DNA-seq of 5fC enriched fragments based
Ontology highlight
ABSTRACT:
INSTRUMENT(S): NextSeq 500
ORGANISM(S): Mus musculus
SUBMITTER: Guillem Portella
PROVIDER: E-MTAB-7307 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:5-methylcytosine (5mC), the predominant epigenetic modification on DNA, plays critical roles in mammalian development and is dysregulated in various human pathologies. In mammals, the TET family of dioxygenases can oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) in a stepwise manner. 5fC and 5caC are selectively recognized and excised by mammalian thymine DNA glycosylase (TDG), and restored to normal cytosine through base excision repair (BER). Once 5mC/5hmC is converted to 5fC and/or 5caC, the modified cytosine is committed to demethylation through BER. Thus 5fC and 5caC most likely mark active demethylation in the mammalian genome. Here we introduce a genome-wide approach to obtain single-base resolution maps of 5fC and 5caC, respectively. We show that, in mouse embryonic stem cells (mESCs), 5fC and 5caC are preferentially generated at highly hypomethylated regions and more active enhancers. Moreover, 5caC-marked regions are characterized by the lowest methylation and highest enhancer activity among all modification sites associated with 5hmC, 5fC and 5caC, and are enriched adjacent to pluripotency transcription factor (TF)-binding motifs. These observations, together with the surprising lack of overlap between 5fC and 5caC sites, highlight a gradient of Tet-mediated 5mC oxidation activity at regulatory elements in tuning epigenetic dynamics11. DNA immunoprecipitation coupled chemical-modification assisted bisulfite sequencing (DIP-CAB-Seq) for Tdg fl/fl and Tdg-/- mESCs
2015-01-20 | E-GEOD-56429 | biostudies-arrayexpress
Project description:Methylation of cytosine in DNA (5mC) is an important epigenetic mark that is involved in the regulation of genome function. During early embryonic development in mammals, the DNA methylation landscape is dynamically reprogrammed in part through active demethylation. Recent advances have identified key players involved in active demethylation pathways, including oxidation of 5mC to 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) by the TET family of enzymes and excision of 5fC by the base excision repair enzyme thymine DNA glycosylase (TDG). Here, we provide the first genome-wide distribution map of 5fC in mouse embryonic stem (ES) cells and evaluate potential roles for 5fC in differentiation. Our method exploits the unique reactivity of 5fC to link a biotin tag for pulldown and high-throughput sequencing. Genome-wide mapping revealed 5fC enrichment in CpG islands (CGIs) of promoters and exons. CGI promoters in which 5fC was relatively more enriched than 5mC or 5hmC corresponded to transcriptionally active genes. Accordingly, 5fC-rich promoters had elevated H3K4me3 levels, a histone mark associated with active transcription, and were frequently bound by RNA Polymerase II. Downregulation of TDG led to accumulation of 5fC in CGIs in ES cells, which correlates with increased methylation in these genomic regions during differentiation and in mouse embryonic fibroblasts derived from TDG knockout embryos. Collectively, our data suggest that 5fC plays a role in epigenetic reprogramming. The formation and removal of this cytosine modification are confined to specific genomic regions, which are in part controlled by TDG. Notably, 5fC excision in ES cells is necessary for the correct establishment of CGI methylation patterns during differentiation, and hence, for appropriate patterns of gene expression during development. We devised a method to map 5-formylcytosine (5fC) by linking a biotin tag to 5fC for pulldown and high-throughput sequencing. We mapped 5fC in the following samples of mouse embryonic stem cells (J1): Wild-type ES cells (two replicates); ES cells transfected with siRNA targeting TDG (two replicates); ES cells transfected with non-targeting siRNA (two replicates). One genomic input library was also sequenced to detect and correct biases in fragment enrichment.
2012-08-17 | E-GEOD-40148 | biostudies-arrayexpress
Project description:5'-complete cDNA sequencing on ribosome-depleted total RNA from the human K562 cell line. Provides high-quality, genome-wide single-base resolution profiling of transcription start sites and their expression levels. This dataset represents a whole-genome, single-base resolution profiling of transcription start site (TSS) expression in the human K562 cell line. These profiles were established using RAMPAGE, a high-throughput, high-accuracy 5'-complete cDNA sequencing method implemented on the Illumina platform. The data was analyzed using custom scripts and algorithms that are all available upon request.
