The Epigenetic Mechanisms Of Renal Injury Induced By Xylene

Part 1. Genome-wide DNA methylation analysis of xylene induced renal injuries in ratsObjective:To observe the DNA methylation change in xylene induced renal tubular and glomerular injuries in rats.Methodolgy:Renal tubular and glomerular tissues from rat models with xylene induced renal injuries were selected with standard sieving method. The reduced-representation-bisulfite-sequencing was used to detect the whole-genome scale methylation analysis of the renal tubular and glomeruli. Promoter and CGI coverage and sequencing depth, promoter and CGI methylation level on average of renal tubular and glomerular tissue were analyzed. The differences between DNA methylation area (DMR), gene ontology (GO) analysis and Kyoto encyclopedia (KEGG) gene were analyzed. Pyrophosphate sequencing were used to detect the methylation of PPARy promoter region in CpG sites the binding site of transcription factors ADR1. The expression of PPAR y were detected by using Western blotting and RT-PCR.Results:Three kinds of cytosine methylation (CG/CHG/CHH) were mainly distributed in the promoter region and CGI of rat renal tubular and glomerular tissue. The average methylation level of promoter and CGI region of renal tubule or rat models was 2.32% and 3.91% respectively. Most methylation of cytosine was happened on CG. The number and distribution of differentially methylated region (DMR) are different between the model rats and controls. The results of Kyoto encyclopedia (KEGG) gene and genome analysis showed that upregulated DMR-related genes of tubular tissue in the models were associated with PPARy pathways. Pyrophosphate sequencing results showed that the methylation level of transcription factors ADR1 binding CpG site of PPARy promoter region were varied in the rats with xylene induced renal injuries. Three of eight rats were increased with 32%,38%,36% respectively while the methylation level was steady with 30±1.87% in the control group. PPARy expressions in the renal tubules of rats with xylene induced injuries were reduced at 4 weeks, however, were significantly increased at 6, and 12weeks. Conclusion:The whole-genome methylation profiles of tubules and glomeruli of the rats with xylene induced renal injuries were obtained. PPARy pathway was involved in the renal tubular injuries induced by xylene. The change of methylation level of ADR1 binding CpG site in the PPARy promoter region was related with xylene induced injuries.Part 2. Genome-wide histone methylation analysis of xylene induced renal injuries in ratsObjective:To observe the histone methylation change in xylene induced renal tubular injuries in rats.Methodolgy:ChIP-sequencing analysis of H3K9 and H3K27 in renal tubular tissues was performed. After the sequencing platform generated the sequencing images, the stages of image analysis and base calling were performed using Off-Line Basecaller software (OLB V1.8). After passing Solexa CHASTITY quality filter, the clean reads were aligned to Rat genome (UCSC RN5) using BOWTIE software (V2.1.0). Aligned reads were used for peak calling of the ChIP regions using MACS V2.09. Statistically significant ChIP-enriched regions (peaks) were identified by comparison to a Poisson background model (Cut-off p-value=10-5). And differentially enriched regions were performed by diffReps with p-value threshold of 10-3. The peaks/differentially enriched region in samples were annotated and analyzed by GO and KEGG pathway.Results:All ChIP-seq data were aligned well with the reference genome. Peak enrichment (H3K9, H3K27) of tubular tissues in the model rats were distributed at the intergenic regions, intron, upstream and promoter of genes, but rarely at the exon region. ChIP-Seq signals were mainly distributed at the transcription start site (TSS) around 2kb. GO analysis showed that the H3K9 enrichment peak (down) of the experimental group were related with the detection and identification of chemical stimulation, whereas the H3K9 enrichment peak (up) were related with metabolism, apoptosis and MAPK signaling pathway. The H3K27 peak (down) of the were related with metabolism, apoptosis and Wnt signaling pathway, whereas the H3K27 peak (up) were related to metabolism, gene expression and so on. KEGG analysis showed that H3K9, H3K27 were involved in the proteasome, MAPK, apoptosis, Wnt, renal cell carcinoma and glucose metabolism signaling pathways. It was found by visualizing the ChIP-Seq profiles of each sample in UCSC Genome Browser that there was H3K9 H3K27 enrichment peak in the PPARy promoter region of chromosome 18.Conclusion:The whole-genome histone methylation profiles (H3K9/H3K27) of tubules and glomeruli of the rats with xylene induced renal injuries were obtained. There was H3K9, H3K27 enrichment peak in the PPARy promoter region, indicating that histone methylation was also involved in the xylene induced injuries.