Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Cellular generation of 5-Hydroxymethylcytosine by redox-active chemicals via an unprecedent non-enzymatic mechanism

ABSTRACT: We proposed that besides TET family dioxygenase oxidizing 5mC to 5hmC, there is a non-enzyme pathway which is due to hydroxyl radica l(OH) or OH-like species also involvement in demethylation of 5mC forming 5hmC. This pathway includes classical fenton reagents such as H2O2 and Fe2+, and more important redox-activity quinoid compounds, especially, tetrachloro-1,4-benzoquinone (TCBQ), which was reported producing hydroxyl radicals independent of transition metal Examination of 5hmC and transcriptome levels with TCBQ and DMSO in human MRC-5 cell lines

ORGANISM(S): Homo sapiens

SUBMITTER: Zechen Chong

PROVIDER: E-GEOD-44457 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Redox-active quinones induces genome-wide DNA methylation changes by an iron-mediated and Tet-dependent mechanism.

Zhao Bailin B Yang Ying Y Wang Xiaoli X Chong Zechen Z Yin Ruichuan R Song Shu-Hui SH Zhao Chao C Li Cuiping C Huang Hua H Sun Bao-Fa BF Wu Danni D Jin Kang-Xuan KX Song Maoyong M Zhu Ben-Zhan BZ Jiang Guibin G Rendtlew Danielsen Jannie M JM Xu Guo-Liang GL Yang Yun-Gui YG Wang Hailin H

Nucleic acids research 20131108 3

DNA methylation has been proven to be a critical epigenetic mark important for various cellular processes. Here, we report that redox-active quinones, a ubiquitous class of chemicals found in natural products, cancer therapeutics and environment, stimulate the conversion of 5 mC to 5 hmC in vivo, and increase 5 hmC in 5751 genes in cells. 5 hmC increase is associated with significantly altered gene expression of 3414 genes. Interestingly, in quinone-treated cells, labile iron-sensitive protein f ...[more]

PMID: 24214992

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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.

Project description:DNA hydroxymethylation (5hmC) represents a new layer of epigenetic regulation in addition to DNA methylation (5mC). The genome-wide patterns of 5hmC distribution in many tissues and cells have recently been revealed by hydroxymethylated DNA immunoprecipitation (hMeDIP) followed by high throughput sequencing or tiling arrays. However, the DNA hydroxymethylome data generated by the conventional hMeDIP-seq method can not be used for direct quantitative comparison across different samples. In this study, we report a new quantitative hMeDIP-seq method, which uses barcode technology to label different DNA samples and performs hMeDIP-seq for multiple samples in one reaction system. Using this new method, we profiled and compared the DNA hydroxymethylomes of mouse ES cells (ESCs) and mouse ESCs-derived neural progenitor cells (NPCs). 5hmC levels around TSS in either ESCs or NPCs had negative correlation with gene expression levels，while 5hmC levels at gene body regions had different correlation with gene expression, depending on cell types. We identified differential hydroxymethylated regions (DHMRs) by comparing the 5hmC density of all 5hmC peaks between ESCs and NPCs. Many selected DHMRs (e.g., Ankrd23, Hist1h2aa, Fbxw7, and Epha2) were validated by hMeDIP-qPCR. Furthermore, we analyzed the relationship between the alteration of DNA hydroxymethylation and the gene expression change during neural differentiation, and our data suggest that both up- and down-regulated genes exhibited dramatic decrease in 5hmC during neural differentiation while the alteration of 5hmC had weak positive correlation with the changes in gene expression. Taken together, our data demonstrate that quantitative hMeDIP-seq is a powerful approach for genome-wide comparison of DNA hydroxymethylation across multiple samples. Importantly, the DHMRs between ESCs and NPCs uncovered in this study may provide clues for further investigation of the function of 5hmC in gene regulation and neural differentiation. Comparision of the genome-wide distribution of 5hmC in mouse embryonic stem cells and mouse ES-derived neural progenitor cells.

Dataset Information

Cellular generation of 5-Hydroxymethylcytosine by redox-active chemicals via an unprecedent non-enzymatic mechanism

Publications

Redox-active quinones induces genome-wide DNA methylation changes by an iron-mediated and Tet-dependent mechanism.

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