Base-Resolution Maps of 5-Methylcytosine and 5-Hydroxymethylcytosine in Dahl S Rats

Abstract

Analysis of 5-hydroxymethylcytosine (5hmC) at single-base resolution has been largely limited to studies of stem cells or developmental stages. Given the potential importance of epigenetic events in hypertension, we have analyzed 5hmC and 5-methylcytosine (5mC) at single-base resolution in the renal outer medulla of the Dahl salt-sensitive SS rat and examined the effect of disease-relevant genetic or environmental alterations on 5hmC and 5mC patterns. Of CpG sites that fell within CpG islands, 11% and 1%contained significant 5mC and 5hmC, respectively. 5mC levels were substantially higher for genes with lower mRNA abundance and showed a prominent nadir around the transcription start site. In contrast, 5hmC levels were higher in genes with higher expression. Substitution of a 12.9 Mbp region of chromosome 13, which attenuates the hypertensive and renal injury phenotypes in SS rats, or exposure to a high-salt diet, which accelerates the disease phenotypes, was associated with differential 5mC or 5hmC in several hundred CpG islands. Nearly 80% of the CpG islands that were differentially methylated in response to salt and associated with differential mRNA abundance were intragenic CpG islands. The substituted genomic segment had significant cis effects on mRNA abundance but not DNA methylation. The study established base-resolution maps of 5mC and 5hmC in an in vivo model of disease and revealed several characteristics of 5mC and 5hmC important for understanding the role of epigenetic modifications in the regulation of organ systems function and complex diseases.

Stability of Global Methylation Profiles of Whole Blood and Extracted DNA Under Different Storage Durations and Conditions

“…A total of 28 DNA samples were prepared as 33 libraries with five samples from G1, G2, or G3 replicated to control for the variation across sequencing lanes (Table 1). Libraries for RRBS were generated according to protocols described by Gu et al [12], followed by sequencing (per Illumina protocols) with modifications to allow multiplexing [13]. The libraries underwent cluster generation using TruSeq Analysis of methylation sequencing data Methylation sequence reads were obtained from Illumina HiSeq 2500 with paired-end reads of 100 bp in read length.…”

“…The libraries underwent cluster generation using TruSeq Analysis of methylation sequencing data Methylation sequence reads were obtained from Illumina HiSeq 2500 with paired-end reads of 100 bp in read length. Prior to the analysis, the raw sequences were processed and trimmed as previously described [13,14]. The trimmed short reads were mapped to the human reference genome (hg19) downloaded from the UCSC Genome Browser (http://hgdownload.soe.ucsc.edu/goldenPath/hg19/database/) using Bismark (v0.16.1) [15].…”

MicroRNA-489 Induction by Hypoxia–Inducible Factor–1 Protects against Ischemic Kidney Injury

“…60 Briefly, mRNA (cDNA) libraries were built with 5 mg RNA followed by cluster generation and sequencing. The RNA-seq data were analyzed by an in-house pipeline, which included reads mapping and alignment, transcript construction, quantification of transcript abundance, and identification of differential expression using Bowtie (http:// bowtie-bio.sourceforge.net/index.shtml), Tophat v2 (http://tophat.cbcb.umd.edu/), and Cufflinks (http://cufflinks.cbcb.umd.edu/).…”

Gut microbiota in hypertension

“…Diet, including salt and gut microbiota can influence epigenetics (116–119); salt can increase oxidative stress (120, 121) and oxidative stress can influence epigenetics (e.g., histone deacetylase activity) (122). Lysine-specific demethylase 1 regulates histone methylation by demethylating histone H3 at lysine residues 4 and 9 and is involved in salt-sensitive hypertension (7, 8, 114, 118, 123). Certain miRs have been implicated in salt sensitivity and inverse salt sensitivity of blood pressure (114, 124–128).…”