The Dynamic Change Of5-hydroxymethylcytosine In Mouse Kidney Insulted By Ischemia-reperfusion Injury And Its Relationship With Gene Transcription

Background:DNA methylation(5mC) and DNA hydroxymethylation(5hmC) are important forms of epigenetic modifications. As5mC and5hmC are in tight connection with modulation of gene transcription, several literatures focused on the disparity of5mC and5hmC in genomic DNA from various tissues and organs recently. Known that the global levels of5mC and5hmC are tissue specific, still there is little knowledge about the distribution of5mC or5hmC in gonomic-feature regions. In this context, we validated the tissue-specificity of global content of5mC and5hmC first. Based on that, we sequenced the DNA methylome and hydroxymethlome to explore the pattern of5mC/5hmC distribution in different mouse organs. Methods:The different organs of normal male C57mice were collected for genomic DNA extraction. Using DNA dot blot assay, the global content of5mC and5hmC were quantified. The DNA fragments enriched5mC or5hmC were immunoprecipitated by specific antibodies and sequenced genome-widely. Compare of DNA methylome and hydroxymethylome were performed by bioinformatic analysis software.Results:The global5hmC content of normal mouse organs is quite variable, does not correlate with global5mC content, which were proven by genome-wide sequencing. The distribution of5hmC was compared to that of5mC. We demonstrated that5hmC is particularly enriched in gene bodies, but5mC is more selectively targeted to intergenic regions. The enrichment tendency of5mC or5hmC in gene body shows no discrepancy among different mouse organs. Similar results have also been obtained in promoter and enhancer regions.Conclusions:The global5hmC level of normal mouse organs is much more variable than5mC and the genome wide distribution of5hmC shows distinguishing feature comparing to5mC. However, the enrichment pattern of5mC or5hmC in genomic-feature regions is stable, showing no tissue specificity. Background:Ischemia reperfusion (IR) is a frequent pathological injury to the perioperative patients. The molecularmechanism underlying IR injury is still not well characterized. In this study, we investigated the effectof IR injury on DNA hydroxymethylation in mouse kidney.Methods:The left kidney of male C57mice was subjected to40min ischemia and the right kidneys was exposed as control. According to the time of reperfusion, ten mice were equally divided into4hours group and24hours group. Dot blot and immunochemistry analysis were used to compare the content of5-methylcytosine (5mC) and5-hydroxymethylcytosine (5hmC). Using hMeDIP-qPCR analysis, the locus-specific distribution of5hmC were quantified. The mRNA expression of tested genes were measured by RT-qPCR analysis.Results:Dot blot and immunochemistry analysisshowed that the global level of5hmC was reduced in mouse kidney insultedby IR; however, the5mC level had no significant change. hMeDIP-qPCR validated thatIR injury also decreased the5hmC enrichment at promoter regions of Cxc110and Ifngr2genes. RT-qPCRanalysis revealed that the mRNA levels of Cxc110and Ifngr2increased in IR-injured kidney. In addition, mRNA expression of both Tet1and Tet2but not Tet3was dramatically downregulated in IR-injured kidney.Conclusions:Taken together, our data provided the first evidence that IR injury influences DNA hydroxymethyla-tion and Tet gene expression in mouse kidney, which may contribute to the regulation of genetranscription during renal IR injury. Background:The number of studies addressing dynamic changes and effects of epigenetic modifications in ischemia reperfusion(IR) injury has steadily increased. It is proven that epigenetic mechanisms are greatly involved in the alterations of gene transcription. As shown in Part2, the global5hmC content of mouse kidney insulted by IR was significantly reduced, without change of global5mC level. The change of locus-specific5hmC content was in correlation with modulation of gene expression. So we observed the impact of IR on genome-wide distribution of5mC and5hmC and explore the relationship between5mC/5hmC and gene expression by genome-wide bioinformatic analysis combined with cDNA microarray data.Methods:The cDNA microarray technique was used for assessing the differential expression of mouse kidney insulted by IR. The DNA fragements with5mC or5hmC was enriched by DNA-antibody immunoprecipatation and sequenced, respectively. The seq data was mapped with reference genome. The correlation between the enrichment of5mC/5hmC and gene expression was analyzed by bioinformatic software(eg. MAT) genome-widely.Results:IR has great impact on gene expression of mouse kidney and induces gene transcription reprogram in a wide range. The differential expression of gene was in agreement with the process of IR injury. But the distribution pattern of5mC/5hmC was not affected by IR. Correlation analysis of gene expression level and seq data showed that the5mC content in transcription start site and the5hmC content in gene body was abundant in actively transcribed genes. Comparison of dynamic change of gene expression and5mC/5hmC content in genomic-specific regions indicated that genes with high level of5hmC in gene body regions tend to change their expression levels, but there is no such correlation between5mC content and gene expression.Conclusions:Though IR accompanied with great alteration of gene transcription, the distribution pattern of5mC/5hmC was not changed. In mouse kidney injured by IR, the genes with high content of5hmC in the upstream of transcription start sites and gene body regions tend to change their expression levels.