Dataset Information

Jump-seq: Genome-Wide Capture and Amplification of 5-Hydroxymethylcytosine Sites.

ABSTRACT: 5-Hydroxymethylcytosine (5hmC) arises from the oxidation of 5-methylcytosine (5mC) by Fe2+ and 2-oxoglutarate-dependent 10-11 translocation (TET) family proteins. Substantial levels of 5hmC accumulate in many mammalian tissues, especially in neurons and embryonic stem cells, suggesting a potential active role for 5hmC in epigenetic regulation beyond being simply an intermediate of active DNA demethylation. 5mC and 5hmC undergo dynamic changes during embryogenesis, neurogenesis, hematopoietic development, and oncogenesis. While methods have been developed to map 5hmC, more efficient approaches to detect 5hmC at base resolution are still highly desirable. Herein, we present a new method, Jump-seq, to capture and amplify 5hmC in genomic DNA. The principle of this method is to label 5hmC by the 6- N3-glucose moiety and connect a hairpin DNA oligonucleotide carrying an alkyne group to the azide-modified 5hmC via Huisgen cycloaddition (click) chemistry. Primer extension starts from the hairpin motif to the modified 5hmC site and then continues to "land" on genomic DNA. 5hmC sites are inferred from genomic DNA sequences immediately spanning the 5-prime junction. This technology was validated, and its utility in 5hmC identification was confirmed.

SUBMITTER: Hu L

PROVIDER: S-EPMC7061342 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Publications

Jump-seq: Genome-Wide Capture and Amplification of 5-Hydroxymethylcytosine Sites.

Hu Lulu L Hu Lulu L Liu Yuwen Y Han Shengtong S Yang Lei L Cui Xiaolong X Gao Yawei Y Dai Qing Q Lu Xingyu X Kou Xiaochen X Zhao Yanhong Y Sheng Wenhui W Gao Shaorong S He Xin X He Chuan C

Journal of the American Chemical Society 20190523 22

5-Hydroxymethylcytosine (5hmC) arises from the oxidation of 5-methylcytosine (5mC) by Fe<sup>2+</sup> and 2-oxoglutarate-dependent 10-11 translocation (TET) family proteins. Substantial levels of 5hmC accumulate in many mammalian tissues, especially in neurons and embryonic stem cells, suggesting a potential active role for 5hmC in epigenetic regulation beyond being simply an intermediate of active DNA demethylation. 5mC and 5hmC undergo dynamic changes during embryogenesis, neurogenesis, hemato ...[more]

PMID: 31117646

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Project description:ChIP-Seq, which combines chromatin immunoprecipitation (ChIP) with ultra high-throughput massively parallel sequencing, is increasingly being used for mapping protein-DNA interactions in-vivo on a genome scale. Typically, short sequence reads from ChIP-Seq are mapped to a reference genome for further analysis. Although genomic regions enriched with mapped reads could be inferred as approximate binding regions, short read lengths (approximately 25-50 nt) pose challenges for determining the exact binding sites within these regions. Here, we present SISSRs (Site Identification from Short Sequence Reads), a novel algorithm for precise identification of binding sites from short reads generated from ChIP-Seq experiments. The sensitivity and specificity of SISSRs are demonstrated by applying it on ChIP-Seq data for three widely studied and well-characterized human transcription factors: CTCF (CCCTC-binding factor), NRSF (neuron-restrictive silencer factor) and STAT1 (signal transducer and activator of transcription protein 1). We identified 26 814, 5813 and 73 956 binding sites for CTCF, NRSF and STAT1 proteins, respectively, which is 32, 299 and 78% more than that inferred previously for the respective proteins. Motif analysis revealed that an overwhelming majority of the identified binding sites contained the previously established consensus binding sequence for the respective proteins, thus attesting for SISSRs' accuracy. SISSRs' sensitivity and precision facilitated further analyses of ChIP-Seq data revealing interesting insights, which we believe will serve as guidance for designing ChIP-Seq experiments to map in vivo protein-DNA interactions. We also show that tag densities at the binding sites are a good indicator of protein-DNA binding affinity, which could be used to distinguish and characterize strong and weak binding sites. Using tag density as an indicator of DNA-binding affinity, we have identified core residues within the NRSF and CTCF binding sites that are critical for a stronger DNA binding.

Dataset Information

Jump-seq: Genome-Wide Capture and Amplification of 5-Hydroxymethylcytosine Sites.

Publications

Jump-seq: Genome-Wide Capture and Amplification of 5-Hydroxymethylcytosine Sites.

Similar Datasets

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