2012-08-30 | E-GEOD-36200 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the following subset Series: GSE36200: RAMPAGE dataset for the human K562 cell line GSE36212: Promoter activity profiling throughout the Drosophila life cycle reveals role of transposons in regulatory innovation Refer to individual Series
2012-08-29 | E-GEOD-36213 | biostudies-arrayexpress
Project description:Organoid technology provides the possibility to culture human colontissue and patient-derived colorectal cancers (CRC) while maintainingall functional and phenotypic characteristics. Labeling of human colonstem cells (CoSCs), especially in normal and benign tumor organoids, ischallenging and therefore limits usability of multi-patient organoidlibraries for CoSC research. Here, we developed STAR (STem cell Ascl2Reporter), a minimal enhancer/promoter element that reportstranscriptional activity of ASCL2, a master regulator of LGR5+ CoSCfate. Among others via lentiviral infection, STAR minigene labels stemcells in normal as well as in multiple engineered and patient-derivedCRC organoids of different stage and genetic make-up. STAR revealed thatstem cell driven differentiation hierarchies and the capacity of cellfate plasticity (de-differentiation) are present at all stages of humanCRC development. The flexible and user-friendly nature of STARapplications in combination with organoid technology will facilitatebasic research on human adult stem cell biology.
2018-01-29 | PXD006575 | Pride
Project description:Since their discovery, transposable elements have been proposed to play a central role in the evolution of their host genomes through their ability to regulate gene expression, in particular by providing transcription start sites (TSSs) for host genes. To investigate their contribution to developmental gene expression, we developed RAMPAGE, a high-throughput 5'-complete cDNA sequencing approach to accurately discover TSSs, characterize their transcripts, and quantify their expression. This strategy, which directly delineates the expression profiles of individual promoters and was designed to offer optimal sample multiplexing capabilities, represents an advantageous alternative to standard RNA-Seq for a wide range of transcriptome profiling applications. We used RAMPAGE in a genome-wide study of promoter activity throughout 36 stages of the life cycle of Drosophila melanogaster, and describe here a comprehensive dataset that represents the first developmental timecourse of promoter usage. We found that over 40% of developmentally expressed genes have at least 2 promoters, and that alternative promoters generally implement distinct regulatory programs. Transposons harbor TSSs driving the expression of hundreds of annotated genes, and they often impart their own expression specificity upon the genes they regulate. Detailed analysis of particular transposons identified sequence elements encoding these regulatory properties. Our results show that transposable elements contribute significantly to the generation of standing variation and to the evolution of gene regulatory networks, by distributing stereotyped regulatory modules throughout the genome. This dataset represents a whole-genome, single-base resolution profiling of transcription start site (TSS) expression throughout 36 stages of the life cycle of Drosophila melanogaster. These profiles were established using RAMPAGE, a high-throughput, high-accuracy 5'-complete cDNA sequencing method implemented on the Illumina platform. Embryos, larvae, pupae and adult flies were collected at specific stages of development, and RAMPAGE profiles were established for pools of whole organisms. The data was analyzed using custom scripts and algorithms that are all available upon request. Supplementary files: Dmel_Combined_+.bw: bigWig coverage by cDNA 5' ends (+ strand). Dmel_Combined_-.bw: bigWig coverage by cDNA 5' ends (- strand). Dmel_All_RAMPAGE_peaks.bed: BED file describing all RAMPAGE peaks. Dmel_GeneTSS_RAMPAGE_peaks.bed: BED file describing all peaks attributed to annotated genes. GeneTSS_expression_RAMPAGE_RPM.txt: Expression matrix for all genic peaks (RPM: reads per million). Transposon_expression_RAMPAGE_RPM.txt: Expression matrix for all RepeatMasker-annotated transposon classes (RPM: reads per million). Genome build: dm3
2012-08-30 | E-GEOD-36212 | biostudies-arrayexpress
Project description:Active DNA demethylation in mammals involves TET-mediated oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC). However, genome-wide detection of 5fC at single-base resolution remains challenging. Here we present a bisulfite-free method for the whole-genome analysis of 5fC, based on a selective chemical labeling of 5fC and subsequent C-to-T transition during PCR. Base-resolution 5fC maps reveal limited overlap with 5hmC, with 5fC-marked regions more active than 5hmC-marked ones. Utilization of cyclization-enabled C-to-T transition of 5fC (hence “fC-CET”) to obtain genome-wide map of 5fC at single-base resolution WT and TdgKO mES cell lines. Two non-enriched input DNAs (Input: preAI), two AI labeled samples (Input: AI), two pull-down output samples.
