Unknown,Transcriptomics,Genomics,Proteomics

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Transcription factor occupancy is linked to DNA methylation turnover at active regulatory regions [MeDIP-Seq]

ABSTRACT: Genome wide analysis revealed that distal regulatory elements form Low Methylated Regions (LMRs). Even though transcription factor binding is required for LMR formation, we show for the test case CTCF that actual occupancy does not distinguish DNA methylation states. However, in line with a dynamic model of binding and DNA methylation turnover, we find that the product of active demethylation, 5-hydroxymethylcytosine (5hmC), is enriched at LMRs. 5hmC is present at active regulatory regions in stem and somatic cells and as a result a substantial fraction of changes in 5hmC occurs at LMRs. This suggests that transcription factor binding mediates active turnover of DNA methylation as an integral part of reprogramming of regulatory regions. hMeDIP sequencing in mouse embryonic stem cells (ES) and derived neuronal progenitors (NP).

ORGANISM(S): Mus musculus

SUBMITTER: Dirk Schuebeler

PROVIDER: E-GEOD-39738 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Gene-environment interactions mediated at the epigenetic level may provide an initial step in delivering an appropriate response to environmental changes. 5-hydroxymethylcytosine (5hmC), a DNA base derived from 5-methylcytosine (5mC), accounts for ~40% of modified cytosine in brain and has been implicated in DNA methylation-related plasticity. To identify the role of 5hmC in gene-environment interactions, we exposed both young (6-week-old) and aged (18-month-old) mice to both an enriched environment and a standard environment. Exposure to EE significantly improves learning and memory in aged mice and reduces 5hmC abundance in mouse hippocampus. Furthermore, we mapped the genome-wide distribution of 5hmC and found that the alteration of 5hmC modification occurred mainly at gene bodies. In particular, genes involved in axon guidance are enriched among the genes with altered 5hmC modification. These results together suggest that environmental enrichment could modulate the dynamics of 5hmC in hippocampus, which could potentially contribute to improved learning and memory in aged animals. To identify the role of 5hmC in gene-environment interactions, we exposed both young (6-week-old) and aged (18-month-old) C57B/6 mice to both an enriched environment (EE) and a standard environment. Exposure of mice to EE was achieved by keeping a group of mice in the EE chamber, which is larger than the standard cage, includes novel objects, such as toys of varied color and texture, tunnels, an exercise wheel for voluntary exercise, and extra bedding material, along with free access to food and water. Daily exposure to EE was kept at 5 hours during the daylight cycle for 4 consecutive weeks. Control animals were kept in their small cages that are used as standard housing cages (CE) containing bedding, food and access to water. YC, young mice exposing to control environment, YE, young mice exposing to enriched environment, AC, aged mice exposing to control environment, AE, aged mice exposed to enriched environment. Please note that 5hmC-containing DNA enrichment method was inspired by a unique character of beta-glucotransferase that can specifically add glucose to 5hmC modification. With a modified glucose conjugated with biotin, we are able to purify the 5hmC-containing DNA by biotin-streptavidin-based immunoprecipitation.

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.

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Transcription factor occupancy is linked to DNA methylation turnover at active regulatory regions [MeDIP-Seq]

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