient method generating NGS-ready libraries from individually amplified PCR products.•This protocol provides a bioinformatics strategy sorting NGS-derived raw sequence reads.•This protocol provides deep bisulfite sequencing results that can measure DNA methylation levels and patterns of individual loci.