2015-09-07 | E-GEOD-66144 | biostudies-arrayexpress
Project description:TET proteins oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5fC and 5caC are excised by mammalian DNA glycosylase TDG, implicating 5mC oxidation in DNA demethylation. Here we show that the genomic locations of 5fC can be determined by coupling chemical reduction with biotin tagging. Genome-wide mapping of 5fC in mouse embryonic stem cells (mESCs) reveals that 5fC preferentially occurs at poised enhancers among other gene regulatory elements. Application to Tdg null mESCs further suggests that 5fC production coordinates with p300 in remodeling epigenetic states of enhancers. This process, which is not influenced by 5hmC, appears to be associated with further oxidation of 5hmC and commitment to demethylation through 5fC. Finally, we resolved 5fC at base-resolution by hydroxylamine-based protection from bisulfite-mediated deamination, thereby confirming sites of 5fC accumulation. Our results reveal roles of active 5mC/5hmC oxidation and TDG-mediated demethylation in epigenetic tuning at regulatory elements. We report here a chemical labeling method that effectively differentiates 5fC from 5mC, 5hmC, and 5caC in genomic DNA. First, we quantitatively protect endogenous 5hmC with a regular glucose using b-glucosytransferase-catalyzed 5hmC glucosylation. Then, we selectively reduce 5fC with NaBH4 to 5hmC, and chemically label the resulting 5hmC (from 5fC) with an azide-modified glucose. Biotin can be installed subsequently for specific enrichment of 5fC. Our method thereby provides an effective tool of general utility for the genomic localization of 5fC. Here we provide genome-wide profiles of 5hmC, 5fC, and p300 in Tdg fl/fl and Tdg-/- mESCs as well as a 5fC control (Non-NaBH4) and polyA RNA-Seq expression data. Genome-wide profiles of 5hmC and 5fC in mESCs differentiated to embryoid bodies are also included. We also report the development and application of a single-base resolution method for the detection of 5fC in genomic DNA by hydroylamine mediated protection of 5fC from deamination during bisulfite treatment, or 5fC Chemical Assisted Bisulfite Sequencing (fCAB-Seq). We applied this method in parallel with conventional ChIP-Methyl-Seq to H3K4me1 ChIP enriched DNA from Tdg fl/fl and Tdg-/- mice.
2013-04-18 | E-GEOD-41545 | biostudies-arrayexpress
Project description:The discovery of TET proteins, enzymes that oxidize 5-methylcytosine (5mC) in DNA, has revealed novel mechanisms for the regulation of DNA methylation. We have mapped 5-hydroxymethylcytosine (5hmC) at different stages of T cell development in the thymus and T cell differentiation in the periphery. We show that 5hmC is enriched in the gene body of highly expressed genes at all developmental stages, and that its presence correlates positively with gene expression. Further emphasizing the connection with gene expression, we find that 5hmC is enriched in active thymus-specific enhancers, and that genes encoding key transcriptional regulators display high intragenic 5hmC levels in precursor cells at those developmental stages where they exert a positive effect. Our data constitute a valuable resource that will facilitate detailed analysis of the role of 5hmC in T cell development and differentiation. Map 5hmC in DP T cells and ES cells
2014-08-04 | E-GEOD-59122 | biostudies-arrayexpress
Project description:Gene expression is driven by the binding of transcription factors to regulatory elements in the genome, such as enhancers and promoters. A powerful technique to study DNA-protein interactions is affinity purification followed by mass spectrometry. Classic affinity purifications coupled to quantitative mass spectrometry provide information about binding specificity. Binding of transcription factors to regulatory elements in vivo, however, also depends on binding affinity, so the strength of an interaction. To obtain this information, we recently developed a technique called PAQMAN that uses a series of DNA affinity purifications to quantify apparent binding affinities proteome-wide. Here, we expand our PAQMAN workflow to obtain information about binding specificity and affinity in a single experiment. To this end, we combine quantitation at the MS1 level with quantitation at the MS2 level, a strategy that is known as higher order multiplexing. This is, to our knowledge, the first time that higher order multiplexing is applied to affinity purification - mass spectrometry experiments. In the future, we anticipate that this new workflow will be a useful tool to investigate transcription factor biology.
2023-10-08 | PXD041674 | Pride