Project description:Epigenetic modifications, such as cytosine methylation and histone modification, have been shown involved in the pathology of ischemic brain injury. Recent works have implicated 5-hydroxymethylcytosine (5hmC), a DNA base derived from 5-methylcytosine (5mC) through the oxidation by Ten-Eleven Translocation (TET) enzymes, in DNA methylation-related plasticity. In this study we show that 5hmC abundance could be induced to increase by ischemia injury. Genome-wide profiling of 5hmC identified differentially hydroxymethylated regions (DhMRs) associated with ischemic injury and DhMRs were found enriched among the genes involved in cell junction, neuronal morphogenesis and neurodevelopment. These data together suggest that 5hmC modification could serve as a potential therapeutic target for the treatment of ischemic stroke. To determine the genome-wide 5hmC distribution in both ischemic injury (I/R) and control mice (C57BL/6), we employed a previously established chemical labeling and affinity purification method, coupled with high-throughput sequencing (Song et al, Nature Biotechnology, 2011). The ischemic or matched control brain tissues from three pairs of ischemic mice and control mice were used for the analyses.
Project description:Epigenetic modifications, such as cytosine methylation and histone modification, have been shown involved in the pathology of ischemic brain injury. Recent works have implicated 5-hydroxymethylcytosine (5hmC), a DNA base derived from 5-methylcytosine (5mC) through the oxidation by Ten-Eleven Translocation (TET) enzymes, in DNA methylation-related plasticity. In this study we show that 5hmC abundance could be induced to increase by ischemia injury. Genome-wide profiling of 5hmC identified differentially hydroxymethylated regions (DhMRs) associated with ischemic injury and DhMRs were found enriched among the genes involved in cell junction, neuronal morphogenesis and neurodevelopment. These data together suggest that 5hmC modification could serve as a potential therapeutic target for the treatment of ischemic stroke.
Project description:Our results revealed that hypoxic-ischemic brain injury decreased the overall 5hmC abundance in rat temporal cortex, and these results suggest that 5hmC modifications are involved in the cerebral palsy pathogenesis.
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.
Project description:Methylation at the 5-position of cytosine is a well-studied epigenetic pathway. In addition to 5-methylcytosine (5mC), substantial amounts of 5-hydroxymethylcytosine (5hmC) also referred to as the 6th DNA base, have been detected in certain tissues, most notably the brain. However, the genomic distribution of this cytosine modification is unknown. Here, we have developed an immunoprecipitation technique (5hmC-IP) to examine the occurrence of 5hmC in human DNA from brain frontal lobe tissue. The distribution of 5hmC was compared to that of 5mC. We show that 5hmC is more selectively targeted to genes than is 5mC. 5hmC is found at promoters and is particularly enriched in intragenic regions (gene bodies) but is largely absent from non-gene regions. 5hmC peaks at transcription start sites did not correlate with gene expression levels or H3K4me3 peaks. However, presence of 5hmC in gene bodies was more positively correlated with gene expression levels than was presence of 5mC. Promoters of testis-specific genes showed strong 5mC peaks in brain DNA but were almost completely devoid of 5hmC. Our data provide a first overview of the genomic distribution of 5hmC in human brain and will set the stage for further functional characterization of this novel DNA modification. Comparison between 5hmC and 5mC profiles
Project description:Ten-Elven Translocation (TET) proteins oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytonsie (5hmC). Our recent work found a decline in global 5hmC level in mouse kidney insulted by ischemia reperfusion (IR). However, the genomic distribution of 5hmC in mouse kidney and its relationship with gene expression remain elusive. Here, we profiled the DNA hydroxymethylome of mouse kidney by hMeDIP-seq and revealed that 5hmC is enriched in genic regions but depleted from intergenic regions. Correlation analyses showed that 5hmC enrichment in gene body is positively associated with gene expression level in mouse kidney. Moreover, IR injury-associated genes (both up- and down-regulated genes during renal IR injury) in mouse kidney exhibit significantly higher 5hmC enrichment in their gene body regions when compared to those un-changed genes. Collectively, our study not only provides the first DNA hydroxmethylome of kidney tissues but also suggests that DNA hyper-hydroxymethylation in gene body may be a novel epigenetic mark of IR injury-associated genes. Eamination of the genome-wide distribution of 5-hydroxymethylcytosine in mouse kidney tissues
Project description:We developed a novel approach, J-binding protein 1 sequencing (JBP1-seq), that combines the benefits of an improved recombinant JBP1 protein, Nextera-based library construction, and nextgeneration sequencing (NGS) for genome-wide profiling of 5-hydroxymethylcytosine (5hmC). Compared with the original JBP1, this new recombinant JBP1 was biotinylatedin vivo and conjugated to magnetic beads via biotin-streptavidin interactions. These modifications allowed a more efficient and consistent pull-down of β-glucosyl-5-hydroxymethylcytosine (β-glu-5hmC), and sequence-ready libraries can be generated within 4.5 hours from DNA inputs as low as 50 ng. 5hmC enrichment of human brain DNA using the new JBP1 resulted in over 25,000 peaks called, which is significantly higher than the 4,003 peaks enriched using the old JBP1. Comparison of the technical duplicates and validations with other platforms indicated the results are reproducible and reliable. Thus, JBP1-seq provides a fast, efficient, cost-effective method for accurate 5hmC genome-wide profiling. An improvement of JBP1-Seq
Project description:Methylation at the 5-position of cytosine is a well-studied epigenetic pathway. In addition to 5-methylcytosine (5mC), substantial amounts of 5-hydroxymethylcytosine (5hmC) also referred to as the 6th DNA base, have been detected in certain tissues, most notably the brain. However, the genomic distribution of this cytosine modification is unknown. Here, we have developed an immunoprecipitation technique (5hmC-IP) to examine the occurrence of 5hmC in human DNA from brain frontal lobe tissue. The distribution of 5hmC was compared to that of 5mC. We show that 5hmC is more selectively targeted to genes than is 5mC. 5hmC is found at promoters and is particularly enriched in intragenic regions (gene bodies) but is largely absent from non-gene regions. 5hmC peaks at transcription start sites did not correlate with gene expression levels or H3K4me3 peaks. However, presence of 5hmC in gene bodies was more positively correlated with gene expression levels than was presence of 5mC. Promoters of testis-specific genes showed strong 5mC peaks in brain DNA but were almost completely devoid of 5hmC. Our data provide a first overview of the genomic distribution of 5hmC in human brain and will set the stage for further functional characterization of this novel DNA modification.
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.